**3. Thermodynamic and kinetic considerations on bioremediation**

The fact that most organic pollutants are composed of molecules with high embodied energies is an interesting starting point for any consideration about the best practice for sustainable soil remediation, especially in remote locations and developing countries where energy-intensive project are cost-prohibitive. Economic incentives are generally small for any kind of remedia‐ tion practice in such regions and, furthermore, time constraints may not be as limiting as in highly developed urban environments. Technologies that manage to optimize the use of ecosystem services provided by indigenous organisms, in combination with a minimum input of extra energy and material resources, are potentially more sustainable in such conditions.

Figure 1 illustrates some basic principles including thermodynamic driving forces (from molecules with high to low embodied energy) and available transformation pathways in air and soil. In the atmosphere, abiotic photochemical reactions rapidly oxidize organic vapours. The rate determining step in these complex photochemical reactions is usually the initial attack on the primary pollutant by hydroxyl radical. Typical turnover times of i.e. reactive hydro‐ carbons are only a few hours in the sunlit atmosphere while lack of photochemistry in the subsurface of soils leads to significantly lower reaction rates and hence to much longer turnover times of pollutants.

In soils where conditions for microbial activity are unfavourable, degradation relies on abiotic chemical reactions. In such cases organic pollutants may persist for years to decades or longer and secondary, incompletely degraded products may accumulate. On the other hand if conditions are favourable (temperature, electron acceptors, nutrients, moisture, available

primary pollutant by hydroxyl radical. Typical turnover times of i.e. reactive hydrocarbons are only a few hours in the sunlit atmosphere while lack of photochemistry in the subsurface of soils leads to significantly lower reaction rates and hence to much longer turnover times of pollutants. Sustainability Aspects of In-Situ Bioremediation of Polluted Soil in Developing Countries and Remote Regions http://dx.doi.org/10.5772/57315 61

Furthermore it resembles what Jørgensen & Mitsch [5] call a *shell game*, in which pollutants are moved from one location to another. In sustainable bioremediation external energy input is ideally used only in the initiation phase to start a process that is subsequently powered by solar energy and the embodied chemical energy of the pollutant itself. The engineer's role is

The ability to cycle nutrients is a fundamental function of all ecosystems. Within the mattercycling function lies the catabolic capacity to degrade and mineralise molecules of varying complexity, including pollutants. This function alone gives a nonmarket value to ecosystems, not always recognised by society. The role of ecological engineers is to identify ecosystem functions that are adoptable to human needs and apply them in ways that doesn't create any further degradation of nature. Since bioremediation projects aims at eliminating pollution that exerts stress on the ecosystem, it is by definition an ecosystem conservation approach. However, if large amounts of soil are physically removed from the site in ex-situ operations the remediation itself might be a threat to the ecosystem. Bioremediation projects are typically involved in the restoration of disturbed ecosystems but measures to prevent toxic compounds

to facilitate the proper conditions in which such a process can take place.

from entering sound ecosystems are likewise part of the bioremediation agenda.

**3. Thermodynamic and kinetic considerations on bioremediation**

The fact that most organic pollutants are composed of molecules with high embodied energies is an interesting starting point for any consideration about the best practice for sustainable soil remediation, especially in remote locations and developing countries where energy-intensive project are cost-prohibitive. Economic incentives are generally small for any kind of remedia‐ tion practice in such regions and, furthermore, time constraints may not be as limiting as in highly developed urban environments. Technologies that manage to optimize the use of ecosystem services provided by indigenous organisms, in combination with a minimum input of extra energy and material resources, are potentially more sustainable in such conditions. Figure 1 illustrates some basic principles including thermodynamic driving forces (from molecules with high to low embodied energy) and available transformation pathways in air and soil. In the atmosphere, abiotic photochemical reactions rapidly oxidize organic vapours. The rate determining step in these complex photochemical reactions is usually the initial attack on the primary pollutant by hydroxyl radical. Typical turnover times of i.e. reactive hydro‐ carbons are only a few hours in the sunlit atmosphere while lack of photochemistry in the subsurface of soils leads to significantly lower reaction rates and hence to much longer

In soils where conditions for microbial activity are unfavourable, degradation relies on abiotic chemical reactions. In such cases organic pollutants may persist for years to decades or longer and secondary, incompletely degraded products may accumulate. On the other hand if conditions are favourable (temperature, electron acceptors, nutrients, moisture, available

**2.5. Ecosystem conservation**

60 Environmental Risk Assessment of Soil Contamination

turnover times of pollutants.

Figure 1. Chemical and biochemical transformations and mineralisation of organic substances in polluted soil and in air. The broad arrow indicates the thermodynamic driving force for the complete degradation of the pollutant in an oxidising environment. Typical average residence times of organic pollutants (**τ)** in air and soil are indicated. **Figure 1.** Chemical and biochemical transformations and mineralisation of organic substances in polluted soil and in air. The broad arrow indicates the thermodynamic driving force for the complete degradation of the pollutant in an oxidising environment. Typical average residence times of organic pollutants (τ) in air and soil are indicated.

carbon sources, low toxicity to microorganisms, pH, to mention some of the most determinant variables), microorganisms such as bacteria and fungi may use the organic pollutant as a source of energy and carbon. Enzymatic degradation is generally much faster than abiotic oxidation reactions and the organic pollutant that enters the metabolic pathways of soil microorganisms may be partly or completely mineralized. The driving force for such an enzymatic process is again the free energy potential that can be used by the organism. For typical hydrocarbon and oxygenate pollutants the free energy that is available by complete oxidation with oxygen as electron acceptor, is in the range of 3-12 x 103 KJ mol-1 or 37-46 MJ kg-1, as can be seen in table 1. This energy potential may be used by a wide range of heterotrophic microorganisms. In soils where conditions for microbial activity are unfavourable, degradation relies on abiotic chemical reactions. In such cases organic pollutants may persist for years to decades or longer and secondary, incompletely degraded products may accumulate. On the other hand if conditions are favourable (temperature, electron acceptors, nutrients, moisture, available carbon sources, low toxicity to microorganisms, pH, to mention some of the most determinant variables), microorganisms such as bacteria and fungi may use the organic pollutant as a source of energy and carbon. Enzymatic degradation is generally much faster than abiotic oxidation reactions and the organic pollutant that enters the metabolic pathways of soil microorganisms may be partly or completely mineralized. The driving force for such an enzymatic process is again the free energy potential that can be used by the organism. For typical hydrocarbon and oxygenate pollutants the free energy that is available by complete oxidation with oxygen as electron acceptor, is in the range of 3-12 x 10<sup>3</sup> KJ mol-1 or 37-46 MJ kg-1, as can be seen in table 1. This energy potential may be used by a wide range of heterotrophic microorganisms. In areas of hydrocarbon contamination the most commonly found heterotrophic bacteria are of the genera Pseudomonas, Achromobacter, Arthrobacter, Micrococcus, Vibrio, Acinetobacter, Brevibacterium, Corynebacterium, Flavobacterium,

Mycobacterium and Nocardia [7]. Some soil microorganisms that are incapable of utilizing the full embodied energy potential

In areas of hydrocarbon contamination the most commonly found heterotrophic bacteria are of the genera Pseudomonas, Achromobacter, Arthrobacter, Micrococcus, Vibrio, Acinetobact‐ er, Brevibacterium, Corynebacterium, Flavobacterium, Mycobacterium and Nocardia [7]. Some soil microorganisms that are incapable of utilizing the full embodied energy potential of the pollutant may still be able to initiate catabolic processes that ultimately lead to complete mineralisation if oxygen is present at sufficient concentration. Filamentous fungi, such as e.g., white-rot fungi, can oxidize recalcitrant high molecular weight polycyclic aromatic hydrocar‐ bons (PAH:s) with their extracellular enzymes to more water soluble substances. Both lignolytic and non lignolytic fungi has been shown to initiate degradation of high molecularweight PAH and the oxidation products formed are thereby made available for further degradation by bacteria [8]. Such interactions between soil microorganisms is an important factor to consider in sustainable soil bioremediation and remediation practices that favour a high soil microbial biodiversity are likely to be more sustainable. of the pollutant may still be able to initiate catabolic processes that ultimately lead to complete mineralisation if oxygen is present at sufficient concentration. Filamentous fungi, such as e.g., white-rot fungi, can oxidize recalcitrant high molecular weight polycyclic aromatic hydrocarbons (PAH:s) with their extracellular enzymes to more water soluble substances. Both lignolytic and non lignolytic fungi has been shown to initiate degradation of high molecular-weight PAH and the oxidation products formed are thereby made available for further degradation by bacteria [8]. Such interactions between soil microorganisms is an important factor to consider in sustainable soil bioremediation and remediation practices that favour a high soil microbial biodiversity are likely to be more sustainable. 

3


*#NIST, National Institute of Standards and Technology website. Available at: http://webbook.nist.gov/chemistry/nameser.html*

**Table 1.** Thermodynamic properties and free energy of mineralisation of some representative "high energy" organic substances commonly found in polluted soil. Standard entropy of formation (∆S<sup>o</sup> f) was calculated from standard entropy values (So) of the substances and their elements (phase indicated in table). Standard free energy of formation (∆G<sup>o</sup> f) was calculated from standard enthalpy of formation (∆H<sup>o</sup> f) and standard entropy of formation. Standard free energy of mineralisation (∆Go) was calculated for the complete oxidative degradation of the organic compounds to the thermodynamically most stable end products according to the reaction: 1 mol of substance + a O2 = b CO2 + c H2O.

In oxygen depleted soils microorganisms are unable to mineralise organic compounds to inorganic molecules only. Complete degradation leaves carbon dioxide and methane as end products. Intermediate fermentation products, that may be toxic to microorganisms and plants, may accumulate if they are not converted to methane [7]. Since methane and fermen‐ tation products has retained a substantial part of the embodied energy, the energy gain by microorganisms by anaerobic degradation is less than under aerobic conditions and the thermodynamic driving force for biodegradation is thus lower. Methane is also a highly potent greenhouse gas [9] and its unintended production should, from a sustainability point of view, be avoided. It is therefore in many cases preferable to maintain aerobic conditions in the soil during in-situ bioremediation projects. There are however some important exceptions. Xenobiotic and toxic chlorinated organic compounds such as e.g. highly chlorinated PCB:s, pentachlorophenol, tri- and perchloroethylene, gamma hexachlorocyclohexane (lindane) and many others are highly persistent to biodegradation under aerobic conditions. The reason for this may be lack of metabolic pathways available to common soil microorganisms that can generate energy gain for the organism. Persistence is also caused by high lipophilicity of many of these substances which reduce their bioavailability.

Chlorinated substances may be efficiently degraded by microorganisms capable of halorespi‐ ration under anaerobic conditions [10]. The degradation mechanism is characterized as reductive dechlorination. In contrast to aerobic co-metabolism which does not provide any energetic benefit for the degrading organism, reductive dechlorination may provide a substantial free energy yield. The thermodynamic driving force of such reactions is demon‐ strated by the fact that hexachloroethane for example, may be a stronger electron acceptor than oxygen [11].

### **3.1. Kinetic considerations**

**Category Substance Formula Phase**

62 Environmental Risk Assessment of Soil Contamination

Gasoline additive

Mineralisation product

Mineralisation product

*ser.html*

(∆G<sup>o</sup>

Carbon dioxide

Element Carbon C

**M So ∆H<sup>o</sup> <sup>f</sup> ∆S<sup>o</sup> <sup>f</sup> ∆G<sup>o</sup> <sup>f</sup>**

MTBE C5H12O Liquid 88.15 265.3# -315.4# -650.52 -121.45 5 6 -3273 -37.14

Gasoline Benzene C6H6 Liquid 78.11 173.26# 49.0# -253.78 124.66 6 3 -3202 -41.00 Gasoline Toluene C7H8 Liquid 92.14 220.96# 12.0# -342.59 114.14 7 4 -3823 -41.50 Gasoline m-xylene C8H10 Liquid 106.16 253.80# -25.4# -446.26 107.65 8 5 -4448 -41.90

Diesel Hexadecane C16H34 Liquid 226.44 586.18# -456.3# -1728.71 59.11 16 17 -10401 -45.93 Diesel Octadecane C18H38 Solid 254.49 480.2# -457.4# -2107.71 171.01 18 19 -11776 -46.27 Diesel Phenanthrene C14H10 Solid 178.23 215.06# 110.1# -520.00 265.14 14 5 -6972 -39.12 Diesel Perylene C20H12 Solid 252.31 264.6# 182.4# -636.14 372.07 20 6 -9682 -38.37

CO2 Gas 44.01 213.79# -393.5# 2.81 -394.36

*#NIST, National Institute of Standards and Technology website. Available at: http://webbook.nist.gov/chemistry/name-*

**Table 1.** Thermodynamic properties and free energy of mineralisation of some representative "high energy" organic

entropy values (So) of the substances and their elements (phase indicated in table). Standard free energy of formation

energy of mineralisation (∆Go) was calculated for the complete oxidative degradation of the organic compounds to the thermodynamically most stable end products according to the reaction: 1 mol of substance + a O2 = b CO2 + c H2O.

In oxygen depleted soils microorganisms are unable to mineralise organic compounds to inorganic molecules only. Complete degradation leaves carbon dioxide and methane as end products. Intermediate fermentation products, that may be toxic to microorganisms and plants, may accumulate if they are not converted to methane [7]. Since methane and fermen‐ tation products has retained a substantial part of the embodied energy, the energy gain by microorganisms by anaerobic degradation is less than under aerobic conditions and the thermodynamic driving force for biodegradation is thus lower. Methane is also a highly potent greenhouse gas [9] and its unintended production should, from a sustainability point of view, be avoided. It is therefore in many cases preferable to maintain aerobic conditions in the soil during in-situ bioremediation projects. There are however some important exceptions. Xenobiotic and toxic chlorinated organic compounds such as e.g. highly chlorinated PCB:s, pentachlorophenol, tri- and perchloroethylene, gamma hexachlorocyclohexane (lindane) and many others are highly persistent to biodegradation under aerobic conditions. The reason for

12.01 5.83# 0 0 0

Water H2O Liquid 18.02 69.95# -285.8# -163.30 -237.19

Solid (graphite)

substances commonly found in polluted soil. Standard entropy of formation (∆S<sup>o</sup>

f) was calculated from standard enthalpy of formation (∆H<sup>o</sup>

Element Hydrogen H2 Gas 2.02 130.68# 0 0 0 Element Oxygen O2 Gas 32.00 205.15# 0 0 0

**g mol-1 J mol-1K-1 KJ mol-1 KJ mol-1 KJ mol-1 KJ mol-1 MJ kg-1**

**b c**

f) was calculated from standard

f) and standard entropy of formation. Standard free

**∆Go(mineralisation)**

> Biodegradation kinetics tells us not only about the rate of degradation of target compounds and of rate dependence on ambient factors, but by studying the shape of the degradation curves we may also get valuable information about degradation mechanisms. It is preferable to use models that have a theoretical basis rather than just empirical mathematical modelling that have limited predictive ability.

> Natural attenuation of organic pollutants are typically mediated by slow growing organisms and if the concentration of pollutants is much lower than the concentration required for maximum growth of degrading organism (which is often the case in many polluted soils) then first order kinetics may apply, i.e. degradation rate is directly dependent on substrate concen‐ tration (Equation 1).

$$-\mathbf{dC/dt} = \mathbf{kC} \tag{1}$$

C is the substrate concentration and k is the first order rate constant. First order kinetics is often applied in degradation studies because of its simplicity but have little predictive power when conditions for growth change.

In situations with rapid growth of degrading organisms or when other factors than the substrate concentration controls the rate of degradation, first order kinetics will not describe the degradation process accurately. According to the earlier mentioned criteria for sustainable soil bioremediation, efforts should be made to stimulate growth of indigenous soil microor‐ ganisms that have or can achieve the ability to degrade target pollutants. The addition of organic waste products from the dairy industry that promotes growth of degrading organisms has been proved to be an efficient means to increase both rate and total degradation of some representative diesel range hydrocarbons in soil [12-14]. Although the addition of readily available carbon sources, such as lactose, lactate and amino acids (components of whey) to contaminated soil would be expected to trigger exponential growth of organisms, a common phenomenon in liquid culture media, it seems that the soil matrix itself does not readily promote exponential growth of degrading organisms. Growth of soil microorganisms is limited by a number of factors caused by the properties of the soil matrix. Among the limiting factors is diffusion of substrate and nutrients but also the specific area available for bacterial sorption on to soil particles. When a large fraction of this area is occupied by bacteria, further growth might be increasingly restricted by diffusion of substrate and nutrients into multilayers of bacteria. Kinetic models that take into consideration slow or restricted (linear growth) may more accurately describe degradation patterns in real life situations. Figure 2 illustrates the application of a three-half-order kinetic model (II) originally proposed by Brunner and Focht [15]. The model assumes linear biomass growth rate and simulates the accumulated 14CO2 production from phenanthrene-9-14C mineralisation in soil microcosms.

$$P\left(t\right) = S\_0 \left(1 - \exp\left(-k\_1 t - k\_2 t^2 / 2\right)\right) + k\_0 t \tag{2}$$

P[t] is the cumulative 14CO2-production (%) where t is the time (days), S0 is the amount of substrate (%) that is "highly bioavailable", k1 is the first-order rate constant (day-1), k2 is a linear growth term (day-2), and k0 is the zero order rate constant describing the mineralisation of "less bioavailable" substrate and indigenous mineralisation (% day-1).The lag time was defined as the initial time until 1% of the added 14C was detected as 14CO2 [16]. From the end of the lag phase, iterative non-linear least squares regression was performed to fit data to the model by using the SOLVER function in Microsoft Excel 2000 [17].

The effects of applications of different amendments on phenanthrene biodegradation are clearly seen in figure 2. Kinetic parameters such as the linear growth term (k2) and S0 was significantly affected by the addition of the different amendments (Table 2). It should be noted that the stimulating effect of complex organic amendments such as whey and fermented whey may not only be an effect of the addition of a readily available carbon source (that promotes growth) but also of available micronutrients, such as B-vitamins, that is required to maintain efficient catabolic pathways for degradation of i.e. polycyclic aromatic hydrocarbons.


**Table 2.** Tree-half-order kinetic constants for phenanthrene mineralisation in soil, at 25 °C. Lag time is expressed in units of days, S0 and mineralisation in %, k1 in day-1, k2 in day-2, k0 in % day-1 and R is the correlation coefficient. Values having the same superscript letters are not significantly different at a 95 % level. (From [18]. Reprinted with permission from Elsevier).

as the initial time until 1% of the added 14C was detected as 14CO2 [16]. From the end of the lag phase, iterative non-linear least squares regression was performed to fit data to the model by using the SOLVER function in Microsoft Excel 2000 [17]. Sustainability Aspects of In-Situ Bioremediation of Polluted Soil in Developing Countries and Remote Regions http://dx.doi.org/10.5772/57315 65

<sup>0</sup> <sup>21</sup> exp1 2 [2]

C is the substrate concentration and k is the first order rate constant. First order kinetics is often applied in degradation studies

In situations with rapid growth of degrading organisms or when other factors than the substrate concentration controls the rate of degradation, first order kinetics will not describe the degradation process accurately. According to the earlier mentioned criteria for sustainable soil bioremediation, efforts should be made to stimulate growth of indigenous soil microorganisms that have or can achieve the ability to degrade target pollutants. The addition of organic waste products from the dairy industry that promotes growth of degrading organisms has been proved to be an efficient means to increase both rate and total degradation of some representative diesel range hydrocarbons in soil [12-14]. Although the addition of readily available carbon sources, such as lactose, lactate and amino acids (components of whey) to contaminated soil would be expected to trigger exponential growth of organisms, a common phenomenon in liquid culture media, it seems that the soil matrix itself does not readily promote exponential growth of degrading organisms. Growth of soil microorganisms is limited by a number of factors caused by the properties of the soil matrix. Among the limiting factors is diffusion of substrate and nutrients but also the specific area available for bacterial sorption on to soil particles. When a large fraction of this area is occupied by bacteria, further growth might be increasingly restricted by diffusion of substrate and nutrients into multilayers of bacteria. Kinetic models that take into consideration slow or restricted (linear growth) may more accurately describe degradation patterns in real life situations. Figure 2 illustrates the application of a three-half-order kinetic model (II) originally proposed by Brunner and Focht [15]. The model assumes linear biomass growth rate and simulates the accumulated 14CO2-production from phenanthrene-9-14C mineralisation

because of its simplicity but have little predictive power when conditions for growth change.

*StP tktktk* <sup>0</sup>

2

P[t] is the cumulative 14CO2-production (%) where t is the time (days), S0 is the amount of substrate (%) that is "highly bioavailable", k1 is the first-order rate constant (day-1), k2 is a linear growth term (day-2), and k0 is the zero order rate constant describing the mineralisation of "less bioavailable" substrate and indigenous mineralisation (% day-1).The lag time was defined

whey 210 mg dw kg-1 soil dw; FW high = Fermented whey 2100 mg dw kg-1 soil dw; SW low = Sweet whey 210 mg dw kg-1 soil dw; SW high = Sweet whey 2100 mg dw kg-1 soil dw, all added at the beginning of the experiment. FW multi = Fermented whey 210 mg dw kg-1 soil dw added at the beginning of the experiment and then every 14th day. The curves are fitted to the data by use of the three-half-order kinetic model. Kinetic parameters are given in table 2 (From [18]. Reprinted with permission from Elsevier). The effects of applications of different amendments on phenanthrene biodegradation are clearly seen in figure 2. Kinetic parameters such as the linear growth term (k2) and S0 was significantly affected by the addition of the different amendments **Figure 2.** Effect of added fermented whey and sweet whey on 14C-phenanthrene mineralisation to 14CO2 in soil at 25 oC. FW low = Fermented whey 210 mg dw kg-1 soil dw; FW high = Fermented whey 2100 mg dw kg-1 soil dw; SW low = Sweet whey 210 mg dw kg-1 soil dw; SW high = Sweet whey 2100 mg dw kg-1 soil dw, all added at the beginning of the experiment. FW multi = Fermented whey 210 mg dw kg-1 soil dw added at the beginning of the experiment and then every 14th day. The curves are fitted to the data by use of the three-half-order kinetic model. Kinetic parameters are given in table 2 (From [18]. Reprinted with permission from Elsevier).

C. FW low = Fermented

Figure 2. Effect of added fermented whey and sweet whey on 14C-phenanthrene mineralisation to 14CO2 in soil at 25 o

5

#### **4. Waste recycling**

in soil microcosms.

factors is diffusion of substrate and nutrients but also the specific area available for bacterial sorption on to soil particles. When a large fraction of this area is occupied by bacteria, further growth might be increasingly restricted by diffusion of substrate and nutrients into multilayers of bacteria. Kinetic models that take into consideration slow or restricted (linear growth) may more accurately describe degradation patterns in real life situations. Figure 2 illustrates the application of a three-half-order kinetic model (II) originally proposed by Brunner and Focht [15]. The model assumes linear biomass growth rate and simulates the accumulated 14CO2-

production from phenanthrene-9-14C mineralisation in soil microcosms.

using the SOLVER function in Microsoft Excel 2000 [17].

**S0 (%)**

Control 21 51.3a 0 4.35a

SW low 21 48.6a 0 4.09a

SW high 18 63.2b 0 20.8c

**Lag time (days)**

64 Environmental Risk Assessment of Soil Contamination

permission from Elsevier).

( ) ( ( )) <sup>2</sup>

P[t] is the cumulative 14CO2-production (%) where t is the time (days), S0 is the amount of substrate (%) that is "highly bioavailable", k1 is the first-order rate constant (day-1), k2 is a linear growth term (day-2), and k0 is the zero order rate constant describing the mineralisation of "less bioavailable" substrate and indigenous mineralisation (% day-1).The lag time was defined as the initial time until 1% of the added 14C was detected as 14CO2 [16]. From the end of the lag phase, iterative non-linear least squares regression was performed to fit data to the model by

The effects of applications of different amendments on phenanthrene biodegradation are clearly seen in figure 2. Kinetic parameters such as the linear growth term (k2) and S0 was significantly affected by the addition of the different amendments (Table 2). It should be noted that the stimulating effect of complex organic amendments such as whey and fermented whey may not only be an effect of the addition of a readily available carbon source (that promotes growth) but also of available micronutrients, such as B-vitamins, that is required to maintain

> **k2 (day-2)**

FW low 24 53.8a 0 5.83b x 10-5 0 0.997 52.1a FW high 87 - - - - - 14.4c

FW multi 15 65.0b 0 63.6d x 10-5 0.014a 0.992 70.8e

**Table 2.** Tree-half-order kinetic constants for phenanthrene mineralisation in soil, at 25 °C. Lag time is expressed in units of days, S0 and mineralisation in %, k1 in day-1, k2 in day-2, k0 in % day-1 and R is the correlation coefficient. Values having the same superscript letters are not significantly different at a 95 % level. (From [18]. Reprinted with

**k0 (% day-1)**

**R**

x 10-5 0 0.973 46.3ab

x 10-5 0 0.974 43.4b

x 10-5 0.0046a 0.983 66.3d

**Mineralisation after 391 days (%)**

efficient catabolic pathways for degradation of i.e. polycyclic aromatic hydrocarbons.

**k1 (day-1)**

0 12 0 *P t S kt kt kt* = - -- + 1 exp 2 (2)

Organic by-products are produced globally on a massive scale. Economic operations such as agriculture, livestock, fishing and forestry [19-21] generate a number of by-products that can be seen in Figure 3. Frequently unexploited organic waste products create serious environ‐ mental problems when disposed of [20]. Agriculture is the economic base of most tropical developing countries, accounting for 50 % of the employment [21]. Organic by-products from agriculture, forestry, fishery and livestock operations are thus available in abundance and are typically of limited economical value [19]. Animal feed has been proposed as an outlet for some of these by-products but due to low levels of protein, high levels of moisture and some antinutritional factors i.e. presence of tannins and polyphenols, this practice is limited [22]. However, being inexpensive and easily available sources of carbon, nutrients and bioactive compounds, many of these by-products can potentially be used as sources of readily available carbon for bioremediation purposes [23].

**Figure 3.** Some organic by-products that potentially can be used as amendments for bioremediation.

A number of organic by-products have the ability to enhance the degradation of different toxic compounds including lignocellulosic wastes such as sugarcane, bagasse and sawdust [26], crop residues such as coffee pulp [27] and molasses [28] and whey, a by-product of the dairy industry [12-14]. Three examples of inexpensive by-products with potential or confirmed bioremediation capacity are described in more detail below: pyroligneous acid, compost teas and whey. **4.1 Pyroligneous acid** Pyroligneous acid (PA) is a by-product of charcoal production [29] with a limited economic value that is also referred to as wood vinegar, pyrolysis oil, smoke extract, bio-oil etc. [30, 31]. PA is typically disposed of as waste [31] but in Japan it has A number of organic by-products have the ability to enhance the degradation of different toxic compounds including lignocellulosic wastes such as sugarcane, bagasse and sawdust [26], crop residues such as coffee pulp [27] and molasses [28] and whey, a by-product of the dairy industry [12-14]. Three examples of inexpensive by-products with potential or confirmed bioremediation capacity are described in more detail below: pyroligneous acid, compost teas and whey.

been used for centuries to increase crop production and to combat agricultural pests [29, 32]. At present, the majority of the

#### charcoal consumed worldwide is produced in traditional kilns in developing countries [33]. Such charcoal production lacks appropriate control of the gas and vapours produced in the process and causes land and air pollution [34]. Production systems **4.1. Pyroligneous acid**

exist however that recover these condensate co-products or use them as fuel in the pyrolysis process, reducing the emission to a minimum [30]. Several low tech kilns that recover the PA from the charcoal production have been developed including *the advanced Brazilian beehive kiln* and the *Casamance kiln* [35]. In recent years the research and technology concerning PA have advanced significantly [30], and the current trend in charcoal production is to improve the environmental performance while maintaining or improving charcoal yield and quality. The chemical composition of PA varies depending on factors such as type of feedstock, pyrolysis temperature and duration etc. [25, 30]. The most frequently identified compounds in PA include methanol, acetic acid, methyl acetone, acetaldehyde, furan, furfural and volatile organic acids. PA contains phenolic compounds that are known to have antimicrobial properties [36] and PA has successfully been used to control fungal growth on wood [30] and bacterial decay of foods [36, 37]. At low concentrations however, PA has been shown to stimulate germination, growth and yield in a wide range of plants [37]. Although the growth promoting effect of PA is not yet clearly understood it has been attributed to nutritious components such as nitrates of Ca, Mg, K, magnesium sulphate etc. but also to its stimulatory effect on symbiotic fungi and bacteria [31]. The formation of fruit bodies of hydrocarbon-degrading fungi such as Lentinus edodes and Pleurotus ostreatus was significantly increased after applications of PA to the growth medium [38]. Furthermore PA has been shown to significantly increase basal respiration and microbial biomass in highly weathered tropical soils suggesting that microbes used PA for their metabolism [29]. **4.2 Compost teas** Compost teas (CT) are liquid compost extracts obtained when compost is soaked in water for a determined number of days. Optionally additives (derived from by-products) such as molasses, fish hydrolysate, rock dust, yeast extract, humic acids etc. Pyroligneous acid (PA) is a by-product of charcoal production [29] with a limited economic value that is also referred to as wood vinegar, pyrolysis oil, smoke extract, bio-oil etc. [30, 31]. PA is typically disposed of as waste [31] but in Japan it has been used for centuries to increase crop production and to combat agricultural pests [29, 32]. At present, the majority of the charcoal consumed worldwide is produced in traditional kilns in developing countries [33]. Such charcoal production lacks appropriate control of the gas and vapours produced in the process and causes land and air pollution [34]. Production systems exist however that recover these condensate co-products or use them as fuel in the pyrolysis process, reducing the emission to a minimum [30]. Several low tech kilns that recover the PA from the charcoal production have been developed including *the advanced Brazilian beehive kiln* and the *Casamance kiln* [35]. In recent years the research and technology concerning PA have advanced signifi‐ cantly [30], and the current trend in charcoal production is to improve the environmental performance while maintaining or improving charcoal yield and quality. The chemical

7

are used to add nutrients and stimulate microbial activity. To date virtually all research on CT has focused on its potential to control plant diseases in agriculture. However, the fact that CT have proved to significantly increase soil microbial respiration and dehydrogenase activity [39] makes it an interesting candidate for bioremediation. A number of production parameters (e.g. aeration, compost source, nutrient additives etc.) have been manipulated in order to optimize plant disease suppression [40]. To date, consensus on optimum production parameters for disease suppression hasn't been reached and some studies report

composition of PA varies depending on factors such as type of feedstock, pyrolysis tempera‐ ture and duration etc. [25, 30]. The most frequently identified compounds in PA include methanol, acetic acid, methyl acetone, acetaldehyde, furan, furfural and volatile organic acids. PA contains phenolic compounds that are known to have antimicrobial properties [36] and PA has successfully been used to control fungal growth on wood [30] and bacterial decay of foods [36, 37]. At low concentrations however, PA has been shown to stimulate germination, growth and yield in a wide range of plants [37]. Although the growth promoting effect of PA is not yet clearly understood it has been attributed to nutritious components such as nitrates of Ca, Mg, K, magnesium sulphate etc. but also to its stimulatory effect on symbiotic fungi and bacteria [31]. The formation of fruit bodies of hydrocarbon-degrading fungi such as *Lentinus edodes* and *Pleurotus ostreatus* was significantly increased after applications of PA to the growth medium [38]. Furthermore PA has been shown to significantly increase basal respiration and microbial biomass in highly weathered tropical soils suggesting that microbes used PA for their metabolism [29].

#### **4.2. Compost teas**

7

Compost teas (CT) are liquid compost extracts obtained when compost is soaked in water for a determined number of days. Optionally additives (derived from by-products) such as molasses, fish hydrolysate, rock dust, yeast extract, humic acids etc. are used to add nutrients and stimulate microbial activity. To date virtually all research on CT has focused on its potential to control plant diseases in agriculture. However, the fact that CT have proved to significantly increase soil microbial respiration and dehydrogenase activity [39] makes it an interesting candidate for bioremediation. A number of production parameters (e.g. aeration, compost source, nutrient additives etc.) have been manipulated in order to optimize plant disease suppression [40]. To date, consensus on optimum production parameters for disease suppression hasn't been reached and some studies report

Figure 3. Some organic by-products that potentially can be used as amendments for bioremediation.

**Figure 3.** Some organic by-products that potentially can be used as amendments for bioremediation.

Processed residues from wood industry

Abbatoir Waste

Dairy Industry Waste

66 Environmental Risk Assessment of Soil Contamination

**4.1 Pyroligneous acid**

and whey.

**4.1. Pyroligneous acid**

**4.2 Compost teas**

suggesting that microbes used PA for their metabolism [29].

A number of organic by-products have the ability to enhance the degradation of different toxic compounds including lignocellulosic wastes such as sugarcane, bagasse and sawdust [26], crop residues such as coffee pulp [27] and molasses [28] and whey, a by-product of the dairy industry [12-14]. Three examples of inexpensive by-products with potential or confirmed

Pyroligneous acid (PA) is a by-product of charcoal production [29] with a limited economic value that is also referred to as wood vinegar, pyrolysis oil, smoke extract, bio-oil etc. [30, 31]. PA is typically disposed of as waste [31] but in Japan it has been used for centuries to increase crop production and to combat agricultural pests [29, 32]. At present, the majority of the charcoal consumed worldwide is produced in traditional kilns in developing countries [33]. Such charcoal production lacks appropriate control of the gas and vapours produced in the process and causes land and air pollution [34]. Production systems exist however that recover these condensate co-products or use them as fuel in the pyrolysis process, reducing the emission to a minimum [30]. Several low tech kilns that recover the PA from the charcoal production have been developed including *the advanced Brazilian beehive kiln* and the *Casamance kiln* [35]. In recent years the research and technology concerning PA have advanced significantly [30], and the current trend in charcoal production is to improve the environmental performance while maintaining or improving charcoal yield and quality. The chemical composition of PA varies depending on factors such as type of feedstock, pyrolysis temperature and duration etc. [25, 30]. The most frequently identified compounds in PA include methanol, acetic acid, methyl acetone, acetaldehyde, furan, furfural and volatile organic acids. PA contains phenolic compounds that are known to have antimicrobial properties [36] and PA has successfully been used to control fungal growth on wood [30] and bacterial decay of foods [36, 37]. At low concentrations however, PA has been shown to stimulate germination, growth and yield in a wide range of plants [37]. Although the growth promoting effect of PA is not yet clearly understood it has been attributed to nutritious components such as nitrates of Ca, Mg, K, magnesium sulphate etc. but also to its stimulatory effect on symbiotic fungi and bacteria [31]. The formation of fruit bodies of hydrocarbon-degrading fungi such as Lentinus edodes and Pleurotus ostreatus was significantly increased after applications of PA to the growth medium [38]. Furthermore PA has been shown to significantly increase basal respiration and microbial biomass in highly weathered tropical soils

bioremediation capacity are described in more detail below: pyroligneous acid, compost teas and whey.

A number of organic by-products have the ability to enhance the degradation of different toxic compounds including lignocellulosic wastes such as sugarcane, bagasse and sawdust [26], crop residues such as coffee pulp [27] and molasses [28] and whey, a by-product of the dairy industry [12-14]. Three examples of inexpensive by-products with potential or confirmed bioremediation capacity are described in more detail below: pyroligneous acid, compost teas

Pyroligneous acid (PA) is a by-product of charcoal production [29] with a limited economic value that is also referred to as wood vinegar, pyrolysis oil, smoke extract, bio-oil etc. [30, 31]. PA is typically disposed of as waste [31] but in Japan it has been used for centuries to increase crop production and to combat agricultural pests [29, 32]. At present, the majority of the charcoal consumed worldwide is produced in traditional kilns in developing countries [33]. Such charcoal production lacks appropriate control of the gas and vapours produced in the process and causes land and air pollution [34]. Production systems exist however that recover these condensate co-products or use them as fuel in the pyrolysis process, reducing the emission to a minimum [30]. Several low tech kilns that recover the PA from the charcoal production have been developed including *the advanced Brazilian beehive kiln* and the *Casamance kiln* [35]. In recent years the research and technology concerning PA have advanced signifi‐ cantly [30], and the current trend in charcoal production is to improve the environmental performance while maintaining or improving charcoal yield and quality. The chemical

Inexpensive organic byproducts

> processed wood residues

Manure

Residues from fishery

Non‐ligno‐ cellulosic

Un‐ crop waste

Ligno‐ cellulosic crop waste

> Compost teas (CT) are liquid compost extracts obtained when compost is soaked in water for a determined number of days. Optionally additives (derived from by-products) such as molasses, fish hydrolysate, rock dust, yeast extract, humic acids etc. are used to add nutrients and stimulate microbial activity. To date virtually all research on CT has focused on its potential to control plant diseases in agriculture. However, the fact that CT have proved to significantly increase soil microbial respiration and dehydrogenase activity [39] makes it an interesting candidate for bioremediation. A number of production parameters (e.g. aeration, compost source, nutrient additives etc.) have been manipulated in order to optimize plant disease suppression [40]. To date, consensus on optimum production parameters for disease suppression hasn't been reached and some studies report inconsistency in the performance [41]. However a number of reports have shown that aeration and additions of nutrients lead to significant increases of the cell mass of active bacteria [39, 40, 42] and the addition of humic acids and yeast extracts significantly increased the fungal populations [43]. Plate counts and microscopic examination studies have revealed that aerated CT has bacterial population dominated by *Bacillus sp*., *Lactobacillus sp*., *Micrococcus lutues*, *Staphylococcus sciuri*, *Pseudomonas putida*, *Burkholderia glumae* and *Clavibacter agropyri* while species of *Aspergillus*, *Penicillium* and *Trichoderma* dominated the fungal communities [43]. Many of these organisms have the capacity to degrade a number of toxic substances including PAH:s and organochlorine compounds [44, 45].

> In a recent study, applications of compost teas made from vermicompost caused significantly greater dehydrogenase activity and microbial respiration [39] which makes it an interesting candidate for bioremediation. Vermicompost generally has higher concentrations of available nutrients (NO3 - , exchangeable Ca, P and soluble K) and significantly larger and more diverse microbial populations than common compost. Microorganisms found in worm castings of *Eisenina fetida* have caused increased catabolic activity and enhanced degradation of PAHs in a number of experiments [46, 47].

#### **4.3. Whey**

Wheyisproducedwhenmilkcaseinis removedfromthemilkindairyoperations tomakecheese and other products. This liquid by-product constitutes between 85-95% of the milk volume and 55% of milk nutrients remain in the whey. About half of the yearly global production of 145 million tonnes is utilized for animal feed etc. The remaining large volumes end up as potential contaminants in the environment [48]. The chemical content of whey is characterised by lactose, anumberofessentialandnon-essentialaminoacidsindifferentproportions,vitaminB1,2,6,7,12, folic acid and lactic acid [12, 13]. Relatively few studies have been performed on the effect of whey on biodegradation of organic pollutants [12]. However, research at Mid Sweden Univer‐ sity has showed that the degradation of aliphatic and aromatic hydrocarbons can be significant‐ ly enhanced by the addition of whey and fermented whey [12, 13]. The biodegradationenhancingeffectofwheywasprimarilyattributedtoanincreasedmicrobialbiomass stimulated bythereadilyavailablecarbonsource[12].Thedegradationstudiesalsoshowedamorecomplex dependence of carbon sources and growth factors, such as B-vitamins, on the degradation of an aromatic compound (phenanthrene) compared to an aliphatic compound (hexadecane) [18]. These results indicate that the presence of co-factors such as vitamins and micronutrients may beimportanttoconsiderwhenevaluatingthesuitabilityoforganicamendmentsforbioremedia‐ tion of soil contaminated by various types of organic pollutants.

## **5. Case studies**

Different technologies and strategies are necessary to remediate the present and future soil pollution.Asourunderstandingofthedegradationmechanismsofdifferentpollutants evolves, so do our chances to develop alternative and complementary strategies that can take the concentrations of toxic compounds to a sustainable level. Since 2006, a research group at Mid Sweden University has undertaken research dedicated to the development of bioremediation methods for low priority remediation sites including, remote locations and tropical develop‐ ing countries. Ecological engineering provide an appealing theoretical framework in such locations where energy and material costs are limiting factors. The research has implied laboratoryexperimentsaswellaspilotscaleandin-situprojectsintropicalandtemperateclimate. It includes pilot scale experiments in an experimental station that was developed in Nicaragua (se figure 4) and field trials in remote parts of northern Sweden where transport distances are long and the cold climate constitutes an additional challenge to in-situ bioremediation projects.

#### **5.1. Field station in Nicaragua, designed for bioremediation experiments in a tropical climate**

Waste management programs in developing countries are often deficient and the inappropri‐ ate disposal of hazardous waste creates pollution problems. Furthermore the use of pesticides and other toxic products is minimally restricted in many developing countries which leads to extensive contamination with persistent substances such as polycyclic aromatic hydrocarbons and organochlorine pesticides [49, 50]. The experimental station in Nicaragua (figure 5) is Sustainability Aspects of In-Situ Bioremediation of Polluted Soil in Developing Countries and Remote Regions http://dx.doi.org/10.5772/57315 69

**4.3. Whey**

68 Environmental Risk Assessment of Soil Contamination

**5. Case studies**

**climate**

Wheyisproducedwhenmilkcaseinis removedfromthemilkindairyoperations tomakecheese and other products. This liquid by-product constitutes between 85-95% of the milk volume and 55% of milk nutrients remain in the whey. About half of the yearly global production of 145 million tonnes is utilized for animal feed etc. The remaining large volumes end up as potential contaminants in the environment [48]. The chemical content of whey is characterised by lactose, anumberofessentialandnon-essentialaminoacidsindifferentproportions,vitaminB1,2,6,7,12, folic acid and lactic acid [12, 13]. Relatively few studies have been performed on the effect of whey on biodegradation of organic pollutants [12]. However, research at Mid Sweden Univer‐ sity has showed that the degradation of aliphatic and aromatic hydrocarbons can be significant‐ ly enhanced by the addition of whey and fermented whey [12, 13]. The biodegradationenhancingeffectofwheywasprimarilyattributedtoanincreasedmicrobialbiomass stimulated bythereadilyavailablecarbonsource[12].Thedegradationstudiesalsoshowedamorecomplex dependence of carbon sources and growth factors, such as B-vitamins, on the degradation of an aromatic compound (phenanthrene) compared to an aliphatic compound (hexadecane) [18]. These results indicate that the presence of co-factors such as vitamins and micronutrients may beimportanttoconsiderwhenevaluatingthesuitabilityoforganicamendmentsforbioremedia‐

Different technologies and strategies are necessary to remediate the present and future soil pollution.Asourunderstandingofthedegradationmechanismsofdifferentpollutants evolves, so do our chances to develop alternative and complementary strategies that can take the concentrations of toxic compounds to a sustainable level. Since 2006, a research group at Mid Sweden University has undertaken research dedicated to the development of bioremediation methods for low priority remediation sites including, remote locations and tropical develop‐ ing countries. Ecological engineering provide an appealing theoretical framework in such locations where energy and material costs are limiting factors. The research has implied laboratoryexperimentsaswellaspilotscaleandin-situprojectsintropicalandtemperateclimate. It includes pilot scale experiments in an experimental station that was developed in Nicaragua (se figure 4) and field trials in remote parts of northern Sweden where transport distances are long and the cold climate constitutes an additional challenge to in-situ bioremediation projects.

**5.1. Field station in Nicaragua, designed for bioremediation experiments in a tropical**

Waste management programs in developing countries are often deficient and the inappropri‐ ate disposal of hazardous waste creates pollution problems. Furthermore the use of pesticides and other toxic products is minimally restricted in many developing countries which leads to extensive contamination with persistent substances such as polycyclic aromatic hydrocarbons and organochlorine pesticides [49, 50]. The experimental station in Nicaragua (figure 5) is

tion of soil contaminated by various types of organic pollutants.

**Figure 4.** Left picture: Map of Central America and Nicaragua. Right picture: The red marked area is Chontales, the region where Mid Sweden University's pilot scale experimental station is located. (Map of Central America by Виктор В licensed under Creative Commons Attribution-Share Alike 2.0 Generic, Mapa de Nicaragua mostrando al departamen‐ to de Chontales by Vrysxy licensed under Creative Commons Attribution-Share Alike 3.0 Unported.)

**Figure 5.** Mid Sweden University´s experimental station at Casa Montesano in Nicaragua.

located on the agro ecological farm *Casa Montesano,* (11˚59'70˝ N 84˚53'06˝ E), where CIPP, a local NGO, dedicated to sustainable rural development projects has its headquarters. The region has a humid tropical climate with an average precipitation of 2000 mm per year and an annual average temperature between 25 and 28 ˚C. The soil on the experimental site is classified as ultisol, a common soil in the humid tropics occupying about 8 % of the global ice-free land area and supporting 18 % of the world's population.

Figure 6 shows the 24 compartments that were built in concrete with a smooth cement plaster in two sets of twelve compartments each. The compartments were built to fit 150 L each of agriculture soil. The purpose of the closed compartments is to test sustainable remediation methods in conditions as similar to in-situ remediation as possible without risks for pollutants to escape into nature. All technologies assessed in the experimental station are designed to be low cost/low technology methods, appropriate to economically marginalised people in developing countries. Notable is the use of locally available organic waste or by-products such as whey, pyroligneous acid and compost teas that can be achieved at a very low cost. The pilot scale research aim to find practical solutions to the obstacles that emerge when positive results from the laboratory are to be reproduced in the field. This implies dealing with issues like chemotaxis limitations, i.e. the importance of biodegrading microorganisms to be in close proximity to the pollutant. That can be achieved by the use of surfactants and solvents and by avoiding clogging of the soil pores.

The first experiment conducted in the station was on soil spiked with diesel at a concentration of 5.00 g diesel dw-1 kg-1 soil dw. Whey, pyroligneous acid and compost teas were applied at a high and a low dose on day 3, 18, 32, 46, 60, 74 and 88 after diesel contamination. The doses were adjusted to reflect the expected ideal dose (high) and an economically more viable dose (low). Soil samples were taken on day 2, 7, 12, 17, 31, 45, 59, 73, 87 and 101 after diesel contamination. Leachate water samples were collected by the end of the experiment in order to detect presence of metabolic by-products.

When this chapter was written the soil samples were not yet analyzed. The soil samples will be analysed by GC/FID to determine the effect on degradation from the different amendments. The amendments' effect on the microbial composition will subsequently be analysed by molecular methods. Whey has engendered significant enhancement of the degradation of aliphatic and aromatic hydrocarbons in previous laboratory experiments within our research group but compost tea and pyroligneous acid have not been previously tested.

In an upcoming project the research group envisages to use the experimental station for novel research on relevant pesticides. Many of the least biodegradable pesticides are relatively persistent to aerobic degradation [27] which poses additional challenges for in-situ remedia‐ tion. These challenges will be addressed constructively in trials with redox barriers and other strategies.

#### **5.2. Experiences from in-situ remediation trials in remote areas of Northern Sweden**

#### *5.2.1. Bioremediation field trial using whey treatment of diesel contaminated soil at a former gas station in Northern Sweden*

A field experiment was carried out at a diesel contaminated site in Gäddede, in the county of Jämtland, north-western Sweden (65°30'N, 14°09'E) [51], see figure 7. The contaminated site is an old fuel station, established in the 1960:s. The contaminated site is about 10 m2 in area Sustainability Aspects of In-Situ Bioremediation of Polluted Soil in Developing Countries and Remote Regions http://dx.doi.org/10.5772/57315 71

**Figure 6.** The compartments filled with soil at the experimental station in Nicaragua.

as ultisol, a common soil in the humid tropics occupying about 8 % of the global ice-free land

Figure 6 shows the 24 compartments that were built in concrete with a smooth cement plaster in two sets of twelve compartments each. The compartments were built to fit 150 L each of agriculture soil. The purpose of the closed compartments is to test sustainable remediation methods in conditions as similar to in-situ remediation as possible without risks for pollutants to escape into nature. All technologies assessed in the experimental station are designed to be low cost/low technology methods, appropriate to economically marginalised people in developing countries. Notable is the use of locally available organic waste or by-products such as whey, pyroligneous acid and compost teas that can be achieved at a very low cost. The pilot scale research aim to find practical solutions to the obstacles that emerge when positive results from the laboratory are to be reproduced in the field. This implies dealing with issues like chemotaxis limitations, i.e. the importance of biodegrading microorganisms to be in close proximity to the pollutant. That can be achieved by the use of surfactants and solvents and by

The first experiment conducted in the station was on soil spiked with diesel at a concentration of 5.00 g diesel dw-1 kg-1 soil dw. Whey, pyroligneous acid and compost teas were applied at a high and a low dose on day 3, 18, 32, 46, 60, 74 and 88 after diesel contamination. The doses were adjusted to reflect the expected ideal dose (high) and an economically more viable dose (low). Soil samples were taken on day 2, 7, 12, 17, 31, 45, 59, 73, 87 and 101 after diesel contamination. Leachate water samples were collected by the end of the experiment in order

When this chapter was written the soil samples were not yet analyzed. The soil samples will be analysed by GC/FID to determine the effect on degradation from the different amendments. The amendments' effect on the microbial composition will subsequently be analysed by molecular methods. Whey has engendered significant enhancement of the degradation of aliphatic and aromatic hydrocarbons in previous laboratory experiments within our research

In an upcoming project the research group envisages to use the experimental station for novel research on relevant pesticides. Many of the least biodegradable pesticides are relatively persistent to aerobic degradation [27] which poses additional challenges for in-situ remedia‐ tion. These challenges will be addressed constructively in trials with redox barriers and other

**5.2. Experiences from in-situ remediation trials in remote areas of Northern Sweden**

is an old fuel station, established in the 1960:s. The contaminated site is about 10 m2

*5.2.1. Bioremediation field trial using whey treatment of diesel contaminated soil at a former gas station*

A field experiment was carried out at a diesel contaminated site in Gäddede, in the county of Jämtland, north-western Sweden (65°30'N, 14°09'E) [51], see figure 7. The contaminated site

in area

group but compost tea and pyroligneous acid have not been previously tested.

area and supporting 18 % of the world's population.

70 Environmental Risk Assessment of Soil Contamination

avoiding clogging of the soil pores.

to detect presence of metabolic by-products.

strategies.

*in Northern Sweden*

and about 0.5 m in depth. Previous investigations had pointed out an area with relatively high loads of aliphatic and aromatic hydrocarbons C5-C35 (approximately 2200 mg kg-1 dw soil) dominated by diesel range aliphatics.

This site is representative of the many small contaminated areas that are spread out in rural areas, and are typically considered low priority sites. According to a study by the Swedish EPA in 2006 excavation and transport was the dominating remediation technology in Sweden [52] which is also the case in the county of Jämtland. In this case the distance from Gäddede to Gräfsåsen, the nearest approved treatment plant for contaminated soil, is 230 km. The alternative of excavation and transport will therefore be expensive and contribute to air pollution and greenhouse gas emissions. For such areas, a low cost, easy to apply in-situ remediation technique would be appropriate. In-situ whey treatment might be such a method. Addition of whey to increase biological activity in soil for remediation purposes has been previously demonstrated in laboratory studies [12, 13, 18].

#### *5.2.1.1. Life cycle assessment of in-situ bioremediation with whey treatment in Gäddede in comparison to traditional excavation and composting*

In order to compare the two alternative treatment strategies, a screening life cycle assessment model was set up. The purpose was to investigate the environmental performance of the whey method, to benchmark the whey method towards the excavation and composting practice and

**Figure 7.** The test spots for in-situ bioremediation trials with whey in northern Sweden. (Location map of Scandinavia by NordNordWest licensed under the Creative Commons Attribution-Share Alike 3.0 Unported) Top picture: Diesel contaminated site at former gasoline station in Gäddede (Village). Bottom picture: Oil spot at lumberyard in Sikås (Vil‐ lage). © Lantmäteriet 2012/1067.

to identify environmental hot spots in the whey treatment life cycle [53]. The functional unit of this study was the treatment of 5 m3 of contaminated soil reaching a diesel concentration of 500 mg kg-1 dw (upper limit in generic guidelines for "non sensitive areas"). For impact assessment the results are characterized as global warming potential (GWP), acidification potential (AP), eutrophication potential (EP) and photo oxidant creation potential (POCP). It was assumed that whey is added together with commercial fertilizer to avoid macronutrient deficit. Since the biological activity only takes place when it is warm enough (this mountainous region has a cold climate with at least 6 months of winter and a yearly average temperature of around 0o C) the total treatment time was estimated to two summers. The alternative treatment assumed in this study is to excavate the 5 m3 of contaminated soil at the site in Gäddede, transport it to the waste treatment site in Gräfsåsen, Östersund, Sweden, and compost the soil in windrows after mixing the soil with manure. The windrows are turned using an excavator and when the concentrations of contaminants are low enough the compost is used as top cover of a landfill. Figure 8 illustrates a simplified model of the life-cycle process flow diagram for the whey treatment scenario.

**Figure 8.** The life cycle inventory model of the whey scenario (From [53]).

to identify environmental hot spots in the whey treatment life cycle [53]. The functional unit of this study was the treatment of 5 m3 of contaminated soil reaching a diesel concentration of 500 mg kg-1 dw (upper limit in generic guidelines for "non sensitive areas"). For impact assessment the results are characterized as global warming potential (GWP), acidification potential (AP), eutrophication potential (EP) and photo oxidant creation potential (POCP). It was assumed that whey is added together with commercial fertilizer to avoid macronutrient deficit. Since the biological activity only takes place when it is warm enough (this mountainous region has a cold climate with at least 6 months of winter and a yearly average temperature of

500 km 0,5 km

**Figure 7.** The test spots for in-situ bioremediation trials with whey in northern Sweden. (Location map of Scandinavia by NordNordWest licensed under the Creative Commons Attribution-Share Alike 3.0 Unported) Top picture: Diesel contaminated site at former gasoline station in Gäddede (Village). Bottom picture: Oil spot at lumberyard in Sikås (Vil‐

**Gädded**

**Sikås**

C) the total treatment time was estimated to two summers. The alternative treatment

transport it to the waste treatment site in Gräfsåsen, Östersund, Sweden, and compost the soil in windrows after mixing the soil with manure. The windrows are turned using an excavator and when the concentrations of contaminants are low enough the compost is used as top cover

of contaminated soil at the site in Gäddede,

around 0o

lage). © Lantmäteriet 2012/1067.

72 Environmental Risk Assessment of Soil Contamination

assumed in this study is to excavate the 5 m3

The hydrocarbon pollution in the 5 m3 soil to be treated (over an area of 10m2 ) was assumed to be the same in the whole mass of soil i.e. 2200 mg kg-1 dw. Comparisons with laboratory results [12] indicate that an addition of 210 mg dw whey kg-1 diesel polluted soil might give a sufficient reduction in diesel oil contamination to reach the higher target value of 500 mg kg-1 dw [54]. This gives a calculated need of about 2 kg dw whey, equivalent to 367 L of whey (5.5 g dw L-1) for the area to be treated. The laboratory tests also indicate that a number of successive additions of whey will give a better degradation compared to one large addition, and eight additions (46 L of wet whey per addition) spaced out with 14 days in between was assumed to be a good practice. The total time needed for the degradation is from the laboratory tests estimated to 167 days. Since on-site biodegradation will only take place when it is warm enough [13] the degradation was assumed to take place during two summer months each year for two years. To ensure that biodegradation is not limited by macronutrient deficiencies addition of commercial fertilizer was assumed. It was estimated that a one-time addition of 1.5 kg YaraMila 18-4-14 would provide the necessary nutrients. During the remediation process whey and fertilizer was assumed to be manually added to the contaminated area. Whey additions of 46 L every 14 day over a period of two subsequent summers were assumed.

For the intended bioremediation a total of 2 kg dw whey is needed. Data on cheese and whey production was obtained from Milko dairy in Östersund. To produce 0.9 ton of Edam cheese and 1 ton of dry whey the cheese dairy on average requires 11000 L of milk. Inventory data was gathered to describe the use of average Swedish electricity, water and auxiliary chemicals [55]. Inventory data for milk production, including farming activities, transports and dairy operations was based on a LCI by the Swedish Diary Association.

Since whey is a by-product of cheese production, economic allocation was used giving an allocation of 80 % on cheese and 20 % on whey. In a sensitivity analysis the results were investigated, assuming that all production environmental impact is allocated to the whey. The fertilizer production is described using emissions data on the production of 1 kg of fertilizer. Whey and fertilizer will have to be transported to the contaminated site. Whey is produced in Sweden by Milko AB is transported over a distance of 230 km from Östersund to Gäddede. Fertilizer is obtained from commercial fertilizer store and transported the same distance. Environmental impacts from the transports were calculated using emissions factors assuming a light distribution truck, Euro 2, vehicle manufactured during 1996-2000.

Figure 9 illustrates a simplified model of the life-cycle process flow diagram for the excavation and composting treatment scenario. Assuming a soil density of 1922 kg m-3 for the 5 m3 soil to be treated (over an area of 10 m2 ) the calculated mass of contaminated soil was 9.6 ton.

**Figure 9.** The life cycle inventory model of the excavation and composting scenario (From [53]).

It was assumed that the Gäddede case would need a 14 ton excavator for 15 minutes and use 2 L of diesel. Production of the diesel oil was described by inventory data from [56]. The excavated soil will then have to be transported 230 km from Gäddede to Östersund to a composting plant. Impacts from the transport is described using emission factors for a medium sized distribution truck with a payload of 8.5-14 ton, Euro class 2, produced between 1996 – 2000.

investigated, assuming that all production environmental impact is allocated to the whey. The fertilizer production is described using emissions data on the production of 1 kg of fertilizer. Whey and fertilizer will have to be transported to the contaminated site. Whey is produced in Sweden by Milko AB is transported over a distance of 230 km from Östersund to Gäddede. Fertilizer is obtained from commercial fertilizer store and transported the same distance. Environmental impacts from the transports were calculated using emissions factors assuming

Figure 9 illustrates a simplified model of the life-cycle process flow diagram for the excavation and composting treatment scenario. Assuming a soil density of 1922 kg m-3 for the 5 m3

) the calculated mass of contaminated soil was 9.6 ton.

soil to

a light distribution truck, Euro 2, vehicle manufactured during 1996-2000.

**Figure 9.** The life cycle inventory model of the excavation and composting scenario (From [53]).

It was assumed that the Gäddede case would need a 14 ton excavator for 15 minutes and use 2 L of diesel. Production of the diesel oil was described by inventory data from [56]. The excavated soil will then have to be transported 230 km from Gäddede to Östersund to a composting plant. Impacts from the transport is described using emission factors for a medium sized distribution truck with a payload of 8.5-14 ton, Euro class 2, produced between 1996 –

be treated (over an area of 10 m2

74 Environmental Risk Assessment of Soil Contamination

2000.

Figure 10. Results from the LCA models for the in-situ whey treatment scenario – with two levels of allocation of the whey production – and the excavation and off-site composting scenario. GWP 100, Global Warming Potential, 100 year time horizon; AP, Acidification Potential; EP, Eutrophication Potential; POCP, Photochemical Ozone Creation Potential. The allocation scenarios mean that respectively 20 and 100 % of the environmental effect of the cheese production was allocated to the whey. Normalized data, the highest result in each category is set to 100% (From [53])*.* **Figure 10.** Results from the LCA models for the in-situ whey treatment scenario – with two levels of allocation of the whey production – and the excavation and off-site composting scenario. GWP 100, Global Warming Potential, 100 year time horizon; AP, Acidification Potential; EP, Eutrophication Potential; POCP, Photochemical Ozone Creation Po‐ tential. The allocation scenarios mean that respectively 20 and 100 % of the environmental effect of the cheese pro‐ duction was allocated to the whey. Normalized data, the highest result in each category is set to 100% (From [53])*.*

At Gräfsåsen, the waste treatment and landfill in Östersund, the contaminated soil is mixed with horse manure and placed in windrows in a string-like arrangement. Over the period 2005 to 2009 some 2320 tons of contaminated soil and 1560 tons of manure have been received at the composting plant in Gräfsåsen, giving an average manure use of 0.67 ton/ton. For the manure transport we assume the same emission factors as for the soil transport and an average transport distance of 50 km. The manure generations are totally allocated to the horse keeping. Every one month during the summer the windrows are mixed using a mixer mounted on a wheel loader. For this process half the environmental impact from the excavation in Gäddede was assumed. Use of the treated compost as cover of the land fill is not assumed to be connected to any environmental impacts. The results from the whey treatment scenario and the com‐ posting scenario for the four impact categories GWP, AP, EP and POCP is shown in Figure 10, together with the results from the sensitivity analysis where all environmental impacts from milk production and the cheese dairy are allocated to the whey. When comparing the allocated and the unallocated whey treatment scenarios the milk-cheese chain has a rather large impact, especially for eutrophication (EP in diagram). It should be noted that the transport of whey and fertilizer for the whey scenario is described with emission factors of a truck transport, that means it is assumed to have the necessary whey and fertilizer as part of the load of the truck and the off-loading will be part of a delivery route where other parts of the load will be off-loaded in other places. Arranging good logistics is thus important both for the whey treatment and for the excavation and transport to offsite composting. *5.2.1.2 Results of bioremediation field trials and microcosm experiments using whey treatment of the contaminated soil in Gäddede* The result from whey treatment of the polluted site during a 3 ½ months field experiment was not promising. The experiment was initiated 10 May when the snow had melted and the soil temperature at 18 cm depth was 5 o C. The polluted area was given an initial dose of 545 g m-2 NPK fertilizer (18 % N; 4 % P; 10 % K) followed by bi-weekly doses of 3.2 L m-2 milk whey [51]. During the course of the summer the soil temperature rose to a maximum of 15 <sup>o</sup> C in the beginning of August and dropped thereafter. When the experiment was ended in mid August no detectable reduction of soil hydrocarbons was observed. In parallel to the field experiment, microcosm studies were carried out on soil samples, from the vicinity of the contaminated site that had been artificially contaminated with diesel fuel. In the microcosms the soil was subjected to the same treatment as in the field study with the only difference that the soil was kept at 22 <sup>o</sup> C. During these conditions the diesel range aliphatics was almost completely degraded within 60 days. This is in accordance with previous studies on hexadecane that showed

For the GWP, AP and POCP impact categories the whey scenarios give lower results than the excavation and off-site composting scenario, whereas the EP category shows higher results. The EP result for the whey treatment is totally dependent on the whey production chain and originates at the farm (such as nutrient runoff, ruminating cattle, vaporization of ammonia from manure) or by the production of materials used at the farm (such as fertilizers). The milk farm activity is the most contributing in the whey scenario for all four impact categories shown. for in-situ bioremediation in cold regions and the short period of warmth during 3 summer months is rarely enough to bring the soil temperature to levels that give sufficient rates of biodegradation. A possible way to circumvent this problem would be to heat up the soil with heat injection. Preliminary calculations carried out at our department reveal that the electric power needed to heat up a 100 x 100 x 2 m contaminated volume of polluted soil from 8 to 25 o C by e.g. a sufficient number of heat rods, would be 166 kW during a one month period corresponding to an input heating energy of 98 x 103 kWh. The calculation assumed that the soil surface was insulated and that there were no vertical and downwards heat losses during the heat-up period. The energy needed to maintain the temperature at 25 o C for another 11 month period assuming horizontal and vertical (downwards) heat losses would amount to 235 x 103 kWh. Total energy need for a one year period is then 333 x 103 kWh. The following input data was used: A linear temperature drop distance of 9.1 m to ambient temperatures, heat conductivity 1.5 W m-1 K-1, specific heat capacity 800 J kg-1 K-1 and soil density 1300 kg m-3. These are rough calculations based on some

C respectively [13]. Soil temperature is probably the most limiting factor

by 0.5 m of the Gäddede

significant difference in degradation rates at 7 and 22 <sup>o</sup>

14

simplified assumptions about heat transfer in soil but may still give an estimate of the magnitude of the input energy needed for

diesel oil plot the corresponding figures would be 27 and 387 kWh respectively for the heating and 11 months maintenance input energy need respectively giving a total energy need for a 12 month period of roughly 414 kWh. Part of this energy need

a temperature enhanced in-situ soil bioremediation project in cold climate. Downscaled to the 10 m2

When comparing the allocated and the unallocated whey treatment scenarios the milk-cheese chain has a rather large impact, especially for eutrophication (EP in diagram). It should be noted that the transport of whey and fertilizer for the whey scenario is described with emission factors of a truck transport, that means it is assumed to have the necessary whey and fertilizer as part of the load of the truck and the off-loading will be part of a delivery route where other parts of the load will be off-loaded in other places. Arranging good logistics is thus important both for the whey treatment and for the excavation and transport to off-site composting.

#### *5.2.1.2. Results of bioremediation field trials and microcosm experiments using whey treatment of the contaminated soil in Gäddede*

The result from whey treatment of the polluted site during a 3 ½ months field experiment was not promising. The experiment was initiated 10 May when the snow had melted and the soil temperature at 18 cm depth was 5 o C. The polluted area was given an initial dose of 545 g m-2 NPK fertilizer (18 % N; 4 % P; 10 % K) followed by bi-weekly doses of 3.2 L m-2 milk whey [51]. During the course of the summer the soil temperature rose to a maximum of 15 o C in the beginning of August and dropped thereafter. When the experiment was ended in mid August no detectable reduction of soil hydrocarbons was observed.

In parallel to the field experiment, microcosm studies were carried out on soil samples, from the vicinity of the contaminated site that had been artificially contaminated with diesel fuel. In the microcosms the soil was subjected to the same treatment as in the field study with the only difference that the soil was kept at 22 o C. During these conditions the diesel range aliphatics was almost completely degraded within 60 days. This is in accordance with previous studies on hexadecane that showed significant difference in degradation rates at 7 and 22 o C respectively [13]. Soil temperature is probably the most limiting factor for in-situ bioremedia‐ tion in cold regions and the short period of warmth during 3 summer months is rarely enough to bring the soil temperature to levels that give sufficient rates of biodegradation. A possible way to circumvent this problem would be to heat up the soil with heat injection. Preliminary calculations carried out at our department reveal that the electric power needed to heat up a 100 x 100 x 2 m contaminated volume of polluted soil from 8 to 25 o C by e.g. a sufficient number of heat rods, would be 166 kW during a one month period corresponding to an input heating energy of 98 x 103 kWh. The calculation assumed that the soil surface was insulated and that there were no vertical and downwards heat losses during the heat-up period. The energy needed to maintain the temperature at 25 o C for another 11 month period assuming horizontal and vertical (downwards) heat losses would amount to 235 x 103 kWh. Total energy need for a one year period is then 333 x 103 kWh. The following input data was used: A linear temper‐ ature drop distance of 9.1 m to ambient temperatures, heat conductivity 1.5 W m-1 K-1, specific heat capacity 800 J kg-1 K-1 and soil density 1300 kg m-3. These are rough calculations based on some simplified assumptions about heat transfer in soil but may still give an estimate of the magnitude of the input energy needed for a temperature enhanced in-situ soil bioremediation project in cold climate. Downscaled to the 10 m2 by 0.5 m of the Gäddede diesel oil plot the corresponding figures would be 27 and 387 kWh respectively for the heating and 11 months maintenance input energy need respectively giving a total energy need for a 12 month period of roughly 414 kWh. Part of this energy need could easily be provided by solar panels. Furthermore, taking into consideration the very rapid increase in degradation rate even at a moderately elevated temperature such as 22 o C it is possible that a successful temperature enhanced remediation of the Gäddede plot could even be carried out within 6 months during a period when solar panels can provide the total need of input energy. Solar panels can easily be moved from one project to another so the technology described here should have a potential for application in in-situ soil bioremediation in remote places and in cold regions.
