**Petroleum Hydrocarbon Biodegradability in Soil – Implications for Bioremediation**

Snežana Maletić, Božo Dalmacija and Srđan Rončević

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/50108

## **1. Introduction**

42 Hydrocarbon

1065–1076.

Mizobe, K. (2007) Geochemical characteristics of natural gases from mud volcanoes in Tokamachi City, Niigata Prefecture. Master Thesis, Graduate School of Science and

Nakada, R., Takahashi, Y., Tsunogai, U., Zheng, G., Shimizu, H., and Hattori, K. H. (2011) A geochemical study on mud volcanoes in the Junggar Basin, China. *Appl. Geochem.*, 26,

Nansheng, Q., Zhihuan, Z. and Ershe, X. (2008) Geothermal regime and Jurassic source rock maturity of the Junggar Basin, northwest China. *J. Asian Earth Sci.*, 31, 464−478. Pallasser, R. J. (2000) Recognising biodegradation in gas/oil accumulations through the 13C

Waseda, A., Iwano, H. (2008). Characterization of natural gases in Japan based on molecular

Schoell, M. (1983) Genetic characterization of natural gases. *AAPG Bull.*, 67, 2225−2238. Valyaev, B.M., Grinchenko, Y.I., Erokhin, V.E., Prokhorov, V.S., Titkov, G.A. (1985) Isotopic

composition of gases from mud volcanoes. *Lithol. Miner. Resour.*, 20, 62–75.

compositions of gas components. *Org. Geochem.*, 31, 1363−1373.

and carbon isotope compositions. *Geofluids*, 8, 286–292.

Engineering, Yamaguchi University, pp. 44.

The development of human civilization throughout history has led to growing disruption of the natural balance and the occurrence of different types of pollution. The world depends on oil, and the use of oil as fuel has led to intensive economic development worldwide. The great need for this energy source has led to the gradual exhaustion of natural oil reserves. However, mankind will witness the results of oil consumption for centuries after its cessation. Environmental pollution with petroleum and petrochemical products has been recognized as a significant and serious problem (Alexander, 1995, 2000). Most components of oil are toxic to humans and wildlife in general, as it is easy to incorporate into the food chain. This fact has increased scientific interest in examining the distribution, fate and behaviour of oil and its derivatives in the environment (Alexander, 1995, 2000; Semple et al., 2001, 2003; Stroud et al., 2007, 2009). Oil spills in the environment cause long-term damage to aquatic and soil ecosystems, human health and natural resources.

Petroleum oil spills tend to be associated with offshore oil rigs and tankers in marine-related accidents. In contrast, land oil spills often go unnoticed by everyone except environmentalists, yet land oil spills contribute to the pollution of our water supply and soil. Typical sources of land oil spills include accidents as well as oil from vehicles on the road.

Characterization of spilled oil and its derivatives is very important in order to predict the behaviour of oil and its long-term effects on the environment, and in order to select the proper cleaning methods. The potential danger which petroleum hydrocarbons pose to humans and the environment makes testing and characterization of the biodegradation and biotransformation processes of hydrocarbons in contaminated soil necessary in order to develop bioremediation techniques for cleaning such soils to levels that ensures its safe disposal or reuse. Biodegradation is the metabolic ability of microorganisms to transform or mineralize organic contaminants into less harmful, non-hazardous substances, which are

© 2013 Maletić et al., licensee InTech. This is an open access chapter 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. © 2013 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.

then integrated into natural biogeochemical cycles. Petroleum hydrocarbon biodegradability in soil is influenced by complex arrays of factors, such as nutrients, oxygen, pH value, composition, concentration and bioavailability of the contaminants, and the soil's chemical and physical characteristics.

Petroleum Hydrocarbon Biodegradability in Soil – Implications for Bioremediation 45

necessary to add substances that stimulate microorganisms. The first one is more attractive because of its low cost, minimum of maintenance and minimal environmental impact. Still, this technology is applicable only in cases when the natural level of biodegradation is higher than the degree of pollution migration. Nevertheless, this technology is more often used as a supplement to the other technologies, or after finished engineered bioremediation in order to prevent migration of pollution from the treated area. Engineered bioremediation is faster than natural attenuation because it includes microbial degradation stimulation, by controlling the concentrations of pollution, oxygen, nutrients, moisture, pH, temperature, etc. (Rahman et al., 2003; Yerushalmi et al., 2003). Engineered bioremediation is applied when it is essential to carry out cleaning in a short time or when the pollution is very rapidly expanding. Its application reduces the costs due to the shorter treatment of land and lower number of sampling and analysis, and it is important for political and psychological needs when the community is exposed to pollution. Engineered bioremediation can be divided in two main groups (1) in situ and (2) ex situ bioremediation techniques, with the most applicable of these and their main characteristics given in Tables 1 and 2. In situ techniques are generally the most desirable options due to lower cost and fewer disturbances since they provide treatment in place, avoiding excavation and transport of contaminants (Vidali, 2001). In situ techniques are limited by the depth of the soil that can be effectively treated. In contrast, ex situ techniques involve the excavation or removal of contaminated soil from the

**3. Hydrocarbon biodegradation mechanisms and products** 

Biodegradation is the process by which microorganisms transform or mineralize organic contaminants, through metabolic or enzymatic processes, into less harmful, non-hazardous substances, which are then integrated into natural biogeochemical cycles. Organic material can be degraded by two biodegradation mechanisms: (1) aerobically, with oxygen, or (2)

Anaerobic processes are conducted by anaerobic microorganisms and this pathway of biodegradation is very slow. Originally thought to contribute marginally to overall biodegradation, anaerobic biodegradation mechanisms have been gaining more attention in recent years due to increased information regarding contaminant site conditions and rapid oxygen depletion (Burland & Edwards, 1999). Anaerobic biodegradation follows different biochemical pathways dependent on the electron acceptor utilized by the microorganism. Petroleum-based contaminants have been shown to degrade under various anaerobic conditions, including nitrate reduction, sulphate reduction, ferric iron reduction, manganese reduction and methanogenic conditions. The metabolic pathways behind anaerobic alkane biodegradation are not well understood. Most of the reports related to the anaerobic mineralization of aliphatic hydrocarbons are studies with pure cultures or enrichment cultures in laboratory scale experiments. Hence, the significance of these results in the environment e.g. in contaminated soils and sediments, is not yet known and evidence for the anaerobic degradation of alkanes in environmental samples has been reported in only a

ground.

anaerobically, without oxygen.

few cases (Salminen, 2004).

Bioremediation is considered a non-destructive, cost-effective, and sometimes logistically favourable cleanup technology, which attempts to accelerate the naturally occurring biodegradation of contaminants through the optimization of limiting conditions. In order to choose the appropriate bioremediation strategy it is extremely important to investigate and understand all factors which affect biodegradation efficiency. In order to better explain those factors, 4 examples of bioremediation studies (conducted 1 year, 5 years and 8 years after contamination) on soil which was directly contaminated with various petroleum products and their combustion products, are described along with their similarities and differences.

## **2. Bioremediation**

Bioremediation can be briefly defined as the use of biological agents, such as bacteria, fungi, or green plants (phytoremediation), to remove or neutralize hazardous substances in polluted soil or water. Bacteria and fungi generally work by breaking down contaminants such as petroleum into less harmful substances. Plants can be used to aerate polluted soil and stimulate microbial action. They can also absorb contaminants such as salts and metals into their tissues, which are then harvested and disposed of. Bioremediation is a complex process, with biological degradation taking place in the cells of microorganisms which absorb pollutants, where if they have specific enzymes, the degradation of pollutants and their corresponding metabolites will take place. Hydrocarbons from oil are used as a source of nutrients and energy for microorganism growth, and at the same time, microorganisms decompose them to naphthenic acids, alcohols, phenols, hydroperoxides, carbonyl compounds, esters, and eventually to carbon dioxide and water (Eglinnton, 1975; Marković et al., 1996).

Bioremediation is considered a non-destructive, cost- and treatment-effective and sometimes logistically favourable cleanup technology, which attempts to accelerate the naturally occurring biodegradation of contaminants through the optimization of limiting conditions. Bioremediation is an option that offers the possibility to destroy or render harmless various contaminants using natural biological activity. As such, it uses relatively low-cost, lowtechnology techniques, which generally have a high public acceptance and can often be carried out on site (Alexander, 1995). It will not always be suitable, however, as the range of contaminants on which it is effective is limited, the time scales involved are relatively long, and the residual contaminant levels achievable may not always be appropriate (Maletić et al., 2009; Rončević et al., 2005).

Bioremediation can be divided into two basic types: (1) natural attenuation, which can be applied when the natural conditions are suitable for the performance of bioremediation without human intervention, and (2) engineered bioremediation, which is used when is necessary to add substances that stimulate microorganisms. The first one is more attractive because of its low cost, minimum of maintenance and minimal environmental impact. Still, this technology is applicable only in cases when the natural level of biodegradation is higher than the degree of pollution migration. Nevertheless, this technology is more often used as a supplement to the other technologies, or after finished engineered bioremediation in order to prevent migration of pollution from the treated area. Engineered bioremediation is faster than natural attenuation because it includes microbial degradation stimulation, by controlling the concentrations of pollution, oxygen, nutrients, moisture, pH, temperature, etc. (Rahman et al., 2003; Yerushalmi et al., 2003). Engineered bioremediation is applied when it is essential to carry out cleaning in a short time or when the pollution is very rapidly expanding. Its application reduces the costs due to the shorter treatment of land and lower number of sampling and analysis, and it is important for political and psychological needs when the community is exposed to pollution. Engineered bioremediation can be divided in two main groups (1) in situ and (2) ex situ bioremediation techniques, with the most applicable of these and their main characteristics given in Tables 1 and 2. In situ techniques are generally the most desirable options due to lower cost and fewer disturbances since they provide treatment in place, avoiding excavation and transport of contaminants (Vidali, 2001). In situ techniques are limited by the depth of the soil that can be effectively treated. In contrast, ex situ techniques involve the excavation or removal of contaminated soil from the ground.

#### **3. Hydrocarbon biodegradation mechanisms and products**

44 Hydrocarbon

differences.

et al., 1996).

al., 2009; Rončević et al., 2005).

**2. Bioremediation** 

and physical characteristics.

then integrated into natural biogeochemical cycles. Petroleum hydrocarbon biodegradability in soil is influenced by complex arrays of factors, such as nutrients, oxygen, pH value, composition, concentration and bioavailability of the contaminants, and the soil's chemical

Bioremediation is considered a non-destructive, cost-effective, and sometimes logistically favourable cleanup technology, which attempts to accelerate the naturally occurring biodegradation of contaminants through the optimization of limiting conditions. In order to choose the appropriate bioremediation strategy it is extremely important to investigate and understand all factors which affect biodegradation efficiency. In order to better explain those factors, 4 examples of bioremediation studies (conducted 1 year, 5 years and 8 years after contamination) on soil which was directly contaminated with various petroleum products and their combustion products, are described along with their similarities and

Bioremediation can be briefly defined as the use of biological agents, such as bacteria, fungi, or green plants (phytoremediation), to remove or neutralize hazardous substances in polluted soil or water. Bacteria and fungi generally work by breaking down contaminants such as petroleum into less harmful substances. Plants can be used to aerate polluted soil and stimulate microbial action. They can also absorb contaminants such as salts and metals into their tissues, which are then harvested and disposed of. Bioremediation is a complex process, with biological degradation taking place in the cells of microorganisms which absorb pollutants, where if they have specific enzymes, the degradation of pollutants and their corresponding metabolites will take place. Hydrocarbons from oil are used as a source of nutrients and energy for microorganism growth, and at the same time, microorganisms decompose them to naphthenic acids, alcohols, phenols, hydroperoxides, carbonyl compounds, esters, and eventually to carbon dioxide and water (Eglinnton, 1975; Marković

Bioremediation is considered a non-destructive, cost- and treatment-effective and sometimes logistically favourable cleanup technology, which attempts to accelerate the naturally occurring biodegradation of contaminants through the optimization of limiting conditions. Bioremediation is an option that offers the possibility to destroy or render harmless various contaminants using natural biological activity. As such, it uses relatively low-cost, lowtechnology techniques, which generally have a high public acceptance and can often be carried out on site (Alexander, 1995). It will not always be suitable, however, as the range of contaminants on which it is effective is limited, the time scales involved are relatively long, and the residual contaminant levels achievable may not always be appropriate (Maletić et

Bioremediation can be divided into two basic types: (1) natural attenuation, which can be applied when the natural conditions are suitable for the performance of bioremediation without human intervention, and (2) engineered bioremediation, which is used when is Biodegradation is the process by which microorganisms transform or mineralize organic contaminants, through metabolic or enzymatic processes, into less harmful, non-hazardous substances, which are then integrated into natural biogeochemical cycles. Organic material can be degraded by two biodegradation mechanisms: (1) aerobically, with oxygen, or (2) anaerobically, without oxygen.

Anaerobic processes are conducted by anaerobic microorganisms and this pathway of biodegradation is very slow. Originally thought to contribute marginally to overall biodegradation, anaerobic biodegradation mechanisms have been gaining more attention in recent years due to increased information regarding contaminant site conditions and rapid oxygen depletion (Burland & Edwards, 1999). Anaerobic biodegradation follows different biochemical pathways dependent on the electron acceptor utilized by the microorganism. Petroleum-based contaminants have been shown to degrade under various anaerobic conditions, including nitrate reduction, sulphate reduction, ferric iron reduction, manganese reduction and methanogenic conditions. The metabolic pathways behind anaerobic alkane biodegradation are not well understood. Most of the reports related to the anaerobic mineralization of aliphatic hydrocarbons are studies with pure cultures or enrichment cultures in laboratory scale experiments. Hence, the significance of these results in the environment e.g. in contaminated soils and sediments, is not yet known and evidence for the anaerobic degradation of alkanes in environmental samples has been reported in only a few cases (Salminen, 2004).

The most rapid and complete degradation of the majority of organic pollutants is brought about under aerobic conditions. The initial intracellular attack of organic pollutants is an oxidative process, and the activation and the incorporation of oxygen is the enzymatic key reaction catalyzed by oxygenases and peroxidases. Degradation pathways convert organic pollutants step by step into intermediates of the central intermediary metabolism, for example, the tricarboxylic acid cycle. Biosynthesis of cell biomass occurs from the central precursor metabolites, for example, acetyl-CoA, succinate, pyruvate. Sugars required for various biosyntheses and growth are synthesized by gluconeogenesis. The degradation of petroleum hydrocarbons can be mediated by specific enzyme systems. Other mechanisms involved are (1) attachment of microbial cells to the substrates and (2) production of biosurfactants. The uptake mechanism linked to the attachment of cell to oil droplet is still unknown but the production of biosurfactants has been well studied (Nilanjana & Chandran, 2011).

Petroleum Hydrocarbon Biodegradability in Soil – Implications for Bioremediation 47

Volatile organic compounds, gasoline, heating and lubricating oil, diesel oil, PAH etc.

Petroleum products, nonhalogenated and halogenated VOC and SVOC,

PAH.

Petroleum products, nonhalogenated and halogenated VOC and SVOC, PAH, PCB and explosives, pesticides.

Petroleum products, nonhalogenated and halogenated VOC and SVOC, PAH, PCB and explosives.

Large amount of land

migration into the environment,

migration into the environment, difficult to expect a pollutants concentration reduction > 95%.

VOC must be pre-treated not efficient for the heavy components of petroleum, possibility of contamination

difficult to expect a reduction in the concentration of pollutants greater than 95%.

Not efficient for the heavy components of petroleum, possibility of contamination

VOC must be pre-treated possibility of contamination

Non-homogeneous and clayey soils can handling

free product removal is

expensive soil dewatering

disposal method is needed

problems,

necessary,

Bioremediation processes are time consuming and as a consequence, many studies have addressed the determination of bioremediation process kinetics. The kinetics for modelling the bioremediation of contaminated soils can be extremely complicated. This is largely due

after treatment,

for wastewater, extensive site and contaminant investigation.

migration into the environment, composting/compost processes to "lock up"' pollutants, the long-term stability of such "stabilized" matrices is uncertain.

required,

Technique / Definition Advantages Disadvantages Applicability

The most cost effective, takes less time and money to remediate, leads to complete destruction of pollutants,

suitable for treating large volumes of contaminated soil.

May be constructed to suit a variety of terrain

the treatment time - 6 months to 2 years, advantages over landfarming: takes up less space, possibility of aeration, VOC control is

conditions,

possible.

Cost effective, takes less time and money to remediate leads to complete destruction of pollutants,

suitable for treating large volumes of contaminated soil.

Control of temperature, moisture, pH, oxygen, nutrients, VOC emission,

addition of surfactants, addition of microorganisms,

monitoring of reaction

**Table 2.** The most applicable **ex situ** techniques and their main characteristics

conditions.

**4. Bioremediation process kinetics** 

Landfarming - spreading of contaminated soils in a thin layer on the ground surface of a treatment site and stimulating aerobic microbial activity within the soils through aeration and addition of nutrients, minerals, and water (Hejazi et al., 2003; Khan et al., 2004).

A hybrid of landfarming and composting engineered cells are constructed

as aerated composted piles (Jorgensen et al.,

Composting - combining contaminated soil with non-hazardous organic materials which support the development of a rich microbial population and elevated temperature for composting (Semple et

Biopile

2000).

al., 2001).

Bioslurry systems the soil is treated in a controlled bioreactor where the slurry is mixed to keep the solids suspended and microorganisms in contact with the contaminants (Nano &Rota, 2003).



Petroleum Hydrocarbon Biodegradability in Soil – Implications for Bioremediation 47


**Table 2.** The most applicable **ex situ** techniques and their main characteristics

## **4. Bioremediation process kinetics**

46 Hydrocarbon

Biosparging - Involves the injection of air under pressure below the water table to increase groundwater oxygen concentrations and enhance the rate of biological degradation of contaminants by naturally occurring bacteria (Baker & Moor, 2000;

Khan et al., 2004).

maximize in situ biodegradation

Phytoremediation -

2006).

Bioventing - injection of air into the contaminated media at a rate designed to

and minimize or eliminate the off-gassing of volatilized contaminants to the

atmosphere (Khan et al., 2004).

application of green plants to remove pollutants and other harmful components from the environment (Joner et al.,

The most rapid and complete degradation of the majority of organic pollutants is brought about under aerobic conditions. The initial intracellular attack of organic pollutants is an oxidative process, and the activation and the incorporation of oxygen is the enzymatic key reaction catalyzed by oxygenases and peroxidases. Degradation pathways convert organic pollutants step by step into intermediates of the central intermediary metabolism, for example, the tricarboxylic acid cycle. Biosynthesis of cell biomass occurs from the central precursor metabolites, for example, acetyl-CoA, succinate, pyruvate. Sugars required for various biosyntheses and growth are synthesized by gluconeogenesis. The degradation of petroleum hydrocarbons can be mediated by specific enzyme systems. Other mechanisms involved are (1) attachment of microbial cells to the substrates and (2) production of biosurfactants. The uptake mechanism linked to the attachment of cell to oil droplet is still unknown but the

production of biosurfactants has been well studied (Nilanjana & Chandran, 2011).

Equipment is readily available and easy to

little disturbance to site

treatment times from 6 months to 2 years, low injection rates reduce the need for vapour capture and treatment.

Equipment is readily available and easy to

short treatment times, from 6 months to 2 years. easy to combine with other

may not require off-gas

Cost-effective for large areas, no impact on the

formation of secondary waste is minimal, post-treatment soil can remain in the treated area and can be used in agriculture, uses solar energy no formation of toxic compounds.

install,

install,

technologies,

environment,

**Table 1.** The most applicable **in situ** techniques and their main characteristics

treatment.

operations,

Technique / Definition Advantages Disadvantages Applicability

Can only be used in areas where air sparging

High concentrations of contaminants may be toxic to organisms. cannot always reach low

Longer period required than one growing season, climate and hydro-logical conditions such may limit plant growth and the plant species that can be used, pollutants can enter the food chain, requires special disposal of

cleanup limits. is effective only in unsaturated soils; other methods are needed for the saturated zone.

plants.

Most types of petroleum contaminated sites, but it is least effective on

heavy petroleum because of the length of time required.

Mid-weight petroleum products like diesel.

Heavy metals, radionuclides, chlorinated solvents, petroleum hydrocarbons, insecticides, explosives and surfactants.

is suitable, complex chemical, physical and biological processes are not well

understood potential for the migration of contaminants.

> Bioremediation processes are time consuming and as a consequence, many studies have addressed the determination of bioremediation process kinetics. The kinetics for modelling the bioremediation of contaminated soils can be extremely complicated. This is largely due

to the fact that the primary function of microbial metabolism is not for the remediation of environmental contaminants. Instead the primary metabolic function, whether bacterial or fungal in nature, is to grow and sustain more of the microorganism. Because of the involvement of adverse factors and the complexity of the process, it is not possible to predict the duration of bioremediation. Therefore, the formulation of a kinetic model must start with the active biomass and factors, such as supplemental nutrients and oxygen source that are necessary for subsequent biomass growth (Maletić et al., 2009; Rončević et al., 2005).

Studies of the kinetics of the bioremediation process proceed in two directions: (1) the first is concerned with factors influencing the amount of transformed compounds with time, and (2) the other approach seeks the types of curves describing the transformation and determines which of them fits the degradation of the given compounds by the microbiologic culture in the laboratory microcosm and sometimes, in the field.

Determinations based on the literature data for values of the degradation degree are useful but less exact, because they do not take into account all the specific characteristics of the soil such as temperature, moisture, and—most often—the adaptation of bacteria to the specific contaminants. A literature survey has shown that studies of biodegradation kinetics in the natural environment are often empiric, reflecting only a basic level of knowledge about the microbiologic population and its activity in a given environment. One such example of the empirical approach is the simple model:

$$\frac{d\mathbf{C}}{dt} = k\mathbf{C}^n\tag{1}$$

Petroleum Hydrocarbon Biodegradability in Soil – Implications for Bioremediation 49

*kt C Ce* (or 0 ln ln *C C kt* ) (2)

et al., 2002; Hohener et al., 2003; Pala et al., 2006; Pollard et al., 2008). First order kinetics enables the prediction of hydrocarbon concentrations at any time from biodegradation halftimes. If the optimal conditions are established, remediation time depends on the biodegradation half-time, initial hydrocarbon concentration in the polluted soil, and the end point concentration which needs to be achieved. Many researchers assume first order kinetics because of the easier presentation and data analysis, simplicity of graphical presentation, and the easier prediction of concentration once half-life has been determined [26, 28]. This approach is least reliable at very high and very low levels of contaminants. Taking the same initial values of concentration, different kinetic models will give significantly different final amounts of unreacted compound (Maletić et al., 2009; Rončević, 2002; Rončević et al., 2005):

where C - concentration of hydrocarbons (g kg-1), t - time of removal (day), C0 - initial concentration of hydrocarbons (g kg-1), and k - rate constant of the change in the

In the simple model, depending on the nature of the substrate and experimental conditions, various investigators obtain different values for the rate constant of substrate degradation: for n-alkanes, 0.14 to 0.61 day-1; for crude oil, 0.0051 to 0.0074 day-1; and for PAHs, 0.01 to 0.14 day-1 (Roncević et al., 2005). Reported rates for the degradation of hydrocarbon compounds under field or field-simulated conditions differ by up to two orders of magnitude. The selection of appropriate kinetics and rate constants is essential for accurate predictions or reconstructions

A more reliable prediction of pollution biodegradation can be obtained from more complex models such as the BIOPLUME II model (BIOPLUME is a two-dimensional computer model that simulates the transport of dissolved hydrocarbons under the influence of oxygen-limited biodegradation). Additionally, in recent years, the state of the art in modelling technology allows for even more reliable prediction using the 3D software MODFLOW, which is available in several versions: MODFLOW, MODPATH, MT3D, RT3D and MODFLOW-SURFACT).

For the ex-situ treatment of soil, remediation time generally does not depend on the transport of nutrients and oxygen and can be roughly determined from the degree of degradation, determined in laboratory tests of samples taken from the field. The following factors often interfere with a simple extrapolation of the kinetics described above in natural conditions:

1. Different barriers may limit or prevent contact between microbial cells and their organic substrates. Many organic molecules sorb to clay or soil humus or sediment, and the kinetics of the decomposition of sorbed substrate can be completely different from that

2. The presence of other organic molecules, which can be metabolized by biodegrading species can reduce or increase the consumption of the examined compounds. 3. Application of inorganic nutrients, oxygen, or growth factors, can affect the speed of transformation and then the process will be governed by diffusion of nutrients or the

speed of their formation or regeneration of the other residents of the community.

0

of the concentrations of hydrocarbons with time in soil after a spill.

of the same compound free in solution.

hydrocarbon content of the soil (day-1).

where C is the concentration of the substrate, t is time, and k is the degradation rate constant of the compound and n is a fitting parameter (most often taken to be unity) (Wethasinghe et al., 2006). Using this model, one can fit the curve of substrate removal by varying n and k until a satisfactory fit is obtained. It is evident from this equation that the rate is proportional to the exponent of substrate concentration. Researchers involved in kinetic studies do not always report whether the model they used was based on theory or experience and whether the constants in the equation have a physical meaning or if they just serve as fitting parameters (Maletić et al., 2009; Rončević et al., 2005).

With the complex array of factors that influence the biodegradation of hydrocarbons noted previously, it is not realistic to expect a simple kinetic model to provide precise and accurate descriptions of concentrations during different seasons and in different environments. The results of short-term degradation experiments are sometimes presented with the implicit assumption of zero-order kinetics (i.e., degradation in mass per unit time or in turnover time). However, short-term degradation experiments may not be adequate to discern the appropriate kinetics. In experiments with a number of samples taken during a length of time sufficient for considerable biodegradation to take place, the concentration of hydrocarbons with time is better described by first-order kinetics, eq. 2 (Collina et al., 2005; Grossi et al., 2002; Hohener et al., 2003; Pala et al., 2006; Rončević et al., 2005).

First order kinetics, such as the well known Michaelis-Menton kinetic model, is the most often used equation for the representation of degradation kinetics (Collina et al., 2005; Grossi et al., 2002; Hohener et al., 2003; Pala et al., 2006; Pollard et al., 2008). First order kinetics enables the prediction of hydrocarbon concentrations at any time from biodegradation halftimes. If the optimal conditions are established, remediation time depends on the biodegradation half-time, initial hydrocarbon concentration in the polluted soil, and the end point concentration which needs to be achieved. Many researchers assume first order kinetics because of the easier presentation and data analysis, simplicity of graphical presentation, and the easier prediction of concentration once half-life has been determined [26, 28]. This approach is least reliable at very high and very low levels of contaminants. Taking the same initial values of concentration, different kinetic models will give significantly different final amounts of unreacted compound (Maletić et al., 2009; Rončević, 2002; Rončević et al., 2005):

48 Hydrocarbon

to the fact that the primary function of microbial metabolism is not for the remediation of environmental contaminants. Instead the primary metabolic function, whether bacterial or fungal in nature, is to grow and sustain more of the microorganism. Because of the involvement of adverse factors and the complexity of the process, it is not possible to predict the duration of bioremediation. Therefore, the formulation of a kinetic model must start with the active biomass and factors, such as supplemental nutrients and oxygen source that are necessary for subsequent biomass growth (Maletić et al., 2009; Rončević et al., 2005).

Studies of the kinetics of the bioremediation process proceed in two directions: (1) the first is concerned with factors influencing the amount of transformed compounds with time, and (2) the other approach seeks the types of curves describing the transformation and determines which of them fits the degradation of the given compounds by the microbiologic

Determinations based on the literature data for values of the degradation degree are useful but less exact, because they do not take into account all the specific characteristics of the soil such as temperature, moisture, and—most often—the adaptation of bacteria to the specific contaminants. A literature survey has shown that studies of biodegradation kinetics in the natural environment are often empiric, reflecting only a basic level of knowledge about the microbiologic population and its activity in a given environment. One such example of the

*dC <sup>n</sup> kC*

where C is the concentration of the substrate, t is time, and k is the degradation rate constant of the compound and n is a fitting parameter (most often taken to be unity) (Wethasinghe et al., 2006). Using this model, one can fit the curve of substrate removal by varying n and k until a satisfactory fit is obtained. It is evident from this equation that the rate is proportional to the exponent of substrate concentration. Researchers involved in kinetic studies do not always report whether the model they used was based on theory or experience and whether the constants in the equation have a physical meaning or if they just

With the complex array of factors that influence the biodegradation of hydrocarbons noted previously, it is not realistic to expect a simple kinetic model to provide precise and accurate descriptions of concentrations during different seasons and in different environments. The results of short-term degradation experiments are sometimes presented with the implicit assumption of zero-order kinetics (i.e., degradation in mass per unit time or in turnover time). However, short-term degradation experiments may not be adequate to discern the appropriate kinetics. In experiments with a number of samples taken during a length of time sufficient for considerable biodegradation to take place, the concentration of hydrocarbons with time is better described by first-order kinetics, eq. 2 (Collina et al., 2005; Grossi et al.,

First order kinetics, such as the well known Michaelis-Menton kinetic model, is the most often used equation for the representation of degradation kinetics (Collina et al., 2005; Grossi

*dt* (1)

culture in the laboratory microcosm and sometimes, in the field.

serve as fitting parameters (Maletić et al., 2009; Rončević et al., 2005).

2002; Hohener et al., 2003; Pala et al., 2006; Rončević et al., 2005).

empirical approach is the simple model:

$$\mathbf{C} = \mathbf{C}\_0 e^{-kt} \text{ (or } \ln \mathbf{C} = \ln \mathbf{C}\_0 - kt \text{ )}\tag{2}$$

where C - concentration of hydrocarbons (g kg-1), t - time of removal (day), C0 - initial concentration of hydrocarbons (g kg-1), and k - rate constant of the change in the hydrocarbon content of the soil (day-1).

In the simple model, depending on the nature of the substrate and experimental conditions, various investigators obtain different values for the rate constant of substrate degradation: for n-alkanes, 0.14 to 0.61 day-1; for crude oil, 0.0051 to 0.0074 day-1; and for PAHs, 0.01 to 0.14 day-1 (Roncević et al., 2005). Reported rates for the degradation of hydrocarbon compounds under field or field-simulated conditions differ by up to two orders of magnitude. The selection of appropriate kinetics and rate constants is essential for accurate predictions or reconstructions of the concentrations of hydrocarbons with time in soil after a spill.

A more reliable prediction of pollution biodegradation can be obtained from more complex models such as the BIOPLUME II model (BIOPLUME is a two-dimensional computer model that simulates the transport of dissolved hydrocarbons under the influence of oxygen-limited biodegradation). Additionally, in recent years, the state of the art in modelling technology allows for even more reliable prediction using the 3D software MODFLOW, which is available in several versions: MODFLOW, MODPATH, MT3D, RT3D and MODFLOW-SURFACT).

For the ex-situ treatment of soil, remediation time generally does not depend on the transport of nutrients and oxygen and can be roughly determined from the degree of degradation, determined in laboratory tests of samples taken from the field. The following factors often interfere with a simple extrapolation of the kinetics described above in natural conditions:



Petroleum Hydrocarbon Biodegradability in Soil – Implications for Bioremediation 51

Experiment duration was 325 days.

day−1.

Bioremediation study 2 – Simulation of in situ bioremediation in a laboratory bioreactor Cylinder reactor, length 3.2 m and 0.8 m in diameter, with 4 piezometers placed in the soil.

the beginning of the reactor. 306 days, changeable water flow 1.8-22 x10-7 m/s.

microbial suspension.

 A layer of sand 10-15 cm thick was first placed in the reactor, then a layer of soil polluted with oil derivatives (thickness of 45-50 cm, 1150 kg of soil). 1 m3 of groundwater from the site was added.

 I - in-situ biostimulation feeding with aerated water - 2.7 dm3 water was discharged into the aerator, where it was saturated with the maximum amount of oxygen, and poured over the surface of the soil at

 II - in-situ biostimulation with magnesium peroxide, the fourth piezometer was filled with magnesium peroxide, whose decomposition provides oxygen in the soil layer. 147 days, water flow 22 x10-7 m/s III - in-situ biostimulation with ex situ biologically treated groundwater - water from the reactor was drained to a system consisting of three separators of the oil-free phase, a bioreactor, settler and sludge conditioner, and then recirculated over the surface of the soil at the beginning of the reactor. 232 days,

 Two samples of 170 and 180 kg were introduced into separate reactors which were filled to a height of 20 cm with water sampled from the piezometers from the refinery area.

 Each reactor was reinoculated daily by replacing 250 ml of the water phase with 250 ml of a suspension of adapted bacteria. One reactor (a) had continuous circulation of the water phase, with the aid of an air lift, at a flow rate of approximately 7 l

 In the second (b), circulation of the water phase was carried out over a short period once a day to give a flow rate of 0.5 l day−1. After percolating through the soil, the water phase was passed through a separator in which water insoluble components (free crude oil plus oil derivatives) were separated out by gravity. The separated-oily layer was removed periodically and fed into a third bioreactor that was used to prepare the adapted

Bioremediation study 1 – Laboratory trial bioremediation

3 versions of the technical bioremediation were performed:

changeable water flows 2.5-16 x10-7 m/s.

**Figure 1.** Experimental conditions for Bioremediation studies 1 and 2


#### **4.1. Bioremediation study – Our experiences**

In order to close this issue for readers, experience from four different bioremediation treatments of petroleum contaminated soil are given as examples (Fig. 1, Fig. 2). Thus, as a consequence of the accidental oil spill in the Novi Sad Oil Refinery (Serbia) in 1999, soil was directly contaminated with various petroleum products (gasoline, crude oil, kerosene, diesel fuel, black oil, etc.) and products of their combustion from frequent fires. Bioremediation studies on this soil were conducted 1 year after contamination (Rončević, 2002; Rončević et al., 2005), after 5 years (Rončević, 2007) and after 8 years (Maletić et al., 2009; Maletić, 2010; Maletić at al., 2011), and the bioremediation kinetics which were determined are compared here and discussed.

The obtained data from these four studies show changes and differences in the bioremediation kinetic rate, depending on the applied technology and stage of weathering (Fig 3.).

In study 1, bioremediation was carried out on a relatively freshly petroleum contaminated soil (one year after contamination), with a start concentration three times greater than in the other case studies, and the % of removed hydrocarbon is the highest. A slight difference was noticed between the two approaches applied (reactor with continuous and discontinuous flow). Namely, in the reactor with discontinuous flow, the hydrocarbon biodegradation rate in the aerobic part of the reactor was lower, indicating that the aerobic bioremediation conditions are favourable for this type of oil contaminated soil. Generally, satisfactory hydrocarbon degradation and removal rates were established by this technology.

As explained above, in study 2, the initial hydrocarbons concentration is three times lower, due to the different environmental conditions to which this soil was exposed during 5 years of weathering. Three varieties of in situ bioremediation technology were applied (Fig. 1). The first two used in-situ biostimulation feeding with aerated water and magnesium peroxide, and did not provide satisfactory results. The biodegradation kinetic rate constant could not be calculated, since no removal of hydrocarbons was observed during the bioremediation.

With the third variation, which used in situ biostimulation with ex situ biologically treated groundwater, the situation was changed drastically. Hydrocarbon content decreased rapidly, by about 60% in 232 days. Even so, the biodegradation kinetic rate constant is twice as low as the rate constant in study 1. This is probably because the degradation of the easily removable hydrocarbon fraction from the soil already occurred during the weathering process. Thus, only the heavier and less degradable fractions remained in the soil.

#### Bioremediation study 1 – Laboratory trial bioremediation

50 Hydrocarbon

4. Many species can metabolize the same organic compounds simultaneously.

growth, population size or activity responsible for biodegradation.

between the occurrence of compounds and their total destruction.

of sorbed or microcolonies is still unresolved.

**4.1. Bioremediation study – Our experiences** 

5. Protozoa or possible species that parasitize on the biodegrading population can manage

6. Many synthetic chemicals have insufficient solubility in water, and the kinetics of their transformation can be completely different from compounds in the aqueous phase. 7. Cells of the active population may be sorbed or can develop microcolonies, and kinetics

8. Many organic compounds disappear only after a period of acclimatization, and there is no method that can predict the length of this period or the expected percentage of time

In order to close this issue for readers, experience from four different bioremediation treatments of petroleum contaminated soil are given as examples (Fig. 1, Fig. 2). Thus, as a consequence of the accidental oil spill in the Novi Sad Oil Refinery (Serbia) in 1999, soil was directly contaminated with various petroleum products (gasoline, crude oil, kerosene, diesel fuel, black oil, etc.) and products of their combustion from frequent fires. Bioremediation studies on this soil were conducted 1 year after contamination (Rončević, 2002; Rončević et al., 2005), after 5 years (Rončević, 2007) and after 8 years (Maletić et al., 2009; Maletić, 2010; Maletić at al., 2011), and the bioremediation kinetics which were determined are compared here and discussed.

The obtained data from these four studies show changes and differences in the bioremediation

In study 1, bioremediation was carried out on a relatively freshly petroleum contaminated soil (one year after contamination), with a start concentration three times greater than in the other case studies, and the % of removed hydrocarbon is the highest. A slight difference was noticed between the two approaches applied (reactor with continuous and discontinuous flow). Namely, in the reactor with discontinuous flow, the hydrocarbon biodegradation rate in the aerobic part of the reactor was lower, indicating that the aerobic bioremediation conditions are favourable for this type of oil contaminated soil. Generally, satisfactory

As explained above, in study 2, the initial hydrocarbons concentration is three times lower, due to the different environmental conditions to which this soil was exposed during 5 years of weathering. Three varieties of in situ bioremediation technology were applied (Fig. 1). The first two used in-situ biostimulation feeding with aerated water and magnesium peroxide, and did not provide satisfactory results. The biodegradation kinetic rate constant could not be calculated, since no removal of hydrocarbons was observed during the bioremediation.

With the third variation, which used in situ biostimulation with ex situ biologically treated groundwater, the situation was changed drastically. Hydrocarbon content decreased rapidly, by about 60% in 232 days. Even so, the biodegradation kinetic rate constant is twice as low as the rate constant in study 1. This is probably because the degradation of the easily removable hydrocarbon fraction from the soil already occurred during the weathering

kinetic rate, depending on the applied technology and stage of weathering (Fig 3.).

hydrocarbon degradation and removal rates were established by this technology.

process. Thus, only the heavier and less degradable fractions remained in the soil.


Bioremediation study 2 – Simulation of in situ bioremediation in a laboratory bioreactor

	- I in-situ biostimulation feeding with aerated water 2.7 dm3 water was discharged into the aerator, where it was saturated with the maximum amount of oxygen, and poured over the surface of the soil at the beginning of the reactor. 306 days, changeable water flow 1.8-22 x10-7 m/s.
	- II in-situ biostimulation with magnesium peroxide, the fourth piezometer was filled with magnesium peroxide, whose decomposition provides oxygen in the soil layer. 147 days, water flow 22 x10-7 m/s
	- III in-situ biostimulation with ex situ biologically treated groundwater water from the reactor was drained to a system consisting of three separators of the oil-free phase, a bioreactor, settler and sludge conditioner, and then recirculated over the surface of the soil at the beginning of the reactor. 232 days, changeable water flows 2.5-16 x10-7 m/s.

**Figure 1.** Experimental conditions for Bioremediation studies 1 and 2


Petroleum Hydrocarbon Biodegradability in Soil – Implications for Bioremediation 53

Case study 1a – 0-10 cm

Case study 1a – 40-50 cm

Case study 1b – 0-10 cm

Case study 1b – 20-30 cm

Case study 1b – 40-50 cm

Case study 2 - I

Case study 2 - II

Case study 2 - III

Case study 3 – 20 cm

Case study 3 – 40 cm

Case study 3 – 60 cm

Case study 3 – centre

Case study 3 – average

Case study 4

Co – start concentration k – rate constant r – correlation coefficient

Study lnCo k (day-1) <sup>r</sup>

4.5 0.0052 0.90

4.5 0.0046 0.96

4.6 0.0057 0.98

4.6 0.0046 0.95

4.5 0.0045 0.92



3.3 0.0083 0.97

3.2 0.00052 0.82

3.2 0.00080 0.85

3.3 0.00093 0.94

3.3 0.00078 0.90

3.2 0.00077 0.96

3.1 0.00065 0.79

Total petroleum hydrocarbon Kinetic parameters

**Figure 3.** Experimental results from the bioremediation studies 1-4



**Figure 2.** Experimental conditions for Bioremediation studies 3 and 4

straw.

bioreactor.

laboratory bioreactor.

water for weathering the landfarm.

Bioremediation study 3 – Biopile bioremediation

 The contaminated soil (2.7 m3) was placed in a 2.2 m prismatic hole dug to a depth of 0.4 m, and covered with resistant polypropylene foil to prevent contamination spreading

 The layer of contaminated soil above the drainage system was in the form of a 1 m tall truncated pyramid composted with straw, and had a

 The contaminated soil (2.7 m3) was placed in a 3x3 m wide and 0.4 m deep prismatic hole, and covered with resistant polypropylene foil to prevent contamination spreading from the landfarm. Experiment

 To facilitate oxygen and water transport through the soil, the soil was composted with straw.

duration 710 days.

from the biopile.

total volume of 2.7 m3.

To facilitate oxygen and water transport through the soil, the contaminated soil was composted with

 The biopile was watered twice a week, and moisture was maintained at approximately 50-80% water holding capacity during the experiment. Leaching water from the biopile was collected in a separate

The landfarm was turned twice a month and watered twice a week; moisture was maintained at

 In addition to the stimulation of native microflora by soil aeration and irrigation, bioaugmentation was also carried out with microorganisms separated from the contaminated soil and cultivated in a

Approximately 25 dm3 of the inoculated water from the bioreactor was used together with leaching

reservoir and used for watering the biopile. Experiment duration 710 days.

approximately 50-80% water holding capacity during the experiment.

**Figure 2.** Experimental conditions for Bioremediation studies 3 and 4

Bioremediation study 4 – Landfarming bioremediation

 At three different heights on the pyramid structure, perforated PVC aeration tubes were placed. To accelerate microbiological activity air was additionally piped through the biopile once a week. As well as stimulation of native microflora by soil aeration and irrigation, bioaugmentation was also carried out with microorganisms separated from the contaminated soil and cultivated in a laboratory

**Figure 3.** Experimental results from the bioremediation studies 1-4

Studies 3 and 4 had similar hydrocarbon concentrations at the beginning of the experiment as study 2; even so, the biodegradation constant rate for both case studies is one order of magnitude lower than in study 2. The reason for this could be hydrocarbon complexation with the soil organic material and also its sorption and sequestration in the soil nanopores with further weathering of the oil contaminated soil (8 years). In this manner the hydrocarbons become recalcitrant and resistant to biodegradation. In study 3 (biopile) the hydrocarbon biodegradation removals were also monitored at different heights in the biopile.

Petroleum Hydrocarbon Biodegradability in Soil – Implications for Bioremediation 55

Petroleum hydrocarbons contain a complex mixture of compounds; all the components of petroleum do not degrade at the same rate. The rate by which microorganisms degrade hydrocarbons depends upon their chemical structure and concentration. Petroleum hydrocarbons can be categorized into four fractions: saturates, aromatics, resins and asphaltene. Of the various petroleum fractions, n-alkanes of intermediate length (C10-C25) are the preferred substrates for microorganisms and tend to be the most readily degradable, whereas shorter chain compounds are rather more toxic. Longer chain alkanes (C25-C40) are hydrophobic solids and consequently are difficult to degrade due to their poor water solubility and bioavailability, and branched chain alkanes and cycloalkanes are also degraded more slowly than the corresponding normal alkanes. Highly condensed aromatic and cycloparaffinic structures, tars, bitumen and asphaltic materials, have the highest boiling points and exhibit the greatest resistance to biodegradation. It has been suggested that the residual material from oil degradation is analogous to, and can even be regarded as, humic material (Balba et al., 1998; Loeher et al., 2001; Ivančev-Tumbas et al., 2004;

Even if the optimal conditions for hydrocarbon biodegradation are provided at the field, it has been shown that a residual fraction of hydrocarbon remains undegraded. Namely, after its arrival in the soil, an organic contaminant may be lost by biodegradation, leaching or volatilization, or it may accumulate within the soil biota or be sequestered and complex within the soil's mineral and organic matter fractions. The rate at which hydrocarbondegrading microorganisms can convert chemicals depends on the rate of transfer to the cell and the rate of uptake and metabolism by the microorganisms. It is controlled by a number of physical-chemical processes such as sorption/desorption, diffusion, and dissolution. (Brassington et al., 2007; Cuypers et al., 2002; Maletić et al., 2011; Semple et al., 2003). The mass transfer of a contaminant determines microbial bioavailability. The term "bioavailability" refers to the fraction of chemicals in soil that can be utilized or transformed by living organisms. The bioavailability of a compound is defined as the ratio of mass transfer and soil biota intrinsic activities. Most soil contaminants show biphasic behaviour, whereby in the initial phase of hydrocarbon biodegradation, the rate of removal is high and removal is primarily limited by microbial degradation kinetics. In the second phase, the rate of hydrocarbon removal is low and removal is generally limited by slow desorption. Altogether, the poorly bioavailable fraction of hydrocarbon contamination is formed by hydrocarbons which desorb slowly in the second phase of bioremediation (Loeher et al., 2001). The biodegradation of an oil-contaminated soil can also be seriously affected by the contamination time, due to weathering processes, which decrease the bioavailability of pollutants to microorganisms. Weathering refers to the results of biological, chemical and physical processes that can affect the type of hydrocarbons that remain in a soil (Maletić et al., 2011; Loeher et al., 2001; Semple et al.,2005). Those processes enhance the sorption of hydrophobic organic contaminants to the soil matrix, decreasing

**5.2. Contaminant characteristics** 

Brassington et al., 2007; Stroud et al., 2007).

**5.3. Bioavailability** 

Similarly to study 1, the lowest biodegradation rate constant was obtained for the lowest layer of the biopile, where the oxygen concentration is limited and anaerobic conditions developed. This confirms the facts from study 1 that aerobic degradation of hydrocarbons is the favourable degradation pathway. It is worth mentioning that in general, greater rate constants were obtained in the biopile than in the landfarming, indicating that the biopile is a better technology choice for bioremediation of this type of soil contamination.

## **5. Factors affecting oil hydrocarbon biodegradation processes**

Successful implementation of bioremediation technologies on contaminated areas depends on the characteristics of the contaminated site and a complex system of many factors that affect the petroleum hydrocarbons biodegradation processes (Jain et al., 2011). The main factors which limit the overall biodegradation rate can be grouped as: soil characteristics, contaminant characteristics, bioavailability, microorganisms number and catabolism evolution (Alexander, 1995). In order to adopt and implement some bioremediation strategy it is extremely important to consider and understand those limiting factors.

#### **5.1. Soil characteristics**

Soil characteristics are especially important for successful hydrocarbon biodegradation, some of the main limiting factors are: soil texture, permeability, pH, water holding capacity, soil temperature, nutrient content and oxygen content. Soil texture affects permeability, water content and the bulk density of soil. Soil with low permeability (such as clays) hinders transportation and the distribution of water, nutrients and oxygen. To enable the bioremediation of such soil, it should be mixed with amendments or bulking materials (straw, sawdust etc.), as the bioremediation processes rely on microbial activity, and microorganisms require oxygen inorganic nutrients, water and optimal temperature and pH to support cell growth and sustain biodegradation (Alexander, 1995; Jain et al., 2011). The optimal conditions for microbial growth and hydrocarbon biodegradation are given in table 3.


**Table 3.** Optimal conditions for microbial growth and hydrocarbon biodegradation

#### **5.2. Contaminant characteristics**

54 Hydrocarbon

**5.1. Soil characteristics** 

Studies 3 and 4 had similar hydrocarbon concentrations at the beginning of the experiment as study 2; even so, the biodegradation constant rate for both case studies is one order of magnitude lower than in study 2. The reason for this could be hydrocarbon complexation with the soil organic material and also its sorption and sequestration in the soil nanopores with further weathering of the oil contaminated soil (8 years). In this manner the hydrocarbons become recalcitrant and resistant to biodegradation. In study 3 (biopile) the hydrocarbon biodegradation removals were also monitored at different heights in the biopile. Similarly to study 1, the lowest biodegradation rate constant was obtained for the lowest layer of the biopile, where the oxygen concentration is limited and anaerobic conditions developed. This confirms the facts from study 1 that aerobic degradation of hydrocarbons is the favourable degradation pathway. It is worth mentioning that in general, greater rate constants were obtained in the biopile than in the landfarming, indicating that the biopile is a better

technology choice for bioremediation of this type of soil contamination.

**5. Factors affecting oil hydrocarbon biodegradation processes** 

it is extremely important to consider and understand those limiting factors.

for microbial growth and hydrocarbon biodegradation are given in table 3.

Water holding capacity 25 -28 40-80 pH 5.5-8.8 6.5-8.0 Temperature (oC) 10-45 20-30 Oxygen (air-filled pore space) 10% 10-40% C:N:P 100:10:1(0.5) 100:10:1(0.5)

Heavy metals <2000 ppm <700 ppm

**Table 3.** Optimal conditions for microbial growth and hydrocarbon biodegradation

Successful implementation of bioremediation technologies on contaminated areas depends on the characteristics of the contaminated site and a complex system of many factors that affect the petroleum hydrocarbons biodegradation processes (Jain et al., 2011). The main factors which limit the overall biodegradation rate can be grouped as: soil characteristics, contaminant characteristics, bioavailability, microorganisms number and catabolism evolution (Alexander, 1995). In order to adopt and implement some bioremediation strategy

Soil characteristics are especially important for successful hydrocarbon biodegradation, some of the main limiting factors are: soil texture, permeability, pH, water holding capacity, soil temperature, nutrient content and oxygen content. Soil texture affects permeability, water content and the bulk density of soil. Soil with low permeability (such as clays) hinders transportation and the distribution of water, nutrients and oxygen. To enable the bioremediation of such soil, it should be mixed with amendments or bulking materials (straw, sawdust etc.), as the bioremediation processes rely on microbial activity, and microorganisms require oxygen inorganic nutrients, water and optimal temperature and pH to support cell growth and sustain biodegradation (Alexander, 1995; Jain et al., 2011). The optimal conditions

Parameter Microbial growth HC biodegradation

Contaminants Not too toxic HC 5–10% of dry weight of soil

Petroleum hydrocarbons contain a complex mixture of compounds; all the components of petroleum do not degrade at the same rate. The rate by which microorganisms degrade hydrocarbons depends upon their chemical structure and concentration. Petroleum hydrocarbons can be categorized into four fractions: saturates, aromatics, resins and asphaltene. Of the various petroleum fractions, n-alkanes of intermediate length (C10-C25) are the preferred substrates for microorganisms and tend to be the most readily degradable, whereas shorter chain compounds are rather more toxic. Longer chain alkanes (C25-C40) are hydrophobic solids and consequently are difficult to degrade due to their poor water solubility and bioavailability, and branched chain alkanes and cycloalkanes are also degraded more slowly than the corresponding normal alkanes. Highly condensed aromatic and cycloparaffinic structures, tars, bitumen and asphaltic materials, have the highest boiling points and exhibit the greatest resistance to biodegradation. It has been suggested that the residual material from oil degradation is analogous to, and can even be regarded as, humic material (Balba et al., 1998; Loeher et al., 2001; Ivančev-Tumbas et al., 2004; Brassington et al., 2007; Stroud et al., 2007).

#### **5.3. Bioavailability**

Even if the optimal conditions for hydrocarbon biodegradation are provided at the field, it has been shown that a residual fraction of hydrocarbon remains undegraded. Namely, after its arrival in the soil, an organic contaminant may be lost by biodegradation, leaching or volatilization, or it may accumulate within the soil biota or be sequestered and complex within the soil's mineral and organic matter fractions. The rate at which hydrocarbondegrading microorganisms can convert chemicals depends on the rate of transfer to the cell and the rate of uptake and metabolism by the microorganisms. It is controlled by a number of physical-chemical processes such as sorption/desorption, diffusion, and dissolution. (Brassington et al., 2007; Cuypers et al., 2002; Maletić et al., 2011; Semple et al., 2003). The mass transfer of a contaminant determines microbial bioavailability. The term "bioavailability" refers to the fraction of chemicals in soil that can be utilized or transformed by living organisms. The bioavailability of a compound is defined as the ratio of mass transfer and soil biota intrinsic activities. Most soil contaminants show biphasic behaviour, whereby in the initial phase of hydrocarbon biodegradation, the rate of removal is high and removal is primarily limited by microbial degradation kinetics. In the second phase, the rate of hydrocarbon removal is low and removal is generally limited by slow desorption. Altogether, the poorly bioavailable fraction of hydrocarbon contamination is formed by hydrocarbons which desorb slowly in the second phase of bioremediation (Loeher et al., 2001). The biodegradation of an oil-contaminated soil can also be seriously affected by the contamination time, due to weathering processes, which decrease the bioavailability of pollutants to microorganisms. Weathering refers to the results of biological, chemical and physical processes that can affect the type of hydrocarbons that remain in a soil (Maletić et al., 2011; Loeher et al., 2001; Semple et al.,2005). Those processes enhance the sorption of hydrophobic organic contaminants to the soil matrix, decreasing

the rate and extent of biodegradation. Moreover, a weathered oil-contaminated soil normally contains a recalcitrant fraction of compounds composed basically of high molecular weight hydrocarbons, which cannot be degraded by indigenous microorganisms (Balba et al., 1998; Maletić et al., 2011; Loeher et al., 2001). In contrast, a recently oilcontaminated soil contains a higher amount of saturated and aliphatic compounds, which are the most susceptible to microbial degradation. However, the pollutant compounds in a recently contaminated soil are potentially more toxic to the native microorganisms, leading to a longer adaptation time (lag phase) before degradation of the pollutant and even to an inhibition of the biodegradation process (Margesin et al., 2000; Loeher et al., 2001; Petrović et al., 2008).

Petroleum Hydrocarbon Biodegradability in Soil – Implications for Bioremediation 57

With the aim of better understanding the factors which affect hydrocarbon biodegradation, results from the bioremediation studies described above are also given here, along with a comparison and discussion of changes in hydrocarbon composition and bioavailability over the years (Ivančev-Tumbas et al., 2004; Maletić, 2010; Maletić at al., 2011; Rončević, 2002; Rončević, 2007). The compounds detected by GC-MS analysis of extracts of the various soil samples taken at the start and end of bioremediation studies are given in Fig. 1, with only the main compounds from the hit lists of the probability-based matching (PBM ≥ 60%) search given.

The data reflect the fact that the soil used in this investigation was sampled from the dumping area of a refinery where the initial pollutants were of very diverse composition, i.e. a mixture of crude oil, mazut, diesel, middle distillates, heavy distillates, kerosene, etc. The untreated soil samples contained a large variety of straight-chain hydrocarbons and their methyl derivatives (those with both even and odd numbers of C atoms), many of which persisted during the treatment. However, if we compare the untreated soil samples at the start of study 1 (1 year after contamination), and study 4 (8 years after contamination) the difference is significant. Namely, in study 1, the soil mostly contains n-alkanes and derivates of aromatic hydrocarbons, and few compounds of iso-alkanes, whereas the soil in study 4 contains mostly n-alkanes and iso-alkanes, with only a few aromatics derivatives detected, with PBM<50%, and few cycloalkanes. The fact that mainly substituted polycyclic aromatic hydrocarbons were not detected in the weathered soil samples (study 4), shows their lower persistence than alkanes. Additionally, the greater number of iso-alkanes in weathered soil indicates their persistence. The cycloalkanes detected

represent one of the main hydrocarbon residual fractions in weathered contaminated soil.

In both studies, at the end of the experiment, the number of detected compounds is significantly reduced. In study 1, the aromatic hydrocarbons were almost completely removed in both reactors, while the number of n-alkanes detected was reduced, but they are still present in significant numbers in the soil at the end. This is a because the aromatics have lower persistence than n-alkanes, but is also due to the higher n-alkanes concentration at the beginning. It is worth mentioning that in the reactor with continuous flow (aerobic), the number of removed n-

**5.5. Bioremediation study – Our experiences** 

**Figure 4.** GC-MS SCAN qualitative analysis of soil samples

As was mentioned above, sequestration and weathering of organic contaminants in the soil reduces the bioavailability of organic compounds and results in non-degraded residues in the soil. Contaminants that have been weathered and sequestrated in soil are not available for biodegradation in soil, even though freshly added compounds are still biodegradable (Alexander, 1995). Sorption is a major factor preventing the complete bioremediation of hydrocarbons in soil. Slow sorption leads to the hydrocarbon fraction becoming resistant to desorption and increases its persistence within the soil organic matrix. The following hypotheses have been proposed as a explanation for weathering: (1) weathering results in a slow diffusion of the hydrocarbon fraction in the solid fraction of the organic matter in the soil; (2) the contaminant slowly diffuses through the soil and becomes sorbed and trapped in the soil nano-and micropores (Semple et al., 2003; Trinidade et al., 2005).

#### **5.4. Microorganisms number and catabolism evolution**

The ability of the soil's microbial community to degrade hydrocarbons depends on the microbes number and its catabolic activity. Microorganisms can be isolated from almost all environmental conditions. Soil microflora contain numbers of different microorganisms including bacteria, algae, fungi, protozoa and actinomycetes, which have a diverse capacity for attacking hydrocarbons. The main factors which affect the rate of microbial decomposition of hydrocarbons are: the availability of the contaminants to the microorganisms that have the catabolic ability to degrade them; the numbers of degrading microorganisms present in the soil; the activity of degrading microorganisms, and the molecular structure of the contaminant (Semple et al., 2003). The soil microorganisms number is usually in the range 104 to 107 CFU, for successful biodegradation this number should not be lower than 103 per gram of soil. Microorganism numbers lower than 103 CFU per gram of soil indicate the presence of toxic concentrations of organic or inorganic contaminants (Margesin et al., 2000; Petrović et al., 2008). The activity of soil microflora can be controlled by the factors discussed above - pH, temperature, nutrients, oxygen etc. For successful biodegradation, it is also necessary that the microorganisms can develop catabolic activity, by the following activities: induction of specific enzymes, development of new metabolic capabilities through genetic changes, and selective enrichment of organisms able to transform the target contaminant (Margesin et al., 2000, Semple et al., 2003).

#### **5.5. Bioremediation study – Our experiences**

56 Hydrocarbon

et al., 2008).

Trinidade et al., 2005).

**5.4. Microorganisms number and catabolism evolution** 

the rate and extent of biodegradation. Moreover, a weathered oil-contaminated soil normally contains a recalcitrant fraction of compounds composed basically of high molecular weight hydrocarbons, which cannot be degraded by indigenous microorganisms (Balba et al., 1998; Maletić et al., 2011; Loeher et al., 2001). In contrast, a recently oilcontaminated soil contains a higher amount of saturated and aliphatic compounds, which are the most susceptible to microbial degradation. However, the pollutant compounds in a recently contaminated soil are potentially more toxic to the native microorganisms, leading to a longer adaptation time (lag phase) before degradation of the pollutant and even to an inhibition of the biodegradation process (Margesin et al., 2000; Loeher et al., 2001; Petrović

As was mentioned above, sequestration and weathering of organic contaminants in the soil reduces the bioavailability of organic compounds and results in non-degraded residues in the soil. Contaminants that have been weathered and sequestrated in soil are not available for biodegradation in soil, even though freshly added compounds are still biodegradable (Alexander, 1995). Sorption is a major factor preventing the complete bioremediation of hydrocarbons in soil. Slow sorption leads to the hydrocarbon fraction becoming resistant to desorption and increases its persistence within the soil organic matrix. The following hypotheses have been proposed as a explanation for weathering: (1) weathering results in a slow diffusion of the hydrocarbon fraction in the solid fraction of the organic matter in the soil; (2) the contaminant slowly diffuses through the soil and becomes sorbed and trapped in the soil nano-and micropores (Semple et al., 2003;

The ability of the soil's microbial community to degrade hydrocarbons depends on the microbes number and its catabolic activity. Microorganisms can be isolated from almost all environmental conditions. Soil microflora contain numbers of different microorganisms including bacteria, algae, fungi, protozoa and actinomycetes, which have a diverse capacity for attacking hydrocarbons. The main factors which affect the rate of microbial decomposition of hydrocarbons are: the availability of the contaminants to the microorganisms that have the catabolic ability to degrade them; the numbers of degrading microorganisms present in the soil; the activity of degrading microorganisms, and the molecular structure of the contaminant (Semple et al., 2003). The soil microorganisms number is usually in the range 104 to 107 CFU, for successful biodegradation this number should not be lower than 103 per gram of soil. Microorganism numbers lower than 103 CFU per gram of soil indicate the presence of toxic concentrations of organic or inorganic contaminants (Margesin et al., 2000; Petrović et al., 2008). The activity of soil microflora can be controlled by the factors discussed above - pH, temperature, nutrients, oxygen etc. For successful biodegradation, it is also necessary that the microorganisms can develop catabolic activity, by the following activities: induction of specific enzymes, development of new metabolic capabilities through genetic changes, and selective enrichment of organisms able

to transform the target contaminant (Margesin et al., 2000, Semple et al., 2003).

With the aim of better understanding the factors which affect hydrocarbon biodegradation, results from the bioremediation studies described above are also given here, along with a comparison and discussion of changes in hydrocarbon composition and bioavailability over the years (Ivančev-Tumbas et al., 2004; Maletić, 2010; Maletić at al., 2011; Rončević, 2002; Rončević, 2007). The compounds detected by GC-MS analysis of extracts of the various soil samples taken at the start and end of bioremediation studies are given in Fig. 1, with only the main compounds from the hit lists of the probability-based matching (PBM ≥ 60%) search given.

**Figure 4.** GC-MS SCAN qualitative analysis of soil samples

The data reflect the fact that the soil used in this investigation was sampled from the dumping area of a refinery where the initial pollutants were of very diverse composition, i.e. a mixture of crude oil, mazut, diesel, middle distillates, heavy distillates, kerosene, etc. The untreated soil samples contained a large variety of straight-chain hydrocarbons and their methyl derivatives (those with both even and odd numbers of C atoms), many of which persisted during the treatment. However, if we compare the untreated soil samples at the start of study 1 (1 year after contamination), and study 4 (8 years after contamination) the difference is significant. Namely, in study 1, the soil mostly contains n-alkanes and derivates of aromatic hydrocarbons, and few compounds of iso-alkanes, whereas the soil in study 4 contains mostly n-alkanes and iso-alkanes, with only a few aromatics derivatives detected, with PBM<50%, and few cycloalkanes. The fact that mainly substituted polycyclic aromatic hydrocarbons were not detected in the weathered soil samples (study 4), shows their lower persistence than alkanes. Additionally, the greater number of iso-alkanes in weathered soil indicates their persistence. The cycloalkanes detected represent one of the main hydrocarbon residual fractions in weathered contaminated soil.

In both studies, at the end of the experiment, the number of detected compounds is significantly reduced. In study 1, the aromatic hydrocarbons were almost completely removed in both reactors, while the number of n-alkanes detected was reduced, but they are still present in significant numbers in the soil at the end. This is a because the aromatics have lower persistence than n-alkanes, but is also due to the higher n-alkanes concentration at the beginning. It is worth mentioning that in the reactor with continuous flow (aerobic), the number of removed nalkanes is almost the same, while in the reactor with discontinuous flows (partially anaerobic), the number of removed n-alkanes progressively reduced with depth, as a consequence of the lack of oxygen for microbial degradation, indicating that for this type of hydrocarbon, aerobic conditions are favourable. No such observation was noticed for aromatics. In study 4, only 3 nalkanes compounds were detected at the end, also the number of poorly degradable iso-alkanes was also significantly reduced; this could be consequence of the lack of more degradable substrate which was probably removed during the weathering process.

Petroleum Hydrocarbon Biodegradability in Soil – Implications for Bioremediation 59

The biodegradation study showed there was little difference between the respiration of the original and diluted samples of weathered oil contaminated soil (Table 5.). The evolved CO2 from those samples could originate from basal microbial respiration and from the very slow degradation of poorly biodegradable hydrocarbon compounds. This is confirmed by the removed amount of TPH in the samples. In contrast, in the freshly contaminated soil, respiration and the amount of TPH removed both strongly depended on the TPH concentration and origin. Thus, the highest quantity of evolved CO2 was produced by the soil contaminated with diesel oil (16 mg TPH/g), with the sample contaminated with crude oil (13 mg TPH/g) producing a slightly lower cumulative quantity of evolved CO2. The sample which contained the highest TPH concentration in the soils contaminated with diesel or crude oil had a lower respiration, which is a consequence of the high level of soluble hydrocarbons and the possible generation of toxic biodegradation products which can be toxic to the microorganisms present. Likewise, the sample with soil contaminated with the highest amount of diesel oil produced the second smallest amount of CO2 in the range of diesel contaminated soils. Thus, the diesel oil contains mostly midrange alkanes which have varying solubility and can cause toxic effects. The smallest amount of evolved CO2 was obtained for the samples with the lowest TPH concentrations of diesel and crude oil, where the biodegradable fraction was readily degraded. The amounts of TPH removed were in general agreement with the respiration rate, but less TPH was removed from the samples with crude oil contaminated soil. This could be due to the higher amounts of polar hydrocarbons (which are not included in the TPH fraction) in crude oil which can be

From comparing the end TPH concentration in the biodegradation sample on the original weathered oil contaminated soil (Table 5), and the predicted bioavailable fraction (Table 4), it can be concluded that a small amount of bioavailable substrate remained at the end of the treatment. Nevertheless, it should be borne in mind that the bioavailability test was conducted at the beginning of the experiment, and that as well as the biodegradation processes during the experiment, the sorption and sequestration of hydrocarbons also took place. These processes reduced the bioavailable hydrocarbon fraction during the treatment. Additionally, it is worth mentioning that during the 2 years of bioremediation study 4, the TPH concentration was reduced by 53% (21% in the first year and 32% in the second year), indicating that all of the biodegradable TPH fraction was removed during

From the above discussion it can be concluded that the lack of hydrocarbon biodegradation was due to highly sorbed and sequestrated hydrocarbons in the soil pores and soil organic matter as a consequence of weathering, and not due to high hydrocarbon concentrations or accumulation of toxic products in the soil. This soil is therefore not suitable for further bioremediation, and if further removal of hydrocarbons is required, other technologies must

degraded faster than the TPH.

the treatment.

be applied.

Although the number of detected compounds (Fig. 4) and TPH concentration (Fig. 3) at the end was significant, the bioremediation rate was too slow to suggest that further bioremediation was possible. With the aim of investigating whether the lack of further hydrocarbon biodegradation was a consequence of the absence of the bioavailable hydrocarbon fraction for microbial degradation, the accumulation of toxic hydrocarbon degradation by-products, or the high concentration of hydrocarbons, a laboratory trial on the soil from study 4 was conducted (Maletić, 2010; Maletić et al., 2011). Study 4 was carried out for almost 2 years, however, after about one year, the biodegradation process slowed down significantly; at that point, some of the soil from study 4 was taken for the laboratory trial. The laboratory trials aimed in two directions: (1) bioavailability and (2) biodegradability investigation. Additionally, in order to test the impact of concentration, chemical composition and weathering on the biodegradation processes, the same tests were conducted on soil freshly contaminated by crude oil and diesel oil [36]. The bioavailability test was done by extraction of hydrocarbon contaminated soil with Tween 80. Table 4 shows the main results obtained from this test. To test whether high concentration or the accumulation of toxic byproducts was the reason for the lack of biodegradation, the same soil sample was diluted with clean soil and then subjected to biodegradation under laboratory conditions (48 days). To ensure the process was not limited by other factors, the optimal conditions was provided, with respect to pH, temperature, water holding capacity, nutrients and oxygen content. The biodegradation process was monitored by measuring daily CO2 production and TPH concentrations at the beginning and at the end of the experiment (Table 5).

The obtained results show that only 33% of the total amount of TPH is bioavailable in the weathered oil contaminated soil (soil taken from study 4). In the freshly contaminated soil, the bioavailable TPH fraction was three times larger, clearly indicating that in the weathered contaminated soil, the hydrocarbon is highly sequestrated in the soil pores and complexed with soil organic matter. As a result of these processes, petroleum hydrocarbons become resistant and unavailable for biodegradation.


**Table 4.** Laboratory bioavailability trial results

The biodegradation study showed there was little difference between the respiration of the original and diluted samples of weathered oil contaminated soil (Table 5.). The evolved CO2 from those samples could originate from basal microbial respiration and from the very slow degradation of poorly biodegradable hydrocarbon compounds. This is confirmed by the removed amount of TPH in the samples. In contrast, in the freshly contaminated soil, respiration and the amount of TPH removed both strongly depended on the TPH concentration and origin. Thus, the highest quantity of evolved CO2 was produced by the soil contaminated with diesel oil (16 mg TPH/g), with the sample contaminated with crude oil (13 mg TPH/g) producing a slightly lower cumulative quantity of evolved CO2. The sample which contained the highest TPH concentration in the soils contaminated with diesel or crude oil had a lower respiration, which is a consequence of the high level of soluble hydrocarbons and the possible generation of toxic biodegradation products which can be toxic to the microorganisms present. Likewise, the sample with soil contaminated with the highest amount of diesel oil produced the second smallest amount of CO2 in the range of diesel contaminated soils. Thus, the diesel oil contains mostly midrange alkanes which have varying solubility and can cause toxic effects. The smallest amount of evolved CO2 was obtained for the samples with the lowest TPH concentrations of diesel and crude oil, where the biodegradable fraction was readily degraded. The amounts of TPH removed were in general agreement with the respiration rate, but less TPH was removed from the samples with crude oil contaminated soil. This could be due to the higher amounts of polar hydrocarbons (which are not included in the TPH fraction) in crude oil which can be degraded faster than the TPH.

58 Hydrocarbon

alkanes is almost the same, while in the reactor with discontinuous flows (partially anaerobic), the number of removed n-alkanes progressively reduced with depth, as a consequence of the lack of oxygen for microbial degradation, indicating that for this type of hydrocarbon, aerobic conditions are favourable. No such observation was noticed for aromatics. In study 4, only 3 nalkanes compounds were detected at the end, also the number of poorly degradable iso-alkanes was also significantly reduced; this could be consequence of the lack of more degradable

Although the number of detected compounds (Fig. 4) and TPH concentration (Fig. 3) at the end was significant, the bioremediation rate was too slow to suggest that further bioremediation was possible. With the aim of investigating whether the lack of further hydrocarbon biodegradation was a consequence of the absence of the bioavailable hydrocarbon fraction for microbial degradation, the accumulation of toxic hydrocarbon degradation by-products, or the high concentration of hydrocarbons, a laboratory trial on the soil from study 4 was conducted (Maletić, 2010; Maletić et al., 2011). Study 4 was carried out for almost 2 years, however, after about one year, the biodegradation process slowed down significantly; at that point, some of the soil from study 4 was taken for the laboratory trial. The laboratory trials aimed in two directions: (1) bioavailability and (2) biodegradability investigation. Additionally, in order to test the impact of concentration, chemical composition and weathering on the biodegradation processes, the same tests were conducted on soil freshly contaminated by crude oil and diesel oil [36]. The bioavailability test was done by extraction of hydrocarbon contaminated soil with Tween 80. Table 4 shows the main results obtained from this test. To test whether high concentration or the accumulation of toxic byproducts was the reason for the lack of biodegradation, the same soil sample was diluted with clean soil and then subjected to biodegradation under laboratory conditions (48 days). To ensure the process was not limited by other factors, the optimal conditions was provided, with respect to pH, temperature, water holding capacity, nutrients and oxygen content. The biodegradation process was monitored by measuring daily CO2 production and TPH

substrate which was probably removed during the weathering process.

concentrations at the beginning and at the end of the experiment (Table 5).

resistant and unavailable for biodegradation.

TPH g/kg residual after Tween

**Table 4.** Laboratory bioavailability trial results

The obtained results show that only 33% of the total amount of TPH is bioavailable in the weathered oil contaminated soil (soil taken from study 4). In the freshly contaminated soil, the bioavailable TPH fraction was three times larger, clearly indicating that in the weathered contaminated soil, the hydrocarbon is highly sequestrated in the soil pores and complexed with soil organic matter. As a result of these processes, petroleum hydrocarbons become

Parameter Type of the soil contaminant

TPH g/kg at the beginning 12 (±1.2) 26 (±2.6) 28 (±2.8)

extraction 8 (±0.8) 3.6 (±0.4) 1.2 (±0.1) %removed by Tween extraction 33 86 96

Weathered oil Crude oil Diesel oil

From comparing the end TPH concentration in the biodegradation sample on the original weathered oil contaminated soil (Table 5), and the predicted bioavailable fraction (Table 4), it can be concluded that a small amount of bioavailable substrate remained at the end of the treatment. Nevertheless, it should be borne in mind that the bioavailability test was conducted at the beginning of the experiment, and that as well as the biodegradation processes during the experiment, the sorption and sequestration of hydrocarbons also took place. These processes reduced the bioavailable hydrocarbon fraction during the treatment. Additionally, it is worth mentioning that during the 2 years of bioremediation study 4, the TPH concentration was reduced by 53% (21% in the first year and 32% in the second year), indicating that all of the biodegradable TPH fraction was removed during the treatment.

From the above discussion it can be concluded that the lack of hydrocarbon biodegradation was due to highly sorbed and sequestrated hydrocarbons in the soil pores and soil organic matter as a consequence of weathering, and not due to high hydrocarbon concentrations or accumulation of toxic products in the soil. This soil is therefore not suitable for further bioremediation, and if further removal of hydrocarbons is required, other technologies must be applied.


Petroleum Hydrocarbon Biodegradability in Soil – Implications for Bioremediation 61

This research was financed by the Ministry of Education and Science (Projects No. III43005

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**Table 5.** Laboratory biodegradability results

## **6. Conclusion**

The cleaning up of petroleum hydrocarbons in the soil environment is a real world problem. Better understanding of the mechanisms and factors which affect biodegradation is of great ecological significance, since the choice of bioremediation strategy depends on it. Microbial degradation processes aid the elimination of spilled oil from the environment, together with various physical and chemical methods. This is possible because microorganisms have enzyme systems to degrade and utilize different hydrocarbons as a source of carbon and energy. Even if the optimal conditions for microbial degradation are provided, the extent of hydrocarbon removal is strongly affected by its bioavailability and stages of weathering. As a consequence, some fractions of hydrocarbons remain undegraded. This residual fraction of hydrocarbon in soil can represent an acceptable end point for bioremediation if (1) hydrocarbon biodegradation is too slow to allow further bioremediation, in which case other technologies must be applied; (2) those concentrations are unable to release from the soil and pose adverse effects to the environment and human health, like those presented in the given case studies. Such residual material from oil degradation is analogous to, and could even be regarded as, humic material. Its inert characteristics, insolubility and similarity to humic materials mean it is unlikely to be environmentally hazardous.

## **Author details**

Snežana Maletić, Božo Dalmacija and Srđan Rončević *University of Novi Sad Faculty of Sciences, Department of Chemistry, Biochemistry and Environmental Protection, Republic of Serbia* 

#### **Acknowledgement**

This research was financed by the Ministry of Education and Science (Projects No. III43005 and TR37004) and the Novi Sad Oil Refinery.

#### **7. References**

60 Hydrocarbon

Weathered oil contaminated oil

Crude oil

Diesel oil

contaminated soil

contaminated soil

**6. Conclusion** 

environmentally hazardous.

Snežana Maletić, Božo Dalmacija and Srđan Rončević

*Biochemistry and Environmental Protection,* 

*University of Novi Sad Faculty of Sciences, Department of Chemistry,* 

**Author details** 

*Republic of Serbia* 

**Table 5.** Laboratory biodegradability results

Contaminated soil TPH g/kg after

12 (±1.2) (original

dilution Evolved CO2 mg/g g/kg removed TPH

soil) 6.1 (±0.9) 2.2 (±0.2) 4.9 (±0.5) 6.8 (±1.0) 1.3 (±0.1) 3.8 (±0.4) 5.2 (±0.8) 1.5 (±0.2) 2.3 (±0.2) 4.6 (±0.7) 0.76 (±0.1)

26 (±2.6) 15 (±2.2) 16 (±1.6) 13 (±1.3) 20 (±3.0) 11 (±1.1) 7.5 (±0.8) 11 (±1.7) 6.5 (±0.7) 5.5 (±0.6) 5.3 (±0.8) 4.4 (±0.4)

28 (±2.8) 14 (±2.2) 11 (±1.1) 16 (±1.6) 23 (±3.4) 11 (±1.1) 9.2 (±0.9) 17 (±2.6) 6.8 (±0.7) 7.0 (±0.7) 7.9 (±1.2) 5.1 (±0.5)

The cleaning up of petroleum hydrocarbons in the soil environment is a real world problem. Better understanding of the mechanisms and factors which affect biodegradation is of great ecological significance, since the choice of bioremediation strategy depends on it. Microbial degradation processes aid the elimination of spilled oil from the environment, together with various physical and chemical methods. This is possible because microorganisms have enzyme systems to degrade and utilize different hydrocarbons as a source of carbon and energy. Even if the optimal conditions for microbial degradation are provided, the extent of hydrocarbon removal is strongly affected by its bioavailability and stages of weathering. As a consequence, some fractions of hydrocarbons remain undegraded. This residual fraction of hydrocarbon in soil can represent an acceptable end point for bioremediation if (1) hydrocarbon biodegradation is too slow to allow further bioremediation, in which case other technologies must be applied; (2) those concentrations are unable to release from the soil and pose adverse effects to the environment and human health, like those presented in the given case studies. Such residual material from oil degradation is analogous to, and could even be regarded as, humic material. Its inert characteristics, insolubility and similarity to humic materials mean it is unlikely to be


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**Chapter 4** 

© 2013 Dadrasnia et al., licensee InTech. This is an open access chapter 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.

© 2013 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,

**Remediation of Contaminated Sites** 

Oil pollution in the environment is now being taken seriously by the oil industries and as such, these companies are always looking for cost-effective methods of dealing with this pollution. The global environment is under great stress due to urbanization and industrialization as well as population pressure on the limited natural resources. The problems are compounded by drastic changes that have been taking place in the lifestyle and habits of people. The environmental problems are diverse and sometimes specific with reference to time and space. The nature and the magnitude of the problems are ever changing, bringing new challenges and creating a constant need for developing newer and

In this context, biotechnology has tremendous potential to cater for the needs and holds hope for environmental protection, sustainability and management [1-2] While some applications such as bioremediation are direct applications of biotechnology [3-4][5], there are many which are indirectly beneficial for environmental remediation, pollution prevention and waste treatment. Large-scale pollution due to man-made chemical substances and to some extent by natural substances is of global concern now. Seepage and run-offs due to the mobile nature, and continuous cycling of volatilization and condensation of many organic chemicals such as pesticides have even led to their presence in rain, fog and

Every year, about 1.7 to 8.8 million metric tons of oil is released into the world's water. More than 90% of this oil pollution is directly related to accidents due to human failures and activities including deliberate waste disposal [7]. PAHs are present at levels varying from 1 μg to 300 g kg -1 soil, depending on the sources of contamination like combustion of fossil fuels, gasification and liquefaction of coal, incineration of wastes, and wood treatment processes [8]. Incomplete combustion of organic substances gives out about 100 different

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

polycyclic aromatic hydrocarbons (PAHs) which are the ubiquitous pollutants.

Arezoo Dadrasnia, N. Shahsavari and C. U. Emenike

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/51591

more appropriate technologies.

**1. Introduction** 

snow [6].


## **Remediation of Contaminated Sites**

Arezoo Dadrasnia, N. Shahsavari and C. U. Emenike

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/51591

## **1. Introduction**

64 Hydrocarbon

1163–1172, ISSN 1365-3075.

Vidali, M. (2001). Bioremediation. An overview. *Pure and Applied Cheistry*, Vol. 73, No. 7, pp.

Wethasinghe, C.; Yuen, S.T.S.; Kaluarachchi, J.J. & Hughes, R. (2006). Uncertainty in biokinetic parameters on bioremediation: Health risks and economic implications.

Yerushalmi, L. Rocheleau, S.; Cimpoia, R.; Sarrazin, M.; Sunahara, G.; Peisajovich, A; Leclair, G. & Guiot, S.R. (2003). Enhanced Biodegradation of Petroleum Hydrocarbons in Contaminated Soil. *Bioremediation Journal*, Vol. 7, No. 1, pp. 37–51, ISSN 1088-9868.

*Environment International*, Vol. 32, No. 3, pp. 312 – 323, ISSN 0160-4120.

Oil pollution in the environment is now being taken seriously by the oil industries and as such, these companies are always looking for cost-effective methods of dealing with this pollution. The global environment is under great stress due to urbanization and industrialization as well as population pressure on the limited natural resources. The problems are compounded by drastic changes that have been taking place in the lifestyle and habits of people. The environmental problems are diverse and sometimes specific with reference to time and space. The nature and the magnitude of the problems are ever changing, bringing new challenges and creating a constant need for developing newer and more appropriate technologies.

In this context, biotechnology has tremendous potential to cater for the needs and holds hope for environmental protection, sustainability and management [1-2] While some applications such as bioremediation are direct applications of biotechnology [3-4][5], there are many which are indirectly beneficial for environmental remediation, pollution prevention and waste treatment. Large-scale pollution due to man-made chemical substances and to some extent by natural substances is of global concern now. Seepage and run-offs due to the mobile nature, and continuous cycling of volatilization and condensation of many organic chemicals such as pesticides have even led to their presence in rain, fog and snow [6].

Every year, about 1.7 to 8.8 million metric tons of oil is released into the world's water. More than 90% of this oil pollution is directly related to accidents due to human failures and activities including deliberate waste disposal [7]. PAHs are present at levels varying from 1 μg to 300 g kg -1 soil, depending on the sources of contamination like combustion of fossil fuels, gasification and liquefaction of coal, incineration of wastes, and wood treatment processes [8]. Incomplete combustion of organic substances gives out about 100 different polycyclic aromatic hydrocarbons (PAHs) which are the ubiquitous pollutants.

© 2013 Dadrasnia et al., licensee InTech. This is an open access chapter 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. © 2013 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.

Except for a few PAHs used in medicines, dyes, plastics and pesticides, they are rarely of industrial use [9]. Some PAHs and their epoxides are highly toxic, and mutagenic even to microorganisms. About six specific PAHs are listed among the top 126 priority pollutants by the US Environmental Protection Agency. As much as the diversity in sources and chemical complexities in organic pollutants exists, there is probably more diversity in microbial members and their capabilities to synthesize or degrade organic compounds [10-11]-[12]. There are three main approaches in dealing with contaminated sites: identification of the problem, assessment of the nature and degree of the hazard, and the best choice of remedial action. The need to remediate these sites has led to the development of new technologies that emphasize the detoxification and destruction of the contaminants [13-14]-[15] rather than the conventional approach of disposal.

Remediation of Contaminated Sites 67

**2.2. Environmental pollution and biological treatments** 

biosphere [5]-[21]-[22].

The problems of environment can be classified into the following subheads as most of the problems can be traced to one or more of the following either directly or indirectly: Waste generation (sewage, wastewater, kitchen waste, industrial waste, effluents, agricultural waste, food waste) and use of chemicals for various purposes in the form of insecticides, pesticides, chemical fertilizers, toxic products and by-products from chemical industries (Fig 1). Waste generation is a side effect of consumption and production activities and tends to increase with economic advance. What is of concern is the increased presence of toxic chemicals such as halogen aliphatics, aromatics, polychlorinated biphenyls and other organic and inorganic pollutants which may reach air, water or soil and affect the environment in several ways, ultimately threatening the self-regulating capacity of the

They may be present in high levels at the points of discharge or may remain low but can be highly toxic for the receiving bodies. The underground water sources are increasingly becoming contaminated. For example, the underground water sources have been permanently abandoned in the valley of the River Po in north Italy due to industrial pollution. Some substances may reach environment in small concentrations but may be subjected to biomagnification or bioaccumulation up the food chain, wherein their

All the more, rapid developments in understanding activated sludge processes and wastewater remediation warrant exploitation of different strategies for studying their degradation and some of the biological remediation terminologies such as bioleaching, biosorption, bioaugmentation, biostimulation, biopulping, biodeterioration, biobleaching, bioaccumulation, biotransformation and bioattenuation are being actively researched on [27]. Enzyme technology has equally been receiving increased attention. Hussain et al. (2009) have reviewed the biotechnological approaches for enhancing the capability of microorganisms and plants through the characterization and transfer of pesticidedegrading genes, induction of catabolic pathways, and display of cell surface enzymes[28], while Theron et al. (2008) have performed a thorough review of nanotechnology, the engineering and art of manipulating matter at the nanoscale (1–100 nm), and have highlighted the potential of novel nanomaterials for treatment of surface water, groundwater, and wastewater contaminated by toxic metal ions, organic and inorganic solutes, and microorganisms [29]. Husain et al. (2009) have analyzed the role of peroxidases

concentrations increase as they pass through the food chain [23]-[24]-[25-26].

in the remediation and treatment of a wide spectrum of aromatic pollutants[28].

destruction of soil function, will be referred to only in passing.

Remediation approaches encompass applied physical, chemical and biological environmental sciences. The aim of this chapter will be to illustrate current understanding of the scientific principles underlying soil remediation and some of the challenges to their successful application. Remediation approaches that isolate treated soils are site rather than soil remediation technologies. These approaches, and the treatments that result in the

Remediation, whether by biological, chemical or a combination of both means, is the only option as the problem of pollution has to be solved without transferring to the future.

## **2. Manuscript**

#### **2.1. Measuring pollutant concentrations**

The setting of soil pollution limits assumes an agreed method for measuring the concentration of a pollutant that is relevant to risk assessment across differing soil types. Limits are generally expressed in terms of 'total' concentrations as there is no consensus on alternative [16] methods more directly related to biological or environmental risk. Yet, assessing the bioavailability of soil pollutants is an essential part of the process of risk assessment and of determining the most appropriate approach to remediation [17]. With developing non exhaustive solvent extraction procedures that consistently predict the bioavailability of organic contaminants across a range of soil conditions [17].

As an alternative to extraction, solid-phase micro-extraction uses adsorbents added to soil– water slurries aiming to mimic the accessibility of organic contaminants to microorganisms. In relation to the assessment of risks to human health, much work is currently underway to develop physiologically based extraction tests; however, progress made in this respect for inorganic pollutants has not been matched by that for organic pollutants [18]. In recent years, there has been a growth in the use of onsite assays to improve decision making regarding the extent of pollution in batches of potentially polluted materials and, therefore, the need for treatment or disposal. In many cases, these new measurements are based on enzyme-linked immunosorbent assays linked to spectroscopy.

Specific assays have, for example, been developed for pentachlorophenol [19] and PAHs [20].Whilst these methodologies can provide useful supplementary and 'real-time' information on pollutant concentration variability in the field, care must be taken when extrapolating findings from the very small samples used in these assays to bulk soil properties.

Various microbiological assays have been proposed as indicators of pollutant bioavailability. Biosensors have been widely deployed to provide fast, cost-effective monitoring of pollutants and their biological toxicity.

## **2.2. Environmental pollution and biological treatments**

66 Hydrocarbon

**2. Manuscript** 

than the conventional approach of disposal.

**2.1. Measuring pollutant concentrations** 

Except for a few PAHs used in medicines, dyes, plastics and pesticides, they are rarely of industrial use [9]. Some PAHs and their epoxides are highly toxic, and mutagenic even to microorganisms. About six specific PAHs are listed among the top 126 priority pollutants by the US Environmental Protection Agency. As much as the diversity in sources and chemical complexities in organic pollutants exists, there is probably more diversity in microbial members and their capabilities to synthesize or degrade organic compounds [10-11]-[12]. There are three main approaches in dealing with contaminated sites: identification of the problem, assessment of the nature and degree of the hazard, and the best choice of remedial action. The need to remediate these sites has led to the development of new technologies that emphasize the detoxification and destruction of the contaminants [13-14]-[15] rather

Remediation, whether by biological, chemical or a combination of both means, is the only option as the problem of pollution has to be solved without transferring to the future.

The setting of soil pollution limits assumes an agreed method for measuring the concentration of a pollutant that is relevant to risk assessment across differing soil types. Limits are generally expressed in terms of 'total' concentrations as there is no consensus on alternative [16] methods more directly related to biological or environmental risk. Yet, assessing the bioavailability of soil pollutants is an essential part of the process of risk assessment and of determining the most appropriate approach to remediation [17]. With developing non exhaustive solvent extraction procedures that consistently predict the

As an alternative to extraction, solid-phase micro-extraction uses adsorbents added to soil– water slurries aiming to mimic the accessibility of organic contaminants to microorganisms. In relation to the assessment of risks to human health, much work is currently underway to develop physiologically based extraction tests; however, progress made in this respect for inorganic pollutants has not been matched by that for organic pollutants [18]. In recent years, there has been a growth in the use of onsite assays to improve decision making regarding the extent of pollution in batches of potentially polluted materials and, therefore, the need for treatment or disposal. In many cases, these new measurements are based on

Specific assays have, for example, been developed for pentachlorophenol [19] and PAHs [20].Whilst these methodologies can provide useful supplementary and 'real-time' information on pollutant concentration variability in the field, care must be taken when extrapolating

Various microbiological assays have been proposed as indicators of pollutant bioavailability. Biosensors have been widely deployed to provide fast, cost-effective

findings from the very small samples used in these assays to bulk soil properties.

bioavailability of organic contaminants across a range of soil conditions [17].

enzyme-linked immunosorbent assays linked to spectroscopy.

monitoring of pollutants and their biological toxicity.

The problems of environment can be classified into the following subheads as most of the problems can be traced to one or more of the following either directly or indirectly: Waste generation (sewage, wastewater, kitchen waste, industrial waste, effluents, agricultural waste, food waste) and use of chemicals for various purposes in the form of insecticides, pesticides, chemical fertilizers, toxic products and by-products from chemical industries (Fig 1). Waste generation is a side effect of consumption and production activities and tends to increase with economic advance. What is of concern is the increased presence of toxic chemicals such as halogen aliphatics, aromatics, polychlorinated biphenyls and other organic and inorganic pollutants which may reach air, water or soil and affect the environment in several ways, ultimately threatening the self-regulating capacity of the biosphere [5]-[21]-[22].

They may be present in high levels at the points of discharge or may remain low but can be highly toxic for the receiving bodies. The underground water sources are increasingly becoming contaminated. For example, the underground water sources have been permanently abandoned in the valley of the River Po in north Italy due to industrial pollution. Some substances may reach environment in small concentrations but may be subjected to biomagnification or bioaccumulation up the food chain, wherein their concentrations increase as they pass through the food chain [23]-[24]-[25-26].

All the more, rapid developments in understanding activated sludge processes and wastewater remediation warrant exploitation of different strategies for studying their degradation and some of the biological remediation terminologies such as bioleaching, biosorption, bioaugmentation, biostimulation, biopulping, biodeterioration, biobleaching, bioaccumulation, biotransformation and bioattenuation are being actively researched on [27]. Enzyme technology has equally been receiving increased attention. Hussain et al. (2009) have reviewed the biotechnological approaches for enhancing the capability of microorganisms and plants through the characterization and transfer of pesticidedegrading genes, induction of catabolic pathways, and display of cell surface enzymes[28], while Theron et al. (2008) have performed a thorough review of nanotechnology, the engineering and art of manipulating matter at the nanoscale (1–100 nm), and have highlighted the potential of novel nanomaterials for treatment of surface water, groundwater, and wastewater contaminated by toxic metal ions, organic and inorganic solutes, and microorganisms [29]. Husain et al. (2009) have analyzed the role of peroxidases in the remediation and treatment of a wide spectrum of aromatic pollutants[28].

Remediation approaches encompass applied physical, chemical and biological environmental sciences. The aim of this chapter will be to illustrate current understanding of the scientific principles underlying soil remediation and some of the challenges to their successful application. Remediation approaches that isolate treated soils are site rather than soil remediation technologies. These approaches, and the treatments that result in the destruction of soil function, will be referred to only in passing.

Remediation of Contaminated Sites 69

land decontamination. Even in the most contaminated soils, indigenous microbial activity can be enough to clean the soil effectively. Bioremediation techniques are cost effective as compared to other technologies. Biological treatments compare favorably with alternative methods. Treatment periods generally last from 2 to 48 months, about the same for chemical or thermal methods. Physical processes (soil washing and soil vapour extraction) are faster,

Bioremediation (when used in solution) does not require environmentally damaging processes such as chemicals or heat treatment. It has beneficial effects upon soil structure and fertility, but with limitation on its effectiveness. These limitations may be summarized

Bioremediation technologies can be broadly classified as ex situ or in situ. Table 1 summarizes the most commonly used bioremediation technologies. Ex situ technologies are those treatment modalities which involve the physical removal of the contaminated to

Bioreactors, land farming, anaerobic digestion, composting, biosorption and some forms of solid-phase treatment are all examples of ex situ treatment techniques. In contrast, in situ techniques involve treatment of the contaminated material in place. Bioventing for the treatment of the contaminated soil and biostimulation of indigenous aquifer microorganisms are examples of these treatment techniques. Although some sites may be

Bioaugmentation Addition of bacterial cultures to a contaminated medium frequently

Biostimulation Stimulation of indigenous microbial populations in soils and/or ground

Bioventing Method of treating contaminated soils by drawing oxygen through the soil to stimulate microbial growth and activity

For example, many sites are located in industrial/ commercial areas, and these sites normally consist of numerous structures interconnected by concrete and asphalt. These physical barriers would make excavation extremely difficult, and if the contamination is deep in the subsurface, excavation becomes too expensive. As a result of these physical barriers, the required excavation efforts may make ex situ biotreatment impracticable. Other

used in bioreactors and ex situ systems Biofilters Use of microbial stripping columns to treat air emission

rarely lasting more than 1 year. Solidification is almost instantaneous.

more easily controlled and maintained with ex situ configurations [34].

as follows:


*2.2.1.1. Bioremediation technologies* 

another area (possibly within the site) for treatment.

water

**Table 1.** Bioremediation treatment technologies

Bioreactors Biodegradation in a container or reactor

**Figure 1.** Summary of environmental fates on organic pollutants in soil.

#### *2.2.1. Bioremediation*

Interest in the microbial biodegradation of pollutants has intensified in recent years as humanity strives to find sustainable ways to clean up contaminated environments. Bioremediation, which is the use of microorganisms consortia or microbial processes to degrade and detoxify environmental contaminants [30]. It is also amongst these new technologies which derives its scientific justification from the emerging concept of Green Chemistry and Green Engineering, and is a fast growing promising remediation technique increasingly being studied and applied in practical use for pollutant clean-up.

Bioremediation techniques have been used for decontamination of surface and subsurface soils, freshwater and marine systems, soils, groundwater and contaminated land ecosystems. However, the majority of bioremediation technologies initially developed were to treat petroleum hydrocarbon contamination to immobilize contaminants or to transform them to chemical products no longer hazardous to human health and the environment. Where contaminants pose no significant risk to water supply or surface water bodies, biodegradation products will include carbon dioxide, water and other compounds with little deleterious effects on the environment [31].

Bioremediation of soils or any site may be enhanced by fertilizing (adding nutrients such as carbon, nitrogen and phosphorous) and/or seeding with suitable microbial populations. These days, using organic wastes is bioremediation process is going to be new method as a option of enhancing and motivating of microorganism to break down of organic compounds [32-33]. This is enhanced or engineered bioremediation. Intrinsic bioremediation, which utilizes existing microbial communities, is often the most cost effective method available for land decontamination. Even in the most contaminated soils, indigenous microbial activity can be enough to clean the soil effectively. Bioremediation techniques are cost effective as compared to other technologies. Biological treatments compare favorably with alternative methods. Treatment periods generally last from 2 to 48 months, about the same for chemical or thermal methods. Physical processes (soil washing and soil vapour extraction) are faster, rarely lasting more than 1 year. Solidification is almost instantaneous.

Bioremediation (when used in solution) does not require environmentally damaging processes such as chemicals or heat treatment. It has beneficial effects upon soil structure and fertility, but with limitation on its effectiveness. These limitations may be summarized as follows:


68 Hydrocarbon

*2.2.1. Bioremediation* 

deleterious effects on the environment [31].

**Figure 1.** Summary of environmental fates on organic pollutants in soil.

Interest in the microbial biodegradation of pollutants has intensified in recent years as humanity strives to find sustainable ways to clean up contaminated environments. Bioremediation, which is the use of microorganisms consortia or microbial processes to degrade and detoxify environmental contaminants [30]. It is also amongst these new technologies which derives its scientific justification from the emerging concept of Green Chemistry and Green Engineering, and is a fast growing promising remediation technique

Bioremediation techniques have been used for decontamination of surface and subsurface soils, freshwater and marine systems, soils, groundwater and contaminated land ecosystems. However, the majority of bioremediation technologies initially developed were to treat petroleum hydrocarbon contamination to immobilize contaminants or to transform them to chemical products no longer hazardous to human health and the environment. Where contaminants pose no significant risk to water supply or surface water bodies, biodegradation products will include carbon dioxide, water and other compounds with little

Bioremediation of soils or any site may be enhanced by fertilizing (adding nutrients such as carbon, nitrogen and phosphorous) and/or seeding with suitable microbial populations. These days, using organic wastes is bioremediation process is going to be new method as a option of enhancing and motivating of microorganism to break down of organic compounds [32-33]. This is enhanced or engineered bioremediation. Intrinsic bioremediation, which utilizes existing microbial communities, is often the most cost effective method available for

increasingly being studied and applied in practical use for pollutant clean-up.


#### *2.2.1.1. Bioremediation technologies*

Bioremediation technologies can be broadly classified as ex situ or in situ. Table 1 summarizes the most commonly used bioremediation technologies. Ex situ technologies are those treatment modalities which involve the physical removal of the contaminated to another area (possibly within the site) for treatment.

Bioreactors, land farming, anaerobic digestion, composting, biosorption and some forms of solid-phase treatment are all examples of ex situ treatment techniques. In contrast, in situ techniques involve treatment of the contaminated material in place. Bioventing for the treatment of the contaminated soil and biostimulation of indigenous aquifer microorganisms are examples of these treatment techniques. Although some sites may be more easily controlled and maintained with ex situ configurations [34].


**Table 1.** Bioremediation treatment technologies

For example, many sites are located in industrial/ commercial areas, and these sites normally consist of numerous structures interconnected by concrete and asphalt. These physical barriers would make excavation extremely difficult, and if the contamination is deep in the subsurface, excavation becomes too expensive. As a result of these physical barriers, the required excavation efforts may make ex situ biotreatment impracticable. Other

factors could also have an impact on the type of treatment. At a typical site, the contamination is basically trapped below the surface.

Remediation of Contaminated Sites 71

(less than 5 m), although in some situations with deeper contamination it may be used in conjunction with other technologies. Third, since the time requirements for phytoremediation are sometimes long relative to some conventional technologies such as land filling and incineration, it is not suitable for situations requiring rapid treatment. Plants

facilitate remediation via several mechanisms (Fig 2):

**Figure 2.** Phytoremediation mechanisms.

to sustain the microbial population [41-42].

*2.2.4. Biopiling* 

*2.2.5. Composting* 

1. Direct uptake, and incorporation of contaminants into plant biomass 2. Immobilization, or Phytostabilization of contaminants in the subsurface

4. Stimulation of microbial mediated degradation in the rhizosphere

3. Release plant enzymes into the rhizosphere that act directly on the contaminants

Biopiles piles are a form of soil treatment where bulking agents, nutrients, and water are added. However, static piles are not mixed and temperatures are usually near ambient. Aeration can be passive or forced by applying a vacuum or blowing air through the pile. Bulking agents used are usually made up of manure or compost, which supports a larger microbial population than soil and provides inorganic nutrients, and relatively inert materials such as sawdust, wood chips, or compost. Water is added periodically, as needed

Composting is an aerobic process that relies on the actions of microorganisms to degrade organic materials, resulting in the thermo genesis and production of organic and inorganic compounds. The metabolically generated heat is trapped within the compost matrix, which

To expose the contamination to the open environment through excavation can result in potential health and safety risks [34]. In addition, the public's perception of the excavation of contaminants could be negative, depending on the situation. All of these conditions clearly favor in situ biotreatment. Nonetheless, the key is to carefully consider the parameters involved with each site before evaluating which technique to use [34].

#### *2.2.2. Land farming*

This technology involves the application of contaminated material that has been excavated onto the soil surface and periodically tilled to mix and aerate the material [35-36]. The contaminants are degraded, transformed and immobilized by means of biotic and abiotic reactions. Sometimes, in cases of very shallow contamination, the top layer of the site may simply be tilled without requiring any excavation. Liners or other methods may be used to control leachate. This technology is designed primarily to treat soil contamination by fuels, PAHs, non-halogenated VOCs, SVOCs, pesticides, and herbicides. The process may be applied to halogenated organics, but is less effective.

simple and inexpensive, it does require large space, and reduction in contaminant concentrations may sometimes be due to volatilization rather than biodegradation [37-38]. Mar´n et al. (2005) assessed the ability land farming to reduce the total hydrocarbon content added to soil with refinery sludge in low rain and high temperature conditions [39]. It was seen that 80% of the hydrocarbons were eliminated in 11 months, half of this reduction taking place during the first 3 months.

#### *2.2.3. Phytoremediation*

Using plants in soil and groundwater remediation (i.e., phytoremediation) is a relatively new concept and the technology has yet to be extensively proven in the marketplace. However, the potential of phytoremediation for cheap, simple and effective soil and groundwater remediation is generating considerable interest.

Phytoremediation may be used for remediation of soil and groundwater contaminated with toxic heavy metals, radio nuclides, and organic contaminants such as chlorinated solvents, BTEX compounds, non-aromatic petroleum hydrocarbons, nitro toluene ammunition wastes, and excess nutrients [40]. Other applications of phytoremediation include Land fill caps, buffer zones for agricultural runoff and even drinking water and industrial wastewater treatment. Phytoremediation may also be used as a final polishing step, in conjunction with other treatment technologies. While indeed promising, the applicability of phytoremediation is limited by several factors. First, it is essential that the contaminated site of interest is able to support plant growth. This requires suitable climate, soil characteristics such as pH and texture, and adequate water and nutrients. Second, because plant roots only go so deep, phytoremediation is practical only in situations where contamination is shallow (less than 5 m), although in some situations with deeper contamination it may be used in conjunction with other technologies. Third, since the time requirements for phytoremediation are sometimes long relative to some conventional technologies such as land filling and incineration, it is not suitable for situations requiring rapid treatment. Plants facilitate remediation via several mechanisms (Fig 2):


**Figure 2.** Phytoremediation mechanisms.

#### *2.2.4. Biopiling*

70 Hydrocarbon

*2.2.2. Land farming* 

factors could also have an impact on the type of treatment. At a typical site, the

To expose the contamination to the open environment through excavation can result in potential health and safety risks [34]. In addition, the public's perception of the excavation of contaminants could be negative, depending on the situation. All of these conditions clearly favor in situ biotreatment. Nonetheless, the key is to carefully consider the

This technology involves the application of contaminated material that has been excavated onto the soil surface and periodically tilled to mix and aerate the material [35-36]. The contaminants are degraded, transformed and immobilized by means of biotic and abiotic reactions. Sometimes, in cases of very shallow contamination, the top layer of the site may simply be tilled without requiring any excavation. Liners or other methods may be used to control leachate. This technology is designed primarily to treat soil contamination by fuels, PAHs, non-halogenated VOCs, SVOCs, pesticides, and herbicides. The process may be

simple and inexpensive, it does require large space, and reduction in contaminant concentrations may sometimes be due to volatilization rather than biodegradation [37-38]. Mar´n et al. (2005) assessed the ability land farming to reduce the total hydrocarbon content added to soil with refinery sludge in low rain and high temperature conditions [39]. It was seen that 80% of the hydrocarbons were eliminated in 11 months, half of this reduction

Using plants in soil and groundwater remediation (i.e., phytoremediation) is a relatively new concept and the technology has yet to be extensively proven in the marketplace. However, the potential of phytoremediation for cheap, simple and effective soil and

Phytoremediation may be used for remediation of soil and groundwater contaminated with toxic heavy metals, radio nuclides, and organic contaminants such as chlorinated solvents, BTEX compounds, non-aromatic petroleum hydrocarbons, nitro toluene ammunition wastes, and excess nutrients [40]. Other applications of phytoremediation include Land fill caps, buffer zones for agricultural runoff and even drinking water and industrial wastewater treatment. Phytoremediation may also be used as a final polishing step, in conjunction with other treatment technologies. While indeed promising, the applicability of phytoremediation is limited by several factors. First, it is essential that the contaminated site of interest is able to support plant growth. This requires suitable climate, soil characteristics such as pH and texture, and adequate water and nutrients. Second, because plant roots only go so deep, phytoremediation is practical only in situations where contamination is shallow

parameters involved with each site before evaluating which technique to use [34].

contamination is basically trapped below the surface.

applied to halogenated organics, but is less effective.

groundwater remediation is generating considerable interest.

taking place during the first 3 months.

*2.2.3. Phytoremediation* 

Biopiles piles are a form of soil treatment where bulking agents, nutrients, and water are added. However, static piles are not mixed and temperatures are usually near ambient. Aeration can be passive or forced by applying a vacuum or blowing air through the pile. Bulking agents used are usually made up of manure or compost, which supports a larger microbial population than soil and provides inorganic nutrients, and relatively inert materials such as sawdust, wood chips, or compost. Water is added periodically, as needed to sustain the microbial population [41-42].

#### *2.2.5. Composting*

Composting is an aerobic process that relies on the actions of microorganisms to degrade organic materials, resulting in the thermo genesis and production of organic and inorganic compounds. The metabolically generated heat is trapped within the compost matrix, which

leads to elevations in temperature, a characteristic of composting. In deed composting is the biochemical degradation of organic materials to a sanitary, nuisance-free, humus-like material [43]. Composting has been defined as a controlled microbial aerobic decomposition process with the formation of stabilized organic materials that may be used as soil conditioner [44]. The main factors in the control of a composting process include environmental parameters (temperature, moisture content, pH and aeration) and substrate nature parameters (C/N ratio, particle size, and nutrient content) [45-46].

Remediation of Contaminated Sites 73

benzo(a)pyrene being degraded the fastest. Nonetheless, variation in TiO2 concentration from 0.5 to 3wt. % did not provide any significant effect on PAH degradation. Under distinct UV wavelengths, photocatalytic degradation rates of PAHs were different. Soil pH was discovered to affect the process whereby the highest pyrene and benzo(a)pyrene degradation rates were obtained at acidic conditions, while phenanthrene was most significantly degraded at alkaline conditions. Additionally, the presence of humic acid in soil was found to enhance PAH photocatalytic degradation by sensitising radicals capable of

Rababah and Matsuzawa [52]developed a recirculating-type photocatalytic reactor assisted by the oxidising agent H2O2 solution to treat soil spiked with fluranthene. It was observed that the degradation efficiency of fluoranthene was 99% in the presence of both TiO2 and H2O2 compared to a lower degradation efficiency of 83% in the presence of TiO2 alone.

Solidification/Stabilization (S/S) is one of the top five source control treatment technologies used at Superfund remedial sites, having been used at more than 160 sites. "Solidification" refers to a process in which materials are added to the waste to produce an immobile mass. This may or may not involve a chemical bonding between the toxic contaminant and the additive. "Stabilization" refers to converting a waste to a more chemically stable form. This conversion may include solidification, but it almost always includes use of physicochemical

Solidification is a technique that encapsulates hazardous waste into a solid material of high structural integrity. Solidifying fine waste particles is termed microencapsulation; macro encapsulation solidifies wastes in large blocks or containers. Stabilization technologies reduce a hazardous wastes solubility, mobility, or toxicity. Solidification and stabilization are effective for treating soils containing metals, asbestos, radioactive materials, in organics, corrosive and cyanide compounds, and semi-volatile organics. Solidification eliminates free liquids, reduces hazardous constituent mobility by lowering waste permeability, minimizes constituent leach ability, and provides stability for handling, transport, and disposal [55].

In cases where the contaminants are volatile, a venting and ex-situ gas treatment system can be applied. Soil vapour extraction is a technology that has been proven effective in reducing concentrations of VOC and certain semi-volatile organic compounds. Principally, a vacuum is applied to the soil matrix to create a negative pressure gradient that causes movement of vapors toward extraction wells. Volatile contaminants are readily removed from the subsurface through the extraction wells. The collected vapors are then treated and discharged to the atmosphere or where permitted, reinjected to the subsurface [56-57].

reaction to transform the contaminants to a less toxic form [53-54].

oxidizing PAHs.

**2.3. Physico- chemical treatments** 

*2.3.1. Solidification and Stabilization* 

*2.3.2. Soil vapour extraction* 

Various factors correlate with each other physically, chemically and biologically in complicated composting processes. A slight change in a single factor may cause a drastic avalanche of metabolic and physical changes in the overall process. In other words, there may be extremely strong non-linearities involved in these processes [47]. These processes occur in matrix of organic particles and interconnected pores, and the pores are partially filled with air, aqueous solution, or a combination of the two. A multitude of microorganisms and their enzymes is responsible for the biodegradation process [48], resulting in a complex biochemical–microbial system.

#### *2.2.6. Electrokinetic remediation*

Electrokinetic treatment is emerging and innovative technology to complement traditional technology limitations and to treat fine-grained soils. Electrokinetic technology evaluated most suitable to remove contaminants effectively from low permeability clayey soil.

In situ electrokinetic remediation can be applied to treat low permeable soils contaminated with heavy metals, radionuclides and selected organic pollutants. The principle behind this method is the application of a low level direct current electric potential through electrodes, which are placed into the contaminated soil. Ionic contaminants are transported to the oppositely charged electrode by electromigration. Additionally, electroosmotic flow provides a driving force for the movement of soluble contaminants [49].

Although the technology has been known and utilised for more than a decade, application to removal of hydrophobic and strongly adsorbed pollutants such as PAHs especially from low permeability soils is recent. Solubilising agents are therefore used in these cases to enhance the removal efficiency of PAHs [50].

#### *2.2.7. Photocatalytic degradation*

The photocatalytic degradation process uses photocatalysts to promote oxidising reactions which destroy organic contaminants in the presence of light radiation. The technology has been widely established for treatment of wastewater, and recently, its application has extended to treatment of contaminated soils.

Zhang et al. [51]conducted a comprehensive study of the photocatalytic degradation of phenanthrene, pyrene and benzo(a)pyrene on soil surfaces using titanium dioxide (TiO2) under UV light. Compared to the absence of catalyst, the addition of TiO2 as catalyst revealed that TiO2 accelerated the photodegradation process of all three PAHs, with benzo(a)pyrene being degraded the fastest. Nonetheless, variation in TiO2 concentration from 0.5 to 3wt. % did not provide any significant effect on PAH degradation. Under distinct UV wavelengths, photocatalytic degradation rates of PAHs were different. Soil pH was discovered to affect the process whereby the highest pyrene and benzo(a)pyrene degradation rates were obtained at acidic conditions, while phenanthrene was most significantly degraded at alkaline conditions. Additionally, the presence of humic acid in soil was found to enhance PAH photocatalytic degradation by sensitising radicals capable of oxidizing PAHs.

Rababah and Matsuzawa [52]developed a recirculating-type photocatalytic reactor assisted by the oxidising agent H2O2 solution to treat soil spiked with fluranthene. It was observed that the degradation efficiency of fluoranthene was 99% in the presence of both TiO2 and H2O2 compared to a lower degradation efficiency of 83% in the presence of TiO2 alone.

#### **2.3. Physico- chemical treatments**

72 Hydrocarbon

leads to elevations in temperature, a characteristic of composting. In deed composting is the biochemical degradation of organic materials to a sanitary, nuisance-free, humus-like material [43]. Composting has been defined as a controlled microbial aerobic decomposition process with the formation of stabilized organic materials that may be used as soil conditioner [44]. The main factors in the control of a composting process include environmental parameters (temperature, moisture content, pH and aeration) and substrate

Various factors correlate with each other physically, chemically and biologically in complicated composting processes. A slight change in a single factor may cause a drastic avalanche of metabolic and physical changes in the overall process. In other words, there may be extremely strong non-linearities involved in these processes [47]. These processes occur in matrix of organic particles and interconnected pores, and the pores are partially filled with air, aqueous solution, or a combination of the two. A multitude of microorganisms and their enzymes is responsible for the biodegradation process [48],

Electrokinetic treatment is emerging and innovative technology to complement traditional technology limitations and to treat fine-grained soils. Electrokinetic technology evaluated

In situ electrokinetic remediation can be applied to treat low permeable soils contaminated with heavy metals, radionuclides and selected organic pollutants. The principle behind this method is the application of a low level direct current electric potential through electrodes, which are placed into the contaminated soil. Ionic contaminants are transported to the oppositely charged electrode by electromigration. Additionally, electroosmotic flow

Although the technology has been known and utilised for more than a decade, application to removal of hydrophobic and strongly adsorbed pollutants such as PAHs especially from low permeability soils is recent. Solubilising agents are therefore used in these cases to

The photocatalytic degradation process uses photocatalysts to promote oxidising reactions which destroy organic contaminants in the presence of light radiation. The technology has been widely established for treatment of wastewater, and recently, its application has

Zhang et al. [51]conducted a comprehensive study of the photocatalytic degradation of phenanthrene, pyrene and benzo(a)pyrene on soil surfaces using titanium dioxide (TiO2) under UV light. Compared to the absence of catalyst, the addition of TiO2 as catalyst revealed that TiO2 accelerated the photodegradation process of all three PAHs, with

most suitable to remove contaminants effectively from low permeability clayey soil.

provides a driving force for the movement of soluble contaminants [49].

nature parameters (C/N ratio, particle size, and nutrient content) [45-46].

resulting in a complex biochemical–microbial system.

enhance the removal efficiency of PAHs [50].

extended to treatment of contaminated soils.

*2.2.7. Photocatalytic degradation* 

*2.2.6. Electrokinetic remediation* 

#### *2.3.1. Solidification and Stabilization*

Solidification/Stabilization (S/S) is one of the top five source control treatment technologies used at Superfund remedial sites, having been used at more than 160 sites. "Solidification" refers to a process in which materials are added to the waste to produce an immobile mass. This may or may not involve a chemical bonding between the toxic contaminant and the additive. "Stabilization" refers to converting a waste to a more chemically stable form. This conversion may include solidification, but it almost always includes use of physicochemical reaction to transform the contaminants to a less toxic form [53-54].

Solidification is a technique that encapsulates hazardous waste into a solid material of high structural integrity. Solidifying fine waste particles is termed microencapsulation; macro encapsulation solidifies wastes in large blocks or containers. Stabilization technologies reduce a hazardous wastes solubility, mobility, or toxicity. Solidification and stabilization are effective for treating soils containing metals, asbestos, radioactive materials, in organics, corrosive and cyanide compounds, and semi-volatile organics. Solidification eliminates free liquids, reduces hazardous constituent mobility by lowering waste permeability, minimizes constituent leach ability, and provides stability for handling, transport, and disposal [55].

#### *2.3.2. Soil vapour extraction*

In cases where the contaminants are volatile, a venting and ex-situ gas treatment system can be applied. Soil vapour extraction is a technology that has been proven effective in reducing concentrations of VOC and certain semi-volatile organic compounds. Principally, a vacuum is applied to the soil matrix to create a negative pressure gradient that causes movement of vapors toward extraction wells. Volatile contaminants are readily removed from the subsurface through the extraction wells. The collected vapors are then treated and discharged to the atmosphere or where permitted, reinjected to the subsurface [56-57].

#### *2.3.3. Soil washing*

Soil washing is an *ex situ* treatment technology for the remediation of contaminated soil. It has been applied to a variety of inorganically, organically, and even radioactively contaminated soils. Although it is a well established technology in continental Europe and North America, there are very few applications in the UK.

Remediation of Contaminated Sites 75

**Figure 3.** Schematic diagram of soil washing.

Air sparging is an in situ technology in which air is injected through a contaminated aquifer. Air-sparing stimulates aerobic biodegradation of contaminated groundwater by delivery of oxygen to the subsurface [60]-[61]. This is accomplished by injecting air below the water table. This technology is designed primarily to treat groundwater contamination by fuels,

Air sparing has also been demonstrated to be an innovative groundwater remediation technology capable of restoring aquifers that have been polluted by volatile and (or) biodegradable contaminants, such as petroleum hydrocarbons. The process may be applied

Air-sparing can cost less than \$1 per 1,000 l in favorable situations and tends to be among the cheapest remedial alternatives when applicable. The technology uses simple, inexpensive, low-maintenance equipment that can be left unattended for long periods of time. Also, the technology tends to enjoy good public acceptance. The technology requires

non-halogenated VOCs, SVOCs, pesticides, organics, and herbicides.

to halogenated organics, but is less effective (Fig 4).

**Figure 4.** A schematic diagram illustrating method of air sparging.

*2.3.4. Air-sparging* 

The selection of soil washing for a particular contamination problem will depend on a variety of factors. Particularly important is whether the contamination is specific to particular groups of particles within the soil and whether these particles can be removed from the contaminant-free bulk of particles by physical or physico-chemical processes [58].

Contamination can occur on or in soil particles in a variety of ways. Six types of association are identified:


Soil washing technology involves mixing the solvent (water) and contaminated soil in an extractor vessel [41, 59]. The mixing dissolves the organic contaminant into the solvent. Solvent and dissolved contaminants are then placed in a separator where the solute and solvent are separated and treated. The soils can be stockpiled, tested and used as inert material (Fig 3).

**Figure 3.** Schematic diagram of soil washing.

#### *2.3.4. Air-sparging*

74 Hydrocarbon

*2.3.3. Soil washing* 

are identified:

particles.

Soil washing is an *ex situ* treatment technology for the remediation of contaminated soil. It has been applied to a variety of inorganically, organically, and even radioactively contaminated soils. Although it is a well established technology in continental Europe and

The selection of soil washing for a particular contamination problem will depend on a variety of factors. Particularly important is whether the contamination is specific to particular groups of particles within the soil and whether these particles can be removed from the contaminant-free bulk of particles by physical or physico-chemical processes [58].

Contamination can occur on or in soil particles in a variety of ways. Six types of association

 *Adsorbed contamination*. Contaminants may be adsorbed to particles and, in many cases, this adsorption may be preferential to particular particle types. For example, the adsorption of inorganic or organic contaminants on peaty organic fraction or on clay

 *Discrete particles*. Some contaminants may occur within the soil matrix as discrete particles that are not necessarily associated with soil particles. Contaminants of this type can include discrete metal grains or metal oxides, tar balls and some waste

 *Coatings*. Contaminants may occur as coatings on individual particles that have resulted from precipitation of the contaminant from solution. For example, metal salts and iron

 *Liquid or semi-liquid coating*. Liquid or semi-liquid viscous substances may occur as coatings around individual soil particles. Contaminants of this type can include oils,

 *Liquid or semi-liquid coating*. Liquid or semi-liquid viscous substances may occur as coatings around individual soil particles. Contaminants of this type can include oils,

 *Internal contamination within pores*. Contamination may also occur inside individual grains but within the pore structure. Here it may be adsorbed (e.g. heavy metal or organic contamination), occur as a coating to the pore walls (e.g. inorganic compound precipitated from solution) or occur within and possibly occlude the pores (e.g.

 *Part of individual grains*. Contamination may occur within the matrix of an individual grain, or as part of an individual grain. Heavy metal contamination may occur in this way, for example, in slags where the heavy metal can occur within the vitrified matrix

Soil washing technology involves mixing the solvent (water) and contaminated soil in an extractor vessel [41, 59]. The mixing dissolves the organic contaminant into the solvent. Solvent and dissolved contaminants are then placed in a separator where the solute and solvent are separated and treated. The soils can be stockpiled, tested and used as inert material (Fig 3).

or associated with specific mineral phases such as magnetite.

North America, there are very few applications in the UK.

materials (e.g. used catalyst fragments).

oxides can precipitate on sand particles.

tars and some other organic contaminants.

tars and some other organic contaminants.

contaminants such as mineral oils).

Air sparging is an in situ technology in which air is injected through a contaminated aquifer. Air-sparing stimulates aerobic biodegradation of contaminated groundwater by delivery of oxygen to the subsurface [60]-[61]. This is accomplished by injecting air below the water table. This technology is designed primarily to treat groundwater contamination by fuels, non-halogenated VOCs, SVOCs, pesticides, organics, and herbicides.

Air sparing has also been demonstrated to be an innovative groundwater remediation technology capable of restoring aquifers that have been polluted by volatile and (or) biodegradable contaminants, such as petroleum hydrocarbons. The process may be applied to halogenated organics, but is less effective (Fig 4).

**Figure 4.** A schematic diagram illustrating method of air sparging.

Air-sparing can cost less than \$1 per 1,000 l in favorable situations and tends to be among the cheapest remedial alternatives when applicable. The technology uses simple, inexpensive, low-maintenance equipment that can be left unattended for long periods of time. Also, the technology tends to enjoy good public acceptance. The technology requires

the presence of indigenous organisms capable of degrading the contaminants of interest, as well as nutrients necessary for growth. Also, it is necessary that the contaminants be available to the organisms, and not tightly sorbed to soil particles. Air sparing is not applicable in sites where high concentrations of inorganic salts, heavy metals, or organic compounds are present, as hinder microbial growth.

Remediation of Contaminated Sites 77

emissions and noise [64]. Incineration technology is intended to permanently destroy organic contaminants. Incineration is a complex system of interacting pieces of equipment and is not just a simple furnace. It is an integrated system of components for waste preparation, feeding, combustion, and emissions control. Central to the system is the combustion chamber, or the incinerator. There are four major types of incinerator: rotary

Nanotechnology has contributed to the development of a great diversity of materials as those used in electronic, optoelectronic, biomedical, pharmaceutical, cosmetic, energy, catalytic, and materials applications. As a general definition, nanotechnology is involved with objects on the nano scale, or materials measuring between 1 and 100 nm [65]. In future, modification and adaptation of nanotechnology will extend the quality and length of life [66]. The social benefits are significant from nanomaterials and the new products are applicable to information technology, medicine, energy, and environment. The emergence of

The steam stripping method is based on a mass transfer concept, which is used to move volatile contaminants from water to air. Steam is injected through an injection well into the soil to vaporize volatile and semi-volatile contaminants [67]. The contaminated vapour steam is removed by vacuum extraction, and the contaminants are then captured through

Dehalogenation of organic compounds is chemical displacement of a chlorine molecule and

Chemical reduction/oxidation is a chemical conversion of hazardous contaminants to nonhazardous or less toxic compounds. The result is a more stable, less mobile and/or inert

Ultraviolet (UV) oxidation technology uses UV radiation, ozone, or hydrogen peroxide to destroy or detoxify organic contaminants as water flows into a treatment tank. The reaction

nanotechnology presents a number of potential environmental benefits.

kiln, fluidized bed, liquid injection, and infrared.

condensation and phased separation processes [68].

products are dechlorinated materials and chlorine gas [70-71].

**2.5. Novel remediation techniques** 

*2.5.1. Nanotechnology and remediation* 

*2.5.2. Steam stripping* 

*2.5.3. Dehalogenation* 

material [69].

resulting reduction of toxicity.

*2.5.4. Chemical reduction/oxidation* 

*2.5.5. Ultraviolet (UV) oxidation* 

Excavation (and removal) is a fundamental remediation method involving the removal of contaminated soil/media, which can be shipped off-site for treatment and/or disposal, or treated on-site when contaminants are amenable to reliable remediation techniques.

Excavation is generally utilized for localized contamination and point source and is also used for the removal of underground structures that are out of compliance or have been identified as a potential or actual point source of contamination. The limiting factor for the use of excavation is often represented by the high unit cost for transportation and final offsite disposal. EPA (1991) further stated some limiting factors that may limit the applicability and effectiveness of the process to include:


In this respect, the on-site removal and treatment can often yield significant savings and, in addition, the treated soil may have beneficial secondary use (e.g. as construction fill or road base material) at the same site.

#### **2.4. Thermal treatment**

#### *2.4.1. Thermal desorption*

Thermal desorption technology is based on a physical separation system. The process desorbs (physically separates) organics from the soil without decomposition. Volatile and semi-volatile organics are removed from contaminated soil in thermal desorbers at 95-315°C for low-temperature thermal desorption (also called soil roasting), or at 315-340°C for hightemperature thermal desorption. To transport the volatilized organics and water to the gas treatment system, the process uses an inert carrier gas. The gas treatment units can be condensers or carbon adsorption units, which will trap organic compounds for subsequent treatment or disposal. The units can also be afterburners or catalytic oxidizers that destroy the organic constituents. The bed temperatures and residence times of the desorbers are designed to volatilize selected contaminants, not to oxidize them. Certain less volatile compounds may not be volatilized at low temperatures [62-63].

#### *2.4.2. Incineration*

For the remediation of soils polluted with organic compounds, incineration is the most widely used method. This method is very expensive and generates problems with air emissions and noise [64]. Incineration technology is intended to permanently destroy organic contaminants. Incineration is a complex system of interacting pieces of equipment and is not just a simple furnace. It is an integrated system of components for waste preparation, feeding, combustion, and emissions control. Central to the system is the combustion chamber, or the incinerator. There are four major types of incinerator: rotary kiln, fluidized bed, liquid injection, and infrared.

#### **2.5. Novel remediation techniques**

#### *2.5.1. Nanotechnology and remediation*

Nanotechnology has contributed to the development of a great diversity of materials as those used in electronic, optoelectronic, biomedical, pharmaceutical, cosmetic, energy, catalytic, and materials applications. As a general definition, nanotechnology is involved with objects on the nano scale, or materials measuring between 1 and 100 nm [65]. In future, modification and adaptation of nanotechnology will extend the quality and length of life [66]. The social benefits are significant from nanomaterials and the new products are applicable to information technology, medicine, energy, and environment. The emergence of nanotechnology presents a number of potential environmental benefits.

#### *2.5.2. Steam stripping*

76 Hydrocarbon

the presence of indigenous organisms capable of degrading the contaminants of interest, as well as nutrients necessary for growth. Also, it is necessary that the contaminants be available to the organisms, and not tightly sorbed to soil particles. Air sparing is not applicable in sites where high concentrations of inorganic salts, heavy metals, or organic

Excavation (and removal) is a fundamental remediation method involving the removal of contaminated soil/media, which can be shipped off-site for treatment and/or disposal, or

Excavation is generally utilized for localized contamination and point source and is also used for the removal of underground structures that are out of compliance or have been identified as a potential or actual point source of contamination. The limiting factor for the use of excavation is often represented by the high unit cost for transportation and final offsite disposal. EPA (1991) further stated some limiting factors that may limit the

ii. The distance from the contaminated site to the nearest disposal facility will affect cost.

iv. Transportation of the soil through populated areas may affect community acceptability. In this respect, the on-site removal and treatment can often yield significant savings and, in addition, the treated soil may have beneficial secondary use (e.g. as construction fill or road

Thermal desorption technology is based on a physical separation system. The process desorbs (physically separates) organics from the soil without decomposition. Volatile and semi-volatile organics are removed from contaminated soil in thermal desorbers at 95-315°C for low-temperature thermal desorption (also called soil roasting), or at 315-340°C for hightemperature thermal desorption. To transport the volatilized organics and water to the gas treatment system, the process uses an inert carrier gas. The gas treatment units can be condensers or carbon adsorption units, which will trap organic compounds for subsequent treatment or disposal. The units can also be afterburners or catalytic oxidizers that destroy the organic constituents. The bed temperatures and residence times of the desorbers are designed to volatilize selected contaminants, not to oxidize them. Certain less volatile

For the remediation of soils polluted with organic compounds, incineration is the most widely used method. This method is very expensive and generates problems with air

iii. Depth and composition of the media requiring excavation must be considered.

treated on-site when contaminants are amenable to reliable remediation techniques.

i. Generation of fugitive emissions may be a problem during operations.

compounds are present, as hinder microbial growth.

applicability and effectiveness of the process to include:

compounds may not be volatilized at low temperatures [62-63].

base material) at the same site.

**2.4. Thermal treatment** 

*2.4.1. Thermal desorption* 

*2.4.2. Incineration* 

The steam stripping method is based on a mass transfer concept, which is used to move volatile contaminants from water to air. Steam is injected through an injection well into the soil to vaporize volatile and semi-volatile contaminants [67]. The contaminated vapour steam is removed by vacuum extraction, and the contaminants are then captured through condensation and phased separation processes [68].

#### *2.5.3. Dehalogenation*

Dehalogenation of organic compounds is chemical displacement of a chlorine molecule and resulting reduction of toxicity.

#### *2.5.4. Chemical reduction/oxidation*

Chemical reduction/oxidation is a chemical conversion of hazardous contaminants to nonhazardous or less toxic compounds. The result is a more stable, less mobile and/or inert material [69].

#### *2.5.5. Ultraviolet (UV) oxidation*

Ultraviolet (UV) oxidation technology uses UV radiation, ozone, or hydrogen peroxide to destroy or detoxify organic contaminants as water flows into a treatment tank. The reaction products are dechlorinated materials and chlorine gas [70-71].

#### *2.5.6. Supercritical fluids extraction*

Supercritical fluids are materials at elevated temperature and pressure that have properties between those of a gas and a liquid. Under these conditions, the organic contaminant readily dissolves in the supercritical fluid. Supercritical fluids processes represent emerging technologies in the site remediation field. Few full-scale applications of Supercritical fluids are currently in existence [72-73].

Remediation of Contaminated Sites 79

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## **3. Conclusion**

A number of organic pollutants, such as PAHs, PCBs and pesticides, and inorganic pollutants are resistant to degradation and represent an ongoing toxicological threat to both wildlife and human beings. Bioremediation has grown into a green, attractive and promising alternative to traditional physico-chemical techniques for the remediation of hydrocarbons at a contaminated site, as it can be more cost-effective and it can selectively degrade the pollutants without damaging the site or its indigenous flora and fauna. However, bioremediation technologies have had limited applications due to the constraints imposed by substrate and environmental variability, and the limited biodegradative potential and viability of naturally occurring microorganisms. For the development of remediation processes to succeed commercially, it is essential to link different disciplines such as microbial ecology, biochemistry and microbial physiology, together with biochemical and bioprocess engineering.

In short, the key to successful remediation resides in continuing to develop the scientific and engineering work that provides the real bases for both the technology and its evaluation; and simultaneously in explaining and justifying the valid reasons which allow scientists and engineeres to actually use these technologies for the welfare and safety of a public which is more and more concerned about the environment and its protection.

## **Author details**

Arezoo Dadrasnia and C. U. Emenike *Institute of Biological Science, University of Malaya, Kuala Lumpur, Malaysia* 

N. Shahsavari *School of Biological Science, University Sains Malaysia, Penang, Malaysia Hajiabad branch, Islamic Azad University, Hajiabad, Hormozgan, Iran* 

## **Acknowledgement**

We wish to express our deepest gratitude to all the researchers whose valuable data as reported in their respective publications and cited in this chapter have been of considerable significance in adding substance. We are also grateful to our other colleagues and the anonymous reviewers whose constructive criticisms have benefited the manuscript, and brought it to its present form.

#### **4. References**

78 Hydrocarbon

*2.5.6. Supercritical fluids extraction* 

are currently in existence [72-73].

biochemical and bioprocess engineering.

Arezoo Dadrasnia and C. U. Emenike

**3. Conclusion** 

**Author details** 

N. Shahsavari

**Acknowledgement** 

brought it to its present form.

Supercritical fluids are materials at elevated temperature and pressure that have properties between those of a gas and a liquid. Under these conditions, the organic contaminant readily dissolves in the supercritical fluid. Supercritical fluids processes represent emerging technologies in the site remediation field. Few full-scale applications of Supercritical fluids

A number of organic pollutants, such as PAHs, PCBs and pesticides, and inorganic pollutants are resistant to degradation and represent an ongoing toxicological threat to both wildlife and human beings. Bioremediation has grown into a green, attractive and promising alternative to traditional physico-chemical techniques for the remediation of hydrocarbons at a contaminated site, as it can be more cost-effective and it can selectively degrade the pollutants without damaging the site or its indigenous flora and fauna. However, bioremediation technologies have had limited applications due to the constraints imposed by substrate and environmental variability, and the limited biodegradative potential and viability of naturally occurring microorganisms. For the development of remediation processes to succeed commercially, it is essential to link different disciplines such as microbial ecology, biochemistry and microbial physiology, together with

In short, the key to successful remediation resides in continuing to develop the scientific and engineering work that provides the real bases for both the technology and its evaluation; and simultaneously in explaining and justifying the valid reasons which allow scientists and engineeres to actually use these technologies for the welfare and safety of a public which is

We wish to express our deepest gratitude to all the researchers whose valuable data as reported in their respective publications and cited in this chapter have been of considerable significance in adding substance. We are also grateful to our other colleagues and the anonymous reviewers whose constructive criticisms have benefited the manuscript, and

more and more concerned about the environment and its protection.

*Institute of Biological Science, University of Malaya, Kuala Lumpur, Malaysia* 

*School of Biological Science, University Sains Malaysia, Penang, Malaysia Hajiabad branch, Islamic Azad University, Hajiabad, Hormozgan, Iran* 


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**Chapter 5** 

© 2013 Pampanin and Sydnes, licensee InTech. This is an open access chapter 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.

© 2013 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,

Crude oil is a complex mixture of hydrocarbons containing more than 17000 compounds [1]. Among the constituents of crude oil there is a group of substances called polycyclic aromatic hydrocarbons (PAHs). PAHs are aromatic compounds containing from two to eight conjugated ring systems. They can have a range of substituents such as alkyl, nitro, and amino groups in their structure [2]. Nitrogen, sulfur, and oxygen atoms can also be incorporated into their ring system [2,3]. The precursors for PAHs found in crude oil are natural products, such as steroids, that have been chemically converted to aromatic

The PAHs that are present in the marine environment in relevant concentrations are divided into two groups depending on their origin, namely pyrogenic and petrogenic [5]. Pyrogenic PAHs are formed by incomplete combustion of organic material while the petrogenic PAHs are present in oil and some oil products [4,6,7]. In general the pyrogenic PAHs are composed of larger ring systems then the petrogenic PAHs. Sources for pyrogenic PAHs are forest fires [6,7,8], incomplete combustion of fossil fuels [6,7,8], and tobacco smoke [6,7]. A range of PAHs are naturally present in crude oil [4,9,10] and coal [10,11] and these compounds are referred to as petrogenic PAHs. In the costal zones PAHs enters the water primarily from sewage, runoff from roads [12], the smelter industry [13,14,15] and oil spills [16,17], while offshore PAHs chiefly enter the water through oil seeps [18], oil spills [16], and produced water discharge from offshore oil installations

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

**Polycyclic Aromatic Hydrocarbons** 

**a Constituent of Petroleum:** 

**Presence and Influence in** 

**the Aquatic Environment** 

Daniela M. Pampanin and Magne O. Sydnes

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/48176

hydrocarbons over time [4].

[19].

**1. Introduction** 

