**Nitrogen and Environment**

**Chapter 1**

**Provisional chapter**

**Inter‐ and Intra‐Annual Variability of Nitrogen**

**Inter**‐ **and Intra**‐**Annual Variability of Nitrogen** 

M. Mercedes Taboada‐Castro, Ricardo Arias and

Taboada-Castro, Ricardo Arias and M. Teresa

Additional information is available at the end of the chapter

nitrogen (TN), nitrate‐nitrogen (N‐NO<sup>3</sup>

the period, and lower values in summer.

Additional information is available at the end of the chapter

M. Luz Rodríguez‐Blanco, M. Mercedes

M. Luz Rodríguez‐Blanco,

Taboada-Castro

**Abstract**

tions of TN, N‐NO<sup>3</sup>

NW Spain

**1. Introduction**

M. Teresa Taboada‐Castro

http://dx.doi.org/10.5772/intechopen.69996

**Concentrations in the Headwaters of the Mero River**

This study examines the inter‐ and intra‐annual variability of different forms of N [total

waters of a rural headwater catchment in Galicia (NW Spain) during a 5‐year period, cov‐ ering 2004–2009 water years (October–September). Daily time series were used to verify the temporal variability and to characterize the nitrogen pollution. The TN concentrations were low, although the values constantly exceeded the critical range (0.5–1.0 mg L−1) over which potential risk of eutrophication of water systems exists. Nitrate was the predomi‐ nant form of nitrogen in the river throughout the study period, accounting for 82–85% of the TN. Significant differences were found for different forms of N between water years and seasons, indicative of wide inter‐ and intra‐annual variability of nitrogen concentra‐ tions, mainly related to rainfall and flow oscillations. The seasonal pattern in the concentra‐

catchments, with higher concentrations in winter, when variability was also the highest in

**Keywords:** nitrogen, variability, rural headwater catchment, temperate humid climate,

) and total Kjeldahl nitrogen (TKN)] in stream

**Concentrations in the Headwaters of the Mero River**

DOI: 10.5772/intechopen.69996

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

© 2018 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

and TKN in stream water was similar to many humid and temperate

and reproduction in any medium, provided the original work is properly cited.

A large number of biological, chemical and physical processes are involved in the produc‐ tion, transformation and translocation of N compound in terrestrial and aquatic systems [1]. The most important natural factors controlling nitrogen losses from soils to surface

**Provisional chapter**

## **Inter‐ and Intra‐Annual Variability of Nitrogen Concentrations in the Headwaters of the Mero River Concentrations in the Headwaters of the Mero River**

**Inter**‐ **and Intra**‐**Annual Variability of Nitrogen** 

DOI: 10.5772/intechopen.69996

M. Luz Rodríguez‐Blanco, M. Mercedes Taboada‐Castro, Ricardo Arias and M. Teresa Taboada‐Castro Taboada-Castro, Ricardo Arias and M. Teresa Taboada-Castro Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

M. Luz Rodríguez‐Blanco, M. Mercedes

http://dx.doi.org/10.5772/intechopen.69996

#### **Abstract**

This study examines the inter‐ and intra‐annual variability of different forms of N [total nitrogen (TN), nitrate‐nitrogen (N‐NO<sup>3</sup> ) and total Kjeldahl nitrogen (TKN)] in stream waters of a rural headwater catchment in Galicia (NW Spain) during a 5‐year period, cov‐ ering 2004–2009 water years (October–September). Daily time series were used to verify the temporal variability and to characterize the nitrogen pollution. The TN concentrations were low, although the values constantly exceeded the critical range (0.5–1.0 mg L−1) over which potential risk of eutrophication of water systems exists. Nitrate was the predomi‐ nant form of nitrogen in the river throughout the study period, accounting for 82–85% of the TN. Significant differences were found for different forms of N between water years and seasons, indicative of wide inter‐ and intra‐annual variability of nitrogen concentra‐ tions, mainly related to rainfall and flow oscillations. The seasonal pattern in the concentra‐ tions of TN, N‐NO<sup>3</sup> and TKN in stream water was similar to many humid and temperate catchments, with higher concentrations in winter, when variability was also the highest in the period, and lower values in summer.

**Keywords:** nitrogen, variability, rural headwater catchment, temperate humid climate, NW Spain

## **1. Introduction**

A large number of biological, chemical and physical processes are involved in the produc‐ tion, transformation and translocation of N compound in terrestrial and aquatic systems [1]. The most important natural factors controlling nitrogen losses from soils to surface

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons

waters are climatology, topography and hydrology, and soil characteristics. For example, the temperature acts on N transformation processes, whereas rainfall distribution and intensity plays a decisive role in the leachate. The distribution of water flow over and through the soil has a great influence on nitrate discharges into the waters [2, 3]. These factors are dynamic and highly variable in space and time, and their action can result in heavy discharges of nitrogen into the watercourses. The impact of natural factors on nitro‐ gen losses can be modified by anthropogenic factors, including soil use and crop rotation, the application of slurry and other fertilizers, as well as the amount and method of appli‐ cation [4–6]. The diffuse pollution of water resources by nitrogen is a problem that affects the whole world, with agriculture being the main primary source of diffuse pollution of water resources [6, 7]. Ultimately, the interaction between the factors involved in the N loss from the soils and those related to N transport determine the final losses of nitrogen to surface waters, as well as the N concentrations and N forms in the rivers; hence, these interactions must be understood in order to manage diffuse losses of agricultural nutrients efficiently.

In this context, the aim of this study was to investigate the inter‐annual and seasonal varia‐

Inter- and Intra-Annual Variability of Nitrogen Concentrations in the Headwaters of the Mero...

ters of Mero River basin, a typical groundwater‐dominated rural catchment in Galicia (NW Spain) located upstream from the Cecebre reservoir, which has unique characteristics, since it is important both strategically (the only drinking water source of the city of A Coruña and its metropolitan area, approximately 450,000 inhabitants) and ecologically (site of commu‐ nity importance included in the Nature 2000 network). Additionally, the data collected in the

The study was conducted on a small headwater catchment of the Mero River basin (Galicia,

approximately 10 km. The geology of the catchment is homogeneous and dominated by basic metamorphic schist of the Órdenes Complex [17]. The main soil types are umbrisols and cambisols [18], which are relatively deep because of heavy weathering. The surface soil layer has a silt and silt‐loam texture, high organic content (2.5–11.5%) and acid pH (4.5–5.6).

and TKN) concentrations in the headwa‐

http://dx.doi.org/10.5772/intechopen.69996

5

and a total length of

tions of different forms of nitrogen (TN, N‐NO<sup>3</sup>

**2. Material and methods**

**2.1. Study site description**

catchment study are of great use in evaluating of several models.

NW Spain; **Figure 1**). The Corbeira has a catchment area of 16 km<sup>2</sup>

**Figure 1.** Localization of the study area, land‐use map and general view of the Corbeira catchment.

The dynamics and distribution of N forms in the aquatic system is not easy to understand since N concentrations vary markedly from one catchment to another, depending not only on land use, agricultural management, the type and quantity of fertilizer applied but also on the climatological and hydrological characteristics of the basins [8]. Several authors relate variability in N concentrations to catchment size and, in general, attribute small spatial and temporal variability to small river basins [9, 10]. In addition, the N concentrations display wide variability within the same catchment. For example, for a given rainfall event, the N concentrations may differ greatly at different times of the year [11, 12]. Climate and land use change is expected to alter the transfer of nutrients from land to water, although the results are controversial [13–16]. Some studies show that nitrogen loads will increase under climate change and may be affected equally by climate and land use change [15]. However, other studies indicate that nitrogen loads will decrease under climate change mainly due to decreases in runoff, which may be runoff, may be more affected by climate change than changes in land use [13, 16].

A good knowledge of the temporal variation of nitrogen concentrations is a key for under‐ standing the N transport at the catchment scale, for modelling their behaviour and, ulti‐ mately for designing agricultural practices management to reduce potential N losses in a more effective manner under current and changing climate and land uses conditions. Therefore, it is essential to have good data on N for long‐term periods. Most of studies on temporal changes of N concentration were mainly focused on catchments with a marked anthropogenic influence and only on one form of N, generally nitrate [9, 10]. Less attention has been paid to understanding the N dynamics at headwater streams with minimal human impact, even though they serve as a reference point against which data from more disturbed catchments can be compared. All this makes clear the need for more detailed long‐term stud‐ ies in small, minimally disturbed, headwater catchments to clarify the behaviour of different forms of N and understand the factors governing the temporal variability of N concentra‐ tions. Therefore, there is a clear need to monitor N concentrations in detail over a long‐term period to investigate the N dynamics.

In this context, the aim of this study was to investigate the inter‐annual and seasonal varia‐ tions of different forms of nitrogen (TN, N‐NO<sup>3</sup> and TKN) concentrations in the headwa‐ ters of Mero River basin, a typical groundwater‐dominated rural catchment in Galicia (NW Spain) located upstream from the Cecebre reservoir, which has unique characteristics, since it is important both strategically (the only drinking water source of the city of A Coruña and its metropolitan area, approximately 450,000 inhabitants) and ecologically (site of commu‐ nity importance included in the Nature 2000 network). Additionally, the data collected in the catchment study are of great use in evaluating of several models.

## **2. Material and methods**

#### **2.1. Study site description**

waters are climatology, topography and hydrology, and soil characteristics. For example, the temperature acts on N transformation processes, whereas rainfall distribution and intensity plays a decisive role in the leachate. The distribution of water flow over and through the soil has a great influence on nitrate discharges into the waters [2, 3]. These factors are dynamic and highly variable in space and time, and their action can result in heavy discharges of nitrogen into the watercourses. The impact of natural factors on nitro‐ gen losses can be modified by anthropogenic factors, including soil use and crop rotation, the application of slurry and other fertilizers, as well as the amount and method of appli‐ cation [4–6]. The diffuse pollution of water resources by nitrogen is a problem that affects the whole world, with agriculture being the main primary source of diffuse pollution of water resources [6, 7]. Ultimately, the interaction between the factors involved in the N loss from the soils and those related to N transport determine the final losses of nitrogen to surface waters, as well as the N concentrations and N forms in the rivers; hence, these interactions must be understood in order to manage diffuse losses of agricultural nutrients

The dynamics and distribution of N forms in the aquatic system is not easy to understand since N concentrations vary markedly from one catchment to another, depending not only on land use, agricultural management, the type and quantity of fertilizer applied but also on the climatological and hydrological characteristics of the basins [8]. Several authors relate variability in N concentrations to catchment size and, in general, attribute small spatial and temporal variability to small river basins [9, 10]. In addition, the N concentrations display wide variability within the same catchment. For example, for a given rainfall event, the N concentrations may differ greatly at different times of the year [11, 12]. Climate and land use change is expected to alter the transfer of nutrients from land to water, although the results are controversial [13–16]. Some studies show that nitrogen loads will increase under climate change and may be affected equally by climate and land use change [15]. However, other studies indicate that nitrogen loads will decrease under climate change mainly due to decreases in runoff, which may be runoff, may be more affected by climate change than

A good knowledge of the temporal variation of nitrogen concentrations is a key for under‐ standing the N transport at the catchment scale, for modelling their behaviour and, ulti‐ mately for designing agricultural practices management to reduce potential N losses in a more effective manner under current and changing climate and land uses conditions. Therefore, it is essential to have good data on N for long‐term periods. Most of studies on temporal changes of N concentration were mainly focused on catchments with a marked anthropogenic influence and only on one form of N, generally nitrate [9, 10]. Less attention has been paid to understanding the N dynamics at headwater streams with minimal human impact, even though they serve as a reference point against which data from more disturbed catchments can be compared. All this makes clear the need for more detailed long‐term stud‐ ies in small, minimally disturbed, headwater catchments to clarify the behaviour of different forms of N and understand the factors governing the temporal variability of N concentra‐ tions. Therefore, there is a clear need to monitor N concentrations in detail over a long‐term

efficiently.

4 Nitrogen in Agriculture - Updates

changes in land use [13, 16].

period to investigate the N dynamics.

The study was conducted on a small headwater catchment of the Mero River basin (Galicia, NW Spain; **Figure 1**). The Corbeira has a catchment area of 16 km<sup>2</sup> and a total length of approximately 10 km. The geology of the catchment is homogeneous and dominated by basic metamorphic schist of the Órdenes Complex [17]. The main soil types are umbrisols and cambisols [18], which are relatively deep because of heavy weathering. The surface soil layer has a silt and silt‐loam texture, high organic content (2.5–11.5%) and acid pH (4.5–5.6).

**Figure 1.** Localization of the study area, land‐use map and general view of the Corbeira catchment.

The C/N of the upper layer is almost 10 (9.2–12). The Corbeira catchment is minimally disturbed by human activity; it is predominantly covered by forest (65%) and agricultural land (30%). The agricultural area is primarily used for meadows and natural pastures (83% of agricultural land), with the latter being located in more or less humid areas, mainly in flood areas, where they produce fodder for consumption in green, dry or as silage. Croplands comprise 3.8% of the catchment area and are mainly used for fodder crops, such as maize, and for winter cereals. Other crops—potatoes, turnips, vegetables or fruit trees are consigned to small family farms oriented to self‐consumption. Finally, a small percent‐ age (about 5%) of the catchment is made up of areas occupied by buildings and roads. Agriculture and forestry are interspersed throughout the catchment (**Figure 1**). Forest areas (dominated mainly by commercial eucalyptus and pines) occupy areas with steep slopes. Two distinct agricultural areas can be distinguished in the catchment (**Figure 1**): one is located in the upper part of the catchment, where the slopes are moderately steep, with some cropland near the drainage network and the largest cattle load in the study area, which presents a great risk of erosion, with sediments and nutrients delivery to the stream. The other zone, located on the right margin of the river, in the middle‐lower part of the catchment, is where most agricultural activity in the catchment takes place and is where the widest area of cultivated land is found, some with good connectivity to the drainage network. It also highlights the mosaic vegetation in agricultural areas, with most of the croplands bordered by meadows and even, in some cases, by small stone walls, which can obstruct connectivity of croplands with the drainage network, resulting in a decrease in the potential pollutant effect in surface waters.

hydrological regime is pluvial, with a seasonal pattern in hydrological response characterized by a dry summer period lasting up to first rainfall events in autumn, when the soil reserves recover, followed by a wet period that extends through the autumn and winter seasons [19]. Consequently, the maximum monthly mean discharge is observed in March (0.31 m<sup>3</sup>

Inter- and Intra-Annual Variability of Nitrogen Concentrations in the Headwaters of the Mero...

baseflow index, a measure of the proportion of stream flow delivered from groundwater, is about 0.80, indicative of a high contribution of groundwater to stream flow. A more detailed description of the study catchment characteristics can be found in the study of Rodríguez‐

Stream discharge and N concentrations have been measured at the catchment outlet from October 2004 to September 2009. Discharge was calculated from measuring water levels (ISCO 720) using the rating curve. Water level data were measured continuously (every min) and recorded at 10‐min intervals. Water samples were collected during both baseflow and runoff events. Under baseflow conditions, water samples were manually collected every 10–15 days, whereas under runoff events, they were collected using an automatic sampler (ISCO 6712‐FS) at short‐time intervals, depending on the characteristics of the runoff events

Stream water samples were analysed for total Kjeldahl nitrogen (TKN), nitrate (NO<sup>3</sup>

centrations were measured using an ammonia‐selective electrode. The NO<sup>2</sup>

variability (four levels: autumn, winter, spring and summer) on N (N‐NO<sup>3</sup>

niacal and organic nitrogen, were determined by Kjeldahl digestion of unfiltered samples, following the American Public Health Association method [22]. After filtration (0.45 µm), NO<sup>3</sup>

concentrations were analysed by capillary electrophoresis, whereas the NH<sup>4</sup>

Daily flow‐weighted mean N concentrations were used to analyse the temporal variability and to characterize the N pollution. Data were organized into four seasons: autumn (October, November and December), winter (January, February and March), spring (April, May and June) and summer (July, August and September). For annual data, the hydrological year was used instead of the calendar year. The flow‐weighted mean concentrations were calculated

An analysis of variance (ANOVA) was performed to examine the effect of inter‐annual variability (five levels: 2004/05, 2005/06, 2006/07, 2007/08 and 2008/09) and intra‐annual

centrations in the stream. The Tukey test was used as a *post‐hoc* test whenever the ANOVA indicated a significance. For all analyses, a significance value of 0.05 was used. All statisti‐ cal analyses were carried out using the PASW Statistics 18 for Windows program package

). The TKN concentrations, which represent the sum of ammo‐

whereas the minimum monthly mean discharge is registered in September (0.06 m<sup>3</sup>

**3. Hydrological monitoring, sampling and water analysis**

Blanco et al. [19–21].

(magnitude, duration).

) and ammonium (NH<sup>4</sup>

trations were below the detection limit in all cases.

for each year or season by dividing the daily load (N‐NO<sup>3</sup>

(NO<sup>2</sup>

and NO<sup>2</sup>

(SPSS Inc.).

s−1),

7

s−1). The

http://dx.doi.org/10.5772/intechopen.69996

), nitrite

+ con‐

concen‐

and TKN) con‐

and NH<sup>4</sup>

and TKN) by the daily stream flow.

The Corbeira catchment is a rural area characterized by the presence of small population centres (population density 35 inhabitants km<sup>2</sup> ). The vast majority of households lack sewer‐ age, so domestic sewage, mainly faecal water, is stored in individual septic tanks. Livestock density was estimated at 0.29 livestock units ha−1, with a predominance of cattle heads (75%). Organic wastes (slurries and manures) generated by agricultural and livestock activity are the most frequently used fertilizers in agriculture in the catchment, with mineral fertilizers supporting these organic ones. Slurries (mainly cattle) are primarily used in meadows and grasslands several times a year, even in the rainy season (October–December), whereas manures are used on croplands, especially in small orchards and in croplands given to maize production, generally prior to planting. Forest areas do not receive fertilizers.

The climate of the study area is Atlantic (temperate oceanic). The mean annual rainfall for the 1983–2009 period reaches a value of 1050 mm. Rainfall is distributed quite evenly through‐ out the year, although it is concentrated in the autumn and winter months, with October, November and December being the rainiest months and July and August the driest. In gen‐ eral, long‐term rainfall causes high rainfall volume but rarely achieves high rainfall inten‐ sity, although this can vary substantially, depending on the type of front passing over. The temperature of the study area is characterized by its evenness, with a mean annual of 13°C and a thermal amplitude of 10°C for the period 1983–2009. Concerning the monthly tempera‐ ture evolution throughout the year, the minimum values occurred in January and the maxi‐ mum in July. The mean annual discharge of the Corbeira stream amounts to 0.20 m<sup>3</sup> s−1. The hydrological regime is pluvial, with a seasonal pattern in hydrological response characterized by a dry summer period lasting up to first rainfall events in autumn, when the soil reserves recover, followed by a wet period that extends through the autumn and winter seasons [19]. Consequently, the maximum monthly mean discharge is observed in March (0.31 m<sup>3</sup> s−1), whereas the minimum monthly mean discharge is registered in September (0.06 m<sup>3</sup> s−1). The baseflow index, a measure of the proportion of stream flow delivered from groundwater, is about 0.80, indicative of a high contribution of groundwater to stream flow. A more detailed description of the study catchment characteristics can be found in the study of Rodríguez‐ Blanco et al. [19–21].

## **3. Hydrological monitoring, sampling and water analysis**

The C/N of the upper layer is almost 10 (9.2–12). The Corbeira catchment is minimally disturbed by human activity; it is predominantly covered by forest (65%) and agricultural land (30%). The agricultural area is primarily used for meadows and natural pastures (83% of agricultural land), with the latter being located in more or less humid areas, mainly in flood areas, where they produce fodder for consumption in green, dry or as silage. Croplands comprise 3.8% of the catchment area and are mainly used for fodder crops, such as maize, and for winter cereals. Other crops—potatoes, turnips, vegetables or fruit trees are consigned to small family farms oriented to self‐consumption. Finally, a small percent‐ age (about 5%) of the catchment is made up of areas occupied by buildings and roads. Agriculture and forestry are interspersed throughout the catchment (**Figure 1**). Forest areas (dominated mainly by commercial eucalyptus and pines) occupy areas with steep slopes. Two distinct agricultural areas can be distinguished in the catchment (**Figure 1**): one is located in the upper part of the catchment, where the slopes are moderately steep, with some cropland near the drainage network and the largest cattle load in the study area, which presents a great risk of erosion, with sediments and nutrients delivery to the stream. The other zone, located on the right margin of the river, in the middle‐lower part of the catchment, is where most agricultural activity in the catchment takes place and is where the widest area of cultivated land is found, some with good connectivity to the drainage network. It also highlights the mosaic vegetation in agricultural areas, with most of the croplands bordered by meadows and even, in some cases, by small stone walls, which can obstruct connectivity of croplands with the drainage network, resulting in a decrease in the

The Corbeira catchment is a rural area characterized by the presence of small population

age, so domestic sewage, mainly faecal water, is stored in individual septic tanks. Livestock density was estimated at 0.29 livestock units ha−1, with a predominance of cattle heads (75%). Organic wastes (slurries and manures) generated by agricultural and livestock activity are the most frequently used fertilizers in agriculture in the catchment, with mineral fertilizers supporting these organic ones. Slurries (mainly cattle) are primarily used in meadows and grasslands several times a year, even in the rainy season (October–December), whereas manures are used on croplands, especially in small orchards and in croplands given to

maize production, generally prior to planting. Forest areas do not receive fertilizers.

mum in July. The mean annual discharge of the Corbeira stream amounts to 0.20 m<sup>3</sup>

The climate of the study area is Atlantic (temperate oceanic). The mean annual rainfall for the 1983–2009 period reaches a value of 1050 mm. Rainfall is distributed quite evenly through‐ out the year, although it is concentrated in the autumn and winter months, with October, November and December being the rainiest months and July and August the driest. In gen‐ eral, long‐term rainfall causes high rainfall volume but rarely achieves high rainfall inten‐ sity, although this can vary substantially, depending on the type of front passing over. The temperature of the study area is characterized by its evenness, with a mean annual of 13°C and a thermal amplitude of 10°C for the period 1983–2009. Concerning the monthly tempera‐ ture evolution throughout the year, the minimum values occurred in January and the maxi‐

). The vast majority of households lack sewer‐

s−1. The

potential pollutant effect in surface waters.

6 Nitrogen in Agriculture - Updates

centres (population density 35 inhabitants km<sup>2</sup>

Stream discharge and N concentrations have been measured at the catchment outlet from October 2004 to September 2009. Discharge was calculated from measuring water levels (ISCO 720) using the rating curve. Water level data were measured continuously (every min) and recorded at 10‐min intervals. Water samples were collected during both baseflow and runoff events. Under baseflow conditions, water samples were manually collected every 10–15 days, whereas under runoff events, they were collected using an automatic sampler (ISCO 6712‐FS) at short‐time intervals, depending on the characteristics of the runoff events (magnitude, duration).

Stream water samples were analysed for total Kjeldahl nitrogen (TKN), nitrate (NO<sup>3</sup> ), nitrite (NO<sup>2</sup> ) and ammonium (NH<sup>4</sup> ). The TKN concentrations, which represent the sum of ammo‐ niacal and organic nitrogen, were determined by Kjeldahl digestion of unfiltered samples, following the American Public Health Association method [22]. After filtration (0.45 µm), NO<sup>3</sup> and NO<sup>2</sup> concentrations were analysed by capillary electrophoresis, whereas the NH<sup>4</sup> + con‐ centrations were measured using an ammonia‐selective electrode. The NO<sup>2</sup> and NH<sup>4</sup> concen‐ trations were below the detection limit in all cases.

Daily flow‐weighted mean N concentrations were used to analyse the temporal variability and to characterize the N pollution. Data were organized into four seasons: autumn (October, November and December), winter (January, February and March), spring (April, May and June) and summer (July, August and September). For annual data, the hydrological year was used instead of the calendar year. The flow‐weighted mean concentrations were calculated for each year or season by dividing the daily load (N‐NO<sup>3</sup> and TKN) by the daily stream flow.

An analysis of variance (ANOVA) was performed to examine the effect of inter‐annual variability (five levels: 2004/05, 2005/06, 2006/07, 2007/08 and 2008/09) and intra‐annual variability (four levels: autumn, winter, spring and summer) on N (N‐NO<sup>3</sup> and TKN) con‐ centrations in the stream. The Tukey test was used as a *post‐hoc* test whenever the ANOVA indicated a significance. For all analyses, a significance value of 0.05 was used. All statisti‐ cal analyses were carried out using the PASW Statistics 18 for Windows program package (SPSS Inc.).

## **4. Results and discussion**

#### **4.1. Weather and discharge**

Rainfall and stream discharge were highly variable during the study period (**Table 1**). Annual rainfall for 2004/2005–2008/2009 averaged 1147 mm, 97 mm above the mean of the 1983–2009 (1050 mm), and ranged from 895 mm in 2004/2005 to 1397 mm in 2006/2007. Rainfall distribu‐ tion throughout the year was also variable. For example, in the hydrological year 2005/2006, more than 80% (963 mm) of annual rainfall occurred in the first half of the year, whereas in 2006/2007 about 55% (750.4 mm) was registered during autumn (October, November and December), reaching record autumn rainfall in the study area. Dry conditions occurred in the second half of the year 2007 and in the autumn 2007/2008, giving rise to the most severe drought episode registered in north‐western of Spain over the last half century. Rainfall evo‐ lution throughout 2007/2008 was also complex. A dry autumn was followed by a very wet period, mainly January, March, April and May (62% of annual rainfall), in which rainfall events of great magnitude and intensity were registered.

Temperatures during the study period were also variable, although less than for rainfall. The 5‐year study period mean temperature was 13.2°C, i.e. 0.2°C above the mean of the 1983–2009 period. Note that 2006/2007 was, together with 2000/2001, the hottest hydrological year in the his‐ torical record, whereas 2008/2009 was the coldest in the past 20 years. The annual thermal ampli‐ tude, i.e. the difference between the mean temperature of the warmest and the coldest month, varied substantially between years. Thus, the amplitude of the year 2005/2006 (14.1°C) was more than 50% of that registered in the year 2006/2007 (9.2°C). There is a clear seasonality, reaching the lowest mean temperatures in winter and the highest temperatures during the summer (**Figure 2**).

Annual flow, as with rainfall, has a high inter‐annual variability, oscillating between 234 mm in 2007/2008 and 449 mm in 2005/2006, with a mean flow during the study period of 364 mm. Direct runoff makes up only a small proportion of streamflow, since it only represents between 20 and 38% of the annual flow. Although the annual flow was highest in 2005/2006, direct runoff was higher in 2006/2007 and 2008/2009 than in 2005/2006, i.e. in the wettest hydrological years, reflecting the role of soil water content in the runoff generation, as has been shown in a previ‐ ous study in this catchment [19]. Streamflow also showed a clear seasonality, with maximum flows occurring in January–February and minimum flows occurring in September–October (**Figure 2**). This behaviour is typical of baseflow‐dominated catchments, such as the Corbeira, resulting from the seasonality in rainfall distribution, although there was a lack of linearity between rainfall and streamflow attributed to differences in soil water balance [19].

**4.2. Inter‐annual variation of N concentrations**

0.26 mg L−1 for TN, N‐NO<sup>3</sup>

period.

1.88 and 0.35 mg L−1 for TN, N‐NO<sup>3</sup>

Over the study period, the mean annual flow‐weighted concentrations were 1.40, 1.15 and

**Figure 2.** Monthly variation of rainfall, temperature and streamflow during the five hydrological years of the study

typical of streams draining minimally perturbed catchments, i.e. with low N deposition, high percentage of forest area and low agriculture activity, as is the case of Corbeira catch‐ ment, so the concentrations there were lower than typical concentrations for catchments with more intense land use, such as agriculture and urban. They were also lower than those reported by Serrano et al. [23] for the Mero basin headwater (mean concentrations of 2.23,

characteristic, landform and soil management of the study area, but with a higher percentage of agricultural land and livestock density. However, they were considerably higher than the levels in minimally disturbed, natural water draining areas in the north‐western region of Spain [24] as well as the reference values proposed by Meybeck et al. [25] for pristine river systems, suggesting some N enrichment in the stream water, probably as a result of the agri‐ cultural practices in the catchment. In the Corbeira catchment, in addition to its presence as a natural component, N derives from agriculture, mainly because agricultural lands receive

and TKN, respectively (**Table 2**). These low N concentrations are

Inter- and Intra-Annual Variability of Nitrogen Concentrations in the Headwaters of the Mero...

http://dx.doi.org/10.5772/intechopen.69996

9

and TKN, respectively), which shows similar climatic


**Table 1.** Annual variability of rainfall, temperature and streamflow during the five hydrological years of the study period.

Inter- and Intra-Annual Variability of Nitrogen Concentrations in the Headwaters of the Mero... http://dx.doi.org/10.5772/intechopen.69996 9

**Figure 2.** Monthly variation of rainfall, temperature and streamflow during the five hydrological years of the study period.

#### **4.2. Inter‐annual variation of N concentrations**

**4. Results and discussion**

events of great magnitude and intensity were registered.

Rainfall and stream discharge were highly variable during the study period (**Table 1**). Annual rainfall for 2004/2005–2008/2009 averaged 1147 mm, 97 mm above the mean of the 1983–2009 (1050 mm), and ranged from 895 mm in 2004/2005 to 1397 mm in 2006/2007. Rainfall distribu‐ tion throughout the year was also variable. For example, in the hydrological year 2005/2006, more than 80% (963 mm) of annual rainfall occurred in the first half of the year, whereas in 2006/2007 about 55% (750.4 mm) was registered during autumn (October, November and December), reaching record autumn rainfall in the study area. Dry conditions occurred in the second half of the year 2007 and in the autumn 2007/2008, giving rise to the most severe drought episode registered in north‐western of Spain over the last half century. Rainfall evo‐ lution throughout 2007/2008 was also complex. A dry autumn was followed by a very wet period, mainly January, March, April and May (62% of annual rainfall), in which rainfall

Temperatures during the study period were also variable, although less than for rainfall. The 5‐year study period mean temperature was 13.2°C, i.e. 0.2°C above the mean of the 1983–2009 period. Note that 2006/2007 was, together with 2000/2001, the hottest hydrological year in the his‐ torical record, whereas 2008/2009 was the coldest in the past 20 years. The annual thermal ampli‐ tude, i.e. the difference between the mean temperature of the warmest and the coldest month, varied substantially between years. Thus, the amplitude of the year 2005/2006 (14.1°C) was more than 50% of that registered in the year 2006/2007 (9.2°C). There is a clear seasonality, reaching the lowest mean temperatures in winter and the highest temperatures during the summer (**Figure 2**). Annual flow, as with rainfall, has a high inter‐annual variability, oscillating between 234 mm in 2007/2008 and 449 mm in 2005/2006, with a mean flow during the study period of 364 mm. Direct runoff makes up only a small proportion of streamflow, since it only represents between 20 and 38% of the annual flow. Although the annual flow was highest in 2005/2006, direct runoff was higher in 2006/2007 and 2008/2009 than in 2005/2006, i.e. in the wettest hydrological years, reflecting the role of soil water content in the runoff generation, as has been shown in a previ‐ ous study in this catchment [19]. Streamflow also showed a clear seasonality, with maximum flows occurring in January–February and minimum flows occurring in September–October (**Figure 2**). This behaviour is typical of baseflow‐dominated catchments, such as the Corbeira, resulting from the seasonality in rainfall distribution, although there was a lack of linearity

between rainfall and streamflow attributed to differences in soil water balance [19].

**Rainfall (mm)** 895 1192 1397 1054 1196 **Temperature (°C)** 13.2 13.5 13.7 13.0 12.6 **Streamflow (mm)** 298 449 412 240 421

**2004/2005 2005/2006 2006/2007 2007/2008 2008/2009**

**Table 1.** Annual variability of rainfall, temperature and streamflow during the five hydrological years of the study

**4.1. Weather and discharge**

8 Nitrogen in Agriculture - Updates

period.

Over the study period, the mean annual flow‐weighted concentrations were 1.40, 1.15 and 0.26 mg L−1 for TN, N‐NO<sup>3</sup> and TKN, respectively (**Table 2**). These low N concentrations are typical of streams draining minimally perturbed catchments, i.e. with low N deposition, high percentage of forest area and low agriculture activity, as is the case of Corbeira catch‐ ment, so the concentrations there were lower than typical concentrations for catchments with more intense land use, such as agriculture and urban. They were also lower than those reported by Serrano et al. [23] for the Mero basin headwater (mean concentrations of 2.23, 1.88 and 0.35 mg L−1 for TN, N‐NO<sup>3</sup> and TKN, respectively), which shows similar climatic characteristic, landform and soil management of the study area, but with a higher percentage of agricultural land and livestock density. However, they were considerably higher than the levels in minimally disturbed, natural water draining areas in the north‐western region of Spain [24] as well as the reference values proposed by Meybeck et al. [25] for pristine river systems, suggesting some N enrichment in the stream water, probably as a result of the agri‐ cultural practices in the catchment. In the Corbeira catchment, in addition to its presence as a natural component, N derives from agriculture, mainly because agricultural lands receive


of eutrophication of water systems [31], highlighting a possible negative effect on agricultural

variability (**Figure 3**), with the highest concentrations (and standard deviation) in winter, when the supply of N on both landscape and streamflow is usually abundant (**Figure 4**). Fertilization in the catchment takes place in autumn and spring. The lowest concentrations were obtained in summer, in combination with low streamflow, the maximum baseflow contribution to stream‐ flow and higher biological activity within the catchment and stream, reducing the flushing of

for TKN between autumn and spring (*p* > 0.05) and between spring and summer (0.05). This pattern was also observed in many other rivers draining forest and agricultural catchments, irrespective of the importance of groundwater to river flow volume [32], and it is frequently related to water moving through the soil, less plant nitrogen uptake and microbial immobili‐ sation during winter in addition to more intensive erosion, which all contribute to higher N levels during the winter. However, this general pattern varied among years depending on the weather and discharge patterns. For examples, in the 2008/2009 hydrological year, TN, N‐NO<sup>3</sup> and TKN concentrations were significantly lower in spring than summer, when it reached the highest daily flow‐weighted mean TKN concentration, which was notably influenced by two large rainfall‐runoff events in July of 2009. The summer of the 2008/2009 water year was characterized by elevated rainfall in July (170% higher than mean rainfall for July in the period 1983–2009) concentrated in two intense rainfall events, which caused high surface runoff and particulate material delivery to the stream [11] and consequently high TKN concentrations were observed. However, the very dry autumn 2007/2008 led to very low TKN concentrations, very similar to those observed in summer. These results suggest that temperature‐dependent biological uptake is not the more important factor controlling the seasonal nitrogen dynamics as frequently it occurs in more natural systems [28], but that delivery of nitrogen to the stream is driving the dynamics, with maximum concentrations when N availability and flow, and

**Figure 3.** Box plot showing nitrogen seasonal variations. Aut: autumn, Win: winter, Spr: spring, Sum: summer.

and TKN concentrations were characterized by a marked intra‐annual

Inter- and Intra-Annual Variability of Nitrogen Concentrations in the Headwaters of the Mero...

and thus reducing the N concentrations in the stream. Significant differences

between autumn and winter (*p* > 0.05) and

http://dx.doi.org/10.5772/intechopen.69996

11

practices in the stream waters.

The daily TN, N‐NO<sup>3</sup>

leaching soil NO<sup>3</sup>

**4.3. Intra‐annual variation of N concentrations**

were detected between seasons, except for N‐NO<sup>3</sup>

thus the transport capacity, is at their highest.

**Table 2.** Flow‐weighted TN, N‐NO<sup>3</sup> , TKN concentrations and standard deviation during the five hydrological years of study period.

a contribution of nitrogen fertilizers considerably higher than that of the forest areas and the N entry of N coming from the population nuclei is low.

The TN, N‐NO<sup>3</sup> and TKN concentrations differed among the observed years (**Table 2**). The con‐ centrations of TN ranged from 1.30 to 1.49 mg L−1, whereas N‐NO<sup>3</sup> and NTKN contents var‐ ied from 1.09 to 1.21 mg L−1 and from 0.21 and 0.29 mg L−1, respectively. For all N forms, the minimum values were registered in 2004/2005, an exceptionally dry water year with rainfall 17% lower than the mean rainfall for the period 1983–2009. Maximum concentrations were recorded in 2006/2007 for N‐NO<sup>3</sup> and TN, i.e. in the rainiest water year of the study period, and in 2008/2009 for TKN (highest SS yield, data not shown). The observed changes in stream N concentrations most likely reflected the effects of change in hydro‐climatic factors, such as hydrology of the catchment and temperature, because potential drivers of N, such as land use and soil management have not varied substantially in the area over the last few decades and, in consequence, fertilization rates have been stable during the monitoring period. However, the trend in mean annual stream N (both N‐NO<sup>3</sup> and TKN) concentrations were not synchronous with changes in stream flow (**Table 1**), suggesting that changes in the timing and magnitude of rainfall episodes could lead to changes in the relationship between N transport per unit of water volume, in agreement with other studies [9, 26], as they affect N availability and transformation processes.

All of the water samples had higher N‐NO<sup>3</sup> concentrations than TKN, so the N‐NO<sup>3</sup> was the predominant form of N in the study catchment, accounting for, on average, 82% of the TN (80–85%). These rates are consistent with those commonly recorded in agricultural and rural groundwater‐dominated catchments worldwide, with organic nitrogen comprising 10–20% of TN [23, 27]. However, the fractionation in this study contrasts with results obtained in a widely variety of catchments minimally disturbed catchments with TN concentrations lower than 2 mg L−1, where frequently more than 80–90% of TN is commonly delivered in the form of organic N [28, 29], constituting a substantial component of TN in many ecosystems receiv‐ ing low N enrichment. Mean annual N‐NO<sup>3</sup> concentrations as a percentage of TN decreased from 85% in 2006/2007 to 80% in 2008/2009, the water year of highest particulate material which could favour higher N transport in organic form.

Daily flow‐weighted TN concentrations were consistently below the threshold level of 2 mg L−1, threshold identified in the European Nitrogen Assessment [30] as an appropriate target for the delivery of good ecological status in European water. However, daily flow‐weighted TN con‐ centrations exceeded the critical range of 0.5–1.0 mg N L−1 above which there is a potential risk of eutrophication of water systems [31], highlighting a possible negative effect on agricultural practices in the stream waters.

#### **4.3. Intra‐annual variation of N concentrations**

a contribution of nitrogen fertilizers considerably higher than that of the forest areas and the

**2004/2005 2005/2006 2006/2007 2007/2008 2008/2009**

**TN (mg L** −1**)** 1.30 (0.36) 1.50 (0.57) 1.37 (0.40) 1.39 (0.55) 1.45 (0.34)

 **(mg L**−1**)** 1.09 (0.17) 1.21 (0.29) 1.16 (0.22) 1.13 (0.27) 1.15 (0.22) **TKN (mg L**−1**)** 0.21 (0.29) 0.29 (0.38) 0.21 (0.24) 0.28 (0.38) 0.30 (0.22)

ied from 1.09 to 1.21 mg L−1 and from 0.21 and 0.29 mg L−1, respectively. For all N forms, the minimum values were registered in 2004/2005, an exceptionally dry water year with rainfall 17% lower than the mean rainfall for the period 1983–2009. Maximum concentrations were

and in 2008/2009 for TKN (highest SS yield, data not shown). The observed changes in stream N concentrations most likely reflected the effects of change in hydro‐climatic factors, such as hydrology of the catchment and temperature, because potential drivers of N, such as land use and soil management have not varied substantially in the area over the last few decades and, in consequence, fertilization rates have been stable during the monitoring period. However, the

with changes in stream flow (**Table 1**), suggesting that changes in the timing and magnitude of rainfall episodes could lead to changes in the relationship between N transport per unit of water volume, in agreement with other studies [9, 26], as they affect N availability and transformation

predominant form of N in the study catchment, accounting for, on average, 82% of the TN (80–85%). These rates are consistent with those commonly recorded in agricultural and rural groundwater‐dominated catchments worldwide, with organic nitrogen comprising 10–20% of TN [23, 27]. However, the fractionation in this study contrasts with results obtained in a widely variety of catchments minimally disturbed catchments with TN concentrations lower than 2 mg L−1, where frequently more than 80–90% of TN is commonly delivered in the form of organic N [28, 29], constituting a substantial component of TN in many ecosystems receiv‐

from 85% in 2006/2007 to 80% in 2008/2009, the water year of highest particulate material

Daily flow‐weighted TN concentrations were consistently below the threshold level of 2 mg L−1, threshold identified in the European Nitrogen Assessment [30] as an appropriate target for the delivery of good ecological status in European water. However, daily flow‐weighted TN con‐ centrations exceeded the critical range of 0.5–1.0 mg N L−1 above which there is a potential risk

and TKN concentrations differed among the observed years (**Table 2**). The con‐

and TN, i.e. in the rainiest water year of the study period,

, TKN concentrations and standard deviation during the five hydrological years of

concentrations than TKN, so the N‐NO<sup>3</sup>

and TKN) concentrations were not synchronous

concentrations as a percentage of TN decreased

and NTKN contents var‐

was the

N entry of N coming from the population nuclei is low.

centrations of TN ranged from 1.30 to 1.49 mg L−1, whereas N‐NO<sup>3</sup>

The TN, N‐NO<sup>3</sup>

**N‐NO**<sup>3</sup>

study period.

processes.

recorded in 2006/2007 for N‐NO<sup>3</sup>

**Table 2.** Flow‐weighted TN, N‐NO<sup>3</sup>

10 Nitrogen in Agriculture - Updates

trend in mean annual stream N (both N‐NO<sup>3</sup>

All of the water samples had higher N‐NO<sup>3</sup>

ing low N enrichment. Mean annual N‐NO<sup>3</sup>

which could favour higher N transport in organic form.

The daily TN, N‐NO<sup>3</sup> and TKN concentrations were characterized by a marked intra‐annual variability (**Figure 3**), with the highest concentrations (and standard deviation) in winter, when the supply of N on both landscape and streamflow is usually abundant (**Figure 4**). Fertilization in the catchment takes place in autumn and spring. The lowest concentrations were obtained in summer, in combination with low streamflow, the maximum baseflow contribution to stream‐ flow and higher biological activity within the catchment and stream, reducing the flushing of leaching soil NO<sup>3</sup> and thus reducing the N concentrations in the stream. Significant differences were detected between seasons, except for N‐NO<sup>3</sup> between autumn and winter (*p* > 0.05) and for TKN between autumn and spring (*p* > 0.05) and between spring and summer (0.05). This pattern was also observed in many other rivers draining forest and agricultural catchments, irrespective of the importance of groundwater to river flow volume [32], and it is frequently related to water moving through the soil, less plant nitrogen uptake and microbial immobili‐ sation during winter in addition to more intensive erosion, which all contribute to higher N levels during the winter. However, this general pattern varied among years depending on the weather and discharge patterns. For examples, in the 2008/2009 hydrological year, TN, N‐NO<sup>3</sup> and TKN concentrations were significantly lower in spring than summer, when it reached the highest daily flow‐weighted mean TKN concentration, which was notably influenced by two large rainfall‐runoff events in July of 2009. The summer of the 2008/2009 water year was characterized by elevated rainfall in July (170% higher than mean rainfall for July in the period 1983–2009) concentrated in two intense rainfall events, which caused high surface runoff and particulate material delivery to the stream [11] and consequently high TKN concentrations were observed. However, the very dry autumn 2007/2008 led to very low TKN concentrations, very similar to those observed in summer. These results suggest that temperature‐dependent biological uptake is not the more important factor controlling the seasonal nitrogen dynamics as frequently it occurs in more natural systems [28], but that delivery of nitrogen to the stream is driving the dynamics, with maximum concentrations when N availability and flow, and thus the transport capacity, is at their highest.

**Figure 3.** Box plot showing nitrogen seasonal variations. Aut: autumn, Win: winter, Spr: spring, Sum: summer.

hydrology processes. These findings have particular relevance in the current global context of climate change, which is predicted to bring more frequent heat waves and an increase extreme rainfall events (mainly during winter) in the study area. These changes will have impacts on the hydrological cycle and, consequently, in nitrogen concentrations, and may increase the transfer of nitrogen from soils to stream water during winter and impact on

Inter- and Intra-Annual Variability of Nitrogen Concentrations in the Headwaters of the Mero...

http://dx.doi.org/10.5772/intechopen.69996

13

This chapter is a contribution to the projects 10MDS103031 of the Xunta de Galicia and CGL2014‐56907‐R of the Programa Estatal de Investigación, Desarrollo e InnovaciónOrientada a los Retos de la Sociedad, which was funded by the Spanish Ministry of Economy and Competitiveness. M.L. Rodríguez‐Blanco has been awarded a post‐doctoral research contract (Juan de la Cierva Programme), which was funded by the Spanish Ministry of Economy and

Faculty of Sciences, Centre for Advanced Scientific Research (CICA), University of A Coruna,

[1] Qualls RG, Richarson CJ. Factors controlling concentration, export, and decomposi‐ tion of dissolved organic nutrients in the Everglades of Florida. Biogeochemistry.

[2] Jordan TE, Correl DL, Weller DE. Relating nutrient discharges from watersheds to land use and streamflow variability. Water Resources Research. 1997;**33**(11):2579‐2590

[3] Outram FN, Cooper RJ, Sünnenberg G, Hiscock KM, Lovett AA. Antecedent conditions, hydrological connectivity and anthropogenic inputs: Factors affecting nitrate and phos‐ phorus transfers to agricultural headwater streams. Science of the Total Environment.

[4] Myrbeck A, Stenberg M, Arvidson J, Rydberg T. Effects of autumn tillage of clay soil on mineral N content, spring cereal yield and soil structure over time. European Journal of

M. Luz Rodríguez‐Blanco\*, M. Mercedes Taboada‐Castro, Ricardo Arias and

\*Address all correspondence to: mrodriguezbl@udc.es

stream water quality.

Competitiveness.

**Author details**

A Coruña, Spain

**References**

M. Teresa Taboada‐Castro

2003;**62**(2):197‐229

2016;**545‐546**:184‐199

Agronomy. 2012;**37**:96‐104

**Acknowledgements**

**Figure 4.** Variation of monthly flow‐weighted nitrogen (N‐NO<sup>3</sup> and TKN) concentrations during the five hydrological years of study period.

The contribution of the individual fractions made to the TN also showed an intra‐annual variability. Thus, N‐NO<sup>3</sup> contributed to more than 83% of TN during autumn and spring, representing about 86% in summer, but this contribution decreases during winter (77%), i.e. in the season with higher streamflow. This reflects an increase in the organic nitrogen during winter, probably due to more intense erosion during high flow periods.

In all seasons, a positive relationship exists between daily N concentrations and flow, sug‐ gesting a predominantly diffuse source of N. However, the relationship was highly variable between seasons. Thus, in autumn and winter, N‐NO<sup>3</sup> was more strongly related to stream flow, whereas in spring and summer, TKN was more strongly related to flow, as expected in view of the role of shear stress in moving particles. These results suggest that although N‐NO<sup>3</sup> and TKN may display a similar seasonal pattern, controls of N‐NO<sup>3</sup> differ substantially from controls on TKN. Nitrate is originated from subsurface paths whereas TKN is delivered from surface flow paths.

#### **5. Conclusions**

Total nitrogen concentrations were relatively low, consistent with the lack of significant anthropogenic pressure in the catchment. However, the N concentrations were always over the critical range over which there is a potential risk of eutrophication of water systems. Most of the N flushed from the Corbeira catchment is in the form of nitrate (82–85%), so manage‐ ment practices could target nitrate, particularly that from fertilizer. In the study area, rainfall and stream flow change annually and seasonally, so the total nitrogen, nitrate and Kjeldahl nitrogen concentrations also showed an important inter‐ and intra‐annual variability linked to rainfall and flow oscillations; showing that N concentrations are mainly controlled by hydrology processes. These findings have particular relevance in the current global context of climate change, which is predicted to bring more frequent heat waves and an increase extreme rainfall events (mainly during winter) in the study area. These changes will have impacts on the hydrological cycle and, consequently, in nitrogen concentrations, and may increase the transfer of nitrogen from soils to stream water during winter and impact on stream water quality.

## **Acknowledgements**

This chapter is a contribution to the projects 10MDS103031 of the Xunta de Galicia and CGL2014‐56907‐R of the Programa Estatal de Investigación, Desarrollo e InnovaciónOrientada a los Retos de la Sociedad, which was funded by the Spanish Ministry of Economy and Competitiveness. M.L. Rodríguez‐Blanco has been awarded a post‐doctoral research contract (Juan de la Cierva Programme), which was funded by the Spanish Ministry of Economy and Competitiveness.

## **Author details**

The contribution of the individual fractions made to the TN also showed an intra‐annual

representing about 86% in summer, but this contribution decreases during winter (77%), i.e. in the season with higher streamflow. This reflects an increase in the organic nitrogen during

In all seasons, a positive relationship exists between daily N concentrations and flow, sug‐ gesting a predominantly diffuse source of N. However, the relationship was highly variable

flow, whereas in spring and summer, TKN was more strongly related to flow, as expected in view of the role of shear stress in moving particles. These results suggest that although N‐NO<sup>3</sup>

controls on TKN. Nitrate is originated from subsurface paths whereas TKN is delivered from

Total nitrogen concentrations were relatively low, consistent with the lack of significant anthropogenic pressure in the catchment. However, the N concentrations were always over the critical range over which there is a potential risk of eutrophication of water systems. Most of the N flushed from the Corbeira catchment is in the form of nitrate (82–85%), so manage‐ ment practices could target nitrate, particularly that from fertilizer. In the study area, rainfall and stream flow change annually and seasonally, so the total nitrogen, nitrate and Kjeldahl nitrogen concentrations also showed an important inter‐ and intra‐annual variability linked to rainfall and flow oscillations; showing that N concentrations are mainly controlled by

winter, probably due to more intense erosion during high flow periods.

and TKN may display a similar seasonal pattern, controls of N‐NO<sup>3</sup>

between seasons. Thus, in autumn and winter, N‐NO<sup>3</sup>

**Figure 4.** Variation of monthly flow‐weighted nitrogen (N‐NO<sup>3</sup>

contributed to more than 83% of TN during autumn and spring,

was more strongly related to stream

and TKN) concentrations during the five hydrological

differ substantially from

variability. Thus, N‐NO<sup>3</sup>

years of study period.

12 Nitrogen in Agriculture - Updates

surface flow paths.

**5. Conclusions**

M. Luz Rodríguez‐Blanco\*, M. Mercedes Taboada‐Castro, Ricardo Arias and

M. Teresa Taboada‐Castro

\*Address all correspondence to: mrodriguezbl@udc.es

Faculty of Sciences, Centre for Advanced Scientific Research (CICA), University of A Coruna, A Coruña, Spain

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**Chapter 2**

**Provisional chapter**

**O** 

O) emissions.

O emissions.

O emis-

O emissions in agricultural practice

O emissions. Once all effective strategies would

**Nitrogen Transformations Associated with N2O**

**Nitrogen Transformations Associated with N<sup>2</sup>**

DOI: 10.5772/intechopen.71922

Nitrogen (N) is one of the most important plant nutrient, and its availability and transformations are vital for net primary production. Soil N transformations include mineralization, nitrification and denitrification processes. Nitrogen mineralization transforms organic N into inorganic N, providing available N for crops. Both nitrification and deni-

O emissions from agricultural soils decrease N fertilization efficiency and potentially

is essential for sustainable development of agriculture considering the environmental

sions, including the application of nitrification inhibitors, such as dicyandiamide (DCD) and 3,4-dimethylpyrazole phosphate (DMPP). Recently, biological nitrification inhibitors have also attracted researchers' attention, which may be more environment-friendly. In addition, biochar commonly used as soil ameliorant to improve soil quality and C

O. Various strategies have been proposed for the mitigation of N2

Nitrification inhibitors have been demonstrated to be useful in decreasing soil N<sup>2</sup>

be widely implemented, more environment-friendly agriculture could be expected.

**Keywords:** fertilizer efficiency, global change, mitigation, nitrogen emissions, nutrition

Nitrogen (N) is one of the most important elements for plant growth and hence it is vital for ecosystem's primary production [1]. In the background of global climate change, understanding soil N balance in agricultural soils is important for sustaining the development of modern agriculture and mitigation of climate change considering the substantial contribution made

trification are microbe-driven processes associated with nitrous oxide (N<sup>2</sup>

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

© 2018 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

**Emissions in Agricultural Soils**

**Emissions in Agricultural Soils**

Additional information is available at the end of the chapter

induce global warming. The mitigation of soil N<sup>2</sup>

sequestration could also mitigate soil N2

Additional information is available at the end of the chapter

Ling Zhang and Xiaojun Liu

**Abstract**

N2

effect of N2

**1. Introduction**

by agriculture to climate change [2–4].

Ling Zhang and Xiaojun Liu

http://dx.doi.org/10.5772/intechopen.71922

[32] Harrington ST, Harrington JR. Dissolved and particulate nutrient transport dynamics of a small Irish catchment: The River Owenabue. Hydrology and Earth System Sciences. 2014;**18**:2191‐2200

**Provisional chapter**

**O** 

## **Nitrogen Transformations Associated with N2O Emissions in Agricultural Soils Emissions in Agricultural Soils**

**Nitrogen Transformations Associated with N<sup>2</sup>**

DOI: 10.5772/intechopen.71922

Ling Zhang and Xiaojun Liu Additional information is available at the end of the chapter

Ling Zhang and Xiaojun Liu

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.71922

#### **Abstract**

[31] Camargo JA, Alonso A. Ecological and toxicological effects of inorganic nitrogen pollu‐ tion in aquatic ecosystems: A global assessment. Environment International. 2006;**32**:831‐

[32] Harrington ST, Harrington JR. Dissolved and particulate nutrient transport dynamics of a small Irish catchment: The River Owenabue. Hydrology and Earth System Sciences.

849. DOI: 10.1016/j.envint.2006.05.002

2014;**18**:2191‐2200

16 Nitrogen in Agriculture - Updates

Nitrogen (N) is one of the most important plant nutrient, and its availability and transformations are vital for net primary production. Soil N transformations include mineralization, nitrification and denitrification processes. Nitrogen mineralization transforms organic N into inorganic N, providing available N for crops. Both nitrification and denitrification are microbe-driven processes associated with nitrous oxide (N<sup>2</sup> O) emissions. N2 O emissions from agricultural soils decrease N fertilization efficiency and potentially induce global warming. The mitigation of soil N<sup>2</sup> O emissions in agricultural practice is essential for sustainable development of agriculture considering the environmental effect of N2 O. Various strategies have been proposed for the mitigation of N2 O emissions. Nitrification inhibitors have been demonstrated to be useful in decreasing soil N<sup>2</sup> O emissions, including the application of nitrification inhibitors, such as dicyandiamide (DCD) and 3,4-dimethylpyrazole phosphate (DMPP). Recently, biological nitrification inhibitors have also attracted researchers' attention, which may be more environment-friendly. In addition, biochar commonly used as soil ameliorant to improve soil quality and C sequestration could also mitigate soil N2 O emissions. Once all effective strategies would be widely implemented, more environment-friendly agriculture could be expected.

**Keywords:** fertilizer efficiency, global change, mitigation, nitrogen emissions, nutrition

## **1. Introduction**

Nitrogen (N) is one of the most important elements for plant growth and hence it is vital for ecosystem's primary production [1]. In the background of global climate change, understanding soil N balance in agricultural soils is important for sustaining the development of modern agriculture and mitigation of climate change considering the substantial contribution made by agriculture to climate change [2–4].

Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons

While organic N accounts for larger part of total soil N, only soil inorganic N (mainly including ammonium and nitrate N) derived from mineralization of organic N is readily available for plant. Thereby, organic N mineralization is an important factor controlling levels of soil inorganic N availability, which is vital for crop production. In agricultural soils, most crop residuals were harvested by agricultural activities, while some of them were returned into soils as roots, rhizospheric deposition or aboveground litter. During decomposition of these organic matters, most inorganic N would be released into surrounding soils via mineralization of organic N. In these processes, N

uptake by plant from soils was returned into soils again, during N mineralization process.

nitrate N, causing new balance among different N components [2, 4, 7].

tion processes (both produce greenhouse gas N2

*2.2.1. Net and gross N transformation rates*

for the calculation were as follows:

ammonium concentration, as mg NH<sup>4</sup>

Net N mineralization rate:

Net nitrification rate:

Net ammonification rate:

**2.2. General methods in studying soil N transformations**

final ammonium concentration, expressed as mg NH<sup>4</sup>

*<sup>N</sup>nitrified* <sup>=</sup> (Nitrate*<sup>f</sup>* <sup>−</sup> Nitrate0)

*<sup>N</sup>ammonification* <sup>=</sup> (Ammonium*<sup>f</sup>* <sup>−</sup> Ammonium0)

Nitrogen produced by mineralization process, including both ammonium and nitrate N, was readily available for crop growth. If there was no leaching or loss via runoffs, all available N will be adsorbed by plant roots and used for growth. However, nitrification and denitrifica-

In addition, soil ammonium N could also be immobilized by soil particles, and both important inorganic N may loss with surface runoff, further decreasing its availability level. Before mineralized by microbes, soluble organic N was also reliable to be removed by runoffs.

Due to complicated interactions among all soil N transformation process, net N transformation rates have been widely studied [10, 11]. Once plant roots were excluded, changes in soil N levels could be considered as results of transformations. Specifically, when soil mineralization rates were examined, both ammonium and nitrate concentrations were determined both at the beginning and at the end of the study. Net N mineralization rates were considered as changes in concentration of both inorganic N per given time unit [11, 12]. The equations used

where *Nmineralized* = net N mineralization rate, expressed as mg N kg−1 soil day−1, Ammonium*<sup>f</sup>*

+

 \_\_\_\_\_\_\_\_\_\_\_\_\_\_ *Tdays*

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ *Tdays*


*<sup>N</sup>*mineralized <sup>=</sup> [(Nitrate*<sup>f</sup>* <sup>+</sup> Ammonium*f*)–(Nitrate0 <sup>+</sup> Ammonium0)] \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ *Tdays*

+

Similarly, net nitrification rates and ammonification rates were calculated as follows:

O) also occurred between ammonium and

http://dx.doi.org/10.5772/intechopen.71922

19

Nitrogen Transformations Associated with N2O Emissions in Agricultural Soils

. (1)


, (2)

, (3)

=

**Figure 1.** Simple diagram showing N cycle between atmosphere and agricultural soil. Note: (A) N deposition; (B) N fixation; (C) fertilization; (D) denitrification; (E) nitrification; (F) organic N mineralization; (G) plant uptake; (H) litter decomposition; (I) soil immobilization; (J) N volatilization, leaching and crops harvest.

In general, soil N mainly originates from atmospheric N deposition (both dry and wet deposition) (**Figure 1**) and biological fixation in natural ecosystems [5]. In agricultural soils, however, N input via anthropogenic activities (e.g., fertilization practice) has been considered as the most important way [6]. Fertilization in agricultural practice could introduce many kinds of N into soil ecosystems. For example, both organic and inorganic N, in liquid or particle status, could often be observed in agricultural fertilization practice. In addition to N input by anthropogenic activities, N output via crops harvest or human-induced runoffs with irrigation could occur simultaneously in agricultural soils (**Figure 1**).

Moreover, the complicated N transformations going on in soil ecosystems also account for large part of N output from soils [7]. For example, N mineralization associated with transforming organic N into inorganic N might induce more N leaching with runoffs or underground water; nitrification process could increase N loss from soils via gas emissions [2, 7, 8] (**Figure 1**).

Thereby, understanding the process and mechanisms underlining N transformations in agricultural soils would be vital for more efficient fertilization practice and agriculture management.

## **2. Nitrogen transformations in agricultural soils**

#### **2.1. Overview of soil N transformations**

Except for N transformation process ongoing during litter decomposition process at the littersoil interface, N was also transformed by processes including mineralization, nitrification, denitrification, and so on, in agricultural soils [9].

While organic N accounts for larger part of total soil N, only soil inorganic N (mainly including ammonium and nitrate N) derived from mineralization of organic N is readily available for plant. Thereby, organic N mineralization is an important factor controlling levels of soil inorganic N availability, which is vital for crop production. In agricultural soils, most crop residuals were harvested by agricultural activities, while some of them were returned into soils as roots, rhizospheric deposition or aboveground litter. During decomposition of these organic matters, most inorganic N would be released into surrounding soils via mineralization of organic N. In these processes, N uptake by plant from soils was returned into soils again, during N mineralization process.

Nitrogen produced by mineralization process, including both ammonium and nitrate N, was readily available for crop growth. If there was no leaching or loss via runoffs, all available N will be adsorbed by plant roots and used for growth. However, nitrification and denitrification processes (both produce greenhouse gas N2 O) also occurred between ammonium and nitrate N, causing new balance among different N components [2, 4, 7].

In addition, soil ammonium N could also be immobilized by soil particles, and both important inorganic N may loss with surface runoff, further decreasing its availability level. Before mineralized by microbes, soluble organic N was also reliable to be removed by runoffs.

#### **2.2. General methods in studying soil N transformations**

#### *2.2.1. Net and gross N transformation rates*

Due to complicated interactions among all soil N transformation process, net N transformation rates have been widely studied [10, 11]. Once plant roots were excluded, changes in soil N levels could be considered as results of transformations. Specifically, when soil mineralization rates were examined, both ammonium and nitrate concentrations were determined both at the beginning and at the end of the study. Net N mineralization rates were considered as changes in concentration of both inorganic N per given time unit [11, 12]. The equations used for the calculation were as follows:

Net N mineralization rate:

In general, soil N mainly originates from atmospheric N deposition (both dry and wet deposition) (**Figure 1**) and biological fixation in natural ecosystems [5]. In agricultural soils, however, N input via anthropogenic activities (e.g., fertilization practice) has been considered as the most important way [6]. Fertilization in agricultural practice could introduce many kinds of N into soil ecosystems. For example, both organic and inorganic N, in liquid or particle status, could often be observed in agricultural fertilization practice. In addition to N input by anthropogenic activities, N output via crops harvest or human-induced runoffs with irriga-

**Figure 1.** Simple diagram showing N cycle between atmosphere and agricultural soil. Note: (A) N deposition; (B) N fixation; (C) fertilization; (D) denitrification; (E) nitrification; (F) organic N mineralization; (G) plant uptake; (H) litter

Moreover, the complicated N transformations going on in soil ecosystems also account for large part of N output from soils [7]. For example, N mineralization associated with transforming organic N into inorganic N might induce more N leaching with runoffs or underground water; nitrification process could increase N loss from soils via gas emissions [2, 7, 8]

Thereby, understanding the process and mechanisms underlining N transformations in agricultural soils would be vital for more efficient fertilization practice and agriculture

Except for N transformation process ongoing during litter decomposition process at the littersoil interface, N was also transformed by processes including mineralization, nitrification,

tion could occur simultaneously in agricultural soils (**Figure 1**).

decomposition; (I) soil immobilization; (J) N volatilization, leaching and crops harvest.

**2. Nitrogen transformations in agricultural soils**

**2.1. Overview of soil N transformations**

denitrification, and so on, in agricultural soils [9].

(**Figure 1**).

18 Nitrogen in Agriculture - Updates

management.

or une carbaauon wee as rouows:

Net N mineralization rate:

$$N\_{\text{mhoralload}} = \frac{\left[\left(\text{Nitrate}\_{/} + \text{Ammonium}\_{/}\right) \cdot \left(\text{Nitrate}\_{0} + \text{Ammonium}\_{0}\right)\right]}{T\_{day}}.\tag{1}$$

where *Nmineralized* = net N mineralization rate, expressed as mg N kg−1 soil day−1, Ammonium*<sup>f</sup>* = final ammonium concentration, expressed as mg NH<sup>4</sup> + -N kg−1 soil, Ammonium0 = initial ammonium concentration, as mg NH<sup>4</sup> + -N kg−1 soil, T*days* = incubation time, days.

Similarly, net nitrification rates and ammonification rates were calculated as follows:

Net nitrification rate:

Net initialization rate: 
$$N\_{\text{utilidad}} = \frac{\text{(Nitrate}\_{\text{f}} - \text{Nitrate}\_{0})}{T\_{\text{dap}}} \tag{2}$$

Net ammonification rate:

Net annonification rate: 
$$N\_{annuplicate} = \frac{(\text{Ammonium}\_f - \text{Ammonium}\_0)}{T\_{dep}} \tag{3}$$

where *Nnitrified* = net nitrification rate, expressed as mg NO<sup>3</sup> − -N kg−1 soil day−1, Nitrate*<sup>f</sup>* = final nitrate concentration, expressed as mg NO<sup>3</sup> − -N kg−1 soil, Nitrate*<sup>0</sup>* = initial nitrate concentration, as mg NO3 − -N kg−1 soil, Ammonium*<sup>f</sup>* = final ammonium concentration, expressed as mg NH<sup>4</sup> + -N kg−1 soil, Ammonium0 = initial ammonium concentration, as mg NH<sup>4</sup> + -N kg−1 soil, T*days* = incubation time, days.

global warming potential of N<sup>2</sup>

N2

N2

Soil N2

to N2

nitrate N to N2

O is 265 times that of CO<sup>2</sup>

those fertilized by N fertilization in agricultural ecosystems. Hence, studying the way N<sup>2</sup>

ing potential [3, 8]. Importantly, most of the atmospheric N2

O emissions and hence for mitigation of atmospheric N2

spheric chemistry and global climate [26–29].

*E* = *P* × *V* × \_\_*dc*

where *E* refers to emission rates of soil N2

*3.1.2. Measurement of soil N2*

cess by previous studies [20]. Specifically, the main process producing N<sup>2</sup>

*O emissions*

rate of incubated soil could be described as follows [17, 30, 31]:

(Pa), *V* is headspace volume of the incubation flask (cm<sup>3</sup>

**Figure 2.** Simple diagram showing the production process of soil N<sup>2</sup>

Methods being developed for the measurement of soil N<sup>2</sup>

ferent sets. This first one was used to measure soil N<sup>2</sup>

O might have contributed substantially to global warming considering its larger global warm-

produced, emitted and adsorbed in agricultural soils would be important for mitigation of soil

namely nitrification and denitrification process, transformed ammonium N to nitrate N, and

produced by complete nitrification process and was produced during denitrification process. In both processes, nitric oxide (NO) was also produced by both nitrification and denitrification processes, which was also considered as an important GHG [23–25]. Recently, in addition

O, NO has also attracted increasing attention due to their role played in altering atmo-

, respectively [21, 22]. The processes associated with N<sup>2</sup>

studies. Different to that conducted *in situ*, incubation studies monitored soil N2

sion rate based on soil mass per time. Equation developed for the calculation of N2

based on soil mass rather than soil surface area. Thereby, those studies measured N<sup>2</sup>

*dt* <sup>×</sup> \_\_\_1

*RT* <sup>×</sup> *<sup>M</sup>* <sup>×</sup> \_\_1

O.

*<sup>m</sup>* <sup>×</sup> \_\_1

described by hole in the pipe model (HIP, **Figure 2**) [20]. The model showed that N<sup>2</sup>

O has been demonstrated to be the by-product or production of N transformation pro-

, while CH<sup>4</sup>

Nitrogen Transformations Associated with N2O Emissions in Agricultural Soils

is only 28 times [3]. Thereby,

O in soil ecosystems,

O production could be

O is

21

O was

O emission

O emission

O emis-

O (ppb),

O was emitted from soils, especially

http://dx.doi.org/10.5772/intechopen.71922

O in this climate changing world.

O emissions rate including two dif-

*<sup>t</sup>* (4)

O (ng g−1 h−1), *P* is standard atmospheric pressure

), *c* is the concentration of N2

O emission rate in laboratory incubation

Meanwhile, with the development of isotope labeling strategy, more studies have been conducted to determine the gross N transformation rates in agriculture and forestry soils [13–16]. By labeling N in ammonium or nitrate N, N element could be traced during the complicated transformation process. Thereby, the gross N transformation rates could be obtained using the isotope labeling method.

#### *2.2.2. In situ and ex situ studies on N transformations*

Studies on N transformations could also be sorted by study place or site, into *in situ* or *ex situ* studies. Laboratory soil incubation studies were widely used to examine N transformations in environmental science [10, 17]. During laboratory incubation, the environmental factors could be easily altered to check their role played on N transformation rates. Thereby, studies conducted in incubators are more operable than those *in situ*. For example, incubation studies could be manipulated with different soil water content, incubated temperature, or even aeration status. However, not all N transformation studies could be conducted in the laboratory. In agricultural ecosystems, the dynamics of soil N might be important for fertilization practice in field crops research and management. In this condition, laboratory incubation studies are no longer applied. Instead, *in situ* soil core incubation is more suitable. Similar to laboratory incubations, both concentrations of soil inorganic N at the beginning and at the monitored date should be determined of the soil column. Moreover, the soil column incubated *in situ* for study should be isolated from the surrounding soils to prevent potential N uptake by roots of crops.

In recent years, ion-exchange resins (Unibest PST-1, Unibest, Bozeman, MT, USA) have been widely used for *in situ* studies on soil N availability [18, 19]. In these studies, incubated soils cores were isolated from surrounding soil. Exchangeable anion/cation resin was used to capture any inorganic N moved into or out of the incubated soil core [19]. Changes in soil inorganic N in soil core and the resin relative to that of the initial soil core with time were considered as net N mineralization rate. Similarly, changes in soil nitrate N with time were considered as net nitrification rate.

#### **3. Nitrogen transformations associated with N2 O emissions**

#### **3.1. Illustration of N2 O emissions**

#### *3.1.1. Soil N2 O production*

N2 O is one important component of greenhouse gas emitted from soil. The global warming potential of N2 O is much larger than that of methane and carbon dioxide (CH<sup>4</sup> and CO<sup>2</sup> ). According to the latest report, even though not so much as CH<sup>4</sup> and CO<sup>2</sup> in atmospheric environment, the global warming potential of N<sup>2</sup> O is 265 times that of CO<sup>2</sup> , while CH<sup>4</sup> is only 28 times [3]. Thereby, N2 O might have contributed substantially to global warming considering its larger global warming potential [3, 8]. Importantly, most of the atmospheric N2 O was emitted from soils, especially those fertilized by N fertilization in agricultural ecosystems. Hence, studying the way N<sup>2</sup> O is produced, emitted and adsorbed in agricultural soils would be important for mitigation of soil N2 O emissions and hence for mitigation of atmospheric N2 O in this climate changing world.

Soil N2 O has been demonstrated to be the by-product or production of N transformation process by previous studies [20]. Specifically, the main process producing N<sup>2</sup> O in soil ecosystems, namely nitrification and denitrification process, transformed ammonium N to nitrate N, and nitrate N to N2 , respectively [21, 22]. The processes associated with N<sup>2</sup> O production could be described by hole in the pipe model (HIP, **Figure 2**) [20]. The model showed that N<sup>2</sup> O was produced by complete nitrification process and was produced during denitrification process. In both processes, nitric oxide (NO) was also produced by both nitrification and denitrification processes, which was also considered as an important GHG [23–25]. Recently, in addition to N2 O, NO has also attracted increasing attention due to their role played in altering atmospheric chemistry and global climate [26–29].

#### *3.1.2. Measurement of soil N2 O emissions*

where *Nnitrified* = net nitrification rate, expressed as mg NO<sup>3</sup>


nitrate concentration, expressed as mg NO<sup>3</sup>

*2.2.2. In situ and ex situ studies on N transformations*

−

tion, as mg NO3

incubation time, days.

20 Nitrogen in Agriculture - Updates

the isotope labeling method.

considered as net nitrification rate.

*O production*

**3.1. Illustration of N2**

*3.1.1. Soil N2*

tial of N2

N2

**3. Nitrogen transformations associated with N2**

**O emissions**

to the latest report, even though not so much as CH<sup>4</sup>

NH<sup>4</sup> + −

−

Meanwhile, with the development of isotope labeling strategy, more studies have been conducted to determine the gross N transformation rates in agriculture and forestry soils [13–16]. By labeling N in ammonium or nitrate N, N element could be traced during the complicated transformation process. Thereby, the gross N transformation rates could be obtained using

Studies on N transformations could also be sorted by study place or site, into *in situ* or *ex situ* studies. Laboratory soil incubation studies were widely used to examine N transformations in environmental science [10, 17]. During laboratory incubation, the environmental factors could be easily altered to check their role played on N transformation rates. Thereby, studies conducted in incubators are more operable than those *in situ*. For example, incubation studies could be manipulated with different soil water content, incubated temperature, or even aeration status. However, not all N transformation studies could be conducted in the laboratory. In agricultural ecosystems, the dynamics of soil N might be important for fertilization practice in field crops research and management. In this condition, laboratory incubation studies are no longer applied. Instead, *in situ* soil core incubation is more suitable. Similar to laboratory incubations, both concentrations of soil inorganic N at the beginning and at the monitored date should be determined of the soil column. Moreover, the soil column incubated *in situ* for study should be isolated from the surrounding soils to prevent potential N uptake by roots of crops. In recent years, ion-exchange resins (Unibest PST-1, Unibest, Bozeman, MT, USA) have been widely used for *in situ* studies on soil N availability [18, 19]. In these studies, incubated soils cores were isolated from surrounding soil. Exchangeable anion/cation resin was used to capture any inorganic N moved into or out of the incubated soil core [19]. Changes in soil inorganic N in soil core and the resin relative to that of the initial soil core with time were considered as net N mineralization rate. Similarly, changes in soil nitrate N with time were

O is one important component of greenhouse gas emitted from soil. The global warming poten-

and CO<sup>2</sup>

O is much larger than that of methane and carbon dioxide (CH<sup>4</sup>



+


= final ammonium concentration, expressed as mg

**O emissions**

and CO<sup>2</sup>

in atmospheric environment, the

). According

= final


Methods being developed for the measurement of soil N<sup>2</sup> O emissions rate including two different sets. This first one was used to measure soil N<sup>2</sup> O emission rate in laboratory incubation studies. Different to that conducted *in situ*, incubation studies monitored soil N2 O emission based on soil mass rather than soil surface area. Thereby, those studies measured N<sup>2</sup> O emission rate based on soil mass per time. Equation developed for the calculation of N2 O emission rate of incubated soil could be described as follows [17, 30, 31]:

$$E = P \times V \times \frac{dc}{dt} \times \frac{1}{RT} \times M \times \frac{1}{m} \times \frac{1}{t} \tag{4}$$

where *E* refers to emission rates of soil N2 O (ng g−1 h−1), *P* is standard atmospheric pressure (Pa), *V* is headspace volume of the incubation flask (cm<sup>3</sup> ), *c* is the concentration of N2 O (ppb),

**Figure 2.** Simple diagram showing the production process of soil N<sup>2</sup> O.

*t* is the time between two sample collections (h), *R* is the universal gas constant, *T* is the absolute air temperature (K), *M* is the molecular mass of N2 O (g mol−1), and *m* is incubated soil mass by dry weight basis (g).

Relative to laboratory incubation studies, *in situ* studies could capture effects of various factors on soil N2 O emissions in natural environment. These studies generally calculated soil N<sup>2</sup> O emission rate based on soil surface area. In general, *in situ* measurements could be conducted using static opaque chamber/gas chromatography method. To be specific, circular or square grooved collars should be buried into soil, with groove filled with water to seal the gas collection chamber [32, 33]. When soil N2 O emission rate would be measured, open-bottom cylindrical or cubic PVC gas sampling chamber would be fit into the groove. To exclude the potential effects of temperature variation during gas collection process, gas chambers were usually wrapped by foam and aluminum foil. Inside each chamber, battery-powered fans were used during gas accumulation process to mix air samples. Gas samples were usually collected manually or automatically using single-use syringes or sir bags, respectively. After gas collection, gas chromatograph with electron capture detector was used to measure N<sup>2</sup> O concentrations. Soil N2 O emission rates were usually determined by the equation as follows [31–33]:

$$F = P \times V \times \frac{dN\_tO}{dt} \times \frac{1}{RT} \times M \times \frac{1}{A} \times \frac{M\_n}{M} \tag{5}$$

amelioration, vegetation or crop types [37], land use change and soil pH [36, 38]. While soil

For example, soil temperature is the key factor controlling microbial activities. Since both nitrification and denitrification processes have been demonstrated as being driven by nitrification and denitrification bacteria, soil temperature could impact both transformation processes via its effects on bacterial activities. According to previous studies, nitrification process preferred temperature between 25 and 35°C, while it will be inhibited when soil temperature decreased below 5°C or increased above 50°C [39]. Moreover, the favorable temperature for

In addition, soil acidification levels as shown by soil pH are also important for microbial

tively lower soil pH, indicating enhancement of denitrification bacteria activities. Similarly,

well-ventilated soil environment, nitrification process could be complete while denitrification

O production, under the same environmental conditions, more N2

more complicated compared with those in natural soils [4]. Agricultural practice generally including fertilization, tillage, water regime [34], and so on, all of which could alter soil physi-

First, agricultural soils received much more N input via fertilization, increasing N availabilities for nitrification and denitrification process [2, 4, 8]. Except for increasing N availability directly, fertilization types, quantity of fertilizations, fertilization method and the time when soil was fertilized together regulated N transformation process. In general, N fertilization including inorganic N or organic N input in agricultural management. In organic agriculture, activities of denitrification bacteria were higher, potentially facilitating the denitrification process,

Second, farmland with decreased-tillage or non-tillage management potentially enhance the accumulation of soil organic C [43]. Due to the balance between C and N regulated by C to N ratio, increased soil organic C might be accompanied by increased fixation of soil N and hence

also observed in studies on decreased tillage farmland [34, 44, 45]. Moreover, soil tillage could

microbial activities. Thereby, soil tillage and other disturbance management may be impor-

O productions [4].

activities of some nitrification bacteria would also increase in response to lower pH.

*O production in agricultural soils*

In agricultural soils with intensive anthropogenic disturbance, soil N<sup>2</sup>

O productions [41]. Indeed, soil N2

also impact soil aeration conditions and indirectly regulate soil N2

O emissions [34, 45, 46].

agriculture than conventional agriculture in another study by Phillips [42].

O emissions from agricultural field [36]. However, increased soil N<sup>2</sup>

O in denitrification process would increase with rela-

Nitrogen Transformations Associated with N2O Emissions in Agricultural Soils

O. Since complete nitrification process was also accom-

O by nitrification or denitrification process. In

O emissions were found lower in organic

O, other factors regulated

23

http://dx.doi.org/10.5772/intechopen.71922

O would be

O productions became

O emissions were

O emissions via effects on

N provided substrate for transformation process producing soil N2

denitrification falls within 30 and 67°C [40].

Soil aeration also controls the quantity of N2

produced in upland soils relative to flooded soils.

cal and chemical properties, impacting soil N2

activities. To be specific, the ratio of N<sup>2</sup>

process posed at stage producing N2

panied by N2

*3.2.2. Factors impacting N<sup>2</sup>*

decreasing soil N2

tant in impacting soil N2

less N2

O production process mainly via indirect effects on soil microbial activities.

N2

where *F* refers to soil N2 O emission rates (mg N2 O m−2 h−1), *P* is the standard atmospheric pressure (Pa), *V* and *A* are the volume (m3 ) and interior bottom area (m<sup>2</sup> ) of the gas collection chamber, *R* stands for universal gas constant, *T* is the absolute air temperature (K) when the gas sample was aspirated and *Mn* and *M* are the molecular masses of N and N2 O (g mol−1), respectively.

When cumulative emissions were needed for study purpose, total soil N<sup>2</sup> O emissions within a given time could be obtained by multiplying average soil N2 O emission rate and the corresponding time span [17, 33].

It should be noted that both methods were used to obtain the net soil N<sup>2</sup> O emission rates. During measurement, soil might be source or sink of N<sup>2</sup> O depends on soils used for studies. However, when results were positive based on two equations, it could be determined that soils were emitting N<sup>2</sup> O. Similarly, when values were negative, soil could be adsorbing N<sup>2</sup> O in the corresponding studies.

#### **3.2. Factors impacting soil N2 O productions**

#### *3.2.1. General factors impacting soil N<sup>2</sup> O productions*

According to the model shown in **Figure 2**, factors impacting nitrification and denitrification could also be able to influence the production and emission of soil N<sup>2</sup> O. As have been reported by previous studies, factors impacting nitrification process including quantity and quality of soil N input, soil moisture (water holding capacity) [34, 35], soil temperature [10, 17, 30], irrigation and tillage practices, soil type, soil oxygen concentration, dissolved organic C availability (controlling substrate availability of soil microbes) [17, 36], additives for soil amelioration, vegetation or crop types [37], land use change and soil pH [36, 38]. While soil N provided substrate for transformation process producing soil N2 O, other factors regulated N2 O production process mainly via indirect effects on soil microbial activities.

For example, soil temperature is the key factor controlling microbial activities. Since both nitrification and denitrification processes have been demonstrated as being driven by nitrification and denitrification bacteria, soil temperature could impact both transformation processes via its effects on bacterial activities. According to previous studies, nitrification process preferred temperature between 25 and 35°C, while it will be inhibited when soil temperature decreased below 5°C or increased above 50°C [39]. Moreover, the favorable temperature for denitrification falls within 30 and 67°C [40].

In addition, soil acidification levels as shown by soil pH are also important for microbial activities. To be specific, the ratio of N<sup>2</sup> O in denitrification process would increase with relatively lower soil pH, indicating enhancement of denitrification bacteria activities. Similarly, activities of some nitrification bacteria would also increase in response to lower pH.

Soil aeration also controls the quantity of N2 O by nitrification or denitrification process. In well-ventilated soil environment, nitrification process could be complete while denitrification process posed at stage producing N2 O. Since complete nitrification process was also accompanied by N2 O production, under the same environmental conditions, more N2 O would be produced in upland soils relative to flooded soils.

#### *3.2.2. Factors impacting N<sup>2</sup> O production in agricultural soils*

*t* is the time between two sample collections (h), *R* is the universal gas constant, *T* is the abso-

Relative to laboratory incubation studies, *in situ* studies could capture effects of various fac-

emission rate based on soil surface area. In general, *in situ* measurements could be conducted using static opaque chamber/gas chromatography method. To be specific, circular or square grooved collars should be buried into soil, with groove filled with water to seal the gas collec-

cal or cubic PVC gas sampling chamber would be fit into the groove. To exclude the potential effects of temperature variation during gas collection process, gas chambers were usually wrapped by foam and aluminum foil. Inside each chamber, battery-powered fans were used during gas accumulation process to mix air samples. Gas samples were usually collected manually or automatically using single-use syringes or sir bags, respectively. After gas collection,

O emission rates were usually determined by the equation as follows [31–33]:

chamber, *R* stands for universal gas constant, *T* is the absolute air temperature (K) when the

However, when results were positive based on two equations, it could be determined that

*<sup>d</sup> <sup>N</sup>*<sup>2</sup> *<sup>O</sup>* \_\_\_\_\_ *dt* <sup>×</sup> \_\_\_1

gas chromatograph with electron capture detector was used to measure N<sup>2</sup>

O emission rates (mg N2

When cumulative emissions were needed for study purpose, total soil N<sup>2</sup>

It should be noted that both methods were used to obtain the net soil N<sup>2</sup>

**O productions**

tion could also be able to influence the production and emission of soil N<sup>2</sup>

*O productions*

According to the model shown in **Figure 2**, factors impacting nitrification and denitrifica-

reported by previous studies, factors impacting nitrification process including quantity and quality of soil N input, soil moisture (water holding capacity) [34, 35], soil temperature [10, 17, 30], irrigation and tillage practices, soil type, soil oxygen concentration, dissolved organic C availability (controlling substrate availability of soil microbes) [17, 36], additives for soil

a given time could be obtained by multiplying average soil N2

During measurement, soil might be source or sink of N<sup>2</sup>

O emissions in natural environment. These studies generally calculated soil N<sup>2</sup>

*RT* <sup>×</sup> *<sup>M</sup>* <sup>×</sup> \_\_1

) and interior bottom area (m<sup>2</sup>

and *M* are the molecular masses of N and N2

O. Similarly, when values were negative, soil could be adsorbing N<sup>2</sup>

*<sup>A</sup>* <sup>×</sup> \_\_\_

O emission rate would be measured, open-bottom cylindri-

O (g mol−1), and *m* is incubated soil

O

O concentrations.

) of the gas collection

O emissions within

O emission rates.

O. As have been

O

O emission rate and the cor-

O depends on soils used for studies.

O (g mol−1),

*MnM* (5)

O m−2 h−1), *P* is the standard atmospheric

lute air temperature (K), *M* is the molecular mass of N2

mass by dry weight basis (g).

22 Nitrogen in Agriculture - Updates

tion chamber [32, 33]. When soil N2

*F* = *P* × *V* ×

pressure (Pa), *V* and *A* are the volume (m3

gas sample was aspirated and *Mn*

responding time span [17, 33].

in the corresponding studies.

**3.2. Factors impacting soil N2**

*3.2.1. General factors impacting soil N<sup>2</sup>*

soils were emitting N<sup>2</sup>

where *F* refers to soil N2

tors on soil N2

Soil N2

respectively.

In agricultural soils with intensive anthropogenic disturbance, soil N<sup>2</sup> O productions became more complicated compared with those in natural soils [4]. Agricultural practice generally including fertilization, tillage, water regime [34], and so on, all of which could alter soil physical and chemical properties, impacting soil N2 O productions [4].

First, agricultural soils received much more N input via fertilization, increasing N availabilities for nitrification and denitrification process [2, 4, 8]. Except for increasing N availability directly, fertilization types, quantity of fertilizations, fertilization method and the time when soil was fertilized together regulated N transformation process. In general, N fertilization including inorganic N or organic N input in agricultural management. In organic agriculture, activities of denitrification bacteria were higher, potentially facilitating the denitrification process, decreasing soil N2 O productions [41]. Indeed, soil N2 O emissions were found lower in organic agriculture than conventional agriculture in another study by Phillips [42].

Second, farmland with decreased-tillage or non-tillage management potentially enhance the accumulation of soil organic C [43]. Due to the balance between C and N regulated by C to N ratio, increased soil organic C might be accompanied by increased fixation of soil N and hence less N2 O emissions from agricultural field [36]. However, increased soil N<sup>2</sup> O emissions were also observed in studies on decreased tillage farmland [34, 44, 45]. Moreover, soil tillage could also impact soil aeration conditions and indirectly regulate soil N2 O emissions via effects on microbial activities. Thereby, soil tillage and other disturbance management may be important in impacting soil N2 O emissions [34, 45, 46].

Water regime, especially that in paddy field, plays an important role in controlling soil N<sup>2</sup> O production and emissions [47–49]. When paddy field was flooded, soil aeration was inhibited, anaerobic microenvironment was hence cultivated. In this kind of condition, N<sup>2</sup> became the main production of denitrification process, soil could be considered as the sink of N<sup>2</sup> O, as almost no emissions were observed. Meanwhile, flooded soil was generally favorable to methanogens, which were associated with soil CH<sup>4</sup> production. Indeed, flooded soil, generally in paddy field, has been demonstrated to be the main source of CH<sup>4</sup> [47]. However, during drainage time, paddy field was not flooded any more, especially during time when soils were humid (i.e., wetting and drying cycles), aerobiotic soil environment was formed, and soil N<sup>2</sup> O emission rate could reach a peak [31]. In this kind of environment, soil is not completely dry but experiencing wetting and drying cycles, allowing more oxygen in soil pores, increasing the production of N2 O [50]. Yan et al. [51] studied the correlations between soil water content and soil N2 O emissions, reporting the largest soil N2 O emission rate when soil water content was equivalent to water holding capacity. Thereby, soil water content and soil aeration condition interact in impacting soil N2 O emissions in agricultural soil.

be used with N fertilization and were effective in mitigation of soil N<sup>2</sup>

first negative effect is the potentially increased ammonia (NH<sup>3</sup>

efficacy of both inhibitors in mitigation of soil N<sup>2</sup>

their potential driving effect on soil N<sup>2</sup>

fertilizer in banana plantations [56].

reported decreased yield-scaled N2

*3.3.2. Biological nitrification inhibitors*

and NBPT could also decrease the yield-scaled N<sup>2</sup>

dent, which should be considered in future applications.

been demonstrated to be less phytotoxic and used at lower rate relative to DCD in general. The

soil chemical and physical characteristics, and so on. However, the difference in mitigation efficacy could also have been induced by the mobility of inhibitors in soil environment.

However, it should be noted that attentions should be paid to the negative effect on soil N availability [55] or food security [54] induced by applications of nitrification inhibitors. The

nitrification inhibitors [55]. Indeed, nitrification inhibitors decreased the rate of nitrification and denitrification process, potentially prolonged the retention time of ammonium N in soil environment, increasing the possibilities of more ammonia volatilization. Increased ammonia emissions would on one hand decrease the efficacy of N fertilization practice, and importantly, on the other hand, have economic and environmental consequences [53] considering

O emissions.

To reduce potentially increased ammonia volatilization after application of nitrification inhibitors, manufactures developed urease inhibitor N-(n-butyl) thiophosphoric triamide (NBPT) [56, 57]. Application of NBPT has been proved effective in reducing pasture soil urease activity and mitigating ammonia volatilization [58, 59]. The combination of nitrification inhibitor

The second caution is the grain yield and quality following alteration in soil N components [54]. While it is rational to expect that grain N concentration may increase in response to soil with longer N retention time, there are studies demonstrating no such effect in grain N following DMPP application [54]. A recent study on banana plantations in tropical China

ence between N fertilization treatment and N with inhibitors [56]. Thereby, the efficiency of N fertilization practice with nitrification inhibitors might be crops or vegetation-type depen-

There are also cautions on N fertilizer types that could be used combining with the application of nitrification inhibitors. To maintain the efficiency of nitrification inhibitors, only several N fertilizers could be widely used with nitrification inhibitors. Due to higher cost of theses fertilizers, wide adoption in agricultural practice became more difficult [59]. Recently, new compounds with higher nitrification inhibitor efficiencies have been developed, such as 3,4-dimethylpyrazole succinic (DMPSA), which is more stable when applied combined with

In tropical grassland and forest ecosystems, nitrification rate was found much slower relative to that in other similar soils. In further studies, it was found that some plant species synthesized important organic compounds and released these compounds into surrounding soils via roots [60]. These compounds were found being able to inhibit nitrification process, imposing similar

other basic fertilizers (e.g., calcium ammonium nitrate) at basic conditions [54, 59].

O emissions, DMPP has

25

) volatilization induced by

O emissions could depend on temperature,

http://dx.doi.org/10.5772/intechopen.71922

Nitrogen Transformations Associated with N2O Emissions in Agricultural Soils

O emissions relative to treatments only with

O emission, but banana yield showed no significant differ-

#### **3.3. Mitigation of N2 O emissions from agricultural soils**

Various strategies have been developed for the mitigation of soil N2 O emissions, especially in agricultural ecosystems. Agricultural management including fertilization, tillage, crop rotations, and so on has been employed in mitigation of soil GHG, especially N<sup>2</sup> O emissions. Not only because N2 O induce global warming, but also N losses that accompanied the production and emission process of N2 O. To increase fertilization efficiency, the economy of agriculture management, and the benefit for environment, more efficient mitigation strategies are still needed. Presently, nitrification inhibitor has been widely used in agricultural management and has been demonstrated to be much more effective.

Nitrification inhibitor interrupted the transformation process from ammonium N to nitrate N, which could decrease N losses from soil (emissions or leaching, for example, Marsden et al. [52]) and increase N availability level for crops and hence the adsorption of ammonium N. Using nitrification inhibitors could potentially decrease soil emissions via interruptions on both nitrification and denitrification processes simultaneously. Chemical nitrification inhibitor and biological nitrification inhibitor are two important choices in recent studies.

#### *3.3.1. Chemical nitrification inhibitors*

Chemical nitrification inhibitors are human-synthesized materials. By decreasing soil N loss induced by nitrification and denitrification process, nitrification inhibitors could enhance efficiency of N fertilization. Thereby, nitrification inhibitors were also used as additives for N fertilizers. Nitrogen fertilizers with these additives (or similar additives like urease inhibitors, etc.) were usually used and called as enhanced efficiency nitrogen fertilizers [53].

Dicyandiamide (DCD) and 3,4-dimethylpyrazole phosphate (DMPP) are two most widely used nitrification inhibitors [54]. Both inhibitors interrupted the oxidation of ammonium N, limiting the important step occurred in nitrification process. Even though both inhibitors could be used with N fertilization and were effective in mitigation of soil N<sup>2</sup> O emissions, DMPP has been demonstrated to be less phytotoxic and used at lower rate relative to DCD in general. The efficacy of both inhibitors in mitigation of soil N<sup>2</sup> O emissions could depend on temperature, soil chemical and physical characteristics, and so on. However, the difference in mitigation efficacy could also have been induced by the mobility of inhibitors in soil environment.

However, it should be noted that attentions should be paid to the negative effect on soil N availability [55] or food security [54] induced by applications of nitrification inhibitors. The first negative effect is the potentially increased ammonia (NH<sup>3</sup> ) volatilization induced by nitrification inhibitors [55]. Indeed, nitrification inhibitors decreased the rate of nitrification and denitrification process, potentially prolonged the retention time of ammonium N in soil environment, increasing the possibilities of more ammonia volatilization. Increased ammonia emissions would on one hand decrease the efficacy of N fertilization practice, and importantly, on the other hand, have economic and environmental consequences [53] considering their potential driving effect on soil N<sup>2</sup> O emissions.

To reduce potentially increased ammonia volatilization after application of nitrification inhibitors, manufactures developed urease inhibitor N-(n-butyl) thiophosphoric triamide (NBPT) [56, 57]. Application of NBPT has been proved effective in reducing pasture soil urease activity and mitigating ammonia volatilization [58, 59]. The combination of nitrification inhibitor and NBPT could also decrease the yield-scaled N<sup>2</sup> O emissions relative to treatments only with fertilizer in banana plantations [56].

The second caution is the grain yield and quality following alteration in soil N components [54]. While it is rational to expect that grain N concentration may increase in response to soil with longer N retention time, there are studies demonstrating no such effect in grain N following DMPP application [54]. A recent study on banana plantations in tropical China reported decreased yield-scaled N2 O emission, but banana yield showed no significant difference between N fertilization treatment and N with inhibitors [56]. Thereby, the efficiency of N fertilization practice with nitrification inhibitors might be crops or vegetation-type dependent, which should be considered in future applications.

There are also cautions on N fertilizer types that could be used combining with the application of nitrification inhibitors. To maintain the efficiency of nitrification inhibitors, only several N fertilizers could be widely used with nitrification inhibitors. Due to higher cost of theses fertilizers, wide adoption in agricultural practice became more difficult [59]. Recently, new compounds with higher nitrification inhibitor efficiencies have been developed, such as 3,4-dimethylpyrazole succinic (DMPSA), which is more stable when applied combined with other basic fertilizers (e.g., calcium ammonium nitrate) at basic conditions [54, 59].

#### *3.3.2. Biological nitrification inhibitors*

Water regime, especially that in paddy field, plays an important role in controlling soil N<sup>2</sup>

main production of denitrification process, soil could be considered as the sink of N<sup>2</sup>

anaerobic microenvironment was hence cultivated. In this kind of condition, N<sup>2</sup>

in paddy field, has been demonstrated to be the main source of CH<sup>4</sup>

O emissions, reporting the largest soil N2

Various strategies have been developed for the mitigation of soil N2

and has been demonstrated to be much more effective.

anogens, which were associated with soil CH<sup>4</sup>

the production of N2

24 Nitrogen in Agriculture - Updates

**3.3. Mitigation of N2**

only because N2

and emission process of N2

*3.3.1. Chemical nitrification inhibitors*

tion interact in impacting soil N2

and soil N2

production and emissions [47–49]. When paddy field was flooded, soil aeration was inhibited,

almost no emissions were observed. Meanwhile, flooded soil was generally favorable to meth-

drainage time, paddy field was not flooded any more, especially during time when soils were humid (i.e., wetting and drying cycles), aerobiotic soil environment was formed, and soil N<sup>2</sup>

emission rate could reach a peak [31]. In this kind of environment, soil is not completely dry but experiencing wetting and drying cycles, allowing more oxygen in soil pores, increasing

was equivalent to water holding capacity. Thereby, soil water content and soil aeration condi-

agricultural ecosystems. Agricultural management including fertilization, tillage, crop rota-

management, and the benefit for environment, more efficient mitigation strategies are still needed. Presently, nitrification inhibitor has been widely used in agricultural management

Nitrification inhibitor interrupted the transformation process from ammonium N to nitrate N, which could decrease N losses from soil (emissions or leaching, for example, Marsden et al. [52]) and increase N availability level for crops and hence the adsorption of ammonium N. Using nitrification inhibitors could potentially decrease soil emissions via interruptions on both nitrification and denitrification processes simultaneously. Chemical nitrification inhibi-

Chemical nitrification inhibitors are human-synthesized materials. By decreasing soil N loss induced by nitrification and denitrification process, nitrification inhibitors could enhance efficiency of N fertilization. Thereby, nitrification inhibitors were also used as additives for N fertilizers. Nitrogen fertilizers with these additives (or similar additives like urease inhibitors,

Dicyandiamide (DCD) and 3,4-dimethylpyrazole phosphate (DMPP) are two most widely used nitrification inhibitors [54]. Both inhibitors interrupted the oxidation of ammonium N, limiting the important step occurred in nitrification process. Even though both inhibitors could

tor and biological nitrification inhibitor are two important choices in recent studies.

etc.) were usually used and called as enhanced efficiency nitrogen fertilizers [53].

**O emissions from agricultural soils**

tions, and so on has been employed in mitigation of soil GHG, especially N<sup>2</sup>

O emissions in agricultural soil.

O [50]. Yan et al. [51] studied the correlations between soil water content

O induce global warming, but also N losses that accompanied the production

O. To increase fertilization efficiency, the economy of agriculture

O

O, as

O

became the

[47]. However, during

O emissions, especially in

O emissions. Not

production. Indeed, flooded soil, generally

O emission rate when soil water content

In tropical grassland and forest ecosystems, nitrification rate was found much slower relative to that in other similar soils. In further studies, it was found that some plant species synthesized important organic compounds and released these compounds into surrounding soils via roots [60]. These compounds were found being able to inhibit nitrification process, imposing similar effects on nitrification process as chemical nitrification inhibitors. They were called biological nitrification inhibitors as they were not human synthesized. Thereby, biological nitrification inhibitors are organic materials that have similar negative effects on nitrification process exuded by plant.

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Nitrogen Transformations Associated with N2O Emissions in Agricultural Soils

http://dx.doi.org/10.5772/intechopen.71922

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[2] Bouwman AF. Direct emission of nitrous oxide from agricultural soils. Nutrient Cycling

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[7] Zhu T, Zhang J, Cai Z. The contribution of nitrogen transformation processes to total

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[10] Jiang L, Zhang L, Deng B, Liu X, Yi H, Xiang H, et al. Alpine meadow restorations by non-dominant species increased soil nitrogen transformation rates but decreased their

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sensitivity to warming. Journal of Soils and Sediments. 2017;**17**(9):2329-2337

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Biological nitrification inhibitors generally including phenolic compounds, alkaloid, isothiocyanate and terpenoid [60, 61]. For example, chemicals produced by *Arbutus unedo*, including phenolic compounds gallocatechin and catechin were able to decrease soil N<sup>2</sup> O emissions [62]. In tropical grassland of Africa, both *Brachiaria humidicola* and *Brachiaria decumbens* were found be able to release biological nitrification inhibitors by producing linoleic, and so on, which enable them to survive in the low-N south Africa Savannas [63].

Compared with chemical nitrification inhibitors, biological inhibitors were environmentfriendly in their producing process and application area. However, due to varying ability in synthesizing these compounds, further studies in how to cultivate species with stronger ability in producing these compounds are still needed.

## *3.3.3. Other gradients incorporated into agricultural soils*

Other efforts have also been tried in decreasing soil N<sup>2</sup> O emissions. In agricultural and forest soils, biochar has been used to improve soil quality and C sequestration [64–67]. Biochar is produced by slow pyrolysis of crop residues, household garbage, poultry litter, wood chips, or some other similar materials at high temperature (pyrolysis temperature generally between 400 and 600°C) without oxygen [67]. The physical characteristics of biochar enable it to be an ideal soil ameliorant. By applying biochar, soil aeration could be improved significantly, providing more oxygen and hence enhancing soil microbial activities. Due to its special physical characteristics, biochar could also prevent soil N leaching by adsorbing nitrate N (temporary immobilization, which would not impose negative effect on plant nutrient availability forever).

## **Acknowledgements**

We acknowledge funding support by National Natural Science Foundation of China (Award number: 41501317), China Postdoctoral Science Foundation (Award number: 2017M612153), and Key Project of Jiangxi Education Department (Award number: GJJ160348).

## **Author details**

Ling Zhang\* and Xiaojun Liu

\*Address all correspondence to: lingzhang09@126.com

Jiangxi Key Laboratory of Silviculture, Co-Innovation Center of Jiangxi Typical Trees Cultivation and Utilization, College of Forestry, Jiangxi Agricultural University, Nanchang, China

## **References**

effects on nitrification process as chemical nitrification inhibitors. They were called biological nitrification inhibitors as they were not human synthesized. Thereby, biological nitrification inhibitors are organic materials that have similar negative effects on nitrification process exuded by plant. Biological nitrification inhibitors generally including phenolic compounds, alkaloid, isothiocyanate and terpenoid [60, 61]. For example, chemicals produced by *Arbutus unedo*, including

[62]. In tropical grassland of Africa, both *Brachiaria humidicola* and *Brachiaria decumbens* were found be able to release biological nitrification inhibitors by producing linoleic, and so on,

Compared with chemical nitrification inhibitors, biological inhibitors were environmentfriendly in their producing process and application area. However, due to varying ability in synthesizing these compounds, further studies in how to cultivate species with stronger abil-

soils, biochar has been used to improve soil quality and C sequestration [64–67]. Biochar is produced by slow pyrolysis of crop residues, household garbage, poultry litter, wood chips, or some other similar materials at high temperature (pyrolysis temperature generally between 400 and 600°C) without oxygen [67]. The physical characteristics of biochar enable it to be an ideal soil ameliorant. By applying biochar, soil aeration could be improved significantly, providing more oxygen and hence enhancing soil microbial activities. Due to its special physical characteristics, biochar could also prevent soil N leaching by adsorbing nitrate N (temporary immobilization, which would not impose negative effect on plant nutrient availability forever).

We acknowledge funding support by National Natural Science Foundation of China (Award number: 41501317), China Postdoctoral Science Foundation (Award number: 2017M612153),

and Key Project of Jiangxi Education Department (Award number: GJJ160348).

Jiangxi Key Laboratory of Silviculture, Co-Innovation Center of Jiangxi Typical Trees Cultivation and Utilization, College of Forestry, Jiangxi Agricultural University, Nanchang,

O emissions

O emissions. In agricultural and forest

phenolic compounds gallocatechin and catechin were able to decrease soil N<sup>2</sup>

which enable them to survive in the low-N south Africa Savannas [63].

ity in producing these compounds are still needed.

*3.3.3. Other gradients incorporated into agricultural soils*

Other efforts have also been tried in decreasing soil N<sup>2</sup>

**Acknowledgements**

26 Nitrogen in Agriculture - Updates

**Author details**

China

Ling Zhang\* and Xiaojun Liu

\*Address all correspondence to: lingzhang09@126.com


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tural N2


**Chapter 3**

**Provisional chapter**

**Controlled-Release Fertilizers as a Means to Reduce**

**Nitrogen Leaching and Runoff in Container-Grown** 

**Controlled-Release Fertilizers as a Means to Reduce** 

DOI: 10.5772/intechopen.73055

**Nitrogen Leaching and Runoff in Container-Grown**

Container-grown plants refer to plants produced in confined volume filled with substrates. The substrates endogenously have limited nutrients and low water-holding capacity. Plants grown in the containers must be fertilized and watered frequently varying from daily to weekly. Frequent fertilization and irrigation can result in nutrient leaching and/or runoff. Since nitrogen (N) is a key component of the majority of fertilizers, container plant production has been viewed as a source of N leaching and/or runoff. The leaching and runoff, if in large quantities on a year-round basis, could affect surface and ground water quality. Application of controlled-release fertilizers (CRFs) has been reported to have less N leaching than plants fertilized with water-soluble fertilizers (WSFs). However, there are different types of CRFs with different compositions and longevities on the market. Container plants also differ greatly in their growth and development and in N requirement. Thus, production of high-quality container plants with minimum N leaching using CRFs still remains challenging. This article is intended to discuss characteristics of container plant production and N leaching and runoff during production, and to document that CRF application can reduce N leaching and/or runoff.

Certain requirements for future development of CRFs are also discussed.

**Keywords:** container-grown plants, controlled-release fertilizers, nitrogen leaching

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

© 2018 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

Container-grown plants refer to those grown from seedlings, liners, rooted cuttings or grafted plants in containers or pots filled with substrates to marketable sizes or harvestable stages. Substrates or growing media are comprised of peat, perlite, soil, vermiculate or other organic

**Plant Production**

**Abstract**

and runoff, nitrate

**1. Introduction**

**Plant Production**

Jianjun Chen and Xiangying Wei

Jianjun Chen and Xiangying Wei

http://dx.doi.org/10.5772/intechopen.73055

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

**Provisional chapter**

## **Controlled-Release Fertilizers as a Means to Reduce Nitrogen Leaching and Runoff in Container-Grown Plant Production Nitrogen Leaching and Runoff in Container-Grown Plant Production**

**Controlled-Release Fertilizers as a Means to Reduce** 

DOI: 10.5772/intechopen.73055

Jianjun Chen and Xiangying Wei Jianjun Chen and Xiangying Wei

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.73055

#### **Abstract**

Container-grown plants refer to plants produced in confined volume filled with substrates. The substrates endogenously have limited nutrients and low water-holding capacity. Plants grown in the containers must be fertilized and watered frequently varying from daily to weekly. Frequent fertilization and irrigation can result in nutrient leaching and/or runoff. Since nitrogen (N) is a key component of the majority of fertilizers, container plant production has been viewed as a source of N leaching and/or runoff. The leaching and runoff, if in large quantities on a year-round basis, could affect surface and ground water quality. Application of controlled-release fertilizers (CRFs) has been reported to have less N leaching than plants fertilized with water-soluble fertilizers (WSFs). However, there are different types of CRFs with different compositions and longevities on the market. Container plants also differ greatly in their growth and development and in N requirement. Thus, production of high-quality container plants with minimum N leaching using CRFs still remains challenging. This article is intended to discuss characteristics of container plant production and N leaching and runoff during production, and to document that CRF application can reduce N leaching and/or runoff. Certain requirements for future development of CRFs are also discussed.

**Keywords:** container-grown plants, controlled-release fertilizers, nitrogen leaching and runoff, nitrate

## **1. Introduction**

Container-grown plants refer to those grown from seedlings, liners, rooted cuttings or grafted plants in containers or pots filled with substrates to marketable sizes or harvestable stages. Substrates or growing media are comprised of peat, perlite, soil, vermiculate or other organic

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

components in different proportions. Many plants can be produced in containers including floriculture, nursery, fruit and vegetable crops. According to the United States Department of Agriculture (USDA) National Agriculture Statistics Service [1], floriculture crops are ornamental plants without woody stems, such as annual and perennial bedding and garden plants, cut flowers, cut cultivated greenery, potted flowering plants, tropical foliage plants and unfinished propagative material. Nursery crops are finished ornamental plants and trees with woody stems that are used for outdoor landscaping. Nursery crops also include ornamental vines, turfgrass sod and other groundcovers. Fruit and vegetable crops can also be produced in containers. Container fruit crops commonly include apple, blueberry, cherry, citrus, fig, orange, peach, pear and plum trees. Container vegetables include basil, beet, carrot, cucumber, ginger, lettuce, radish, onion, strawberry and tomato.

[13], there were over 2069 commercial nursery and greenhouse farms in Florida, with total sales of \$1.796 billion, and \$3.291 billion in capital assets in land, buildings and equipment.

The rapid increase in container plant production, however, has been under increasing scrutiny because of potential contamination of surface and/or ground water by nutrient ele-

2000 kg N/ha, were found in soil depth of 100 cm underlying commercial greenhouses [14]. In

old greenhouses [15]. A survey conducted in six states in the US such as Alabama, Florida, New Jersey, North Carolina, Ohio and Virginia suggested that the levels of runoff NO<sup>3</sup>

varied from 0.5 to 33 mg/L for container nurseries using controlled-released fertilizers (CRFs) and 0.1–135 mg/L for those using CRFs supplemented with water soluble fertilizers (WSFs) [16]. Also a survey completed from 11 nurseries in southern California showed that media

gation runoff in a foliage plant production nursery in southern Florida ranged from 41 to

Nitrate N is also leached from container substrates during crop production. In a container production of *Ilex crenata* Thunb. 'Compacta', Fare et al. [19] reported that the percentage of

to 63% when 13-mm irrigation was applied in a single cycle. Broschat [20] investigated N leaching from a container substrate comprised of 50% pine bark, 40% sedge peat and 10% sand

per hectare. Container production of poinsettia (*Euphorbia pulcherrima* Willd. ex Klotzsch), a potted floriculture crop, fertilized with a solution containing 210 mg/L of N showed that 40 and 60% of applied N was leached from containers when fertigated with leaching fractions of 0.2 and 0.4, respectively (leaching fraction is defined as the volume of leachate divided by the irrigation solution applied) [21]. Production of container azalea (*Rhododendron* L. 'Karen') with a weekly application of N at 250 mg/L could result in the loss of N at 924 kg/ha [22]. Container production of a bedding plant *Impatiens walleriana* Hook. f. by overhead irrigation resulted in 25.6% of the total applied water leaching out of the container and 34% fell between containers, and weekly N concentrations ranged from 137 to 153 mg/L in leachate and 165–256 in runoff

*vera* L., *Kalanchoe blossfeldiana* Poelln. and *Gazania splendens* Lem. ranged from 15 to 90 mg/L when plants were watered in 45% of the container capacity using nutrient solutions contain-

Nitrate N resulted from leaching and runoff could enter rivers, lakes and estuaries contributing to water eutrophication. N concentrations greater than 0.4 mg/L have been shown to

is a major human health concern, particularly to infants when nitrate is transformed to nitrite

─N and different concentrations of sodium.

tion of *Spathiphyllum* Schott. This could be translated to the annual loss of 666 kg of NO<sup>3</sup>

─N concentrations in runoff exceeded 10 mg/L in most nurseries [17]. NO<sup>3</sup>

─N accumulation over 2300 kg/ha was recorded in soil under decades-

Controlled-Release Fertilizers as a Means to Reduce Nitrogen Leaching and Runoff…

─N ranged from 46% when 13-mm irrigation was applied in 3 cycles

─N could be leached per container during a 6-month produc-

─N in leachates of container-grown *Aloe* 

─N contamination of groundwater

─N concentrations, up to

http://dx.doi.org/10.5772/intechopen.73055

─N

35

─N

─N in irri-

**2. Nitrogen loss during container plant production**

ments, particularly nitrogen (N). In Europe, extremely high NO<sup>3</sup>

386 mg/L depending on irrigation methods [18].

water during a 6-week production [23]. In Spain, NO<sup>3</sup>

accelerate eutrophication, causing algal blooms [24]. NO<sup>3</sup>

Connecticut, US, NO<sup>3</sup>

applied N leached as NO<sup>3</sup>

ing 372 mg/L NO<sup>3</sup>

and reported 3710 mg of NO<sup>3</sup>

NO<sup>3</sup>

Container crop production has become increasingly popular over the past 50 years [2, 3]. This is because container plant production has several advantages over traditional field production: (1) container plants are grown in substrates, not in soil, their production does not rely on arable land; (2) container sizes, substrate types and pH, pest, disease, water and nutrient management are easier to control or modify in container plant production than field production [4]; (3) plants grown in containers have a greater fine root mass compared to field-grown plants [5, 6]. Root surface area of holly plants (*Ilex x attenuata* Ashe 'East Palatka') grown in containers increased more than twofold than those grown in ground, and plant leaf dry weight and total top dry weight were 22.5 and 15% greater, respectively, when grown in containers [5]; (4) container plants are more convenient for moving and shipping, allowing more operational flexibility and improving shipping efficiency; (5) containerization allows growers to sell plants throughout the year regardless of soil conditions or plant growth stage, which increases productivity per unit area; (6) container-grown plants exhibit much less transplant shock and higher survival rates after transplanting compared to field-grown plants [7]; (7) plant spacing for containers ranges from 17,300 to 247,000 plants per hectare in nurseries and 99,000–865,000 plants per hectare in greenhouse production compared to 1480–12,360 plants per hectare in field production [2], thus, much more plants are produced per hectare by container production and more profit is made per unit area and (8) containergrown plants can be consolidated to provide space for growing additional plants after inventories are sold. However, such consolidation will not be possible for field-grown plants. More plants per unit area of container-grown crops means higher revenue compared with field production [8].

Currently, approximately 90% of greenhouse, nursery and floriculture crops in the USA are produced in containers [9]. The floriculture and nursery industries are strong and fast-growing sectors of US agriculture. Together, it accounts for a total of \$11.7 billion in sales in 2009, a 10.7% increase since 1998. Floriculture and nursery crops comprise almost 30% of the specialty crops grown in the USA [10]. Since floriculture and nursery crops are used largely for decoration of the surrounding environment, they are produced in every state in the USA. The leading floriculture and nursery states are California, Florida, Michigan, Texas and New York [11]. The floriculture and nursery industries generate 170,000 jobs worth \$3.78 billion to California's economy [12]. Floriculture and nursery crops are among the largest agricultural commodity groups in Florida. According to the Census of Horticulture Specialties for 2014 [13], there were over 2069 commercial nursery and greenhouse farms in Florida, with total sales of \$1.796 billion, and \$3.291 billion in capital assets in land, buildings and equipment.

## **2. Nitrogen loss during container plant production**

components in different proportions. Many plants can be produced in containers including floriculture, nursery, fruit and vegetable crops. According to the United States Department of Agriculture (USDA) National Agriculture Statistics Service [1], floriculture crops are ornamental plants without woody stems, such as annual and perennial bedding and garden plants, cut flowers, cut cultivated greenery, potted flowering plants, tropical foliage plants and unfinished propagative material. Nursery crops are finished ornamental plants and trees with woody stems that are used for outdoor landscaping. Nursery crops also include ornamental vines, turfgrass sod and other groundcovers. Fruit and vegetable crops can also be produced in containers. Container fruit crops commonly include apple, blueberry, cherry, citrus, fig, orange, peach, pear and plum trees. Container vegetables include basil, beet, carrot,

Container crop production has become increasingly popular over the past 50 years [2, 3]. This is because container plant production has several advantages over traditional field production: (1) container plants are grown in substrates, not in soil, their production does not rely on arable land; (2) container sizes, substrate types and pH, pest, disease, water and nutrient management are easier to control or modify in container plant production than field production [4]; (3) plants grown in containers have a greater fine root mass compared to field-grown plants [5, 6]. Root surface area of holly plants (*Ilex x attenuata* Ashe 'East Palatka') grown in containers increased more than twofold than those grown in ground, and plant leaf dry weight and total top dry weight were 22.5 and 15% greater, respectively, when grown in containers [5]; (4) container plants are more convenient for moving and shipping, allowing more operational flexibility and improving shipping efficiency; (5) containerization allows growers to sell plants throughout the year regardless of soil conditions or plant growth stage, which increases productivity per unit area; (6) container-grown plants exhibit much less transplant shock and higher survival rates after transplanting compared to field-grown plants [7]; (7) plant spacing for containers ranges from 17,300 to 247,000 plants per hectare in nurseries and 99,000–865,000 plants per hectare in greenhouse production compared to 1480–12,360 plants per hectare in field production [2], thus, much more plants are produced per hectare by container production and more profit is made per unit area and (8) containergrown plants can be consolidated to provide space for growing additional plants after inventories are sold. However, such consolidation will not be possible for field-grown plants. More plants per unit area of container-grown crops means higher revenue compared with field

Currently, approximately 90% of greenhouse, nursery and floriculture crops in the USA are produced in containers [9]. The floriculture and nursery industries are strong and fast-growing sectors of US agriculture. Together, it accounts for a total of \$11.7 billion in sales in 2009, a 10.7% increase since 1998. Floriculture and nursery crops comprise almost 30% of the specialty crops grown in the USA [10]. Since floriculture and nursery crops are used largely for decoration of the surrounding environment, they are produced in every state in the USA. The leading floriculture and nursery states are California, Florida, Michigan, Texas and New York [11]. The floriculture and nursery industries generate 170,000 jobs worth \$3.78 billion to California's economy [12]. Floriculture and nursery crops are among the largest agricultural commodity groups in Florida. According to the Census of Horticulture Specialties for 2014

cucumber, ginger, lettuce, radish, onion, strawberry and tomato.

production [8].

34 Nitrogen in Agriculture - Updates

The rapid increase in container plant production, however, has been under increasing scrutiny because of potential contamination of surface and/or ground water by nutrient elements, particularly nitrogen (N). In Europe, extremely high NO<sup>3</sup> ─N concentrations, up to 2000 kg N/ha, were found in soil depth of 100 cm underlying commercial greenhouses [14]. In Connecticut, US, NO<sup>3</sup> ─N accumulation over 2300 kg/ha was recorded in soil under decadesold greenhouses [15]. A survey conducted in six states in the US such as Alabama, Florida, New Jersey, North Carolina, Ohio and Virginia suggested that the levels of runoff NO<sup>3</sup> ─N varied from 0.5 to 33 mg/L for container nurseries using controlled-released fertilizers (CRFs) and 0.1–135 mg/L for those using CRFs supplemented with water soluble fertilizers (WSFs) [16]. Also a survey completed from 11 nurseries in southern California showed that media NO<sup>3</sup> ─N concentrations in runoff exceeded 10 mg/L in most nurseries [17]. NO<sup>3</sup> ─N in irrigation runoff in a foliage plant production nursery in southern Florida ranged from 41 to 386 mg/L depending on irrigation methods [18].

Nitrate N is also leached from container substrates during crop production. In a container production of *Ilex crenata* Thunb. 'Compacta', Fare et al. [19] reported that the percentage of applied N leached as NO<sup>3</sup> ─N ranged from 46% when 13-mm irrigation was applied in 3 cycles to 63% when 13-mm irrigation was applied in a single cycle. Broschat [20] investigated N leaching from a container substrate comprised of 50% pine bark, 40% sedge peat and 10% sand and reported 3710 mg of NO<sup>3</sup> ─N could be leached per container during a 6-month production of *Spathiphyllum* Schott. This could be translated to the annual loss of 666 kg of NO<sup>3</sup> ─N per hectare. Container production of poinsettia (*Euphorbia pulcherrima* Willd. ex Klotzsch), a potted floriculture crop, fertilized with a solution containing 210 mg/L of N showed that 40 and 60% of applied N was leached from containers when fertigated with leaching fractions of 0.2 and 0.4, respectively (leaching fraction is defined as the volume of leachate divided by the irrigation solution applied) [21]. Production of container azalea (*Rhododendron* L. 'Karen') with a weekly application of N at 250 mg/L could result in the loss of N at 924 kg/ha [22]. Container production of a bedding plant *Impatiens walleriana* Hook. f. by overhead irrigation resulted in 25.6% of the total applied water leaching out of the container and 34% fell between containers, and weekly N concentrations ranged from 137 to 153 mg/L in leachate and 165–256 in runoff water during a 6-week production [23]. In Spain, NO<sup>3</sup> ─N in leachates of container-grown *Aloe vera* L., *Kalanchoe blossfeldiana* Poelln. and *Gazania splendens* Lem. ranged from 15 to 90 mg/L when plants were watered in 45% of the container capacity using nutrient solutions containing 372 mg/L NO<sup>3</sup> ─N and different concentrations of sodium.

Nitrate N resulted from leaching and runoff could enter rivers, lakes and estuaries contributing to water eutrophication. N concentrations greater than 0.4 mg/L have been shown to accelerate eutrophication, causing algal blooms [24]. NO<sup>3</sup> ─N contamination of groundwater is a major human health concern, particularly to infants when nitrate is transformed to nitrite in the digestive system [25, 26]. The nitrite can oxidize the iron in hemoglobin of red blood cells, resulting in the formation of methemoglobin. Because methemoglobin lacks the ability to bind (or release) oxygen, blood will be unable to carry sufficient oxygen to the individual body cells, causing the veins and skin to appear blue. This is a condition known as methemoglobinemia (sometimes referred to as "blue baby syndrome") [27]. Most humans over 1 year of age have the ability to rapidly convert methemoglobin back to oxyhemoglobin. Thus, the total amount of methemoglobin within red blood cells remains low despite relatively high levels of nitrate/nitrite uptake. In infants under 6 months of age, however, the enzyme systems responsible for reducing methemoglobin to oxyhemoglobin are incompletely developed and methemoglobinemia can occur. This also may happen in older individuals who have genetically impaired enzyme systems for metabolizing methemoglobin. Furthermore, prolonged nitrate and nitrite ingestion could increase risks of certain cancers [28].

The rationales for the solutions in Chen et al. [36] were as follows: (1) plants are generally inefficient in N utilization. It has been well documented that crops directly utilize less than half (rarely more than 40%) of applied N [37]. Moreover, overall N-use efficiency (NUE) declined with increasing N-fertilizer application [38]. However, recommended fertilizer rates for container-grown plants are often much higher than actual plant needs. As shown by Chen et al. [36], N rates for some container-grown crops ranged from 1067 to 2354 kg per hectare per year, which is 10–15 times higher than those recommended for many agronomic field crops. Such high recommendation rates, along with extensive irrigation further enhance N leaching and runoff. In addition, different plant species and even their different cultivars differ in N requirement. Thus, a nursery operation should have different fertilizer programs suited to each species or a group of species [36, 39]. (2) Since the commercialization of container substrates after the World War II, substrate components have been predominantly pine bark, peat, vermiculite and perlite. Components newly introduced are coconut coir and polymer gel [36]. Accumulated research evidence indicates that specific zeolites and biochars have an added adsorption capacity for nutrient elements, including N [36, 40, 41]. Incorporating selected zeolites and/or engineered biochars into substrate formation should improve nutrient holding capacity and reduce nutrient leaching. (3) N is the most abundant element in most fertilizer formulation. This is due to the fact that N is the most important nutrient to plant growth and development and a plant generally absorbs more N than other element.

Common N compounds in fertilizer formulations include ammonium (NH<sup>4</sup>

, most plant species prefer NO<sup>3</sup>

+

−

−

+

−

is why CRFs have been developed [42]. (4) As water and fertilizer are interrelated in container plant production, one way to avoid N runoff or leaching into groundwater is to use zero runoff subirrigation [36]. Growers in Florida adopting either ebb-and-flow or capillary mat irrigation reported 20% reduction of fertilizer use and 75% reduction of water consumption in containerized plant production. Another irrigation method, which can achieve minimal runoff and less salt buildup in substrates, is to use surface irrigation systems, but to also capture, retain and recycle the runoff and stormwater within the boundaries of the production facility [43]. This is exemplified by whole greenhouse/nursery recycling system, called the total nursery recycling system. This recycling system includes (1) stormwater and/or irrigation runoff collection, (2) sedimentation, flocculation, filtration and disinfection, if necessary and (3) irrigation. Skimina [44] tested more than 100 species of landscape ornamental plants using this system and found that the range of plant growth response was 73–171% relative to control plants. However, few nurseries have used this total nursery recycle system for the production of greenhouse container plants. Growers were concerned about the feasibility and reliability of the water sources for the production of high-quality plants. As a result, the use of CRFs is considered to be a more convenient method for container plant production, while potentially

and NO<sup>3</sup>

Controlled-Release Fertilizers as a Means to Reduce Nitrogen Leaching and Runoff…

over NH<sup>4</sup>

, encapsulated N fertilizers should be a better choice, and this

−

+

can also be nitrified by soil bacteria to NO<sup>3</sup>

does not bind readily to the predominantly

]. Plants can directly absorb NH<sup>4</sup>

by microorganisms. NH<sup>4</sup>

. Additionally, as an anion, NO<sup>3</sup>

−

negatively charged soil and substrate colloids. Thus, NO<sup>3</sup>

and urea [CO-(NH<sup>2</sup>

Between NH<sup>4</sup>

cies prefer NH<sup>4</sup>

hydrolyzed into NH<sup>4</sup>

+

+

To reduce the mobility of NO<sup>3</sup>

reducing N leaching and runoff.

) 2

and NO<sup>3</sup>

+

−

+

http://dx.doi.org/10.5772/intechopen.73055

is highly mobile in soil or substrate.

, but not urea. Urea in soil is

although a few plant spe-

), nitrate (NO<sup>3</sup>

− ) 37

− .

The US Public Health Service adopted drinking water standards and set the recommended limit for NO<sup>3</sup> ─N at 10 mg/L in 1962 [29]. This drinking water standard was established to protect the health of infants, children, pregnant women, the elderly and immune-compromised individuals. The potential health hazard for others depends on the individual's reaction to NO<sup>3</sup> ─N and the total ingestion of NO<sup>3</sup> ─N and nitrites from all sources. From 1970 to 1992, the US Geological Survey found that 9% of the private wells that were tested exceed the recommended limit of 10 mg/L NO<sup>3</sup> ─N [30]. The US Environmental Protection Agency (USEPA) [31] has since adopted the 10 mg/L standard as the maximum contaminant level (MCL) for NO<sup>3</sup> ─N and 1 mg/L for nitrite-N for regulated public water systems. Subsequent reviews of this standard have not resulted in any changes.

Applied N can also be evolved as ammonia (NH<sup>3</sup> ) or nitrous oxide (N<sup>2</sup> O) gases. It was estimated the 10% of manufactured N fertilizers could be volatilized as NH<sup>3</sup> gas [32] and 1% of N applied in inorganic forms was lost to the atmosphere as N<sup>2</sup> O [33]. The volatilization of both NH<sup>3</sup> and N<sup>2</sup> O are serious environmental concern as NH<sup>3</sup> contributes to photochemical smog [34] and N<sup>2</sup> O is a potent greenhouse gas with a global warming potential of 310 times greater than carbon dioxide [35].

#### **3. Methods for reducing N loss**

Different strategies and methods have been proposed and used for reducing NO<sup>3</sup> ─N leaching and runoff during the production of container-grown plants. Chen et al. [36] suggested that approaches to NO<sup>3</sup> ─N leaching and runoff should take plant species, fertilizer application rates, container substrate and irrigation methods into consideration for developing best management practices (BMPs), which include (1) understanding plant species requirement for N and application of N based on plant need; (2) improving physical and chemical properties of container substrates and increasing their holding capacities for water and nutrients, particularly NO<sup>3</sup> ─N; (3) using controlled-release fertilizers to reduce NO<sup>3</sup> ─N leaching; and (4) irrigation system improvement by using either drip irrigation or subirrigation to reduce leaching and runoff.

The rationales for the solutions in Chen et al. [36] were as follows: (1) plants are generally inefficient in N utilization. It has been well documented that crops directly utilize less than half (rarely more than 40%) of applied N [37]. Moreover, overall N-use efficiency (NUE) declined with increasing N-fertilizer application [38]. However, recommended fertilizer rates for container-grown plants are often much higher than actual plant needs. As shown by Chen et al. [36], N rates for some container-grown crops ranged from 1067 to 2354 kg per hectare per year, which is 10–15 times higher than those recommended for many agronomic field crops. Such high recommendation rates, along with extensive irrigation further enhance N leaching and runoff. In addition, different plant species and even their different cultivars differ in N requirement. Thus, a nursery operation should have different fertilizer programs suited to each species or a group of species [36, 39]. (2) Since the commercialization of container substrates after the World War II, substrate components have been predominantly pine bark, peat, vermiculite and perlite. Components newly introduced are coconut coir and polymer gel [36]. Accumulated research evidence indicates that specific zeolites and biochars have an added adsorption capacity for nutrient elements, including N [36, 40, 41]. Incorporating selected zeolites and/or engineered biochars into substrate formation should improve nutrient holding capacity and reduce nutrient leaching. (3) N is the most abundant element in most fertilizer formulation. This is due to the fact that N is the most important nutrient to plant growth and development and a plant generally absorbs more N than other element. Common N compounds in fertilizer formulations include ammonium (NH<sup>4</sup> + ), nitrate (NO<sup>3</sup> − ) and urea [CO-(NH<sup>2</sup> ) 2 ]. Plants can directly absorb NH<sup>4</sup> + and NO<sup>3</sup> − , but not urea. Urea in soil is hydrolyzed into NH<sup>4</sup> + by microorganisms. NH<sup>4</sup> + can also be nitrified by soil bacteria to NO<sup>3</sup> − . Between NH<sup>4</sup> + and NO<sup>3</sup> − , most plant species prefer NO<sup>3</sup> − over NH<sup>4</sup> + although a few plant species prefer NH<sup>4</sup> + . Additionally, as an anion, NO<sup>3</sup> − does not bind readily to the predominantly negatively charged soil and substrate colloids. Thus, NO<sup>3</sup> − is highly mobile in soil or substrate. To reduce the mobility of NO<sup>3</sup> − , encapsulated N fertilizers should be a better choice, and this is why CRFs have been developed [42]. (4) As water and fertilizer are interrelated in container plant production, one way to avoid N runoff or leaching into groundwater is to use zero runoff subirrigation [36]. Growers in Florida adopting either ebb-and-flow or capillary mat irrigation reported 20% reduction of fertilizer use and 75% reduction of water consumption in containerized plant production. Another irrigation method, which can achieve minimal runoff and less salt buildup in substrates, is to use surface irrigation systems, but to also capture, retain and recycle the runoff and stormwater within the boundaries of the production facility [43]. This is exemplified by whole greenhouse/nursery recycling system, called the total nursery recycling system. This recycling system includes (1) stormwater and/or irrigation runoff collection, (2) sedimentation, flocculation, filtration and disinfection, if necessary and (3) irrigation. Skimina [44] tested more than 100 species of landscape ornamental plants using this system and found that the range of plant growth response was 73–171% relative to control plants. However, few nurseries have used this total nursery recycle system for the production of greenhouse container plants. Growers were concerned about the feasibility and reliability of the water sources for the production of high-quality plants. As a result, the use of CRFs is considered to be a more convenient method for container plant production, while potentially reducing N leaching and runoff.

in the digestive system [25, 26]. The nitrite can oxidize the iron in hemoglobin of red blood cells, resulting in the formation of methemoglobin. Because methemoglobin lacks the ability to bind (or release) oxygen, blood will be unable to carry sufficient oxygen to the individual body cells, causing the veins and skin to appear blue. This is a condition known as methemoglobinemia (sometimes referred to as "blue baby syndrome") [27]. Most humans over 1 year of age have the ability to rapidly convert methemoglobin back to oxyhemoglobin. Thus, the total amount of methemoglobin within red blood cells remains low despite relatively high levels of nitrate/nitrite uptake. In infants under 6 months of age, however, the enzyme systems responsible for reducing methemoglobin to oxyhemoglobin are incompletely developed and methemoglobinemia can occur. This also may happen in older individuals who have genetically impaired enzyme systems for metabolizing methemoglobin. Furthermore, pro-

The US Public Health Service adopted drinking water standards and set the recommended

tect the health of infants, children, pregnant women, the elderly and immune-compromised individuals. The potential health hazard for others depends on the individual's reaction to

US Geological Survey found that 9% of the private wells that were tested exceed the recom-

[31] has since adopted the 10 mg/L standard as the maximum contaminant level (MCL) for

─N and 1 mg/L for nitrite-N for regulated public water systems. Subsequent reviews of

O is a potent greenhouse gas with a global warming potential of 310 times greater

─N at 10 mg/L in 1962 [29]. This drinking water standard was established to pro-

─N and nitrites from all sources. From 1970 to 1992, the

O) gases. It was esti-

gas [32] and 1% of N

─N leach-

─N leaching; and

O [33]. The volatilization of both

contributes to photochemical smog

─N [30]. The US Environmental Protection Agency (USEPA)

) or nitrous oxide (N<sup>2</sup>

─N leaching and runoff should take plant species, fertilizer applica-

longed nitrate and nitrite ingestion could increase risks of certain cancers [28].

mated the 10% of manufactured N fertilizers could be volatilized as NH<sup>3</sup>

Different strategies and methods have been proposed and used for reducing NO<sup>3</sup>

ing and runoff during the production of container-grown plants. Chen et al. [36] suggested

tion rates, container substrate and irrigation methods into consideration for developing best management practices (BMPs), which include (1) understanding plant species requirement for N and application of N based on plant need; (2) improving physical and chemical properties of container substrates and increasing their holding capacities for water and nutrients,

─N; (3) using controlled-release fertilizers to reduce NO<sup>3</sup>

(4) irrigation system improvement by using either drip irrigation or subirrigation to reduce

O are serious environmental concern as NH<sup>3</sup>

limit for NO<sup>3</sup>

36 Nitrogen in Agriculture - Updates

─N and the total ingestion of NO<sup>3</sup>

this standard have not resulted in any changes.

Applied N can also be evolved as ammonia (NH<sup>3</sup>

applied in inorganic forms was lost to the atmosphere as N<sup>2</sup>

mended limit of 10 mg/L NO<sup>3</sup>

NO<sup>3</sup>

NO<sup>3</sup>

NH<sup>3</sup>

and N<sup>2</sup>

than carbon dioxide [35].

that approaches to NO<sup>3</sup>

particularly NO<sup>3</sup>

leaching and runoff.

**3. Methods for reducing N loss**

[34] and N<sup>2</sup>

## **4. Controlled-release fertilizers and their applications**

Controlled-release fertilizers are granules that are purposely designed to release nutrients in a controlled, delayed manner in synchrony with plant requirements for nutrients. CRFs belong to enhanced-efficiency fertilizers (EEFs), which is defined as "fertilizer products with characteristics that allow increased plant uptake and reduce the potential of nutrient losses to the environment (e.g., gaseous losses, leaching or runoff) when compared to an appropriate reference product" [45]. EEFs include CRFs, slow-release fertilizers (SRFs), stabilized N fertilizers, nitrification inhibitors and urease inhibitors. The terms, CRFs and SRFs, are generally considered analogous. However, Trenkel [42] and Shaviv [46] clearly defined their differences. In SRFs, the pattern of nutrient release is generally unpredictable and remains subject to change by soil type and climatic conditions. In contrary, the pattern, quantity and time of release can be predicted, within limits, for CRFs. This review, as indicated by the title, is intended to focus on CRFs only.

#### **4.1. Common CRFs used in container plant production**

**Table 1** lists the leading producers and/or suppliers of CRFs including Agrium Inc., Calgary, Alberta, Canada; Chisso Asahi Fertilizer Co., Tokyo, Japan; Everris NA, Inc., a subsidiary of Israel Chemicals Ltds; Haifa Group, Haifa, Israel; Shandong Kingenta, Shandong, China; and J.R. Simplot, Boise, Idaho, US. CRFs produced by Agrium includes those with trade names: ESN, Polyon, Duration and XCU in which urea is coated by polymer. Popular CRFs include Nutricote and Meister are manufactured by Chisso Asahi Fertilizer, and urea is coated by resin. Everris, Inc. produces Agrocote, Osmocote and Poly-S where urea is coated by sulfur/polymer and resin, resin and sulfur and polymer, respectively. Urea in Multicote produced by Haifa Group is coated by resin, and Florikote produced by J.R. Simplot is coated by polymer [47].

Urea is a major N source for formulation of CRFs. Urea is actually the most widely used fertilizer globally because of its high N content (46%). Urea has the lowest transportation costs per unit of N and ease of application [32, 33]. Additionally, urea is highly soluble in water and has much lower risk of causing fertilizer burn to crops. Other N sources used in the formulation of CRFs include ammonium nitrate, ammonium phosphate and potassium nitrate. Sulfur was initially used as a material for coating urea. The Tennessee Valley Authority developed the production process for sulfur-coated urea more than 50 years ago [48] in which preheated urea granules were coated with molten sulfur and wax. The sulfur coating is an impermeable layer which can be slowly degraded through microbial activities and soil chemical and physical processes. The uniformity in coating coverage and thickness of coating determine the speed and effectiveness of urea release. Incompletely coated or cracked prills are immediately amenable to dissolution in soil water and hydrolysis by urease. However, due to its amorphous nature, sulfur alone cannot be used to produce well controlled-release urea. Subsequently, many other materials, such as binders, plasticizers and sealants were evaluated for reducing the immediate burst effect. Some tested materials reduced the burst effect but increased the cost and complexity [48]. As a result, sulfur alone has not been used as a coating agent. If used, it is in combination with some polymers. Polymer coating is a more sophisticated technology, and it consists of a core of soluble nutrients surrounded by a polymer coating. Each coated

particle is known as a prill and nutrient release is controlled by the chemical composition and thickness of the polymer coating. Polymers could be thermosetting, thermoplastic or biodegradable. Some of the common thermoset polymers include urethane resin, epoxy resin, alkyd resin, unsaturated polyester resin, phenol resin, urea resin, melamine resin, phenol resin and silicon resin [49]. Among them, urethane resin is very commonly used [50]. Polyacrylamide is known to reduce soil erosion, and more studies should be conducted for its use in CRFs [46, 51]. Thermoplastic resins are not very commonly used because they are either not soluble in a solvent or make a very viscous solution which is not suitable for spraying; however, polyolefin is used for coating the fertilizer granules. Biodegradable polymers are naturally available and are known to be environmentally friendly because they decompose in bioactive environments and degrade by the enzymatic action of microorganisms, such as bacteria, fungi and algae and their polymer chains may also be broken down by nonenzymatic processes, such as chemical hydrolysis. Commercially, polymers used for coating urea include alkyd resin (Osmocote), polyurethane (Polyon, Multicote and Plantacote) and thermoplastic polymers.

**Table 1.** Common controlled-release fertilizers (CRFs) used for production of container-grown plants, vegetables and

and sulfur

**Trade name Manufacturer Type of CRFs Coating materials Selected commercial products**

technology

Resin-coated Granular urea coated with a

polymeric shell

with organic resin

Coated with polymer/sulfur and resin coatings

Controlled-Release Fertilizers as a Means to Reduce Nitrogen Leaching and Runoff…

Urea is coated with flexible micro-thin polymer

polymer composition of natural products, resin and additives

Polymer coating with a special chemical release agent

Granule contains NPK coated

Layers Coating" (ultra-thin ployurethane coating)

Urea coated with sulfur followed by polymer

Urea coated with polymer

polymer membrane

Agrocote® 19-6-12, Agrocote®

39

ESN® 44-0-0 (Environmentally

Florikote® 40-0-0), Florikote® 12-0-40, Florikote® 19-6-13,

Meister® 15-5-15, Meister®

Multicote® Agri 6 22-8-13, Multicote® Agri 6 34-0-7, Multicote® Agri 8 34-0-7

Nutricote® NPK 20-7-10

Osmocote® Start

Poly-S® 37-0-0

Trikote® 42-0-0

20-6-13

Osmocote® Exact, Osmocote® Exact Mini, Osmocote® Pro,

Polyon® 41-0-0, Polyon® NPK

Duration®CR, Duration® 44-0-0, Duration® 19-6-13

39-0-0 + 11% S

http://dx.doi.org/10.5772/intechopen.73055

smart nitrogen)

19-5-14

Agrocote® Everris, Inc. Polymer/

Meister® Chisso-Asahi

Nutricote® Chisso-Asahi

turfgrass.

ESN® Agrium, Inc. Polymer-coated

Fertilizer Co.

Fertilizer Co.

Poly-S® Everris, Inc. Polymer-/sulfur-

TriKote® Agrium, Inc. Polymer-/sulfur-

Osmocote® Everris, Inc. Organic

resin-coated

urea

Florikote J.R. Simplot Polymer-coated Coated with dual layer

Multicote® Haifa Group Resin-coated Nutrients encapsulated in a

Polymer-coated

resin-coated

coated urea

coated urea

Polyon® Agrium, Inc. Polymer-coated Coated with patented "Reactive

NPK

Duration® Agrium, Inc. Polymer-coated Clay-coated PCU or micro-thin


**4. Controlled-release fertilizers and their applications**

38 Nitrogen in Agriculture - Updates

**4.1. Common CRFs used in container plant production**

Controlled-release fertilizers are granules that are purposely designed to release nutrients in a controlled, delayed manner in synchrony with plant requirements for nutrients. CRFs belong to enhanced-efficiency fertilizers (EEFs), which is defined as "fertilizer products with characteristics that allow increased plant uptake and reduce the potential of nutrient losses to the environment (e.g., gaseous losses, leaching or runoff) when compared to an appropriate reference product" [45]. EEFs include CRFs, slow-release fertilizers (SRFs), stabilized N fertilizers, nitrification inhibitors and urease inhibitors. The terms, CRFs and SRFs, are generally considered analogous. However, Trenkel [42] and Shaviv [46] clearly defined their differences. In SRFs, the pattern of nutrient release is generally unpredictable and remains subject to change by soil type and climatic conditions. In contrary, the pattern, quantity and time of release can be predicted, within limits, for CRFs. This review, as indicated by the title, is intended to focus on CRFs only.

**Table 1** lists the leading producers and/or suppliers of CRFs including Agrium Inc., Calgary, Alberta, Canada; Chisso Asahi Fertilizer Co., Tokyo, Japan; Everris NA, Inc., a subsidiary of Israel Chemicals Ltds; Haifa Group, Haifa, Israel; Shandong Kingenta, Shandong, China; and J.R. Simplot, Boise, Idaho, US. CRFs produced by Agrium includes those with trade names: ESN, Polyon, Duration and XCU in which urea is coated by polymer. Popular CRFs include Nutricote and Meister are manufactured by Chisso Asahi Fertilizer, and urea is coated by resin. Everris, Inc. produces Agrocote, Osmocote and Poly-S where urea is coated by sulfur/polymer and resin, resin and sulfur and polymer, respectively. Urea in Multicote produced by Haifa Group is coated by resin, and Florikote produced by J.R. Simplot is coated by polymer [47].

Urea is a major N source for formulation of CRFs. Urea is actually the most widely used fertilizer globally because of its high N content (46%). Urea has the lowest transportation costs per unit of N and ease of application [32, 33]. Additionally, urea is highly soluble in water and has much lower risk of causing fertilizer burn to crops. Other N sources used in the formulation of CRFs include ammonium nitrate, ammonium phosphate and potassium nitrate. Sulfur was initially used as a material for coating urea. The Tennessee Valley Authority developed the production process for sulfur-coated urea more than 50 years ago [48] in which preheated urea granules were coated with molten sulfur and wax. The sulfur coating is an impermeable layer which can be slowly degraded through microbial activities and soil chemical and physical processes. The uniformity in coating coverage and thickness of coating determine the speed and effectiveness of urea release. Incompletely coated or cracked prills are immediately amenable to dissolution in soil water and hydrolysis by urease. However, due to its amorphous nature, sulfur alone cannot be used to produce well controlled-release urea. Subsequently, many other materials, such as binders, plasticizers and sealants were evaluated for reducing the immediate burst effect. Some tested materials reduced the burst effect but increased the cost and complexity [48]. As a result, sulfur alone has not been used as a coating agent. If used, it is in combination with some polymers. Polymer coating is a more sophisticated technology, and it consists of a core of soluble nutrients surrounded by a polymer coating. Each coated

**Table 1.** Common controlled-release fertilizers (CRFs) used for production of container-grown plants, vegetables and turfgrass.

particle is known as a prill and nutrient release is controlled by the chemical composition and thickness of the polymer coating. Polymers could be thermosetting, thermoplastic or biodegradable. Some of the common thermoset polymers include urethane resin, epoxy resin, alkyd resin, unsaturated polyester resin, phenol resin, urea resin, melamine resin, phenol resin and silicon resin [49]. Among them, urethane resin is very commonly used [50]. Polyacrylamide is known to reduce soil erosion, and more studies should be conducted for its use in CRFs [46, 51]. Thermoplastic resins are not very commonly used because they are either not soluble in a solvent or make a very viscous solution which is not suitable for spraying; however, polyolefin is used for coating the fertilizer granules. Biodegradable polymers are naturally available and are known to be environmentally friendly because they decompose in bioactive environments and degrade by the enzymatic action of microorganisms, such as bacteria, fungi and algae and their polymer chains may also be broken down by nonenzymatic processes, such as chemical hydrolysis. Commercially, polymers used for coating urea include alkyd resin (Osmocote), polyurethane (Polyon, Multicote and Plantacote) and thermoplastic polymers.

#### **4.2. N release patterns from CRFs**

Different models have been proposed for explaining nutrient release patterns of CRFs [45, 52, 53]. It is generally agreed that nutrient release is governed by diffusion mechanisms. Shaviv [46] and Liu [54] proposed a multi-stage diffusion model. According to this model, after application of a coated fertilizer, irrigation water penetrates the coating to condense on the solid fertilizer core followed by partial nutrient dissolution. As osmotic pressure builds within the containment, the granule swells and causes the occurrence of two processes. One could be "catastrophic release". When osmotic pressure surpasses threshold membrane resistance, the coating bursts and the entire core are spontaneously released. This is also referred to as the "failure mechanism". In the second, if the membrane withstands the developing pressure, core fertilizer is thought to be released slowly via diffusion for which the driving force may be a concentration or pressure gradient, or combination thereof called the "diffusion mechanism". The failure mechanism is generally observed in frail coatings (e.g. sulfur or modified sulfur), while polymer coatings (e.g. polyolefin) are expected to exhibit the diffusion release mechanism [48]. Nutrient release from CRFs is generally classified into linear and sigmoidal patterns [42, 55]. In most cases, the energy of activation of the release, EArel, is calculated on the basis of estimates of the rate of the release (percentage release per day) during the linear period obtained from the release curves [52]. Nutrient release profiles are established in both laboratory and field tests. Laboratory tests include extraction of nutrients at 25, 40 and 100 °C. Field tests include the placement of net bags in the ploughed layer or soil in the actual production soil [42]. Shaviv [56] reported that nutrient release consists of three stages: the initial stage or lag period during which little release is observed; the constant release stage characterized with an increasing release; and the last or mature stage where nutrient release is gradually reduced.

N release or loss. More N leached from substrates incorporated with CRFs than those topdressed [59]. Furthermore, substrate moisture is a key factor influencing nutrient release from CRFs.

Controlled-Release Fertilizers as a Means to Reduce Nitrogen Leaching and Runoff…

Due to their controlled-release characteristics, research has been conducted since the 1960s on the feasibility of the use of CRFs for container plant production [61, 62]. With the increasing availability of CRF types and awareness of N leaching and runoff in the 1980s, research has shifted attention towards N release patterns and N leaching and runoff. **Table 2** presents some representative studies conducted in container-grown ornamental plants, turfgrass, citrus and field crops such as potato. At least six conclusions can be drawn from these studies: (1) the use of CRFs reduces N leaching and/or runoff. Depending on fertilizer types, plant species, application methods and environmental conditions, N in leachates or runoff resulting from CRF application could be approximately 50% less than WSF application. Mello et al. [63] showed that polymer-coated urea reduced N leaching by 64.5% compared to conventional urea in container production of *Lantan camara* L. Broschat [20] showed that 48 and 54% of applied N were leached from a liquid WSF and a granular WSF, respectively, in container production of *Spathiphyllum*, while N leached from two CRFs were 29 and 35%, respectively. N concentrations in runoff derived from container greenhouse production facilities was 43.1 mg/L compared to 4.4 mg/L after the same facilities switched from WSF application to the use of CRFs [64]. (2) CRF applica-

eters 30 cm below potato production bed ranged from 7 to 45.1 mg/L from 39 to 95 days after

atmosphere compared to 45–73% emission by using a CRF [66]. (4) Plant growth or yield resulting from CRF application are equal to or better than those produced by WSF including ornamental plants [16, 20], field crops [65, 67] and turfgrass [66]. (5) CRFs vary in N release and thus N leaching. N concentrations in leachates varied from 60 to 275 mg/L in container production of *Vibrunum* [16] and from 50 to 400 mg/L in other container ornamental plant production [68] due in part to the application of different CRFs. (6) CRF application may improve the rhizhosphere microbial community. A study conducted in Japan showed that application of ureaformaldehyde fertilizers to onion bulbs and main roots of sugar beet changed the diversity of

Furthermore, the use of CRFs has been shown to increase nutrient use efficiency and decrease fertilizer application. Trenkel [42] suggested CRFs can potentially decrease fertilizer use by 20–30% of the recommended rate of a conventional fertilizer while obtaining the same yield. In several field trials in Florida, young or non-bearing citrus trees fertilized with CRFs at a 50% of the recommended rate performed equally well compared to 100% of the recommended rate with WSF [70]. The same magnitude of reduction happened in potato production in Florida [71]. Applying CRFs generally reduces salt accumulation, thus minimizing the possibility of leaf burning. The use of CRFs reduces labor costs. Depending on plant species, one application of appropriate amount of CRFs will ensure plant growth until marketable size, while WSF fertilizers have to be applied as fertigation weekly, and sometimes daily.

planting compared to 15.6–172 mg/L fertilized with a WSF [65]. (3) CRFs reduce N<sup>2</sup>

Application of urea in turfgrass production resulted in 127–476% more N<sup>2</sup>

the microbial community and the abundances of certain bacterial species [69].

─N in soil water collected by lysim-

http://dx.doi.org/10.5772/intechopen.73055

41

O emission.

O emission into the

**4.3. CRF application reduces N leaching and runoff in container plant production**

tion also reduces N leaching in field crop production. NO<sup>3</sup>

Nitrogen release profiles from CRFs have been studied during container plant production. CRFs are either top dressed (granules are placed on the surface of container substrate) or incorporated (granules are mixed with container substrate before being used for potting). Plants are watered in a specific leaching fraction. Leachates are captured and collected weekly. NO<sup>3</sup> ─N and NH<sup>4</sup> ─N in each collected leachate are analyzed. This method is not designed to determine the amount of N released from a CRF over a period of time since N leaching, volatilization and absorption by plants occur simultaneously. It is intended to use the leached N as an indicator for analyzing N release patterns. Leached N can be plotted based on the cumulative N leached (the percentage of N leached in reference of total N applied) at a specific production time or period [57, 58] or simply plotted as concentration of N per container against time (days or weeks) sampled [20, 59]. Depending on the types and formulation of CRFs, container substrate components, production temperature and irrigation volume and frequency, different N release profiles have been reported. Based on the cumulative N leached, the release curves can be generalized to two types: linear [57, 60] and sigmoidal [58] curves. Regardless of N sources in CRFs, NO<sup>3</sup> ─N is the main N leached, accounting for 80–90%, suggesting that nitrification is active in container substrates [59]. Temperature is a force driving N release from CRFs. Cumulative N leached from both sand and bark substrates incorporated with an Osmocote fertilizer in Florida was much greater than in Ohio [58]. The methods of CRF application affect N release or loss. More N leached from substrates incorporated with CRFs than those topdressed [59]. Furthermore, substrate moisture is a key factor influencing nutrient release from CRFs.

#### **4.3. CRF application reduces N leaching and runoff in container plant production**

**4.2. N release patterns from CRFs**

40 Nitrogen in Agriculture - Updates

is gradually reduced.

and NH<sup>4</sup>

in CRFs, NO<sup>3</sup>

Different models have been proposed for explaining nutrient release patterns of CRFs [45, 52, 53]. It is generally agreed that nutrient release is governed by diffusion mechanisms. Shaviv [46] and Liu [54] proposed a multi-stage diffusion model. According to this model, after application of a coated fertilizer, irrigation water penetrates the coating to condense on the solid fertilizer core followed by partial nutrient dissolution. As osmotic pressure builds within the containment, the granule swells and causes the occurrence of two processes. One could be "catastrophic release". When osmotic pressure surpasses threshold membrane resistance, the coating bursts and the entire core are spontaneously released. This is also referred to as the "failure mechanism". In the second, if the membrane withstands the developing pressure, core fertilizer is thought to be released slowly via diffusion for which the driving force may be a concentration or pressure gradient, or combination thereof called the "diffusion mechanism". The failure mechanism is generally observed in frail coatings (e.g. sulfur or modified sulfur), while polymer coatings (e.g. polyolefin) are expected to exhibit the diffusion release mechanism [48]. Nutrient release from CRFs is generally classified into linear and sigmoidal patterns [42, 55]. In most cases, the energy of activation of the release, EArel, is calculated on the basis of estimates of the rate of the release (percentage release per day) during the linear period obtained from the release curves [52]. Nutrient release profiles are established in both laboratory and field tests. Laboratory tests include extraction of nutrients at 25, 40 and 100 °C. Field tests include the placement of net bags in the ploughed layer or soil in the actual production soil [42]. Shaviv [56] reported that nutrient release consists of three stages: the initial stage or lag period during which little release is observed; the constant release stage characterized with an increasing release; and the last or mature stage where nutrient release

Nitrogen release profiles from CRFs have been studied during container plant production. CRFs are either top dressed (granules are placed on the surface of container substrate) or incorporated (granules are mixed with container substrate before being used for potting). Plants are watered in a specific leaching fraction. Leachates are captured and collected weekly. NO<sup>3</sup>

the amount of N released from a CRF over a period of time since N leaching, volatilization and absorption by plants occur simultaneously. It is intended to use the leached N as an indicator for analyzing N release patterns. Leached N can be plotted based on the cumulative N leached (the percentage of N leached in reference of total N applied) at a specific production time or period [57, 58] or simply plotted as concentration of N per container against time (days or weeks) sampled [20, 59]. Depending on the types and formulation of CRFs, container substrate components, production temperature and irrigation volume and frequency, different N release profiles have been reported. Based on the cumulative N leached, the release curves can be generalized to two types: linear [57, 60] and sigmoidal [58] curves. Regardless of N sources

is active in container substrates [59]. Temperature is a force driving N release from CRFs. Cumulative N leached from both sand and bark substrates incorporated with an Osmocote fertilizer in Florida was much greater than in Ohio [58]. The methods of CRF application affect

─N in each collected leachate are analyzed. This method is not designed to determine

─N is the main N leached, accounting for 80–90%, suggesting that nitrification

─N

Due to their controlled-release characteristics, research has been conducted since the 1960s on the feasibility of the use of CRFs for container plant production [61, 62]. With the increasing availability of CRF types and awareness of N leaching and runoff in the 1980s, research has shifted attention towards N release patterns and N leaching and runoff. **Table 2** presents some representative studies conducted in container-grown ornamental plants, turfgrass, citrus and field crops such as potato. At least six conclusions can be drawn from these studies: (1) the use of CRFs reduces N leaching and/or runoff. Depending on fertilizer types, plant species, application methods and environmental conditions, N in leachates or runoff resulting from CRF application could be approximately 50% less than WSF application. Mello et al. [63] showed that polymer-coated urea reduced N leaching by 64.5% compared to conventional urea in container production of *Lantan camara* L. Broschat [20] showed that 48 and 54% of applied N were leached from a liquid WSF and a granular WSF, respectively, in container production of *Spathiphyllum*, while N leached from two CRFs were 29 and 35%, respectively. N concentrations in runoff derived from container greenhouse production facilities was 43.1 mg/L compared to 4.4 mg/L after the same facilities switched from WSF application to the use of CRFs [64]. (2) CRF application also reduces N leaching in field crop production. NO<sup>3</sup> ─N in soil water collected by lysimeters 30 cm below potato production bed ranged from 7 to 45.1 mg/L from 39 to 95 days after planting compared to 15.6–172 mg/L fertilized with a WSF [65]. (3) CRFs reduce N<sup>2</sup> O emission. Application of urea in turfgrass production resulted in 127–476% more N<sup>2</sup> O emission into the atmosphere compared to 45–73% emission by using a CRF [66]. (4) Plant growth or yield resulting from CRF application are equal to or better than those produced by WSF including ornamental plants [16, 20], field crops [65, 67] and turfgrass [66]. (5) CRFs vary in N release and thus N leaching. N concentrations in leachates varied from 60 to 275 mg/L in container production of *Vibrunum* [16] and from 50 to 400 mg/L in other container ornamental plant production [68] due in part to the application of different CRFs. (6) CRF application may improve the rhizhosphere microbial community. A study conducted in Japan showed that application of ureaformaldehyde fertilizers to onion bulbs and main roots of sugar beet changed the diversity of the microbial community and the abundances of certain bacterial species [69].

Furthermore, the use of CRFs has been shown to increase nutrient use efficiency and decrease fertilizer application. Trenkel [42] suggested CRFs can potentially decrease fertilizer use by 20–30% of the recommended rate of a conventional fertilizer while obtaining the same yield. In several field trials in Florida, young or non-bearing citrus trees fertilized with CRFs at a 50% of the recommended rate performed equally well compared to 100% of the recommended rate with WSF [70]. The same magnitude of reduction happened in potato production in Florida [71]. Applying CRFs generally reduces salt accumulation, thus minimizing the possibility of leaf burning. The use of CRFs reduces labor costs. Depending on plant species, one application of appropriate amount of CRFs will ensure plant growth until marketable size, while WSF fertilizers have to be applied as fertigation weekly, and sometimes daily.


**Plant species** A greenhouse

Peat/vermiculite/

Nutricote 18-6-8

Osmocote 18-6-12

Osmocote 18-6-12 FS

Osmocote 24-4-8 HN

Polyon 25-4-12

Prokote Plus 20-3-10

Woodave 20-4-11

Container

Peat/pine bark/sand

Osmocote

Polyon Multicote Nutricote

> Potato

Foliage plants

Turfgrass

**Table 2.** (WSFs) used in crop production or leaching experiments.

A Timpanogos

Polymer-coated urea

1.25 mg N O2

2.22 mg N O2

─N/m2/h

Nitrogen lost in leachates, runoff water or emitted into the atmosphere when controlled-release fertilizers (CRFs) only or CRFs with water soluble fertilizers

─N/m2/h

Polymer-coated urea emitted significantly

Lemonte et al.

http://dx.doi.org/10.5772/intechopen.73055

43

[66]

low amount of N

 O2 ─N

loam soil

Urea

Canadian peat/pine

WSF CRF

43.1

4.4

mg/L in runoff

mg/L in runoff

bark/lava rock

Loamy sand

Polymer-coated urea

Soluble N

21.3 kg NO ─3 N/ha

26.9 kg NO ─3 N/ha

400 mg/L

400 mg/L

50 mg/L 200 mg/L

Osmocote steadily release of N

N release reached a peak on week 9 then

N release reached a peak on week 8 then

N release reached a peak on week 9 then

Apparent fertilizer N recovery with PCU

Wilson et al. [67]

Controlled-Release Fertilizers as a Means to Reduce Nitrogen Leaching and Runoff…

(65% averaged over four rates) tended

to be higher than split-applied soluble N

(55%) at equivalent rates

Plant growth was not affected by

Wilson and

Albabo [64]

switching from a WSF to a CRF

stabilized

stabilized

stabilized

Merhaut et al.

[68]

ornamental plants

leaching study

sand

without plants

**Growing substrate**

**Fertilizer**

**N leached or N conc. in** 

**Plant growth or comments**

**References**

**leachates/runoff**

32% of applied N leached

36% of applied N leached

51% of applied N leached

49% of applied N leached

45% of applied N leached

50% of applied N leached

30% of applied N leached

Osmocote 18-6-12, Nutricote, and

Cabrera [59]

Woodave exhibited less response to

temperature increase and thus less N

leaching


**Plant species** *Spathiphyllum* spp.

Pine bark/peat/sand

Liquid WSF Dry granular WSF

Lightly-coated CRF

Heavily-coat CRF

*Lantana camara L.*

Krome soil

Urea

Schott

**Growing substrate**

**Fertilizer**

**N leached or N conc. in** 

**Plant growth or comments**

**References**

**leachates/runoff**

48% of applied N leached

54% of applied N leached

29% of applied N leached

35% of applied N leached

N leached from containers

More flowers were produced by plants

Mello et al. [63]

fertilized with polymer-coated urea than

fertilized with polymer-coated

urea was 64.5% lower than

urea

those fertilized with urea

Polymer-coated urea

A leaching

Florida sandy soil

Ammonium nitrate

Isobutylidene diurea

Meister polyolefin

resin-coated urea

100% of applied N leached

32% of applied N leached

12% of applied N leached

28% of applied N leached

12% of applied N leached

6% of applied N leached

5% of applied N leached

N than urea

Meister and Osmocote leached much less

Paramasivam and

Alva [80]

Much less N was leached from Meister

Wang and Alva

[79]

than isobutylidene coated urea, and all N

was leached from ammonium nitrate.

column study

without plants

A leaching

Fine sandy soil

Urea Poly-S Meister polyolefin

resin-coated urea

Osmocote

Different foliage

Peat/pine bark/sand

(41-0-0)

Trikote (42-0-0)

Regalite Nitroform (38-0-0)

 27.6 mg/L

275 mg/L

220 mg/L 125 mg/L

60 mg/L

64.9 mg/L

Polymer-coated urea

23.1 mg/L

Polymer-coat urea provided stable and

long-last release of NO3

−

than the other two. Trikote released more

+ than the other two. Plant growth

was not significantly affect by treatments.

The highest concentrations of NO3

leached from CRFs during a 4.5-month

production period. Plant growth indices

were not significantly affected by CRFs


Yeager and

Cashion [16]

NH4

─N and NH4

+

Blythe et al. [81]

and flowering

crops

Containergrown *Viburnum* 

Pine bark/peat/sand

Nutricote Osmocote Prokote Plus

Woodace

*odoratissimum*

Ker-Gawl

column study

without plants

No plant dry weigh differences among

Broschat [20]

42 Nitrogen in Agriculture - Updates

fertilizer treatments

**Table 2.** Nitrogen lost in leachates, runoff water or emitted into the atmosphere when controlled-release fertilizers (CRFs) only or CRFs with water soluble fertilizers (WSFs) used in crop production or leaching experiments.

#### **4.4. Problems associated with the use of CRFs in container plant production**

Several problems are associated with the use of CRFs in production of container-grown plants. Some are due to CRF design and formulation: (1) CRFs cost considerably more to manufacture than conventional fertilizers, thus they are more expensive. For example, one ton of a CRF (44% N) could be \$650 compared to one ton of urea (46% N) at \$481 [72]. (2) CRFs may not release nutrients based on plant requirements. This could be due to several factors: the formulation of nutrient elements, the permeability and durability of coating materials, plant species and growth stage difference, and inappropriate placement of CRFs, substrate moisture levels and microbial effects as well as production environmental conditions. The N release pattern of CRFs in laboratory tests is generally represented by a sigmoidal curve (**Figure 1**). Such release pattern is appropriate for field-grown crops, such as corn, wheat and tomato, as the lag phase is appropriate for seedling growth or allow transplants to get recovered and established from transplanting shock; log phase is designed for rapidly vegetative growth and the transition from vegetative growth to reproductive growth; and the stationery phase would allow nutrients absorbed or stored in vegetative organs to translocate to reproductive organs. The sigmoidal curve, however, may not be an ideal pattern for producing container-grown plants. Container plants are initiated with rooted cuttings or liners which already have well established root systems. Once the liners are planted in containers, they grow in an accelerated speed and require a steady supply of nutrient without lag phase. Thus, we propose here that CRFs for container-grown plants should have a nutrient release pattern, called "the expected curve for container plants" presented in **Figure 1**, not a sigmoidal curve. Many CRFs were predominantly developed based on the sigmoidal release curve, thus, they may not be ideally suitable for producing container-grown plants. (3) Thus far, nutrient formulations of few CRFs are developed according to specific groups of plant species in nutrient requirements. Some species have low nutrient requirements. For example, ornamental foliage plants largely originate from the rainforest floor, and they inherently require low light levels and low nutrient supply for slow growth. This group of plants should be fertilized by CRFs that have complete nutrient elements with a rather slower release pattern. CRFs designed for use in subtropics and tropics should be different from those to be used in temperate regions. As shown by Birrenkott et al. [58], the same CRF for growing the same

Controlled-Release Fertilizers as a Means to Reduce Nitrogen Leaching and Runoff…

http://dx.doi.org/10.5772/intechopen.73055

45

Other problems with the use of CRFs are related to inappropriate application. The first is the misuse of CRFs. A CRF that is supposed to be used in the Southern USA, but used in the Northern USA, which may cause reduced release of required nutrients; as a result plant growth will be slow. If a CRF designed for container-woody ornamental plants is used for production of annual bedding plants, plant growth may slow down due to limited release of nutrients. The second problem is to apply either too little or too much CRFs. The use of an extra amount is the most common problem in container plant production. This practice not only wastes fertilizers and increases production costs, but also causes N leaching and runoff after excessive irrigation. A large number of plant species are produced in containers, but few species have been studied for N requirements [39]. Those studied were based on a particular substrate in a specific environmental condition. In reality, however, a wide range of substrates have been used in container plant production, and different substrates have different physical and chemical properties. Thus, the established N requirements may not be well suitable for plants to be produced in a different substrate. However, such information does provide reference guides for N application. Nevertheless, the use of extra amount practice must be changed, otherwise, even with the best CRFs available, N leaching and runoff could still occur in container plant production. Third, the methods of placing CRFs significantly affect N release or leaching. Several studies have shown that more N is leached by incorporation of CRFs with substrates, while topdressing had significantly less amount of N leaching [59]. The explanation is that the time for transfer of nutrients through membranes in topdressed CRFs is presumably extended over incorporation due to intermittent drying of the upper growing

It is certain that CRFs are needed, and the need is increasing. Since the world population keeps growing, it requires more food. Food production requires fertilizers. Meanwhile, container plant production has been growing at a fast pace. The production of container plants also requires fertilizers. As this article documented, container plant production is associated

been well studied in container plant production. This does not mean that the volatilization and emission are not a problem since fertilization is estimated to account for 78% of the

only pay attention to N leaching but also take emission problems into consideration in the

and emission of N<sup>2</sup>

O at the global scale [35]. Therefore, manufacturers should not

O have not

crop released different amount of N in Florida and Ohio.

substrate between irrigation [73].

total emission of NH<sup>3</sup>

**5. Future development of CRFs**

with N leaching and runoff. So far, the volatilization of NH<sup>3</sup>

and N<sup>2</sup>

**Figure 1.** A proposed nutrient release curve versus the commonly preferred sigmoidal curve used for developing controlled-release fertilizers. Controlled-release fertilizer with the proposed curve could be more suitable for production of container-grown plants than those with a sigmoidal curve.

nutrient formulations of few CRFs are developed according to specific groups of plant species in nutrient requirements. Some species have low nutrient requirements. For example, ornamental foliage plants largely originate from the rainforest floor, and they inherently require low light levels and low nutrient supply for slow growth. This group of plants should be fertilized by CRFs that have complete nutrient elements with a rather slower release pattern. CRFs designed for use in subtropics and tropics should be different from those to be used in temperate regions. As shown by Birrenkott et al. [58], the same CRF for growing the same crop released different amount of N in Florida and Ohio.

Other problems with the use of CRFs are related to inappropriate application. The first is the misuse of CRFs. A CRF that is supposed to be used in the Southern USA, but used in the Northern USA, which may cause reduced release of required nutrients; as a result plant growth will be slow. If a CRF designed for container-woody ornamental plants is used for production of annual bedding plants, plant growth may slow down due to limited release of nutrients. The second problem is to apply either too little or too much CRFs. The use of an extra amount is the most common problem in container plant production. This practice not only wastes fertilizers and increases production costs, but also causes N leaching and runoff after excessive irrigation. A large number of plant species are produced in containers, but few species have been studied for N requirements [39]. Those studied were based on a particular substrate in a specific environmental condition. In reality, however, a wide range of substrates have been used in container plant production, and different substrates have different physical and chemical properties. Thus, the established N requirements may not be well suitable for plants to be produced in a different substrate. However, such information does provide reference guides for N application. Nevertheless, the use of extra amount practice must be changed, otherwise, even with the best CRFs available, N leaching and runoff could still occur in container plant production. Third, the methods of placing CRFs significantly affect N release or leaching. Several studies have shown that more N is leached by incorporation of CRFs with substrates, while topdressing had significantly less amount of N leaching [59]. The explanation is that the time for transfer of nutrients through membranes in topdressed CRFs is presumably extended over incorporation due to intermittent drying of the upper growing substrate between irrigation [73].

## **5. Future development of CRFs**

**4.4. Problems associated with the use of CRFs in container plant production**

44 Nitrogen in Agriculture - Updates

Several problems are associated with the use of CRFs in production of container-grown plants. Some are due to CRF design and formulation: (1) CRFs cost considerably more to manufacture than conventional fertilizers, thus they are more expensive. For example, one ton of a CRF (44% N) could be \$650 compared to one ton of urea (46% N) at \$481 [72]. (2) CRFs may not release nutrients based on plant requirements. This could be due to several factors: the formulation of nutrient elements, the permeability and durability of coating materials, plant species and growth stage difference, and inappropriate placement of CRFs, substrate moisture levels and microbial effects as well as production environmental conditions. The N release pattern of CRFs in laboratory tests is generally represented by a sigmoidal curve (**Figure 1**). Such release pattern is appropriate for field-grown crops, such as corn, wheat and tomato, as the lag phase is appropriate for seedling growth or allow transplants to get recovered and established from transplanting shock; log phase is designed for rapidly vegetative growth and the transition from vegetative growth to reproductive growth; and the stationery phase would allow nutrients absorbed or stored in vegetative organs to translocate to reproductive organs. The sigmoidal curve, however, may not be an ideal pattern for producing container-grown plants. Container plants are initiated with rooted cuttings or liners which already have well established root systems. Once the liners are planted in containers, they grow in an accelerated speed and require a steady supply of nutrient without lag phase. Thus, we propose here that CRFs for container-grown plants should have a nutrient release pattern, called "the expected curve for container plants" presented in **Figure 1**, not a sigmoidal curve. Many CRFs were predominantly developed based on the sigmoidal release curve, thus, they may not be ideally suitable for producing container-grown plants. (3) Thus far,

**Figure 1.** A proposed nutrient release curve versus the commonly preferred sigmoidal curve used for developing controlled-release fertilizers. Controlled-release fertilizer with the proposed curve could be more suitable for production

of container-grown plants than those with a sigmoidal curve.

It is certain that CRFs are needed, and the need is increasing. Since the world population keeps growing, it requires more food. Food production requires fertilizers. Meanwhile, container plant production has been growing at a fast pace. The production of container plants also requires fertilizers. As this article documented, container plant production is associated with N leaching and runoff. So far, the volatilization of NH<sup>3</sup> and emission of N<sup>2</sup> O have not been well studied in container plant production. This does not mean that the volatilization and emission are not a problem since fertilization is estimated to account for 78% of the total emission of NH<sup>3</sup> and N<sup>2</sup> O at the global scale [35]. Therefore, manufacturers should not only pay attention to N leaching but also take emission problems into consideration in the development of CRFs. Future fertilizers must be environmentally friendly and have minimal loss to the air and leaching and/or runoff of N to ground and surface water systems.

**Author details**

\* and Xiangying Wei1,2

Fujian Agriculture and Forestry University, Fuzhou, China

Publications/Flo/2007/09Sep/FLO2007.pdf

Environmental Horticulture. 1996;**14**:88-91

1 University of Florida, Institute of Food and Agricultural Sciences, Mid-Florida Research

Controlled-Release Fertilizers as a Means to Reduce Nitrogen Leaching and Runoff…

http://dx.doi.org/10.5772/intechopen.73055

47

2 Fujian University Key Laboratory of Plant-Microbe Interaction, College of Life Sciences,

[1] Jerardo A. Floriculture and Nursery Crops Yearbook: A Report from Economic Research Service. Washington, DC: US Department of Agriculture; 2007 http://www.ers.usda.gov/

[2] Majsztrik JC, Ristvey AG, Lea-Cox JD. Water and nutrient management in the produc-

[3] USDA (U.S. Department of Agriculture). Nursery crops 2006 summary. In National

[4] Ruter JM. Growth and landscape performance of three landscape plants produced in conventional and pot-in-pot production systems. Journal of Environmental Horticulture.

[5] Harris JR, Gilman EF. Production method affects growth and post-transplant establishment of 'East Palatka' holly. Journal of the American Society for Horticultural Science.

[6] Gilman EF, Beeson RC Jr. Nursery production method affects root growth. Journal of

[7] Judd LA, Jackson BE, Fonteno WC. Advancements in root growth measurement technologies and observation capabilities for container-grown plants. Plants. 2015;**4**:369-392

[8] Majsztrik JC, Fernandez RT, Fisher PR, Hitchcock DR, Lea-Cox J, Owen JS, et al. Water use and treatment in container-grown specialty crop production: A review. Water Air

[9] USDA (U.S. Department of Agriculture). Census of agriculture, 2007 summary and state data. USDA, Washington, DC; 2009. Available online: http://www.agcensus.usda.gov/

[10] Parrella MP, Wagner A, Fujino DW. The floriculture and nursery industry's struggle

Publications/2007/Full\_Report/usv1.pdf [Accessed 3 December, 2017]

with invasive species. American Entomologist. 2015;**61**:39-50

tion of container-grown ornamentals. Horticultural Reviews. 2011;**38**:253

Agricultural Statistics Service, USDA, Washington, DC; 2007. p. 63

\*Address all correspondence to: jjchen@ufl.edu

and Education Center, Apopka, FL, USA

Jianjun Chen<sup>1</sup>

**References**

1993;**11**:124-127

1993;**118**:194-200

Soil Pollution. 2017;**228**:151

The development of CRFs has evolved from a sulfur-coating technology to a polymer-coated technology. With the advance of nanotechnology, future CRFs should integrate nanotechnology components for improving controlled-release characteristics [74]. The future CRFs should be biodegradable; materials used for producing CRFs should be capable of decomposing naturally in most common environmental conditions. Nutrient composition and formulations should be developed based on (1) different groups of plants: annual, perennial and evergreen; (2) the purpose of plant production: growth for fruit, grain or biomass increase (ornamental plants) and/or (3) their inherent needs for nutrients: low, medium and high requirements for major nutrient elements, particularly N. New coating materials that have better permeability and duration as well as biodegradability should be used for coating the nutrient elements. Depending on plant groups and production regions, appropriate coating materials should be used to ensure that nutrients are release largely based on plant requirements. Some natural polymers should be considered including chitosan, xanthan gum, carrageenan, pectin and modified clays [49]. Polymer-clay superabsorbent composites have been reported to be promising as their production costs are low with high water absorbency [75]. Additionally, future CRFs should consider the incorporation of beneficial microbes, such as plant growth promoting bacteria [76] and mycorrhizal fungi [77, 78] to maximize nutrient use efficiency and minimize negative impact on the environment.

## **6. Conclusion**

There is an increasing trend for producing plants in containers worldwide. Container plant production, however, poses mounting concern over N leaching and/or runoff. This is due to the fact that plants are grown in confined substrates that are highly permeable and have low water and nutrient holding capacities, and a large amount of N and water are required for sustaining plant growth. In addition to N leaching and/or runoff, applied N may be volatilized as NH<sup>3</sup> and emitted as N<sup>2</sup> O into the atmosphere, contributing to climate changes. This article documents that the use of CRFs can reduce N leaching and runoff and raises the question about NH<sup>3</sup> volatilization and N<sup>2</sup> O emission in container plant production. It is firmly believed that the use of CRFs is an effective way of reducing N leaching and runoff and possibly NH<sup>3</sup> volatilization and N<sup>2</sup> O emission. With the increase need for food and ornamental plants, the need for fertilizers, particularly CRFs will continuously increase. New environment friendly CRFs should be developed and used for crop and container plant production. On the other hand, since the amount of N lost is a function of fertilizer source, timing, soil infiltration and percolation rate, micropore flow, root density, soil moisture, and precipitation/irrigation rate and intensity, CRFs alone cannot resolve N loss problem. The application of CRFs along with integrated production practices should be carried out for minimizing N loss. Integration includes the application of CRFs based on plant species types and production purpose, irrigation of substrate according to plant need and appropriate methods of applying CRFs to the substrate.

## **Author details**

development of CRFs. Future fertilizers must be environmentally friendly and have minimal

The development of CRFs has evolved from a sulfur-coating technology to a polymer-coated technology. With the advance of nanotechnology, future CRFs should integrate nanotechnology components for improving controlled-release characteristics [74]. The future CRFs should be biodegradable; materials used for producing CRFs should be capable of decomposing naturally in most common environmental conditions. Nutrient composition and formulations should be developed based on (1) different groups of plants: annual, perennial and evergreen; (2) the purpose of plant production: growth for fruit, grain or biomass increase (ornamental plants) and/or (3) their inherent needs for nutrients: low, medium and high requirements for major nutrient elements, particularly N. New coating materials that have better permeability and duration as well as biodegradability should be used for coating the nutrient elements. Depending on plant groups and production regions, appropriate coating materials should be used to ensure that nutrients are release largely based on plant requirements. Some natural polymers should be considered including chitosan, xanthan gum, carrageenan, pectin and modified clays [49]. Polymer-clay superabsorbent composites have been reported to be promising as their production costs are low with high water absorbency [75]. Additionally, future CRFs should consider the incorporation of beneficial microbes, such as plant growth promoting bacteria [76] and mycorrhizal fungi [77, 78] to maximize nutrient use efficiency and mini-

There is an increasing trend for producing plants in containers worldwide. Container plant production, however, poses mounting concern over N leaching and/or runoff. This is due to the fact that plants are grown in confined substrates that are highly permeable and have low water and nutrient holding capacities, and a large amount of N and water are required for sustaining plant growth. In addition to N leaching and/or runoff, applied N may be volatil-

article documents that the use of CRFs can reduce N leaching and runoff and raises the ques-

believed that the use of CRFs is an effective way of reducing N leaching and runoff and pos-

plants, the need for fertilizers, particularly CRFs will continuously increase. New environment friendly CRFs should be developed and used for crop and container plant production. On the other hand, since the amount of N lost is a function of fertilizer source, timing, soil infiltration and percolation rate, micropore flow, root density, soil moisture, and precipitation/irrigation rate and intensity, CRFs alone cannot resolve N loss problem. The application of CRFs along with integrated production practices should be carried out for minimizing N loss. Integration includes the application of CRFs based on plant species types and production purpose, irrigation of substrate according to plant need and appropriate methods of applying

O into the atmosphere, contributing to climate changes. This

O emission. With the increase need for food and ornamental

O emission in container plant production. It is firmly

loss to the air and leaching and/or runoff of N to ground and surface water systems.

mize negative impact on the environment.

and emitted as N<sup>2</sup>

volatilization and N<sup>2</sup>

volatilization and N<sup>2</sup>

**6. Conclusion**

46 Nitrogen in Agriculture - Updates

ized as NH<sup>3</sup>

sibly NH<sup>3</sup>

tion about NH<sup>3</sup>

CRFs to the substrate.

Jianjun Chen<sup>1</sup> \* and Xiangying Wei1,2

\*Address all correspondence to: jjchen@ufl.edu

1 University of Florida, Institute of Food and Agricultural Sciences, Mid-Florida Research and Education Center, Apopka, FL, USA

2 Fujian University Key Laboratory of Plant-Microbe Interaction, College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, China

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**Chapter 4**

**Provisional chapter**

**An Overview of the Effects of Heat Treatments on the**

Sewage sludge is often heat-dried to eliminate water and pathogens. However, heat-drying can also change the form of nitrogen (N). To improve our understanding of this phenomenon, we examined the heat-induced changes in the rate of N mineralization from soils and organic wastes. Published results revealed that the response to the heating temperature differed between soils and organic wastes. As the heating temperature increased to 200°C, the rate of N mineralization increased in soils but decreased in organic wastes. In organic wastes such as sewage sludge, the content of mineralized N tended to decrease sharply when heating temperatures increased to 150–200°C. Furthermore, our results obtained from heat-drying of sewage sludge at 180°C indicated that the rate of carbon (C) mineralization decreased with increasing heating period after the sludge temperature reached 180°C. The C in sewage sludge heated at 180°C for 120 hours after complete drying contained more humin and aromatic C than that in sludge that was heat-dried at 180°C without the additional heating period. These results suggest that the heat-drying treatment can be divided into the drying and denaturing periods and that the tempera-

ture of the sludge, not that of the reactor, affects the quality of the end-product. **Keywords:** carbon, heat-drying, nitrogen mineralization, organic wastes, sewage

**An Overview of the Effects of Heat Treatments on the** 

DOI: 10.5772/68163

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

© 2018 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

The treatment of raw organic wastes increases their potential range of beneficial uses. The most conventional treatment is composting, which is typically conducted at ambient temperatures. It is generally recognized that composted organic matter is a good soil amendment that releases inorganic nitrogen (N) relatively slowly. Besides composting, heating has become a

**Quality of Organic Wastes as a Nitrogen Fertilizer**

**Quality of Organic Wastes as a Nitrogen Fertilizer**

Naoki Moritsuka and Kaori Matsuoka

Naoki Moritsuka and Kaori Matsuoka

http://dx.doi.org/10.5772/68163

sludge, soil, stabilization

popular method to enhance the quality of organic wastes.

**1. Introduction**

**Abstract**

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter


**Provisional chapter**

## **An Overview of the Effects of Heat Treatments on the Quality of Organic Wastes as a Nitrogen Fertilizer Quality of Organic Wastes as a Nitrogen Fertilizer**

**An Overview of the Effects of Heat Treatments on the** 

DOI: 10.5772/68163

Naoki Moritsuka and Kaori Matsuoka Additional information is available at the end of the chapter

Naoki Moritsuka and Kaori Matsuoka

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/68163

#### **Abstract**

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by graphene oxide films. Chemical Engineering Journal. 2014;**255**:107-113

2011;**23**:81-89

52 Nitrogen in Agriculture - Updates

**255**:571-586

2016;**7**:1327

1049-1061

Sewage sludge is often heat-dried to eliminate water and pathogens. However, heat-drying can also change the form of nitrogen (N). To improve our understanding of this phenomenon, we examined the heat-induced changes in the rate of N mineralization from soils and organic wastes. Published results revealed that the response to the heating temperature differed between soils and organic wastes. As the heating temperature increased to 200°C, the rate of N mineralization increased in soils but decreased in organic wastes. In organic wastes such as sewage sludge, the content of mineralized N tended to decrease sharply when heating temperatures increased to 150–200°C. Furthermore, our results obtained from heat-drying of sewage sludge at 180°C indicated that the rate of carbon (C) mineralization decreased with increasing heating period after the sludge temperature reached 180°C. The C in sewage sludge heated at 180°C for 120 hours after complete drying contained more humin and aromatic C than that in sludge that was heat-dried at 180°C without the additional heating period. These results suggest that the heat-drying treatment can be divided into the drying and denaturing periods and that the temperature of the sludge, not that of the reactor, affects the quality of the end-product.

**Keywords:** carbon, heat-drying, nitrogen mineralization, organic wastes, sewage sludge, soil, stabilization

## **1. Introduction**

The treatment of raw organic wastes increases their potential range of beneficial uses. The most conventional treatment is composting, which is typically conducted at ambient temperatures. It is generally recognized that composted organic matter is a good soil amendment that releases inorganic nitrogen (N) relatively slowly. Besides composting, heating has become a popular method to enhance the quality of organic wastes.

Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons

To eliminate water and pathogens, sewage sludge is heat-dried by various methods such as convective drying, conductive drying, and solar drying [1]. Several researchers have also reported that heat-drying of sewage sludge can change the form of N [2]. For example, Smith and Durham [3] evaluated the content of N in five sewage sludges in paired treatments, both anaerobically digested and one additionally heat-dried for pelleting or granulation. During heat-drying, more than 75% of inorganic ammonium was lost by volatilization. However, during the aerobic incubation at 25°C for 56 days, the production of nitrate in soil amended with heat-dried sludge was similar to, or even larger than, that from the corresponding sludge without heat-drying. These results suggest that heat-drying of sewage sludge greatly increased the content of mineralizable organic N.

Both forms of N were increased by the heat treatments (**Figure 1**). The average content of inorganic N was 39.8 mg kg−1 (130°C) and 199.3 mg kg−1 (200°C) as compared to 25.2 mg kg−1 (original). The average content of mineralizable N was 89.4 mg kg−1 (130°C) and 111.1 mg kg−1 (200°C) as compared to 28.0 mg kg−1 (original). The amount of N mineralized by heating at 200°C and subsequent incubation was positively correlated with the content of total N in the original soil (*r* = 0.55\*\*), suggesting that the soil heating effect was greater for humus-rich soils. The findings of Mitsui [8] were extended by Sakamoto et al. [9]. They tried to reveal the origin of the N mineralized by heating and measuring the amount of N mineralized from two soils fumigated with chloroform or heated at different temperatures. Chloroform fumigation can kill most of soil microbes, so it has been used to extract the elements such as carbon (C), nitro-

An Overview of the Effects of Heat Treatments on the Quality of Organic Wastes as a Nitrogen Fertilizer

http://dx.doi.org/10.5772/68163

55

The amount of N mineralized from the fumigated soil was similar to that from the soil heated at 50 and 100°C, but it was much less than that from the soil heated at 150 and 200°C (**Figure 2**).

**Figure 1.** Frequency distributions of inorganic and mineralizable N in Japanese agricultural soils (*n* = 44) before and after heating at 130 or 200°C for 4 hours (adapted from Ref. [8]). Mineralizable N was evaluated by aerobic incubation

gen (N), and phosphorus (P) in soil microbial biomass.

at 26°C for 31 days.

The occurrence of such heat-induced N transformations is not surprising, because a similar phenomenon has been seen for a variety of soils. In 1901, Daikuhara [4] reported that heating of soil samples in a pan for 20 minutes increased the content of N that could be extracted by dilute acid solutions. His forgotten research is summarized briefly in our recent paper [5].

In contrast with soils, little information is available for organic wastes. In particular, limited attention has been paid to the relationship between the heating temperature and the resulting changes in the form of N. Case et al. [6] recently reported that heat-drying of sewage sludge at temperatures ranging from 130 to 250°C significantly decreased the rate of N mineralization. Their findings differ from previous results including ours [7], which showed increased N mineralization by heat-drying.

In this chapter, we briefly review what is known about this topic by examining data from previous papers in which the heating temperature was specified. The focus is on the effect of heating on the quality of organic wastes as a source of N for crops. The content is not limited to sewage sludge but covers other organic wastes and soils. For a more comprehensive review of sewage sludge, please refer to Rigby et al. [2]. Since Daikuhara's pioneering work, there have been many relevant publications from Japan. We introduce some of these papers to make them available to the international research community and our own unpublished results.

## **2. Previous publications from Japan on heat-induced changes in the quality of soils and organic wastes**

#### **2.1. Soils**

Around 1940, the early findings of Daikuhara [4] were re-evaluated by Mitsui [8]. A series of his experiments were carried out before and during the Pacific War (World War II), when supplies of inorganic fertilizer ran short [5]. By heat-treating two soils at several temperatures (from 65 to 400°C, for 4 hours), Mitsui found that the content of mineralized N (initial inorganic N plus mineralizable N) reached a maximum with heating around 200°C. To generalize this result, he collected 44 types of soils from paddy and upland fields throughout Japan and treated them at 130 or 200°C for 4 hours. He then evaluated the contents of inorganic N and mineralizable N by means of aerobic incubation at 26°C for 31 days.

Both forms of N were increased by the heat treatments (**Figure 1**). The average content of inorganic N was 39.8 mg kg−1 (130°C) and 199.3 mg kg−1 (200°C) as compared to 25.2 mg kg−1 (original). The average content of mineralizable N was 89.4 mg kg−1 (130°C) and 111.1 mg kg−1 (200°C) as compared to 28.0 mg kg−1 (original). The amount of N mineralized by heating at 200°C and subsequent incubation was positively correlated with the content of total N in the original soil (*r* = 0.55\*\*), suggesting that the soil heating effect was greater for humus-rich soils.

To eliminate water and pathogens, sewage sludge is heat-dried by various methods such as convective drying, conductive drying, and solar drying [1]. Several researchers have also reported that heat-drying of sewage sludge can change the form of N [2]. For example, Smith and Durham [3] evaluated the content of N in five sewage sludges in paired treatments, both anaerobically digested and one additionally heat-dried for pelleting or granulation. During heat-drying, more than 75% of inorganic ammonium was lost by volatilization. However, during the aerobic incubation at 25°C for 56 days, the production of nitrate in soil amended with heat-dried sludge was similar to, or even larger than, that from the corresponding sludge without heat-drying. These results suggest that heat-drying of sewage sludge greatly

The occurrence of such heat-induced N transformations is not surprising, because a similar phenomenon has been seen for a variety of soils. In 1901, Daikuhara [4] reported that heating of soil samples in a pan for 20 minutes increased the content of N that could be extracted by dilute acid solutions. His forgotten research is summarized briefly in our recent paper [5].

In contrast with soils, little information is available for organic wastes. In particular, limited attention has been paid to the relationship between the heating temperature and the resulting changes in the form of N. Case et al. [6] recently reported that heat-drying of sewage sludge at temperatures ranging from 130 to 250°C significantly decreased the rate of N mineralization. Their findings differ from previous results including ours [7], which showed increased

In this chapter, we briefly review what is known about this topic by examining data from previous papers in which the heating temperature was specified. The focus is on the effect of heating on the quality of organic wastes as a source of N for crops. The content is not limited to sewage sludge but covers other organic wastes and soils. For a more comprehensive review of sewage sludge, please refer to Rigby et al. [2]. Since Daikuhara's pioneering work, there have been many relevant publications from Japan. We introduce some of these papers to make them

available to the international research community and our own unpublished results.

**2. Previous publications from Japan on heat-induced changes in the** 

mineralizable N by means of aerobic incubation at 26°C for 31 days.

Around 1940, the early findings of Daikuhara [4] were re-evaluated by Mitsui [8]. A series of his experiments were carried out before and during the Pacific War (World War II), when supplies of inorganic fertilizer ran short [5]. By heat-treating two soils at several temperatures (from 65 to 400°C, for 4 hours), Mitsui found that the content of mineralized N (initial inorganic N plus mineralizable N) reached a maximum with heating around 200°C. To generalize this result, he collected 44 types of soils from paddy and upland fields throughout Japan and treated them at 130 or 200°C for 4 hours. He then evaluated the contents of inorganic N and

increased the content of mineralizable organic N.

N mineralization by heat-drying.

54 Nitrogen in Agriculture - Updates

**quality of soils and organic wastes**

**2.1. Soils**

The findings of Mitsui [8] were extended by Sakamoto et al. [9]. They tried to reveal the origin of the N mineralized by heating and measuring the amount of N mineralized from two soils fumigated with chloroform or heated at different temperatures. Chloroform fumigation can kill most of soil microbes, so it has been used to extract the elements such as carbon (C), nitrogen (N), and phosphorus (P) in soil microbial biomass.

The amount of N mineralized from the fumigated soil was similar to that from the soil heated at 50 and 100°C, but it was much less than that from the soil heated at 150 and 200°C (**Figure 2**).

**Figure 1.** Frequency distributions of inorganic and mineralizable N in Japanese agricultural soils (*n* = 44) before and after heating at 130 or 200°C for 4 hours (adapted from Ref. [8]). Mineralizable N was evaluated by aerobic incubation at 26°C for 31 days.

**Figure 2.** Amount of mineralized N in fumigated or heat-treated soils during incubation (adapted from Ref. [9]). Soils were fumigated with chloroform or heated at 50–200°C for 24 hours and then were incubated aerobically at 25°C for 20 days.

Yoshida [13] confirmed and extended those findings in 1970s. We are not sure if he was aware of Yoshimura's results, as he did not cite them. But whether by chance or not the materials that he selected were rapeseed oil cake and food sludge. Food sludge was dewatered by centrifugation, and both materials were dried at 90–95°C and crushed. They were then heattreated at 150°C for 2 hours, 165°C for 1 hour, 175°C for 1 hour, or 200°C for 0.5 hour. These materials were subjected to the aerobic incubation at 30°C for 50 days, and the content of

**Figure 3.** Effects of the application of rapeseed oil cake (ROC) on the growth of tobacco (reprinted from Ref. [11]).

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was applied at 1.5 gN pot−1. Underlined values indicate the percentage of total N in the

When rapeseed oil cake was heat-dried at 175°C for 1 hour or at 200°C for 0.5 hour, the rate of N mineralization became much lower than that of the control (**Figure 4**). The results were similar for food sludge, indicating that the decrease of N mineralization caused by high temperatures was not limited to rapeseed oil cake. The influence of heating at 175°C was quite different from that of heating at 165°C for both materials. This difference suggests that there is a threshold temperature between 165 and 175°C after which the rate of N mineralization

**Figure 4.** Patterns of N mineralization from a sandy loam soil to which rapeseed oil cake and food sludge treated at different temperatures were applied at a rate of 0.2 gN kg−1 soil (created by the authors from the data in Ref. [13]). Aerobic incubation was carried out at 30°C for 50 days. "No application" represents the original soil without

mineralized N produced during the incubation was evaluated.

decreases with increasing temperature.

application.

Rapeseed oil cake or (NH4

) 2 SO4

ROC that was extractable with 50 mM NaOH.

Sakamoto et al. also counted the number of microbes after the treatments by the dilution agar plate method. They found that the decrease in the number of bacteria caused by the fumigation was comparable to that by heating at 50°C, and the decrease in the number of actinomycetes and fungi by the fumigation was comparable to that caused by heating at 100°C. From these results, they concluded that the N mineralized by heating at 50 and 100°C derived mainly from the microbial biomass fraction, whereas the N mineralized at temperatures above 100°C derived mainly from the nonbiomass fraction.

#### **2.2. Rapeseed oil cake and sewage sludge**

In addition to soils, organic wastes have been heat-treated before recycling for use as fertilizer. In 1932, Yoshimura et al. [10] reported why the rate of N mineralization from rapeseed oil cake (the residue that remains after oil extraction) imported from China was lower than that from oil cake produced in Japan. At that time, rapeseed oil cake was produced by roasting, crushing, steam-heating, and squeezing. Because the Chinese rapeseed oil cakes were darker, they hypothesized that the rapeseed was roasted for a longer period and thus at a higher temperature and evaluated the relationship between the roasting conditions and the rate of N mineralization.

Yoshimura et al. found that the rate of N mineralization decreased with increasing roasting period and temperature. **Figure 3** clearly shows that the rate of extraction of N with 50 mM NaOH and the growth of tobacco decreased as the roasting temperature increased from 150 to 170°C [11]. The growth of tobacco supplied with rapeseed oil cakes imported from China was smaller than that with the domestic cakes. These results contrast with those of soils. Probably because of the uniqueness, we found no evidence that their paper had been cited until we rediscovered it recently [12].

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**Figure 3.** Effects of the application of rapeseed oil cake (ROC) on the growth of tobacco (reprinted from Ref. [11]). Rapeseed oil cake or (NH4 )2 SO4 was applied at 1.5 gN pot−1. Underlined values indicate the percentage of total N in the ROC that was extractable with 50 mM NaOH.

Yoshida [13] confirmed and extended those findings in 1970s. We are not sure if he was aware of Yoshimura's results, as he did not cite them. But whether by chance or not the materials that he selected were rapeseed oil cake and food sludge. Food sludge was dewatered by centrifugation, and both materials were dried at 90–95°C and crushed. They were then heattreated at 150°C for 2 hours, 165°C for 1 hour, 175°C for 1 hour, or 200°C for 0.5 hour. These materials were subjected to the aerobic incubation at 30°C for 50 days, and the content of mineralized N produced during the incubation was evaluated.

Sakamoto et al. also counted the number of microbes after the treatments by the dilution agar plate method. They found that the decrease in the number of bacteria caused by the fumigation was comparable to that by heating at 50°C, and the decrease in the number of actinomycetes and fungi by the fumigation was comparable to that caused by heating at 100°C. From these results, they concluded that the N mineralized by heating at 50 and 100°C derived mainly from the microbial biomass fraction, whereas the N mineralized at temperatures above 100°C

**Figure 2.** Amount of mineralized N in fumigated or heat-treated soils during incubation (adapted from Ref. [9]). Soils were fumigated with chloroform or heated at 50–200°C for 24 hours and then were incubated aerobically at 25°C for 20

In addition to soils, organic wastes have been heat-treated before recycling for use as fertilizer. In 1932, Yoshimura et al. [10] reported why the rate of N mineralization from rapeseed oil cake (the residue that remains after oil extraction) imported from China was lower than that from oil cake produced in Japan. At that time, rapeseed oil cake was produced by roasting, crushing, steam-heating, and squeezing. Because the Chinese rapeseed oil cakes were darker, they hypothesized that the rapeseed was roasted for a longer period and thus at a higher temperature and evaluated the relationship between the roasting conditions and the rate of

Yoshimura et al. found that the rate of N mineralization decreased with increasing roasting period and temperature. **Figure 3** clearly shows that the rate of extraction of N with 50 mM NaOH and the growth of tobacco decreased as the roasting temperature increased from 150 to 170°C [11]. The growth of tobacco supplied with rapeseed oil cakes imported from China was smaller than that with the domestic cakes. These results contrast with those of soils. Probably because of the uniqueness, we found no evidence that their paper had been cited until we

derived mainly from the nonbiomass fraction.

**2.2. Rapeseed oil cake and sewage sludge**

N mineralization.

days.

56 Nitrogen in Agriculture - Updates

rediscovered it recently [12].

When rapeseed oil cake was heat-dried at 175°C for 1 hour or at 200°C for 0.5 hour, the rate of N mineralization became much lower than that of the control (**Figure 4**). The results were similar for food sludge, indicating that the decrease of N mineralization caused by high temperatures was not limited to rapeseed oil cake. The influence of heating at 175°C was quite different from that of heating at 165°C for both materials. This difference suggests that there is a threshold temperature between 165 and 175°C after which the rate of N mineralization decreases with increasing temperature.

**Figure 4.** Patterns of N mineralization from a sandy loam soil to which rapeseed oil cake and food sludge treated at different temperatures were applied at a rate of 0.2 gN kg−1 soil (created by the authors from the data in Ref. [13]). Aerobic incubation was carried out at 30°C for 50 days. "No application" represents the original soil without application.

In the same year, Kurihara and Watanabe [14] reported that heat-drying of sewage sludge at 130°C for 1 hour could increase the rate of N mineralization by around 30%. In their experiment, mineralizable N was evaluated by the aerobic incubation at 29°C for 35 days. Their result is similar to the findings of Smith and Durham [3]. In addition to heat-drying, Kurihara and Watanabe reported that freeze-drying of sewage sludge decreased the rate of N mineralization by around 30%.

## **3. Heat-induced changes in mineralized N in sewage sludge**

We have also examined the effect of heating of sewage sludge on the rate of N mineralization [7] and the growth of komatsuna (*Brassica campestris* L. var. *rapa*), a leafy vegetable [15]. We introduce our results in this section. For heating of sewage sludge, we used a pilot-scale conductive dryer ([16]; Krosaki Harima, K-10, Fukuoka, Japan). Sewage sludge in the reactor is heated indirectly by means of a surrounding oil heater in which the temperature can be regulated up to 200°C (**Figure 5**). During the treatment, the sludge is also mixed and crushed by a rotary stirrer to homogenize and granulate the products.

Dewatered sewage sludge collected from a wastewater treatment plant in Shimane Prefecture, Japan, was used in the experiment. We compared two heat treatments such as dry-heating of air-dried sludge (AD) and heat-drying of moist sludge without a preliminary air-drying step (HD). Using these treatments, we prepared four types of materials: AD dry-heated at 120°C

(AD-120), AD dry-heated at 180°C (AD-180), moist sludge heat-dried at 120°C (HD-120), and moist sludge heat-dried at 180°C (HD-180). Dry-heating requires an air-drying pretreatment and is not practical in terms of time and cost. We nonetheless used it to evaluate the effect of heating on the sludge properties more directly by comparing AD-120 and AD-180 with AD.

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These materials were mixed with three soils (an Andosol, a Fluvisol and an Arenosol) at a rate of 1% w/w, and the soils were aerobically incubated at 30°C for 84 days. Regardless of the soil type, the rate of N mineralization was increased significantly by heating of the air-dried sludge at 120°C and it was decreased significantly by heating of the air-dried sludge at 180°C (**Figure 6**). The conventional treatment based on heat-drying of moist sludge at 120 or 180°C exerted similar but less pronounced effects. These heat-induced changes were attributed to the transformation of sludge organic N, because volatilization of N during the heating treat-

Using the same combination of sludges and soils, we carried out two successive pot experiments. Komatsuna, a popular test vegetable in Japan, was grown after a basal application of sludges at a rate of 20 Mg ha−1 (dry matter base). Sludge application was carried out only once

**Figure 6.** Patterns of N mineralization from three soil types amended with heat-treated sewage sludge (adapted from Ref. [7]). The heat treatment was followed by aerobic incubation at 30°C for 84 days. AD: air-drying, AD-120: dry-heating at 120°C, AD-180: dry-heating at 180°C, HD-120: heat-drying at 120°C, and HD-180: heat-drying at 180°C. Each treatment

The heating period was fixed at 16 hours.

before the start of the first experiment.

ments was negligible [7].

was duplicated.

**Figure 5.** Reactor used in the pilot-scale conductive dryer system in our previous research [7, 16].

(AD-120), AD dry-heated at 180°C (AD-180), moist sludge heat-dried at 120°C (HD-120), and moist sludge heat-dried at 180°C (HD-180). Dry-heating requires an air-drying pretreatment and is not practical in terms of time and cost. We nonetheless used it to evaluate the effect of heating on the sludge properties more directly by comparing AD-120 and AD-180 with AD. The heating period was fixed at 16 hours.

In the same year, Kurihara and Watanabe [14] reported that heat-drying of sewage sludge at 130°C for 1 hour could increase the rate of N mineralization by around 30%. In their experiment, mineralizable N was evaluated by the aerobic incubation at 29°C for 35 days. Their result is similar to the findings of Smith and Durham [3]. In addition to heat-drying, Kurihara and Watanabe reported that freeze-drying of sewage sludge decreased the rate of N mineral-

We have also examined the effect of heating of sewage sludge on the rate of N mineralization [7] and the growth of komatsuna (*Brassica campestris* L. var. *rapa*), a leafy vegetable [15]. We introduce our results in this section. For heating of sewage sludge, we used a pilot-scale conductive dryer ([16]; Krosaki Harima, K-10, Fukuoka, Japan). Sewage sludge in the reactor is heated indirectly by means of a surrounding oil heater in which the temperature can be regulated up to 200°C (**Figure 5**). During the treatment, the sludge is also mixed and crushed

Dewatered sewage sludge collected from a wastewater treatment plant in Shimane Prefecture, Japan, was used in the experiment. We compared two heat treatments such as dry-heating of air-dried sludge (AD) and heat-drying of moist sludge without a preliminary air-drying step (HD). Using these treatments, we prepared four types of materials: AD dry-heated at 120°C

**3. Heat-induced changes in mineralized N in sewage sludge**

**Figure 5.** Reactor used in the pilot-scale conductive dryer system in our previous research [7, 16].

by a rotary stirrer to homogenize and granulate the products.

ization by around 30%.

58 Nitrogen in Agriculture - Updates

These materials were mixed with three soils (an Andosol, a Fluvisol and an Arenosol) at a rate of 1% w/w, and the soils were aerobically incubated at 30°C for 84 days. Regardless of the soil type, the rate of N mineralization was increased significantly by heating of the air-dried sludge at 120°C and it was decreased significantly by heating of the air-dried sludge at 180°C (**Figure 6**). The conventional treatment based on heat-drying of moist sludge at 120 or 180°C exerted similar but less pronounced effects. These heat-induced changes were attributed to the transformation of sludge organic N, because volatilization of N during the heating treatments was negligible [7].

Using the same combination of sludges and soils, we carried out two successive pot experiments. Komatsuna, a popular test vegetable in Japan, was grown after a basal application of sludges at a rate of 20 Mg ha−1 (dry matter base). Sludge application was carried out only once before the start of the first experiment.

**Figure 6.** Patterns of N mineralization from three soil types amended with heat-treated sewage sludge (adapted from Ref. [7]). The heat treatment was followed by aerobic incubation at 30°C for 84 days. AD: air-drying, AD-120: dry-heating at 120°C, AD-180: dry-heating at 180°C, HD-120: heat-drying at 120°C, and HD-180: heat-drying at 180°C. Each treatment was duplicated.

In the first experiment, the amount of N uptake by the plants increased significantly by heating air-dried sludge at 120°C and decreased significantly by heating at 180°C, when the sludge was applied to the Fluvisol or Arenosol (**Figure 7**). Heat-drying of the sludge at 120 or 180°C also increased N uptake significantly. In the Andosol, on the other hand, N uptake was not so much influenced by the rate of N mineralization from sewage sludge as that observed in the other two soils (**Figure 6**). Because both Andosol and sewage sludge (which contained ferrous polysulfate as a coagulant) adsorbed P in soil solution (data not shown), we considered that the beneficial effect of N supply from sewage sludge on plant growth was offset by the limited supply of P [15].

The second pot experiment was carried out without an additional sludge application after complete removal of the plants from the first experiment. The plant growth in the second experiment became smaller than that in the first one, and the difference among the treatments decreased (**Figure 8**). The supply of N from the sludge did not last long. Thus, at least for the komatsuna plants used in our experiment, frequent application of sludge is required to sustain plant growth. However, it will also lead to the accumulation of sludge-derived N and P in soil, because less than 40 and 15% of the sludge N and P, respectively, were apparently recovered by two harvests of the plants [15]. From these results, we concluded that heated sludges can act as an effective organic N fertilizer, provided that they are applied to a suitable type of soil and that the short-term effects on soil productivity are balanced with the long-term effects on environmental quality.

Case et al. [6] re-evaluated previous results, including ours. On the hypothesis that the effect of heat-drying on N mineralization from sewage sludge would differ among heating temperatures, they heat-dried anaerobically digested sewage sludge at different temperatures (70, 130, 190, or 250°C) until the water content reached less than 5%. Heat-drying treatment was carried out with a laboratory oven (laboratory-drying), and the product was abbreviated as LD. The sludge materials were applied to a sandy loam soil (Luvisol) and incubated aerobically at 15°C for 160 days. During the incubation, the production of exchangeable ammonium,

**Figure 8.** Sustainability of the effects of an initial application of sewage sludge on the growth of a leafy vegetable (komatsuna) grown in an Arenosol (adapted from Ref. [15]). The second experiment was carried out without sludge application. AD: air-drying, AD-120: dry-heating at 120°C and AD-180: dry-heating at 180°C. Values are the dry matter

An Overview of the Effects of Heat Treatments on the Quality of Organic Wastes as a Nitrogen Fertilizer

The production of nitrate during the whole incubation period was largest for the original sewage sludge (Original), followed by LD-70, LD-130 > LD-190 > LD-250 (**Figure 9**). Thus, nitrate production decreased with increasing temperature from 130 to 250°C. Because the content of exchangeable ammonium was almost zero at 80 days after the incubation (data not shown), the amount of nitrate produced after day 80 can be regarded as the amount of mineralized N

lower than that from LD-70 and LD-130, indicating that microbial decomposition of LD-70 and LD-130 occurred more rapidly. From these results, Case et al. concluded that heat-drying temperature significantly influenced the rate of N mineralization from sewage sludge but that heatdrying did not improve the rate of N mineralization at any temperatures they examined. They also emphasized that the heat-drying treatment in the laboratory produced different results from the treatment in a large-scale wastewater treatment plant (**Figure 9**) and the heat-drying temperature is one of the several factors that potentially affect the rate of N mineralization.

was also largest in Original, LD-70, and LD-130, followed by

emission from Original was

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) was monitored.

nitrate, and carbon dioxide (CO2

weight (g pot−1, *n* = 3).

The overall production of CO2

(initial inorganic N plus mineralizable N).

LD-190 and LD-250 (data not shown). But the initial rate of CO2


**Figure 7.** Effect of the application of heat-treated sewage sludge on the growth of a leafy vegetable (komatsuna) grown in three types of soils (adapted from Ref. [15]). This first experiment was carried out for 88 days in the winter. AD: air-drying, AD-120: dry-heating at 120°C, AD-180: dry-heating at 180°C, HD-120: heat-drying at 120°C, and HD-180: heat-drying at 180°C. Upper and lower values indicate the dry matter weight (g pot−1, *n* = 3) and N uptake (mg pot−1, *n* = 3), respectively. All pictures were taken from the same angle.

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In the first experiment, the amount of N uptake by the plants increased significantly by heating air-dried sludge at 120°C and decreased significantly by heating at 180°C, when the sludge was applied to the Fluvisol or Arenosol (**Figure 7**). Heat-drying of the sludge at 120 or 180°C also increased N uptake significantly. In the Andosol, on the other hand, N uptake was not so much influenced by the rate of N mineralization from sewage sludge as that observed in the other two soils (**Figure 6**). Because both Andosol and sewage sludge (which contained ferrous polysulfate as a coagulant) adsorbed P in soil solution (data not shown), we considered that the beneficial effect of N supply from sewage sludge on plant growth was offset by the limited

The second pot experiment was carried out without an additional sludge application after complete removal of the plants from the first experiment. The plant growth in the second experiment became smaller than that in the first one, and the difference among the treatments decreased (**Figure 8**). The supply of N from the sludge did not last long. Thus, at least for the komatsuna plants used in our experiment, frequent application of sludge is required to sustain plant growth. However, it will also lead to the accumulation of sludge-derived N and P in soil, because less than 40 and 15% of the sludge N and P, respectively, were apparently recovered by two harvests of the plants [15]. From these results, we concluded that heated sludges can act as an effective organic N fertilizer, provided that they are applied to a suitable type of soil and that the short-term effects on soil productivity are balanced with the

**Figure 7.** Effect of the application of heat-treated sewage sludge on the growth of a leafy vegetable (komatsuna) grown in three types of soils (adapted from Ref. [15]). This first experiment was carried out for 88 days in the winter. AD: air-drying, AD-120: dry-heating at 120°C, AD-180: dry-heating at 180°C, HD-120: heat-drying at 120°C, and HD-180: heat-drying at 180°C. Upper and lower values indicate the dry matter weight (g pot−1, *n* = 3) and N uptake (mg pot−1, *n*

supply of P [15].

60 Nitrogen in Agriculture - Updates

long-term effects on environmental quality.

= 3), respectively. All pictures were taken from the same angle.

**Figure 8.** Sustainability of the effects of an initial application of sewage sludge on the growth of a leafy vegetable (komatsuna) grown in an Arenosol (adapted from Ref. [15]). The second experiment was carried out without sludge application. AD: air-drying, AD-120: dry-heating at 120°C and AD-180: dry-heating at 180°C. Values are the dry matter weight (g pot−1, *n* = 3).

Case et al. [6] re-evaluated previous results, including ours. On the hypothesis that the effect of heat-drying on N mineralization from sewage sludge would differ among heating temperatures, they heat-dried anaerobically digested sewage sludge at different temperatures (70, 130, 190, or 250°C) until the water content reached less than 5%. Heat-drying treatment was carried out with a laboratory oven (laboratory-drying), and the product was abbreviated as LD. The sludge materials were applied to a sandy loam soil (Luvisol) and incubated aerobically at 15°C for 160 days. During the incubation, the production of exchangeable ammonium, nitrate, and carbon dioxide (CO2 ) was monitored.

The production of nitrate during the whole incubation period was largest for the original sewage sludge (Original), followed by LD-70, LD-130 > LD-190 > LD-250 (**Figure 9**). Thus, nitrate production decreased with increasing temperature from 130 to 250°C. Because the content of exchangeable ammonium was almost zero at 80 days after the incubation (data not shown), the amount of nitrate produced after day 80 can be regarded as the amount of mineralized N (initial inorganic N plus mineralizable N).

The overall production of CO2 was also largest in Original, LD-70, and LD-130, followed by LD-190 and LD-250 (data not shown). But the initial rate of CO2 emission from Original was lower than that from LD-70 and LD-130, indicating that microbial decomposition of LD-70 and LD-130 occurred more rapidly. From these results, Case et al. concluded that heat-drying temperature significantly influenced the rate of N mineralization from sewage sludge but that heatdrying did not improve the rate of N mineralization at any temperatures they examined. They also emphasized that the heat-drying treatment in the laboratory produced different results from the treatment in a large-scale wastewater treatment plant (**Figure 9**) and the heat-drying temperature is one of the several factors that potentially affect the rate of N mineralization.

**Figure 9.** Patterns of nitrate production from a sandy loam soil (a Luvisol) amended with heat-treated sewage sludge at 2% (w/w). Aerobic (pF 2) incubation was carried out at 15°C for 160 days. Anaerobically digested sewage sludge (Original) was heated in a laboratory oven at 70, 130, 190, or 250°C until the water content was less than 5% (LD-70, LD-130, LD-190, and LD-250, respectively). Reproduced with permission from Case et al. [6], *Environ. Sci. Pollut. Res*., Springer International Publishing AG.

In addition to Case et al. [6], the positive ([17]; 60°C for 13 hours) or negative ([18]; 250°C until reaching constant weight) effects of heat-drying on the amount of mineralized N in sewage sludge have been reported by comparing dewatered sludge with sewage sludge heat-dried at a single temperature. Maki et al. [19] also found that the content of chemically extractable organic N in cow dung manure was decreased by heating at temperatures from 80 to 180°C for 2 hours in a laboratory oven. As summarized by Rigby et al. [2] and Case et al. [6], several researchers have reported N mineralization from heat-dried sludge without specifying the heating conditions. According to the literature review by Rigby et al. [2], the percentage of mineralizable N to total organic N in heat-dried sewage sludge ranged from 26 to 71% (40% in average). This value was similar to aerobically digested sewage sludge whose value ranged from 32 to 58% (47% in average).

120 to 200°C, N mineralization increased in soil but decreased in organic wastes. The reason for the difference remains to be elucidated. Second, the content of mineralized N in organic wastes decreased sharply by heating at 150–200°C except for Case et al. [6] who reported a more gradual decrease. The reason for this difference is also uncertain. The release of CO<sup>2</sup> from the sludge of Case et al. during the incubation decreased slightly with heating at 130°C, but decreased sharply with heating at 190°C [6], which indicates that the threshold temperature for the stabilization of sludge C did exist between 130 and 190°C. On the other hand, we could not estimate the suitable temperature to increase N mineralization from organic wastes due to the insufficient number of available data. Lastly, the heating effect on N mineralization was influenced by both the heating temperature and by the initial water content of the sewage sludge. A typical example can be found in the difference between our samples at 180°C

**Figure 10.** Relationship between temperature at which organic wastes were heated and heat-induced changes in mineralized N (initial inorganic N plus mineralizable N). The data from Mitsui [8] represent the average value obtained

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from 44 soils. Our data [7] also represent the average value obtained from three soils (**Figure 6**).

We will have a closer look at the last point. **Figure 11** shows the temporal changes in the water content and the temperature of sewage sludge during the production of AD-180 and HD-180. Samples were heated for 16 hours with the reactor temperature set at 180°C. For both materials, the heat-drying process can be divided into two periods: the drying period (sludge < 100°C), in which most of the heat is consumed to dry the sludge, and the denaturing period (sludge > 100°C). Because of the difference in the initial water content, it took about 5 hours for AD-180 to reach 180°C, and about 14 hours for HD-180. As a result, HD-180 stayed at 180°C for only 2–3 hours. This clearly indicates that the temperature of sewage sludge during the heat-drying

(AD-180 and HD-180).

#### **4. Summary of the heating effects on mineralized N in organic wastes**

By compiling the abovementioned reports, we plotted the heat-induced changes of mineralized N (initial inorganic N plus mineralizable N) in organic wastes as a function of the heating temperature (**Figure 10**). It should be noted that only a rough comparison of these results among the studies is possible, because the materials used as a control, the heating conditions and the incubation conditions differed.

Nevertheless, the figure suggests certain trends. First, the response to the heating temperature differed between soils and organic wastes. As the heating temperature increased from around

An Overview of the Effects of Heat Treatments on the Quality of Organic Wastes as a Nitrogen Fertilizer http://dx.doi.org/10.5772/68163 63

**Figure 10.** Relationship between temperature at which organic wastes were heated and heat-induced changes in mineralized N (initial inorganic N plus mineralizable N). The data from Mitsui [8] represent the average value obtained from 44 soils. Our data [7] also represent the average value obtained from three soils (**Figure 6**).

In addition to Case et al. [6], the positive ([17]; 60°C for 13 hours) or negative ([18]; 250°C until reaching constant weight) effects of heat-drying on the amount of mineralized N in sewage sludge have been reported by comparing dewatered sludge with sewage sludge heat-dried at a single temperature. Maki et al. [19] also found that the content of chemically extractable organic N in cow dung manure was decreased by heating at temperatures from 80 to 180°C for 2 hours in a laboratory oven. As summarized by Rigby et al. [2] and Case et al. [6], several researchers have reported N mineralization from heat-dried sludge without specifying the heating conditions. According to the literature review by Rigby et al. [2], the percentage of mineralizable N to total organic N in heat-dried sewage sludge ranged from 26 to 71% (40% in average). This value was similar to aerobically digested sewage sludge whose value ranged

**Figure 9.** Patterns of nitrate production from a sandy loam soil (a Luvisol) amended with heat-treated sewage sludge at 2% (w/w). Aerobic (pF 2) incubation was carried out at 15°C for 160 days. Anaerobically digested sewage sludge (Original) was heated in a laboratory oven at 70, 130, 190, or 250°C until the water content was less than 5% (LD-70, LD-130, LD-190, and LD-250, respectively). Reproduced with permission from Case et al. [6], *Environ. Sci. Pollut. Res*.,

**4. Summary of the heating effects on mineralized N in organic wastes**

By compiling the abovementioned reports, we plotted the heat-induced changes of mineralized N (initial inorganic N plus mineralizable N) in organic wastes as a function of the heating temperature (**Figure 10**). It should be noted that only a rough comparison of these results among the studies is possible, because the materials used as a control, the heating conditions

Nevertheless, the figure suggests certain trends. First, the response to the heating temperature differed between soils and organic wastes. As the heating temperature increased from around

from 32 to 58% (47% in average).

Springer International Publishing AG.

62 Nitrogen in Agriculture - Updates

and the incubation conditions differed.

120 to 200°C, N mineralization increased in soil but decreased in organic wastes. The reason for the difference remains to be elucidated. Second, the content of mineralized N in organic wastes decreased sharply by heating at 150–200°C except for Case et al. [6] who reported a more gradual decrease. The reason for this difference is also uncertain. The release of CO<sup>2</sup> from the sludge of Case et al. during the incubation decreased slightly with heating at 130°C, but decreased sharply with heating at 190°C [6], which indicates that the threshold temperature for the stabilization of sludge C did exist between 130 and 190°C. On the other hand, we could not estimate the suitable temperature to increase N mineralization from organic wastes due to the insufficient number of available data. Lastly, the heating effect on N mineralization was influenced by both the heating temperature and by the initial water content of the sewage sludge. A typical example can be found in the difference between our samples at 180°C (AD-180 and HD-180).

We will have a closer look at the last point. **Figure 11** shows the temporal changes in the water content and the temperature of sewage sludge during the production of AD-180 and HD-180. Samples were heated for 16 hours with the reactor temperature set at 180°C. For both materials, the heat-drying process can be divided into two periods: the drying period (sludge < 100°C), in which most of the heat is consumed to dry the sludge, and the denaturing period (sludge > 100°C). Because of the difference in the initial water content, it took about 5 hours for AD-180 to reach 180°C, and about 14 hours for HD-180. As a result, HD-180 stayed at 180°C for only 2–3 hours. This clearly indicates that the temperature of sewage sludge during the heat-drying

was sampled from a wastewater treatment plant in Chiba Prefecture, Japan. The sludge was air-dried at room temperature or heat-dried at 180°C by the conductive dryer in **Figure 5**. The air-dried sludge without heating (AD) contained C and N at 36 and 5.6%, respectively. The sludge was heated for 14 hours until its temperature reached 180°C, or it was continued for an additional 24 hours (a total of 38 hours) or 120 hours (a total of 134 hours). The heat-dried materials treated at 180°C for the additional 0, 24, or 120 hours are referred to as HD-180 (0

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The difference between HD-180 (0 hour) and other two HD-180 materials is originated solely from the dry-heating process at 180°C. The total C content was about 37% in all HD-180 materials. However, the rate of C mineralization decreased significantly with increasing dry-heating period (**Figure 12**). The percentage of total C that was mineralized during the 35-days incubation was 12.5% in HD-180 (0 hour), which was slightly lower than AD (13.1%). It decreased to 5.8% in HD-180 (24 hours) and to 4.1% for HD-180 (120 hours). These results indicate that the stabilization of C occurred mainly during the initial 24 hours of the dry-heating period and

The color of the heat-dried sewage sludge was also quite different from that of the air-dried sludge. For example, HD-180 (30 hours) was darker than AD (**Figure 13**). This suggests that the stabilization of C and N in sewage sludge was concomitant with the Maillard reaction, which is a series of nonenzymatic browning reactions occurring when virtually all foods are heated [22]. This reaction starts from the condensation of the carbonyl group of a reducing sugar with a free amino group of an amino acid [22]. The color of powdered materials can be measured easily and precisely with a color sensor [23]. Thus, the color of the end-products can be a useful proxy for the temperature of the sludge throughout the heat-drying

**Figure 12.** Average percentage of sludge C mineralized during aerobic incubation at 30°C for 35 days. The sludge materials were applied to an Acrisol from Shimane University at Honjo at 1% (v/v), and the soil was incubated at a

moisture content of 60% of maximum water holding capacity. Each treatment was triplicated.

hour), HD-180 (24 hours), or HD-180 (120 hours), respectively.

proceeded slowly thereafter.

treatment.

**Figure 11.** Changes in the water content and the temperature of sewage sludge during heating at 180°C with (AD-180) or without (HD-180) air-drying as a pretreatment (adapted from Ref. [7]). The heating treatment was carried out for 16 hours in the pilot-scale conductive dryer illustrated in **Figure 5**.

treatment rather than the temperature of the reactor is a most important factor that affects the availability of N in the heat-dried products. In the case of heat-drying of sewage sludge at temperatures higher than 150°C, it is plausible that the presence and the length of the denaturing period are key factors that determine whether the rate of N mineralization decreases or not.

Different from our samples treated beyond the drying period, the water content of the heatdried sewage sludge produced at wastewater treatment plants varies from less than 10% [3, 6, 20] to more than 60% [21]. If sewage sludge was heat-dried homogeneously and the content of water in the end-product was relatively high, we can assume that the sludge temperature during the heat-drying treatment did not exceed 100°C.

## **5. Heat-induced stabilization of C in sewage sludge**

#### **5.1. Preliminary experiments**

We carried out additional experiments to better understand why N mineralization was decreased significantly by dry-heating at 180°C (**Figure 6**). Since N mineralization is a part of the biological decomposition of organic C, we focused on the chemical form of the C. The heat-induced denaturation of C in sewage sludge (stabilization in the case of AD-180) was evaluated by using biological and chemical methods as follows; aerobic incubation, chemical extraction, and 13C-NMR.

To extend the stabilization period, sewage sludge was heated at 180°C for a much longer period than in our previous experiment. Dewatered sewage sludge made from human waste was sampled from a wastewater treatment plant in Chiba Prefecture, Japan. The sludge was air-dried at room temperature or heat-dried at 180°C by the conductive dryer in **Figure 5**. The air-dried sludge without heating (AD) contained C and N at 36 and 5.6%, respectively. The sludge was heated for 14 hours until its temperature reached 180°C, or it was continued for an additional 24 hours (a total of 38 hours) or 120 hours (a total of 134 hours). The heat-dried materials treated at 180°C for the additional 0, 24, or 120 hours are referred to as HD-180 (0 hour), HD-180 (24 hours), or HD-180 (120 hours), respectively.

The difference between HD-180 (0 hour) and other two HD-180 materials is originated solely from the dry-heating process at 180°C. The total C content was about 37% in all HD-180 materials. However, the rate of C mineralization decreased significantly with increasing dry-heating period (**Figure 12**). The percentage of total C that was mineralized during the 35-days incubation was 12.5% in HD-180 (0 hour), which was slightly lower than AD (13.1%). It decreased to 5.8% in HD-180 (24 hours) and to 4.1% for HD-180 (120 hours). These results indicate that the stabilization of C occurred mainly during the initial 24 hours of the dry-heating period and proceeded slowly thereafter.

The color of the heat-dried sewage sludge was also quite different from that of the air-dried sludge. For example, HD-180 (30 hours) was darker than AD (**Figure 13**). This suggests that the stabilization of C and N in sewage sludge was concomitant with the Maillard reaction, which is a series of nonenzymatic browning reactions occurring when virtually all foods are heated [22]. This reaction starts from the condensation of the carbonyl group of a reducing sugar with a free amino group of an amino acid [22]. The color of powdered materials can be measured easily and precisely with a color sensor [23]. Thus, the color of the end-products can be a useful proxy for the temperature of the sludge throughout the heat-drying treatment.

treatment rather than the temperature of the reactor is a most important factor that affects the availability of N in the heat-dried products. In the case of heat-drying of sewage sludge at temperatures higher than 150°C, it is plausible that the presence and the length of the denaturing period are key factors that determine whether the rate of N mineralization decreases or not.

**Figure 11.** Changes in the water content and the temperature of sewage sludge during heating at 180°C with (AD-180) or without (HD-180) air-drying as a pretreatment (adapted from Ref. [7]). The heating treatment was carried out for 16

Different from our samples treated beyond the drying period, the water content of the heatdried sewage sludge produced at wastewater treatment plants varies from less than 10% [3, 6, 20] to more than 60% [21]. If sewage sludge was heat-dried homogeneously and the content of water in the end-product was relatively high, we can assume that the sludge tem-

We carried out additional experiments to better understand why N mineralization was decreased significantly by dry-heating at 180°C (**Figure 6**). Since N mineralization is a part of the biological decomposition of organic C, we focused on the chemical form of the C. The heat-induced denaturation of C in sewage sludge (stabilization in the case of AD-180) was evaluated by using biological and chemical methods as follows; aerobic incubation, chemical

To extend the stabilization period, sewage sludge was heated at 180°C for a much longer period than in our previous experiment. Dewatered sewage sludge made from human waste

perature during the heat-drying treatment did not exceed 100°C.

hours in the pilot-scale conductive dryer illustrated in **Figure 5**.

**5. Heat-induced stabilization of C in sewage sludge**

**5.1. Preliminary experiments**

64 Nitrogen in Agriculture - Updates

extraction, and 13C-NMR.

**Figure 12.** Average percentage of sludge C mineralized during aerobic incubation at 30°C for 35 days. The sludge materials were applied to an Acrisol from Shimane University at Honjo at 1% (v/v), and the soil was incubated at a moisture content of 60% of maximum water holding capacity. Each treatment was triplicated.

**Figure 15** shows the relative contents of the fractions in our samples. The results of Collard et al. [20] were added to the figure, which were obtained from heat-drying of sewage sludge at 85 and 120°C. However, only a rough comparison between two datasets is possible, because Collard et al. used a slightly different extraction method (the IHSS method) and also removed lipids with a mixture of dichloromethane and methanol before extracting humic substances. In contrast, we did not remove nonhumic substances beforehand (the original method), so our extractable fractions (fulvic and humic acids) may have been overestimated as a result of contamination of nonhumic substances such as lipids and carbohydrates. Here we pay attention to the percentage of humin as an index of the stability of sludge C, since the humin fraction was considered to be

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The percentage of humin in the original sludge without heating suggested that our sample was less stabilized than that of Collard et al. In our samples, the heat-drying at 180°C greatly increased the percentage of humin; 12% in AD, 37% in HD-180 (0 hour), and 50% in HD-180 (120 hours). In the samples of Collard et al. [20], on the other hand, heat-drying at 120°C decreased the percentage of humin; 59% in Original, 59% in HD-85, and 30% in HD-120. These results indicate that the stability of sludge C was unaffected by heat-drying at 85°C, decreased at 120°C and increased at 180°C. The effects of heating on the stability of C differed

Comparing among our samples, however, the rate of C mineralization from AD and HD-180 (0 hour) was not so much different as the percentage of humin (**Figures 12** and **15**). This indicates that the increase of the percentage of humin was not proportional to the decrease of C mineralization during the incubation. In our previous study [7], the chemical forms of organic N in heat-treated materials were also evaluated by sequential extraction. Although organic N in AD-180 was most recalcitrant to chemical extractions, the results could not quantitatively explain the very low rate of N mineralization. These results suggest that mineralization of sludge

**Figure 15.** Relative percentages of fulvic acid, humic acid, and humin in sewage sludge samples. The percentage of lipid is also presented for the results of Collard et al. [20] after adaptation by the authors. In their paper, the activated sludge

(Original) was heat-dried at 85°C (HD-85) and then pelleted at 120°C (HD-120).

between 120 and 180°C, which agreed with our results of N mineralization [7].

least affected by such contamination.

**Figure 13.** Color differences between two samples of finely ground sewage sludge. AD: air-drying, HD-180 (30h): heatdrying at 180°C with a stabilization period of 30 hours.

#### **5.2. Fractionation of organic matter by chemical extraction**

Based on these preliminary observations, we analyzed the composition of organic matter in three samples, i.e., AD, HD-180 (0 hour), and HD-180 (120 hours). The samples were crushed with an agate mortar to pass a 70-mesh sieve with an opening of 0.212 mm for the following analyses. Analyses were performed without replicates.

We used an extraction method known as Nagoya method [24], which is based on the different solubilities of organic matter fractions in alkaline and acid solutions. This method was originally designed to classify humic substances in soils, and we applied it to sewage sludge. **Figure 14** shows the procedures for separating humic substances into fulvic acid, humic acid, and humin fractions.

**Figure 14.** Procedures for the fractionation of soil organic matter into fulvic acid, humic acid, and humin according to the Nagoya method [24].

**Figure 15** shows the relative contents of the fractions in our samples. The results of Collard et al. [20] were added to the figure, which were obtained from heat-drying of sewage sludge at 85 and 120°C. However, only a rough comparison between two datasets is possible, because Collard et al. used a slightly different extraction method (the IHSS method) and also removed lipids with a mixture of dichloromethane and methanol before extracting humic substances. In contrast, we did not remove nonhumic substances beforehand (the original method), so our extractable fractions (fulvic and humic acids) may have been overestimated as a result of contamination of nonhumic substances such as lipids and carbohydrates. Here we pay attention to the percentage of humin as an index of the stability of sludge C, since the humin fraction was considered to be least affected by such contamination.

The percentage of humin in the original sludge without heating suggested that our sample was less stabilized than that of Collard et al. In our samples, the heat-drying at 180°C greatly increased the percentage of humin; 12% in AD, 37% in HD-180 (0 hour), and 50% in HD-180 (120 hours). In the samples of Collard et al. [20], on the other hand, heat-drying at 120°C decreased the percentage of humin; 59% in Original, 59% in HD-85, and 30% in HD-120. These results indicate that the stability of sludge C was unaffected by heat-drying at 85°C, decreased at 120°C and increased at 180°C. The effects of heating on the stability of C differed between 120 and 180°C, which agreed with our results of N mineralization [7].

Comparing among our samples, however, the rate of C mineralization from AD and HD-180 (0 hour) was not so much different as the percentage of humin (**Figures 12** and **15**). This indicates that the increase of the percentage of humin was not proportional to the decrease of C mineralization during the incubation. In our previous study [7], the chemical forms of organic N in heat-treated materials were also evaluated by sequential extraction. Although organic N in AD-180 was most recalcitrant to chemical extractions, the results could not quantitatively explain the very low rate of N mineralization. These results suggest that mineralization of sludge

**Figure 15.** Relative percentages of fulvic acid, humic acid, and humin in sewage sludge samples. The percentage of lipid is also presented for the results of Collard et al. [20] after adaptation by the authors. In their paper, the activated sludge (Original) was heat-dried at 85°C (HD-85) and then pelleted at 120°C (HD-120).

**Figure 14.** Procedures for the fractionation of soil organic matter into fulvic acid, humic acid, and humin according to

Based on these preliminary observations, we analyzed the composition of organic matter in three samples, i.e., AD, HD-180 (0 hour), and HD-180 (120 hours). The samples were crushed with an agate mortar to pass a 70-mesh sieve with an opening of 0.212 mm for the following

**Figure 13.** Color differences between two samples of finely ground sewage sludge. AD: air-drying, HD-180 (30h): heat-

We used an extraction method known as Nagoya method [24], which is based on the different solubilities of organic matter fractions in alkaline and acid solutions. This method was originally designed to classify humic substances in soils, and we applied it to sewage sludge. **Figure 14** shows the procedures for separating humic substances into fulvic acid, humic acid,

**5.2. Fractionation of organic matter by chemical extraction**

drying at 180°C with a stabilization period of 30 hours.

analyses. Analyses were performed without replicates.

the Nagoya method [24].

and humin fractions.

66 Nitrogen in Agriculture - Updates

C and N was determined not only by its chemical solubility but also by other physical factors such as microbial accessibility.

C increased from 31 to 38%, whereas that of O-alkyl C decreased from 35 to 29%. During the stabilization period caused by dry-heating for 120 hours, the percentage of aromatic C increased from 14 to 24%, whereas that of O-alkyl C decreased from 29 to 20%. The proportion

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The transformation of C during the treatments at 180°C could be detected by applying the 13C-NMR method to our crude samples. The transformation occurred not only during the stabilization period but also during the initial drying period, but the functional groups affected by the transformation differed between the periods. These results indicate that the reactions in the drying period differed from those in the stabilization period. A slight decrease of carbonyl C from 19 to 17% during the stabilization period implied the occurrence of the Maillard reaction. However, it is uncertain to what extent the changes observed in the <sup>13</sup>C-NMR spectra were responsible for the different rates of mineralization of C between HD-180 (0 hour) and HD-180 (120 hours). The higher proportion of aromatic C in HD-180 (120 hours) may have contributed to the lower rate of C mineralization, because the aromatic polymers in plant residues and microbial products are regarded as more recalcitrant

Similar to our results, the heating of Susuki (*Miscanthus sinensis* A.) leaves at 250°C for 1 hour in a laboratory oven increased the percentage of aromatic C from 17 to 66% and decreased that of O-alkyl C from 70 to 15% [28]. Heating at 250°C burned the leaves. On the other hand, the 13C-NMR spectrum of the 200°C-heated samples remained unchanged from that of the untreated control. Heating of the leaves at 200°C for 1 hour did not cause burning but turned

As for sewage sludge, Fernández et al. [29] compared the properties of heat-dried sewage sludge with those of composted sludge. Sewage sludge that was heat-dried at the maximum sludge temperature of 75°C contained more fulvic acid than sewage sludge composted in windrows for 3 months. The solid-state 13C-NMR spectroscopy indicated that the humic acid fraction of the heat-dried sludge contained more alkyl C and less aromatic C than that of the composted sludge. These results indicated that heat-drying of sewage sludge at 75°C (sludge temperature) did not increase the stability of C as much as did windrow composting. In their study, the heat-drying was carried out by indirect convection with air heated between 380 and 450°C. The temperature of the sludge during the treatment (<75°C) was therefore much lower than the hot air supplied for drying. This reemphasizes the importance of the temperature of sewage sludge during the drying period as a factor that affects the quality of

By reviewing literature, we suggested that the heat-induced changes in N mineralization differ between soils and organic wastes. As the heating temperature increased to 200°C, the rate of N mineralization increased in soils but decreased in organic wastes. The rate of N mineralization from organic wastes tended to decrease sharply when heating temperatures increased

of carbonyl C was relatively constant throughout the treatments.

to biological decomposition [27].

the end-product.

**6. Conclusions**

them brown possibly owing to the Maillard reaction.

#### **5.3. Analysis of the functional groups of C by 13C-NMR**

We also analyzed the C forms in AD, HD-180 (0 hour), and HD-180 (120 hours) by the solid-state 13C nuclear magnetic resonance (13C-NMR) spectroscopy. The 13C-NMR method provides estimates of the relative percentages of the main functional groups of C. The analytical procedures and machine conditions were the same as those described by Hiradate et al. [25]. When our samples were analyzed without any pretreatment, the 13C-NMR spectra were divided into the four regions. According to Hiradate et al. [25], these regions were assigned to the functional groups as follows (**Figure 16**); alkyl C (0–45 ppm), O-alkyl C (45–110 ppm), aromatic C (110–165 ppm), and carbonyl C (165–210 ppm).

In AD, the percentage of alkyl C plus O-alkyl C exceeded 60%. Fernández et al. [26] also reported the predominance of these functional groups (75%) in sewage sludge heat-dried at 70–80°C. During the drying period from AD to HD-180 (0 hour), the percentage of alkyl

**Figure 16.** Solid-state 13C-NMR (CP/MAS-TOSS, 75.45 MHz) spectra of sewage sludge samples. The 13C-NMR spectrum was divided into the four chemical shift regions: 0–45 ppm (alkyl C), 45–110 ppm (O-alkyl C), 110–165 ppm (aromatic C), and 165–210 ppm (carbonyl C). The values in the spectra indicate the relative percentage of each type of C calculated by integrating the signal intensity.

C increased from 31 to 38%, whereas that of O-alkyl C decreased from 35 to 29%. During the stabilization period caused by dry-heating for 120 hours, the percentage of aromatic C increased from 14 to 24%, whereas that of O-alkyl C decreased from 29 to 20%. The proportion of carbonyl C was relatively constant throughout the treatments.

The transformation of C during the treatments at 180°C could be detected by applying the 13C-NMR method to our crude samples. The transformation occurred not only during the stabilization period but also during the initial drying period, but the functional groups affected by the transformation differed between the periods. These results indicate that the reactions in the drying period differed from those in the stabilization period. A slight decrease of carbonyl C from 19 to 17% during the stabilization period implied the occurrence of the Maillard reaction. However, it is uncertain to what extent the changes observed in the <sup>13</sup>C-NMR spectra were responsible for the different rates of mineralization of C between HD-180 (0 hour) and HD-180 (120 hours). The higher proportion of aromatic C in HD-180 (120 hours) may have contributed to the lower rate of C mineralization, because the aromatic polymers in plant residues and microbial products are regarded as more recalcitrant to biological decomposition [27].

Similar to our results, the heating of Susuki (*Miscanthus sinensis* A.) leaves at 250°C for 1 hour in a laboratory oven increased the percentage of aromatic C from 17 to 66% and decreased that of O-alkyl C from 70 to 15% [28]. Heating at 250°C burned the leaves. On the other hand, the 13C-NMR spectrum of the 200°C-heated samples remained unchanged from that of the untreated control. Heating of the leaves at 200°C for 1 hour did not cause burning but turned them brown possibly owing to the Maillard reaction.

As for sewage sludge, Fernández et al. [29] compared the properties of heat-dried sewage sludge with those of composted sludge. Sewage sludge that was heat-dried at the maximum sludge temperature of 75°C contained more fulvic acid than sewage sludge composted in windrows for 3 months. The solid-state 13C-NMR spectroscopy indicated that the humic acid fraction of the heat-dried sludge contained more alkyl C and less aromatic C than that of the composted sludge. These results indicated that heat-drying of sewage sludge at 75°C (sludge temperature) did not increase the stability of C as much as did windrow composting. In their study, the heat-drying was carried out by indirect convection with air heated between 380 and 450°C. The temperature of the sludge during the treatment (<75°C) was therefore much lower than the hot air supplied for drying. This reemphasizes the importance of the temperature of sewage sludge during the drying period as a factor that affects the quality of the end-product.

## **6. Conclusions**

**Figure 16.** Solid-state 13C-NMR (CP/MAS-TOSS, 75.45 MHz) spectra of sewage sludge samples. The 13C-NMR spectrum was divided into the four chemical shift regions: 0–45 ppm (alkyl C), 45–110 ppm (O-alkyl C), 110–165 ppm (aromatic C), and 165–210 ppm (carbonyl C). The values in the spectra indicate the relative percentage of each type of C calculated

C and N was determined not only by its chemical solubility but also by other physical factors

We also analyzed the C forms in AD, HD-180 (0 hour), and HD-180 (120 hours) by the solid-state 13C nuclear magnetic resonance (13C-NMR) spectroscopy. The 13C-NMR method provides estimates of the relative percentages of the main functional groups of C. The analytical procedures and machine conditions were the same as those described by Hiradate et al. [25]. When our samples were analyzed without any pretreatment, the 13C-NMR spectra were divided into the four regions. According to Hiradate et al. [25], these regions were assigned to the functional groups as follows (**Figure 16**); alkyl C (0–45 ppm), O-alkyl C

In AD, the percentage of alkyl C plus O-alkyl C exceeded 60%. Fernández et al. [26] also reported the predominance of these functional groups (75%) in sewage sludge heat-dried at 70–80°C. During the drying period from AD to HD-180 (0 hour), the percentage of alkyl

(45–110 ppm), aromatic C (110–165 ppm), and carbonyl C (165–210 ppm).

by integrating the signal intensity.

such as microbial accessibility.

68 Nitrogen in Agriculture - Updates

**5.3. Analysis of the functional groups of C by 13C-NMR**

By reviewing literature, we suggested that the heat-induced changes in N mineralization differ between soils and organic wastes. As the heating temperature increased to 200°C, the rate of N mineralization increased in soils but decreased in organic wastes. The rate of N mineralization from organic wastes tended to decrease sharply when heating temperatures increased to 150–200°C. Since the materials examined, heating conditions and analytical methods differed among the researchers, our findings may have been biased by these artifacts. More comparative studies are required to confirm these findings and reveal the processes involved. Furthermore, the results obtained from heat-dried sewage sludge indicated that the solidstate 13C-NMR spectroscopy can be a powerful tool to characterize the heat-induced stabilization of sludge C in addition to the incubation and extraction methods that have been used by soil scientists.

[4] Daikuhara, G. (1901) Studies on heated soil fertilizer. *The Bulletin of the Imp. Agri. Exp.* 

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[11] Murata, K., Sonoda, T. (1934) Influence of thermal changes of nitrogenous compounds in oil-cake during the production on the fertilizer values. *Jpn. J. Soil Sci. Plant Nutr*. **8**,

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[15] Moritsuka, N., Matsuoka, K., Matsumoto, S., Masunaga, T., Matsui, K., Wakatsuki, T. (2006) Effects of the application of heated sewage sludge on soil nutrient supply to

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http://dx.doi.org/10.5772/68163

ing temperature of biosolids reduced nitrogen mineralization and soil N2

*Environ. Sci. Pollut. Res*. **23**, 14383-14392. DOI: 10.1007/s11356-016-6607-3

*Stat. Jpn*. **20**, 1-46. (In Japanese).

203-221, InTech, Croatia. DOI: 10.5772/62526

**52**, 519-527. DOI: 10.1111/j.1747-0765.2006.00061.x

1-48. (In Japanese with an English summary).

10.1080/00380768.1992.10416960

Japanese).

37-38. (In Japanese).

DOI: 10.1002/clen.201100681

## **Acknowledgements**

We thank Dr. Noriko Iwashima (Shimane University) for the analysis of mineralization of C by the incubation method and Dr. Syuntaro Hiradate (Institute for Agro-Environmental Sciences, NARO) for the analysis of C forms by the solid-state 13C-NMR spectroscopy. We also thank Dr. Toshiyuki Wakatsuki, Dr. Tsugiyuki Masunaga, Dr. Shingo Matsumoto, and Dr. Hisao Nakano; the faculty members of Shimane University who gave us valuable comments and technical supports during our research activities at Shimane University. This work was supported by the JSPS KAKENHI Grant Number JP15K00634.

## **Author details**

Naoki Moritsuka1 \* and Kaori Matsuoka2


## **References**


[4] Daikuhara, G. (1901) Studies on heated soil fertilizer. *The Bulletin of the Imp. Agri. Exp. Stat. Jpn*. **20**, 1-46. (In Japanese).

to 150–200°C. Since the materials examined, heating conditions and analytical methods differed among the researchers, our findings may have been biased by these artifacts. More comparative studies are required to confirm these findings and reveal the processes involved. Furthermore, the results obtained from heat-dried sewage sludge indicated that the solidstate 13C-NMR spectroscopy can be a powerful tool to characterize the heat-induced stabilization of sludge C in addition to the incubation and extraction methods that have been used by

We thank Dr. Noriko Iwashima (Shimane University) for the analysis of mineralization of C by the incubation method and Dr. Syuntaro Hiradate (Institute for Agro-Environmental Sciences, NARO) for the analysis of C forms by the solid-state 13C-NMR spectroscopy. We also thank Dr. Toshiyuki Wakatsuki, Dr. Tsugiyuki Masunaga, Dr. Shingo Matsumoto, and Dr. Hisao Nakano; the faculty members of Shimane University who gave us valuable comments and technical supports during our research activities at Shimane University. This work was

[1] Bennamoun, L., Arlabosse, P., Léonard, A. (2013) Review on fundamental aspect of application of drying process to wastewater sludge. *Renew. Sust. Energ. Rev*. **28**, 29-43.

[2] Rigby, H., Clarke, B. O., Pritchard, D. L., Meehan, B., Beshah, F., Smith, S. R., Porter, N. A. (2016) A critical review of nitrogen mineralization in biosolids-amended soil, associated fertilizer value for crop production and potential for emissions to the environment.

[3] Smith, S. R., Durham, E. (2002) Nitrogen release and fertilizer value of thermally-dried biosolids. *Water Environ. J*. **16**, 121-126. DOI: 10.1111/j.1747-6593.2002.tb00382.x

*Sci. Total Environ*. **541**, 1310-1338. DOI: 10.1016/j.scitotenv.2015.08.089

supported by the JSPS KAKENHI Grant Number JP15K00634.

\* and Kaori Matsuoka2

DOI: 10.1016/j.rser.2013.07.043

\*Address all correspondence to: morituka@kais.kyoto-u.ac.jp

1 Graduate School of Agriculture, Kyoto University, Kyoto, Japan 2 Institute of Fruit Tree and Tea Science, NARO, Tsukuba, Japan

soil scientists.

**Acknowledgements**

70 Nitrogen in Agriculture - Updates

**Author details**

Naoki Moritsuka1

**References**


[18] Corrêa, R. S., White, R. E., Weatherley, A. J. (2012) Effects of sewage sludge stabilization on organic-N mineralization in two soils. *Soil Use Manage*. **28**, 12-18. DOI: 10.1111/j. 1475-2743.2012.00387.x

**Chapter 5**

),

**Provisional chapter**

**Nitrogen-Fixation by Endophytic Bacteria in**

**Nitrogen-Fixation by Endophytic Bacteria in** 

DOI: 10.5772/intechopen.71988

Endophytic bacteria represents a unique class of bacteria that can colonize interior tissues of plant and provide a range of benefits to the plant similar to those provided by the rhizospheric bacteria. Certain endophytic bacteria can provide nitrogen to the plants through biological nitrogen fixation, which is an important source of nitrogen input in agriculture and represents a promising substitute for chemical fertilizers, and are known as endophytic diazotrophic bacteria. Besides fixing nitrogen, endophytic bacteria can produce plant growth hormones like auxin and gibberellin, help in nutrient uptake, and increase the plant's tolerance to biotic and abiotic stresses. Various direct and indirect methods have been used to quantify the amount of nitrogen fixed by these bacteria, including the acetylene reduction assay, which is a quick but indirect method, and the 15N isotopic dilution assay, which is a robust and accurate method. Research on endophytic diazotrophic bacteria has come a long way, and in this chapter, we have briefly discussed the mechanisms of biological nitrogen fixation and methods to quantify the fixed nitrogen along with reviewing recent studies focused on evaluating the role of endophytic diazotrophic bacteria in promoting plant growth in both native and nonnative crop hosts. **Keywords:** endophytic bacteria, diazotroph, biological nitrogen fixation, plant growth

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

© 2018 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

Nitrogen (N) is an essential component of all proteins and enzymes, nucleic acids that make up DNA, and chlorophyll that enables the process of photosynthesis in plants [1]. It is a very common element in nature that is present in abundant amounts in atmosphere, lithosphere, and hydrosphere of the earth [2]. However, much of this N is in the form of dinitrogen (N<sup>2</sup>

which is inert and cannot be used by plants. In order for plants to use this dinitrogen, it has

**Agricultural Crops: Recent Advances**

**Agricultural Crops: Recent Advances**

Akshit Puri, Kiran Preet Padda and Chris P. Chanway

Akshit Puri, Kiran Preet Padda and Chris P. Chanway

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.71988

promotion, agricultural crops

**1. Introduction**

**Abstract**


**Provisional chapter**

## **Nitrogen-Fixation by Endophytic Bacteria in Agricultural Crops: Recent Advances Agricultural Crops: Recent Advances**

**Nitrogen-Fixation by Endophytic Bacteria in** 

DOI: 10.5772/intechopen.71988

Akshit Puri, Kiran Preet Padda and Chris P. Chanway Additional information is available at the end of the chapter

Akshit Puri, Kiran Preet Padda and Chris P. Chanway

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.71988

#### **Abstract**

[18] Corrêa, R. S., White, R. E., Weatherley, A. J. (2012) Effects of sewage sludge stabilization on organic-N mineralization in two soils. *Soil Use Manage*. **28**, 12-18. DOI: 10.1111/j.

[19] Maki, H., Kono, S., Nagai, K. (2009) Change in nutrient content and solubility by carbonization or heat treatment of cattle manure. *Jpn. J. Soil Sci. Plant Nutr*. **80**, 257-262. (In

[20] Collard, M., Teychené, B., Lemée, L. (in press) Comparison of three different wastewater sludge and their respective drying processes: Solar, thermal and reed beds – Impact on

[21] Yoshida, H., Nielsen, M. P., Scheutz, C., Jensen, L. S., Christensen, T. H., Nielsen, S., Bruun, S. (2015) Effects of sewage sludge stabilization on fertilizer value and greenhouse gas emissions after soil application. *Acta Agri. Scand. B*. **65**, 506-516. DOI: 10.1080/090647

[22] Ames, J. M. (1990) Control of the Maillard reaction in food systems. *Trends Food Sci.* 

[23] Moritsuka, N., Matsuoka, K., Katsura, K., Sano, S., Yanai, J. (2014) Soil color analysis for statistically estimating total carbon, total nitrogen and active iron contents in Japanese agricultural soils. *Soil Sci. Plant Nutr*. **60**, 475-485. DOI: 10.1080/00380768.2014.906295 [24] Kumada, K., Sato, O., Ohsumi, Y., Ohta, S. (1967) Humus composition of mountain soils in Central Japan with special reference to the distribution of P type humic acid. *Soil Sci.* 

[25] Hiradate, S., Nakadai, T., Shindo, H., Yoneyama, T. (2004) Carbon source of humic substances in some Japanese volcanic ash soils determined by carbon stable isotopic ratio,

[26] Fernández, J. M., Plaza, C., Polo, A., Plante, A. (2012) Use of thermal analysis techniques (TG-DSC) for the characterization of diverse organic municipal wastes streams to predict biological stability prior to land application. *Waste Manage*. **32**, 158-164. DOI:10.1016/

[27] Lützow, M. V., Kögel-Knabner, I., Ekschmitt, K., Matzner, E., Guggenberger, G., Marschner, B., Flessa, H. (2006) Stabilization of organic matter in temperate soils: mechanisms and their relevance under different soil conditions – a review. *Euro. J. Soil Sci*. **57**, 426-445. DOI:

[28] Honma, H., Hiradate, S., Shindo, H. (1999) Analysis of the burning process of Susuki (*Miscanthus sinensis* A.) plants by 13C-NMR, FT-IR and XRD. *Jpn. J. Soil Sci. Plant Nutr*. **70**,

[29] Fernández, J. M., Hockaday, W. C., Plaza, C., Polo, A., Hatcher, P. (2008) Effects of longterm soil amendment with sewage sludges on soil humic acid thermal and molecular

properties. *Chemosphere*. **73**, 1838-1844. Doi:10.1016/j.chemosphere.2008.08.001

organic characteristics. *J. Env. Manage*. DOI: 10.1016/j.jenvman.2016.05.070

*Technol*. **1**, 150-154. DOI: 10.1016/0924-2244(90)90113-D

*Plant Nutr*. **13**, 151-158. DOI: 10.1080/00380768.1967.10431990

δ13C. *Geoderma*, **119**, 133-141. DOI:10.1016/S0016-7061(03)00257-X

1475-2743.2012.00387.x

72 Nitrogen in Agriculture - Updates

10.2015.1027730

S0016-7061(03)00257-X

10.1111/j.1365-2389.2006.00809.x

277-282. (In Japanese with an English summary).

Japanese with an English summary).

Endophytic bacteria represents a unique class of bacteria that can colonize interior tissues of plant and provide a range of benefits to the plant similar to those provided by the rhizospheric bacteria. Certain endophytic bacteria can provide nitrogen to the plants through biological nitrogen fixation, which is an important source of nitrogen input in agriculture and represents a promising substitute for chemical fertilizers, and are known as endophytic diazotrophic bacteria. Besides fixing nitrogen, endophytic bacteria can produce plant growth hormones like auxin and gibberellin, help in nutrient uptake, and increase the plant's tolerance to biotic and abiotic stresses. Various direct and indirect methods have been used to quantify the amount of nitrogen fixed by these bacteria, including the acetylene reduction assay, which is a quick but indirect method, and the 15N isotopic dilution assay, which is a robust and accurate method. Research on endophytic diazotrophic bacteria has come a long way, and in this chapter, we have briefly discussed the mechanisms of biological nitrogen fixation and methods to quantify the fixed nitrogen along with reviewing recent studies focused on evaluating the role of endophytic diazotrophic bacteria in promoting plant growth in both native and nonnative crop hosts.

**Keywords:** endophytic bacteria, diazotroph, biological nitrogen fixation, plant growth promotion, agricultural crops

## **1. Introduction**

Nitrogen (N) is an essential component of all proteins and enzymes, nucleic acids that make up DNA, and chlorophyll that enables the process of photosynthesis in plants [1]. It is a very common element in nature that is present in abundant amounts in atmosphere, lithosphere, and hydrosphere of the earth [2]. However, much of this N is in the form of dinitrogen (N<sup>2</sup> ), which is inert and cannot be used by plants. In order for plants to use this dinitrogen, it has

Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons

to be reduced/fixed into forms like nitrate (NO<sup>3</sup> − ) and ammonium (NH<sup>4</sup> + ). N fixation, the process by which dinitrogen is reduced to plant-available forms, is, therefore, a vital process for the sustenance of life on earth. A major industrial process by which dinitrogen is converted into ammonia is known as the Haber-Bosch process. This artificial N-fixation process was established in 1913 and uses a catalyst (iron with a small amount of aluminum added) at high pressure (as much as 5.06 × 10<sup>7</sup> Pa) and high temperature (600–800 K) consuming large amounts of fossil fuel. Ammonia produced through this highly expensive process is combined with other elements to produce nitrogenous fertilizers like urea and ammonium nitrate. Although the use of these fertilizers is inevitable in meeting rising food demand to sustain the growing global population, their indiscriminate use has set off very negative effects on the environment [3]. Naturally, N is commonly fixed by two processes. The first is atmospheric N fixation by lightning, in which the enormous amount of energy contained in lightning breaks dinitrogen molecules and enables their atoms to combine with oxygen in the air forming N oxides that dissolve in rain. These oxides of N then form nitrates that are carried to the earth in rainfall [4]. The second is biological N fixation (BNF), in which certain prokaryotic microorganisms, known as diazotrophs, fix N by breaking down the triple bond of dinitrogen using a highly specialized enzyme complex called nitrogenase enzyme and convert it to ammonia [4]. This chapter mainly focuses on diazotrophic bacteria that can fix N while living in the internal tissues of plants. In this chapter, only recent developments (from last 5 years) related to this subject have been discussed.

and the FeMo cofactor. The dinitrogenase reductase protein is a homodimer of two identical subunits, with an overall molecular mass of ~60 kDa. It contains two ATP/ADP molecules and

The overall functioning of nitrogenase can be summarized as a key biochemical cycle that involves five steps [6, 7]: (i) the reduction of Fe protein by electron carriers such as flavodoxin or ferredoxin; (ii) association of the reduced Fe protein (including two MgATP complexes) with the Mo-Fe protein in preparation for electron transfer; (iii) hydrolysis of MgATP, which

transfer to dinitrogen and thus its reduction, while it is bound to the active site within the Mo-Fe protein; and (v) dissociation of the two protein molecules, exchange of ATP back into

The structure and function of nitrogenase enzyme are encoded by ~20 genes, known as N-fixation genes (*nif* genes), organized in 7 operons (*nif* cluster) spanning over 24 kb. These genes fall into three categories, structural, regulatory, and supplementary, and can be housed either in genomic DNA or on plasmids. The Fe protein is encoded by the *nifH* gene and the Mo-Fe protein is encoded by *nifD* and *nifK* genes [8, 9]. The *nifD*, *nifH*, and *nifK* genes are recognized as structural *nif* genes since they are responsible for encoding the aforementioned structural subunits [10]. The *nif* cluster of the free-living bacterium *Klebsiella pneumoniae* is the most studied of *nif* genes and serves as a model for understanding the regulation, synthesis,

BNF can be measured using various methods, the most common being: N balance method, xylem solute analysis, acetylene reduction assay, and stable isotope (<sup>15</sup>N) method [12]. In the N balance method, the amount of N fixed is estimated by calculating the difference between total N content of plants inoculated by diazotrophs and those that are not inoculated. In this method, it is assumed that both inoculated and noninoculated plants absorb equal amounts of N from the soil, which is hard to justify as there are differences in root morphology and physiological attributes [12]. In the xylem solute analysis, the composition of N compounds flowing through the xylem sap to the shoot of the plant is determined. The N absorbed by plants from the soil is predominantly nitrate, whereas the fixed N is primarily in the form of amides and ureides [13]. This difference in composition of N compounds is used to make quantitative measurements of N fixation [14]. However, its major disadvantage is that only a very small proportion of N-fixing plants export fixed N in the form of ureides [15]. The acetylene reduction assay is a popular technique used to indirectly measure BNF by estimating the nitrogenase enzyme activity. It is based on the ability of nitrogenase to reduce acetylene (H─C≡C─H) to ethylene by breaking the triple bond between carbon atoms. Samples are incubated in a gas-tight chamber and a portion of the head space is injected with acetylene. After incubation, gas samples are collected from the chamber and analyzed for ethylene production using gas chromatography [16]. It is a simple, low cost, and sensitive assay that can measure BNF in bacterial cultures, detached nodules, plant parts, or even whole plants.

S4

Nitrogen-Fixation by Endophytic Bacteria in Agricultural Crops: Recent Advances

and the P-cluster); (iv) electron

http://dx.doi.org/10.5772/intechopen.71988

75

one Fe<sup>4</sup>


cluster [6, 7].

enables transfer of one electron to the Mo-Fe protein (via Fe<sup>4</sup>

the Fe protein, and rereduction of the Fe protein.

and assembly of nitrogenase enzyme [11].

**2.2. Quantification of biologically fixed N**

## **2. Biological nitrogen fixation (BNF)**

Farmers since ancient Chinese and Roman civilizations practiced crop rotation with legumes to increase soil fertility and agricultural productivity. However, the science behind such practice was first revealed by Boussingault in 1838, who established that legumes can fix N. But it was not until 1886 when Hellriegel and Wilfarth provided a firm evidence that microbes are responsible for N fixation occurring in leguminous plants [5].

#### **2.1. Chemistry and genetics of BNF**

The overall chemical reaction of BNF catalyzed by the nitrogenase enzyme is represented below:

$$\rm N\_{2} + 8H^{+} + 8e^{-} + 16MgATP \rightarrow \rm 2NH\_{3} + H\_{2} + 16MgADP + 16Pi \tag{1}$$

Nitrogenase is a complex enzyme comprised of two metalloproteins: the Mo-Fe protein, also called dinitrogenase protein, and the Fe protein, also called dinitrogenase reductase protein. The dinitrogenase protein is a heterotetramer composed of two α- and two β-subunits with an overall molecular weight of 240kDa. This protein contains two types of metal centers, the FeMo-cofactor and the P-cluster pair, of which the FeMo cofactor is the active site where dinitrogen binds, whereas the P-cluster mediates electron transfer between the Fe protein and the FeMo cofactor. The dinitrogenase reductase protein is a homodimer of two identical subunits, with an overall molecular mass of ~60 kDa. It contains two ATP/ADP molecules and one Fe<sup>4</sup> -S4 cluster [6, 7].

The overall functioning of nitrogenase can be summarized as a key biochemical cycle that involves five steps [6, 7]: (i) the reduction of Fe protein by electron carriers such as flavodoxin or ferredoxin; (ii) association of the reduced Fe protein (including two MgATP complexes) with the Mo-Fe protein in preparation for electron transfer; (iii) hydrolysis of MgATP, which enables transfer of one electron to the Mo-Fe protein (via Fe<sup>4</sup> S4 and the P-cluster); (iv) electron transfer to dinitrogen and thus its reduction, while it is bound to the active site within the Mo-Fe protein; and (v) dissociation of the two protein molecules, exchange of ATP back into the Fe protein, and rereduction of the Fe protein.

The structure and function of nitrogenase enzyme are encoded by ~20 genes, known as N-fixation genes (*nif* genes), organized in 7 operons (*nif* cluster) spanning over 24 kb. These genes fall into three categories, structural, regulatory, and supplementary, and can be housed either in genomic DNA or on plasmids. The Fe protein is encoded by the *nifH* gene and the Mo-Fe protein is encoded by *nifD* and *nifK* genes [8, 9]. The *nifD*, *nifH*, and *nifK* genes are recognized as structural *nif* genes since they are responsible for encoding the aforementioned structural subunits [10]. The *nif* cluster of the free-living bacterium *Klebsiella pneumoniae* is the most studied of *nif* genes and serves as a model for understanding the regulation, synthesis, and assembly of nitrogenase enzyme [11].

## **2.2. Quantification of biologically fixed N**

to be reduced/fixed into forms like nitrate (NO<sup>3</sup>

74 Nitrogen in Agriculture - Updates

subject have been discussed.

**2. Biological nitrogen fixation (BNF)**

**2.1. Chemistry and genetics of BNF**

below:

responsible for N fixation occurring in leguminous plants [5].

−

cess by which dinitrogen is reduced to plant-available forms, is, therefore, a vital process for the sustenance of life on earth. A major industrial process by which dinitrogen is converted into ammonia is known as the Haber-Bosch process. This artificial N-fixation process was established in 1913 and uses a catalyst (iron with a small amount of aluminum added) at high pressure (as much as 5.06 × 10<sup>7</sup> Pa) and high temperature (600–800 K) consuming large amounts of fossil fuel. Ammonia produced through this highly expensive process is combined with other elements to produce nitrogenous fertilizers like urea and ammonium nitrate. Although the use of these fertilizers is inevitable in meeting rising food demand to sustain the growing global population, their indiscriminate use has set off very negative effects on the environment [3]. Naturally, N is commonly fixed by two processes. The first is atmospheric N fixation by lightning, in which the enormous amount of energy contained in lightning breaks dinitrogen molecules and enables their atoms to combine with oxygen in the air forming N oxides that dissolve in rain. These oxides of N then form nitrates that are carried to the earth in rainfall [4]. The second is biological N fixation (BNF), in which certain prokaryotic microorganisms, known as diazotrophs, fix N by breaking down the triple bond of dinitrogen using a highly specialized enzyme complex called nitrogenase enzyme and convert it to ammonia [4]. This chapter mainly focuses on diazotrophic bacteria that can fix N while living in the internal tissues of plants. In this chapter, only recent developments (from last 5 years) related to this

Farmers since ancient Chinese and Roman civilizations practiced crop rotation with legumes to increase soil fertility and agricultural productivity. However, the science behind such practice was first revealed by Boussingault in 1838, who established that legumes can fix N. But it was not until 1886 when Hellriegel and Wilfarth provided a firm evidence that microbes are

The overall chemical reaction of BNF catalyzed by the nitrogenase enzyme is represented

N2 + 8H<sup>+</sup> + 8e− + 16MgATP → 2NH3 + H2 + 16MgADP + 16Pi (1)

Nitrogenase is a complex enzyme comprised of two metalloproteins: the Mo-Fe protein, also called dinitrogenase protein, and the Fe protein, also called dinitrogenase reductase protein. The dinitrogenase protein is a heterotetramer composed of two α- and two β-subunits with an overall molecular weight of 240kDa. This protein contains two types of metal centers, the FeMo-cofactor and the P-cluster pair, of which the FeMo cofactor is the active site where dinitrogen binds, whereas the P-cluster mediates electron transfer between the Fe protein

) and ammonium (NH<sup>4</sup>

+

). N fixation, the pro-

BNF can be measured using various methods, the most common being: N balance method, xylem solute analysis, acetylene reduction assay, and stable isotope (<sup>15</sup>N) method [12]. In the N balance method, the amount of N fixed is estimated by calculating the difference between total N content of plants inoculated by diazotrophs and those that are not inoculated. In this method, it is assumed that both inoculated and noninoculated plants absorb equal amounts of N from the soil, which is hard to justify as there are differences in root morphology and physiological attributes [12]. In the xylem solute analysis, the composition of N compounds flowing through the xylem sap to the shoot of the plant is determined. The N absorbed by plants from the soil is predominantly nitrate, whereas the fixed N is primarily in the form of amides and ureides [13]. This difference in composition of N compounds is used to make quantitative measurements of N fixation [14]. However, its major disadvantage is that only a very small proportion of N-fixing plants export fixed N in the form of ureides [15]. The acetylene reduction assay is a popular technique used to indirectly measure BNF by estimating the nitrogenase enzyme activity. It is based on the ability of nitrogenase to reduce acetylene (H─C≡C─H) to ethylene by breaking the triple bond between carbon atoms. Samples are incubated in a gas-tight chamber and a portion of the head space is injected with acetylene. After incubation, gas samples are collected from the chamber and analyzed for ethylene production using gas chromatography [16]. It is a simple, low cost, and sensitive assay that can measure BNF in bacterial cultures, detached nodules, plant parts, or even whole plants. The major disadvantage is the short-term nature of the assay and the autoinhibition of acetylene conversion to ethylene [17]. The stable isotope method using <sup>15</sup>N is a widely used and accepted method. This method is based on the principle that soil has a noticeably different 15N to 14N ratio as compared to the atmosphere, which has a constant ratio (0.3663%). Therefore, plants absorbing fixed N from the atmosphere will have a different 15N to 14N ratio as compared to the ones absorbing N only from the soil. When plants inoculated with diazotrophs are grown in air labeled with <sup>15</sup>N, they are expected to have an enhanced ratio as compared to the noninoculated ones (15N incorporation method). When available soil N is labeled with <sup>15</sup>N, a reduction in the ratio is expected since the inoculated plants tend to incorporate fixed N from the air as compared to the noninoculated plants, which take up labeled N from the soil (15N isotope dilution method) [17].

activity only occurs in the rhizosphere soil but not in roots [35, 36]. However, later it was determined that rhizospheric N fixation does not occur at sufficient rates to facilitate high sugarcane yields. Cavalcante and Döbereiner [37] were the first to report the isolation of a diazotroph (*Gluconacetobacter diazotrophicus*) from internal tissues of a nonleguminous plant (stem and root tissues of sugarcane) and postulated that this bacterium might be involved in fixing high amounts of N biologically. This bacterium was able to multiply considerably and fix N at high sucrose concentrations [38] and in low pH conditions typically found in internal tissues of sugarcane [38, 39]. This led to the postulation that it can satisfy almost all of the sugarcane N requirements while living inside their tissues. Such bacteria that were able to multiply inside the tissues of a live plant and promote its growth through one or more mechanisms had already been discovered many years ago and are known as 'endophytic bacteria.'

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77

The term 'endophyte' was first coined more than 150 years ago by de Bary [40] for pathogenic fungi entering the internal tissues of leaves. Since then, many authors have redefined this term, but each has its own restrictions. Taken literally, the word endophyte means 'in the plant' (endon = within; phyton = plant) [41]. Since our main focus in this chapter is on 'endophytic bacteria,' we would like to reiterate the definition notated by Chanway et al. [42]: "bacteria that can be detected at a particular moment within the tissue of apparently healthy plant hosts without inducing disease or organogenesis are known as endophytic bacteria." The occurrence of endophytic bacteria in internal tissues was first reported inside a healthy potato plant [43]. Since then, many scientific studies have been focused on isolating the endophytic bacteria from a variety of plant species and evaluating their benefits for agricultural plants [44–47]. In contrast to free-living, rhizosphere or phyllosphere microorganisms, endophytic bacteria are better protected from abiotic stresses such as extreme variations in temperature, pH, nutrient, and water availability as well as biotic stresses such as competition [48–50]. In addition, endophytic bacteria colonize niches that are more conducive to forming mutualistic relationships with plants [51], for example, providing fixed N to the plant and getting photosynthate in return [52–54]. Following the rhizospheric colonization, endophytic bacteria can colonize various plant organs such as roots, stem, leaves, flowers, fruits, and seeds [55–61], indicating different capacities of endophytic bacteria to colonize various plant compartments. They can even colonize legume nodules [62] and tubercles of mycorrhizal fungi [63]. The endophytic bacterial population is extremely variable in different plant organs and tissues and have been shown to vary from as low as hundreds to as high as 10<sup>9</sup> cfu per gram plant

Localization of endophytic bacteria within plant tissues requires techniques that facilitate observation on a tiny spatial scale. Various methods have been used to locate bacteria *in planta* and visualize them at their sites of colonization, but each one has its own limitations. Most methods require either chemical or physical treatment of plant tissues for *in situ* detection and visualization of endophytic bacteria [68]. However, the use of autofluorescent proteins in conjunction with confocal laser scanning microscopy (CLSM) eliminates the need for any

**3. Endophytic bacteria**

tissue [64–67].

#### **2.3. N-fixing organisms**

The ability to fix N, in other words, the presence of nitrogenase enzyme, is only limited to certain bacteria and archaea [18]. Within these groups, it is quite widely distributed revealing considerable phylogenetic diversity among diazotrophs. A comprehensive list of N-fixing bacteria and archaea, under 12 broad phylogenetic groups based on 16S rDNA phylogeny was prepared by Young [19]. Diazotrophs are also widely distributed ecologically. They can be found living in soils and water freely, in the rhizosphere and phyllosphere and inside the plant tissues, in symbiotic association with legumes and actinorhizal association with woody plants, and in cyanobacterial symbiosis with phytoplankton, fungi, and terrestrial plants [19]. Free-living diazotrophs are those that do not associate with plants and are found in soils that are free from the direct influence of plant roots. These microorganisms are ubiquitous in terrestrial and aquatic environments and are physiologically very diverse [20]. Many diazotrophs can be found dwelling in the rhizosphere of a plant. Due to their ability to fix N, diazotrophs can have a competitive advantage over other microbes in the rhizosphere. They prevail in the rhizosphere particularly when soil N is limited [21]. The phyllosphere (leaf surface) is another microsite known to be colonized by diazotrophs [22]. The symbiotic association between legume and *Rhizobium* is a well-known mutualistic relationship involving *Leguminosae* plants and *Rhizobiaceae* bacteria [23]. This symbiosis has been studied widely from ecological, agronomic, and molecular biological perspectives not only to enhance the N-fixing efficacy of existing symbioses but also to determine if similar associations might be developed with nonleguminous plants [24, 25]. The actinorhizal association is functionally analogous to the legume and *Rhizobium* association but is restricted between a small group of woody plant species known as Actinorhizal plants and diazotrophs belonging to a genus, *Frankia* [26]. Many diazotrophic cyanobacteria also form symbiotic association with eukaryotes and are known to contribute a significant portion of N required for growth of both organisms through BNF in N-limited aquatic and terrestrial environments [27, 28].

The presence of diazotrophs in nonleguminous plants was first detected by Brazilian researchers in the rhizosphere and rhizoplane of sugarcane (*Saccharum officinarum*) [29, 30]. In subsequent studies, various diazotrophs like *Azospirillum lipoferum*, *Azospirillum amazonense*, *Bacillus azotofixans*, *Enterobacter cloacae*, *Erwinia herbicola*, and *Bacillus polymyxa* [31–34] were isolated from the rhizosphere of sugarcane. Initially, it was postulated that nitrogenase activity only occurs in the rhizosphere soil but not in roots [35, 36]. However, later it was determined that rhizospheric N fixation does not occur at sufficient rates to facilitate high sugarcane yields. Cavalcante and Döbereiner [37] were the first to report the isolation of a diazotroph (*Gluconacetobacter diazotrophicus*) from internal tissues of a nonleguminous plant (stem and root tissues of sugarcane) and postulated that this bacterium might be involved in fixing high amounts of N biologically. This bacterium was able to multiply considerably and fix N at high sucrose concentrations [38] and in low pH conditions typically found in internal tissues of sugarcane [38, 39]. This led to the postulation that it can satisfy almost all of the sugarcane N requirements while living inside their tissues. Such bacteria that were able to multiply inside the tissues of a live plant and promote its growth through one or more mechanisms had already been discovered many years ago and are known as 'endophytic bacteria.'

## **3. Endophytic bacteria**

The major disadvantage is the short-term nature of the assay and the autoinhibition of acetylene conversion to ethylene [17]. The stable isotope method using <sup>15</sup>N is a widely used and accepted method. This method is based on the principle that soil has a noticeably different 15N to 14N ratio as compared to the atmosphere, which has a constant ratio (0.3663%). Therefore, plants absorbing fixed N from the atmosphere will have a different 15N to 14N ratio as compared to the ones absorbing N only from the soil. When plants inoculated with diazotrophs are grown in air labeled with <sup>15</sup>N, they are expected to have an enhanced ratio as compared to the noninoculated ones (15N incorporation method). When available soil N is labeled with <sup>15</sup>N, a reduction in the ratio is expected since the inoculated plants tend to incorporate fixed N from the air as compared to the noninoculated plants, which take up labeled N from the soil

The ability to fix N, in other words, the presence of nitrogenase enzyme, is only limited to certain bacteria and archaea [18]. Within these groups, it is quite widely distributed revealing considerable phylogenetic diversity among diazotrophs. A comprehensive list of N-fixing bacteria and archaea, under 12 broad phylogenetic groups based on 16S rDNA phylogeny was prepared by Young [19]. Diazotrophs are also widely distributed ecologically. They can be found living in soils and water freely, in the rhizosphere and phyllosphere and inside the plant tissues, in symbiotic association with legumes and actinorhizal association with woody plants, and in cyanobacterial symbiosis with phytoplankton, fungi, and terrestrial plants [19]. Free-living diazotrophs are those that do not associate with plants and are found in soils that are free from the direct influence of plant roots. These microorganisms are ubiquitous in terrestrial and aquatic environments and are physiologically very diverse [20]. Many diazotrophs can be found dwelling in the rhizosphere of a plant. Due to their ability to fix N, diazotrophs can have a competitive advantage over other microbes in the rhizosphere. They prevail in the rhizosphere particularly when soil N is limited [21]. The phyllosphere (leaf surface) is another microsite known to be colonized by diazotrophs [22]. The symbiotic association between legume and *Rhizobium* is a well-known mutualistic relationship involving *Leguminosae* plants and *Rhizobiaceae* bacteria [23]. This symbiosis has been studied widely from ecological, agronomic, and molecular biological perspectives not only to enhance the N-fixing efficacy of existing symbioses but also to determine if similar associations might be developed with nonleguminous plants [24, 25]. The actinorhizal association is functionally analogous to the legume and *Rhizobium* association but is restricted between a small group of woody plant species known as Actinorhizal plants and diazotrophs belonging to a genus, *Frankia* [26]. Many diazotrophic cyanobacteria also form symbiotic association with eukaryotes and are known to contribute a significant portion of N required for growth of both organ-

isms through BNF in N-limited aquatic and terrestrial environments [27, 28].

The presence of diazotrophs in nonleguminous plants was first detected by Brazilian researchers in the rhizosphere and rhizoplane of sugarcane (*Saccharum officinarum*) [29, 30]. In subsequent studies, various diazotrophs like *Azospirillum lipoferum*, *Azospirillum amazonense*, *Bacillus azotofixans*, *Enterobacter cloacae*, *Erwinia herbicola*, and *Bacillus polymyxa* [31–34] were isolated from the rhizosphere of sugarcane. Initially, it was postulated that nitrogenase

(15N isotope dilution method) [17].

**2.3. N-fixing organisms**

76 Nitrogen in Agriculture - Updates

The term 'endophyte' was first coined more than 150 years ago by de Bary [40] for pathogenic fungi entering the internal tissues of leaves. Since then, many authors have redefined this term, but each has its own restrictions. Taken literally, the word endophyte means 'in the plant' (endon = within; phyton = plant) [41]. Since our main focus in this chapter is on 'endophytic bacteria,' we would like to reiterate the definition notated by Chanway et al. [42]: "bacteria that can be detected at a particular moment within the tissue of apparently healthy plant hosts without inducing disease or organogenesis are known as endophytic bacteria." The occurrence of endophytic bacteria in internal tissues was first reported inside a healthy potato plant [43]. Since then, many scientific studies have been focused on isolating the endophytic bacteria from a variety of plant species and evaluating their benefits for agricultural plants [44–47]. In contrast to free-living, rhizosphere or phyllosphere microorganisms, endophytic bacteria are better protected from abiotic stresses such as extreme variations in temperature, pH, nutrient, and water availability as well as biotic stresses such as competition [48–50]. In addition, endophytic bacteria colonize niches that are more conducive to forming mutualistic relationships with plants [51], for example, providing fixed N to the plant and getting photosynthate in return [52–54]. Following the rhizospheric colonization, endophytic bacteria can colonize various plant organs such as roots, stem, leaves, flowers, fruits, and seeds [55–61], indicating different capacities of endophytic bacteria to colonize various plant compartments. They can even colonize legume nodules [62] and tubercles of mycorrhizal fungi [63]. The endophytic bacterial population is extremely variable in different plant organs and tissues and have been shown to vary from as low as hundreds to as high as 10<sup>9</sup> cfu per gram plant tissue [64–67].

Localization of endophytic bacteria within plant tissues requires techniques that facilitate observation on a tiny spatial scale. Various methods have been used to locate bacteria *in planta* and visualize them at their sites of colonization, but each one has its own limitations. Most methods require either chemical or physical treatment of plant tissues for *in situ* detection and visualization of endophytic bacteria [68]. However, the use of autofluorescent proteins in conjunction with confocal laser scanning microscopy (CLSM) eliminates the need for any chemical treatment of plant tissues and requires minimal physical preparation of plant tissue samples before microscopic visualization. The green fluorescent protein (GFP) gene found in the jellyfish *Aequorea aequorea* is the most popular autofluorescent protein used for localization of endophytic bacteria. GFP is a useful biomarker because it does not require any substrate or cofactor in order to fluoresce. GFP cassettes can be integrated into the bacterial

chromosome and expressed through an inducible or constitutive promoter of indigenous or exogenous origin [69–72]. Alternatively, a plasmid-borne GFP gene can be introduced into bacterial cells of interest [73–75]. Bacterial cells expressing GFP can be visualized by epifluorescence microscopy or CLSM [76, 77]. This technique has been used with various agricultural crops including wheat (*Triticum* spp.) [78], rice (*Oryza sativa*) [78–80], corn (*Zea mays*) [78, 81], tomato (*Solanum lycopersicum*) [82], ryegrass (*Lolium multiflorum*) [83], creeping bentgrass

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79

A few years after the discovery of diazotrophs by Cavalcante and Döbereiner [37] in the stem and root tissues of sugarcane plant, Döbereiner [85] coined the term "endophytic diazotrophic bacteria" to designate all diazotrophs able to colonize primarily the root interior of graminaceous plants, survive very poorly in soil and fix N in association with these plants [86]. Since the discovery of endophytic diazotrophic bacteria in sugarcane, other agronomically important crop species like rice [87–89], corn [90–93], wheat [94], canola (*Brassica napus* L.) [95], and Kallar grass (*Leptochloa fusca* L.) [96] have been postulated to receive significant amounts of fixed N in this way. In the following section, recent studies (from last 5 years) about endophytic diazotrophic bacteria and their role in promoting the growth of agricultural crops primarily by providing N nutrition as a result of BNF and secondarily through other plant growth–promotion (PGP) mechanisms have been discussed

**4. Recent studies highlighting the role of endophytic diazotrophic** 

Rice is a major staple crop in many countries around the world. It is a highly N-demanding crop; thus, it becomes extremely important to find alternatives to reduce the use of chemical N fertilizers applied to rice without decreasing the productivity. Endophytic diazotrophic strains were isolated from root, culm, and leaf tissues of traditional rice varieties (Zebu Branco and Manteiga) cultivated traditionally by the local farmers in the Maranhão state, Brazil [97]. Ten strains showing consistent acetylene reduction activity and capable of producing indole-3-acetic acid (IAA) were identified as belonging to the genera *Azospirillum*, *Sphingomonas*, and *Burkholderia*. These endophytic diazotrophic strains were inoculated into 10 different traditional varieties of rice to select the best strain/rice variety interaction by growing them in gnotobiotic, greenhouse, and field conditions. Although a strain belonging to the genus *Azospirillum* showed highest biomass enhancement (48%) under gnotobiotic conditions, *Burkholderia vietnamiensis* strain AR1122 inoculated into a traditional variety Arroz 70 showed best results as compared to other strain/variety combinations when grown under greenhouse and field conditions. The grain yield of Arroz 70 variety was also significantly enhanced when inoculated with the strain AR1122 in comparison to a control treatment that was provided with sufficient amounts of N fertilizer. These results clearly indicate that *Burkholderia vietnamiensis* strain AR1122 is a candidate biofertilizer for traditional rice varieties in Brazil and

(*Agrostis stolonifera*) [84], and grapevine (*Vitis vinifera*) [72].

**3.1. Endophytic diazotrophic bacteria**

in detail (listed in **Table 1** as well).

**bacteria in agricultural crops**


**Table 1.** List of endophytic diazotrophic bacteria recently isolated and associated with agricultural crops.

chromosome and expressed through an inducible or constitutive promoter of indigenous or exogenous origin [69–72]. Alternatively, a plasmid-borne GFP gene can be introduced into bacterial cells of interest [73–75]. Bacterial cells expressing GFP can be visualized by epifluorescence microscopy or CLSM [76, 77]. This technique has been used with various agricultural crops including wheat (*Triticum* spp.) [78], rice (*Oryza sativa*) [78–80], corn (*Zea mays*) [78, 81], tomato (*Solanum lycopersicum*) [82], ryegrass (*Lolium multiflorum*) [83], creeping bentgrass (*Agrostis stolonifera*) [84], and grapevine (*Vitis vinifera*) [72].

#### **3.1. Endophytic diazotrophic bacteria**

chemical treatment of plant tissues and requires minimal physical preparation of plant tissue samples before microscopic visualization. The green fluorescent protein (GFP) gene found in the jellyfish *Aequorea aequorea* is the most popular autofluorescent protein used for localization of endophytic bacteria. GFP is a useful biomarker because it does not require any substrate or cofactor in order to fluoresce. GFP cassettes can be integrated into the bacterial

**Isolated from Colonized into Method used to** 

Wheat (*Triticum aestivum* L.)

Rice (*Oryza sativa* L.) Rice (*Oryza sativa* L.) Acetylene reduction

Rice (*Oryza sativa* var.

Corn (*Zea mays* L.) Corn (*Zea mays* L.) Amplification of

Sugarcane (*Saccharum* 

Rice (*Oryza sativa*) Rice (*Oryza sativa*) Acetylene reduction

Corn (*Zea mays*), canola (*Brassica napus* L.), tomato (*Solanum lycopersicum*)

*officinarum*)

*Japonica*)

**confirm N-fixing** 

Amplification of *nifH* genes; acetylene reduction assay

Amplification of *nifH*

*nifH* genes; acetylene reduction assay

*nifH* genes; acetylene reduction assay

Kjeldahl method; natural abundance of <sup>15</sup>N in leaf samples; isotopic 15N dilution

assay

Amplification of *nifH* genes; acetylene reduction assay; isotopic 15N dilution

**References**

[104]

[97]

[106]

[103]

[102]

[98]

[105]

[109, 113, 117, 119]

**ability**

assay

genes

— Amplification of

**Endophytic diazotrophic** 

78 Nitrogen in Agriculture - Updates

*Azospirillum amazonense* AR3122; *Burkholderia vietnamiensis* AR

*Paenibacillus kribbensis* HS-R01,

*Bacillus aryabhattai* HS-S05; *Bacillus megaterium* KW7-R08; *Klebsiella pneumoniae* KW7-S06, KW7-S22, KW7-S27, KW7- S33; *Bacillus subtilis* CB-R05; *Microbacterium binotii* CB-S18; *Microbacterium trichotecenolyticum* SW521-L21, SW521-L37;

*Bacillus subtilis* EB-04; *Bacillus pumilus* EB-64, EB-169; *Paenibacillus* sp. EB-144

*Bacillus* sp. CNPSo 2476, CNPSo 2477, CNPSo 2478; *Enterobacter*

*Gluconacetobacter diazotrophicus* Pal5T-BR11281; *Amazon Azospirillum* Cbamc-BR11145; *Herbaspirillum seropedicae* HRC54-BR11335;

*Herbaspirillum rubrisubalbicans* HCC103-BR11504; *Burkholderia tropica* PPe8T-BR11366

*Burkholderia* spp.; *Klebsiella* spp.; *Novosphingobium* spp.; *Sphingomonas* spp.

*Paenibacillus polymyxa* P2b-2R Lodgepole pine

sp. CNPSo 2480

*Pseudomonas aeruginosa* PM389 Pearl millet

(*Pennisetum glaucum*)

Rice (*Oryza sativa* var. *Japonica*)

Banana tree cultivar 'Prata Anã' (*Musa acuminata × balbisiana*)

Sugarcane (*Saccharum officinarum*)

(*Pinus contorta var.* 

**Table 1.** List of endophytic diazotrophic bacteria recently isolated and associated with agricultural crops.

*latifolia*)

**bacteria**

1122;

HS-R14;

A few years after the discovery of diazotrophs by Cavalcante and Döbereiner [37] in the stem and root tissues of sugarcane plant, Döbereiner [85] coined the term "endophytic diazotrophic bacteria" to designate all diazotrophs able to colonize primarily the root interior of graminaceous plants, survive very poorly in soil and fix N in association with these plants [86]. Since the discovery of endophytic diazotrophic bacteria in sugarcane, other agronomically important crop species like rice [87–89], corn [90–93], wheat [94], canola (*Brassica napus* L.) [95], and Kallar grass (*Leptochloa fusca* L.) [96] have been postulated to receive significant amounts of fixed N in this way. In the following section, recent studies (from last 5 years) about endophytic diazotrophic bacteria and their role in promoting the growth of agricultural crops primarily by providing N nutrition as a result of BNF and secondarily through other plant growth–promotion (PGP) mechanisms have been discussed in detail (listed in **Table 1** as well).

## **4. Recent studies highlighting the role of endophytic diazotrophic bacteria in agricultural crops**

Rice is a major staple crop in many countries around the world. It is a highly N-demanding crop; thus, it becomes extremely important to find alternatives to reduce the use of chemical N fertilizers applied to rice without decreasing the productivity. Endophytic diazotrophic strains were isolated from root, culm, and leaf tissues of traditional rice varieties (Zebu Branco and Manteiga) cultivated traditionally by the local farmers in the Maranhão state, Brazil [97]. Ten strains showing consistent acetylene reduction activity and capable of producing indole-3-acetic acid (IAA) were identified as belonging to the genera *Azospirillum*, *Sphingomonas*, and *Burkholderia*. These endophytic diazotrophic strains were inoculated into 10 different traditional varieties of rice to select the best strain/rice variety interaction by growing them in gnotobiotic, greenhouse, and field conditions. Although a strain belonging to the genus *Azospirillum* showed highest biomass enhancement (48%) under gnotobiotic conditions, *Burkholderia vietnamiensis* strain AR1122 inoculated into a traditional variety Arroz 70 showed best results as compared to other strain/variety combinations when grown under greenhouse and field conditions. The grain yield of Arroz 70 variety was also significantly enhanced when inoculated with the strain AR1122 in comparison to a control treatment that was provided with sufficient amounts of N fertilizer. These results clearly indicate that *Burkholderia vietnamiensis* strain AR1122 is a candidate biofertilizer for traditional rice varieties in Brazil and should be investigated with other genotypes of rice for a sustainable rice crop production. In Brazil, sugarcane has been one of the fastest growing crops, reaching new frontiers and decisively influencing the economic, social, and cultural development. However, similar to rice, it is also one of the most N-demanding crops that makes it crucial to invest in research on alternatives other than chemical N fertilizers like biofertilizers with diazotrophs, so as to ensure a competitive and sustainable development of sugarcane industry. A study conducted in 2014 reported the effects of inoculating the sugarcane plants with a consortium of five different endophytic diazotrophic bacteria of *Gluconacetobacter diazotrophicus*, *Herbaspirillum*, and *Burkholderia* [98]. In this study, the consortium was evaluated with regard to the agronomic performance and N nutrition of sugarcane in field against chemical N fertilizer and it was found that the consortium of inoculant increased the stalk yield of sugarcane similar to the chemical fertilization. However, authors did not find any evidence of BNF in sugarcane by the consortium of diazotrophic strains, which indicates that the diazotrophic strains used in this study may possess other PGP characteristics that could have resulted in increased yields of sugarcane. In another study, *Gluconacetobacter diazotrophicus* strain PAL 5, which has been studied extensively for its N-fixing and PGP abilities [99], and a strain belonging to the genus *Herbaspirillum* were inoculated into sugarcane plants to evaluate their drought stress recovery [100]. After being subjected to 21 days of drought stress, bacteria-inoculated plants had significantly higher shoot and root dry weight (50 and 70%, respectively) and total N content in leaves (77%). Authors also reported that these diazotrophic strains induce preservation of leaf water potential and relative water content by closing stomata efficiently resulting in plant water preservation during the drought, which highlights the ability of these endophytic diazotrophic bacteria to protect the plant from abiotic stresses. Another type of abiotic stress, that is, salinity, has been recently reported to stimulate the population and diversity of endophytic diazotrophic bacteria in forage cactus (*Opuntia stricta*) [101]. In this study, the population density of endophytic diazotrophic strains in root tissues was evaluated by using the most probable number method (MPN) and strains were characterized phenotypically to evaluate the diversity. Authors reported that the forage cactus plants that received the highest amount of saline water had the highest population density of putative endophytic diazotrophic bacteria with high phenotypic diversity. These findings indicate that endophytic diazotrophic bacteria thrive when conditions are adverse by assisting the host plant through direct or indirect mechanisms to flourish in poor conditions.

germination by 47% and root volume by 44% [102]. In yet another study conducted in Brazil, 40 endophytic strains were isolated from roots of banana (*Musa* L.) tree cultivar 'Prata Anã' [103]. Banana is a very common edible fruit (botanically a berry), produced primarily in the tropics but consumed all around the world. Banana trees grow rapidly and require substantial amount of nutrients in the soil for their development and fruit production. Out of the 40 strains isolated in that study, 20 strains were able to grow on N-free media, but only four isolates showed positive results for N-fixing activity when analyzed using acetylene reduction assay and Kjeldahl method. All four isolates were identified as belonging to the genus *Bacillus* and were also tested positive for *in vitro* phosphate solubilization and IAA production, thus, indicating their potential to be used as growth-promoting microbial inoculants for banana

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Pearl millet (*Pennisetum glaucum* (L.) R. Br.) is a staple cereal crop of the hottest and driest areas of tropics and subtropics. Pearl millet is commonly grown in Rajasthan, India, which has an arid climate and uncertain and erratic rainfall season. In a study reported in 2013, endophytic diazotrophic strains were isolated from pearl millet plants growing in a field with a nutrient-deficient sandy clay loam soil located in Rajasthan [104]. *Pseudomonas aeruginosa* strain PM389 was the most dominant diazotrophic strain in pearl millet plants harvested from this field, whose upward migration and establishment in the stem tissues were later tracked by using enterobacterial repetitive intergenic consensus sequences-PCR (ERIC-PCR) as a biomarker. Efficient reduction of acetylene during the acetylene reduction assay and presence of *nifH* gene indicated the N-fixing potential of the strain PM389. As reported in the study, this strain possesses other PGP characteristics as well, like mineral phosphate solubilization, siderophore production, and antagonistic activity against many pathogenic bacterial and fungal species. In addition, when inoculated into a nonnative plant species (wheat), strain PM389 significantly increased seed germination rate, root and shoot length, and vigor index, which highlights its ability to infect other crop hosts and promote their growth [104]. Local cultivars that have been grown traditionally for many years could serve as a source for potential endophytic diazotrophic bacteria that could be applied to modern commercial varieties as biofertilizers. This theory was proved by scientists from Thailand, who isolated 396 potential endophytic diazotrophic strains from 6 different landraces of rice growing in Chiang Mai, Thailand [105]. Based on the results of acetylene reduction assay, authors chose 21 isolates that were further screened to 10 on the basis of tests conducted for other PGP characteristics. These strains belonged to genera *Burkholderia*, *Klebsiella*, *Novosphingobium*, and *Sphingomonas* and were able to recolonize the tissues of a commercial rice cultivar Khao Dawk Mali 105 along with increasing the N content in the seedlings and promoting seedling length and dry weight. Korean rice cultivars have also been evaluated for the presence of endophytic diazotrophic bacteria [106]. Twelve potential endophytic diazotrophic strains were isolated and identified as belonging to the genera *Paenibacillus* [107], *Bacillus*, *Microbacterium*, and *Klebsiella* and were tested positive for the presence of *nifH* gene. When reinoculated into rice plants, these strains improved plant growth, increased height and dry weight, and showed antagonistic effects against fungal pathogens, thus, establishing their potential role as biofertilizer

trees pending *in vivo* greenhouse or field experiments.

and biocontrol agents for Korean rice cultivars.

Corn is an agriculturally important crop that is extensively grown and consumed by a large population around the world. Szilagyi-Zecchin et al. [102] isolated and identified six endophytic strains from roots of corn growing in the southern Brazilian region of Campo Largo, PR. Out of these six endophytic isolates, four were able to grow on N-free media, consistently reducing acetylene, and were found positive for the presence of *nifH* gene. Apart from showing positive results for N-fixing activity, two out of these four strains (identified as *Bacillus* sp.) also showed other PGP characteristics, like production of IAA, siderophores, and lytic enzymes and antagonism against the common pathogenic fungi. When all endophytic isolates were reinoculated into corn to check for *in vivo* plant growth promotion, another endophytic diazotrophic strain belonging to the genus *Enterobacter* significantly enhanced seed germination by 47% and root volume by 44% [102]. In yet another study conducted in Brazil, 40 endophytic strains were isolated from roots of banana (*Musa* L.) tree cultivar 'Prata Anã' [103]. Banana is a very common edible fruit (botanically a berry), produced primarily in the tropics but consumed all around the world. Banana trees grow rapidly and require substantial amount of nutrients in the soil for their development and fruit production. Out of the 40 strains isolated in that study, 20 strains were able to grow on N-free media, but only four isolates showed positive results for N-fixing activity when analyzed using acetylene reduction assay and Kjeldahl method. All four isolates were identified as belonging to the genus *Bacillus* and were also tested positive for *in vitro* phosphate solubilization and IAA production, thus, indicating their potential to be used as growth-promoting microbial inoculants for banana trees pending *in vivo* greenhouse or field experiments.

should be investigated with other genotypes of rice for a sustainable rice crop production. In Brazil, sugarcane has been one of the fastest growing crops, reaching new frontiers and decisively influencing the economic, social, and cultural development. However, similar to rice, it is also one of the most N-demanding crops that makes it crucial to invest in research on alternatives other than chemical N fertilizers like biofertilizers with diazotrophs, so as to ensure a competitive and sustainable development of sugarcane industry. A study conducted in 2014 reported the effects of inoculating the sugarcane plants with a consortium of five different endophytic diazotrophic bacteria of *Gluconacetobacter diazotrophicus*, *Herbaspirillum*, and *Burkholderia* [98]. In this study, the consortium was evaluated with regard to the agronomic performance and N nutrition of sugarcane in field against chemical N fertilizer and it was found that the consortium of inoculant increased the stalk yield of sugarcane similar to the chemical fertilization. However, authors did not find any evidence of BNF in sugarcane by the consortium of diazotrophic strains, which indicates that the diazotrophic strains used in this study may possess other PGP characteristics that could have resulted in increased yields of sugarcane. In another study, *Gluconacetobacter diazotrophicus* strain PAL 5, which has been studied extensively for its N-fixing and PGP abilities [99], and a strain belonging to the genus *Herbaspirillum* were inoculated into sugarcane plants to evaluate their drought stress recovery [100]. After being subjected to 21 days of drought stress, bacteria-inoculated plants had significantly higher shoot and root dry weight (50 and 70%, respectively) and total N content in leaves (77%). Authors also reported that these diazotrophic strains induce preservation of leaf water potential and relative water content by closing stomata efficiently resulting in plant water preservation during the drought, which highlights the ability of these endophytic diazotrophic bacteria to protect the plant from abiotic stresses. Another type of abiotic stress, that is, salinity, has been recently reported to stimulate the population and diversity of endophytic diazotrophic bacteria in forage cactus (*Opuntia stricta*) [101]. In this study, the population density of endophytic diazotrophic strains in root tissues was evaluated by using the most probable number method (MPN) and strains were characterized phenotypically to evaluate the diversity. Authors reported that the forage cactus plants that received the highest amount of saline water had the highest population density of putative endophytic diazotrophic bacteria with high phenotypic diversity. These findings indicate that endophytic diazotrophic bacteria thrive when conditions are adverse by assisting the host plant through direct or indirect

Corn is an agriculturally important crop that is extensively grown and consumed by a large population around the world. Szilagyi-Zecchin et al. [102] isolated and identified six endophytic strains from roots of corn growing in the southern Brazilian region of Campo Largo, PR. Out of these six endophytic isolates, four were able to grow on N-free media, consistently reducing acetylene, and were found positive for the presence of *nifH* gene. Apart from showing positive results for N-fixing activity, two out of these four strains (identified as *Bacillus* sp.) also showed other PGP characteristics, like production of IAA, siderophores, and lytic enzymes and antagonism against the common pathogenic fungi. When all endophytic isolates were reinoculated into corn to check for *in vivo* plant growth promotion, another endophytic diazotrophic strain belonging to the genus *Enterobacter* significantly enhanced seed

mechanisms to flourish in poor conditions.

80 Nitrogen in Agriculture - Updates

Pearl millet (*Pennisetum glaucum* (L.) R. Br.) is a staple cereal crop of the hottest and driest areas of tropics and subtropics. Pearl millet is commonly grown in Rajasthan, India, which has an arid climate and uncertain and erratic rainfall season. In a study reported in 2013, endophytic diazotrophic strains were isolated from pearl millet plants growing in a field with a nutrient-deficient sandy clay loam soil located in Rajasthan [104]. *Pseudomonas aeruginosa* strain PM389 was the most dominant diazotrophic strain in pearl millet plants harvested from this field, whose upward migration and establishment in the stem tissues were later tracked by using enterobacterial repetitive intergenic consensus sequences-PCR (ERIC-PCR) as a biomarker. Efficient reduction of acetylene during the acetylene reduction assay and presence of *nifH* gene indicated the N-fixing potential of the strain PM389. As reported in the study, this strain possesses other PGP characteristics as well, like mineral phosphate solubilization, siderophore production, and antagonistic activity against many pathogenic bacterial and fungal species. In addition, when inoculated into a nonnative plant species (wheat), strain PM389 significantly increased seed germination rate, root and shoot length, and vigor index, which highlights its ability to infect other crop hosts and promote their growth [104]. Local cultivars that have been grown traditionally for many years could serve as a source for potential endophytic diazotrophic bacteria that could be applied to modern commercial varieties as biofertilizers. This theory was proved by scientists from Thailand, who isolated 396 potential endophytic diazotrophic strains from 6 different landraces of rice growing in Chiang Mai, Thailand [105]. Based on the results of acetylene reduction assay, authors chose 21 isolates that were further screened to 10 on the basis of tests conducted for other PGP characteristics. These strains belonged to genera *Burkholderia*, *Klebsiella*, *Novosphingobium*, and *Sphingomonas* and were able to recolonize the tissues of a commercial rice cultivar Khao Dawk Mali 105 along with increasing the N content in the seedlings and promoting seedling length and dry weight. Korean rice cultivars have also been evaluated for the presence of endophytic diazotrophic bacteria [106]. Twelve potential endophytic diazotrophic strains were isolated and identified as belonging to the genera *Paenibacillus* [107], *Bacillus*, *Microbacterium*, and *Klebsiella* and were tested positive for the presence of *nifH* gene. When reinoculated into rice plants, these strains improved plant growth, increased height and dry weight, and showed antagonistic effects against fungal pathogens, thus, establishing their potential role as biofertilizer and biocontrol agents for Korean rice cultivars.

Our lab group has been working with endophytic diazotrophic bacteria from many years and has published several reports regarding the role of these bacteria in fixing N and promoting plant growth in both agricultural and forest ecosystems [108]. In 2012, our lab discovered an endophytic diazotrophic bacterium, *P. polymyxa* P2b-2R, from stem tissues of lodgepole pine (*Pinus contorta var. latifolia*) trees naturally regenerating at a site located near Williams Lake, BC, Canada [109]. Strain P2b-2R was able to grow on N-free media and consistently reduced significant amounts of acetylene in the acetylene reduction assay [109]. This bacterial strain was able to fix significant amounts of atmospheric N (up to 79%) when reinoculated into lodgepole pine and evaluated using foliar 15N isotope dilution method [110–112]. It was also observed that strain P2b-2R possesses *nif* genes required to encode the nitrogenase enzyme, thus confirming the N-fixing ability of this strain [113].

Endophytic colonization of lodgepole pine by P2b-2R strain was confirmed by constructing a GFP-tagged derivative of P2b-2R and visualizing the sites of colonization using CLSM [75]. It was found that this strain can colonize both intercellular and intracellular spaces of lodgepole pine interior tissues possibly by degrading major cell wall components [75, 114]. Strain P2b-2R was able to colonize internal tissues of another gymnosperm tree species, western red cedar (*Thuja plicata*), and fix considerable amounts of N from the atmosphere along with enhancing seedling length and biomass of cedar [115, 116]. Subsequently, Puri et al. [117] hypothesized that strain P2b-2R could provide similar benefits to angiosperms, specifically agricultural crop species, by colonizing them endophytically. They tested this hypothesis by inoculating strain P2b-2R into agriculturally important crops, namely corn, canola, and tomato, and found that P2b-2R was able to colonize internal tissues of these crop species, fix substantial amounts of atmospheric N, and increase seedling length and biomass (see **Table 2**) [117–119]. These reports indicate the ability of strain P2b-2R to symbiotically associate with a broad range of hosts and promote their growth primarily by fixing atmospheric N. An interesting observation with the GFP-tagged P2b-2R strain (P2b-2R*gfp*) was reported recently where P2b-2R*gfp* inoculation significantly enhanced corn and canola seedling length and biomass as compared to the wild-type P2b-2R inoculation [119–122]. In addition, strain P2b-2R*gfp* fixed significantly higher amounts of N as compared to the wildtype strain. Subsequently, similar results were reported when both strains were inoculated into their original host, that is, lodgepole pine [123]. To the best of our knowledge, these were the very first *in planta* studies in literature reporting that GFP tagging of a bacterial strain could significantly enhance its ability to promote plant growth. Enhancement of these abilities *in vitro* after GFP-tagging were reported previously in *Azospirillum brasilense* [124]. A plausible reason for increased N fixing and plant growth–promoting efficacy of P2b-2R after GFP tagging could be the overexpression of structural *nif* genes (*nifH*, *nifD*, and *nifK*), which play an important role in the N-fixation process [121]. However, it is still unclear how GFP tagging affects the expression of structural *nif* genes of strain P2b-2R. Also, other plausible reasons behind the increased plant growth–promoting efficacy after GFP tagging

Nitrogen-Fixation by Endophytic Bacteria in Agricultural Crops: Recent Advances

http://dx.doi.org/10.5772/intechopen.71988

83

Since their discovery in sugarcane tissues decades ago, endophytic diazotrophic bacteria have been characterized for their role in performing BNF. Studies have suggested that these bacteria can act as N biofertilizer for highly N-demanding crops like sugarcane, corn, and rice. Most recent studies have also focused their attention on testing the PGP characteristics of isolated endophytic diazotrophic strains other than N fixation, which indicates the growing concern of agricultural scientists to develop bacterial inoculants that can enhance plant growth through a variety of mechanisms, so as to decrease the dependence on chemical fertilizers. Endophytic diazotrophic strains like *P. polymyxa* P2b-2R that are able to colonize nonnative host and fix atmospheric N and promote their growth have great potential as biofertilizers for

need to be investigated.

sustainable crop production.

**5. Conclusions**


a Percent nitrogen derived from the atmosphere (%Ndfa).

b Percent increase in foliar nitrogen concentration by inoculation with *P. polymyxa* strains P2b-2R and P2b-2R*gfp*.

c Percent seedling length promoted by inoculation with *P. polymyxa* strains P2b-2R and P2b-2R*gfp*.

<sup>d</sup>Percent seedling biomass promoted by inoculation with *P. polymyxa* strains P2b-2R and P2b-2R*gfp*.

These parameters were calculated using the formulas described in Puri et al. [122].

**Table 2.** Plant growth promotion and biological nitrogen fixation by *Paenibacillus polymyxa* strain P2b-2R and its GFPtagged derivative, P2b-2R*gfp*, when inoculated into agricultural crops, namely, corn, canola, and tomato.

Endophytic colonization of lodgepole pine by P2b-2R strain was confirmed by constructing a GFP-tagged derivative of P2b-2R and visualizing the sites of colonization using CLSM [75]. It was found that this strain can colonize both intercellular and intracellular spaces of lodgepole pine interior tissues possibly by degrading major cell wall components [75, 114]. Strain P2b-2R was able to colonize internal tissues of another gymnosperm tree species, western red cedar (*Thuja plicata*), and fix considerable amounts of N from the atmosphere along with enhancing seedling length and biomass of cedar [115, 116]. Subsequently, Puri et al. [117] hypothesized that strain P2b-2R could provide similar benefits to angiosperms, specifically agricultural crop species, by colonizing them endophytically. They tested this hypothesis by inoculating strain P2b-2R into agriculturally important crops, namely corn, canola, and tomato, and found that P2b-2R was able to colonize internal tissues of these crop species, fix substantial amounts of atmospheric N, and increase seedling length and biomass (see **Table 2**) [117–119]. These reports indicate the ability of strain P2b-2R to symbiotically associate with a broad range of hosts and promote their growth primarily by fixing atmospheric N. An interesting observation with the GFP-tagged P2b-2R strain (P2b-2R*gfp*) was reported recently where P2b-2R*gfp* inoculation significantly enhanced corn and canola seedling length and biomass as compared to the wild-type P2b-2R inoculation [119–122]. In addition, strain P2b-2R*gfp* fixed significantly higher amounts of N as compared to the wildtype strain. Subsequently, similar results were reported when both strains were inoculated into their original host, that is, lodgepole pine [123]. To the best of our knowledge, these were the very first *in planta* studies in literature reporting that GFP tagging of a bacterial strain could significantly enhance its ability to promote plant growth. Enhancement of these abilities *in vitro* after GFP-tagging were reported previously in *Azospirillum brasilense* [124]. A plausible reason for increased N fixing and plant growth–promoting efficacy of P2b-2R after GFP tagging could be the overexpression of structural *nif* genes (*nifH*, *nifD*, and *nifK*), which play an important role in the N-fixation process [121]. However, it is still unclear how GFP tagging affects the expression of structural *nif* genes of strain P2b-2R. Also, other plausible reasons behind the increased plant growth–promoting efficacy after GFP tagging need to be investigated.

## **5. Conclusions**

Our lab group has been working with endophytic diazotrophic bacteria from many years and has published several reports regarding the role of these bacteria in fixing N and promoting plant growth in both agricultural and forest ecosystems [108]. In 2012, our lab discovered an endophytic diazotrophic bacterium, *P. polymyxa* P2b-2R, from stem tissues of lodgepole pine (*Pinus contorta var. latifolia*) trees naturally regenerating at a site located near Williams Lake, BC, Canada [109]. Strain P2b-2R was able to grow on N-free media and consistently reduced significant amounts of acetylene in the acetylene reduction assay [109]. This bacterial strain was able to fix significant amounts of atmospheric N (up to 79%) when reinoculated into lodgepole pine and evaluated using foliar 15N isotope dilution method [110–112]. It was also observed that strain P2b-2R possesses *nif* genes required to encode the nitrogenase enzyme, thus confirming the N-fixing ability of this strain [113].

**%Ndfa<sup>a</sup> % growth promotion References**

**P2b-2R P2b-**

**Seedling lengthc Seedling** 

**2R***gfp*

**biomassd**

**P2b-2R P2b-**

**2R***gfp*

**Foliar nitrogen concentrationb**

**P2b-2R P2b-**

Corn 10 6.65 — 5.42 — 10.0 — 20.9 — [117]

Canola 20 8.08 13.0 28.7 37.8 17.8 37.4 57.0 91.6 [119]

Tomato 20 10.0 8.32 33.3 25.5 40.6 48.4 56.1 44.1 [119]

Percent increase in foliar nitrogen concentration by inoculation with *P. polymyxa* strains P2b-2R and P2b-2R*gfp*.

**Table 2.** Plant growth promotion and biological nitrogen fixation by *Paenibacillus polymyxa* strain P2b-2R and its GFP-

Percent seedling length promoted by inoculation with *P. polymyxa* strains P2b-2R and P2b-2R*gfp*. <sup>d</sup>Percent seedling biomass promoted by inoculation with *P. polymyxa* strains P2b-2R and P2b-2R*gfp*.

tagged derivative, P2b-2R*gfp*, when inoculated into agricultural crops, namely, corn, canola, and tomato.

These parameters were calculated using the formulas described in Puri et al. [122].

 12.9 15.1 18.0 36.1 20.5 48.7 53.7 93.5 [119] 16.2 22.1 23.4 40.8 28.4 69.4 37.1 108 [119] 21.8 — 40.3 — 24.9 — 30.1 — [118] 27.1 35.1 11.7 25.0 70.7 102.5 100.8 159.1 [120]

30 12.3 11.2 30.6 23.2 36.5 37.5 69.0 61.4 [119] 40 18.1 16.7 30.0 22.5 24.9 28.3 93.0 82.9 [119]

**2R***gfp*

 10.8 10.9 13.6 25.0 13.8 41.3 26.1 34.0 [117, 121] 19.6 14.1 14.2 22.6 35.3 36.3 30.9 55.5 [117, 121] 15.7 18.0 17.1 27.6 24.7 27.6 28.4 48.9 [121] 30.2 32.2 27.3 31.8 51.9 68.4 52.7 66.9 [122]

**Host plant**

a

b

c

**Harvest (days)**

82 Nitrogen in Agriculture - Updates

**P2b-2R P2b-**

Percent nitrogen derived from the atmosphere (%Ndfa).

**2R***gfp*

Since their discovery in sugarcane tissues decades ago, endophytic diazotrophic bacteria have been characterized for their role in performing BNF. Studies have suggested that these bacteria can act as N biofertilizer for highly N-demanding crops like sugarcane, corn, and rice. Most recent studies have also focused their attention on testing the PGP characteristics of isolated endophytic diazotrophic strains other than N fixation, which indicates the growing concern of agricultural scientists to develop bacterial inoculants that can enhance plant growth through a variety of mechanisms, so as to decrease the dependence on chemical fertilizers. Endophytic diazotrophic strains like *P. polymyxa* P2b-2R that are able to colonize nonnative host and fix atmospheric N and promote their growth have great potential as biofertilizers for sustainable crop production.

## **Acknowledgements**

Authors would like to dedicate this work to Late Mr. Darshan K. Puri (1956–2014). You were, are and always will be an inspirational figure for us.

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## **Author details**

Akshit Puri<sup>1</sup> , Kiran Preet Padda<sup>1</sup> and Chris P. Chanway1,2\*

\*Address all correspondence to: chris.chanway@ubc.ca

1 Department of Soil Science, Faculty of Land and Food Systems, The University of British Columbia, Vancouver, Canada

2 Department of Forest and Conservation Sciences, Faculty of Forestry, The University of British Columbia, Vancouver, Canada

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**Acknowledgements**

84 Nitrogen in Agriculture - Updates

**Author details**

Akshit Puri<sup>1</sup>

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**Chapter 6**

**Provisional chapter**

) fixation (BNF) is one of the most

transferred to non-N<sup>2</sup>

transfer and management consid-

transfer in agricultural


**Nitrogen Fixation and Transfer in Agricultural**

**Nitrogen Fixation and Transfer in Agricultural** 

DOI: 10.5772/intechopen.71766

There is a consensus within the scientific community that nitrogenous fertilizers are almost indispensable in today's agriculture. However, the geometric increase in nitrogenous fertilizer applications and the associated environmental concerns call for focus on

sustainable approaches to meeting crop nitrogen (N) demands. The BNF is, especially, important in low value crops (e.g., forages) and in developing economies. However, just like synthetic N fertilizers, BNF has issues of its own. Among the issues of great impor-

plants. The proportion of transfer ranges from as low as 0% to as high as 70%, depending on a myriad of factors. Most of the factors (e.g., N fertilizer application, species, and cultivar selection) are management related and can, therefore, be controlled for improved N<sup>2</sup> fixation and transfer. In this chapter, we discuss current trends in BNF in selected legume

fixation and transfer are discussed.

**Keywords:** biological nitrogen fixation, nitrogen transfer, fertilizers, legumes,

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

© 2018 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

Plants require N in relatively large quantities to grow and reproduce. In fact, N is the third most important factor in the growth and development of crop plants [1]. This made N one of the most important nutrients in agricultural production systems. The important role N plays in global food production is evident in the ever-increasing amounts of N fertilizers applied annually. It has been estimated that approximately 100 Tg of synthetic N fertilizers

M. Anowarul Islam and Albert Tetteh Adjesiwor

more sustainable alternatives. Biological dinitrogen (N<sup>2</sup>

tance is the low and highly variable proportion of fixed N<sup>2</sup>

crops, the global economics of BNF, and recent reports on N<sup>2</sup>

production systems. Additionally, factors affecting N<sup>2</sup>

M. Anowarul Islam and Albert Tetteh Adjesiwor

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.71766

erations for improving N<sup>2</sup>

grass-legume mixtures

**1. Introduction**

**Abstract**

**Production Systems**

**Production Systems**


**Provisional chapter**

## **Nitrogen Fixation and Transfer in Agricultural Production Systems Production Systems**

**Nitrogen Fixation and Transfer in Agricultural** 

DOI: 10.5772/intechopen.71766

#### M. Anowarul Islam and Albert Tetteh Adjesiwor Additional information is available at the end of the chapter

M. Anowarul Islam and Albert Tetteh Adjesiwor

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.71766

#### **Abstract**

[121] Padda KP, Puri A, Zeng Q, Chanway CP, Wu X. Effect of GFP-tagging on nitrogen fixation and plant growth promotion of an endophytic diazotrophic strain of *Paenibacillus* 

[122] Puri A, Padda KP, Chanway CP. Seedling growth promotion and nitrogen fixation by a bacterial endophyte *Paenibacillus polymyxa* P2b-2R and its GFP derivative in corn in a

[123] Tang Q, Puri A, Padda KP, Chanway CP. Biological nitrogen fixation and plant growth promotion of lodgepole pine by an endophytic diazotroph *Paenibacillus polymyxa* and its GFP-tagged derivative. Botany. 2017;**95**(6):611-619. DOI: 10.1139/cjb-2016-0300 [124] Rodriguez H, Mendoza A, Cruz MA, Holguin G, Glick BR, Bashan Y. Pleiotropic physiological effects in the plant growth-promoting bacterium *Azospirillum brasilense* following chromosomal labeling in the clpX gene. FEMS Microbiology Ecology. 2006;**57**(2):

long-term trial. Symbiosis. 2016;**69**(2):123-129. DOI: 10.1007/s13199-016-0385-z

*polymyxa*. Botany. 2017;**95**(9):933-942. DOI: 10.1139/cjb-2017-0056

217-225. DOI: 10.1111/j.1574-6941.2006.00111.x

94 Nitrogen in Agriculture - Updates

There is a consensus within the scientific community that nitrogenous fertilizers are almost indispensable in today's agriculture. However, the geometric increase in nitrogenous fertilizer applications and the associated environmental concerns call for focus on more sustainable alternatives. Biological dinitrogen (N<sup>2</sup> ) fixation (BNF) is one of the most sustainable approaches to meeting crop nitrogen (N) demands. The BNF is, especially, important in low value crops (e.g., forages) and in developing economies. However, just like synthetic N fertilizers, BNF has issues of its own. Among the issues of great importance is the low and highly variable proportion of fixed N<sup>2</sup> transferred to non-N<sup>2</sup> -fixing plants. The proportion of transfer ranges from as low as 0% to as high as 70%, depending on a myriad of factors. Most of the factors (e.g., N fertilizer application, species, and cultivar selection) are management related and can, therefore, be controlled for improved N<sup>2</sup> fixation and transfer. In this chapter, we discuss current trends in BNF in selected legume crops, the global economics of BNF, and recent reports on N<sup>2</sup> transfer in agricultural production systems. Additionally, factors affecting N<sup>2</sup> transfer and management considerations for improving N<sup>2</sup> fixation and transfer are discussed.

**Keywords:** biological nitrogen fixation, nitrogen transfer, fertilizers, legumes, grass-legume mixtures

## **1. Introduction**

Plants require N in relatively large quantities to grow and reproduce. In fact, N is the third most important factor in the growth and development of crop plants [1]. This made N one of the most important nutrients in agricultural production systems. The important role N plays in global food production is evident in the ever-increasing amounts of N fertilizers applied annually. It has been estimated that approximately 100 Tg of synthetic N fertilizers

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons

were applied in 2009 [2]. The geometric increase in N fertilizer use worldwide is in part, attributable to the need to produce enough food to feed the over 7 billion people currently living on earth. Although there is a consensus within the scientific community that N fertilizers are almost indispensable in today's agriculture, there are great concerns with the use of N fertilizers. Some of these include pollution of surface and underground waters, greenhouse gas (e.g., nitrous oxide: N<sup>2</sup> O) emissions, and low N use efficiency (NUE). There is, therefore, a multi-pronged approach to N management in global food production. While N fertilizers are being increasingly applied to crops to increase crop productivity, there are calls for more sustainable approaches to meeting N demand of crops such as climate-smart agriculture and sustainable intensification.

The BNF, the process whereby micro-organisms use nitrogenase enzyme to convert atmospheric inert N<sup>2</sup> to plant usable forms [3, 4], was the main source of N prior to the industrial revolution [5]. It is generally agreed that BNF is one of the most sustainable approaches to meeting crop N demands. For example, it has been estimated that NUE increases exponentially with increasing levels of biologically fixed N<sup>2</sup> in soils while NUE decreases linearly with increasing levels of applied synthetic N fertilizers [2]. There are concerns about the best approach for quantifying inputs of fixed N<sup>2</sup> . Conservative estimates based on harvested areas and yields from 2005 Food and Agricultural Organization (FAO) database on world crop production (FAOSTAT) showed that 2.95 and 18.5 Tg N was fixed annually by pulses and oilseed crops, respectively [6]. Soybean (*Glycine max* (L.)) fixed 16.4 Tg N, representing 77% of total N2 fixation by legume crops in 2005 [6]. Although BNF contributes ~25 Tg N which is dwarfed by the ~100 Tg contributed by synthetic N fertilizers [2], the importance of BNF to the global N budget is substantial.

and developing economies, where farmers either have limited access to synthetic N fertilizers or are unable to afford N fertilizers. In fact, forage accumulation and profitability from grasslegume mixtures have been reported to be equal or greater than N-fertilized grass monocultures [13–15]. Aside direct N input from BNF, N from BNF reduces the amount of synthetic N fertilizers applied in agriculture and natural ecosystems. This, in turn, reduces cost of production, greenhouse gas (GHG) emissions, and pollution of surface and underground waters. Low NUE and N recovery are major issues associated with use of N fertilizers [16, 17]. In a comprehensive analysis, Lassaletta et al. [2] showed that the efficiency of N use of biologically fixed

Rhizobia Symbiosis with roots of legumes (nodules); important source of N for legumes; proper Rhizobia strains required for effective nodulation and N<sup>2</sup>

Anabaena Autotrophic; mostly aquatic but can be terrestrial; symbiosis with non-legumes (e.g.,

*Azolla* sp.); important in paddy rice (*Oryza sativa* L.) production; can be utilized as



Frankia (Actinomycetes) Symbiosis with non-legume angiosperms (e.g., *Alnus*, *Myrica*, *Alder*, *Casuarina*);

can be important source of N for non-legumes

*officinarum* L.) and some tropical grasses

lichens (fungi), cycads, etc.

Acetobacter Heterotrophic; endophytic, can be important source of N for sugarcane (*Saccharum* 

important source of N in agroforestry

fixation

http://dx.doi.org/10.5772/intechopen.71766

Nitrogen Fixation and Transfer in Agricultural Production Systems


fixation in agriculture and natural ecosystems†


*Rhizobia*-legume symbiosis is a significant source of N in agriculture. Needless to say, BNF from associative and free-living bacteria and diazotrophs are important in natural ecosystems and

factors. These include, but not limited to, plant species and cultivar, residual soil N, *Rhizobia* strains, and environmental conditions. Generally, perennial forages fix greater amounts of N<sup>2</sup> compared to annual forages since they live longer in the field [18]. For example, estimated total BNF from alfalfa (*Medicago sativa* L.), red clover (*Trifolium pratense* L.), and white clover (*Trifolium repens* L.) are 465, 252, and 102 kg N ha−1 year−1 while from faba bean (*Vicia faba* L.), field pea (*Pisum sativum* L.), and lentil (*Lens culinaris* Medik.) are 165, 111, and 52 kg N ha−1 year−1,

fixed from *Rhizobia*-legume symbiosis varies greatly depending on many

fixation from selected crops has shown that in 2014, up to 29

fixation from

.

97

is greater than synthetic N. Among the micro-organisms involved in BNF, N<sup>2</sup>

 **fixed by legumes**

water-logged production areas (e.g., paddy fields) [6].

**Micro-organism Properties and importance**

green manure

Azospirillum Microaerophilic; heterotrophic; free-living N<sup>2</sup>

Bradyrhizobium Aerobic, heterotrophic, free-living N<sup>2</sup>

Azotobacter Aerobic; heterotrophic; free-living N<sup>2</sup>

**Table 1.** Properties of selected micro-organisms involved in biological N<sup>2</sup>

Cyanobacteria Autotrophic; free-living N<sup>2</sup>

**2.1. Amount and value of N<sup>2</sup>**

respectively [19]. Estimates of N<sup>2</sup>

The amount of N<sup>2</sup>

N2

†

Modified from [3, 7, 18].

Just like synthetic fertilizers, BNF has issues of its own. Among the issues of great importance is the transfer of fixed N<sup>2</sup> to non-N<sup>2</sup> -fixing plants. The proportion of biologically fixed N<sup>2</sup> transferred to neighboring plants can range from as low as 0% to as high as 73%, depending on a myriad of factors [1]. The biology, chemistry, and processes involved in BNF have been extensively described in the literature [7–12]. Therefore, in this chapter, we discuss briefly the organisms involved in BNF and then proceed to current trends in global N<sup>2</sup> fixation and value of BNF transfer in agricultural production systems with special emphasis on N<sup>2</sup> fixation from *Rhizobia*-legume symbiosis. Finally, we summarize current findings on N transfer in agricultural systems, discuss the factors responsible for low and variable transfer of biologically fixed N2, and provide some suggestions for improved transfer of fixed N<sup>2</sup> .

#### **2. Biological dinitrogen fixation: importance and economics**

Several micro-organisms can convert inert atmospheric N<sup>2</sup> to plant usable forms. These organisms may exist in association and symbiosis with host plants or independent of a host plant (**Table 1**). Organisms relying solely on atmospheric N<sup>2</sup> as their N source for growth are referred to as diazotrophs [7]. Biological N<sup>2</sup> fixation is a significant source of N in agricultural and natural ecosystems. The N input from BNF is particularly important in low value crops (e.g., forages)


**Table 1.** Properties of selected micro-organisms involved in biological N<sup>2</sup> fixation in agriculture and natural ecosystems† .

and developing economies, where farmers either have limited access to synthetic N fertilizers or are unable to afford N fertilizers. In fact, forage accumulation and profitability from grasslegume mixtures have been reported to be equal or greater than N-fertilized grass monocultures [13–15]. Aside direct N input from BNF, N from BNF reduces the amount of synthetic N fertilizers applied in agriculture and natural ecosystems. This, in turn, reduces cost of production, greenhouse gas (GHG) emissions, and pollution of surface and underground waters. Low NUE and N recovery are major issues associated with use of N fertilizers [16, 17]. In a comprehensive analysis, Lassaletta et al. [2] showed that the efficiency of N use of biologically fixed N2 is greater than synthetic N. Among the micro-organisms involved in BNF, N<sup>2</sup> fixation from *Rhizobia*-legume symbiosis is a significant source of N in agriculture. Needless to say, BNF from associative and free-living bacteria and diazotrophs are important in natural ecosystems and water-logged production areas (e.g., paddy fields) [6].

#### **2.1. Amount and value of N<sup>2</sup> fixed by legumes**

were applied in 2009 [2]. The geometric increase in N fertilizer use worldwide is in part, attributable to the need to produce enough food to feed the over 7 billion people currently living on earth. Although there is a consensus within the scientific community that N fertilizers are almost indispensable in today's agriculture, there are great concerns with the use of N fertilizers. Some of these include pollution of surface and underground waters, greenhouse

a multi-pronged approach to N management in global food production. While N fertilizers are being increasingly applied to crops to increase crop productivity, there are calls for more sustainable approaches to meeting N demand of crops such as climate-smart agriculture and

The BNF, the process whereby micro-organisms use nitrogenase enzyme to convert atmo-

revolution [5]. It is generally agreed that BNF is one of the most sustainable approaches to meeting crop N demands. For example, it has been estimated that NUE increases exponen-

with increasing levels of applied synthetic N fertilizers [2]. There are concerns about the best

and yields from 2005 Food and Agricultural Organization (FAO) database on world crop production (FAOSTAT) showed that 2.95 and 18.5 Tg N was fixed annually by pulses and oilseed crops, respectively [6]. Soybean (*Glycine max* (L.)) fixed 16.4 Tg N, representing 77% of total

 fixation by legume crops in 2005 [6]. Although BNF contributes ~25 Tg N which is dwarfed by the ~100 Tg contributed by synthetic N fertilizers [2], the importance of BNF to the global

Just like synthetic fertilizers, BNF has issues of its own. Among the issues of great importance

transferred to neighboring plants can range from as low as 0% to as high as 73%, depending on a myriad of factors [1]. The biology, chemistry, and processes involved in BNF have been extensively described in the literature [7–12]. Therefore, in this chapter, we discuss briefly the

*Rhizobia*-legume symbiosis. Finally, we summarize current findings on N transfer in agricultural systems, discuss the factors responsible for low and variable transfer of biologically

isms may exist in association and symbiosis with host plants or independent of a host plant

ecosystems. The N input from BNF is particularly important in low value crops (e.g., forages)

to plant usable forms [3, 4], was the main source of N prior to the industrial

O) emissions, and low N use efficiency (NUE). There is, therefore,

in soils while NUE decreases linearly

.

to plant usable forms. These organ-

as their N source for growth are referred

fixation is a significant source of N in agricultural and natural

fixation and value

fixation from

. Conservative estimates based on harvested areas


gas (e.g., nitrous oxide: N<sup>2</sup>

96 Nitrogen in Agriculture - Updates

sustainable intensification.

N budget is substantial.

is the transfer of fixed N<sup>2</sup>

tially with increasing levels of biologically fixed N<sup>2</sup>

to non-N<sup>2</sup>

organisms involved in BNF and then proceed to current trends in global N<sup>2</sup>

fixed N2, and provide some suggestions for improved transfer of fixed N<sup>2</sup>

**2. Biological dinitrogen fixation: importance and economics**

Several micro-organisms can convert inert atmospheric N<sup>2</sup>

(**Table 1**). Organisms relying solely on atmospheric N<sup>2</sup>

to as diazotrophs [7]. Biological N<sup>2</sup>

of BNF transfer in agricultural production systems with special emphasis on N<sup>2</sup>

approach for quantifying inputs of fixed N<sup>2</sup>

spheric inert N<sup>2</sup>

N2

The amount of N<sup>2</sup> fixed from *Rhizobia*-legume symbiosis varies greatly depending on many factors. These include, but not limited to, plant species and cultivar, residual soil N, *Rhizobia* strains, and environmental conditions. Generally, perennial forages fix greater amounts of N<sup>2</sup> compared to annual forages since they live longer in the field [18]. For example, estimated total BNF from alfalfa (*Medicago sativa* L.), red clover (*Trifolium pratense* L.), and white clover (*Trifolium repens* L.) are 465, 252, and 102 kg N ha−1 year−1 while from faba bean (*Vicia faba* L.), field pea (*Pisum sativum* L.), and lentil (*Lens culinaris* Medik.) are 165, 111, and 52 kg N ha−1 year−1, respectively [19]. Estimates of N<sup>2</sup> fixation from selected crops has shown that in 2014, up to 29 Tg N was fixed by eight crops (**Figure 1**). Soybean (*Glycine max* (L.) Merr.) alone contributed 23.4 Tg, representing 81% of total N<sup>2</sup> fixed by these crops (**Figure 1**). While these might not be precise estimates, there is a clear indication that the contribution N<sup>2</sup> fixation to the global N budget is enormous. Though N<sup>2</sup> fixation from peas, lentils, common bean (*Phaseolus vulgaris* L.), faba bean, cowpea (*Vigna unguiculata* (L.) Walp.), chickpeas (*Cicer arietinum* L.), and groundnut

**Figure 1.** Estimates of global trends in biological N<sup>2</sup> fixation for selected legume crops. The N<sup>2</sup> was estimated based on harvested areas and yield data from Food and Agricultural Organization (FAO) database on world crop production (FAOSTAT) [21]. This follows the procedure described by [6].

(*Arachis hypogaea* L.) is dwarfed by soybean (because of the larger area planted to soybean)

fixation (**Figure 1**) and price of urea fertilizer from 2005 to 2014 reported by the World Bank [20].

fixation. Value of fixed N<sup>2</sup>

farmers in developing countries is substantial. Unlike forages, grains from grain legumes are harvested and removed from the field. Thus, grain legumes usually remove more soil N than

grazed savannas, sugarcane (*Saccharum officinarum* L.), and rice (*Oryza sativa* L.) production

fixation from these crops (e.g., cowpea) to

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99

fixation from forages, extensively

was calculated based on estimated N<sup>2</sup>

based on these estimates, the contribution of N<sup>2</sup>

**Figure 2.** Trends in global economics of biological N<sup>2</sup>

forages [18]. The uncertainties associated with estimating N<sup>2</sup>

Tg N was fixed by eight crops (**Figure 1**). Soybean (*Glycine max* (L.) Merr.) alone contributed

faba bean, cowpea (*Vigna unguiculata* (L.) Walp.), chickpeas (*Cicer arietinum* L.), and groundnut

precise estimates, there is a clear indication that the contribution N<sup>2</sup>

fixed by these crops (**Figure 1**). While these might not be

fixation from peas, lentils, common bean (*Phaseolus vulgaris* L.),

fixation for selected legume crops. The N<sup>2</sup>

harvested areas and yield data from Food and Agricultural Organization (FAO) database on world crop production

was estimated based on

fixation to the global N

23.4 Tg, representing 81% of total N<sup>2</sup>

**Figure 1.** Estimates of global trends in biological N<sup>2</sup>

(FAOSTAT) [21]. This follows the procedure described by [6].

budget is enormous. Though N<sup>2</sup>

98 Nitrogen in Agriculture - Updates

**Figure 2.** Trends in global economics of biological N<sup>2</sup> fixation. Value of fixed N<sup>2</sup> was calculated based on estimated N<sup>2</sup> fixation (**Figure 1**) and price of urea fertilizer from 2005 to 2014 reported by the World Bank [20].

(*Arachis hypogaea* L.) is dwarfed by soybean (because of the larger area planted to soybean) based on these estimates, the contribution of N<sup>2</sup> fixation from these crops (e.g., cowpea) to farmers in developing countries is substantial. Unlike forages, grains from grain legumes are harvested and removed from the field. Thus, grain legumes usually remove more soil N than forages [18]. The uncertainties associated with estimating N<sup>2</sup> fixation from forages, extensively grazed savannas, sugarcane (*Saccharum officinarum* L.), and rice (*Oryza sativa* L.) production systems have been acknowledged [6]. Nonetheless, the estimated annual N<sup>2</sup> fixation from these systems are 5 Tg from rice, <4 Tg from non-legume crops, 12–25 Tg from pasture and fodder legumes, 0.5 Tg from sugarcane, and <14 Tg from extensive savannas. It is worth mentioning that biologically fixed N<sup>2</sup> must be transferred to neighboring and subsequent non-N<sup>2</sup> -fixing crops in the cropping systems for optimum benefits. Nitrogen transfer in cropping systems is often low. Thus, all the estimated N<sup>2</sup> fixed (**Figure 1**) may not be transferred to neighboring and subsequent non-N<sup>2</sup> -fixing crops.

The economic value of N<sup>2</sup> fixation is extraordinarily large. Of course, the value of biologically fixed N<sup>2</sup> is directly related to the amount N<sup>2</sup> fixed. Using estimates of N<sup>2</sup> fixation from **Figure 1** and cost of urea N fertilizer from the World Bank [20], it is estimated that in 2014, the value of N fixed by these eight crops is about 18.5 billion US dollars (**Figure 2**). Of this amount, about 14.9 billion (81%) is contributed by soybeans.

## **3. Management considerations for improving biological dinitrogen fixation**

There are several management practices that influence BNF in agricultural production systems. These include but not limited to N-fertilization [22], species [23], genotype and cultivar [24], and seeding ratios (intercropping systems). Adopting best management practices can, therefore, improve N<sup>2</sup> fixation. In mixed swards, perennial ryegrass (*Lolium perenne* L.) competition for available soil N was reported to be important in determining N<sup>2</sup> fixation in birdsfoot trefoil (*Lotus corniculatus* L.), alfalfa, and white clover [25]. Species may differ in their reliance on soil N and fixed N<sup>2</sup> . In a red clover-grass-forbs mixture, grass relied mostly on fixed N2 , while forbs relied on soil N [23]. Selecting compatible cultivars (**Figure 3**) and species may improve N<sup>2</sup> fixation and N<sup>2</sup> transfer in agricultural production systems [26]. For example, the proportion of N<sup>2</sup> derived from BNF was 75–94% in white clover monoculture compared to 85–97% in white clover-ryegrass mixtures [27]. The relatively greater N<sup>2</sup> -fixation in grasslegume mixtures compared to legume monocultures might be attributable to greater competition for soil N from non-N<sup>2</sup> -fixing plants [28]. In an extensive review, Rouquette and Smith [29] asserted that BNF in forage legumes may vary depending on the legume cultivar, species, soil nutrient composition, prevailing environmental conditions, and climate. The myriad of factors influencing BNF might explain the varied amounts of N<sup>2</sup> fixed by legumes even at same locations reported by many researchers [30–35]. For example, at the same location, the proportion of plant total N derived from BNF was reported to range from 12 to 96% on grazed plots [36]. Application of N fertilizers has been found to suppress BNF in legumes [22]. For example, the application of N fertilizer decreased atmospheric derived N<sup>2</sup> of clover from 77 to 43% [37].

mixtures and these include: low forage yield, low proportion of legumes in mixtures, and low

**Figure 3.** Established stands of meadow bromegrass (*Bromus biebersteinii* Roem. & Schult.) monoculture (a) and 50% meadow bromegrass: 50% sainfoin (*Onobrychis viciifolia* Scop.) (b), 50% meadow bromegrass:50% birdsfoot trefoil (c), 50% meadow bromegrass:16.7% sainfoin:16.7% birdsfoot trefoil:16.7% alfalfa (d) seeding ratios in 2014 at the University

fixation [40]. To maintain optimal N<sup>2</sup>

satisfies the immediate N needs of the host plants. However, the fixed N<sup>2</sup>

populations must be maintained in grass-legume stands. This might be difficult to achieve because of the selective grazing of legumes by livestock (in grazing systems), poor soil conditions, and pest and disease problems [28]. However, using optimal seed mass ratios and good

grazing and haying practices may help maintain optimal legume proportions [15].

**4. Transfer of biologically fixed nitrogen in agricultural production** 

transfer is accomplished through three main routes, viz.: decomposition of nodules and secondary roots that are not thickened, exudates of soluble N compounds, and transfer mediated by mycorrhizal fungi [1, 41–43]. The transfer of N through nodule and root decomposition and exudation of N compounds is termed as rhizodeposition [44]. The proportion of biologically

can be transferred to other crops in the cropping system, especially non-N<sup>2</sup>

fixation, sufficient legume

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101


reliance of the legume on N<sup>2</sup>

of Wyoming Sheridan Research and Extension Center, WY, USA.

**systems**

Biologically fixed N<sup>2</sup>

The strain of *Rhizobia* also determines the level of N<sup>2</sup> fixation [38]. Most of these *Rhizobia* strains are highly specialized and due to this specialization and the intricacy of interaction between N2 -fixing plant species and bacteria involved in N<sup>2</sup> fixation, any disturbance or manipulation may be detrimental to the amount of N<sup>2</sup> fixed [39]. Thus, inoculation with the right strains of *Rhizobia* would improve N<sup>2</sup> fixation. There are three major constraints to BNF in grass-legume

systems have been acknowledged [6]. Nonetheless, the estimated annual N<sup>2</sup>

that biologically fixed N<sup>2</sup>

100 Nitrogen in Agriculture - Updates

The economic value of N<sup>2</sup>

subsequent non-N<sup>2</sup>

fixed N<sup>2</sup>

**fixation**

N2

N2

therefore, improve N<sup>2</sup>

may improve N<sup>2</sup>

the proportion of N<sup>2</sup>

ance on soil N and fixed N<sup>2</sup>

tion for soil N from non-N<sup>2</sup>

often low. Thus, all the estimated N<sup>2</sup>


is directly related to the amount N<sup>2</sup>

14.9 billion (81%) is contributed by soybeans.

fixation and N<sup>2</sup>

influencing BNF might explain the varied amounts of N<sup>2</sup>

application of N fertilizer decreased atmospheric derived N<sup>2</sup>

The strain of *Rhizobia* also determines the level of N<sup>2</sup>


may be detrimental to the amount of N<sup>2</sup>

*Rhizobia* would improve N<sup>2</sup>

systems are 5 Tg from rice, <4 Tg from non-legume crops, 12–25 Tg from pasture and fodder legumes, 0.5 Tg from sugarcane, and <14 Tg from extensive savannas. It is worth mentioning

crops in the cropping systems for optimum benefits. Nitrogen transfer in cropping systems is

and cost of urea N fertilizer from the World Bank [20], it is estimated that in 2014, the value of N fixed by these eight crops is about 18.5 billion US dollars (**Figure 2**). Of this amount, about

There are several management practices that influence BNF in agricultural production systems. These include but not limited to N-fertilization [22], species [23], genotype and cultivar [24], and seeding ratios (intercropping systems). Adopting best management practices can,

trefoil (*Lotus corniculatus* L.), alfalfa, and white clover [25]. Species may differ in their reli-

legume mixtures compared to legume monocultures might be attributable to greater competi-

asserted that BNF in forage legumes may vary depending on the legume cultivar, species, soil nutrient composition, prevailing environmental conditions, and climate. The myriad of factors

tions reported by many researchers [30–35]. For example, at the same location, the proportion of plant total N derived from BNF was reported to range from 12 to 96% on grazed plots [36]. Application of N fertilizers has been found to suppress BNF in legumes [22]. For example, the

are highly specialized and due to this specialization and the intricacy of interaction between

, while forbs relied on soil N [23]. Selecting compatible cultivars (**Figure 3**) and species

**3. Management considerations for improving biological dinitrogen** 

tition for available soil N was reported to be important in determining N<sup>2</sup>

to 85–97% in white clover-ryegrass mixtures [27]. The relatively greater N<sup>2</sup>

must be transferred to neighboring and subsequent non-N<sup>2</sup>

fixation is extraordinarily large. Of course, the value of biologically

fixed. Using estimates of N<sup>2</sup>

fixation. In mixed swards, perennial ryegrass (*Lolium perenne* L.) compe-

derived from BNF was 75–94% in white clover monoculture compared

. In a red clover-grass-forbs mixture, grass relied mostly on fixed


transfer in agricultural production systems [26]. For example,

fixed (**Figure 1**) may not be transferred to neighboring and

fixation from these

fixation from **Figure 1**

fixation in birdsfoot


fixed by legumes even at same loca-

of clover from 77 to 43% [37].

fixation [38]. Most of these *Rhizobia* strains

fixation, any disturbance or manipulation

fixed [39]. Thus, inoculation with the right strains of

fixation. There are three major constraints to BNF in grass-legume


**Figure 3.** Established stands of meadow bromegrass (*Bromus biebersteinii* Roem. & Schult.) monoculture (a) and 50% meadow bromegrass: 50% sainfoin (*Onobrychis viciifolia* Scop.) (b), 50% meadow bromegrass:50% birdsfoot trefoil (c), 50% meadow bromegrass:16.7% sainfoin:16.7% birdsfoot trefoil:16.7% alfalfa (d) seeding ratios in 2014 at the University of Wyoming Sheridan Research and Extension Center, WY, USA.

mixtures and these include: low forage yield, low proportion of legumes in mixtures, and low reliance of the legume on N<sup>2</sup> fixation [40]. To maintain optimal N<sup>2</sup> fixation, sufficient legume populations must be maintained in grass-legume stands. This might be difficult to achieve because of the selective grazing of legumes by livestock (in grazing systems), poor soil conditions, and pest and disease problems [28]. However, using optimal seed mass ratios and good grazing and haying practices may help maintain optimal legume proportions [15].

## **4. Transfer of biologically fixed nitrogen in agricultural production systems**

Biologically fixed N<sup>2</sup> satisfies the immediate N needs of the host plants. However, the fixed N<sup>2</sup> can be transferred to other crops in the cropping system, especially non-N<sup>2</sup> -fixing plants. The transfer is accomplished through three main routes, viz.: decomposition of nodules and secondary roots that are not thickened, exudates of soluble N compounds, and transfer mediated by mycorrhizal fungi [1, 41–43]. The transfer of N through nodule and root decomposition and exudation of N compounds is termed as rhizodeposition [44]. The proportion of biologically fixed N<sup>2</sup> transferred to neighboring or succeeding crop plants is highly variable [45]. This can range from as low as 0% to as high as 73%, depending on a myriad of factors [1]. In an extensive review, rhizodeposition was reported to vary from 4 to 71% [44]. Review of literature from 2015 to 2017 on transfer of N in selected crops has shown that N transfer ranged from 0 to 70% (**Table 2**). Among the three main N transfer routes, rhizodeposition through decomposition of the nodules and roots represents the main pathway of N transfer.

from N<sup>2</sup>

non-N<sup>2</sup>

form of N<sup>2</sup>

with non-N<sup>2</sup>

fixing crop to the non-N<sup>2</sup>

dients [47], close proximity between N<sup>2</sup>

compared to 2 m from the shelterbelt.



plant organs or parts, not all the N<sup>2</sup>

**5. Factors affecting nitrogen transfer**

transfer of N from N<sup>2</sup>

5.4 to 15.7% [49]. Proportion of fixed N<sup>2</sup>


transferred from faba bean to wheat (*Triticum aestivum*

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103

fixed by plants will be transferred to neighboring plants


and uptake

fixation and transfer. For




cropping study, arbuscular mycorrhizal fungi (AMF) inoculation increased N transfer from

L.) was 50% when inoculated with AMF compared 15% in uninoculated stands [52]. Similar results were also reported in garden pea-barley (*Hordeum vulgare* L.) and soybean-maize (*Zea mays* L.) intercropping studies [48, 53]. The AMF-mediated transfer of N can be both unidirectional and bidirectional [48, 54] and often along with a concentration gradient [47]. Thus,

It has long been acknowledged that since plant N composition is partitioned into various

or succeeding plants in cropping systems [56]. However, there are a number of biotic and abiotic factors influencing N transfer in agricultural production systems [1]. Environmental factors such as water, temperature, and light have direct and indirect effects on N transfer in cropping systems. Soil moisture has a great influence on decomposition and it is required for

of mineralized N by plants. However, moisture stress promotes nodule senescence, implying that more nodule biomass will be available for mineralization during moisture stress conditions [57]. Nitrogen is highly soluble. Thus, excess water can result in N leaching out of the rooting zone of plants making it unavailable for uptake. Flooding (e.g., low land rice production systems) results in anaerobic conditions, and thus could result in gaseous N losses in the

example, nodule activity and N exudation from roots of soybean and sesbania (*Sesbania cannabina* (Retz.) Poir.) were the greatest at 30 and 35°C day and night temperatures, respectively [58]. Prolonged dark treatment affected nodule functioning in barrel medic (*Medicago truncatula* Gaertn.) and induced nodule senescence [59]. This condition is common in intercropping systems (e.g., grass-legume mixtures) [1], especially in species with varied canopy heights.

(e.g., forages) and in developing countries. In intercropping systems, the proximity of the N<sup>2</sup>

N in the rhizosphere is the greatest closer to the root surface [60]. Therefore, N transfer predominantly occurs in upper soil layers [23]. Since N uptake is along with concentration gra-

distance of travel for dissolved N compounds [1]. Close proximity is achieved either through direct root contact or mycorrhizal hyphae connections [61]. However, Issah et al. [62] reported that maximum oat productivity was obtained when grown 4 m from caragana shelterbelt

O [18]. Optimum light conditions (quality, quantity, and duration) and temperature




the uptake of N. Thus, moisture stress affects both the mineralization of fixed N<sup>2</sup>

have a direct effect on photosynthesis and hence, promote both N<sup>2</sup>

A common practice in agricultural production systems is intercropping N<sup>2</sup>



Nitrogen transfer from signal grass (*Brachiaria decumbens* Stapf.) to stylo (*Stylosanthes guianensis* (Aublet) Sw.) was reported to be mainly through decomposition of roots compared to root exudates and transfer mediated by mycorrhizae [46]. This might be particularly true for forage species since aboveground biomass is the economic part of the plant. Additionally, non-tree legumes have relatively greater proportion of fine roots that have faster turnover rate. It must be noted that despite the greater contribution of decomposition of the nodules and roots to N transfer, this transfer route is relatively slower compared to exudates of soluble N compounds and transfer mediated by mycorrhizae [1]. Nitrogen transfer from the tropical legume, gliricidia (*Gliricidia sepium* (Jacq.) Kunth ex Walp.) to yellow-blue stem (*Dichanthium aristatum* (Poir.) C.E. Hubb.) was reported to be mainly via root exudates [47]. In a short-term rhizodeposition study, 3.5 and 5.3% N was rhizodeposited through root exudates in white clover monocrop and white clover-perennial ryegrass mixture, respectively, over a 3-day period [42]. This significant N transfer within a short period is an indication of the importance of exudation of N compounds in meeting N needs of crops, especially during early growing stages [42]. It is well documented that mycorrhizae can facilitate the transfer of biologically fixed N<sup>2</sup>


**Table 2.** Amount of nitrogen (N) fixed and proportion transferred to soil or neighboring plants in agricultural systems.

from N<sup>2</sup> -fixers to non-N<sup>2</sup> -fixing plants [48–51]. In a rice and mung bean (*Vigna radiata* L.) intercropping study, arbuscular mycorrhizal fungi (AMF) inoculation increased N transfer from 5.4 to 15.7% [49]. Proportion of fixed N<sup>2</sup> transferred from faba bean to wheat (*Triticum aestivum* L.) was 50% when inoculated with AMF compared 15% in uninoculated stands [52]. Similar results were also reported in garden pea-barley (*Hordeum vulgare* L.) and soybean-maize (*Zea mays* L.) intercropping studies [48, 53]. The AMF-mediated transfer of N can be both unidirectional and bidirectional [48, 54] and often along with a concentration gradient [47]. Thus, transfer of N from N<sup>2</sup> -fixing plants to non-N<sup>2</sup> -fixers is often expected to be greater than from non-N<sup>2</sup> -fixing plants to N<sup>2</sup> -fixers [55].

## **5. Factors affecting nitrogen transfer**

fixed N<sup>2</sup>

102 Nitrogen in Agriculture - Updates

transferred to neighboring or succeeding crop plants is highly variable [45]. This can

range from as low as 0% to as high as 73%, depending on a myriad of factors [1]. In an extensive review, rhizodeposition was reported to vary from 4 to 71% [44]. Review of literature from 2015 to 2017 on transfer of N in selected crops has shown that N transfer ranged from 0 to 70% (**Table 2**). Among the three main N transfer routes, rhizodeposition through decompo-

Nitrogen transfer from signal grass (*Brachiaria decumbens* Stapf.) to stylo (*Stylosanthes guianensis* (Aublet) Sw.) was reported to be mainly through decomposition of roots compared to root exudates and transfer mediated by mycorrhizae [46]. This might be particularly true for forage species since aboveground biomass is the economic part of the plant. Additionally, non-tree legumes have relatively greater proportion of fine roots that have faster turnover rate. It must be noted that despite the greater contribution of decomposition of the nodules and roots to N transfer, this transfer route is relatively slower compared to exudates of soluble N compounds and transfer mediated by mycorrhizae [1]. Nitrogen transfer from the tropical legume, gliricidia (*Gliricidia sepium* (Jacq.) Kunth ex Walp.) to yellow-blue stem (*Dichanthium aristatum* (Poir.) C.E. Hubb.) was reported to be mainly via root exudates [47]. In a short-term rhizodeposition study, 3.5 and 5.3% N was rhizodeposited through root exudates in white clover monocrop and white clover-perennial ryegrass mixture, respectively, over a 3-day period [42]. This significant N transfer within a short period is an indication of the importance of exudation of N compounds in meeting N needs of crops, especially during early growing stages [42]. It is well documented that mycorrhizae can facilitate the transfer of biologically fixed N<sup>2</sup>

sition of the nodules and roots represents the main pathway of N transfer.

**Crop(s) Amount of N transferred** 

na, could not be estimated from data.

Cumulative over 3-year period.

4 m distance from caragana shelterbelt.

§

†

Caragana (*Caragana arborescens* Lam.)-oat (*Avena sativa* L.) 38–45 kg ha−1 (60–70)§ [62] Alfalfa-tall fescue (*Schedonorus arundinaceus* (Schreb.) Dumort.) 0–650 kg ha−1 (0–12)† [66] White clover-perennial ryegrass 0–340 kg ha−1 (0–47)† [66] Mung bean-oat 12.8 mg plant−1 (9.7) [68] Soybean-maize 7.84 mg pot−1 (7.57) [53] Soybean-maize 10.77–13.72 mg pot−1 (1.26–2.17) [55] Faba bean-wheat 0.17 mg plant shoot−1 (14.9) [52] Red clover-bluegrass (*Poa pratensis* L.) 35.85 mg plant−1 (1.5) [24] Pigeon pea (*Cajanus cajan* (L.) Millsp.-coffee (*Coffea arabica* L.) 21.8 g kg−1 (na) [63] Crotalaria-coffee 13.5 g kg−1 (na) [63] Velvet bean (*Mucuna pruriens* (L.) DC.)-coffee 19.7 g kg−1 (na) [63] Red clover-perennial ryegrass and forbs 25–58 kg ha−1 (9.5–15) [23]

**Table 2.** Amount of nitrogen (N) fixed and proportion transferred to soil or neighboring plants in agricultural systems.

**(% of fixed N)**

**Reference(s)**

It has long been acknowledged that since plant N composition is partitioned into various plant organs or parts, not all the N<sup>2</sup> fixed by plants will be transferred to neighboring plants or succeeding plants in cropping systems [56]. However, there are a number of biotic and abiotic factors influencing N transfer in agricultural production systems [1]. Environmental factors such as water, temperature, and light have direct and indirect effects on N transfer in cropping systems. Soil moisture has a great influence on decomposition and it is required for the uptake of N. Thus, moisture stress affects both the mineralization of fixed N<sup>2</sup> and uptake of mineralized N by plants. However, moisture stress promotes nodule senescence, implying that more nodule biomass will be available for mineralization during moisture stress conditions [57]. Nitrogen is highly soluble. Thus, excess water can result in N leaching out of the rooting zone of plants making it unavailable for uptake. Flooding (e.g., low land rice production systems) results in anaerobic conditions, and thus could result in gaseous N losses in the form of N<sup>2</sup> O [18]. Optimum light conditions (quality, quantity, and duration) and temperature have a direct effect on photosynthesis and hence, promote both N<sup>2</sup> fixation and transfer. For example, nodule activity and N exudation from roots of soybean and sesbania (*Sesbania cannabina* (Retz.) Poir.) were the greatest at 30 and 35°C day and night temperatures, respectively [58]. Prolonged dark treatment affected nodule functioning in barrel medic (*Medicago truncatula* Gaertn.) and induced nodule senescence [59]. This condition is common in intercropping systems (e.g., grass-legume mixtures) [1], especially in species with varied canopy heights.

A common practice in agricultural production systems is intercropping N<sup>2</sup> -fixing legumes with non-N<sup>2</sup> -fixing crops (**Figure 3**) [15]. This is particularly important in low value crops (e.g., forages) and in developing countries. In intercropping systems, the proximity of the N<sup>2</sup> fixing crop to the non-N<sup>2</sup> -fixing determines the amount of N transferred. The concentration of N in the rhizosphere is the greatest closer to the root surface [60]. Therefore, N transfer predominantly occurs in upper soil layers [23]. Since N uptake is along with concentration gradients [47], close proximity between N<sup>2</sup> -fixing legumes and non-N<sup>2</sup> -fixing crops reduces the distance of travel for dissolved N compounds [1]. Close proximity is achieved either through direct root contact or mycorrhizal hyphae connections [61]. However, Issah et al. [62] reported that maximum oat productivity was obtained when grown 4 m from caragana shelterbelt compared to 2 m from the shelterbelt.

Aside proximity, species (**Table 2**) of N<sup>2</sup> -fixing legumes as well as the non-N<sup>2</sup> -fixing crops (when grown in mixtures) influence the amount of N<sup>2</sup> fixed and transferred to neighboring crops. The amount of N transferred to Arabian coffee (*Coffea arabica* L.) ranged from 13.5 to 21.8 g kg−1 depending on the N<sup>2</sup> -fixing legume (**Table 2**) [63]. There was no observable N transfer from berseem clover (*Trifolium alexandrinum* L.) to annual ryegrass (*L. perenne* L. subsp. *multiflorum* [Lam.] Husnot) when gown in mixtures [64]. This was attributed to the greater efficiency of annual ryegrass in the uptake of available soil N which resulted in berseem clover becoming reliant on fixed N<sup>2</sup> [64]. In an alfalfa-Bermudagrass (*Cynodon dactylon* (L.) Pers.) intercrop, alfalfa fixed 80 to 222 kg N ha−1 year−1 and transferred about 18 kg N ha−1 year−1 to Bermudagrass [65]. Alfalfa fixed twice as much N as white clover but transferred only 59 kg N ha−1 compared to 147 kg N ha−1 transferred by white clover over a 3-year period [66]. Although decomposed alfalfa roots released greater N than that of birdsfoot trefoil, the opposite was true for decomposed nodules [41]. There was no transfer of N from any of seven legumes [snail medick (*M. scutellata* L.), common vetch (*V. sativa* L.), squarrosum clover (*T. squarrosum* L.), hairy vetch (*V. villosa* Roth), sulla (*Hedysarum coronarium* L.), and fenugreek (*Trigonella foenum-graecum* L.)] to annual ryegrass under Mediterranean conditions [67]. N transfer is also influenced by crop cultivars. For example, red clover cultivars differed in amount of N transferred to Kentucky bluegrass [24]. Compatibility of species grown in mixed swards affects the amount of N<sup>2</sup> fixed and the proportion transferred. A recent study has shown that grass N demand in grass-legume mixtures might be more important than legume N supply in determining N transfer efficiency [26].

contribution of N<sup>2</sup>

plants. Proportion of fixed N<sup>2</sup>

controlled for improved N<sup>2</sup>

optimal legume proportions.

**Author details**

**References**

grass-legume systems for optimal N<sup>2</sup>

M. Anowarul Islam\* and Albert Tetteh Adjesiwor

\*Address all correspondence to: mislam@uwyo.edu

Department of Plant Sciences, University of Wyoming, Laramie, WY, USA

to cropland. Environmental Research Letters. 2014;**9**:1-9

N2

fixation to the global N budget is enormous. Though N<sup>2</sup>

transferred to non-N<sup>2</sup>

importance is the low and highly variable proportion of fixed N<sup>2</sup>

lentils, common bean, faba bean, cowpea, chickpeas, and groundnut is dwarfed by soybean (because of the larger area planted to soybean) based on these estimates, the contribution of

to as high as 70%, depending on a myriad of factors. This was not different than the range of values reported from previous reviews. However, most of the factors (e.g., N fertilizer application, species, and cultivar selection) are management related and can, therefore, be

and due to this specialization, inoculation with the right strains of *Rhizobia* would improve N<sup>2</sup> fixation. One of the constraints to BNF in grass-legume mixtures is low proportion of legumes in the mixtures. It is, therefore, important to maintain sufficient legume populations in the

because of the selective grazing of legumes by livestock (in grazing systems). Nonetheless, using optimal seed mass ratios and good grazing and haying practices may help maintain

[1] Thilakarathna MS, McElroy MS, Chapagain T, Papadopoulos YA, Raizada MN. Belowground nitrogen transfer from legumes to non-legumes under managed herbaceous cropping systems. A review. Agronomy for Sustainable Development. 2016;**36**:1-16

[2] Lassaletta L, Billen G, Grizzetti B, Anglade J, Garnier J. 50 year trends in nitrogen use efficiency of world cropping systems: The relationship between yield and nitrogen input

[3] Unkovich M, Herridge D, Peoples M, Cadisch G, Boddey B, Giller K, Alves B, Chalk P. Measuring Plant-Associated Nitrogen Fixation in Agricultural Systems. Canberra, Australia: Australian Centre for International Agricultural Research (ACIAR); 2008. p. 258

 fixation from these crops (e.g., cowpea) to farmers in developing countries is substantial. Unlike forages, grains from grain legumes are harvested and removed from the field. Thus, grain legumes usually remove more soil N than forages. There are, however, several issues related to BNF that are of concern to the scientific community. Among the issues of great

fixation from peas,


105

transferred to non-N<sup>2</sup>

http://dx.doi.org/10.5772/intechopen.71766


Nitrogen Fixation and Transfer in Agricultural Production Systems

fixation and transfer. Most *Rhizobia* strains are highly specialized

fixation. This might, however, be difficult to achieve

Other factors such as age or stage of growth [68], season or year [69–71], proportion of N-fixing species [71], compatibility [45], and stand persistence [35] affect N transfer in cropping systems. For example, N in naked oats (*Avena nuda* L.) derived from N<sup>2</sup> fixed by mung bean was 7.6% at pod setting and increased to 9.7% at maturity [68]. The proportion of N transferred from red clover to Kentucky bluegrass was reported to have increased over time [24]. This is particularly true for perennial forages because of relatively low N<sup>2</sup> fixation in establishment year compared to well-established stands [1]. It is generally expected that as the proportion of legumes in mixed swards increases, N<sup>2</sup> fixation and transfer increases [1]. However, in a continental-scale field study with two perennial N<sup>2</sup> -fixing legumes (red clover and white clover) and four perennial grasses (perennial ryegrass, Timothy (*Phleum pratense* L.), Kentucky bluegrass, and orchardgrass (*Dactylis glomerata* L.)), it was reported that N gained in mixed swards increased with increasing legume proportion up to 30% [71]. This supports the assertion by [26] that grass N demand in grass-legume mixtures might be more important than legume N supply in determining N transfer efficiency. In an annual garden pea-barley intercropping system, greatest N transfer was obtained in 1:1 garden pea: barley compared to 2:1 system [72].

#### **6. Conclusions**

It is generally agreed that BNF is one of the most sustainable sources of N in agricultural production systems. The BNF is especially important in low value crops (e.g., forages) and in developing economies. Estimated N<sup>2</sup> fixation from selected crops showed that the contribution of N<sup>2</sup> fixation to the global N budget is enormous. Though N<sup>2</sup> fixation from peas, lentils, common bean, faba bean, cowpea, chickpeas, and groundnut is dwarfed by soybean (because of the larger area planted to soybean) based on these estimates, the contribution of N2 fixation from these crops (e.g., cowpea) to farmers in developing countries is substantial. Unlike forages, grains from grain legumes are harvested and removed from the field. Thus, grain legumes usually remove more soil N than forages. There are, however, several issues related to BNF that are of concern to the scientific community. Among the issues of great importance is the low and highly variable proportion of fixed N<sup>2</sup> transferred to non-N<sup>2</sup> -fixing plants. Proportion of fixed N<sup>2</sup> transferred to non-N<sup>2</sup> -fixing plants ranges from as low as 0% to as high as 70%, depending on a myriad of factors. This was not different than the range of values reported from previous reviews. However, most of the factors (e.g., N fertilizer application, species, and cultivar selection) are management related and can, therefore, be controlled for improved N<sup>2</sup> fixation and transfer. Most *Rhizobia* strains are highly specialized and due to this specialization, inoculation with the right strains of *Rhizobia* would improve N<sup>2</sup> fixation. One of the constraints to BNF in grass-legume mixtures is low proportion of legumes in the mixtures. It is, therefore, important to maintain sufficient legume populations in the grass-legume systems for optimal N<sup>2</sup> fixation. This might, however, be difficult to achieve because of the selective grazing of legumes by livestock (in grazing systems). Nonetheless, using optimal seed mass ratios and good grazing and haying practices may help maintain optimal legume proportions.

## **Author details**

Aside proximity, species (**Table 2**) of N<sup>2</sup>

21.8 g kg−1 depending on the N<sup>2</sup>

104 Nitrogen in Agriculture - Updates

ver becoming reliant on fixed N<sup>2</sup>

affects the amount of N<sup>2</sup>

**6. Conclusions**

in determining N transfer efficiency [26].

legumes in mixed swards increases, N<sup>2</sup>

tinental-scale field study with two perennial N<sup>2</sup>

and in developing economies. Estimated N<sup>2</sup>

(when grown in mixtures) influence the amount of N<sup>2</sup>



[64]. In an alfalfa-Bermudagrass (*Cynodon dactylon* (L.) Pers.)

fixed and the proportion transferred. A recent study has shown that

fixation and transfer increases [1]. However, in a con-


fixation from selected crops showed that the

crops. The amount of N transferred to Arabian coffee (*Coffea arabica* L.) ranged from 13.5 to

fer from berseem clover (*Trifolium alexandrinum* L.) to annual ryegrass (*L. perenne* L. subsp. *multiflorum* [Lam.] Husnot) when gown in mixtures [64]. This was attributed to the greater efficiency of annual ryegrass in the uptake of available soil N which resulted in berseem clo-

intercrop, alfalfa fixed 80 to 222 kg N ha−1 year−1 and transferred about 18 kg N ha−1 year−1 to Bermudagrass [65]. Alfalfa fixed twice as much N as white clover but transferred only 59 kg N ha−1 compared to 147 kg N ha−1 transferred by white clover over a 3-year period [66]. Although decomposed alfalfa roots released greater N than that of birdsfoot trefoil, the opposite was true for decomposed nodules [41]. There was no transfer of N from any of seven legumes [snail medick (*M. scutellata* L.), common vetch (*V. sativa* L.), squarrosum clover (*T. squarrosum* L.), hairy vetch (*V. villosa* Roth), sulla (*Hedysarum coronarium* L.), and fenugreek (*Trigonella foenum-graecum* L.)] to annual ryegrass under Mediterranean conditions [67]. N transfer is also influenced by crop cultivars. For example, red clover cultivars differed in amount of N transferred to Kentucky bluegrass [24]. Compatibility of species grown in mixed swards

grass N demand in grass-legume mixtures might be more important than legume N supply

Other factors such as age or stage of growth [68], season or year [69–71], proportion of N-fixing species [71], compatibility [45], and stand persistence [35] affect N transfer in cropping sys-

7.6% at pod setting and increased to 9.7% at maturity [68]. The proportion of N transferred from red clover to Kentucky bluegrass was reported to have increased over time [24]. This is

year compared to well-established stands [1]. It is generally expected that as the proportion of

and four perennial grasses (perennial ryegrass, Timothy (*Phleum pratense* L.), Kentucky bluegrass, and orchardgrass (*Dactylis glomerata* L.)), it was reported that N gained in mixed swards increased with increasing legume proportion up to 30% [71]. This supports the assertion by [26] that grass N demand in grass-legume mixtures might be more important than legume N supply in determining N transfer efficiency. In an annual garden pea-barley intercropping system, greatest N transfer was obtained in 1:1 garden pea: barley compared to 2:1 system [72].

It is generally agreed that BNF is one of the most sustainable sources of N in agricultural production systems. The BNF is especially important in low value crops (e.g., forages)

tems. For example, N in naked oats (*Avena nuda* L.) derived from N<sup>2</sup>

particularly true for perennial forages because of relatively low N<sup>2</sup>


fixed and transferred to neighboring

fixed by mung bean was

fixation in establishment

M. Anowarul Islam\* and Albert Tetteh Adjesiwor

\*Address all correspondence to: mislam@uwyo.edu

Department of Plant Sciences, University of Wyoming, Laramie, WY, USA

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[31] Ritchie ME, Raina R. Effects of herbivores on nitrogen fixation by grass endophytes, legume symbionts and free-living soil surface bacteria in the Serengeti. Pedobiologia.

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[33] Denton MD, Phillips LA, Peoples MB, Pearce DJ, Swan AD, Mele PM, Brockwell J.Legume inoculant application methods: Effects on nodulation patterns, nitrogen fixation, crop growth and yield in narrow-leaf lupin and faba bean. Plant and Soil. 2017;**419**:25-39 [34] Dey SK, Chakrabarti B, Prasanna R, Pratap D, Singh SD, Purakayastha TJ, Pathak H. Elevated carbon dioxide level along with phosphorus application and cyanobacterial inoculation enhances nitrogen fixation and uptake in cowpea crop. Archives of

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[41] Dubach M, Russelle MP. Forage legume roots and nodules and their role in nitrogen

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exudation by white clover and transfer to companion ryegrass of symbiotically fixed


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nitrogen fixation. Journal of Plant Nutrition and Soil Science. 2017;**180**:199-211

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**Section 2**

**Nitrogen and Crops**


**Section 2**

**Nitrogen and Crops**

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[60] zu Schweinsberg-Mickan MS, Joergensen RG, Müller T. Fate of <sup>13</sup>C- and <sup>15</sup>N-labelled rhizodeposition of *Lolium perenne* as function of the distance to the root surface. Soil Biology

[61] Jalonen R, Nygren P, Sierra J. Root exudates of a legume tree as a nitrogen source for a

[62] Issah G, Kimaro AA, Kort J, Knight JD. Nitrogen transfer to forage crops from a Caragana

[63] Mendonca ED, de Lima PC, Guimaraes GP, Moura WD, Andrade FV. Biological nitrogen fixation by legumes and N uptake by coffee plants. Revista Brasileira de Ciencia do

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[66] Louarn G, Pereira-Lopes E, Fustec J, Mary B, Voisin AS, Carvalho PCD, Gastal F. The amounts and dynamics of nitrogen transfer to grasses differ in alfalfa and white cloverbased grass-legume mixtures as a result of rooting strategies and rhizodeposit quality.

[67] Saia S, Urso V, Amato G, Frenda AS, Giambalvo D, Ruisi P, Di Miceli G. Mediterranean forage legumes grown alone or in mixture with annual ryegrass: Biomass production, N<sup>2</sup>

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communities including a deep-rooted legume or non-legume species. Plant and Soil.

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annual ryegrass under different managements. Plant and Soil. 2011;**342**:379-391 [65] Haby VA, Stout SA, Hons FM, Leonard AT. Nitrogen fixation and transfer in a mixed

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110 Nitrogen in Agriculture - Updates

N2

**Chapter 7**

**Provisional chapter**

**Field Scale Simulation of Nitrogen Dynamics Using**

Computer models have been used extensively to study the dynamics of nitrogen (N) at effluent‐irrigated land treatment systems (LTS). Nitrogen version of leaching estimation and chemistry (LEACHN) model and OVERSEER® are two such models that have the ability to simulate N movement through the soil‐water‐plant system. This chapter covers brief description of two models, that is, LEACHN and OVERSEER® that were used in this study. This is the third phase of previously conducted studies, and the focus of this third phase was (i) to use the LEACHN model (as optimised based on best N transformation rate constants in a previous study) to simulate N dynamics (under different irrigation sce‐ narios, that is, natural rainfall only, rainfall and irrigation with no N, and rainfall with irri‐ gation containing N) for the medium effluent irrigation treatment plot at an existing land disposal site and (ii) to use another model (i.e., OVERSEER®) to simulate N movement at the same land disposal site and compare its prediction with LEACHN model's predic‐ tions (for the low effluent irrigation treatment at the site). This study showed that the LEACHN model has the ability to simulate the fate and transport of N (under different irrigation scenarios) at field scale level. Also, OVERSEER® model could be used to simu‐ late N dynamics at an effluent‐irrigated land disposal site. The amount of N leached as predicted by OVERSEER® was reasonably close to LEACHN model predictions. **Keywords:** LEACHN model, OVERSEER® model, N dynamics at field scale level,

**Field Scale Simulation of Nitrogen Dynamics Using** 

DOI: 10.5772/intechopen.69100

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

© 2018 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

As we know that it is impractical to directly measure the nutrient losses to the environ‐ ment and therefore simulation models could be the best alternative to assess the potential loss of nutrients to the local environment. Over the past decade, the development and use

**LEACHN and OVERSEER® Models**

**LEACHN and OVERSEER® Models**

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.69100

Babar Mahmood

**Abstract**

nitrate‐N leaching

**1. Introduction**

Babar Mahmood

**Provisional chapter**

## **Field Scale Simulation of Nitrogen Dynamics Using LEACHN and OVERSEER® Models LEACHN and OVERSEER® Models**

**Field Scale Simulation of Nitrogen Dynamics Using** 

DOI: 10.5772/intechopen.69100

#### Babar Mahmood Additional information is available at the end of the chapter

Babar Mahmood

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.69100

#### **Abstract**

Computer models have been used extensively to study the dynamics of nitrogen (N) at effluent‐irrigated land treatment systems (LTS). Nitrogen version of leaching estimation and chemistry (LEACHN) model and OVERSEER® are two such models that have the ability to simulate N movement through the soil‐water‐plant system. This chapter covers brief description of two models, that is, LEACHN and OVERSEER® that were used in this study. This is the third phase of previously conducted studies, and the focus of this third phase was (i) to use the LEACHN model (as optimised based on best N transformation rate constants in a previous study) to simulate N dynamics (under different irrigation sce‐ narios, that is, natural rainfall only, rainfall and irrigation with no N, and rainfall with irri‐ gation containing N) for the medium effluent irrigation treatment plot at an existing land disposal site and (ii) to use another model (i.e., OVERSEER®) to simulate N movement at the same land disposal site and compare its prediction with LEACHN model's predic‐ tions (for the low effluent irrigation treatment at the site). This study showed that the LEACHN model has the ability to simulate the fate and transport of N (under different irrigation scenarios) at field scale level. Also, OVERSEER® model could be used to simu‐ late N dynamics at an effluent‐irrigated land disposal site. The amount of N leached as predicted by OVERSEER® was reasonably close to LEACHN model predictions.

**Keywords:** LEACHN model, OVERSEER® model, N dynamics at field scale level, nitrate‐N leaching

## **1. Introduction**

As we know that it is impractical to directly measure the nutrient losses to the environ‐ ment and therefore simulation models could be the best alternative to assess the potential loss of nutrients to the local environment. Over the past decade, the development and use

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons

of simulation models for predicting nutrient and pesticide behaviour in the root zone of agricultural land systems, and in the underlying unsaturated zone, have received consider‐ able attention. The models currently available for predicting the fate and transpiration of pesticides and nutrients in soils and groundwater have been critically reviewed and used, among others [1–7].

(as optimised using the best N transformation rate constant values in the previous study [14]) to refine our understanding of the predicted fate of N at the existing effluent‐irrigated land treat‐ ment site under different irrigation scenarios. Also, it was decided to use another model (i.e., OVERSEER®) to simulate N dynamics for the same site and then compare its predictions with LEACHN model's predictions for one of the effluent irrigation treatments (i.e., the low efflu‐ ent irrigation treatment for pasture plot was chosen). The details of optimisation of LEACHN model are not given here, and can be found in [14] as recently published. Thus, the specific objectives of this study were to (i) undertake irrigation scenario analysis (i.e., rainfall alone with no effluent irrigation, rainfall and irrigation of effluent containing no N, and rainfall and irriga‐ tion with effluent containing N) using the optimised model in order to understand the predicted fate of N added in effluent at a land disposal site and (ii) explore the use of OVERSEER® model at land treatment systems (LTS), and then compare its predictions of the amount of N leached

that the movement of N through the soil‐water matrix is a very complex process. Therefore, in terms of novelty, the work presented here in this chapter refines our knowledge and under‐ standing of N dynamics movement at a land treatment facility. This work explores and com‐ pares the abilities of two different models (i.e., LEACHN—a process‐based models, and OVERSEER®—a farm management tool), which has not been undertaken earlier. Further, the testing of OVERSEER® model for a LTS was challenging and it was done in an innovative way

As noted above, the work described in this study (i.e., *third* phase) covers two main areas.

**2.** To use the OVERSEER® model to simulate N movement at the CDC land disposal site.

LEACHN is the nitrogen version of the LEACHM model, which has evolved from model‐ ling efforts in the last 20 years and has successfully been used by several workers to describe nitrate and pesticide movement in the field [4, 14–19, 24]. It is a research model that can be used for management purposes. There are five modules of LEACHM. One of these modules, LEACHN, describes nitrogen transport and transformation and that was optimised in the second phase [14] of the study. The details of other modules and the soil hydrological proper‐ ties, interactions, transport and transformation of N in LEACHN are *not* provided here, as it

is covered in more detail in the second phase of this study [14, 24].

**1.** To use the optimised LEACHN model to gain a greater understanding of the dynamics of N within an existing effluent‐irrigated land disposal site in Carterton District Council

as this model is not designed to predict N leaching at a LTS (refer to section 2.4).

‐N concentrations with optimised LEACHN model predictions. As we know

Field Scale Simulation of Nitrogen Dynamics Using LEACHN and OVERSEER® Models

http://dx.doi.org/10.5772/intechopen.69100

115

and average NO<sup>3</sup>

**2. Material and methods**

**2.1. Model descriptions**

*2.1.1. LEACHN*

(CDC) of Wellington region, New Zealand.

Cichota and Snow [1] presented an overview of some models used for nutrient loss estimation at farm scale (including OVERSEER®, NPLAS—nitrogen and phosphorus load assessment system, SPASMO—soil‐plant‐atmosphere system model, EcoMod, LUCI—land use change and intensification, and APSIM—agricultural production systems simulator), and at large scales, that is, catchment level (including NLE—nitrogen leaching estimation, SPARROW spatial referenced regression on watershed attributes, ROTAN—rotorua and taupo nitrogen, CLUES—catchment land use and environmental sustainability, and AquiferSim).

They [1] also reviewed some soil process models such as groundwater loading effects of agriculture systems (GLEAMS), Leaching Estimation and Chemistry Mode (LEACHM) and HYDRUS. In their review, they said that most of the models have shown their usefulness to estimate nutrient losses in order to prevent environmental impacts at a small or larger scale levels. However, there is a *lack* of information about how these models work and what is their main focus, and therefore, it is important to know the main purpose, strengths and weaknesses of a model (before it is used to perform any kind of simulation) so that the most appropriate model could be selected.

It is well known that the simulation models are useful to understand the interaction between the transformation and transport of N in the field. The growing concern about the environ‐ mental impact of effluent‐irrigated systems has increased the desire to predict the transport and transformation of N in the soil‐plant system more accurately. Several models simulating N transformations and transport in the soil‐plant system have been developed and tested over time [2–4, 8–13]. Evaluating N transformation and transport models under field condi‐ tions is a complex research challenge. A major difficulty in evaluating these models under field conditions results from the strong interaction between physical and biological factors, plant uptake and N cycling processes.

In recent studies [14, 24], the nitrogen version of leaching estimation and chemistry (LEACHN) model was optimised and an in‐depth analysis of changing the N transformation rate constants (i.e., mineralisation—Kmin – day−1, nitrification—Knit – day−1 and denitrification—Kden – day−1) and bulk density on LEACHN model predictions was undertaken. The in‐depth analysis (based on N transformation rate constants) (i.e., second phase of the study) provided an in‐depth understanding of movement and distribution of effluent N down the profile at the studied land disposal site. The testing of the parameterised/optimised LEACHN model (as part of second phase of the study) showed that this model had the ability to predict the timing and amount of leachate nitrate‐N (NO<sup>3</sup> ‐N) concentrations at an effluent disposal site. The work reported here (i.e., *third* phase) is the continuation of a previous study [14]. The key research *questions* were: (i) Is LEACHN model able to simulate N dynamics (under different irrigation scenarios) at a land disposal site and (ii) Is it possible to use OVERSEER® model to simulate N movement at a land disposal site? Therefore, the *main* focus of this chapter was to use LEACHN model (as optimised using the best N transformation rate constant values in the previous study [14]) to refine our understanding of the predicted fate of N at the existing effluent‐irrigated land treat‐ ment site under different irrigation scenarios. Also, it was decided to use another model (i.e., OVERSEER®) to simulate N dynamics for the same site and then compare its predictions with LEACHN model's predictions for one of the effluent irrigation treatments (i.e., the low efflu‐ ent irrigation treatment for pasture plot was chosen). The details of optimisation of LEACHN model are not given here, and can be found in [14] as recently published. Thus, the specific objectives of this study were to (i) undertake irrigation scenario analysis (i.e., rainfall alone with no effluent irrigation, rainfall and irrigation of effluent containing no N, and rainfall and irriga‐ tion with effluent containing N) using the optimised model in order to understand the predicted fate of N added in effluent at a land disposal site and (ii) explore the use of OVERSEER® model at land treatment systems (LTS), and then compare its predictions of the amount of N leached and average NO<sup>3</sup> ‐N concentrations with optimised LEACHN model predictions. As we know that the movement of N through the soil‐water matrix is a very complex process. Therefore, in terms of novelty, the work presented here in this chapter refines our knowledge and under‐ standing of N dynamics movement at a land treatment facility. This work explores and com‐ pares the abilities of two different models (i.e., LEACHN—a process‐based models, and OVERSEER®—a farm management tool), which has not been undertaken earlier. Further, the testing of OVERSEER® model for a LTS was challenging and it was done in an innovative way as this model is not designed to predict N leaching at a LTS (refer to section 2.4).

## **2. Material and methods**

As noted above, the work described in this study (i.e., *third* phase) covers two main areas.


#### **2.1. Model descriptions**

#### *2.1.1. LEACHN*

of simulation models for predicting nutrient and pesticide behaviour in the root zone of agricultural land systems, and in the underlying unsaturated zone, have received consider‐ able attention. The models currently available for predicting the fate and transpiration of pesticides and nutrients in soils and groundwater have been critically reviewed and used,

Cichota and Snow [1] presented an overview of some models used for nutrient loss estimation at farm scale (including OVERSEER®, NPLAS—nitrogen and phosphorus load assessment system, SPASMO—soil‐plant‐atmosphere system model, EcoMod, LUCI—land use change and intensification, and APSIM—agricultural production systems simulator), and at large scales, that is, catchment level (including NLE—nitrogen leaching estimation, SPARROW spatial referenced regression on watershed attributes, ROTAN—rotorua and taupo nitrogen,

They [1] also reviewed some soil process models such as groundwater loading effects of agriculture systems (GLEAMS), Leaching Estimation and Chemistry Mode (LEACHM) and HYDRUS. In their review, they said that most of the models have shown their usefulness to estimate nutrient losses in order to prevent environmental impacts at a small or larger scale levels. However, there is a *lack* of information about how these models work and what is their main focus, and therefore, it is important to know the main purpose, strengths and weaknesses of a model (before it is used to perform any kind of simulation) so that the most

It is well known that the simulation models are useful to understand the interaction between the transformation and transport of N in the field. The growing concern about the environ‐ mental impact of effluent‐irrigated systems has increased the desire to predict the transport and transformation of N in the soil‐plant system more accurately. Several models simulating N transformations and transport in the soil‐plant system have been developed and tested over time [2–4, 8–13]. Evaluating N transformation and transport models under field condi‐ tions is a complex research challenge. A major difficulty in evaluating these models under field conditions results from the strong interaction between physical and biological factors,

In recent studies [14, 24], the nitrogen version of leaching estimation and chemistry (LEACHN) model was optimised and an in‐depth analysis of changing the N transformation rate constants (i.e., mineralisation—Kmin – day−1, nitrification—Knit – day−1 and denitrification—Kden – day−1) and bulk density on LEACHN model predictions was undertaken. The in‐depth analysis (based on N transformation rate constants) (i.e., second phase of the study) provided an in‐depth understanding of movement and distribution of effluent N down the profile at the studied land disposal site. The testing of the parameterised/optimised LEACHN model (as part of second phase of the study) showed that this model had the ability to predict the timing and amount of

(i.e., *third* phase) is the continuation of a previous study [14]. The key research *questions* were: (i) Is LEACHN model able to simulate N dynamics (under different irrigation scenarios) at a land disposal site and (ii) Is it possible to use OVERSEER® model to simulate N movement at a land disposal site? Therefore, the *main* focus of this chapter was to use LEACHN model

‐N) concentrations at an effluent disposal site. The work reported here

CLUES—catchment land use and environmental sustainability, and AquiferSim).

among others [1–7].

114 Nitrogen in Agriculture - Updates

appropriate model could be selected.

plant uptake and N cycling processes.

leachate nitrate‐N (NO<sup>3</sup>

LEACHN is the nitrogen version of the LEACHM model, which has evolved from model‐ ling efforts in the last 20 years and has successfully been used by several workers to describe nitrate and pesticide movement in the field [4, 14–19, 24]. It is a research model that can be used for management purposes. There are five modules of LEACHM. One of these modules, LEACHN, describes nitrogen transport and transformation and that was optimised in the second phase [14] of the study. The details of other modules and the soil hydrological proper‐ ties, interactions, transport and transformation of N in LEACHN are *not* provided here, as it is covered in more detail in the second phase of this study [14, 24].

## *2.1.2. OVERSEER®*

OVERSEER®is a decision support system (DSS) farm model used by farmers, advisors and policy makers. It estimates the nutrient budget for a farm taking into account all inputs and outputs (including internal cycling of nutrients around the farm) to and from the farm. This model is widely used in New Zealand as a decision support tool by consultants and regional councils. This model was based on the knowledge obtained primarily from New Zealand and in consultation with farmers and consultants, and therefore, it is well suited for handling management practices and environmental conditions specific to New Zealand. Initially, this model was used to examine the impacts of land use and management practices on nutrient losses as a way of estimating fertiliser requirements, but recently the model has been used to monitor farm nutrient losses as a tool for applying new environmental policies [20].

OVERSEER®has proved to be a very useful tool to examine nutrient use and flows within a farm (i.e., as products, fertiliser, effluent, supplements or transfer by animals) and to assess nutrient use efficiency and environmental impacts at the farm scale. It is an empirical model (i.e., based on observations or experiments) and empirical relationships, internal databases and readily available data from existing farms are used by the model to calculate nutrient budgets at a farm scale level [20, 21]. The overall model contains separate sub‐models deal‐ ing with pastoral, cropping and horticulture enterprises. Of these various sub‐models, the pastoral model is probably the most *robust* because of the large quantity of trial data that is available in New Zealand on grazed pasture systems.

The model is designed so that the input data required is meaningful to farmers and is easily available. This is in contrast to LEACHN, which is a data hungry model. For reliable perfor‐ mance, OVERSEER® requires that reasonable input data are given [21]. This means that the amount of fertiliser required to support the given level of production needs to be known. The model assumes that the system is in equilibrium (i.e., model does not account for transition during a change from one practice to another). The OVERSEER®model is designed to predict long‐term average behaviour of the system (as compared to LEACHN that can predict on a daily, weekly and monthly basis), and therefore, it is not suitable for extreme case scenarios or a system in transition, or for estimating nutrient losses from particular years.

In OVERSEER®,a *dairy* farm can be divided into blocks that can include effluent disposal and non‐effluent disposal blocks. This provided the opportunity to assess whether the OVERSEER® model could be used to simulate N leaching losses at a LTS by assuming that it was operating as an effluent‐irrigated block on a dairy farm. If the OVERSEER® model could be *used* at a LTS, this would have considerable advantages as many regional councils already understand and accept the principals and predictions from the OVERSEER®model.

45 (Med) and 100 (High) mm per week. The site was planted with pasture and short rotation trees, that is, *Eucalyptus ovata* and *Eucalyptus nitens* (refer to **Figures 1** and **2**). The total study

**Figure 2.** A layout map of the effluent‐irrigated land treatment site planted with trees and pasture, showing low, medium and high irrigations areas, and the locations of monitoring wells (sourced from Ref. [14], and as modified from

P P P

O N O

N O N

N N O

O O N

W3 High Medium Low

W2 W1

(i.e., one control plot of pasture and two plots of randomly planted trees; **Figure 2**). The loca‐

The experiment was conducted over the time period of 38 weeks (i.e., December 1997– August 1998). Soil moisture content (SMC) measurements were made using the time domain

per block) and each block has three plots

Pasture plots (Control)

http://dx.doi.org/10.5772/intechopen.69100

117

Field Scale Simulation of Nitrogen Dynamics Using LEACHN and OVERSEER® Models

Tree plots O = Ovata N = Nitens P = Pasture

W5

. There were three blocks (i.e., 180 m<sup>2</sup>

tion of monitoring wells is also shown in **Figures 1** and **2**.

**Figure 1.** The CDC effluent‐irrigated land disposal site.

North

W4

area was 540 m<sup>2</sup>

Refs. [15, 24]).

#### **2.2. Experimental site**

A brief detail of experimental site is provided here. Further details can be found in Refs. [14, 15]. A diagram and the layout map of the existing CDC land disposal site are shown in **Figures 1** and **2**. The site was located close to the Carterton's wastewater land treatment facility. An irrigation system was designed to apply effluent at three rates, that is, 30 (Low), Field Scale Simulation of Nitrogen Dynamics Using LEACHN and OVERSEER® Models http://dx.doi.org/10.5772/intechopen.69100 117

**Figure 1.** The CDC effluent‐irrigated land disposal site.

*2.1.2. OVERSEER®*

116 Nitrogen in Agriculture - Updates

OVERSEER®is a decision support system (DSS) farm model used by farmers, advisors and policy makers. It estimates the nutrient budget for a farm taking into account all inputs and outputs (including internal cycling of nutrients around the farm) to and from the farm. This model is widely used in New Zealand as a decision support tool by consultants and regional councils. This model was based on the knowledge obtained primarily from New Zealand and in consultation with farmers and consultants, and therefore, it is well suited for handling management practices and environmental conditions specific to New Zealand. Initially, this model was used to examine the impacts of land use and management practices on nutrient losses as a way of estimating fertiliser requirements, but recently the model has been used to

monitor farm nutrient losses as a tool for applying new environmental policies [20].

available in New Zealand on grazed pasture systems.

OVERSEER®has proved to be a very useful tool to examine nutrient use and flows within a farm (i.e., as products, fertiliser, effluent, supplements or transfer by animals) and to assess nutrient use efficiency and environmental impacts at the farm scale. It is an empirical model (i.e., based on observations or experiments) and empirical relationships, internal databases and readily available data from existing farms are used by the model to calculate nutrient budgets at a farm scale level [20, 21]. The overall model contains separate sub‐models deal‐ ing with pastoral, cropping and horticulture enterprises. Of these various sub‐models, the pastoral model is probably the most *robust* because of the large quantity of trial data that is

The model is designed so that the input data required is meaningful to farmers and is easily available. This is in contrast to LEACHN, which is a data hungry model. For reliable perfor‐ mance, OVERSEER® requires that reasonable input data are given [21]. This means that the amount of fertiliser required to support the given level of production needs to be known. The model assumes that the system is in equilibrium (i.e., model does not account for transition during a change from one practice to another). The OVERSEER®model is designed to predict long‐term average behaviour of the system (as compared to LEACHN that can predict on a daily, weekly and monthly basis), and therefore, it is not suitable for extreme case scenarios or

In OVERSEER®,a *dairy* farm can be divided into blocks that can include effluent disposal and non‐effluent disposal blocks. This provided the opportunity to assess whether the OVERSEER® model could be used to simulate N leaching losses at a LTS by assuming that it was operating as an effluent‐irrigated block on a dairy farm. If the OVERSEER® model could be *used* at a LTS, this would have considerable advantages as many regional councils already understand and

A brief detail of experimental site is provided here. Further details can be found in Refs. [14, 15]. A diagram and the layout map of the existing CDC land disposal site are shown in **Figures 1** and **2**. The site was located close to the Carterton's wastewater land treatment facility. An irrigation system was designed to apply effluent at three rates, that is, 30 (Low),

a system in transition, or for estimating nutrient losses from particular years.

accept the principals and predictions from the OVERSEER®model.

**2.2. Experimental site**

**Figure 2.** A layout map of the effluent‐irrigated land treatment site planted with trees and pasture, showing low, medium and high irrigations areas, and the locations of monitoring wells (sourced from Ref. [14], and as modified from Refs. [15, 24]).

45 (Med) and 100 (High) mm per week. The site was planted with pasture and short rotation trees, that is, *Eucalyptus ovata* and *Eucalyptus nitens* (refer to **Figures 1** and **2**). The total study area was 540 m<sup>2</sup> . There were three blocks (i.e., 180 m<sup>2</sup> per block) and each block has three plots (i.e., one control plot of pasture and two plots of randomly planted trees; **Figure 2**). The loca‐ tion of monitoring wells is also shown in **Figures 1** and **2**.

The experiment was conducted over the time period of 38 weeks (i.e., December 1997– August 1998). Soil moisture content (SMC) measurements were made using the time domain reflectometry (TDR) machine (prior to irrigation and then fortnightly) at 0–150, 0–300, and 0–450 mm soil depths in all pasture and tree plots. Soil‐water samples were collected using ceramic cups installed at 150, 300, and 450 mm depths before the first irrigation and then bi‐weekly. The groundwater samples were also collected before starting effluent irrigation and then bi‐weekly from the monitoring wells. All soil and water samples were analysed for NO<sup>3</sup> ‐N and ammonium‐N (NH<sup>4</sup> ‐N) using the standard methods of water analysis [22]. Please *refer* to [14, 24] for more experimental details.

irrigation treatment on the pasture plot at the CDC LTS. The reason for matching the applica‐

Field Scale Simulation of Nitrogen Dynamics Using LEACHN and OVERSEER® Models

http://dx.doi.org/10.5772/intechopen.69100

119

Although by manipulating the size of the notional effluent area, it was possible in OVERSEER® to adjust the rate of N application on the effluent block to a value similar to that was applied at the land disposal site for the low effluent irrigation pasture plot, there are differences between farm dairy effluent (FDE) and municipal sewage effluent in the concentration of total N (which affects the amount of water added per kg of N) and the ratio of organic to inorganic N. Fortunately, the OVERSEER® model can account for these differences through its capacity

FDE is more concentrated than the municipal sewage effluent at the CDC LTS. Its N concen‐ tration, on average, varies between 200 and 500 mg/L [23]. Also, it has been reported that organic N is the main component of total N in FDE (up to approximately 80% [23]). In this analysis, it was assumed that OVERSEER® would use an average total N concentration for

As noted above, the OVERSEER® model (version 6) has the capacity to specify an amount of irrigation on a monthly basis, and also to specify the nutrient concentration in that irriga‐ tion water. If a N concentration in the irrigation water is specified, the OVERSEER® model assumes that this N is in the inorganic form. By using this feature of the OVERSEER® model, it was possible to mimic the N application regime in the *low* effluent treatment at the CDC LTS exactly, in terms of the depth of water applied, the total N applied and the ratio of organic to

**1.** The quantities of water, organic N and inorganic N applied in the low effluent treatment

**2.** The application rate of FDE needed to apply the same amount of organic N as at the CDC LTS was then calculated (assuming that 80% of total N in FDE was organic N) and the "size" of the notional effluent area was adjusted so that quantity of organic N was being

**3.** This application rate of FDE was then supplying the correct amount of organic N, but the quantities of water and inorganic N applied were much less than at the CDC LTS. These

**4.** The amount of additional irrigation water required to match the total water applied in the

**5.** The inorganic N concentration in this irrigation water needed to ensure that the same quantities of inorganic N were applied as in the low effluent treatment at the CDC LTS

When setting up the notional effluent block in the OVERSEER® model, it was specified in the model that all the pasture grown on the effluent‐irrigated block was taken off as supplements

shortfalls were then added in the OVERSEER® model through irrigation.

low effluent treatment at the CDC LTS was then calculated.

in order to mimic the "cut & carry" system at the CDC LTS.

to apply irrigation water containing inorganic N, as well as applying FDE.

tion rates of organic N in this way is explained below.

FDE of 250 mg/L, of which 80% would be in organic form.

inorganic N. This was done as follows:

at the CDC LTS were calculated.

applied.

was then calculated.

## **2.3. Use of the optimised LEACHN model to explore the effect of effluent irrigation on the dynamics of water and N at the CDC LTS**

It was assumed that the values for Kmin, Knit and Kden (i.e., 0.02, 0.02 and 0.0000017 per day, respectively) *used* in the optimised LEACHN model [14] were adequate to describe the NO<sup>3</sup> ‐N concentrations at the CDC's land disposal site. The optimised model was then used to investi‐ gate in more detail the effect of adding effluent to the soil at the land disposal site. The *medium* irrigation treatment of the tree plot (refer to **Figure 2**) was only used for this analysis, and this treatment was compared first with the model predictions of what would have occurred at the site under natural rainfall with no added effluent and second with the model predictions of what would have occurred if the medium irrigation strategy had been followed, but with pure water, rather than N‐containing effluent. The chosen time period was 260 days (almost 38 weeks) to simulate N dynamics for the medium effluent irrigation treatment pasture plot at the land disposal site.

#### **2.4. The ability of the OVERSEER® nutrient budget model to predict N dynamics at the CDC LTS**

The OVERSEER® model was used to compare the amount of N applied and leached for the low application rate of effluent irrigation treatment on pasture plot (refer to **Figure 2**) at the CDC LTS. This part was interesting, innovative and bit *challenging* as the OVERSEER® model was not originally designed to predict N leaching from a LTS. But, as mentioned earlier that the OVERSEER® model does have the ability to simulate N leaching from effluent disposal areas on *dairy* farms. In doing this, the OVERSEER® model (version 6 that was used for the study) does not have a facility to enter the application rate of effluent N directly into the model. Instead, the OVERSEER® model calculates the application rate of effluent N from the number of dairy cows in the herd on the dairy farm and the area of the effluent disposal area. For a given number of cows, if the size of the effluent area is large, the application rate of effluent N per hectare will be low, and if the area of the effluent area is very small, then the application rate will be correspondingly high.

This feature of OVERSEER® provided the opportunity to use the model to simulate N leach‐ ing on an LTS (for the low effluent irrigation pasture plot) such as that at Carterton. This was done in the OVERSEER® model by setting up the CDC LTS as the effluent disposal block of a *notional* dairy farm. The notional "size" of the effluent block was adjusted so that the applica‐ tion rate of effluent organic N corresponded with the application rate of organic N in the low irrigation treatment on the pasture plot at the CDC LTS. The reason for matching the applica‐ tion rates of organic N in this way is explained below.

reflectometry (TDR) machine (prior to irrigation and then fortnightly) at 0–150, 0–300, and 0–450 mm soil depths in all pasture and tree plots. Soil‐water samples were collected using ceramic cups installed at 150, 300, and 450 mm depths before the first irrigation and then bi‐weekly. The groundwater samples were also collected before starting effluent irrigation and then bi‐weekly from the monitoring wells. All soil and water samples were analysed

**2.3. Use of the optimised LEACHN model to explore the effect of effluent irrigation on the** 

It was assumed that the values for Kmin, Knit and Kden (i.e., 0.02, 0.02 and 0.0000017 per day, respectively) *used* in the optimised LEACHN model [14] were adequate to describe the NO<sup>3</sup>

concentrations at the CDC's land disposal site. The optimised model was then used to investi‐ gate in more detail the effect of adding effluent to the soil at the land disposal site. The *medium* irrigation treatment of the tree plot (refer to **Figure 2**) was only used for this analysis, and this treatment was compared first with the model predictions of what would have occurred at the site under natural rainfall with no added effluent and second with the model predictions of what would have occurred if the medium irrigation strategy had been followed, but with pure water, rather than N‐containing effluent. The chosen time period was 260 days (almost 38 weeks) to simulate N dynamics for the medium effluent irrigation treatment pasture plot

**2.4. The ability of the OVERSEER® nutrient budget model to predict N dynamics at the** 

The OVERSEER® model was used to compare the amount of N applied and leached for the low application rate of effluent irrigation treatment on pasture plot (refer to **Figure 2**) at the CDC LTS. This part was interesting, innovative and bit *challenging* as the OVERSEER® model was not originally designed to predict N leaching from a LTS. But, as mentioned earlier that the OVERSEER® model does have the ability to simulate N leaching from effluent disposal areas on *dairy* farms. In doing this, the OVERSEER® model (version 6 that was used for the study) does not have a facility to enter the application rate of effluent N directly into the model. Instead, the OVERSEER® model calculates the application rate of effluent N from the number of dairy cows in the herd on the dairy farm and the area of the effluent disposal area. For a given number of cows, if the size of the effluent area is large, the application rate of effluent N per hectare will be low, and if the area of the effluent area is very small, then the

This feature of OVERSEER® provided the opportunity to use the model to simulate N leach‐ ing on an LTS (for the low effluent irrigation pasture plot) such as that at Carterton. This was done in the OVERSEER® model by setting up the CDC LTS as the effluent disposal block of a *notional* dairy farm. The notional "size" of the effluent block was adjusted so that the applica‐ tion rate of effluent organic N corresponded with the application rate of organic N in the low

‐N) using the standard methods of water analysis [22].

‐N

for NO<sup>3</sup>

118 Nitrogen in Agriculture - Updates

‐N and ammonium‐N (NH<sup>4</sup>

**dynamics of water and N at the CDC LTS**

application rate will be correspondingly high.

at the land disposal site.

**CDC LTS**

Please *refer* to [14, 24] for more experimental details.

Although by manipulating the size of the notional effluent area, it was possible in OVERSEER® to adjust the rate of N application on the effluent block to a value similar to that was applied at the land disposal site for the low effluent irrigation pasture plot, there are differences between farm dairy effluent (FDE) and municipal sewage effluent in the concentration of total N (which affects the amount of water added per kg of N) and the ratio of organic to inorganic N. Fortunately, the OVERSEER® model can account for these differences through its capacity to apply irrigation water containing inorganic N, as well as applying FDE.

FDE is more concentrated than the municipal sewage effluent at the CDC LTS. Its N concen‐ tration, on average, varies between 200 and 500 mg/L [23]. Also, it has been reported that organic N is the main component of total N in FDE (up to approximately 80% [23]). In this analysis, it was assumed that OVERSEER® would use an average total N concentration for FDE of 250 mg/L, of which 80% would be in organic form.

As noted above, the OVERSEER® model (version 6) has the capacity to specify an amount of irrigation on a monthly basis, and also to specify the nutrient concentration in that irriga‐ tion water. If a N concentration in the irrigation water is specified, the OVERSEER® model assumes that this N is in the inorganic form. By using this feature of the OVERSEER® model, it was possible to mimic the N application regime in the *low* effluent treatment at the CDC LTS exactly, in terms of the depth of water applied, the total N applied and the ratio of organic to inorganic N. This was done as follows:


When setting up the notional effluent block in the OVERSEER® model, it was specified in the model that all the pasture grown on the effluent‐irrigated block was taken off as supplements in order to mimic the "cut & carry" system at the CDC LTS.

The behaviour of N predicted by OVERSEER® for this farm scenario was then compared with the predictions of the LEACHN model. The annual rainfall data for the Carterton land treat‐ ment site were used in the scenarios.

## **3. Results and discussion**

#### **3.1. The ability of the LEACHN model to predict N dynamics at the CDC LTS**

This study used the optimised LEACHN model to investigate the fate of the added water and N in the effluent at the CTC LTS (**Table 1**). The model predicted that under natural rainfall and with no effluent irrigation (Scenario S1 in **Table 1**), there would be very little accumulated leachate from day 1 to 196. However, there was some leaching from day 210 onwards (a total of 177 mm during winter). The predicted leachate NO<sup>3</sup> ‐N concentrations were very low (<1 mg/L) and the cumulative mass of NO<sup>3</sup> ‐N leached for this scenario was <1 kg/ha (**Figure 3**) for the simulation period (260 days). The reason for these low predicted NO<sup>3</sup> ‐N concentra‐ tions in the drainage water is discussed later in this section. The predicted net N mineralised in scenario S1 was 6 kg/ha.

When LEACHN was used to simulate the addition of irrigation water containing no N at the medium effluent application rate (Scenario S2 in **Figure 3**), the irrigation caused drainage from day 28 onward (i.e., during summer). The accumulated drainage for sce‐ nario S2 was 1080 mm as compared to 177 mm for S1. The amount of water added (as rain‐ fall and irrigation water) in scenario S2 was 2256 mm as compared to 681 mm added in scenario S1 as rainfall only. In Scenario S2, the predicted leachate NO<sup>3</sup> ‐N concentrations were high (up to 32.0 mg/L) at the start of the drainage period but then dropped away to much lower concentrations (1 mg/L) towards the end of the simulation period (**Figure 3**). The cumulative mass of NO<sup>3</sup> ‐N leached was 20 kg/ha (refer to **Tables 1** and **2**; **Figures 3** and **4**), and net N mineralised was 9 kg/ha (i.e., 3 kg/ha more than when no irrigation water was applied).

When the normal N‐containing effluent was added (Scenario S3 in **Figure 3**) LEACHN pre‐ dicted that the cumulative drainage would be 1080 mm (the same as in scenario S2). The NO<sup>3</sup>

(S1—rainfall alone and no effluent irrigation, S2—rainfall and irrigation with effluent containing no N, and S3—rainfall

concentrations at the commencement of drainage were similar to those at the start of scenario S2 (32 mg/L) but then increased to a maximum of 68.6 mg/L after about 90 days. From then on,

was greater (i.e., 92 kg/ha—**Tables 1** and **2**; **Figure 4**) than that in scenario 2 (20 kg/ha; **Figure 4**).

which were similar to those in scenario S2 until about day 115. After this date, however, the

than in scenario S2 (20 kg/ha), the difference (i.e., 72 kg/ha) was much less than the amount of N added in the effluent (184 kg/ha; **Table 2**). The net mineralisation was predicted to be the same in both scenarios and so the remainder of the added N was divided between increased volatilisation and plant uptake, and a greater amount of inorganic N remaining in the soil

The predicted differences in plant N uptake and soil mineral N concentrations are interesting. It is apparent from **Tables 2** and **3**, and **Figure 5** that the LEACHN model predicts that the quantities of inorganic N in the profile will decrease during the period of the simulation particularly in scenarios S1 and S2. This is a result of plant uptake being greater than the

eralised was the same as in scenario 2 (i.e., 9 kg/ha) but the cumulative mass of NO<sup>3</sup>

arrival of the first of the effluent N at the drainage depth, that is, 500 mm.

‐N concentrations dropped steadily to a minimum value of 2.75 mg/L. The net N min‐

‐N concentrations over time was reflected in the cumulative loads of NO<sup>3</sup>

‐N in scenario S3 increased rapidly—presumably reflecting the

‐N would be leached in scenario S3 (92 kg/ha)

the NO<sup>3</sup>

The pattern of NO<sup>3</sup>

Pred. leachate nitrate-N

concentraion (mg/L)

7

**Figure 3.** The predicted leachate NO<sup>3</sup>

49

S1

91

133

S A W

175

and irrigation with effluent that contains N) using the LEACHN model.

217

Pred. nitrate-N concentration (mg/L)

259

35

77

S2

119

Time (days)

161

203

S=summer, A=autumn, W=winter

245

21

Field Scale Simulation of Nitrogen Dynamics Using LEACHN and OVERSEER® Models

63

‐N concentrations and cumulative drainage volume for three irrigation scenarios

105

S3

147

189

231

profile (**Table 2**).

cumulative load of leached NO<sup>3</sup>

Although LEACHN predicted that more NO<sup>3</sup>

‐N

‐N

‐N leached

0

200

400

Pred. cumulative drainage (mm)

121

600

800

1000

1200

1400

http://dx.doi.org/10.5772/intechopen.69100


**Table 1.** The amount drainage, NO<sup>3</sup> ‐N leached, and net N mineralised in response to three different irrigation scenarios, as calculated by LEACHN.

The behaviour of N predicted by OVERSEER® for this farm scenario was then compared with the predictions of the LEACHN model. The annual rainfall data for the Carterton land treat‐

This study used the optimised LEACHN model to investigate the fate of the added water and N in the effluent at the CTC LTS (**Table 1**). The model predicted that under natural rainfall and with no effluent irrigation (Scenario S1 in **Table 1**), there would be very little accumulated leachate from day 1 to 196. However, there was some leaching from day 210 onwards (a total

tions in the drainage water is discussed later in this section. The predicted net N mineralised

When LEACHN was used to simulate the addition of irrigation water containing no N at the medium effluent application rate (Scenario S2 in **Figure 3**), the irrigation caused drainage from day 28 onward (i.e., during summer). The accumulated drainage for sce‐ nario S2 was 1080 mm as compared to 177 mm for S1. The amount of water added (as rain‐ fall and irrigation water) in scenario S2 was 2256 mm as compared to 681 mm added in

were high (up to 32.0 mg/L) at the start of the drainage period but then dropped away to much lower concentrations (1 mg/L) towards the end of the simulation period (**Figure 3**).

and **4**), and net N mineralised was 9 kg/ha (i.e., 3 kg/ha more than when no irrigation

**‐N** 

**(kg/ha) (mm) (kg/ha)**

<1 177 6

20 1080 9

92 1080 9

‐N concentrations were very low

‐N concentra‐

‐N concentrations

‐N leached for this scenario was <1 kg/ha (**Figure 3**)

‐N leached was 20 kg/ha (refer to **Tables 1** and **2**; **Figures 3**

‐N leached, and net N mineralised in response to three different irrigation scenarios,

**Cumulative drainage Net N mineralised**

**3.1. The ability of the LEACHN model to predict N dynamics at the CDC LTS**

for the simulation period (260 days). The reason for these low predicted NO<sup>3</sup>

scenario S1 as rainfall only. In Scenario S2, the predicted leachate NO<sup>3</sup>

of 177 mm during winter). The predicted leachate NO<sup>3</sup>

(<1 mg/L) and the cumulative mass of NO<sup>3</sup>

in scenario S1 was 6 kg/ha.

The cumulative mass of NO<sup>3</sup>

**Irrigation scenarios Cumulative mass of NO3**

**leached**

water was applied).

S1—Just rainfall and no

S2—Rainfall and effluent

S3—Rainfall and effluent

as calculated by LEACHN.

**Table 1.** The amount drainage, NO<sup>3</sup>

effluent

with no N

with N

ment site were used in the scenarios.

**3. Results and discussion**

120 Nitrogen in Agriculture - Updates

**Figure 3.** The predicted leachate NO<sup>3</sup> ‐N concentrations and cumulative drainage volume for three irrigation scenarios (S1—rainfall alone and no effluent irrigation, S2—rainfall and irrigation with effluent containing no N, and S3—rainfall and irrigation with effluent that contains N) using the LEACHN model.

When the normal N‐containing effluent was added (Scenario S3 in **Figure 3**) LEACHN pre‐ dicted that the cumulative drainage would be 1080 mm (the same as in scenario S2). The NO<sup>3</sup> ‐N concentrations at the commencement of drainage were similar to those at the start of scenario S2 (32 mg/L) but then increased to a maximum of 68.6 mg/L after about 90 days. From then on, the NO<sup>3</sup> ‐N concentrations dropped steadily to a minimum value of 2.75 mg/L. The net N min‐ eralised was the same as in scenario 2 (i.e., 9 kg/ha) but the cumulative mass of NO<sup>3</sup> ‐N leached was greater (i.e., 92 kg/ha—**Tables 1** and **2**; **Figure 4**) than that in scenario 2 (20 kg/ha; **Figure 4**). The pattern of NO<sup>3</sup> ‐N concentrations over time was reflected in the cumulative loads of NO<sup>3</sup> ‐N which were similar to those in scenario S2 until about day 115. After this date, however, the cumulative load of leached NO<sup>3</sup> ‐N in scenario S3 increased rapidly—presumably reflecting the arrival of the first of the effluent N at the drainage depth, that is, 500 mm.

Although LEACHN predicted that more NO<sup>3</sup> ‐N would be leached in scenario S3 (92 kg/ha) than in scenario S2 (20 kg/ha), the difference (i.e., 72 kg/ha) was much less than the amount of N added in the effluent (184 kg/ha; **Table 2**). The net mineralisation was predicted to be the same in both scenarios and so the remainder of the added N was divided between increased volatilisation and plant uptake, and a greater amount of inorganic N remaining in the soil profile (**Table 2**).

The predicted differences in plant N uptake and soil mineral N concentrations are interesting. It is apparent from **Tables 2** and **3**, and **Figure 5** that the LEACHN model predicts that the quantities of inorganic N in the profile will decrease during the period of the simulation particularly in scenarios S1 and S2. This is a result of plant uptake being greater than the


**Table 2.** A quantitative N balance for day 252 for three irrigation scenarios, as calculated by LEACHN.

supply of inorganic N through mineralisation. By day 56 in scenario S1 (**Table 3**), the amount of inorganic N in the upper layers of the soil had been depleted but most of the inorganic N in the lower layers remained. In contrast, by day 56 in scenario S2, irrigation had moved inorganic N down the soil profile and some had leached beyond the root zone. As a result, inorganic N levels were low throughout the soil profile and plants were unable to access suf‐ ficient N for maximum growth. As a result, plant uptake by day 56 was predicted to be lower

(i.e., S1—just rainfall and no effluent irrigation, S2—rainfall and irrigation without N, and S3—rainfall and irrigation

**‐N for** 

 7.02 7.02 7.02 5.31 5.31 5.31 5.31 5.31 5.31 7.03 7.03 7.03

 2.94 0.47 4.85 4.97 1.50 6.56 5.71 2.94 7.90 6.61 4.36 9.09

 0.00 0.00 3.82 0.02 0.00 5.75 0.10 0.00 7.29 0.18 0.00 8.11

 0.00 0.00 0.81 0.00 0.00 1.10 0.00 0.03 1.18 0.00 0.05 1.21

**Soil NO3**

**) (mg/dm3**

**scenario S2**

Field Scale Simulation of Nitrogen Dynamics Using LEACHN and OVERSEER® Models

**‐N for** 

**Soil NO3**

http://dx.doi.org/10.5772/intechopen.69100

**) (mg/dm3**

) on day 0, 56, 126 and 252 for the three irrigation scenarios

**scenario S3**

**)**

**‐N for** 

123

**scenario S1**

0 100 6.63 6.63 6.63

56 100 1.44 0.12 4.16

126 100 0.00 0.00 2.90

252 100 0.00 0.00 0.68

‐N in the soil profile (mg/dm<sup>3</sup>

By day 126, virtually all the inorganic N in the profile in scenarios S1 and S2 had been exhausted by a combination of plant uptake (S1 and S2) and leaching (S2). As a result, the predicted plant

in scenario S2 than in scenario S1 (data not presented).

**Table 3.** The distribution of NO<sup>3</sup>

with effluent that contains N).

**Day no. Soil depth Soil NO3**

**(mm) (mg/dm3**

**Figure 4.** The cumulative mass of NO<sup>3</sup> ‐N leached over the simulation time period of 260 days for the three irrigation scenarios.



**Table 3.** The distribution of NO<sup>3</sup> ‐N in the soil profile (mg/dm<sup>3</sup> ) on day 0, 56, 126 and 252 for the three irrigation scenarios (i.e., S1—just rainfall and no effluent irrigation, S2—rainfall and irrigation without N, and S3—rainfall and irrigation with effluent that contains N).

supply of inorganic N through mineralisation. By day 56 in scenario S1 (**Table 3**), the amount of inorganic N in the upper layers of the soil had been depleted but most of the inorganic N in the lower layers remained. In contrast, by day 56 in scenario S2, irrigation had moved inorganic N down the soil profile and some had leached beyond the root zone. As a result, inorganic N levels were low throughout the soil profile and plants were unable to access suf‐ ficient N for maximum growth. As a result, plant uptake by day 56 was predicted to be lower in scenario S2 than in scenario S1 (data not presented).

Cumulative mass of nitrate-N

leached (kg/ha)

**Figure 4.** The cumulative mass of NO<sup>3</sup>

scenarios.

Initial NH<sup>4</sup>

Initial NO<sup>3</sup>

‐N in the soil

122 Nitrogen in Agriculture - Updates

‐N in the soil

NH<sup>4</sup>

NO<sup>3</sup>

NH<sup>4</sup>

NO<sup>3</sup>

NH<sup>4</sup> ‐N

NO<sup>3</sup> ‐N

Plant uptake of

Plant uptake of

profile

profile

0 20 40 60 80 100 120 140 160 180 200 220 240 260

‐N leached over the simulation time period of 260 days for the three irrigation

Time (days)

**Irrigation scenarios Units Rainfall alone (S1) Rainfall and irrigation** 

Total N applied (kg/ha) 0 0 184 Humus‐N<sup>i</sup> (kg/ha) 17,922 17,922 17,922 Humus‐N<sup>f</sup> (kg/ha) 17,916 17,913 17,913

‐N (kg/ha) 19 19 19

‐N (kg/ha) 31 31 31

‐N leached (kg/ha) 0.2 1 3

‐N leached (kg/ha) <1 20 92

Volatilised (kg/ha) <1 <1 34 Denitrified (kg/ha) 0.00 0.05 0.22 Cumulative leachate (mm) 177 1080 1080

**Table 2.** A quantitative N balance for day 252 for three irrigation scenarios, as calculated by LEACHN.

(kg/ha) 0.4 0.6 19

(kg/ha) 0.00 0.05 5

(kg/ha) 8 8 25

(kg/ha) 47 30 66

**without N (S2)**

**Rainfall and effluent irrigation with N (S3)**

Scenario S1 Scenario S2 Scenario S3

> By day 126, virtually all the inorganic N in the profile in scenarios S1 and S2 had been exhausted by a combination of plant uptake (S1 and S2) and leaching (S2). As a result, the predicted plant

**Input Unit CDC low effluent irrigation treatment OVERSEER® model**

**Total water** mm/year **1560 1560**

**Total organic N** kg/ha/year **131 132**

kg/ha/year **180 180**

**Table 4.** The amounts of water, inorganic and organic N that were applied as FDE and irrigation in the OVERSEER®

model to create a notional effluent disposal site similar to the low irrigation treatment pasture plot at CDC.

**Inputs Units LEACHN model OVERSEER® model**

1560 FDE application 67

Field Scale Simulation of Nitrogen Dynamics Using LEACHN and OVERSEER® Models

131 FDE application 132

180 FDE application 34

Irrigation 1493

http://dx.doi.org/10.5772/intechopen.69100

125

Irrigation 146

application

application

application

Inorganic N applied kg/ha 180 180 Organic N applied kg/ha (131) 132 N fixation kg/ha ‐ 2 Total N inputs kg/ha 311 313

N leached kg/ha 85 69 Plant N uptake kg/ha 132 133 Loss to atmosphere kg/ha 32 4 Total N outputs kg/ha 249 206

Soil inorganic N kg/ha −39 0 Soil organic N kg/ha (101) 107 Total change in soil N kg/ha (62) 107

Effluent and irrigation mm 1560 1560 Cumulative leachate mm 1629 1682

mg/L 5.2 4.1

**Table 5.** Comparison of the predictions from the LEACHN and OVERSEER® models in terms of N leached (kg/ha/year)

‐N concentrations (mg/L) when the amount of N applied is the same.

Water mm/year Effluent

Organic N kg/ha/year Effluent

Inorganic N kg/ha/year Effluent

**Total inorganic** 

**N**

**Outputs**

**Change in soil N**

Rainfall mm

‐N

**Water**

Average NO<sup>3</sup>

concentration

and average NO<sup>3</sup>

**Figure 5.** Predicted soil solution NO<sup>3</sup> ‐N concentrations down the soil profile on the starting day and day 252 for the three irrigation scenarios (i.e., S1—just rainfall and no effluent irrigation, S2—rainfall and irrigation without N, and S3—rainfall and irrigation with effluent that contains N).

N uptake in scenarios S1 and S2 was considerably less than in scenario S3 in which effluent containing N was irrigated on to the soil. Such a plant response to added N is commonly observed in New Zealand agricultural systems.

## **3.2. The ability of the OVERSEER® nutrient budget model to predict N dynamics at the CDC LTS**

The focus of this exercise was to explore if it is possible to use the OVERSEER® model (with some modifications to input parameters) to simulate N dynamics at an effluent‐irri‐ gated LTS (similar to CDC). As described in methodology section, the amounts of water, organic N and inorganic N applied in the low irrigation treatment of the pasture plot at CDC land disposal site (refer to **Table 4**) were entered into the OVERSEER® model as a notional combination of FDE and irrigation water containing a low concentration of inor‐ ganic N.

The outputs from the OVERSEER® model were compared (**Table 5**) with the outputs from the LEACHN model optimised as described in the previous section. Both models predicted similar annual total leachate (1629 mm and 1682 mm for the LEACHN and OVERSEER®models, respectively). The inputs of inorganic N in the applied effluent (and irrigation) were as measured at the CDC LTS and were therefore the same for both mod‐ els. Similarly, in both models, the plant uptake of N was set at 132 kg N/ha, which was measured at the CDC site. Given these similar inputs, the OVERSEER®model predicted a leaching loss of 69 kg N/ha, which was reasonably similar to the 85 kg N/ha predicted by the LEACHN model. The OVERSEER®model predicted very small emissions of N to the atmosphere (4 kg N/ha), whereas the LEACHN model predicted that 32 kg N/ha would be lost to the atmosphere. Most of the predicted emissions in the LEACHN model were as ammonia volatilisation.


**Table 4.** The amounts of water, inorganic and organic N that were applied as FDE and irrigation in the OVERSEER® model to create a notional effluent disposal site similar to the low irrigation treatment pasture plot at CDC.

N uptake in scenarios S1 and S2 was considerably less than in scenario S3 in which effluent containing N was irrigated on to the soil. Such a plant response to added N is commonly

three irrigation scenarios (i.e., S1—just rainfall and no effluent irrigation, S2—rainfall and irrigation without N, and

‐N concentrations down the soil profile on the starting day and day 252 for the

0 5 10 15 20 25 30 35 40

Pred. soil solution nitrate-N concentration (mg/L**)**

Scenario S1

Scenario S2

Scenario S3

**3.2. The ability of the OVERSEER® nutrient budget model to predict N dynamics at the** 

The focus of this exercise was to explore if it is possible to use the OVERSEER® model (with some modifications to input parameters) to simulate N dynamics at an effluent‐irri‐ gated LTS (similar to CDC). As described in methodology section, the amounts of water, organic N and inorganic N applied in the low irrigation treatment of the pasture plot at CDC land disposal site (refer to **Table 4**) were entered into the OVERSEER® model as a notional combination of FDE and irrigation water containing a low concentration of inor‐

The outputs from the OVERSEER® model were compared (**Table 5**) with the outputs from the LEACHN model optimised as described in the previous section. Both models predicted similar annual total leachate (1629 mm and 1682 mm for the LEACHN and OVERSEER®models, respectively). The inputs of inorganic N in the applied effluent (and irrigation) were as measured at the CDC LTS and were therefore the same for both mod‐ els. Similarly, in both models, the plant uptake of N was set at 132 kg N/ha, which was measured at the CDC site. Given these similar inputs, the OVERSEER®model predicted a leaching loss of 69 kg N/ha, which was reasonably similar to the 85 kg N/ha predicted by the LEACHN model. The OVERSEER®model predicted very small emissions of N to the atmosphere (4 kg N/ha), whereas the LEACHN model predicted that 32 kg N/ha would be lost to the atmosphere. Most of the predicted emissions in the LEACHN model were as

observed in New Zealand agricultural systems.

S3—rainfall and irrigation with effluent that contains N).

Soil depth (mm)

124 Nitrogen in Agriculture - Updates

**Figure 5.** Predicted soil solution NO<sup>3</sup>

**CDC LTS**

ganic N.

ammonia volatilisation.


**Table 5.** Comparison of the predictions from the LEACHN and OVERSEER® models in terms of N leached (kg/ha/year) and average NO<sup>3</sup> ‐N concentrations (mg/L) when the amount of N applied is the same.

The OVERSEER® model predicted that there would be no change in soil inorganic N over the year but that the organic N would increase by 107 kg N/ha. The LEACHN model predicted a decrease in inorganic N, but prediction of changes in organic N by the LEACHN model is diffi‐ cult. At the CDC LTS, the effluent applied contained both inorganic and organic N (**Table 4**) and the organic N was included in the inputs to the OVERSEER® model. It is, however, not easy to include inputs of organic N in the LEACHN model, and therefore only the inorganic N content of the CDC effluent was included in the LEACHN simulations described throughout the study.

**4.** The comparison of two different models showed the amount of N leached, as predicted by OVERSEER® model, was 69 kg/ha/year which was reasonably close to that of predicted by LEACHN model (85 kg/ha/year). The comparison showed that, depending on the level of precision required, the OVERSEER® model could be used to simulate N dynamics at a

**5.** The strength of LEACHN lies in its ability to predict what would happen to soil N down the profile when irrigation with or without N is added at a LTS and when, where and

is likely to happen. At the same time, it provides useful insights into the movements and transformation of soil and effluent N down the profile, which could be very helpful for the operators/managers of effluent land disposal facilities. Therefore, a LEACHN model could be a useful decision‐making tool to design effluent loading rates that minimise the risk of

‐N contamination at the effluent land disposal sites.

(a) In the simulations reported in this study, the focus in the LEACHN model was on the fate of inorganic N added in effluent. There is, however, the capacity within the LEACHN model to include the addition of plant residues, manure and urea. It would be useful in future studies to explore ways in which the ability of LEACHN to include different types

of N input could be used to mimic more exactly the management regimes at LTSs.

(b) The study demonstrated that when using the LEACHN‐based DSS to explore the con‐ sequences of different effluent application strategies care must be taken to ensure that realistic rainfall patterns are input into the model—rather than estimates of long‐term

Overall, the findings of this chapter suggest that LEACHN model has the ability to simulate N dynamics (i.e., movement and transformation down the soil profile) at effluent‐irrigated land disposal sites. In future, LEACHN‐based DSS could be used by LTS managers/environmental engineers/scientists to monitor and manage effluent‐irrigated land treatment sites in a way

I would like to acknowledge that a large portion of this chapter is based on one of the chapters of author's thesis [24]. Some modifications were made to fulfil the purpose of this chapter.

that reduces the risk of groundwater nitrate‐N contamination at the sites.

more accurate useful information on how much NO<sup>3</sup>

‐N is going to leach. It can be concluded that LEACHN model provides

Field Scale Simulation of Nitrogen Dynamics Using LEACHN and OVERSEER® Models

‐N is going to leach and when this

http://dx.doi.org/10.5772/intechopen.69100

127

LTS similar to CDC.

how much NO<sup>3</sup>

groundwater NO<sup>3</sup>

**4.1. Recommendations**

averages.

**4.2. Practicality**

**Acknowledgements**

In **Table 5**, a notional N balance was calculated for the LEACHN model by adding the input of organic N (number in brackets) and then calculating the final increase or decrease in organic N by difference. When this was done, the predicted increase in organic N was less than pre‐ dicted by the OVERSEER® model—reflecting the greater losses by leaching and volatilisation.

It would therefore appear that the existing OVERSEER® model can be adapted to simulate a LTS and gives a similar prediction to a more detailed process‐based model such as LEACHN. This could be of value because regional councils are increasingly recognising the use of OVERSEER® as a monitoring tool to demonstrate compliance with environmental regulations.

## **4. Summary and conclusions**

This study was about the application of two models (i.e., OVERSEER® and LEACHN) to predict the fate of water and N dynamics at an effluent‐irrigated land disposal facility, and therefore the following conclusions could be drawn from this piece of work.


#### **4.1. Recommendations**

The OVERSEER® model predicted that there would be no change in soil inorganic N over the year but that the organic N would increase by 107 kg N/ha. The LEACHN model predicted a decrease in inorganic N, but prediction of changes in organic N by the LEACHN model is diffi‐ cult. At the CDC LTS, the effluent applied contained both inorganic and organic N (**Table 4**) and the organic N was included in the inputs to the OVERSEER® model. It is, however, not easy to include inputs of organic N in the LEACHN model, and therefore only the inorganic N content of the CDC effluent was included in the LEACHN simulations described throughout the study. In **Table 5**, a notional N balance was calculated for the LEACHN model by adding the input of organic N (number in brackets) and then calculating the final increase or decrease in organic N by difference. When this was done, the predicted increase in organic N was less than pre‐ dicted by the OVERSEER® model—reflecting the greater losses by leaching and volatilisation. It would therefore appear that the existing OVERSEER® model can be adapted to simulate a LTS and gives a similar prediction to a more detailed process‐based model such as LEACHN. This could be of value because regional councils are increasingly recognising the use of OVERSEER® as a monitoring tool to demonstrate compliance with environmental regulations.

This study was about the application of two models (i.e., OVERSEER® and LEACHN) to predict the fate of water and N dynamics at an effluent‐irrigated land disposal facility, and

**1.** The irrigation scenario analysis showed that the LEACHN model is able to build up a very detailed picture of what is happening to soil N in the soil profile in response to three irri‐ gation scenarios, that is, no effluent application (i.e., just natural rainfall—S1), application of pure water (i.e., rainfall and effluent without N—S2) and effluent application with N composition (S3). This is very useful piece of information to understand the fate of water

**2.** The irrigation scenario (using the optimised LEACHN model) showed that the accumu‐ lated leachate was almost nil from day 1 to 196 under natural rainfall (with no N effluent irrigation). There was a total of 177 mm leaching during winter from day 210 onwards and

soil depth when more water (i.e., rainfall and irrigation with water containing no N) was added to the system and consequently the soil N was leached and therefore there was a lack of available soil N for plants uptake in irrigation scenario 2. The LEACHN model pre‐

tion would be significantly increased (up to 5.0 mg/L) in irrigation scenario 3 (i.e., rainfall

dicted that more nitrate would be in the soil profile and the soil solution NO<sup>3</sup>

‐N concentration were quite low (<1 mg/L) during that time. The accumulated

‐N leached in the first 177 mm of S1 and S2 was <1 and 20 kg/ha, respectively.

‐N concentrations did not change greatly in the top 500 mm

‐N concentra‐

therefore the following conclusions could be drawn from this piece of work.

**4. Summary and conclusions**

126 Nitrogen in Agriculture - Updates

and N added to a land disposal site.

**3.** The predicted soil solution NO<sup>3</sup>

and irrigation with effluent containing N).

leachate NO<sup>3</sup>

mass of NO<sup>3</sup>


#### **4.2. Practicality**

Overall, the findings of this chapter suggest that LEACHN model has the ability to simulate N dynamics (i.e., movement and transformation down the soil profile) at effluent‐irrigated land disposal sites. In future, LEACHN‐based DSS could be used by LTS managers/environmental engineers/scientists to monitor and manage effluent‐irrigated land treatment sites in a way that reduces the risk of groundwater nitrate‐N contamination at the sites.

## **Acknowledgements**

I would like to acknowledge that a large portion of this chapter is based on one of the chapters of author's thesis [24]. Some modifications were made to fulfil the purpose of this chapter.

I would like to express my sincere gratitude and indebtedness to Professor Russ Tillman (from Massey University, Palmerston North Campus) for his guidance, perceptive questioning and support during this research work. The completion of this study could not have been pos‐ sible without the friendship, inspiration and encouragement that I received from Professor Tillman and for these, I am particularly grateful. I would also like to thank Department of Civil Engineering, Unitec Institute of Technology for providing me with time and resources that I needed to complete this study.

[6] Donigian Jr AS, Rao PSC. Selection, application, and validation of environmental fate models. In: Proceedings of the International Symposium on Water Quality Modelling of Agricultural Non‐Point Sources. Part II, USDA‐ARS‐81, US Government Printing Office,

Field Scale Simulation of Nitrogen Dynamics Using LEACHN and OVERSEER® Models

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129

[7] Wagenet RJ, Rao PSC. Modelling pesticide fate in soils. In: Pesticides in the Environment: Processes, Impacts, and Modelling. Madison, WI: Soil Science Society of America; 1990

[8] Bryant JR, Snow VO, Cichota R, Jolly BH. The effect of situational variability in cli‐ mate and soil, choice of animal type and N fertilisation level on nitrogen leaching from pastoral farming systems around Lake Taupo, New Zealand. Agricultural Systems.

[9] Vanclooster M, Viaene P, Diels J, Freyen J. A deterministic evaluation analysis applied to

[10] Eckersten H, Jansson PE, Johnsson H. SOILN Model, User's Manual. 2nd ed. Division of Agricultural Hydrotechnics Communications. Vol. 94. Uppsala: Department of Soil

[11] Jabro JD, Jemison JM Jr, Lengnick LL, Fox RH, Fritton DD. Field validation and compari‐ son of LEACHM and NCSWAP models for predicting nitrate leaching. Transactions of

[12] Hutson JL, Wagenet RJ. LEACHM (Leaching Estimation and Chemistry Model): A pro‐ cess‐based model of water and solute movement, transformations, plant uptake and chemical reactions in the unsaturated zone. Version 3. Department of Soil, Crop and Atmospheric Sciences, Research Series No. 92‐93. Ithaca, New York: New York State

[13] Johnsson JL, Bergstrom L, Jansson PE, Paustian K. Simulated nitrogen dynamics and losses in a layered agriculture soil. Agriculture, Ecosystems and Environment.

[14] Mahmood B, Tillman R. Sensitivity of LEACHN model's predictions to changes to nitro‐ gen transformation rate constants and bulk density. Transactions of the American Society of Agricultural Engineers (ASAE). 2015;**58**(2):353‐366. DOI: 10.130331/trans.58.10636

[15] Mahmood B, Russell JM, Wall GL. Field scale nitrate simulations. Transactions of the

[16] Sogbedji JM, van Es HM, Hutson JL. N fate and transport under variable cropping history and fertiliser rate on loamy sand and clay loam soils: I. Calibration of the LEACHMN

[17] Ramos C, Carbonell EA. Nitrate leaching and soil moisture prediction with the LEACHM

American Society of Agricultural Engineers (ASAE). 2002;**45**(6):1835‐1842

an integrated soil‐crop model. Ecological Modelling. 1995;**81**:83‐195

American Society of Agricultural Engineers (ASAE). 1993;**36**:1651‐1657

College of Agriculture and Life Sciences, Cornell University; 1992

Science, Swedish University of Agricultural Sciences; 1994. p. 4

Washington, DC. 1990. pp. 557‐600

2011;**104**:271‐280

1987;**18**:333‐356

model. Plant and Soil. 2001;**229**(1):57‐70

model. Fertiliser Research. 1991;**27**(7):171‐180

I am grateful for the moral support and joyful company of my loving wife Saima Babar, my son Daniyal Mahmood, and my two lovely daughters Bismah Mahmood and Zoya Mahmood, who patiently shared in the sacrifices that were a necessary part of my research work. Finally, my deepest gratitude and thanks to my parents (late), brother and sisters whose love, patience and constant encouragement have been invaluable in my life.

## **Author details**

#### Babar Mahmood

Address all correspondence to: bmahmood@unitec.ac.nz

Department of Civil Engineering, Unitec Institute of Technology, Auckland, New Zealand

## **References**


[6] Donigian Jr AS, Rao PSC. Selection, application, and validation of environmental fate models. In: Proceedings of the International Symposium on Water Quality Modelling of Agricultural Non‐Point Sources. Part II, USDA‐ARS‐81, US Government Printing Office, Washington, DC. 1990. pp. 557‐600

I would like to express my sincere gratitude and indebtedness to Professor Russ Tillman (from Massey University, Palmerston North Campus) for his guidance, perceptive questioning and support during this research work. The completion of this study could not have been pos‐ sible without the friendship, inspiration and encouragement that I received from Professor Tillman and for these, I am particularly grateful. I would also like to thank Department of Civil Engineering, Unitec Institute of Technology for providing me with time and resources

I am grateful for the moral support and joyful company of my loving wife Saima Babar, my son Daniyal Mahmood, and my two lovely daughters Bismah Mahmood and Zoya Mahmood, who patiently shared in the sacrifices that were a necessary part of my research work. Finally, my deepest gratitude and thanks to my parents (late), brother and sisters whose love, patience

Department of Civil Engineering, Unitec Institute of Technology, Auckland, New Zealand

[1] Cichota R, Snow VO. Estimating nutrient loss to waterways — an overview of mod‐ els of relevance to New Zealand pastoral farms. New Zealand Journal of Agricultural

[2] Johnson IR, Chapman DF, Snow VO, Eckard RJ, Parsons AJ, Lambert MG, Cullen BR. DairyMod and EcoMod: Biophysical pasture simulation models for Australia and New

[3] Kunjikutty SP, Praher SO, Patel RM, Barrington SF, Kim SH. Simulation of nitrogen transport in soil under municipal wastewater application using LEACHN. Journal of

[4] Mahmood B. Analysis of a LEACHN‐based management technique in predictive mode.

[5] Nolan BT, Bayless ER, Green CT, Garg S, Voss FD, Lampe DC, Barbash JE, Capel PD, Barbara A. Evaluation of Unsaturated‐Zone Solute‐Transport Models for Studies of Agricultural Chemicals [Internet]. Reston, Virginia, USA: Geological Survey; 2005. Available from:

https://pubs.usgs.gov/of/2005/1196/ofr20051196.pdf [Accessed: 18 March 2016]

Zealand. Australian Journal of Experimental Agriculture. 2008;**48**:621‐631

the American Water Resources Association. 2007;**43**:1097‐1107

Agricultural Water Management. 2005;**75**:25‐37

that I needed to complete this study.

Research. 2009;**52**:239‐260

**Author details**

128 Nitrogen in Agriculture - Updates

Babar Mahmood

**References**

and constant encouragement have been invaluable in my life.

Address all correspondence to: bmahmood@unitec.ac.nz


[18] Wagenet RJ, Hutson JL. Predicting the fate of non‐volatile pesticides in the saturated zone. Journal of Environmental Quality. 1986;**15**:315‐322

**Chapter 8**

**Provisional chapter**

**Optimization of Nitrogen in Durum Wheat in the**

**Optimization of Nitrogen in Durum Wheat in the** 

**Greenhouse Gas (GHG) Emissions**

Luigi Tedone, Salem Alhajj Ali and Giuseppe

**Greenhouse Gas (GHG) Emissions**

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

Luigi Tedone, Salem Alhajj Ali and

http://dx.doi.org/10.5772/intechopen.70195

Giuseppe De Mastro

**Abstract**

application.

(GHG)

De Mastro

**Mediterranean Climate: The Agronomical Aspect and**

Durum wheat (*Triticum turgidum* L. subsp. *durum*) is the most cultivated cereal crop in the Mediterranean basin, traditionally grown under rainfed conditions using conventional tillage. Agronomical practices, soil type and climate variables are known to influence crop productivity. Their interaction effect is very complex and the time in which they occur strongly affects yield and quality. The nitrogen supply, in combination with climatic conditions, is the main constraint determining the physiological performance, grain yield and quality response of wheat. In addition, the N formulation, fertilizer management, crop sequence, seasonal trends, and the supply of residual and mineralized N influence the response of wheat to N fertilizer. N fertilizer management must be optimized to prevent N deficiency in the critical crop growth period, to avoid yield and quality losses and also prevent the excessive application of N fertilizer, thus reducing the environmental impact. The split application of N fertilizer is a promising strategy that satisfies plant needs and reduces N losses through improved nitrogen use efficiency (NUE). Such a strategy can result in a remarkable reduction in greenhouse gas (GHG) emissions and the carbon footprint of Italian durum wheat, considering that the highest proportion of the total emissions deriving from N fertilizer production and its

**Mediterranean Climate: The Agronomical Aspect and** 

DOI: 10.5772/intechopen.70195

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

© 2018 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

**Keywords:** durum wheat, nitrogen, nitrogen use efficiency, greenhouse gas emission


**Provisional chapter**

## **Optimization of Nitrogen in Durum Wheat in the Mediterranean Climate: The Agronomical Aspect and Greenhouse Gas (GHG) Emissions Mediterranean Climate: The Agronomical Aspect and Greenhouse Gas (GHG) Emissions**

**Optimization of Nitrogen in Durum Wheat in the** 

DOI: 10.5772/intechopen.70195

Luigi Tedone, Salem Alhajj Ali and Giuseppe De Mastro De Mastro Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

Luigi Tedone, Salem Alhajj Ali and Giuseppe

http://dx.doi.org/10.5772/intechopen.70195

#### **Abstract**

[18] Wagenet RJ, Hutson JL. Predicting the fate of non‐volatile pesticides in the saturated

[19] Wagenet RJ, Hutson JL, Biggar JW. Simulating the fate of a volatile pesticide in unsatu‐ rated soil: A case study with DBCP. Journal of Environmental Quality. 1989;**18**:78‐84 [20] Wheeler DM, Ledgard SF, Monaghan RM, McDowell R, deKlein CAM. Overseer® nutrient budget model—what is it, what it does. In: Proceedings of the Workshop on Implementing Sustainable Nutrient Management Strategies in Agriculture. Occasional Report No. 19. ISSN 0112‐9902. Palmerston North, New Zealand: Massey University;

[21] Wheeler DM. OVERSEER® scenarios—"what if" analysis. In: Nutrient Management in a Rapidly Changing World. Occasional Report No. 22. Palmerston North, New Zealand:

[22] Gillian G. Methods of Soil, Plant, and Water Analysis. Forest Research Institute Bulletin

[23] Longhurst RD, Roberts AHC, Connor MB. Farm dairy effluent: A review of published data on chemical and physical characteristics in New Zealand. New Zealand Journal of

[24] Mahmood B. Decision supports tools to reduce the risk of groundwater nitrate con‐ tamination at land treatment systems [PhD thesis]. Palmerston North Campus, New

Zealand: Institute of Engineering and Technology, Massey University; 2013

zone. Journal of Environmental Quality. 1986;**15**:315‐322

Fertiliser and Lime Research Centre, Massey University; 2009

No. 70. Rotorua, New Zealand: Forest Research Institute; 1984

2006. pp. 231‐236

130 Nitrogen in Agriculture - Updates

Agricultural Research. 2000;**43**:7‐14

Durum wheat (*Triticum turgidum* L. subsp. *durum*) is the most cultivated cereal crop in the Mediterranean basin, traditionally grown under rainfed conditions using conventional tillage. Agronomical practices, soil type and climate variables are known to influence crop productivity. Their interaction effect is very complex and the time in which they occur strongly affects yield and quality. The nitrogen supply, in combination with climatic conditions, is the main constraint determining the physiological performance, grain yield and quality response of wheat. In addition, the N formulation, fertilizer management, crop sequence, seasonal trends, and the supply of residual and mineralized N influence the response of wheat to N fertilizer. N fertilizer management must be optimized to prevent N deficiency in the critical crop growth period, to avoid yield and quality losses and also prevent the excessive application of N fertilizer, thus reducing the environmental impact. The split application of N fertilizer is a promising strategy that satisfies plant needs and reduces N losses through improved nitrogen use efficiency (NUE). Such a strategy can result in a remarkable reduction in greenhouse gas (GHG) emissions and the carbon footprint of Italian durum wheat, considering that the highest proportion of the total emissions deriving from N fertilizer production and its application.

**Keywords:** durum wheat, nitrogen, nitrogen use efficiency, greenhouse gas emission (GHG)

Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons

## **1. Introduction**

Wheat is one of the main sources of carbohydrate foods worldwide, and as such its cultivation is considered strategic, along with other important cereals grown widely across other continents [1].

use of chemicals. The study of energy systems has gained considerable importance in this regard, with the aim of reducing the use of fossil energy and, consequently, the emission of greenhouse gases into the atmosphere. The study of cropping systems that use improved crops and conservative systems becomes crucial for increasing the accumulation of organic matter in the soil.

Optimization of Nitrogen in Durum Wheat in the Mediterranean Climate: The Agronomical...

http://dx.doi.org/10.5772/intechopen.70195

133

The effect of climate change is of particular concern in agriculture, as it significantly affects crop yields and their inter-annual variability, and can potentially affect their traditional ter-

The phenomenon of climate change affects the whole planet but is very much observed at the Mediterranean basin level, where the climate is characterized by hot dry summers and mild wet winters. The main field crops are winter cereals, in particular durum wheat, which grows in the range of 200–600 mm per year [6]. Durum wheat and other cereals are typically sown in

The conditions for growth during this period are favorable for cereal crops, ensuring an optimal course of their cycle until the end of April. After this period, the phenomena of water and

Variable climatic conditions cause water stress with large fluctuations in both grain yield and in the physical property of the grain (hectoliter weight, 1000 seed weight), and also in grain protein content and composition, which has considerable effects on the rheological properties

Nitrogen fertilization is a particularly significant parameter in the technique of wheat cultivation, in terms of its effects on specific agronomic characteristics, the production level, and the quality

In the absence of other limiting factors, primarily the availability of water, wheat response to increasing nitrogen doses in terms of crop production and/or protein content is influenced by the level of chemical fertility of the soil [11]. The combined effect of nitrogen availability and

The effect of nitrogen and water deficiency is then evident in the subsequent phases of heading and spike emission/flowering, when stress conditions can cause an increase in flower

The effect of water stress, combined with high temperatures, contributes to determining the

In Mediterranean environments, the crop response to nitrogen fertilization is conditioned by climate change (precipitation during the March–May period), agronomic practice (normal or conservative soil management), the quantity and number of fertilizer operations, and the type

The effect of nitrogen availability, in optimum water conditions, is evident from the tillering phase. The combined effect of nitrogen and water deficiency is also evident in subsequent phases of heading and spikelet initiation/flowering, when stress conditions cause an

ritorial distribution.

of semolina [7–9].

and quantity of grain proteins [10].

abortions and a reduction in vegetation.

of fertilizer applied [12–14].

productivity and quality of wheat grain [10].

October to December and harvested in May to June.

thermal stress during the final filling-ripening stage are likely.

optimum water conditions is evident in the tillering phase.

From 1950 to 1990, the production increased exponentially due to the combined effect of genetic improvement and new agronomic techniques, which allowed the crop yield to increase massively [2].

This increase in production is strongly related to the use of nitrogen, which is essential for the life of the crop and is a limiting input factor for the crop productivity [3]. Since the 1960s, there has been a ninefold increase in the use of nitrogen, and this is expected to grow by another 40–50% over the next 40 years [4].

The application of nitrogen fertilizer greatly improves agricultural productivity but increases the risk of aquifer pollution, estimated in the EU alone to be by 20% [5], also due to inefficient use of the fertilizer.

Sustainable agriculture is a very broad concept and includes ecological, economic, and social aspects.

"Sustainable agriculture" definition was defined by Congress in the 1990 "Farm Bill," [Public Law 101–624, Title XVI, Subtitle A, Section 1603 (Government Printing Office, Washington, DC, 1990) NAL Call # KF1692.A31 1990]. Under that law, the term "sustainable agriculture" means an integrated system of plant and animal production practices having a site-specific application that will, over the long term:


The Food and Agriculture Organization [2] has developed a common vision and an integrated approach to sustainability across agriculture, forestry, and fisheries. This unified perspective, valid across all agricultural sectors and taking into account social, economic, and environmental considerations, ensures the effectiveness of action on the ground and is underpinned by knowledge based on the best available science. It can be adapted at community and country levels to ensure local relevance and applicability.

From an environmental point of view, sustainability means a respectful farming system in terms of the use of natural resources such as water, soil fertility, and biodiversity, and a reduction in the use of chemicals. The study of energy systems has gained considerable importance in this regard, with the aim of reducing the use of fossil energy and, consequently, the emission of greenhouse gases into the atmosphere. The study of cropping systems that use improved crops and conservative systems becomes crucial for increasing the accumulation of organic matter in the soil.

**1. Introduction**

132 Nitrogen in Agriculture - Updates

continents [1].

increase massively [2].

use of the fertilizer.

economy depends;

aspects.

40–50% over the next 40 years [4].

application that will, over the long term: • satisfy human food and fiber needs;

Wheat is one of the main sources of carbohydrate foods worldwide, and as such its cultivation is considered strategic, along with other important cereals grown widely across other

From 1950 to 1990, the production increased exponentially due to the combined effect of genetic improvement and new agronomic techniques, which allowed the crop yield to

This increase in production is strongly related to the use of nitrogen, which is essential for the life of the crop and is a limiting input factor for the crop productivity [3]. Since the 1960s, there has been a ninefold increase in the use of nitrogen, and this is expected to grow by another

The application of nitrogen fertilizer greatly improves agricultural productivity but increases the risk of aquifer pollution, estimated in the EU alone to be by 20% [5], also due to inefficient

Sustainable agriculture is a very broad concept and includes ecological, economic, and social

"Sustainable agriculture" definition was defined by Congress in the 1990 "Farm Bill," [Public Law 101–624, Title XVI, Subtitle A, Section 1603 (Government Printing Office, Washington, DC, 1990) NAL Call # KF1692.A31 1990]. Under that law, the term "sustainable agriculture" means an integrated system of plant and animal production practices having a site-specific

• enhance environmental quality and the natural resource base upon which the agricultural

• make the most efficient use of nonrenewable resources and on-farm resources and inte-

The Food and Agriculture Organization [2] has developed a common vision and an integrated approach to sustainability across agriculture, forestry, and fisheries. This unified perspective, valid across all agricultural sectors and taking into account social, economic, and environmental considerations, ensures the effectiveness of action on the ground and is underpinned by knowledge based on the best available science. It can be adapted at community and coun-

From an environmental point of view, sustainability means a respectful farming system in terms of the use of natural resources such as water, soil fertility, and biodiversity, and a reduction in the

grate, where appropriate, natural biological cycles and controls;

• enhance the quality of life for farmers and society as a whole.

• sustain the economic viability of farm operations; and

try levels to ensure local relevance and applicability.

The effect of climate change is of particular concern in agriculture, as it significantly affects crop yields and their inter-annual variability, and can potentially affect their traditional territorial distribution.

The phenomenon of climate change affects the whole planet but is very much observed at the Mediterranean basin level, where the climate is characterized by hot dry summers and mild wet winters. The main field crops are winter cereals, in particular durum wheat, which grows in the range of 200–600 mm per year [6]. Durum wheat and other cereals are typically sown in October to December and harvested in May to June.

The conditions for growth during this period are favorable for cereal crops, ensuring an optimal course of their cycle until the end of April. After this period, the phenomena of water and thermal stress during the final filling-ripening stage are likely.

Variable climatic conditions cause water stress with large fluctuations in both grain yield and in the physical property of the grain (hectoliter weight, 1000 seed weight), and also in grain protein content and composition, which has considerable effects on the rheological properties of semolina [7–9].

Nitrogen fertilization is a particularly significant parameter in the technique of wheat cultivation, in terms of its effects on specific agronomic characteristics, the production level, and the quality and quantity of grain proteins [10].

In the absence of other limiting factors, primarily the availability of water, wheat response to increasing nitrogen doses in terms of crop production and/or protein content is influenced by the level of chemical fertility of the soil [11]. The combined effect of nitrogen availability and optimum water conditions is evident in the tillering phase.

The effect of nitrogen and water deficiency is then evident in the subsequent phases of heading and spike emission/flowering, when stress conditions can cause an increase in flower abortions and a reduction in vegetation.

The effect of water stress, combined with high temperatures, contributes to determining the productivity and quality of wheat grain [10].

In Mediterranean environments, the crop response to nitrogen fertilization is conditioned by climate change (precipitation during the March–May period), agronomic practice (normal or conservative soil management), the quantity and number of fertilizer operations, and the type of fertilizer applied [12–14].

The effect of nitrogen availability, in optimum water conditions, is evident from the tillering phase. The combined effect of nitrogen and water deficiency is also evident in subsequent phases of heading and spikelet initiation/flowering, when stress conditions cause an increase in flower abortions and a reduction in vegetation [15, 16]. Management strategies, including the alteration of sowing time to avoid if possible the stressful period and using resistant wheat varieties would therefore be valuable to sustain productivity. In fact, alternative wheat management options have been proposed and tested worldwide to evaluate their performance with respect to yield [17]. These durum wheat cultivation management systems are necessary to maintain acceptable levels of grain yield with lower economic and environmental costs. One of the most effective and widely spread practices in wheat production is the use of a no-tillage system. This system has been increasingly adopted in dry areas as it enables water to be conserved in low rainfall regions. In addition to improving wheat productivity through improved soil quality [18], no-tillage represents an option to lower the environmental impact. It can save energy (a major direct expense for farmers in terms of fuel costs) through the reduction in mechanical operations for tillage and by lowering the emissions of GHGs, as compared to conventional tillage no-tillage acts as a sink source of soil emissions.

Conservative farming practices enabling the ability to retain water have been applied in countries such as Turkey, particularly in drought seasons, leading to an impressive yield increase

Optimization of Nitrogen in Durum Wheat in the Mediterranean Climate: The Agronomical...

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135

**Species Surface (ha) Total production (tonnes)**

Maize 184,800,969 1,037,791,518 Rice 162,716.862 741,477,711 Wheat 220,417,745 729,012,175

FAO has consequently pushed key producer countries to change their farming practices so

Durum wheat (*Triticum turgidum* L. subsp. *durum*) is a minor cereal crop representing just 5% of all wheat grown. It is thought to have originated in the Fertile Crescent (Mesopotamia) and is an allotetraploid (two genomes: AABB) with a total of 28 chromosomes (2n = 4x = 28), containing the full diploid complement of chromosomes from each of its progenitor species. Durum wheat is mainly cultivated in three basins: the Mediterranean, the northern US and-Canada, and within the desert areas of the south-west US and northern Mexico. Other much smaller areas where durum wheat is cultivated include Russia and Kazakhstan, Australia,

In 2015, global production of durum wheat reached about 36 million tonnes, according to the International Grain Council [19]. Eurostat [20] reported that about 7.5 million tonnes of

1961 1965 1970 1975 1980 1985 1990 1995 2000 2005 2010 2014

Africa Americas Asia Europe

in some regions [1].

they meet sustainability criteria.

**Table 1.** Statistical data of the main cereal crops [2].

India, and Argentina (**Figure 2**).

0

**Figure 1.** Trend of wheat production from 1961 to 2014.

50

100

150

200

250

)s not f o noili m( noitcudor p 300

350

This chapter is a review of international studies that focus on the environmental implications of nitrogen fertilizer application in durum wheat, with respect to yield and quality response. Factors controlling wheat response to N fertilizer application are also discussed. The aim of this chapter is to identify options for enhancing NUE to achieve a more sustainable fertilization management of durum wheat in the Mediterranean environment.

## **2. Wheat diffusion and statistics**

According to data collected between 2008 and 2013, cereal cultivation covered an area of more than 700 million hectare worldwide with a total production of 2.6 billion tonnes.

Wheat, as the main constituent of bread and durum, is the primary global cereal crop in terms of surface area invested, with more than 220 million hectares. By comparison, maize covers 184 million hectares and rice 162 million, according to FAO data.

Corn and rice are the top two crops in terms of productivity, while wheat is third with 729 million tonnes (**Table 1**).

Wheat production is mainly concentrated in Europe and Asia, where the crop's importance is increasing continuously, unlike on other continents where productivity has remained stable, or as in America, where the choice of alternatives crops (i.e., soybean) has led to a contraction in wheat production (**Figure 1**).

Improvements in cultivation, agricultural techniques, and mechanization have led to better wheat cultivars, and thus an increase in productivity.

The adoption of best agronomic practices has also been important in improving the sustainable production of cereals. Precision farming systems for the assessment of the physiological state of plants and conservative cultivation systems that ensure the optimization of inputs have enabled a higher level of soil water storage, resulting in an increase in crop production [18].


**Table 1.** Statistical data of the main cereal crops [2].

increase in flower abortions and a reduction in vegetation [15, 16]. Management strategies, including the alteration of sowing time to avoid if possible the stressful period and using resistant wheat varieties would therefore be valuable to sustain productivity. In fact, alternative wheat management options have been proposed and tested worldwide to evaluate their performance with respect to yield [17]. These durum wheat cultivation management systems are necessary to maintain acceptable levels of grain yield with lower economic and environmental costs. One of the most effective and widely spread practices in wheat production is the use of a no-tillage system. This system has been increasingly adopted in dry areas as it enables water to be conserved in low rainfall regions. In addition to improving wheat productivity through improved soil quality [18], no-tillage represents an option to lower the environmental impact. It can save energy (a major direct expense for farmers in terms of fuel costs) through the reduction in mechanical operations for tillage and by lowering the emissions of GHGs, as compared to conventional tillage no-tillage acts as a sink source of

This chapter is a review of international studies that focus on the environmental implications of nitrogen fertilizer application in durum wheat, with respect to yield and quality response. Factors controlling wheat response to N fertilizer application are also discussed. The aim of this chapter is to identify options for enhancing NUE to achieve a more sustainable fertilization

According to data collected between 2008 and 2013, cereal cultivation covered an area of more

Wheat, as the main constituent of bread and durum, is the primary global cereal crop in terms of surface area invested, with more than 220 million hectares. By comparison, maize covers

Corn and rice are the top two crops in terms of productivity, while wheat is third with 729

Wheat production is mainly concentrated in Europe and Asia, where the crop's importance is increasing continuously, unlike on other continents where productivity has remained stable, or as in America, where the choice of alternatives crops (i.e., soybean) has led to a contraction

Improvements in cultivation, agricultural techniques, and mechanization have led to better

The adoption of best agronomic practices has also been important in improving the sustainable production of cereals. Precision farming systems for the assessment of the physiological state of plants and conservative cultivation systems that ensure the optimization of inputs have enabled a higher level of soil water storage, resulting in an increase in crop production [18].

than 700 million hectare worldwide with a total production of 2.6 billion tonnes.

management of durum wheat in the Mediterranean environment.

184 million hectares and rice 162 million, according to FAO data.

wheat cultivars, and thus an increase in productivity.

**2. Wheat diffusion and statistics**

million tonnes (**Table 1**).

in wheat production (**Figure 1**).

soil emissions.

134 Nitrogen in Agriculture - Updates

Conservative farming practices enabling the ability to retain water have been applied in countries such as Turkey, particularly in drought seasons, leading to an impressive yield increase in some regions [1].

FAO has consequently pushed key producer countries to change their farming practices so they meet sustainability criteria.

Durum wheat (*Triticum turgidum* L. subsp. *durum*) is a minor cereal crop representing just 5% of all wheat grown. It is thought to have originated in the Fertile Crescent (Mesopotamia) and is an allotetraploid (two genomes: AABB) with a total of 28 chromosomes (2n = 4x = 28), containing the full diploid complement of chromosomes from each of its progenitor species.

Durum wheat is mainly cultivated in three basins: the Mediterranean, the northern US and-Canada, and within the desert areas of the south-west US and northern Mexico. Other much smaller areas where durum wheat is cultivated include Russia and Kazakhstan, Australia, India, and Argentina (**Figure 2**).

In 2015, global production of durum wheat reached about 36 million tonnes, according to the International Grain Council [19]. Eurostat [20] reported that about 7.5 million tonnes of

**Figure 1.** Trend of wheat production from 1961 to 2014.

durum wheat was produced in Europe in 2015/16, of which almost half (3.9 million tonnes) was produced in Italy (**Figure 3**), making it the primary durum wheat producer in Europe, followed by Turkey and France with average production of 2.7 and 1.7 million tonnes, respectively.

Smaller producers in the Mediterranean basin are concentrated in Morocco, Algeria, and Tunisia, mainly due to the effect on the crop cycle of the dry climate that often occurs there. The consumption and manufacturing of durum wheat is concentrated around the Mediterranean Sea, where the main food products of durum wheat are consumed: pasta, couscous, bulgur, and bread, which are obtained from four completely different technologies.

An additional 5 million tonnes guaranteed that local production is in fact required, which is mainly imported from North America.

The total production in the Mediterranean Basin under the winter cycle varies significantly, as the agronomic yields are highly influenced by rainfall during the spring period, and the total production can fluctuate between approximately 14 and 20 million tonnes in different years.

Currently, due to immigration to Europe and the growing popularity of pasta worldwide, increasing quantities of couscous are produced in Europe, and an increasing amount of pasta is produced in Northern Africa and in Tunisia.

Turkey is becoming an important exporter to the Middle Eastern and African pasta markets, where durum wheat is used for both bulgur and pasta production.

French breeding companies produce durum wheat varieties from commercial lots, which are very well managed from both agronomic and segregation points of view.

a constant and sufficient supply in the Mediterranean basin, its cultivation and associated agronomic practices must be modified, to cope with the projected change in climate events. Meeting the increasing demand for durum wheat is a challenge even under current conditions, with the reductions from year-to-year variation in grain yield due to irregular and seasonal rainfall distribution. Under the winter cycle of the Mediterranean basin, rain-fed durum wheat production can experience remarkable yield losses due to insufficient rainfall,

2014/2015 2015/2016

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137

) is the main component of our atmosphere, at a percentage of 78.084%. It is essential

for various metabolic reactions and vital processes, and is involved in various reactions that occur through air, soil, and water. It thus undergoes various chemical and biological transformations. Nitrogen is a crucial mineral element and is involved in several fundamental compounds (amino acids, nucleic acids, chlorophyll, cytokines, polyamines, and secondary metabolites)

The agricultural world is heavily dependent on the nitrogen cycle, and sometimes the excessive use of nitrogen results in increased agricultural production [21]. More than 100 million Mt of N year−1 of reactive N is industrially produced by the Haber-Bosch process using fossil

particularly during the spring.

EU

**Figure 3.** Main world producers of durum wheat (Eurostat).

essential for the biological plant cycle [3].

Nitrogen (N<sup>2</sup>

0,0

1,0

2,0

3,0

4,0

tons x 000 5,0

6,0

7,0

8,0

9,0

**3. Nitrogen consumption for wheat production**

Semolina (milled durum wheat) is the raw material for four different food products: pasta, couscous, bulgur, and bread, all of which are typical of the Mediterranean diet. To ensure

**Figure 2.** World map with the territories where durum wheat is grown highlighted in red (Eurostat).

Optimization of Nitrogen in Durum Wheat in the Mediterranean Climate: The Agronomical... http://dx.doi.org/10.5772/intechopen.70195 137

**Figure 3.** Main world producers of durum wheat (Eurostat).

durum wheat was produced in Europe in 2015/16, of which almost half (3.9 million tonnes) was produced in Italy (**Figure 3**), making it the primary durum wheat producer in Europe, followed by Turkey and France with average production of 2.7 and 1.7 million tonnes,

Smaller producers in the Mediterranean basin are concentrated in Morocco, Algeria, and Tunisia, mainly due to the effect on the crop cycle of the dry climate that often occurs there. The consumption and manufacturing of durum wheat is concentrated around the Mediterranean Sea, where the main food products of durum wheat are consumed: pasta, couscous, bulgur,

An additional 5 million tonnes guaranteed that local production is in fact required, which is

The total production in the Mediterranean Basin under the winter cycle varies significantly, as the agronomic yields are highly influenced by rainfall during the spring period, and the total production can fluctuate between approximately 14 and 20 million tonnes in different years. Currently, due to immigration to Europe and the growing popularity of pasta worldwide, increasing quantities of couscous are produced in Europe, and an increasing amount of pasta

Turkey is becoming an important exporter to the Middle Eastern and African pasta markets,

French breeding companies produce durum wheat varieties from commercial lots, which are

Semolina (milled durum wheat) is the raw material for four different food products: pasta, couscous, bulgur, and bread, all of which are typical of the Mediterranean diet. To ensure

and bread, which are obtained from four completely different technologies.

respectively.

136 Nitrogen in Agriculture - Updates

mainly imported from North America.

is produced in Northern Africa and in Tunisia.

where durum wheat is used for both bulgur and pasta production.

very well managed from both agronomic and segregation points of view.

**Figure 2.** World map with the territories where durum wheat is grown highlighted in red (Eurostat).

a constant and sufficient supply in the Mediterranean basin, its cultivation and associated agronomic practices must be modified, to cope with the projected change in climate events. Meeting the increasing demand for durum wheat is a challenge even under current conditions, with the reductions from year-to-year variation in grain yield due to irregular and seasonal rainfall distribution. Under the winter cycle of the Mediterranean basin, rain-fed durum wheat production can experience remarkable yield losses due to insufficient rainfall, particularly during the spring.

#### **3. Nitrogen consumption for wheat production**

Nitrogen (N<sup>2</sup> ) is the main component of our atmosphere, at a percentage of 78.084%. It is essential for various metabolic reactions and vital processes, and is involved in various reactions that occur through air, soil, and water. It thus undergoes various chemical and biological transformations.

Nitrogen is a crucial mineral element and is involved in several fundamental compounds (amino acids, nucleic acids, chlorophyll, cytokines, polyamines, and secondary metabolites) essential for the biological plant cycle [3].

The agricultural world is heavily dependent on the nitrogen cycle, and sometimes the excessive use of nitrogen results in increased agricultural production [21]. More than 100 million Mt of N year−1 of reactive N is industrially produced by the Haber-Bosch process using fossil fuels as energy sources, and of this 50% is applied to the three main cereals (maize, 16%; rice, 16%; and wheat, 18%) that provide the bulk of human food calories and proteins consumed either directly as grain or indirectly through livestock products [22].

Plants with an adequate amount of nitrogen grow rapidly and have dark green leaves and stems that grow very fast. Nitrogen is the mineral element most absorbed by plants and is placed in a plant's organs according to its physiological needs (seeds, leaves, and

Optimization of Nitrogen in Durum Wheat in the Mediterranean Climate: The Agronomical...

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139

Nitrogen promotes digestion and increases vegetative growth, the number of heads per plant

Excess nitrogen may favor lodging, particularly in tall grain varieties, and retard the cycle with stress on grain filling if the season course is dry. The culture is also more susceptible to

Therefore, responses to increasing doses of nitrogen fertilizers on the yields are variable due to the sensitive influence of pedoclimatic conditions and the effect of crop precession. An optimum climatic performance in the filling phase of the kernels can favor both the accumulation of amidaceous and protein substances, so the availability of nitrogen in the soil at this time is

The calculation of N removal from seed durum wheat with 11% protein is [25]: SEED t ha−1 ×

This results in about 25.7 kg/ha of N being removed from 1 tonne of seed wheat. Studies have reported that in general between 24 and 28 kg/tonnes is removed from durum wheat grain.

Nitrogen in the plant is a basilar element, as it is essential to the plant's vital activity. The following distribution of nitrogen in the plant at different stages has been reported in several

Recommendations of the correct dose of N fertilizer to be applied should take into account the different N source (**Figure 4**), which is calculated based on the following equation:

**Plant part Dry matter Nitrogen**

Total (kg/ha) 7644 84.3 Shoot (%) 84 80 Root (%) 17 20

Total (kg/ha) 7422 71.7 Straw (%) 71.4 35.7 Grain (%) 28.6 64.3

**Table 2.** N distribution balance in wheat plant.

and the dimension of the spike, the weight of the kernels and the protein content [24].

stems).

studies:

(**Table 2**)

**Anthesis**

**Harvest**

rust and septoria attacks.

critical to the qualitative improvement of the grain.

0.11 × 2.34 (correction factor) = X kg N ha−1 required.

Most nitrogen is contained in seeds, which have an average protein content of 12%, slightly higher in hard wheat than tender. In straw, the content ranges from 3.5 to 4.8% [23].

The average content of N in protein is about 6%.

In global terms, it is almost certain that additional N fertilizer will be required, which can be offset to some extent by management practices that improve nitrogen use efficiency (NUE).

According to some studies [22], the amount of nitrogen used on maize, rice, and wheat from 1961 to 2010 increased, which has been justified by the linear increase in production and the consequent N asportation.

Considering the balance between the input and output of nitrogen, there has been a strong increase of fertilizer application from 1961 to date, estimated at 10 times, and a consequent increase in fertilizer losses (**Figure 4**), which has an environmental impact.

**Figure 4.** Estimated N inputs and outputs in 1961 and 2010 (from Ladha et al. [22]).

## **4. Agronomical implications of nitrogen use and the system of optimizing nitrogen use on durum wheat**

Wheat is very sensitive to nitrogen deficiency and is highly reactive to the element. One of the most obvious responses to nitrogen deficiency is chlorosis, as a result of the lack of chlorophyll synthesis and reduced cell size and proliferation, leading to a stunted, reduced leaf surface and a yellowish (chlorotic) appearance of the crop.

Chlorosis is particularly apparent initially on mature leaves, and later on, the last growing leaves, as the N is shifted from old leaves to new to support growth. Thus, the older leaves dry out and cause poor plant growth and reduced yield.

Plants with an adequate amount of nitrogen grow rapidly and have dark green leaves and stems that grow very fast. Nitrogen is the mineral element most absorbed by plants and is placed in a plant's organs according to its physiological needs (seeds, leaves, and stems).

Nitrogen promotes digestion and increases vegetative growth, the number of heads per plant and the dimension of the spike, the weight of the kernels and the protein content [24].

Excess nitrogen may favor lodging, particularly in tall grain varieties, and retard the cycle with stress on grain filling if the season course is dry. The culture is also more susceptible to rust and septoria attacks.

Therefore, responses to increasing doses of nitrogen fertilizers on the yields are variable due to the sensitive influence of pedoclimatic conditions and the effect of crop precession. An optimum climatic performance in the filling phase of the kernels can favor both the accumulation of amidaceous and protein substances, so the availability of nitrogen in the soil at this time is critical to the qualitative improvement of the grain.

The calculation of N removal from seed durum wheat with 11% protein is [25]: SEED t ha−1 × 0.11 × 2.34 (correction factor) = X kg N ha−1 required.

This results in about 25.7 kg/ha of N being removed from 1 tonne of seed wheat. Studies have reported that in general between 24 and 28 kg/tonnes is removed from durum wheat grain.

Nitrogen in the plant is a basilar element, as it is essential to the plant's vital activity. The following distribution of nitrogen in the plant at different stages has been reported in several studies:

Recommendations of the correct dose of N fertilizer to be applied should take into account the different N source (**Figure 4**), which is calculated based on the following equation: (**Table 2**)


**Table 2.** N distribution balance in wheat plant.

fuels as energy sources, and of this 50% is applied to the three main cereals (maize, 16%; rice, 16%; and wheat, 18%) that provide the bulk of human food calories and proteins consumed

Most nitrogen is contained in seeds, which have an average protein content of 12%, slightly

In global terms, it is almost certain that additional N fertilizer will be required, which can be offset to some extent by management practices that improve nitrogen use efficiency (NUE).

According to some studies [22], the amount of nitrogen used on maize, rice, and wheat from 1961 to 2010 increased, which has been justified by the linear increase in production and the

Considering the balance between the input and output of nitrogen, there has been a strong increase of fertilizer application from 1961 to date, estimated at 10 times, and a consequent

increase in fertilizer losses (**Figure 4**), which has an environmental impact.

**4. Agronomical implications of nitrogen use and the system of** 

Wheat is very sensitive to nitrogen deficiency and is highly reactive to the element. One of the most obvious responses to nitrogen deficiency is chlorosis, as a result of the lack of chlorophyll synthesis and reduced cell size and proliferation, leading to a stunted, reduced leaf

Chlorosis is particularly apparent initially on mature leaves, and later on, the last growing leaves, as the N is shifted from old leaves to new to support growth. Thus, the older leaves

**optimizing nitrogen use on durum wheat**

**Figure 4.** Estimated N inputs and outputs in 1961 and 2010 (from Ladha et al. [22]).

surface and a yellowish (chlorotic) appearance of the crop.

dry out and cause poor plant growth and reduced yield.

higher in hard wheat than tender. In straw, the content ranges from 3.5 to 4.8% [23].

either directly as grain or indirectly through livestock products [22].

The average content of N in protein is about 6%.

consequent N asportation.

138 Nitrogen in Agriculture - Updates

 N amount(D) = crop N demand(Fc) − environmental inputs(E) + environmental removal(U) (1)

and, in more detail: D = Fc − (P + M + Cp) + (L + V + De + Um)

where


matter. Legumes leave nitrogen in the soil in available form for the crops that follow under a rotation system, but providing an accurate value of the quantity individual species is difficult. However, it can be estimated at about 30 kg/ha on average for perennial legume

**Runoff/erosion Leaching**

**Other form of loses**

**Nitrogen Efficiency**

**N availability** 

**Potential N loses** 

**Soil biological processes Nitrification** Nitrite NO2 -

**Nitrogen (N) source**

**N fertilizers Manures**

**Soil Nitrogen supply**

**Crop N demand**

> **Volatilization** Ammonia NH3

**Figure 5.** Potential fate of nitrogen in the agricultural system (in the case of durum wheat).

**Immobilization**

**Atmospheric depositions**

Optimization of Nitrogen in Durum Wheat in the Mediterranean Climate: The Agronomical...

**Denitrification** Ammonia NH3

Nitrate NO3 -

**Abiotic variables**

**Immobilization**

**Climate variables**  141

**Soil variables** 

http://dx.doi.org/10.5772/intechopen.70195

**Mineralization**

**Organic and inorganic form of Nitrogen**

Leaching is the movement of nitrogen in percolating water along the profile of the soil, when

As a result of leaching, nitrogen is not utilized by the crops and it thus represents both a

Normally leaching affects nitric form, even in sandy soils and it can also affect the ammonia form. The water constitutes the vehicle by which the nitrate N in the soil moves, and nitrogen leaching is, therefore, a phenomenon that, for the parity of nitrates present in the soil, is closely

Leaching is the main, if not the only, type of nitrogen removal affected by the environment.

Nitrogen can also be derived from organic fertilization carried out in previous years.

species and 20 kg/ha for annual species [26].

it exceeds that taken up by the root layer of plants.

**4.2. Leaching (L)**

nutritional and economic loss.

**Best N management practices data base**

**Site specific conditions**

**Plant uptake; N in grain and straw**

dependent on its water balance.


#### **4.1. Contributions from the previous crop (Cp)**

Previous crops such as legumes have certain characteristics that can result in nitrogen production. The legume species are able to establish a high amount of nitrogen for the symbiotic bacteria, which partly remain in the roots and enter into the cycle of organic soil Optimization of Nitrogen in Durum Wheat in the Mediterranean Climate: The Agronomical... http://dx.doi.org/10.5772/intechopen.70195 141

**Figure 5.** Potential fate of nitrogen in the agricultural system (in the case of durum wheat).

matter. Legumes leave nitrogen in the soil in available form for the crops that follow under a rotation system, but providing an accurate value of the quantity individual species is difficult. However, it can be estimated at about 30 kg/ha on average for perennial legume species and 20 kg/ha for annual species [26].

Nitrogen can also be derived from organic fertilization carried out in previous years.

#### **4.2. Leaching (L)**

 N amount(D) = crop N demand(Fc) − environmental inputs(E) + environmental removal(U) (1)

• E represents the amount of nitrogen that can be used from the culture but is not distributed with the fertilizer, and which is derived from: atmospheric precipitation (P); mineralization of soil organic matter (M); contributions arising from the previous crop (Cp); and U represents the amount of N that the environment removes due to the possible utilization

• The amount of nitrogen from rain varies widely due to geographical position and fluctuations throughout the year, which makes it extremely difficult to generalize. However, in the Mediterranean climate, we can estimate a supply of nitrogen from atmospheric precipita-

• Soil nitrogen (N) content ranging between 0.05% and 0.2%. Considering a 30-cm layer of soil depth, this values correspond to an amount of total N between 1,950 to 7,800 kg/ha,

• Approximately 98%, however, is contained in the organic substance soil (humus) and is not absorbable by plants. The organic matter is gradually decomposed by microorganisms and atmospheric agents, and the nitrogen content is transformed into mineral form, which

• This complex set of processes is known as the mineralization of the organic matter. In warmer climates and sandy soils, 3% of nitrogen can be freed each year and 1% in more temperate climates and with clay soils. A 30-cm layer of soil, therefore, releases 40–80 kg/ha of nitrogen.

Previous crops such as legumes have certain characteristics that can result in nitrogen production. The legume species are able to establish a high amount of nitrogen for the symbiotic bacteria, which partly remain in the roots and enter into the cycle of organic soil

tion ranging from 20 kg/ha to 30 kg/ha for the entire crop cycle of wheat.

higher than those necessary for the cultivation of wheat (**Figure 5**).

and, in more detail: D = Fc − (P + M + Cp) + (L + V + De + Um)

where

• Fc is crop N demand

140 Nitrogen in Agriculture - Updates

by plants through:

• leaching (L)

• volatilization (V)

• denitrification (De)

• **Atmospheric precipitation (P)**

is then absorbable by plants.

• immobilization into soil organic matter (Um)

• **Mineralization of soil organic matter (M)**

**4.1. Contributions from the previous crop (Cp)**

Leaching is the movement of nitrogen in percolating water along the profile of the soil, when it exceeds that taken up by the root layer of plants.

As a result of leaching, nitrogen is not utilized by the crops and it thus represents both a nutritional and economic loss.

Normally leaching affects nitric form, even in sandy soils and it can also affect the ammonia form.

The water constitutes the vehicle by which the nitrate N in the soil moves, and nitrogen leaching is, therefore, a phenomenon that, for the parity of nitrates present in the soil, is closely dependent on its water balance.

Leaching is the main, if not the only, type of nitrogen removal affected by the environment.

## **4.3. Volatilization (V)**

Nitrogen volatilization occurs in the soil when it forms gaseous ammonia. The rate of this phenomenon depends on the level of humidity, the temperature, and the pH of the soil.

Here, the term "N available" also refers to the amount of N derived from mineralization of S.O. soil, atmospheric deposition, and bacterial nitrogen fixation. For simplicity of analysis, in many cases, the nitrogen arising from environmental inputs is defined as the absorption of

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The different viable approaches for the enhancement of NUE are explored in more detail in the introductions of each individual experiment. Several tools and strategies are available to improve the NUE, such as systems for genetic breeding that include genetic parameters and

Genetic improvements aimed at optimizing the NUE are crucial for achieving the economic benefits farmers seek, and result in a reduction of the environmental impact of nitrogen.

The increase in yield brought by a new cultivar results in an increase in nitrogen consumption, which apparently is the aspect that has been able to increase the yield from old to new varieties [30]. Indeed, studies have reported that Cimmyt varieties saw a gain in NUE of

Several components affect the level of NUE improvement. In particular, the N Harvest Index (NHI) component contributed to an estimated 0.15% improvement in the NUE per year, which was aimed at reducing the amount of straw produced during harvesting [33–35] (**Table 3**).

Other physiological, metabolic and physical-chemical components can help improve NUE by

Aspects that act on both the cellular scale and the whole plant include root absorption, nitrate assimilation, N distribution, photosynthesis, senescence, nitrogen rebuilding, accumulation, and wheat composition. These have been taken into account in genetic improvement work

between 0.4 and 1.1% annually from 1962 to 1985 [31, 32].

**Period Genotypes N level (kg N/ha) NUE (% per year) References**

75 0.4 150 0.6 300 0.9

200 0.58 1985–2010 195 150 0.37 Cormier et al. [34] 250 0.3

1962–1985 8 0 1.2 Ortiz-Monasterio et al. [31]

1977–2007 24 0 0.35 Sylvester-Bradley and Kindred [30]

**Table 3.** Assessment of yearly percentage genetic gain in nitrogen use efficiency (NUE) from a direct comparison of old

reducing the contribution of nitrogen fertilizer.

[36] and are reported in **Table 4**.

and modern cultivars [35].

a crop not fertilized.

agronomical tools [29].

**4.7. Breeding for NUE**

Volatilization is higher in alkaline soil, when the temperature can reach 30°C and where the ammonia or the sources of it (urea and ammonium sulphate from fertilizers) are applied.

#### **4.4. Denitrification (De)**

Denitrification is the progressive reduction of nitrate to gaseous compounds, such as nitrous oxide and molecular nitrogen, which pass into the atmosphere.

In Italian agricultural soils, nitrogen lost by denitrification does not appear to reach very significant levels, except in special cases such as paddy fields, and has been estimated at 1–5 kg/ha per year.

#### **4.5. Humification**

Humification is the transformation of the organic substance in humus by microorganisms, occurring within the soil. Humification is a process that requires nitrogen, and if there is not an enough amount in the organic matter, the level of microorganisms in the soil is reduced, which reduced N availability to the crop.

To prevent fertility decreasing, the soil humus content must remain unchanged over time, and the humification value must equal that of the mineralization. Sufficient organic matter (crop residues or fertilizers) is, therefore, necessary to restore the proportion of humus and thus the organic nitrogen of the soil.

The response of wheat to N fertilizer is influenced by different factors: soil management, N fertilizer management, and timing of application, soil properties, crop sequence, seasonal trends and the supply of residual and mineralized N.

The agronomic efficiency of N fertilizer in a Mediterranean climate may be lower than that in temperate zones as the climatic conditions are largely outside the control of farmers, and it is difficult to predict the amount of N to apply to attain optimum growth.

#### **4.6. Nitrogen use efficiency**

This is the efficiency ratio of output (economic yield) to input (fertilizers) and increases in environmental and economic pressure make it a priority to optimize nitrogen use efficiency (NUE) in agriculture [27].

NUE has various definitions, but that of Moll et al. [28] is one of the most complete, as it does not only refer to the nitrogen of manure and fertilizers:

$$\text{NUE} = \frac{\text{Nuptake}}{\text{Navailable}} \times \frac{\text{Seed yield}}{\text{Nuptake}} \tag{2}$$

Here, the term "N available" also refers to the amount of N derived from mineralization of S.O. soil, atmospheric deposition, and bacterial nitrogen fixation. For simplicity of analysis, in many cases, the nitrogen arising from environmental inputs is defined as the absorption of a crop not fertilized.

The different viable approaches for the enhancement of NUE are explored in more detail in the introductions of each individual experiment. Several tools and strategies are available to improve the NUE, such as systems for genetic breeding that include genetic parameters and agronomical tools [29].

## **4.7. Breeding for NUE**

**4.3. Volatilization (V)**

142 Nitrogen in Agriculture - Updates

**4.4. Denitrification (De)**

1–5 kg/ha per year.

**4.5. Humification**

which reduced N availability to the crop.

thus the organic nitrogen of the soil.

**4.6. Nitrogen use efficiency**

(NUE) in agriculture [27].

trends and the supply of residual and mineralized N.

not only refer to the nitrogen of manure and fertilizers:

NUE <sup>=</sup> <sup>N</sup> uptake \_\_\_\_\_\_\_\_\_\_\_\_\_\_

difficult to predict the amount of N to apply to attain optimum growth.

Nitrogen volatilization occurs in the soil when it forms gaseous ammonia. The rate of this phenomenon depends on the level of humidity, the temperature, and the pH of the soil.

Volatilization is higher in alkaline soil, when the temperature can reach 30°C and where the ammonia or the sources of it (urea and ammonium sulphate from fertilizers) are applied.

Denitrification is the progressive reduction of nitrate to gaseous compounds, such as nitrous

In Italian agricultural soils, nitrogen lost by denitrification does not appear to reach very significant levels, except in special cases such as paddy fields, and has been estimated at

Humification is the transformation of the organic substance in humus by microorganisms, occurring within the soil. Humification is a process that requires nitrogen, and if there is not an enough amount in the organic matter, the level of microorganisms in the soil is reduced,

To prevent fertility decreasing, the soil humus content must remain unchanged over time, and the humification value must equal that of the mineralization. Sufficient organic matter (crop residues or fertilizers) is, therefore, necessary to restore the proportion of humus and

The response of wheat to N fertilizer is influenced by different factors: soil management, N fertilizer management, and timing of application, soil properties, crop sequence, seasonal

The agronomic efficiency of N fertilizer in a Mediterranean climate may be lower than that in temperate zones as the climatic conditions are largely outside the control of farmers, and it is

This is the efficiency ratio of output (economic yield) to input (fertilizers) and increases in environmental and economic pressure make it a priority to optimize nitrogen use efficiency

NUE has various definitions, but that of Moll et al. [28] is one of the most complete, as it does

<sup>N</sup> available <sup>×</sup> Seed yield \_\_\_\_\_\_\_\_\_\_\_\_\_

<sup>N</sup> uptake (2)

oxide and molecular nitrogen, which pass into the atmosphere.

Genetic improvements aimed at optimizing the NUE are crucial for achieving the economic benefits farmers seek, and result in a reduction of the environmental impact of nitrogen.

The increase in yield brought by a new cultivar results in an increase in nitrogen consumption, which apparently is the aspect that has been able to increase the yield from old to new varieties [30]. Indeed, studies have reported that Cimmyt varieties saw a gain in NUE of between 0.4 and 1.1% annually from 1962 to 1985 [31, 32].

Several components affect the level of NUE improvement. In particular, the N Harvest Index (NHI) component contributed to an estimated 0.15% improvement in the NUE per year, which was aimed at reducing the amount of straw produced during harvesting [33–35] (**Table 3**).

Other physiological, metabolic and physical-chemical components can help improve NUE by reducing the contribution of nitrogen fertilizer.

Aspects that act on both the cellular scale and the whole plant include root absorption, nitrate assimilation, N distribution, photosynthesis, senescence, nitrogen rebuilding, accumulation, and wheat composition. These have been taken into account in genetic improvement work [36] and are reported in **Table 4**.


**Table 3.** Assessment of yearly percentage genetic gain in nitrogen use efficiency (NUE) from a direct comparison of old and modern cultivars [35].


particular soil can influence the microbiological processes responsible for mineralization, fixation, and denitrification of soil nitrogen. The N availability in the soil is known to be largely controlled by bacteria, so the rate of N cycling is dependent on factors such as soil type, soil moisture, temperature, and pH. Generally, the application of N fertilizer in wheat field depends on the availability of soil nitrogen and the potential losses. The effect of soil moisture on the response of wheat to N fertilizer application has been studied extensively, and the research has found that soil moisture must be considered in N fertilizer management for all soil types. The literature review shows that yield response varies with different nitrogen and moisture levels. In rain-fed areas, for example, soil moisture must be reserved when selecting fertilizer rates.

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The application of N to wheat should be timed so an adequate amount is supplied when the crop needs it. As the nitrogen cycle is biologically influenced by prevailing climatic conditions, along with the physical and chemical properties of a particular soil, the choice of a suitable N fertilizer formula to be applied in a specific soil is fundamental. The electrical charges of soil particles vary according to the soil texture, so understanding the chemical properties and therefore, the chemical reactions within the soil will inform recommendations as to when fertilizers can be applied to soils. Soil scientists indicate that these reactions have a major influence on when fertilizer can be applied and how efficiently it is taken up by the crop. Clay particles in the soil have a negative electrical charge, so the amount of clay present in the soil surface is an important factor in these reactions. Clay particles with negative charges will react with components of the fertilizer that dissolve as positively (+) charged particles (cations) when added to soil. For example, the application of N fertilizers that include more ammonium (positive charges) and less nitrate (negative charges) forms

of N can reduce the potential for losses in the short term. Ammonium (NH<sup>4</sup>

−

naturally help prevent losses of ammonium forms of N.

**4.9. Effect of soil management on nitrogen application in wheat**

remaining amount to nitrate (NO<sup>3</sup>

organic matter [38].

bind to soil particles with negative charges and will not be subjected to losses through leaching or denitrification, therefore, increasing N availability for the plant. However, if not all of the applied ammonium is taken up by the plant, soil bacteria will convert the

lost when excessive rainfall leaches or saturates soils. Low soil temperatures can affect the transformation process as they minimize the activity of the nitrifying bacteria, which can

Soil is managed to ensure that sufficient nutrients are available for plant growth and development, and therefore, good soil management is critical for crop productivity. Poor soil management can lead to erosion, loss of fertility, deterioration of soil structure, and poor crop yields [37]. Understanding the nutritional status of the soil is important in any N fertilizer management plan, and involves the knowledge of different soil components such as microorganisms (densities, diversity, and activity), organic matter and the biological processes by which microorganisms make micro- and macroelements available to the plant, through the decomposition of organic matter. Soil microorganisms have many physical and chemical effects on plants. For example, their secretions help dissolve minerals, and they convert inorganic substances into other forms, making them available to plants. They also aerate the soil and help decompose

+

), a form that is not bound to soil particles and can be

) forms of N

**Table 4.** Physiological aspects studied for nitrogen use efficiency [36].

#### **4.8. Effect of soil type**

The soil-plant-environment system is very complex due to the interactions of several factors that affect plant growth, development, and the final yield. Agricultural soil represents the major source of nutrition for crop survival, and therefore, a good balance of macro- and microelements in the soil is fundamental to ensuring a better crop response.

Agriculture intensification has become common practice in different parts of the world to provide sufficient food for the increasing population, and while the high amounts of external inputs such as inorganic fertilizers have led to considerable increases in overall food production worldwide, they have resulted in continuous environmental degradation, particularly of the soil. This deterioration of soil quality and the reduction in agricultural productivity due to nutrient depletion, organic matter losses, and erosion have in turn led to a greater use of chemical inputs, particularly nitrogen. Today, N fertilizer is considered one of the main inputs for crop production, and any lack of N will lead to disruption of plant growth, which will result in economic losses. Nitrogen in the soil is present in soluble form as organic N (Org-N), as ammonium nitrogen (NH<sup>4</sup> + -N), and as nitrate nitrogen (NO<sup>3</sup> − -N). The nitrogen cycle is extremely dynamic and complex, so climatic conditions and the physical and chemical properties of a particular soil can influence the microbiological processes responsible for mineralization, fixation, and denitrification of soil nitrogen. The N availability in the soil is known to be largely controlled by bacteria, so the rate of N cycling is dependent on factors such as soil type, soil moisture, temperature, and pH. Generally, the application of N fertilizer in wheat field depends on the availability of soil nitrogen and the potential losses. The effect of soil moisture on the response of wheat to N fertilizer application has been studied extensively, and the research has found that soil moisture must be considered in N fertilizer management for all soil types. The literature review shows that yield response varies with different nitrogen and moisture levels. In rain-fed areas, for example, soil moisture must be reserved when selecting fertilizer rates.

The application of N to wheat should be timed so an adequate amount is supplied when the crop needs it. As the nitrogen cycle is biologically influenced by prevailing climatic conditions, along with the physical and chemical properties of a particular soil, the choice of a suitable N fertilizer formula to be applied in a specific soil is fundamental. The electrical charges of soil particles vary according to the soil texture, so understanding the chemical properties and therefore, the chemical reactions within the soil will inform recommendations as to when fertilizers can be applied to soils. Soil scientists indicate that these reactions have a major influence on when fertilizer can be applied and how efficiently it is taken up by the crop. Clay particles in the soil have a negative electrical charge, so the amount of clay present in the soil surface is an important factor in these reactions. Clay particles with negative charges will react with components of the fertilizer that dissolve as positively (+) charged particles (cations) when added to soil. For example, the application of N fertilizers that include more ammonium (positive charges) and less nitrate (negative charges) forms of N can reduce the potential for losses in the short term. Ammonium (NH<sup>4</sup> + ) forms of N bind to soil particles with negative charges and will not be subjected to losses through leaching or denitrification, therefore, increasing N availability for the plant. However, if not all of the applied ammonium is taken up by the plant, soil bacteria will convert the remaining amount to nitrate (NO<sup>3</sup> − ), a form that is not bound to soil particles and can be lost when excessive rainfall leaches or saturates soils. Low soil temperatures can affect the transformation process as they minimize the activity of the nitrifying bacteria, which can naturally help prevent losses of ammonium forms of N.

#### **4.9. Effect of soil management on nitrogen application in wheat**

**4.8. Effect of soil type**

N remobilization efficiency

post-anthesis

nium nitrogen (NH<sup>4</sup>

+

The soil-plant-environment system is very complex due to the interactions of several factors that affect plant growth, development, and the final yield. Agricultural soil represents the major source of nutrition for crop survival, and therefore, a good balance of macro- and micro-

Agriculture intensification has become common practice in different parts of the world to provide sufficient food for the increasing population, and while the high amounts of external inputs such as inorganic fertilizers have led to considerable increases in overall food production worldwide, they have resulted in continuous environmental degradation, particularly of the soil. This deterioration of soil quality and the reduction in agricultural productivity due to nutrient depletion, organic matter losses, and erosion have in turn led to a greater use of chemical inputs, particularly nitrogen. Today, N fertilizer is considered one of the main inputs for crop production, and any lack of N will lead to disruption of plant growth, which will result in economic losses. Nitrogen in the soil is present in soluble form as organic N (Org-N), as ammo-

dynamic and complex, so climatic conditions and the physical and chemical properties of a

−


Gregory and Brown (1989), Steele et al. (2006), Reynolds et al. (2007) and Manschadi et al.

Masclaux et al. (2001) and Martin et al. (2006)

Reynolds et al. (2001, 2005), Wang et al. (2002)

Borrell and Hammer (2000), Spano et al. (2003)

(2006)

*Oryza* spp. Hibberd et al. (2008)

*O*. *sativa*, *B*. *napus* Shrawat et al. (2008) and Good et al. (2007)

and Ding et al. (2007)

and Verma et al. (2004)

*S*. *bicolour* Borrell and Hammer (2000)

elements in the soil is fundamental to ensuring a better crop response.

**Traits Species References**

*O*. *sativa*

Leaf posture *T*. *aestivum* Araus et al. (1993)

Stem N storage *T*. *aestivum* Critchley (2001)

Post-anthesis N uptake *T*. *aestivum* Triboı et al. (2006)

*O*. *sativa*

*S*. *bicolor*

Glutamine synthetase (GS) activity *T*. *aestivum*, *Z*. *mays* Habash et al. (2001), Hirel et al. (2001),

specificity factor *G*. *partitida* Uemura et al. (1997)

Specific leaf N content *Triticum* spp. Austin et al. (1982) and Semenov et al. (2007) Vertical N distribution with leaf layer *T*. *aestivum*, *S*. *altissima* Hirose and Werger (1987) and Critchley (2001)

Root length density at depth *T*. *aestivum*, *H*. *vulgare*,

Leaf photosynthetic rate post-anthesis *Z*. *mays*, *T*. *aestivum*,

Stay-green *T*. *aestivum*, *T*. *durum*,

**Table 4.** Physiological aspects studied for nitrogen use efficiency [36].

Alanine aminotransferase (AlaAT)

144 Nitrogen in Agriculture - Updates

Introduction of C4 "Krantz" anatomy

activity

RuBisCo CO<sup>2</sup>

into to C3 species


Soil is managed to ensure that sufficient nutrients are available for plant growth and development, and therefore, good soil management is critical for crop productivity. Poor soil management can lead to erosion, loss of fertility, deterioration of soil structure, and poor crop yields [37]. Understanding the nutritional status of the soil is important in any N fertilizer management plan, and involves the knowledge of different soil components such as microorganisms (densities, diversity, and activity), organic matter and the biological processes by which microorganisms make micro- and macroelements available to the plant, through the decomposition of organic matter. Soil microorganisms have many physical and chemical effects on plants. For example, their secretions help dissolve minerals, and they convert inorganic substances into other forms, making them available to plants. They also aerate the soil and help decompose organic matter [38].

Direct and indirect interventions can improve biological soil management. Direct methods attempt to alter the abundance or activity of specific groups of organisms [39]. Seeds or roots can, for example, be inoculated with rhizobia, mycorrhizae, fungi, and rhizobacteria to enhance soil fertility. Most agricultural practices are in fact indirect methods, such as incorporating plant residues into the soil, introducing legume crops and various tillage methods. They provide important sources of nutrition in the fields, and therefore, must be considered when applying N fertilizers [40]. Many studies have reported that the incorporation of straw, together with the N-rich aboveground biomass of the crops promotes the immobilization of soil mineral N, restores soil organic matter, and improves aeration. The N content in the residue is the input to the soil humus, and thus becomes a part of the supply for future crops. The levels of residual inorganic N in the root profile contribute to the total plant N and should be taken into account when formulating fertilizer recommendations for improving N utilization efficiency. The influence of tillage on the physical, chemical, and microbiological properties of the soil is becoming of great importance when studying issues related to soil fertility and productivity. Several studies suggest that tillage management can have beneficial effects on both grain yield and nutritional quality. Tillage practices such as no-tillage are believed to benefit the soil in terms of improving water retention and the conservation of organic matter in the soil [41].

The large-scale genetic breeding of wheat to reduce the plant size (Rht) has improved efficiency in the redistribution of photosynthesis products (Harvest Index) and has had positive effects on

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As the upper physiological limit of HI (of approximately 60%) has almost been reached, genetic improvement has been directed toward increasing the capacity to produce aboveg-

The maximum yield potential has undoubtedly led to an increase in the general nitrogen needs of non-nitrogen-fixing crops, which have in fact become dependent on substantial levels of

The reduced height of the plants has materially allowed this increase, as the new varieties are

Breeding is an important factor in wheat improvement-related GM production. The overexpression of the French bean in GM wheat has led to an increased grain yield, and consequently NUE, estimated at about 20%. However, difficulties in carrying out experiments with

Another gene studied in terms of the ability to modify NUE was the GS1 (Gln-1-3), whose overexpression was able to improve the rice crop index, N collection rate, and N utilization efficiency.

To be effective, the amount of mineral nutrients applied must be consistent with the steps of development and the absorption capacity of the crop. This is known as the subdivision of the N extra dose fractionation best yield, with respect to a unique single intake. With a distributed total dose, nitrogen fertilization is reduced during the early phases (tillering) in favor of the later phases (stem elongation, earing), which encourages a greater absorption of nutrients and

Even if traditionally and environmentally the entire dose of N is supplied at the sowing, this approach can be considered valid: the correction of the fertilization dose, with contributions between late tillering and stem elongation, is an economically profitable technique (**Figure 6**).

**Figure 6.** Effect of doses and number of N application on yield response in durum wheat [14].

the fertility of the spike [48].

mineral fertilizer.

**4.11. Time of application**

increases the efficiency of the crop [14].

round biomass as part of the crop [49].

much less sensitive to the risk of lodging than the old.

GM materials in Europe have hindered further research into this.

Conservation agriculture (CA) is an ecologically interesting land management technique, aimed at reducing agronomical inputs related to the reduction of ploughing [42, 43].

The technique may have the potential to reduce the loss of important parameters of soil fertility.

Organic matter (OM) is one such fundamental parameter, and any increase in the soil has obvious benefits in terms of reducing greenhouse gas emissions and thus climate change [17, 37, 44]. An increase in OM, particularly in the more superficial layers, results in an improvement in the physical and chemical properties of the soil, its biological activity and, consequently, in N losses [45].

CA-related studies have also found a tendency to better water conservation and adsorption in the soil; however, under special conditions, CA adoption appears to result in lower NUE rates than conventional systems, largely due to N fertilizer immobilization through crop residues.

Fertilization strategies must therefore be appropriately adjusted by combining the rate, timing, splitting, and source of N, to optimize yield and quality [46].

Soil conditions such as temperature must also be considered, which is found to be lower in a CA system compared to conventional systems. Soil conditions are fundamentally important in N fertilizer management, as they strongly influence the nitrogen cycle. Studies have indicated that, even after CA has been applied for over 10 years, slightly higher doses of fertilizers are still needed to achieve significant yields in winter wheat, with the presence of crop residues on the surface layer.

#### **4.10. Varietal assessment**

Developments in wheat genetics since the 1960s have enabled a substantial increase in the yield potential of many crops, particularly cereals [47].

The large-scale genetic breeding of wheat to reduce the plant size (Rht) has improved efficiency in the redistribution of photosynthesis products (Harvest Index) and has had positive effects on the fertility of the spike [48].

As the upper physiological limit of HI (of approximately 60%) has almost been reached, genetic improvement has been directed toward increasing the capacity to produce aboveground biomass as part of the crop [49].

The maximum yield potential has undoubtedly led to an increase in the general nitrogen needs of non-nitrogen-fixing crops, which have in fact become dependent on substantial levels of mineral fertilizer.

The reduced height of the plants has materially allowed this increase, as the new varieties are much less sensitive to the risk of lodging than the old.

Breeding is an important factor in wheat improvement-related GM production. The overexpression of the French bean in GM wheat has led to an increased grain yield, and consequently NUE, estimated at about 20%. However, difficulties in carrying out experiments with GM materials in Europe have hindered further research into this.

Another gene studied in terms of the ability to modify NUE was the GS1 (Gln-1-3), whose overexpression was able to improve the rice crop index, N collection rate, and N utilization efficiency.

#### **4.11. Time of application**

Direct and indirect interventions can improve biological soil management. Direct methods attempt to alter the abundance or activity of specific groups of organisms [39]. Seeds or roots can, for example, be inoculated with rhizobia, mycorrhizae, fungi, and rhizobacteria to enhance soil fertility. Most agricultural practices are in fact indirect methods, such as incorporating plant residues into the soil, introducing legume crops and various tillage methods. They provide important sources of nutrition in the fields, and therefore, must be considered when applying N fertilizers [40]. Many studies have reported that the incorporation of straw, together with the N-rich aboveground biomass of the crops promotes the immobilization of soil mineral N, restores soil organic matter, and improves aeration. The N content in the residue is the input to the soil humus, and thus becomes a part of the supply for future crops. The levels of residual inorganic N in the root profile contribute to the total plant N and should be taken into account when formulating fertilizer recommendations for improving N utilization efficiency. The influence of tillage on the physical, chemical, and microbiological properties of the soil is becoming of great importance when studying issues related to soil fertility and productivity. Several studies suggest that tillage management can have beneficial effects on both grain yield and nutritional quality. Tillage practices such as no-tillage are believed to benefit the soil in terms of

improving water retention and the conservation of organic matter in the soil [41].

splitting, and source of N, to optimize yield and quality [46].

yield potential of many crops, particularly cereals [47].

quently, in N losses [45].

146 Nitrogen in Agriculture - Updates

on the surface layer.

**4.10. Varietal assessment**

aimed at reducing agronomical inputs related to the reduction of ploughing [42, 43].

Conservation agriculture (CA) is an ecologically interesting land management technique,

The technique may have the potential to reduce the loss of important parameters of soil fertility.

Organic matter (OM) is one such fundamental parameter, and any increase in the soil has obvious benefits in terms of reducing greenhouse gas emissions and thus climate change [17, 37, 44]. An increase in OM, particularly in the more superficial layers, results in an improvement in the physical and chemical properties of the soil, its biological activity and, conse-

CA-related studies have also found a tendency to better water conservation and adsorption in the soil; however, under special conditions, CA adoption appears to result in lower NUE rates than conventional systems, largely due to N fertilizer immobilization through crop residues. Fertilization strategies must therefore be appropriately adjusted by combining the rate, timing,

Soil conditions such as temperature must also be considered, which is found to be lower in a CA system compared to conventional systems. Soil conditions are fundamentally important in N fertilizer management, as they strongly influence the nitrogen cycle. Studies have indicated that, even after CA has been applied for over 10 years, slightly higher doses of fertilizers are still needed to achieve significant yields in winter wheat, with the presence of crop residues

Developments in wheat genetics since the 1960s have enabled a substantial increase in the

To be effective, the amount of mineral nutrients applied must be consistent with the steps of development and the absorption capacity of the crop. This is known as the subdivision of the N extra dose fractionation best yield, with respect to a unique single intake. With a distributed total dose, nitrogen fertilization is reduced during the early phases (tillering) in favor of the later phases (stem elongation, earing), which encourages a greater absorption of nutrients and increases the efficiency of the crop [14].

Even if traditionally and environmentally the entire dose of N is supplied at the sowing, this approach can be considered valid: the correction of the fertilization dose, with contributions between late tillering and stem elongation, is an economically profitable technique (**Figure 6**).

**Figure 6.** Effect of doses and number of N application on yield response in durum wheat [14].

The most effective nitrogen enhancement is administered during the phases of more intense vegetative activity (raised) and depends on the greater capacity of interception of plants. An experiment in the Mediterranean area (Spain) with a radioactive tracer (15N) has shown that only a small proportion of nitrogen supplied in presowing is effectively intercepted by wheat (14.1%). Nitrogen raised initially is instead absorbed in a greater proportion (54.8%) [50] (**Figure 7**).

A more extensive definition [53] of PF is "farming management (agriculture, forestry and animal husbandry) based on the observation, measurement and response of the inter- and intrafield quantitative and qualitative variables that influence the production system of the farm." This aims to define, after site-specific data analysis, a complete decision support system for business management, with the aim of optimizing returns in terms of climate, environmental,

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The application of precision agriculture in extensive crops, particularly wheat, has been the subject of various studies [54–57], and involves the acquisition and integration of a large

The first input to consider is soil variability. The physical and chemical characteristics of soil vary, which affects the production response [58]. In arid environments, in particular, the spatial variability of the soil factors, combined with climatic factors, greatly influences productivity [59]. Various techniques can be used to obtain soil data, although the approach of sensor-based gathering of crop and soil data is most common [60, 61]. They reported in their reviews that the widely used electrical and electromagnetic sensors provide valuable information about

These techniques are more intensive and generally cheaper than conventional sampling and

The use of sensors can be considered to spatially diagnose the seasonal pattern of crop conditions during the cycle. Sensors on planes or satellites can potentially collect the reflected electromagnetic radiation from the canopy at small scales of space and time. These remote sensors can evaluate the changes in growth environments from location to location and can potentially gather

Both passive and active-light proximal sensors can be used to collect the reflected radiation. Passive noncontact sensors depend on sunlight, while active sensors, with their own light sources, enable the assessment of the crop status irrespective of ambient light conditions [63, 64].

• SPAD-502 Minolta analytical transmittance, which produces an estimate of leaf chlorophyll

Several studies have been conducted on wheat crops using these systems [65, 66], and the data

The normalized difference vegetation index (NDVI) is the more precise vegetative normalization index and is based on differences in the red (670 nm) and near infrared (780 nm) spectrums.

collected must be converted into measures of vegetation, using vegetative indices.

• The passive Yara N-Sensor®/FieldScan (Yara International, ASA, Oslo, Norway).

information about the field, predicting grain yield and nitrogen status [62].

• FieldSpec® Portable Spectroradiometer (ASD Inc., Boulder, CO, USA).

economic, productive, and social sustainability [54].

amount of information.

field heterogeneity.

content.

analysis of soil or crop variables [56].

Commercial proximal sensors include:

• The active sensor GreenSeeker® (Trimble Inc.). • Crop Circle ™ (Dutch Scientific, Lincoln, NE).

The gross availability of nitrogen resulting from the mineralization of organic matter and from previous crop residues may adversely affect efficiency. Indications have emerged from the study of durum wheat in the same environment [50].

Very late fertilization around the time of earing/flowering, while not having significant effects on the yield, can increase the protein content of kernels and the production of protein per hectare [15, 51].

#### **4.12. Use of precision farming for nitrogen application**

Precision farming can represent a sustainable system, making nitrogen more sustainable while achieving the best economic results.

Although there are several definitions of precision agriculture (PF), Pierce and Nowak [52] have summarized it as "a system that provides the tools to do the right thing, the right place, at the right time," where "the right thing" is an agronomic operation. In fact, recent technological innovations have led to an increase of application opportunities and that definition can therefore be extended.

**Figure 7.** Nitrogen fertilizer recovery (%) of 15N-labeled fertilizer (NR) and N derived from 15N fertilizer (NF) in the whole plant at maturity for hard red spring wheat, as affected by N timing [49].

A more extensive definition [53] of PF is "farming management (agriculture, forestry and animal husbandry) based on the observation, measurement and response of the inter- and intrafield quantitative and qualitative variables that influence the production system of the farm." This aims to define, after site-specific data analysis, a complete decision support system for business management, with the aim of optimizing returns in terms of climate, environmental, economic, productive, and social sustainability [54].

The application of precision agriculture in extensive crops, particularly wheat, has been the subject of various studies [54–57], and involves the acquisition and integration of a large amount of information.

The first input to consider is soil variability. The physical and chemical characteristics of soil vary, which affects the production response [58]. In arid environments, in particular, the spatial variability of the soil factors, combined with climatic factors, greatly influences productivity [59].

Various techniques can be used to obtain soil data, although the approach of sensor-based gathering of crop and soil data is most common [60, 61]. They reported in their reviews that the widely used electrical and electromagnetic sensors provide valuable information about field heterogeneity.

These techniques are more intensive and generally cheaper than conventional sampling and analysis of soil or crop variables [56].

The use of sensors can be considered to spatially diagnose the seasonal pattern of crop conditions during the cycle. Sensors on planes or satellites can potentially collect the reflected electromagnetic radiation from the canopy at small scales of space and time. These remote sensors can evaluate the changes in growth environments from location to location and can potentially gather information about the field, predicting grain yield and nitrogen status [62].

Both passive and active-light proximal sensors can be used to collect the reflected radiation. Passive noncontact sensors depend on sunlight, while active sensors, with their own light sources, enable the assessment of the crop status irrespective of ambient light conditions [63, 64].

Commercial proximal sensors include:

The most effective nitrogen enhancement is administered during the phases of more intense vegetative activity (raised) and depends on the greater capacity of interception of plants. An experiment in the Mediterranean area (Spain) with a radioactive tracer (15N) has shown that only a small proportion of nitrogen supplied in presowing is effectively intercepted by wheat (14.1%). Nitrogen raised initially is instead absorbed in a greater proportion (54.8%)

The gross availability of nitrogen resulting from the mineralization of organic matter and from previous crop residues may adversely affect efficiency. Indications have emerged from

Very late fertilization around the time of earing/flowering, while not having significant effects on the yield, can increase the protein content of kernels and the production of protein per

Precision farming can represent a sustainable system, making nitrogen more sustainable

Although there are several definitions of precision agriculture (PF), Pierce and Nowak [52] have summarized it as "a system that provides the tools to do the right thing, the right place, at the right time," where "the right thing" is an agronomic operation. In fact, recent technological innovations have led to an increase of application opportunities and that definition can

**Figure 7.** Nitrogen fertilizer recovery (%) of 15N-labeled fertilizer (NR) and N derived from 15N fertilizer (NF) in the

whole plant at maturity for hard red spring wheat, as affected by N timing [49].

the study of durum wheat in the same environment [50].

**4.12. Use of precision farming for nitrogen application**

while achieving the best economic results.

[50] (**Figure 7**).

148 Nitrogen in Agriculture - Updates

hectare [15, 51].

therefore be extended.


Several studies have been conducted on wheat crops using these systems [65, 66], and the data collected must be converted into measures of vegetation, using vegetative indices.

The normalized difference vegetation index (NDVI) is the more precise vegetative normalization index and is based on differences in the red (670 nm) and near infrared (780 nm) spectrums.

NDVI has shown good correlation with N absorption in both bread and durum wheat. Correlations were often more stable when they involved single locations or varieties. An asymptotic tendency was found in the NDVI value for N uptake over 200 kg ha−1 [67–69].

The SPAD value showed a good correlation with the N status in bread wheat [70]. Of all observed indexes, the nitrogen nutrition index (NNI) was the most highly correlated. SPAD close to flowering was closely related to the final grain protein content.

### **5. Implication of nitrogen use on greenhouse gas (GHG) emissions**

#### **5.1. Problem statement**

The rapid increase in the world population over recent decades has led to an expansion in agricultural land in order to provide sufficient food. Consequently, agricultural inputs have been extensively used to increase yield, resulting in serious environmental effects. The emission of anthropogenic gases into the atmosphere is a serious environmental burden, and it is generally accepted that agricultural activities are a major source of several of these gases. Since the industrialization of agriculture, or the so-called green revolution, significant increases in the atmospheric concentrations of these gases have been observed globally. These cause environmental change that will have an impact at both regional and global levels. Carbon dioxide (CO<sup>2</sup> ), methane (CH<sup>4</sup> ), and nitrous oxide (N<sup>2</sup> O) are referred to as greenhouse gases (GHGs), as they are considered to contribute most to global warming. The effect of agricultural intensification, in particular the increased use of nitrogen fertilizer, on GHG emissions is likely to increase in the future. The Intergovernmental Panel on Climate Change (IPCC) has indicated that agricultural activities contributed around 10% of the total global GHG emissions [72]. The main agricultural contributors are soil emissions due to inorganic fertilizers and plant residues, in addition to biological processes. N is mainly lost from agriculture soils through nitrous oxide (N<sup>2</sup> O) emissions, which has recently been the subject of considerable attention due to its greenhouse gas effect (300 times worse than carbon dioxide). The increase in population has made it global necessary to produce more foodly, so both the agricultural land area and N<sup>2</sup> O emissions are likely to continue to rise in the coming decades (**Figure 8**). At a global level, agriculture land is estimated to contribute 60% of the N<sup>2</sup> O emissions to the atmosphere annually. Pires et al. [73] derived interesting results from several field experiments. They found that the amount of N fertilizer applied in the agricultural fields is the strongest indicator of N<sup>2</sup> O fluxes in major cropping systems. However, N fertilizer formulation and application timing, and the agronomic practices that determine N availability in the soil such as tillage and waste management can also influence the N<sup>2</sup> O flow, providing an opportunity for mitigation options in agriculture through N management. Several studies [74, 75] have reported that an average of 2% of the N applied to cultivated soils is emitted into the atmosphere as N<sup>2</sup> O. Inappropriate synchronization between N fertilization rate and crop demand would lead to a further increase in emission rates.

atmosphere [76]. According to the IPCC, nitrogen fertilizer application, crop residues, and

excessive levels of N applied, the higher amount fixed by N-fixing crops, and higher concentrations in plant residue. The remaining N not taken up by the plant can be lost indirectly

−

sphere or by biological soil processes such as nitrification and denitrification, all of which

The environmental consequences of N fertilizer application in agriculture have become of great interest to many researchers worldwide. They aim to understand the mechanism of different N pools (both organic and inorganic forms of N in the soil), their interaction, and the

activities, so understanding the biological processes and the factors affecting them will help in the international efforts toward the development of mitigation strategies for greenhouse gas emissions from agricultural soil. In addition, to optimize the trade-off between economic development and the impact of agricultural inputs on GHG emissions, environmental issues must be included in any future political agendas, as they are extremely important in terms of

Nitrogen has been widely studied to find ways of improving its efficiency in terms of producing acceptable grain yield and quality with less environmental impact. Recent research efforts have been directed toward reducing N loss from the soil by improving the absorption mechanism and the metabolism of N in the plant. Most have in fact succeeded in reducing

O emissions in agricultural fields [72] due to the

O-N yr−1) from agricultural soils between 1990 and 2030 (adapted from Reay et al.

Optimization of Nitrogen in Durum Wheat in the Mediterranean Climate: The Agronomical...

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151

) in groundwater, volatilization to the atmo-

O gas is a natural product of soil microbial

N-fixing crops present a direct source of N<sup>2</sup>

O emissions (Tg N<sup>2</sup>

through surface runoff, leached nitrate (NO<sup>3</sup>

processes by which they enter and leave the soil. N<sup>2</sup>

agricultural productivity and food security.

pose environmental concerns.

**Figure 8.** Global N<sup>2</sup>

[71]).

**5.2. Mitigation strategy**

Although the N loss from agricultural soils in different gaseous forms is considered the dominant mechanism in agricultural production systems, the manufacturing processes of agricultural inputs such as N fertilizers are also responsible for direct emissions of CO<sup>2</sup> into the

**Figure 8.** Global N<sup>2</sup> O emissions (Tg N<sup>2</sup> O-N yr−1) from agricultural soils between 1990 and 2030 (adapted from Reay et al. [71]).

atmosphere [76]. According to the IPCC, nitrogen fertilizer application, crop residues, and N-fixing crops present a direct source of N<sup>2</sup> O emissions in agricultural fields [72] due to the excessive levels of N applied, the higher amount fixed by N-fixing crops, and higher concentrations in plant residue. The remaining N not taken up by the plant can be lost indirectly through surface runoff, leached nitrate (NO<sup>3</sup> − ) in groundwater, volatilization to the atmosphere or by biological soil processes such as nitrification and denitrification, all of which pose environmental concerns.

The environmental consequences of N fertilizer application in agriculture have become of great interest to many researchers worldwide. They aim to understand the mechanism of different N pools (both organic and inorganic forms of N in the soil), their interaction, and the processes by which they enter and leave the soil. N<sup>2</sup> O gas is a natural product of soil microbial activities, so understanding the biological processes and the factors affecting them will help in the international efforts toward the development of mitigation strategies for greenhouse gas emissions from agricultural soil. In addition, to optimize the trade-off between economic development and the impact of agricultural inputs on GHG emissions, environmental issues must be included in any future political agendas, as they are extremely important in terms of agricultural productivity and food security.

#### **5.2. Mitigation strategy**

NDVI has shown good correlation with N absorption in both bread and durum wheat. Correlations were often more stable when they involved single locations or varieties. An asymptotic tendency was found in the NDVI value for N uptake over 200 kg ha−1 [67–69].

The SPAD value showed a good correlation with the N status in bread wheat [70]. Of all observed indexes, the nitrogen nutrition index (NNI) was the most highly correlated. SPAD

The rapid increase in the world population over recent decades has led to an expansion in agricultural land in order to provide sufficient food. Consequently, agricultural inputs have been extensively used to increase yield, resulting in serious environmental effects. The emission of anthropogenic gases into the atmosphere is a serious environmental burden, and it is generally accepted that agricultural activities are a major source of several of these gases. Since the industrialization of agriculture, or the so-called green revolution, significant increases in the atmospheric concentrations of these gases have been observed globally. These cause environmental change that will have an impact at both regional and global levels. Carbon dioxide

as they are considered to contribute most to global warming. The effect of agricultural intensification, in particular the increased use of nitrogen fertilizer, on GHG emissions is likely to increase in the future. The Intergovernmental Panel on Climate Change (IPCC) has indicated that agricultural activities contributed around 10% of the total global GHG emissions [72]. The main agricultural contributors are soil emissions due to inorganic fertilizers and plant residues, in addition to biological processes. N is mainly lost from agriculture soils through nitrous oxide

O) emissions, which has recently been the subject of considerable attention due to its greenhouse gas effect (300 times worse than carbon dioxide). The increase in population has made it

are likely to continue to rise in the coming decades (**Figure 8**). At a global level, agriculture land

derived interesting results from several field experiments. They found that the amount of N

ping systems. However, N fertilizer formulation and application timing, and the agronomic practices that determine N availability in the soil such as tillage and waste management can also

N management. Several studies [74, 75] have reported that an average of 2% of the N applied to

Although the N loss from agricultural soils in different gaseous forms is considered the dominant mechanism in agricultural production systems, the manufacturing processes of agri-

N fertilization rate and crop demand would lead to a further increase in emission rates.

cultural inputs such as N fertilizers are also responsible for direct emissions of CO<sup>2</sup>

O flow, providing an opportunity for mitigation options in agriculture through

global necessary to produce more foodly, so both the agricultural land area and N<sup>2</sup>

fertilizer applied in the agricultural fields is the strongest indicator of N<sup>2</sup>

O) are referred to as greenhouse gases (GHGs),

O emissions to the atmosphere annually. Pires et al. [73]

O. Inappropriate synchronization between

O emissions

into the

O fluxes in major crop-

**5. Implication of nitrogen use on greenhouse gas (GHG) emissions**

close to flowering was closely related to the final grain protein content.

), and nitrous oxide (N<sup>2</sup>

**5.1. Problem statement**

150 Nitrogen in Agriculture - Updates

), methane (CH<sup>4</sup>

is estimated to contribute 60% of the N<sup>2</sup>

cultivated soils is emitted into the atmosphere as N<sup>2</sup>

(CO<sup>2</sup>

(N<sup>2</sup>

influence the N<sup>2</sup>

Nitrogen has been widely studied to find ways of improving its efficiency in terms of producing acceptable grain yield and quality with less environmental impact. Recent research efforts have been directed toward reducing N loss from the soil by improving the absorption mechanism and the metabolism of N in the plant. Most have in fact succeeded in reducing the excessive input of N fertilizers, while maintaining profitable grain yield with less environmental impact. The results indicate that efficiency and effectiveness of N use on crops can be achieved [77] through further synchronizing the timing of N fertilizer application and the N rate with plant nitrogen demand, and its capacity to uptake sufficient amounts of available nitrogen. This strategy is believed to be of paramount importance for the reduction of N<sup>2</sup> O and therefore GHG emissions from the cropping system. As N uptake capacity is generally low at the beginning of the growing season, a large quantity of N fertilizer applied at sowing time will lead to a greater potential for increased N<sup>2</sup> O emissions due to slow plant uptake. Bouwman et al. [77] indicated that not only does the N rate significantly influence N2 O emissions from fields but also the N fertilizer type, and the time when the N fertilizer is applied throughout the growing season. Hao et al. [78] found that autumn N fertilizer application resulted in significantly greater N<sup>2</sup> O emissions than spring application. Despite being the key factor affecting total GHG emissions in the wheat production system, several studies in the literature found that the increased N fertilizer rate had no positive correlation with grain yield. Therefore, any wheat management option that reduces or eliminates the N fertilizer application (i.e., through the introduction of an N-fixing crop) would result in a significant reduction in total emissions. Rees et al. [79] highlighted the importance of N supply reduction in an emission mitigation strategy. Mosier et al. [80] indicated that improved soil management and better nutrient use efficiency could result in a reduction of 10–30% of total emissions from the soil.

soil, the choice of a suitable N fertilizer formula for a specific soil is fundamental [86]. The electrical charges of soil particles vary according to the soil texture, so studying the chemical properties and reactions within soil particles will help understanding when and which N fertilizers type can be applied to a specific soil. These reactions are believed to have a significant influence on when fertilizer can be applied and how efficiently the crop takes it up. As explained, clay particles in the soil have a negative electrical charge, so the amount of clay present in the soil surface, which the fertilizer first comes into contact with, is an important factor controlling plant response to the N type and the timing. The clay particles with negative charges will react with components of the fertilizer that dissolve as positively (+) charged particles (cations) when added to soil. For example, the application of N fertilizers that include more ammonium (positively charged) and less nitrate (negatively charged) forms of N can reduce their potential for losses in the short term. The application

Optimization of Nitrogen in Durum Wheat in the Mediterranean Climate: The Agronomical...

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153

**Figure 9.** Timing and application of N fertilizer in winter durum wheat (from Fertilizers Europe).

ing from the application of different nitrogen fertilizers, and **Figure 11** shows N losses via volatilization emissions. In both cases, N losses are significantly higher with urea than other

jected to losses through leaching or denitrification, and therefore, increase the N availability for the plant [87]. However, if the total amount of applied ammonium is not taken up by the

particles and will be lost when excessive rainfall leaches or saturates the soil. As identified, soil conditions are important in the transformation process, and nitrifying bacteria activity is minimal in cold soil temperatures, which can naturally help protect ammonium forms of N

) forms of N binding to soil particles with negative charges will not be sub-

−

O emissions from the soil, according

O soil emissions result-

), a form that cannot bind to soil

of the correct type of N fertilizer can help reduce N<sup>2</sup>

plant, soil bacteria will convert the remainder to nitrate (NO<sup>3</sup>

N fertilizers.

from losses.

Ammonium (NH<sup>4</sup>

+

to the Fertilizers Europe Initiative. **Figure 10** shows comparative N<sup>2</sup>

To tackle the projected changes in climate events and the potential effects on crop yield, farmers should focus on the establishment of sustainable wheat production systems through efficient management of agricultural inputs [81]. As N inputs are the main cause of several environmental problems, a priority when establishing such a system would be to make them more efficient. The calculation of nitrogen use efficiency (NUE) as an agro-environmental indicator is important in the agro-policy context [82], as it informs the economic utility of a specific N fertilizer quantity applied to the field with reference to crop yield. However, to obtain the maximum from N fertilizer application in cereal production, Pires et al. [82] suggested that both the environmental and economic pillars of sustainability must be addressed along with agronomic efficiency. Alternative management strategies have recently been proposed to optimize the NUE and therefore increase crop yield, including N fertilizer timing, split applications, site-specific management, crop rotation, crop diversification, biological N fixation, and improved plant traits by genetic breeding, which is believed to help improve yields with a low input of N fertilizer, reducing its losses from the agriculture system [83]. Options such as the use of biological nitrification inhibition by *Brachiaria* roots exudates [84] and the adoption of alternative farming techniques based on no-till continuous cover cropping [85] are other strategies for improving NUE. However, the response to these management strategies can vary, depending on the complex interaction of soil, crop, and environmental factors.

The literature review shows that the yield response varies with the nitrogen and moisture level. The goal of N application to wheat and its timing is to supply adequate N when the crop needs it (**Figure 9**). As the nitrogen cycle is biologically influenced by prevailing climatic conditions, along with the physical and chemical properties of a particular Optimization of Nitrogen in Durum Wheat in the Mediterranean Climate: The Agronomical... http://dx.doi.org/10.5772/intechopen.70195 153

**Figure 9.** Timing and application of N fertilizer in winter durum wheat (from Fertilizers Europe).

the excessive input of N fertilizers, while maintaining profitable grain yield with less environmental impact. The results indicate that efficiency and effectiveness of N use on crops can be achieved [77] through further synchronizing the timing of N fertilizer application and the N rate with plant nitrogen demand, and its capacity to uptake sufficient amounts of available nitrogen. This strategy is believed to be of paramount importance for the reduction

O and therefore GHG emissions from the cropping system. As N uptake capacity is generally low at the beginning of the growing season, a large quantity of N fertilizer applied

uptake. Bouwman et al. [77] indicated that not only does the N rate significantly influence

being the key factor affecting total GHG emissions in the wheat production system, several studies in the literature found that the increased N fertilizer rate had no positive correlation with grain yield. Therefore, any wheat management option that reduces or eliminates the N fertilizer application (i.e., through the introduction of an N-fixing crop) would result in a significant reduction in total emissions. Rees et al. [79] highlighted the importance of N supply reduction in an emission mitigation strategy. Mosier et al. [80] indicated that improved soil management and better nutrient use efficiency could result in a reduction of 10–30% of total

To tackle the projected changes in climate events and the potential effects on crop yield, farmers should focus on the establishment of sustainable wheat production systems through efficient management of agricultural inputs [81]. As N inputs are the main cause of several environmental problems, a priority when establishing such a system would be to make them more efficient. The calculation of nitrogen use efficiency (NUE) as an agro-environmental indicator is important in the agro-policy context [82], as it informs the economic utility of a specific N fertilizer quantity applied to the field with reference to crop yield. However, to obtain the maximum from N fertilizer application in cereal production, Pires et al. [82] suggested that both the environmental and economic pillars of sustainability must be addressed along with agronomic efficiency. Alternative management strategies have recently been proposed to optimize the NUE and therefore increase crop yield, including N fertilizer timing, split applications, site-specific management, crop rotation, crop diversification, biological N fixation, and improved plant traits by genetic breeding, which is believed to help improve yields with a low input of N fertilizer, reducing its losses from the agriculture system [83]. Options such as the use of biological nitrification inhibition by *Brachiaria* roots exudates [84] and the adoption of alternative farming techniques based on no-till continuous cover cropping [85] are other strategies for improving NUE. However, the response to these management strategies can vary, depending on the complex interaction of soil, crop, and envi-

The literature review shows that the yield response varies with the nitrogen and moisture level. The goal of N application to wheat and its timing is to supply adequate N when the crop needs it (**Figure 9**). As the nitrogen cycle is biologically influenced by prevailing climatic conditions, along with the physical and chemical properties of a particular

O emissions from fields but also the N fertilizer type, and the time when the N fertilizer is applied throughout the growing season. Hao et al. [78] found that autumn N fertilizer

O emissions due to slow plant

O emissions than spring application. Despite

at sowing time will lead to a greater potential for increased N<sup>2</sup>

application resulted in significantly greater N<sup>2</sup>

emissions from the soil.

ronmental factors.

of N<sup>2</sup>

152 Nitrogen in Agriculture - Updates

N2

soil, the choice of a suitable N fertilizer formula for a specific soil is fundamental [86]. The electrical charges of soil particles vary according to the soil texture, so studying the chemical properties and reactions within soil particles will help understanding when and which N fertilizers type can be applied to a specific soil. These reactions are believed to have a significant influence on when fertilizer can be applied and how efficiently the crop takes it up. As explained, clay particles in the soil have a negative electrical charge, so the amount of clay present in the soil surface, which the fertilizer first comes into contact with, is an important factor controlling plant response to the N type and the timing. The clay particles with negative charges will react with components of the fertilizer that dissolve as positively (+) charged particles (cations) when added to soil. For example, the application of N fertilizers that include more ammonium (positively charged) and less nitrate (negatively charged) forms of N can reduce their potential for losses in the short term. The application of the correct type of N fertilizer can help reduce N<sup>2</sup> O emissions from the soil, according to the Fertilizers Europe Initiative. **Figure 10** shows comparative N<sup>2</sup> O soil emissions resulting from the application of different nitrogen fertilizers, and **Figure 11** shows N losses via volatilization emissions. In both cases, N losses are significantly higher with urea than other N fertilizers.

Ammonium (NH<sup>4</sup> + ) forms of N binding to soil particles with negative charges will not be subjected to losses through leaching or denitrification, and therefore, increase the N availability for the plant [87]. However, if the total amount of applied ammonium is not taken up by the plant, soil bacteria will convert the remainder to nitrate (NO<sup>3</sup> − ), a form that cannot bind to soil particles and will be lost when excessive rainfall leaches or saturates the soil. As identified, soil conditions are important in the transformation process, and nitrifying bacteria activity is minimal in cold soil temperatures, which can naturally help protect ammonium forms of N from losses.

**6. Conclusion**

the Mediterranean environment.

**Author details**

Durum wheat is mainly cultivated in Mediterranean countries, but it is also of great importance in other European countries and in Canada and the United States, which are the world's leading exporters. The quality of durum wheat, often considered only in product market and technological terms, is in fact the result of a complex system that must take into account all the components of the supply chain. Climate change and the consequences of greenhouse gas emissions make

Optimization of Nitrogen in Durum Wheat in the Mediterranean Climate: The Agronomical...

http://dx.doi.org/10.5772/intechopen.70195

In addition to aspects related to the nutritional, safety, and technological characteristics of the product, it is also necessary to consider those related to the production environment, through the development of agronomic strategies that can improve precision farming, or techniques

The possibility of using improved varieties with better efficiency and higher quality standards could offer an opportunity to increase Mediterranean production, making it less dependent on imports from foreign countries. The strategy should include changing the phenological model to avoid stressful times during plant growth. In addition, a comprehensive understanding of the

tion, temperature, and drought, alone or in combination, will help the crop plant adapt to these changes. Nitrogen fertilization is an indispensable agronomic technique that can achieve good levels of production, both quantitatively and qualitatively, and has been extensively studied in

The application of precision farming systems (drones, NDVI systems, N sensors, etc.) can provide useful indications for the precise application of nitrogen. Many experiments suggest that the best agronomic technique in nitrogen application is the splitting of nitrogen application (sowing, tillering, stem elongation, and if necessary grain filling), which appears to be more efficient than the application divided once, which gives a 15% increase, or twice, where the increase is 7%. This strategy appears to be effective in reducing the loss of soil nitrates through leaching runoff and denitrification, which is more of a risk during the winter period, when the rainfall in the Mediterranean climate is concentrated. In fact, several types of nitrogen efficiency, and efficiency in N fertilization, increase when the fertilizer is applied at the stem elongation phase, while higher levels of N at the sowing time and tillering result in poor efficiency. The splitting application of nitrogen appears to be an effective method of avoiding environmental problems associated with the potential loss of this element, but more importantly could maximize the efficiency of the fertilization of wheat, increasing the yield in the

concentra-

155

crops very susceptible to climate variations, resulting in a variable yield response.

that can reduce the environmental impact of cultivation (conservative farming).

responses of each plant growth phase to environmental variables such as high CO<sup>2</sup>

Mediterranean region and providing a rational management strategy for the crop.

Luigi Tedone\*, Salem Alhajj Ali and Giuseppe De Mastro

Department of Agricultural and Environmental Science, Bari, Italy

\*Address all correspondence to: luigi.tedone@uniba.it

**Figure 10.** Comparative N<sup>2</sup> O emissions from the application of different nitrogen fertilizers (adapted from Fertilizers Europe). AN, ammonium nitrate; CAN, calcium ammonium nitrate; ANS, ammonium nitrosulphate; CN, calcium nitrate; AS, ammonium sulphate; DAP, ammonium phosphates; Urea, urea; UAN, urea ammonium nitrate; NPK, NPK 15-15-15; TSP, triple superphosphate; MOP, muriate of potash.

**Figure 11.** Volatilization losses (% N) from the application of different N fertilizers in arable land; Adapted from Fertilizers Europe; Data from the official European Emission Inventory (EMEP) as well as from a UK Government Department of Environment, Food and Rural Affairs (Defra) study.

## **6. Conclusion**

Durum wheat is mainly cultivated in Mediterranean countries, but it is also of great importance in other European countries and in Canada and the United States, which are the world's leading exporters. The quality of durum wheat, often considered only in product market and technological terms, is in fact the result of a complex system that must take into account all the components of the supply chain. Climate change and the consequences of greenhouse gas emissions make crops very susceptible to climate variations, resulting in a variable yield response.

In addition to aspects related to the nutritional, safety, and technological characteristics of the product, it is also necessary to consider those related to the production environment, through the development of agronomic strategies that can improve precision farming, or techniques that can reduce the environmental impact of cultivation (conservative farming).

The possibility of using improved varieties with better efficiency and higher quality standards could offer an opportunity to increase Mediterranean production, making it less dependent on imports from foreign countries. The strategy should include changing the phenological model to avoid stressful times during plant growth. In addition, a comprehensive understanding of the responses of each plant growth phase to environmental variables such as high CO<sup>2</sup> concentration, temperature, and drought, alone or in combination, will help the crop plant adapt to these changes. Nitrogen fertilization is an indispensable agronomic technique that can achieve good levels of production, both quantitatively and qualitatively, and has been extensively studied in the Mediterranean environment.

The application of precision farming systems (drones, NDVI systems, N sensors, etc.) can provide useful indications for the precise application of nitrogen. Many experiments suggest that the best agronomic technique in nitrogen application is the splitting of nitrogen application (sowing, tillering, stem elongation, and if necessary grain filling), which appears to be more efficient than the application divided once, which gives a 15% increase, or twice, where the increase is 7%. This strategy appears to be effective in reducing the loss of soil nitrates through leaching runoff and denitrification, which is more of a risk during the winter period, when the rainfall in the Mediterranean climate is concentrated. In fact, several types of nitrogen efficiency, and efficiency in N fertilization, increase when the fertilizer is applied at the stem elongation phase, while higher levels of N at the sowing time and tillering result in poor efficiency. The splitting application of nitrogen appears to be an effective method of avoiding environmental problems associated with the potential loss of this element, but more importantly could maximize the efficiency of the fertilization of wheat, increasing the yield in the Mediterranean region and providing a rational management strategy for the crop.

## **Author details**

**Figure 11.** Volatilization losses (% N) from the application of different N fertilizers in arable land; Adapted from Fertilizers Europe; Data from the official European Emission Inventory (EMEP) as well as from a UK Government

O emissions from the application of different nitrogen fertilizers (adapted from Fertilizers

Europe). AN, ammonium nitrate; CAN, calcium ammonium nitrate; ANS, ammonium nitrosulphate; CN, calcium nitrate; AS, ammonium sulphate; DAP, ammonium phosphates; Urea, urea; UAN, urea ammonium nitrate; NPK, NPK

Department of Environment, Food and Rural Affairs (Defra) study.

**Figure 10.** Comparative N<sup>2</sup>

154 Nitrogen in Agriculture - Updates

15-15-15; TSP, triple superphosphate; MOP, muriate of potash.

Luigi Tedone\*, Salem Alhajj Ali and Giuseppe De Mastro

\*Address all correspondence to: luigi.tedone@uniba.it

Department of Agricultural and Environmental Science, Bari, Italy

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**Chapter 9**

**Provisional chapter**

**The Effect of N Fertilization on Wheat under**

**The Effect of N Fertilization on Wheat under** 

DOI: 10.5772/intechopen.68904

The biological nitrogen fixation (BNF) process in wheat (*Triticum aestivum L.*) occurs by diazotrophic bacteria, particularly *Azospirillum* brasilense. However, researches are lacking on BNF efficiency to define how much mineral nitrogen (N) can be applied to achieve more sustainable high yields, and if urea with the urease enzyme inhibitor is less harmful, benefiting BNF in grasses (cereals). Therefore, the objective was to evaluate the effect of N sources (urea and Super N, urea with urease enzyme inhibitor N‐(n‐butyl thiophosphoric triamide) (NBPT) and N rates (0, 50, 100, 150, and 200 kg ha−1) applied in topdressing associated with inoculation with *A. brasilense*, regarding the leaf N concentration, leaf chlorophyll index (LCI), accumulation of N in the straw and grains, the nitrogen utilization efficiency (NUE), recovery of the applied nitrogen (RAN), physiological efficiency (FE), agronomic efficiency (AE), and wheat grain yield in the Brazilian Cerrado (tropical savanna) region. The N sources provide similar N accumulations in straw and grains, and wheat grain yield. Inoculation with *A. brasilense* afforded higher N grain concentration (increase in protein content more sustainably) by applying less N fertilizer in topdressing. Inoculation with *A. brasilense* increased the

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

© 2018 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

The management of nitrogen fertilization is performed in order to ensure adequate productivity, and depending on the N dynamics in the soil, large amounts of N are added to the soil,

**Keywords:** *Triticum aestivum* L., nitrogen sources, biological nitrogen fixation, urease

**Inoculation with** *Azospirillum brasilense*

**Inoculation with** *Azospirillum brasilense*

Marcelo Carvalho Minhoto Teixeira Filho,

Marcelo Carvalho Minhoto Teixeira Filho, Fernando Shintate Galindo, Salatiér Buzetti and Eduardo Henrique Marcandalli Boleta

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

Eduardo Henrique Marcandalli Boleta

http://dx.doi.org/10.5772/intechopen.68904

**Abstract**

AE, RAN, and NUE.

**1. Introduction**

inhibitor, no‐tillage system

Fernando Shintate Galindo, Salatiér Buzetti and


**Provisional chapter**

## **The Effect of N Fertilization on Wheat under Inoculation with** *Azospirillum brasilense* **Inoculation with** *Azospirillum brasilense*

**The Effect of N Fertilization on Wheat under** 

DOI: 10.5772/intechopen.68904

Marcelo Carvalho Minhoto Teixeira Filho, Fernando Shintate Galindo, Salatiér Buzetti and Eduardo Henrique Marcandalli Boleta Fernando Shintate Galindo, Salatiér Buzetti and Eduardo Henrique Marcandalli Boleta Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

Marcelo Carvalho Minhoto Teixeira Filho,

http://dx.doi.org/10.5772/intechopen.68904

#### **Abstract**

[80] Mosier A, Kroeze C, Nevison C, Oenema O, Seitzinger S, Cleemput O. Mitigation strategies for greenhouse gas emissions from animal production systems: Synergy between measuring and modelling at different scales. Nutrient Cycling in Agroecosystems.

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One. 2015;**10**(8):e0135234. DOI: 10.1371/journalpone.0135234

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513. DOI: 10.3390/agronomy4040497

007-5663-2 pp. 261. DOI: 10.1007/978-94-007-5663-2\_5

44810433.pdf

162 Nitrogen in Agriculture - Updates

2016

The biological nitrogen fixation (BNF) process in wheat (*Triticum aestivum L.*) occurs by diazotrophic bacteria, particularly *Azospirillum* brasilense. However, researches are lacking on BNF efficiency to define how much mineral nitrogen (N) can be applied to achieve more sustainable high yields, and if urea with the urease enzyme inhibitor is less harmful, benefiting BNF in grasses (cereals). Therefore, the objective was to evaluate the effect of N sources (urea and Super N, urea with urease enzyme inhibitor N‐(n‐butyl thiophosphoric triamide) (NBPT) and N rates (0, 50, 100, 150, and 200 kg ha−1) applied in topdressing associated with inoculation with *A. brasilense*, regarding the leaf N concentration, leaf chlorophyll index (LCI), accumulation of N in the straw and grains, the nitrogen utilization efficiency (NUE), recovery of the applied nitrogen (RAN), physiological efficiency (FE), agronomic efficiency (AE), and wheat grain yield in the Brazilian Cerrado (tropical savanna) region. The N sources provide similar N accumulations in straw and grains, and wheat grain yield. Inoculation with *A. brasilense* afforded higher N grain concentration (increase in protein content more sustainably) by applying less N fertilizer in topdressing. Inoculation with *A. brasilense* increased the AE, RAN, and NUE.

**Keywords:** *Triticum aestivum* L., nitrogen sources, biological nitrogen fixation, urease inhibitor, no‐tillage system

## **1. Introduction**

The management of nitrogen fertilization is performed in order to ensure adequate productivity, and depending on the N dynamics in the soil, large amounts of N are added to the soil,

Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons

raising production cost for the farmers. This applies to wheat (*Triticum aestivum* L.) that is an annual cycle plant, considered, among the winter season's cereal, one that has the greater economic importance with large grain yield capacity [1]. This cereal has great relevance in the diet and is cultivated in a wide range of environments and geographic regions. The cereal occupies over 17% of cultivable land in the world and represents approximately 30% of the world's grain production. In the period from 2012 to 2016, the annual average area of wheat cultivated worldwide was approximately 220 million hectares, reaching 734 million tons in the 2015/2016 harvest [2].

plant species. The components released by *Azospirillum brasilense* responsible for stimulating root growth are indole acetic acid (IAA), gibberellins, and cytokinins [18]. Inoculation with *Azospirillum* can improve the leaf photosynthetic parameters, including chlorophyll content and stomata conductance, greater proline content in shoots and roots, improvement in water potential, an increase in water content in the apoplast, more elasticity of the cell wall, more biomass production, and greater plant size as reported by Barassi et al. [19]. Increases in photosynthetic pigments such as chlorophyll a and b, and auxiliary photoprotective pigments, such as violaxantine, zeaxantine, ateroxantine, lutein, neoxantine, and beta‐carotene, which result in greener plants without water‐related stress, were verified by Bashan et al. [20].

The Effect of N Fertilization on Wheat under Inoculation with *Azospirillum brasilense*

http://dx.doi.org/10.5772/intechopen.68904

In addition, the increase in root development caused by inoculation with *Azospirillum* is involved with several other effects. Increases in water and mineral uptake have been reported, as well as greater tolerance to stresses such as salinity and drought, resulting in a more vigorous and productive plant [21, 22]. According to Dobbelaere et al. [23], positive responses to inoculation with *A. brasilense* are obtained even when the crops are grown in soils with high N content, which indicates that the plant responses occur not only due to the fixed N2

also depending on the production of phytohormones growth promoters such as cytokinin, gibberellin, and indole acetic acid. Lemos et al. [24], studying five wheat cultivars, found a positive interaction between *A. brasilense* and N fertilization only for one wheat cultivar (CD 150). Increases in N fertilization efficiency associated with inoculation with *A. brasilense* were

Considering the benefits attributed to several crops by inoculation with *A. brasilense*, with an emphasis on biological nitrogen fixation, greater development of the root system, and, consequently, greater uptake of water and nutrients, the inoculation can improve crop performance allowing greater efficiency of nitrogen fertilization. Thus, more experiments of this type should be carried out in order to evaluate the effect on plant nutrition. In addition, there are still few studies which define how much of N minerals can be applied for BNF to be successful in increasing the yield. It would be interesting to analyze urea with an NBPT urease

The hypothesis of this study was that inoculation with *A. brasilense* can increase the efficiency of N fertilization and N plant nutrition. The objective was to evaluate the effect of N sources and N rates associated, or not, with *A. brasilense* inoculation regarding the leaf N concentration, leaf chlorophyll index (LCI), accumulation of N in the straw and grains, the nitrogen utilization efficiency (NUE), recovery of the applied nitrogen (RAN), physiological efficiency (FE), agronomic efficiency (AE), and wheat grain yield in the *Brazilian Cerrado* (tropical

The wheat experiment was conducted in 2015, in the field in an experimental area that belongs to the Paulista State University (UNESP), Engineering Faculty, located in Selvíria—MS/Brazil,

reported by Galindo et al. [14] but in the corn grain yields in the *Brazilian Cerrado*.

enzyme inhibitor to verify whether it causes damage to BNF in grass.

savanna) region.

**2. Material and methods**

**2.1. Location and soil‐climatic conditions**

but

165

The final crop yield is defined according to the cultivar used, the amount of agricultural supplies and management techniques employed. The increasing use of the high yield potential of wheat has implicated in a more frequent use of agricultural supplies, among which nitrogen fertilization is shown to be important in defining the grain yield [3]. Several authors reported a positive response of nitrogen fertilization on the grain yield of wheat [4–6]. Therefore, there is a need to study wheat cultivars verifying their response in the uptake and utilization of nutrients in the soil and their performance and cultural practices in different environments [7].

Nitrogen fertilization includes one of the highest costs of the production process of non‐leguminous crops [8]. Wheat, corn, and rice crops utilize approximately 60% of the N fertilizers produced in the world [9]. The use of N fertilizers must be carefully controlled to ensure good yield and manage N in the soil; N fertilizer increases production costs for farmers [10]. Also, both nitrogen fertilizer production and application contribute to the emission of gases (CO2 and NO2 ) that contribute to the increase of the greenhouse effect on the Earth. In a report developed by the International Fertilizer Industry Association and the United Nations Environment Program, 873 m3 of natural gas was used to produce 1 metric ton of nitrogen fertilizer synthesized by the Haber‐Bosch process [11].

It is estimated that there may be a reduction in grain yield due to the volatilization of N‐NH3 at the rate of 10 kg ha−1 of grains for each 1% N that is volatilized [12]. In this context, one possibility to increase the nitrogen fertilization efficiency is the use of the N‐(n‐butyl) thiophosphoric triamide (NPBT) inhibitor, which can delay the hydrolysis of urea and significantly reduce NH3 losses depending on the weather, that is, heat and rain as well as the chemical characteristics of the soil [13, 14]. Due to the climate in Brazil, urea with urease enzyme inhibitor and conventional urea are equally effective in terms of N nutrition and grain yield of cereals. Studies in countries with milder weather have had different results [15].

Due to the high cost of fertilizers and awareness in support of sustainable agriculture and less pollution, in which the research is growing, another possibility would be to use inoculants containing bacteria that promote growth and increase the productivity of plants. Studies on biological nitrogen fixation (BNF) by *Azospirillum* species in grass have been carried out in Brazil. Until recently, no commercial inoculants with these bacteria are available in the country [16].

Although the plant genotype performs an essential role in the colonization of bacteria, existing cultivars with high and low potential of association [17]. Several studies have been published confirming that *Azospirillum* produces phytohormones that stimulate root growth in many plant species. The components released by *Azospirillum brasilense* responsible for stimulating root growth are indole acetic acid (IAA), gibberellins, and cytokinins [18]. Inoculation with *Azospirillum* can improve the leaf photosynthetic parameters, including chlorophyll content and stomata conductance, greater proline content in shoots and roots, improvement in water potential, an increase in water content in the apoplast, more elasticity of the cell wall, more biomass production, and greater plant size as reported by Barassi et al. [19]. Increases in photosynthetic pigments such as chlorophyll a and b, and auxiliary photoprotective pigments, such as violaxantine, zeaxantine, ateroxantine, lutein, neoxantine, and beta‐carotene, which result in greener plants without water‐related stress, were verified by Bashan et al. [20].

In addition, the increase in root development caused by inoculation with *Azospirillum* is involved with several other effects. Increases in water and mineral uptake have been reported, as well as greater tolerance to stresses such as salinity and drought, resulting in a more vigorous and productive plant [21, 22]. According to Dobbelaere et al. [23], positive responses to inoculation with *A. brasilense* are obtained even when the crops are grown in soils with high N content, which indicates that the plant responses occur not only due to the fixed N2 but also depending on the production of phytohormones growth promoters such as cytokinin, gibberellin, and indole acetic acid. Lemos et al. [24], studying five wheat cultivars, found a positive interaction between *A. brasilense* and N fertilization only for one wheat cultivar (CD 150). Increases in N fertilization efficiency associated with inoculation with *A. brasilense* were reported by Galindo et al. [14] but in the corn grain yields in the *Brazilian Cerrado*.

Considering the benefits attributed to several crops by inoculation with *A. brasilense*, with an emphasis on biological nitrogen fixation, greater development of the root system, and, consequently, greater uptake of water and nutrients, the inoculation can improve crop performance allowing greater efficiency of nitrogen fertilization. Thus, more experiments of this type should be carried out in order to evaluate the effect on plant nutrition. In addition, there are still few studies which define how much of N minerals can be applied for BNF to be successful in increasing the yield. It would be interesting to analyze urea with an NBPT urease enzyme inhibitor to verify whether it causes damage to BNF in grass.

The hypothesis of this study was that inoculation with *A. brasilense* can increase the efficiency of N fertilization and N plant nutrition. The objective was to evaluate the effect of N sources and N rates associated, or not, with *A. brasilense* inoculation regarding the leaf N concentration, leaf chlorophyll index (LCI), accumulation of N in the straw and grains, the nitrogen utilization efficiency (NUE), recovery of the applied nitrogen (RAN), physiological efficiency (FE), agronomic efficiency (AE), and wheat grain yield in the *Brazilian Cerrado* (tropical savanna) region.

## **2. Material and methods**

raising production cost for the farmers. This applies to wheat (*Triticum aestivum* L.) that is an annual cycle plant, considered, among the winter season's cereal, one that has the greater economic importance with large grain yield capacity [1]. This cereal has great relevance in the diet and is cultivated in a wide range of environments and geographic regions. The cereal occupies over 17% of cultivable land in the world and represents approximately 30% of the world's grain production. In the period from 2012 to 2016, the annual average area of wheat cultivated worldwide was approximately 220 million hectares, reaching 734 million tons in

The final crop yield is defined according to the cultivar used, the amount of agricultural supplies and management techniques employed. The increasing use of the high yield potential of wheat has implicated in a more frequent use of agricultural supplies, among which nitrogen fertilization is shown to be important in defining the grain yield [3]. Several authors reported a positive response of nitrogen fertilization on the grain yield of wheat [4–6]. Therefore, there is a need to study wheat cultivars verifying their response in the uptake and utilization of nutrients in the soil and their performance and cultural practices

Nitrogen fertilization includes one of the highest costs of the production process of non‐leguminous crops [8]. Wheat, corn, and rice crops utilize approximately 60% of the N fertilizers produced in the world [9]. The use of N fertilizers must be carefully controlled to ensure good yield and manage N in the soil; N fertilizer increases production costs for farmers [10]. Also, both nitrogen fertilizer production and application contribute to the emission of gases

report developed by the International Fertilizer Industry Association and the United Nations

It is estimated that there may be a reduction in grain yield due to the volatilization of N‐NH3 at the rate of 10 kg ha−1 of grains for each 1% N that is volatilized [12]. In this context, one possibility to increase the nitrogen fertilization efficiency is the use of the N‐(n‐butyl) thiophosphoric triamide (NPBT) inhibitor, which can delay the hydrolysis of urea and significantly

characteristics of the soil [13, 14]. Due to the climate in Brazil, urea with urease enzyme inhibitor and conventional urea are equally effective in terms of N nutrition and grain yield of cere-

Due to the high cost of fertilizers and awareness in support of sustainable agriculture and less pollution, in which the research is growing, another possibility would be to use inoculants containing bacteria that promote growth and increase the productivity of plants. Studies on biological nitrogen fixation (BNF) by *Azospirillum* species in grass have been carried out in Brazil. Until recently, no commercial inoculants with these bacteria are available in the

Although the plant genotype performs an essential role in the colonization of bacteria, existing cultivars with high and low potential of association [17]. Several studies have been published confirming that *Azospirillum* produces phytohormones that stimulate root growth in many

als. Studies in countries with milder weather have had different results [15].

) that contribute to the increase of the greenhouse effect on the Earth. In a

losses depending on the weather, that is, heat and rain as well as the chemical

of natural gas was used to produce 1 metric ton of nitrogen

the 2015/2016 harvest [2].

164 Nitrogen in Agriculture - Updates

in different environments [7].

Environment Program, 873 m3

fertilizer synthesized by the Haber‐Bosch process [11].

(CO2

reduce NH3

country [16].

and NO2

#### **2.1. Location and soil‐climatic conditions**

The wheat experiment was conducted in 2015, in the field in an experimental area that belongs to the Paulista State University (UNESP), Engineering Faculty, located in Selvíria—MS/Brazil, with the following geographical coordinates 20°22'S, 51°22'W and an altitude of 335 m. Soil in this experimental area was classified as distroferric red oxisol with clay texture (with values of particle sizes as 420, 50, and 530 g kg−1 of sand, silt, and clay, respectively), according to Embrapa [25], which has been cultivated with annual cultures over 27 years and with no‐tillage system in the past 11 years. The area was under corn cultivation before sowing wheat. The annual average temperature was 23.5°C, annual average pluvial precipitation was 1370 mm, and annual average relative air humidity was between 70 and 80%. Weather data recorded during the experimental period are shown in **Figure 1**.

and 200 kg ha−1) applied in topdressing at the growth stage 3.2 on Zadok scale; and two seed inoculation treatments—half of the tests were carried out with seeds inoculated with *A. brasilense*, whereas the other half did not have this inoculation. Each plot consisted of 5 m in length with 12 lines and an inter‐row spacing of 0.17 m. The usable area of the plot was 8

Wheat seeds were inoculated with 300 mL of inoculant liquid of *A. brasilense* bacteria, AbV5

of wheat seeds. The inoculant was mixed with the seeds using a cement mixer, 1 h before planting, and after that, the seed treatments were carried out with carbendazim and thiram fungicides (45 and 105 g a.i. per 100 kg of seeds) and thiodicarb and imidacloprid insecticides

Was applied 350 kg ha−1 of a 08‐28‐16 formulation in the forms of urea, triple superphosphate and potassium chloride, respectively, at wheat sowing. The experiments were conducted in a no‐tillage system. The area was irrigated by a central pivot sprinkler system. The water coverage was 14 mm over a period of around 72 h. The cultivar used was the CD 116 and sowing was done with an experimental machine on May 15, and 80 seeds per meter were being sown. Metsulfuron methyl (3.0 g a.i. ha−1), a post‐emergence herbicide, was applied 20 days after emergence (DAE) to control weeds like *Ipomoea grandifolia*, *Tridax procumbens,* and *Spermacoce* 

The seedling emergence was 6 days after sowing. Topdressing with nitrogen fertilization was performed at 35 DAE, manually distributing the fertilizer on the soil surface (no incorporation), and there was approximately 8 cm of sowing lines in order to avoid the contact of the

The LCI was determined indirectly after application of the treatments and when the plants were in the flowering stage, in 10 plants per plot, through readings in the leaf below the ear

The N leaf concentration (leaf diagnosis) was performed by collecting 20 leaf flags in the flowering of wheat plants, according to the methodology described in Cantarella et al. [27]. The N determination was carried out as described by Malavolta et al. [28]. The N concentrations in the grains and straw (above the soil) of wheat were also measured during the harvest occasion (at the end of the crop cycle), in 10 plants per useful area of the plot, according to the methodology described by Cantarella et al. [27]. By means of these nutrient concentrations and dry matter of plants, nutrient accumulation in the grains and straw was calculated and extrapolated to kg ha−1. The wheat was harvested from the plants in the useful area of each plot and the grain yield was calculated after mechanical threshing. Data were transformed

fertilizer with the plants. The plants were harvested 110 days after wheat emergence.

.

The Effect of N Fertilization on Wheat under Inoculation with *Azospirillum brasilense*

UFC mL−1) per sack of 40 kg

http://dx.doi.org/10.5772/intechopen.68904

167

center lines, excluding 0.5 m extremities. The plot size was 10.20 m2

and AbV6 strains (guaranteed minimum analysis of 2 × 10<sup>8</sup>

**2.3. Wheat crop management**

(45 and 135 g a.i. per 100 kg of seeds).

**2.4. Research evaluations and statistical analysis**

into kg ha−1 and corrected for 13% moisture (on a wet basis).

(in the middle third of each leaf).

*latifolia*.

Glyphosate [1800 g ha−1 of active ingredient (a.i.) and 2,4‐D (670 g ha−1 of a.i.)] herbicides were used for desiccation, applied 2 weeks prior to sowing wheat. Chemical attributes of the soil in the tillable layer were determined before the wheat experiment began. The methods proposed by Raij et al. [26] showed the following results: 13 mg dm−3 of P (resin), 6 mg dm−3 of S‐SO4, 23 g dm−3 of organic matter (OM), pH (CaCl2 ) of 4.8, 2.6 mmol<sup>c</sup> dm−3 of K<sup>+</sup> , 13.0 mmol<sup>c</sup> dm−3 of Ca2+, 8.0 mmol<sup>c</sup> dm−3 of Mg2+, 42.0 mmol<sup>c</sup> dm−3 of H + Al, 5.9 mg dm−3 of Cu, 30.0 mg dm−3 of Fe, 93.9 mg dm−3 of Mn, 1.0 mg dm−3 of Zn (DTPA), 0.24 mg dm−3 of B (hot water), and 36% of base saturation. After soil chemical analysis, 2.5 t ha−1 of dolomitic limestone (with 88% of relative total neutralizing power) was directly applied as topdressing for 80 days before the wheat was sown in 2015 in order to elevate base saturation to 70%, as recommended by Cantarella et al. [27].

#### **2.2. Treatments and experimental design**

A randomized block experimental design in a 2 × 5 × 2 arrangement with four replications was used for both crops. There were two N sources–conventional urea with 45% N and Super N, urea with a urease enzyme inhibitor, NBPT with 45% N; five rates of N (0, 50, 100, 150,

**Figure 1.** Rainfall, air relative humidity, and maximum and minimum average temperature obtained from the weather station located on the Education and Research Farm of FE/UNESP during wheat cultivation in the period from May to September 2015.

and 200 kg ha−1) applied in topdressing at the growth stage 3.2 on Zadok scale; and two seed inoculation treatments—half of the tests were carried out with seeds inoculated with *A. brasilense*, whereas the other half did not have this inoculation. Each plot consisted of 5 m in length with 12 lines and an inter‐row spacing of 0.17 m. The usable area of the plot was 8 center lines, excluding 0.5 m extremities. The plot size was 10.20 m2 .

#### **2.3. Wheat crop management**

with the following geographical coordinates 20°22'S, 51°22'W and an altitude of 335 m. Soil in this experimental area was classified as distroferric red oxisol with clay texture (with values of particle sizes as 420, 50, and 530 g kg−1 of sand, silt, and clay, respectively), according to Embrapa [25], which has been cultivated with annual cultures over 27 years and with no‐tillage system in the past 11 years. The area was under corn cultivation before sowing wheat. The annual average temperature was 23.5°C, annual average pluvial precipitation was 1370 mm, and annual average relative air humidity was between 70 and 80%. Weather data recorded

Glyphosate [1800 g ha−1 of active ingredient (a.i.) and 2,4‐D (670 g ha−1 of a.i.)] herbicides were used for desiccation, applied 2 weeks prior to sowing wheat. Chemical attributes of the soil in the tillable layer were determined before the wheat experiment began. The methods proposed by Raij et al. [26] showed the following results: 13 mg dm−3 of P (resin), 6 mg dm−3 of S‐SO4, 23 g

) of 4.8, 2.6 mmol<sup>c</sup>

mg dm−3 of Mn, 1.0 mg dm−3 of Zn (DTPA), 0.24 mg dm−3 of B (hot water), and 36% of base saturation. After soil chemical analysis, 2.5 t ha−1 of dolomitic limestone (with 88% of relative total neutralizing power) was directly applied as topdressing for 80 days before the wheat was sown in 2015 in order to elevate base saturation to 70%, as recommended by Cantarella et al. [27].

A randomized block experimental design in a 2 × 5 × 2 arrangement with four replications was used for both crops. There were two N sources–conventional urea with 45% N and Super N, urea with a urease enzyme inhibitor, NBPT with 45% N; five rates of N (0, 50, 100, 150,

**Figure 1.** Rainfall, air relative humidity, and maximum and minimum average temperature obtained from the weather station located on the Education and Research Farm of FE/UNESP during wheat cultivation in the period from May to

dm−3 of K<sup>+</sup>

dm−3 of H + Al, 5.9 mg dm−3 of Cu, 30.0 mg dm−3 of Fe, 93.9

, 13.0 mmol<sup>c</sup>

dm−3 of Ca2+,

during the experimental period are shown in **Figure 1**.

dm−3 of organic matter (OM), pH (CaCl2

dm−3 of Mg2+, 42.0 mmol<sup>c</sup>

**2.2. Treatments and experimental design**

8.0 mmol<sup>c</sup>

166 Nitrogen in Agriculture - Updates

September 2015.

Wheat seeds were inoculated with 300 mL of inoculant liquid of *A. brasilense* bacteria, AbV5 and AbV6 strains (guaranteed minimum analysis of 2 × 10<sup>8</sup> UFC mL−1) per sack of 40 kg of wheat seeds. The inoculant was mixed with the seeds using a cement mixer, 1 h before planting, and after that, the seed treatments were carried out with carbendazim and thiram fungicides (45 and 105 g a.i. per 100 kg of seeds) and thiodicarb and imidacloprid insecticides (45 and 135 g a.i. per 100 kg of seeds).

Was applied 350 kg ha−1 of a 08‐28‐16 formulation in the forms of urea, triple superphosphate and potassium chloride, respectively, at wheat sowing. The experiments were conducted in a no‐tillage system. The area was irrigated by a central pivot sprinkler system. The water coverage was 14 mm over a period of around 72 h. The cultivar used was the CD 116 and sowing was done with an experimental machine on May 15, and 80 seeds per meter were being sown. Metsulfuron methyl (3.0 g a.i. ha−1), a post‐emergence herbicide, was applied 20 days after emergence (DAE) to control weeds like *Ipomoea grandifolia*, *Tridax procumbens,* and *Spermacoce latifolia*.

The seedling emergence was 6 days after sowing. Topdressing with nitrogen fertilization was performed at 35 DAE, manually distributing the fertilizer on the soil surface (no incorporation), and there was approximately 8 cm of sowing lines in order to avoid the contact of the fertilizer with the plants. The plants were harvested 110 days after wheat emergence.

## **2.4. Research evaluations and statistical analysis**

The LCI was determined indirectly after application of the treatments and when the plants were in the flowering stage, in 10 plants per plot, through readings in the leaf below the ear (in the middle third of each leaf).

The N leaf concentration (leaf diagnosis) was performed by collecting 20 leaf flags in the flowering of wheat plants, according to the methodology described in Cantarella et al. [27]. The N determination was carried out as described by Malavolta et al. [28]. The N concentrations in the grains and straw (above the soil) of wheat were also measured during the harvest occasion (at the end of the crop cycle), in 10 plants per useful area of the plot, according to the methodology described by Cantarella et al. [27]. By means of these nutrient concentrations and dry matter of plants, nutrient accumulation in the grains and straw was calculated and extrapolated to kg ha−1. The wheat was harvested from the plants in the useful area of each plot and the grain yield was calculated after mechanical threshing. Data were transformed into kg ha−1 and corrected for 13% moisture (on a wet basis).

The accumulation values of N were obtained by the N concentrations in the plant and the dry matter production (DM). With the data of dry mass and accumulation of N, the following indices were calculated:

Similar results were obtained by Theago et al. [6], who observed a linear increase in foliar N concentration with the increment of the doses in coverage (0, 50, 100, 150, and 200 kg ha−1). The increase of N concentration in the wheat leaf, due to the increase of N rates, was also reported by Teixeira Filho et al. [33], with a quadratic response up to the dose of 100 kg ha−1

The Effect of N Fertilization on Wheat under Inoculation with *Azospirillum brasilense*

http://dx.doi.org/10.5772/intechopen.68904

169

**LCI N leaf concentration N straw N grains**

 50.92 38.89 24.12 44.86 52.31 44.20 31.39 78.35 54.50 47.39 37.08 92.22 55.23 48.46 38.59 77.67 56.16 49.95 54.89 96.10

Urea 52.66 b 46.08 aΨ 38.87 a 78.35 a Super N 54.99 a 45.47 a 35.56 a 77.33 a L.S.D. (5%) 1.31 1.33 4.91 9.81

With *Azospirillum* 54.26 a 45.24 a 34.82 79.20 a Without *Azospirillum* 53.39 a 46.31 a 39.60 76.48 a L.S.D. (5%) 1.31 1.33 4.91 9.81 Overall mean 53.82 45.78 37.21 77.84 C.V. (%) 4.64 5.55 25.26 19.04

ΨMeans followed by the same letters in the column do not differ by Tukey at 0.05 probability level.

N rates, and sources and inoculation with *Azospirillum brasilense* in wheat crops.

(A) (B)

**Table 1.** Leaf chlorophyll index (LCI), N leaf concentration, N accumulated in straw and grains of wheat in function of

**Figure 2.** LCI (A) and N leaf concentration (B) in wheat crops in the function of N rates. Selvíria—MS, Brazil, 2015.

**N rates (kg ha−1) (g kg−1)**

**N sources**

**Inoculation**

Selvíria—MS, Brazil, 2015.


The results were subjected to analysis of variance and the Tukey test at 5% probability to compare the averages of N sources and plants that had been inoculated with *A. brasilense* with those that had not been inoculated. Regression equations were fitted for the effect of N rates using the Sisvar program [31].

## **3. Results and discussions**

LCI increased linearly with increasing doses of N (**Table 1**, **Figure 2**). The increase in LCI values, as a consequence of the N rates, resembles those reported by Theago et al. [6], who observed an increase in LCI in wheat up to the dose of 200 kg ha−1, and Teixeira Filho et al. [5] up to 147 kg ha−1 of N. This behavior is due to the increase in chlorophyll concentration, promoted by the greater availability of total N in the tissues. This relationship is attributed mainly to the fact that 50–70% of the total leaf N is integral to enzymes that are associated with chloroplasts [32].

Regarding the sources, the Super N provided greater readings of LCI compared to urea (**Table 1**). However, inoculation with *A. brasilense* did not influence LCI, unlike that reported by Galindo et al. [14], working with the N rates (0, 50, 100, 150 and 200 kg ha−1 in topdressing) and the sources urea and Super N in the corn crop.

The increase of N rates influenced N leaf concentration (**Table 1**). There was an adjustment of the quadratic function, with maximum concentration point obtained of the N rate at 181 kg ha−1 approximately (**Figure 2**). The increase of N concentration in the leaf tissue was expected since by providing more nutrient quantity, the uptake of the crop would be greater, with reflection in the concentration in the leaf and in the aerial part, even in a consolidated system of no tillage in the area of study, over 10 years, which could supply the need of this nutrient due to the decomposition of the straw and as a result of the crop sequence after wheat was the maize crop, the wheat crop response to N rates was more evident.

Similar results were obtained by Theago et al. [6], who observed a linear increase in foliar N concentration with the increment of the doses in coverage (0, 50, 100, 150, and 200 kg ha−1). The increase of N concentration in the wheat leaf, due to the increase of N rates, was also reported by Teixeira Filho et al. [33], with a quadratic response up to the dose of 100 kg ha−1


Selvíria—MS, Brazil, 2015.

The accumulation values of N were obtained by the N concentrations in the plant and the dry matter production (DM). With the data of dry mass and accumulation of N, the following

**1.** Nitrogen utilization efficiency = total dry matter in kg/accumulation of N in g, in kg of DM/

**2.** Recovery of the applied nitrogen = accumulation of N in kg with fertilization−accumulation of N in kg without fertilization/N rate applied in kg × 100, in percentage [30],

**3.** Physiological efficiency (FE) = biological yield (straw and grains) without fertilization in kg/N accumulation with fertilizer (straw and grains) in kg−N accumulation without fertili-

**4.** Agronomic efficiency (AE) = grain yield with fertilization in kg−grain yield without fertili-

The results were subjected to analysis of variance and the Tukey test at 5% probability to compare the averages of N sources and plants that had been inoculated with *A. brasilense* with those that had not been inoculated. Regression equations were fitted for the effect of N rates

LCI increased linearly with increasing doses of N (**Table 1**, **Figure 2**). The increase in LCI values, as a consequence of the N rates, resembles those reported by Theago et al. [6], who observed an increase in LCI in wheat up to the dose of 200 kg ha−1, and Teixeira Filho et al. [5] up to 147 kg ha−1 of N. This behavior is due to the increase in chlorophyll concentration, promoted by the greater availability of total N in the tissues. This relationship is attributed mainly to the fact that 50–70% of the total leaf N is integral to enzymes that are associated

Regarding the sources, the Super N provided greater readings of LCI compared to urea (**Table 1**). However, inoculation with *A. brasilense* did not influence LCI, unlike that reported by Galindo et al. [14], working with the N rates (0, 50, 100, 150 and 200 kg ha−1 in topdressing)

The increase of N rates influenced N leaf concentration (**Table 1**). There was an adjustment of the quadratic function, with maximum concentration point obtained of the N rate at 181 kg ha−1 approximately (**Figure 2**). The increase of N concentration in the leaf tissue was expected since by providing more nutrient quantity, the uptake of the crop would be greater, with reflection in the concentration in the leaf and in the aerial part, even in a consolidated system of no tillage in the area of study, over 10 years, which could supply the need of this nutrient due to the decomposition of the straw and as a result of the crop sequence after wheat was the

zation (straw and grains) in kg, in kg of DM/kg of accumulated N [30], and

zation in kg/N rate in kg, in kg of DM/kg of applied N [30].

indices were calculated:

168 Nitrogen in Agriculture - Updates

kg of accumulated N [29],

using the Sisvar program [31].

**3. Results and discussions**

and the sources urea and Super N in the corn crop.

maize crop, the wheat crop response to N rates was more evident.

with chloroplasts [32].

ΨMeans followed by the same letters in the column do not differ by Tukey at 0.05 probability level.

**Table 1.** Leaf chlorophyll index (LCI), N leaf concentration, N accumulated in straw and grains of wheat in function of N rates, and sources and inoculation with *Azospirillum brasilense* in wheat crops.

**Figure 2.** LCI (A) and N leaf concentration (B) in wheat crops in the function of N rates. Selvíria—MS, Brazil, 2015.

of N, and Teixeira Filho et al. [34], with a point of maximum concentration of N being reached with the application estimate of 163 kg ha−1 of N. It should be noted that the concentration of N in the leaf tissue, even in the absence of nitrogen fertilization (0 kg ha−1), was above that as recommended by Cantarella et al. [27], ranging from 20–34 g kg−1.

N sources did not differ in N concentration, indicating that Super N was not efficient for N nutrition, even in the area with remaining straw of corn or wheat (**Table 1**). Similar results were verified by Meira et al. [35], using ammonium sulfonitrate, ammonium sulfate, and urea sources for the N concentration of irrigated wheat in the *Cerrado*. However, Teixeira Filho et al. [5] verified higher N concentrations in leaf tissue when the sources of ammonium sulfonitrate and ammonium sulfate compared to urea were used.

On the other hand, Megda et al. [36] (ammonium sulfonitrate, ammonium sulfate, and urea), in the corn crop, verified that the ammonium sulfonitrate source had a higher N concentration in the leaf, compared to the other sources, unlike the results obtained in the present research. It should be noted that Super N acts on the inhibition of the urease enzyme, whereas the ammonium sulfonitrate has in its composition dimethylpyrazolophosphate (DMPP) molecules that act to inhibit nitrification. This makes the fertilizer less susceptible to leaching, since there is a longer residence time of N, as ammonium in the soil, and, under conditions of tropical climate and high temperatures, presents different responses to Super N. Also, there is evidence of active urea transport by high affinity transporters (symport) located in the plasmatic membrane of the root epidermis cells, which would allow uptake of some urea applied before urease has acted and NH3 has been formed, especially when urea concentration in the soil and soil pH is low [37].

According to Pankievicz et al. [38], there is a greater development and growth of the root system of the *Setaria viridis* grass inoculated with *A. brasilense* as a function of associative fixers, with greater CO2 fixation and less accumulation of photoassimilated carbon in the leaves, which would favor the plant with greater growth in aerial part, greater accumulation of water, and conditions of less stress caused by the greater accumulation and metabolism of carbon, thus, increasing the concentration of nutrients in the plant. On the other hand, Bashan et al. [22] reported that the production of plant hormones, mainly indole acetic acid by bacteria of the genus *Azospirillum*, plays an essential role in the promotion of plant growth and, according to Hungria et al. [39], can improve the uptake of several macro and micronutrients, increasing the efficiency of the use of the available nutrients; however, this result was not verified in the present work, which raises the question of the difference of efficiency of the inoculation in grass crops.

The greater accumulation of nutrients provided by the increase of the N rates associated or not with the inoculation with *A. brasilense* is a very significant result considering that conservation practices benefit the productive systems and environmental conservation, such as no‐ tillage system, whose straw will provide nutrients to subsequent crops, recycling nutrients,

**Figure 3.** N rates and inoculation with *A. brasilense* interaction in the N accumulation in straw (A) and in grains (B) of

**0 50 100 150 200**

The Effect of N Fertilization on Wheat under Inoculation with *Azospirillum brasilense*

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171

With 22.38 aΨ 30.29 a 35.28 a 34.20 a 51.97 a Without 25.86 a 30.29 a 38.88 a 42.99 a 57.81 a

ΨMeans followed by the same letters in the column do not differ by Tukey at 0.05 probability level.

**Table 2.** Inoculation with *A. brasilense* and N rates interaction in the N accumulation in wheat straw.

N is the nutrient that most interferes in the development and productivity of crops, especially grasses. This mineral nutrient is found in higher concentrations, in vegetative tissues and grains, which characterizes it as being the element most demanded by the wheat plant. Thus, the higher availability of this nutrient in the plants favored the development of the root system, which, by exploiting a larger volume of soil, may have an uptake of a greater amount of nutrients and water, reflecting the accumulation in aerial part, both in the straw and in the grains. Since N is involved in the synthesis of proteins, chlorophyll, coenzymes, phytohormones, nucleic acids, and secondary metabolites (Amines, amides, amino sugars, purines,

The interaction between N rates and inoculation with *A. brasilense* was significant for N accumulation in grains; however, there was no difference between with or without *A. brasilense* inoculation for any of the N rates (**Table 3**). However, in treatments inoculated with *A. brasilense*,

and minimizing losses and environmental pollution, such as nutrient leaching.

pyrimidines and alkaloids) [40].

wheat. Selvíria—MS, Brazil 2015.

**Inoculation N rates (kg ha−1)**

Selvíria—MS, Brazil 2015.

L.S.D. (5%) 10.98

(A) (B)

Analyzing the split between with or without *A. brasilense* inoculation and N rates for N accumulation in the straw (**Table 2**), there was no difference between inoculation and not with *A. brasilense* for any of the N rates. With the increase in N rates, there was a linear increase in the N accumulation in straw (**Figure 3**), regardless of inoculation or not with *A. brasilense*. Dobbelaere et al. [23] also reported positive responses to inoculation with *A. brasilense* even when the crops are grown in soils with high N content available, which indicates that the plant responses occur not only due to the fixed N2 but mainly depending on the production of phytohormone growth promoters such as cytokinin, gibberellin, and indole acetic acid.


ΨMeans followed by the same letters in the column do not differ by Tukey at 0.05 probability level.

of N, and Teixeira Filho et al. [34], with a point of maximum concentration of N being reached with the application estimate of 163 kg ha−1 of N. It should be noted that the concentration of N in the leaf tissue, even in the absence of nitrogen fertilization (0 kg ha−1), was above that as

N sources did not differ in N concentration, indicating that Super N was not efficient for N nutrition, even in the area with remaining straw of corn or wheat (**Table 1**). Similar results were verified by Meira et al. [35], using ammonium sulfonitrate, ammonium sulfate, and urea sources for the N concentration of irrigated wheat in the *Cerrado*. However, Teixeira Filho et al. [5] verified higher N concentrations in leaf tissue when the sources of ammonium sulfoni-

On the other hand, Megda et al. [36] (ammonium sulfonitrate, ammonium sulfate, and urea), in the corn crop, verified that the ammonium sulfonitrate source had a higher N concentration in the leaf, compared to the other sources, unlike the results obtained in the present research. It should be noted that Super N acts on the inhibition of the urease enzyme, whereas the ammonium sulfonitrate has in its composition dimethylpyrazolophosphate (DMPP) molecules that act to inhibit nitrification. This makes the fertilizer less susceptible to leaching, since there is a longer residence time of N, as ammonium in the soil, and, under conditions of tropical climate and high temperatures, presents different responses to Super N. Also, there is evidence of active urea transport by high affinity transporters (symport) located in the plasmatic membrane of the root epidermis cells, which would allow uptake of some urea applied

According to Pankievicz et al. [38], there is a greater development and growth of the root system of the *Setaria viridis* grass inoculated with *A. brasilense* as a function of associative

leaves, which would favor the plant with greater growth in aerial part, greater accumulation of water, and conditions of less stress caused by the greater accumulation and metabolism of carbon, thus, increasing the concentration of nutrients in the plant. On the other hand, Bashan et al. [22] reported that the production of plant hormones, mainly indole acetic acid by bacteria of the genus *Azospirillum*, plays an essential role in the promotion of plant growth and, according to Hungria et al. [39], can improve the uptake of several macro and micronutrients, increasing the efficiency of the use of the available nutrients; however, this result was not verified in the present work, which raises the question of the difference of efficiency of the

Analyzing the split between with or without *A. brasilense* inoculation and N rates for N accumulation in the straw (**Table 2**), there was no difference between inoculation and not with *A. brasilense* for any of the N rates. With the increase in N rates, there was a linear increase in the N accumulation in straw (**Figure 3**), regardless of inoculation or not with *A. brasilense*. Dobbelaere et al. [23] also reported positive responses to inoculation with *A. brasilense* even when the crops are grown in soils with high N content available, which indicates that the

of phytohormone growth promoters such as cytokinin, gibberellin, and indole acetic acid.

has been formed, especially when urea concentration in the

but mainly depending on the production

fixation and less accumulation of photoassimilated carbon in the

recommended by Cantarella et al. [27], ranging from 20–34 g kg−1.

trate and ammonium sulfate compared to urea were used.

before urease has acted and NH3

soil and soil pH is low [37].

170 Nitrogen in Agriculture - Updates

fixers, with greater CO2

inoculation in grass crops.

plant responses occur not only due to the fixed N2

**Table 2.** Inoculation with *A. brasilense* and N rates interaction in the N accumulation in wheat straw.

**Figure 3.** N rates and inoculation with *A. brasilense* interaction in the N accumulation in straw (A) and in grains (B) of wheat. Selvíria—MS, Brazil 2015.

The greater accumulation of nutrients provided by the increase of the N rates associated or not with the inoculation with *A. brasilense* is a very significant result considering that conservation practices benefit the productive systems and environmental conservation, such as no‐ tillage system, whose straw will provide nutrients to subsequent crops, recycling nutrients, and minimizing losses and environmental pollution, such as nutrient leaching.

N is the nutrient that most interferes in the development and productivity of crops, especially grasses. This mineral nutrient is found in higher concentrations, in vegetative tissues and grains, which characterizes it as being the element most demanded by the wheat plant. Thus, the higher availability of this nutrient in the plants favored the development of the root system, which, by exploiting a larger volume of soil, may have an uptake of a greater amount of nutrients and water, reflecting the accumulation in aerial part, both in the straw and in the grains. Since N is involved in the synthesis of proteins, chlorophyll, coenzymes, phytohormones, nucleic acids, and secondary metabolites (Amines, amides, amino sugars, purines, pyrimidines and alkaloids) [40].

The interaction between N rates and inoculation with *A. brasilense* was significant for N accumulation in grains; however, there was no difference between with or without *A. brasilense* inoculation for any of the N rates (**Table 3**). However, in treatments inoculated with *A. brasilense*,


ΨMeans followed by the same letters in the column do not differ by Tukey at 0.05 probability level.

**Table 3.** Inoculation with *A. brasilense* and N rates interaction in the N accumulation in wheat grains.

there was an adjustment to the quadratic function for the dose of 165.3 kg ha−1 of N, whereas in the treatments without inoculation with this bacterium, the adjustment was linearly increasing for N in the grains (**Figure 3**) but reaching lower N concentrations in the highest rate of N.

These bacteria can act on plant growth by producing substances promoting development (auxins, gibberellins, and cytokinins) which provide better root growth [41] and, therefore, help in greater uptake of water and nutrients [42], resulting in a more vigorous and productive plant [16, 22]; to be free‐living organisms with endophytic characteristics, it is possible to perform some of the metabolic and vital use of nutrients in the plant, which would then be made available to reflect in increased concentrations in the grains.

Galindo et al. [14] studied N rates (0, 50, 100, 150, and 200 kg ha−1, in topdressing), N sources (Super N and urea), with and without inoculation with *A. brasilense* in maize, and found positive influence of inoculation on nutrient concentration in leaf tissue, which may be indicative of the phytohormonal effect cited in the literature, confirming that *Azospirillum* produces phytohormones that stimulate root growth of several plant species and that this greater development of the roots may imply several other effects such as increases in the water and nutrient uptakes and greater tolerance to stresses such as salinity and drought, resulting in a more vigorous and productive plant [22]. In addition, Barassi et al. [19] reported improvement in leaf photosynthetic parameters, including chlorophyll content and stomatal conductance, higher proline content in shoots and roots, improvement in water potential, increase in water content of apoplast, greater cell wall elasticity, and higher production of plant biomass.

weight in an area with high availability of N. According to Frank & Bauer (1996), in the period between the emergence phase of the seedlings and the differentiation of the floral primordium, N deficiency reduces the mass of 100‐kernel weight. Therefore, the results obtained can be explained by the fact that N deficiency was not observed in the plants in any of the treatments by the fact of the irrigated wheat crop and because the number of kernels per spike increased as a function of increasing N rates, thereby providing increment on the competition for photo assimilates inside the spike [5] but not to the point of reducing the mass of grains. This can be proved by the high hectoliter weight to be obtained (>78 kg 100 L−1) in the experiment, which

**Table 4.** Number of kernels per spike, number of spikes per meter, hectoliter weight, 100‐kernel weight, and wheat grain

Conventional urea and urea with NBPT provided similar results for number of kernels per spike, number of spikes per meter, hectoliter weight, 100‐kernel weight, and, consequently, for wheat grain yield (**Table 4**) in agreement with Megda et al. [36] and Theago et al. [6]. Prando et al. [44], evaluating the sources of urea, urea + NBPT, and coated urea, in another climatic condition (temperate climate), also did not observe changes in grain yield. It is worth noting that N sources may have presented similar behavior due to irrigation during the entire

classified (by individual analysis) the type 1 wheat with the best quality [10].

**Number of kernels per spike**

**N sources**

**Inoculation**

Without *Azospirillum*

Selvíria—MS, Brazil, 2015.

**Number of spikes per meter**

**N rates (kg ha−1) (kg 100 L−1) (g) (kg ha−1)** 34.40 69.17 87.23 4.20 1906 35.75 76.50 86.83 4.10 3027 42.48 84.42 87.42 4.19 3363 37.88 73.75 86.14 4.13 3167 38.91 74.50 85.96 4.12 3263

Urea 38.60 aΨ 75.50 a 87.14 a 4.17 a 2930 a Super N 37.17 a 75.83 a 86.29 a 4.13 a 2960 a L.S.D. (5%) 2.70 5.90 1.40 0.06 212

With *Azospirillum* 39.04 a 76.00 a 86.61 a 4.14 a 3007

L.S.D. (5%) 2.70 5.90 1.40 0.06 212 Overall mean 37.88 75.67 86.71 4.15 2945 C.V. (%) 13.65 14.92 3.61 3.36 16.08

ΨMeans followed by the same letters in the column do not differ by Tukey at 0.05 probability level.

yield in function of N rates and sources and inoculation with *Azospirillum brasilense* in wheat crop.

36.73 a 75.33 a 86.82 a 4.15 a 2883

**Hectoliter weight 100‐kernel weight Grain yield**

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173

The Effect of N Fertilization on Wheat under Inoculation with *Azospirillum brasilense*

The increase in N rates influenced significantly the number of kernels per spike and spikes per meter (**Table 4**). For the number of kernels per spike, the data adjusted the quadratic function with maximum point in 151 kg ha−1 of N (**Figure 4**). On the other hand, the N rates influenced the number of spikes per meter, adjusted to the quadratic function, up to the dose of 110 kg ha−1 of N (**Figure 4**). These results explain why grain yield was influenced positively by the increase of N rates, independently of the source of N and inoculation with *A. brasilense* (**Figure 4**).

For the hectoliter weight and 100‐kernel weight, there was no influence of N rates (**Table 4**). Similar results were obtained by Teixeira Filho et al. [33] and Theago et al. [6] that did not verify the influence of the N rates on the mentioned parameters in the irrigated wheat crop. Nunes et al. [8] and Souza et al. [43], who found no influence of N rates in topdressing for 100‐kernel


ΨMeans followed by the same letters in the column do not differ by Tukey at 0.05 probability level.

there was an adjustment to the quadratic function for the dose of 165.3 kg ha−1 of N, whereas in the treatments without inoculation with this bacterium, the adjustment was linearly increasing for N in the grains (**Figure 3**) but reaching lower N concentrations in the highest rate of N.

**0 50 100 150 200**

With 40.13 aΨ 78.03 a 99.90 a 75.13 a 102.81 a Without 49.59 a 78.66 a 84.54 a 80.20 a 89.40 a

ΨMeans followed by the same letters in the column do not differ by Tukey at 0.05 probability level.

**Table 3.** Inoculation with *A. brasilense* and N rates interaction in the N accumulation in wheat grains.

These bacteria can act on plant growth by producing substances promoting development (auxins, gibberellins, and cytokinins) which provide better root growth [41] and, therefore, help in greater uptake of water and nutrients [42], resulting in a more vigorous and productive plant [16, 22]; to be free‐living organisms with endophytic characteristics, it is possible to perform some of the metabolic and vital use of nutrients in the plant, which would then be

Galindo et al. [14] studied N rates (0, 50, 100, 150, and 200 kg ha−1, in topdressing), N sources (Super N and urea), with and without inoculation with *A. brasilense* in maize, and found positive influence of inoculation on nutrient concentration in leaf tissue, which may be indicative of the phytohormonal effect cited in the literature, confirming that *Azospirillum* produces phytohormones that stimulate root growth of several plant species and that this greater development of the roots may imply several other effects such as increases in the water and nutrient uptakes and greater tolerance to stresses such as salinity and drought, resulting in a more vigorous and productive plant [22]. In addition, Barassi et al. [19] reported improvement in leaf photosynthetic parameters, including chlorophyll content and stomatal conductance, higher proline content in shoots and roots, improvement in water potential, increase in water content

The increase in N rates influenced significantly the number of kernels per spike and spikes per meter (**Table 4**). For the number of kernels per spike, the data adjusted the quadratic function with maximum point in 151 kg ha−1 of N (**Figure 4**). On the other hand, the N rates influenced the number of spikes per meter, adjusted to the quadratic function, up to the dose of 110 kg ha−1 of N (**Figure 4**). These results explain why grain yield was influenced positively by the increase of N rates, independently of the source of N and inoculation with *A. brasilense* (**Figure 4**).

For the hectoliter weight and 100‐kernel weight, there was no influence of N rates (**Table 4**). Similar results were obtained by Teixeira Filho et al. [33] and Theago et al. [6] that did not verify the influence of the N rates on the mentioned parameters in the irrigated wheat crop. Nunes et al. [8] and Souza et al. [43], who found no influence of N rates in topdressing for 100‐kernel

of apoplast, greater cell wall elasticity, and higher production of plant biomass.

made available to reflect in increased concentrations in the grains.

**Inoculation N rates (kg ha−1)**

172 Nitrogen in Agriculture - Updates

Selvíria—MS, Brazil, 2015.

L.S.D. (5%) 21.94

**Table 4.** Number of kernels per spike, number of spikes per meter, hectoliter weight, 100‐kernel weight, and wheat grain yield in function of N rates and sources and inoculation with *Azospirillum brasilense* in wheat crop.

weight in an area with high availability of N. According to Frank & Bauer (1996), in the period between the emergence phase of the seedlings and the differentiation of the floral primordium, N deficiency reduces the mass of 100‐kernel weight. Therefore, the results obtained can be explained by the fact that N deficiency was not observed in the plants in any of the treatments by the fact of the irrigated wheat crop and because the number of kernels per spike increased as a function of increasing N rates, thereby providing increment on the competition for photo assimilates inside the spike [5] but not to the point of reducing the mass of grains. This can be proved by the high hectoliter weight to be obtained (>78 kg 100 L−1) in the experiment, which classified (by individual analysis) the type 1 wheat with the best quality [10].

Conventional urea and urea with NBPT provided similar results for number of kernels per spike, number of spikes per meter, hectoliter weight, 100‐kernel weight, and, consequently, for wheat grain yield (**Table 4**) in agreement with Megda et al. [36] and Theago et al. [6]. Prando et al. [44], evaluating the sources of urea, urea + NBPT, and coated urea, in another climatic condition (temperate climate), also did not observe changes in grain yield. It is worth noting that N sources may have presented similar behavior due to irrigation during the entire

The *A. brasilense* inoculation associated with the N rates in the 140 kg ha−1 range provided the maximum grain yield of the wheat crop; in contrast, in the absence of inoculation, the magnitude of response to N rates was higher. Worth mentioning that the estimated grain yield of the inoculated treatments was higher, numerically, most of the rates tested in both crops, even using N rates considered high, showing the benefit of inoculation with *A. brasilense* in irrigated wheat crop. In turn, Galindo et al. [45] verified that the co‐inoculation with *A. brasilense,* and Co + Mo application via seeds promote higher grain yield and profitability with the

The Effect of N Fertilization on Wheat under Inoculation with *Azospirillum brasilense*

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175

With regard to grain yield, several authors also reported a positive response to N fertilization on wheat [4, 7, 46]. In similar climatic conditions for wheat crop in the low‐altitude *Cerrado region*, it was reported that the maximum grain yield was 78 [33], 90 [7], and 120 kg ha−1 of N [5]. This difference in rates of N that provides maximum wheat grain yield is due to different N requirements of cultivars as well as the variation in soil and climatic conditions of these researches. Anwar et al. [47] found that maximum spikes m−2, grains per spikes, thousand grains weight, and grain yield (4061 kg ha−1) were produced by 125 kg ha−1 of N for two wheat

Lemos et al. [24] studied five wheat cultivars (CD 104, CD 108, CD 119, CD 120, and CD 150), with and without inoculation and associated with nitrogen rates, and found that response to inoculation with *A. brasilense* in wheat crop occurs satisfactorily when held in conjunction with the nitrogen fertilization, as observed in this study at a dose of 100 kg ha−1 N (**Table 4**). However, *A. brasilense* alone is not effective enough to replace entire nitrogen fertilization but is associated with N fertilization, which makes it possible to achieve the highest yields of

On the other hand, Ferreira et al. [48], working with foliar application of *A. brasilense* and nitrogen rates in the wheat crop in the *Brazilian Cerrado*, observed that inoculation had no effect on grain yield. Similarly, Nunes et al. [8] studied inoculation with *A. brasilense* in soils with high and low availability of N, and Galindo et al. [49] in research with application times by the leaf of *A. brasilense* with the application of 100 kg ha−1 N found no effect of inoculation

It is noteworthy that bacteria of *Azospirillum* genus are native from soil [50], so it is possible that these were at a high population in the soil under study and, therefore, cancel or minimize the effect of inoculation. Moreover, the affinity of the cultivar with the strains of

in the production components and grain yields of wheat in the *Brazilian Cerrado*.

soybean crop in the *Brazilian Cerrado*, being technically and economically viable.

With 1671 aΨ 3036 a 3663 a 3167 a 3497 a Without 2141 a 3018 a 3063 b 3166 a 3029 a

ΨMeans followed by the same letters in the column do not differ by Tukey at 0.05 probability level.

**Table 5.** Inoculation with *A. brasilense* and N rates interaction in the wheat grain yield.

cultivars in Pakistan.

**Inoculation 2015**

L.S.D. (5%) 474 Selvíria—MS, Brazil, 2015.

irrigated wheat grains in *Brazilian Cerrado*.

**Figure 4.** Number of kernels per spike (A) and number of spikes per meter (B) in the function of N rates and N rates and inoculation with *A. brasilense* interaction in the wheat grain yield (C). Selvíria—MS, Brazil 2015.

crop cycle, which would have reduced volatilization losses, mainly in the form of NH3 , which occur at a higher proportion in the first 4 days after urea application.

As regards inoculation with *A. brasilense*, this alone did not significantly interfere in the number of kernels per spike, number of spikes per meter, hectoliter weight, and 100‐kernel weight (**Table 4**). Most probably, there was no effect of this inoculation on these production components because the number of kernels per spike is mainly determined by the genetic characteristics of the cultivar, and from the other evaluations mentioned above, it is due to the cultivation of irrigated wheat, fertilization, and adequate plants stand and soil fertility.

There was a significant interaction between N rates and inoculation with *A. brasilense* for wheat grain yield. At the dose of 100 kg ha−1 of N, the inoculation provided higher productivity than the uninoculated treatment (**Table 5**). Grain yield adjusted the quadratic function for N rates in the treatments with and without inoculation with *A. brasilense*, with a positive response up to 142 and 134 kg ha−1 of N, respectively (**Figure 4**). However, in relation to the control (without N), the optimal N dose of the treatment inoculated with this diazotrophic bacterium provided a higher grain yield of 391 kg ha−1 in relation to the best N rate of the treatment without inoculation; that is, this increase was higher by 7%.

The N rates mentioned above for obtaining the maximum grain yield were high since the wheat was cultivated on corn straw (high C/N ratio), that is, part of the applied N was immobilized by the decomposing/mineralizing of microorganisms.


**Table 5.** Inoculation with *A. brasilense* and N rates interaction in the wheat grain yield.

crop cycle, which would have reduced volatilization losses, mainly in the form of NH3

As regards inoculation with *A. brasilense*, this alone did not significantly interfere in the number of kernels per spike, number of spikes per meter, hectoliter weight, and 100‐kernel weight (**Table 4**). Most probably, there was no effect of this inoculation on these production components because the number of kernels per spike is mainly determined by the genetic characteristics of the cultivar, and from the other evaluations mentioned above, it is due to the cultivation of irrigated wheat, fertilization, and adequate plants stand and soil fertility.

**Figure 4.** Number of kernels per spike (A) and number of spikes per meter (B) in the function of N rates and N rates and

There was a significant interaction between N rates and inoculation with *A. brasilense* for wheat grain yield. At the dose of 100 kg ha−1 of N, the inoculation provided higher productivity than the uninoculated treatment (**Table 5**). Grain yield adjusted the quadratic function for N rates in the treatments with and without inoculation with *A. brasilense*, with a positive response up to 142 and 134 kg ha−1 of N, respectively (**Figure 4**). However, in relation to the control (without N), the optimal N dose of the treatment inoculated with this diazotrophic bacterium provided a higher grain yield of 391 kg ha−1 in relation to the best N rate of the

The N rates mentioned above for obtaining the maximum grain yield were high since the wheat was cultivated on corn straw (high C/N ratio), that is, part of the applied N was immo-

occur at a higher proportion in the first 4 days after urea application.

inoculation with *A. brasilense* interaction in the wheat grain yield (C). Selvíria—MS, Brazil 2015.

(A) (B)

174 Nitrogen in Agriculture - Updates

(C)

treatment without inoculation; that is, this increase was higher by 7%.

bilized by the decomposing/mineralizing of microorganisms.

, which

The *A. brasilense* inoculation associated with the N rates in the 140 kg ha−1 range provided the maximum grain yield of the wheat crop; in contrast, in the absence of inoculation, the magnitude of response to N rates was higher. Worth mentioning that the estimated grain yield of the inoculated treatments was higher, numerically, most of the rates tested in both crops, even using N rates considered high, showing the benefit of inoculation with *A. brasilense* in irrigated wheat crop. In turn, Galindo et al. [45] verified that the co‐inoculation with *A. brasilense,* and Co + Mo application via seeds promote higher grain yield and profitability with the soybean crop in the *Brazilian Cerrado*, being technically and economically viable.

With regard to grain yield, several authors also reported a positive response to N fertilization on wheat [4, 7, 46]. In similar climatic conditions for wheat crop in the low‐altitude *Cerrado region*, it was reported that the maximum grain yield was 78 [33], 90 [7], and 120 kg ha−1 of N [5]. This difference in rates of N that provides maximum wheat grain yield is due to different N requirements of cultivars as well as the variation in soil and climatic conditions of these researches. Anwar et al. [47] found that maximum spikes m−2, grains per spikes, thousand grains weight, and grain yield (4061 kg ha−1) were produced by 125 kg ha−1 of N for two wheat cultivars in Pakistan.

Lemos et al. [24] studied five wheat cultivars (CD 104, CD 108, CD 119, CD 120, and CD 150), with and without inoculation and associated with nitrogen rates, and found that response to inoculation with *A. brasilense* in wheat crop occurs satisfactorily when held in conjunction with the nitrogen fertilization, as observed in this study at a dose of 100 kg ha−1 N (**Table 4**). However, *A. brasilense* alone is not effective enough to replace entire nitrogen fertilization but is associated with N fertilization, which makes it possible to achieve the highest yields of irrigated wheat grains in *Brazilian Cerrado*.

On the other hand, Ferreira et al. [48], working with foliar application of *A. brasilense* and nitrogen rates in the wheat crop in the *Brazilian Cerrado*, observed that inoculation had no effect on grain yield. Similarly, Nunes et al. [8] studied inoculation with *A. brasilense* in soils with high and low availability of N, and Galindo et al. [49] in research with application times by the leaf of *A. brasilense* with the application of 100 kg ha−1 N found no effect of inoculation in the production components and grain yields of wheat in the *Brazilian Cerrado*.

It is noteworthy that bacteria of *Azospirillum* genus are native from soil [50], so it is possible that these were at a high population in the soil under study and, therefore, cancel or minimize the effect of inoculation. Moreover, the affinity of the cultivar with the strains of this bacteria diazotrophic may vary and determine the success or failure of *A. brasilense* inoculation.

The NUE was negatively affected by the increment of N rates with adjustment to the linear function decreasing (**Table 6**, **Figure 5**). This result can be attributed to the losses of N portrayed clearly in the literature. The increase of N rates culminates in greater losses and less use by the crops, since there is a limit in the nutritional demand of the plant, that is, the plants uptake certain amount of nutrients for a given time; thus, the N that is applied and is not taken can be lost, decreasing the efficiency of fertilization with the higher rates of N, as portrayed in the literature by the law of decreasing increments. The results are similar to those reported by Silva et al. [51], studying N rates (0, 100, 200, and 300 kg ha−1) in the Marandu palisadegrass and Sant'Ana et al. [52], working with common bean crop in the rates of 0, 30, 60, 120, and 240 kg ha−1 of N in topdressing.

In relation to the N sources, urea presented higher NUE compared to Super N, differently from what was expected due to the possibility of mitigation of volatilization of the ammonia provided by Super N fertilizer (urea with urease enzyme inhibitor NBPT) (**Table 6**). However, Dupas et al. [53] evaluated in the *Brazilan Cerrado* the dry‐matter yield, RAN, and NUE of palisade grass in response to sources of N (ammonium nitrate, ammonium sulfate, ammonium sulfate‐nitrate, urea, urea with urease inhibitor NBPT, polymer‐coated urea, and control) in seven harvests (100 kg ha−1 N applied after each harvest) and reported for RAN and NUE that the use of N fertilizers that minimizes N loss, such as urea with urease inhibitor NBPT, and polymer‐coated urea, was very promising, especially for minimizing the environmental impact of N fertilization. However, they found no difference in DMY due to N sources.

The Effect of N Fertilization on Wheat under Inoculation with *Azospirillum brasilense*

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177

**Figure 5.** Nitrogen utilization efficiency (NUE) in wheat crop in the function of N rates. Selvíria—MS, Brazil 2015.

The inoculation with *A. brasilense* did not influence the NUE, although it gave 15.4% greater efficiency compared to the non‐inoculated treatments (**Table 6**), which again may be indicative of the phytohormonal effect cited in the literature, confirming that *Azospirillum* produces phytohormones that stimulate root growth of several plant species and that this greater development of the roots may be implied in several other effects, such as increases in the water and

The interaction between N rates and inoculation, and N sources and inoculation, was significant for RAN. Analyzing the split between N rates and inoculation, at 50 and 100 kg ha−1 of

**Table 7.** Inoculation with *A. brasilense* and N rates interaction in the recovery of the applied nitrogen (RAN) in wheat

With 96.29 aΨ 79.21 a 33.11 a 43.83 a Without 61.34 b 44.95 b 29.05 a 35.21 a

ΨMeans followed by the same letters in the column do not differ by Tukey at 0.05 probability level.

**50 100 150 200**

nutrient uptakes like N.

Selvíria—MS, Brazil 2015.

#

crop.

**Inoculation# N rates (kg ha−1)**

Corrected data following equation (*x* + 0.5)0.5.

L.S.D. (5%) 25.56


Selvíria—MS, Brazil 2015.
