Chapter 12

Bioremediation of Oil Spills on Land

L.D. Brown, D.L. Cologgi, K.F. Gee, and A.C. Ulrich     University of Alberta, Edmonton, AB, Canada

Abstract

Bioremediation can be a viable mechanism for treating soils contaminated with petroleum hydrocarbons. Bioremediation strategies range from encouraging natural biodegradation processes (biostimulation), to supplementing the existing system with microorganisms able to degrade the contamination (bioaugmentation), to monitoring and verifying natural processes (natural attenuation). Application of bioremediation technologies are customized to specific site characteristics, as contaminated soil may be excavated for on- or off-site treatment at surface facilities (ex situ) or treated in place (in situ). In situ technologies are often cost-effective, but delivery and mixing of stimulants with the microorganisms and contaminants is challenging. Ex situ technologies allow for greater process control, but soil excavation is costly, disruptive, and increases exposure to contaminants. Microorganisms capable of degrading petroleum hydrocarbons have been found to be prolific in the subsurface. Environmental conditions can also be paramount for effective biodegradation; temperature, pH, salinity, nutrients, moisture, and redox condition may be altered to enhance or accelerate treatment. Biosurfactants may also provide an additional means of improving treatment by increasing the surface area of hydrophobic hydrocarbon compounds, thus increasing exposure to microorganisms. Bioremediation of soil contaminated with petroleum hydrocarbons provides a flexible, cost-effective, environmentally sustainable treatment strategy that can be tailored to site-specific conditions and requirements.

Keywords

Bioaugmentation; Bioremediation; Biostimulation; Hydrocarbons; Oil spill

12.1. Introduction

Biodegradation of hydrocarbons by naturally occurring populations of microorganisms is one of the primary mechanisms by which petroleum is removed from the environment [1]. Biodegradation of petroleum hydrocarbons in soil differs greatly from aquatic environments due to the predominantly vertical infiltration of the oil contamination into the subsurface, where sorption processes may not only reduce the toxicity of oil components, but may also serve as a persistent source of contamination [1]. In addition, volatile organic compounds may become trapped in the soil matrix, potentially causing toxic effects on microorganisms [1]. This chapter focuses on treating the solid, or soil, phase in both unsaturated and saturated zones in the subsurface.

12.2. Brief Overview of Bioremediation Techniques for Land Oil Spills

Bioremediation involves harnessing the natural biodegradation processes of living organisms. This process can be further accelerated or encouraged via alteration of the contaminated media. Bioremediation of contaminated soils has been used worldwide, with many case studies taking place in Europe and the United States [2]. In remote areas, bioremediation may represent the most practical and cost-effective means of treating soils contaminated with petroleum hydrocarbons [3]. A flowchart showing the remediation technologies discussed in this chapter is given in Fig. 12.1.
image
Figure 12.1 Bioremediation techniques for land-based oil spills.

12.2.1. In Situ Versus Ex Situ

Bioremediation technologies may be employed on-site or off-site and in situ or ex situ. In situ bioremediation involves microbial biodegradation of the contamination within the subsurface soil/water matrix. No excavation of the contaminated soil takes place, but techniques such as groundwater pumping or vacuum aeration may be required to distribute constituents, such as nutrients or oxygen, required for enhanced degradation [2]. Ex situ technologies require the excavation of the contaminated soil, which may be treated on- or off-site. Contamination of surface soils may be easily treated by ex situ techniques, but treatment of deeper contamination is more difficult and in situ techniques are likely to be more appropriate [4].
In situ treatment approaches are typically more cost-effective, but it can be challenging to ensure that the required amendments are delivered effectively through the contaminated region, and that the appropriate level of treatment has been achieved. The main advantage of ex situ technologies lies in process control, but excavation of soil is disruptive, costly, and increases exposure to contaminants.

12.2.2. Biostimulation Versus Bioaugmentation

On a fundamental level, bioremediation may be enhanced by either biostimulation and bioaugmentation [2]. Biostimulation involves the alteration of a site's physical or chemical characteristics to enhance the natural biodegradation processes performed by the indigenous (native to the contaminated area) microorganisms [2]. Bioaugmentation entails the addition of microorganisms selected for their ability to biodegrade the contaminants of concern in the system. Both techniques can be used in combination for both in situ and ex situ remediation schemes, and will be discussed in more detail later in the chapter.

12.3. Key Organisms Involved in Biodegradation of Oil Spills on Land

Due to the ubiquitous presence of hydrocarbons in the environment, microorganisms able to biodegrade petroleum hydrocarbons are plentiful in soil. Heterotrophic (organisms that utilize organic carbon as a food source) bacteria and fungi are the predominant microorganisms biodegrading hydrocarbons [1]. Many studies have found that microbial diversity is decreased in contaminated soil, likely due to the selection and subsequent dominance of microorganisms able to degrade petroleum hydrocarbons within the indigenous community, as well as the toxic effects of petroleum hydrocarbons on other organisms [5,6]. These same studies have also shown a significant increase in the proportion of petroleum hydrocarbon-degrading bacteria in contaminated soils. Table 12.1 provides a number of identified bacteria, fungi, and earthworms able to utilize various hydrocarbons as a food source.

Table 12.1

Examples of Hydrocarbon-Degrading Microorganisms Identified in Field and Laboratory Studies

Microorganism
GenusSpeciesRedox ConditionCarbon SourceReferences
PseudomonasFluorescensAerobicOil[7]
sp.aAerobicDiesel[8]
sp.AerobicOil[5]
sp.AerobicCrude oil[9,10]
ETB 001/2, 005/2AerobicRefinery oil[11]
PutidaAerobicCrude oil[12,13]
BacillusMycoidesAerobicOil[7]
sp.AerobicCrude oil[9,10]
SerratiaMarcescensAerobicOil[7]
RhodoccusRhodochrousAerobicOil[14]
sp.AerobicOil[5]
Baikonurensis EN3AerobicDiesel[15]
sp.AerobicRefinery oil[11]
RuberAerobicCrude oil[16]
Ralstoniasp.AerobicOil[5]
CyanobacteriumMastigocladusAerobicGasoline[17]
Micrococcussp.AerobicCrude oil[9,10]
Proteussp.AerobicCrude oil[9]
Acinetobactersp.AerobicDiesel[18]
sp.AerobicRefinery oil[11]
sp.AerobicCrude oil[10]
Mycobacteriumsp.AerobicRefinery oil[11]
Arthrobactersp.AerobicRefinery oil[11]
sp.AerobicCrude oil[10]
Flavobacteriumsp.AerobicCrude oil[10]
Table Continued

image

Microorganism
GenusSpeciesRedox ConditionCarbon SourceReferences
Moraxellasp.AerobicCrude oil[10]
Corynebacteriumsp.AerobicCrude oil[10]
Fungi: PleurotusOstreatusAerobicTar[19]
AerobicOil[20]
Yeast: CandidaTropicalisAerobicCrude oil[21]
Earthworm: EiseniaFetidaAerobicCrude oil[22]
Earthworm: AllolobophoraChloroticaAerobicCrude oil[22]
Earthworm: LumbricusTerrestrisAerobicCrude oil[22]
Fungi: PhanerochaeteChrysosporiumAerobicOil[20]
Fungi: CoriolusVersicolorAerobicOil[20]
Bacillussp.AnaerobicWaste oil[23]
Arthrobactersp.AnaerobicWaste oil[23]
Clavibactersp.AnaerobicWaste oil[23]
Corynebacteriumsp.AnaerobicWaste oil[23]
Nocardiasp.AnaerobicWaste oil[23]
Azoarcus (Thauera)sp.AnaerobicBTEX[24,25]
Geobactersp.AnaerobicToluene[24]
Dechloromonassp.AnaerobicBenzene[24]

image

a Species.

12.3.1. Communities Versus Isolates

Numerous studies have demonstrated that individual organisms can only metabolize a limited number of hydrocarbon compounds, so mixed populations of microorganisms are able to more readily biodegrade the complex mixtures of hydrocarbon compounds found in oil [1,5,6]. Benka-Coker and Ekundayo isolated indigenous microorganisms capable of degrading hydrocarbons, but found that 20% greater degradation of crude oil was achieved with mixed cultures versus a pure culture of Micrococcus sp. after 14 days. In some cases, sequential degradation of compounds may also occur, meaning that degradation of a hydrocarbon compound is done in steps by a number of different species rather than completely mineralized to CO2 by a single organism [2].

12.4. Environmental Factors Affecting Bioremediation

Two principle sources of rate limiting factors for field applications of bioremediation technologies are biochemical/microbiological factors (the presence of the appropriate hydrocarbon-degrading microbial community) and environmental factors (listed in the following sections) [2]. In some cases, changing the environmental conditions of the site may be required to enhance biodegradation, especially with contaminated sites that have been stagnant for extended periods of time.

12.4.1. Temperature

Temperature is an important environmental factor impacting bioremediation because the physical nature and chemical composition of the oil, microbial metabolism, and composition of the microbial community are all temperature dependent [1]. With decreasing temperature, the viscosity of oil increases and potentially toxic volatile compounds become less volatile and more water soluble, thus potentially delaying the onset of biodegradation [1]. In general, microbial metabolism rates decrease and the acclimation period for growth of microorganisms is longer with decreasing temperature. A study of the biodegradation of diesel found that microbial activities peaked in a few days at 20°C versus 3 weeks at 6°C, and carbon dioxide production was almost five times greater at the higher temperature [3]. Another study focusing on benzene, toluene, ethylbenzene, and xylene (BTEX) compounds found that in general, attenuation rates decrease approximately 50% for every 10°C decrease in temperature [26].
Psychrotolerant bacteria, or those that grow optimally at low temperatures, have been readily isolated from hydrocarbon-contaminated polar soils, growing at temperatures less than 10°C [27], and biodegradation has been observed in soils as cold as −1 to −3°C [27]. Although mineralization will occur at low temperatures, the rate, and perhaps even the extent, of biodegradation will be higher at elevated temperatures [27].
Thermophilic, or “heat-loving,” microorganisms grow optimally at temperatures between 40 and 70°C and are dominant in a number of ex situ treatment technologies, including composting and aerated biopiles [28]. Although two cases of temperature-sensitive microorganisms are given here, most of the microbial world in the subsurface flourishes at temperatures between these two extremes, and Mohammed et al. report that optimal petroleum hydrocarbon biodegradation will occur between 10 and 30°C [2].

12.4.2. pH

Most heterotrophic bacteria and fungi prefer neutral pH environments, but terrestrial environments are more variable compared to aquatic ecosystems, where soil pH can vary from 2.5 to 12.0 [1] due to soil chemistry [27]. Optimum hydrocarbon biodegradation occurs around pH 6.5–8.0 [27].
Soil pH may be lowered due to fertilizer application or the production of aliphatic acids during alkane biodegradation [27]. Adjustment of pH can be achieved by the addition of lime or sulfur, for example, for alkaline or acidic correction, respectively.
Margesin and Schinner hypothesize that the persistence of aromatic hydrocarbons in the environment is due to sensitivity of aromatic degraders to pH [28]. Chang et al. observed that the biodegradation rate constants for various polycyclic aromatic hydrocarbons (PAHs) doubled when pH changed from 7.0 or 9.0 to the optimal pH value of 8.0 [29].

12.4.3. Salinity

Although microorganisms that require salt for growth (halophiles) do exist, it is generally assumed that there is an inverse relationship between salinity and the biodegradation of petroleum hydrocarbons [28]. However, there is little information on the effects of salinity on the bioremediation of petroleum-contaminated soils [28]. One study found that artificial salinity at a level comparable to an oilfield brine inhibited oil degradation by 20–44% [28]. Salt concentrations greater than 1% (w/v) inhibited biodegradation of weathered hydrocarbons from production facilities by increasing lag time and decreasing mineralization rate, even reducing the extent of mineralization in some cases [30]. However, at lower levels salinity may not always be inhibitory. Ulrich et al. also found that salt concentrations in the range of 0.5–1% (w/v) stimulated microbial activity [30]. Margesin and Schinner also hypothesize that microorganisms found in natural saline soil environments may be impacted less by high salt concentrations due to acclimation [28].

12.4.4. Nutrients

In order for bioremediation to proceed, nutrients must be present in quantities and proportions necessary for microorganisms to thrive. In general, micronutrients, namely, trace metals such as calcium, magnesium, sulfur, and zinc, tend to be present in quantities sufficient for microbial growth, so no supplementation is required. However, some soil environments may be deficient in nitrogen and/or phosphorus [31], which are the most important macronutrients for microbial growth. While no consensus has been reached in the literature on optimum soil nutrient levels, carbon to nitrogen ratios for enhancing biodegradation of hydrocarbons range from 200:1 to 9:1 [27] and the United States Environmental Protection Agency recommends a carbon to nitrogen to phosphorus ratio ranging from 100:10:1 to 100:1:0.5 for bioremediation technologies [32]. In the event a contaminated site is limited in these nutrients, supplements such as fertilizer and other sources of nitrogen and phosphorous can be added to enhance bioremediation. The addition of these compounds in varying proportions has been successfully utilized in several studies [3,33,34]. However, it is important to note that nitrogen concentrations in the soil that are either too high or too low will inhibit biodegradation [3]. This means that care must be taken when utilizing inorganic fertilizers on soils with low water-holding capacity (the amount of water a soil can hold for crop use) because overfertilization may cause osmotic stress; slow release fertilizers, such as those used for gardening, are recommended [27,35,36].
The use of this technique in conjunction with bioaugmentation will be discussed later in the chapter.

12.4.5. Moisture

Moisture plays an essential role in ensuring that microorganisms maintain growth and metabolic activity [1]. It is therefore unsurprising that biodegradation may be limited in soils with low moisture content (below 30%) [1]. Conversely, excessively high moisture levels such as those found in water-logged soils, may also negatively impact degradation due to low oxygen availability [1]. The moisture content of the contaminated soil impacts the dissolution of petroleum hydrocarbon compounds and thus substrate diffusion and availability, as well as microbial transport. Most studies have indicated that the optimum moisture content is 50–70% of the water-holding capacity of the soil [2]. An additional consideration is that the soil grains coated in oil can render the soil more hydrophobic, reducing water-holding capacity [27].

12.4.6. Redox Environment

In order to gain energy for growth and maintenance of cellular activities, microorganisms carry out an electron-transfer process, removing electrons from the electron donor (in this case, petroleum hydrocarbons), and catalyzing its transfer to an electron acceptor (oxygen, nitrate, sulfate, and iron and other metals). The redox environment, which refers to the terminal electron acceptor reduction process dominant in the area of interest, biochemically influences what microbial populations exist and are active in the area. This means that each redox zone will be enriched for microorganisms able to carry out the reduction of the electron acceptor indicated. Fig. 12.2 shows an example of a redox ladder and zones within the contaminated subsurface. Electron acceptors with a higher redox value (i.e., O2) represent a more aerobic environment with ample oxygen available for use. Electron acceptors with a lower redox value (i.e., CO2) represent a more anaerobic or O2-depleted zone. The amount of oxygen available in the soil matrix is dependent on microbial oxygen consumption and the replenishment of oxygen, which is influenced by the type of soil, and whether the soil is saturated or unsaturated [1].
image
Figure 12.2 Redox zones within a contaminant plume.
Historically, anaerobic biodegradation of petroleum hydrocarbons was thought to occur at negligible rates and thus was considered insignificant [1]. Researches in the late 1980s and 1990s provided evidence that aromatic hydrocarbons are degraded under denitrifying, iron-reducing, sulfate-reducing, and fermentative and methanogenic conditions, with iron reduction, fermentation and methanogenesis dominating the anaerobic processes at several sites [4]. In a soil column study, Boopathy found that total petroleum hydrocarbons (TPH) were removed by 88% in 310 days from diesel-contaminated soil when mixed anaerobic electron acceptors were provided, versus 61% solely under sulfate-reducing conditions [37].
The limited solubility of oxygen in water and the rapid consumption of available dissolved oxygen by microorganisms have resulted in new perspectives on enhancing particular anaerobic environments through the addition of alternative electron acceptors, such as nitrate and sulfate [4]. However, consideration must be given to abiotic reactions that may occur, as well as the impacts of injecting these compounds into the subsurface. Authorities may also be concerned with the potential accumulation in groundwater, where negative effects on drinking water may take place [4].

12.4.7. Soil Type

Soil type greatly influences permeability, which Mohammed et al. identify as one of the most important factors in in situ bioremediation, and the use of in situ treatment for soil with hydraulic conductivity of less than 10−4 cm/s is not recommended [2]. As highlighted later in this chapter, this is because water is required to transport nutrients, microorganisms, substrate, and electron acceptors, and permeabilities less than 10−4 cm/s can inhibit this transport [2].
Properties of soils that impact sorption, such as organic matter and surface charge, also impact bioavailability and biodegradation of hydrocarbon compounds. Hydrophobic petroleum hydrocarbons will partition to those soils with high clay or organic content and be rendered unavailable to microorganisms [38].
A study on diesel-contaminated soil compared the effects of sand and gravel soils on degradation [3]. Cumulative carbon dioxide production for the contaminated sand was double for the gravel and this contrast is attributed to the increased surface area and porosity of the sand, increasing bioavailability of the contaminants and oxygen supply to microorganisms.

12.5. In Situ Bioremediation Strategies

In situ bioremediation techniques focus on initiating or enhancing the degradation of the contaminants of concern in the subsurface. According to Brown and Crosbie [39], unsaturated and residually saturated soils contain the most significant petroleum hydrocarbon contaminant load and makes for a continual source of groundwater contamination, if left untreated. When soil excavation is prohibitively expensive or difficult, as might be the case with deep subsurface contamination (such as leaking underground storage tanks) or sites that are in close proximity to structures, in situ bioremediation strategies are likely a more effective means of remediation.
A key first step in in situ bioremediation is the evaluation of site conditions, the bioavailability of contaminants, and the assessment of limiting factors that may need to be altered during treatment [31].

12.5.1. Monitored Natural Attenuation

According to Jorgensen [4], natural attenuation is defined as “the natural degradation of contaminants without human intervention.” Similarly, monitored natural attenuation is an in situ bioremediation strategy, whereby no human intervention occurs, but extensive monitoring of the natural processes in the soil and groundwater are conducted to establish a case for reduction in mass, toxicity, volume, or concentration of contaminants [4]. This technique is not a “do-nothing” approach. Typically, monitoring requirements are more extensive than with other remediation technologies and a case for treatment within a reasonable timeframe must be established [32]. Often, monitored natural attenuation is utilized after more active treatment techniques have been employed or in the areas of dilute contamination [32].
Intrinsic bioremediation will involve both aerobic and anaerobic biodegradation, as microorganisms in the subsurface exhaust the available supply of each terminal electron acceptor, as shown in Fig. 12.2. In addition to biodegradation, physical and chemical processes such as dilution, sorption, and volatilization can also occur in the subsurface and reduce contaminant concentrations.
Monitoring requirements include a thorough understanding of groundwater conditions, as well as obtaining direct evidence that contaminant biodegradation is occurring. The concentrations of the contaminants of concern are closely monitored, and documented evidence of decreasing concentrations at the site must be provided. Evidence of microbial activity also indicates the occurrence of biodegradation. Observing changes in the geochemical conditions within the contaminant plume must also provide indirect evidence that biodegradation is occurring as well [4]. Contaminant compound stable isotope analysis has also emerged as a technique for demonstrating whether biodegradation or abiotic processes have transformed the contaminant of concern, because the ratio of stable isotopes will change with the extent of degradation [40].
In some situations, natural attenuation is preferred over intervention. Several studies report that enhanced bioremediation techniques will only slightly increase the speed or efficacy of the process [41,42], but the authors suggest that the added time and cost of materials and/or manpower required to carry out biostimulation or bioaugmentation is not worthwhile, when similar results can eventually be obtained via natural attenuation [41,42].
For a more thorough description of the monitoring requirements for natural attenuation, the reader is strongly encouraged to consult Wiedemeier and Haas' 2002 publication on the subject [43].
A field example where monitored natural attenuation was utilized is provided in Table 12.2.

12.5.2. Enhancement of In Situ Bioremediation

In situ bioremediation can be enhanced by providing amendments to improve contaminant biodegradation beyond what may occur naturally. Amendments may include nutrients, terminal electron acceptors, and surfactants, all intended to improve the existing conditions in the subsurface for enhanced bioremediation.
Injection wells are utilized for contamination deep in the subsurface, but infiltration methods, such as spray irrigation or ditches, may suffice for shallow contamination [44]. Recirculation of treated groundwater from the site may be utilized for delivery of amendments.
Nitrogen and phosphorus are often required because contaminated soil has been depleted of these macronutrients due to biodegradation of the increased carbon loading by petroleum hydrocarbons. Oleophilic fertilizers (those with an affinity for oils) are widely used as they adhere to hydrocarbons, providing nutrients at the oil–water interface [31].

Table 12.2

Field Results From Select In Situ Bioremediation Studies

Site DescriptionContaminantTreatment Technique% ReductionReferences
(Analytical Method)
Municipal vehicle maintenance yardDiesel, motor oil, gasoline, automotive fluidsEnhanced bioremediation—hydrogen peroxide and nitrogenTPHa as gasoline 99%[45]
(GCb)
TOGc 84%
(Infrared)
Industrial siteMixture of petroleum hydrocarbonsEnhanced bioremediation—hydrogen peroxide, fertilizer, surfactantOil 100%[31]
Lubricant 99%
Gas oil 100%
(GC-FIDd, GC-MSe)
∗In groundwater
Refinery siteOil hydrocarbonsEnhanced bioremediation—treated groundwater with nutrients, surfactant, microorganismsTotal oil hydrocarbons 86% in 15 weeks[11]
(GC-FID)
Former waste oil refineryOil hydrocarbonsEnhanced bioremediation—nitrogen, phosphorus, oxygen, hydrogen peroxideOil 50–70% in 300 days[23]
(GC)
Alpine skiing areaDiesel oilMonitored natural attenuationTPH 50% in 780 days[46]
(infrared)

image

∗ The analysis was completed on groundwater.

a Total petroleum hydrocarbons.

b Gas chromatography.

c Total oil and grease.

d Gas chromatography–flame ionization detector.

e Gas chromatography–mass spectrometer.

Aerobic biodegradation is generally preferred and most commonly used due to the speed of the process, and because fewer undesirable end products are produced, as can occur with anaerobic degradation [2]. Oxygen may be provided via air sparging, bioventing, or through the injection of chemicals, such as hydrogen peroxide or nitrate [39]. Hydrogen peroxide is more widely used due to its high oxygen-releasing potential [31], but Brown and Crosbie point out that high operating cost may result if soils contain high levels of iron or manganese, because hydrogen peroxide will decompose due to metal catalysis [39]. Anaerobic biodegradation may be enhanced by the injection of nitrate, but in many jurisdictions, regulations exist for maximum allowable nitrate concentrations in groundwater [39].
Surfactants, or surface-active agents that reduce surface tension and stabilize emulsions [10], may be injected into the subsurface, or allowed to infiltrate through the unsaturated zone, to increase bioavailability of contaminants to microorganisms [31]. Bioaugmentation may also be employed to improve biodegradation. Both will be discussed in Section 12.7.
Examples of the successful field application of enhanced bioremediation are provided in Table 12.2.

12.5.3. Bioventing

Bioventing enhances aerobic biodegradation in the subsurface by supplying air or pure oxygen into the unsaturated zone through gas injection or extraction wells installed into the soil (Fig. 12.3) and was one of the first in situ technologies applied at a large scale in the 1990s [4]. In contrast with soil vapor extraction, a physical remediation technique, bioventing employs very low airflow rates to minimize volatilization of hydrocarbons. Mid-weight petroleum products, such as diesel and jet fuel, are particularly well suited to bioventing treatment; gasoline, due to rapid volatilization, can be removed more rapidly via soil vapor extraction [32].
image
Figure 12.3 Typical bioventing system.
The installation and use of bioventing systems is an efficient method for providing oxygen to unsaturated contaminated soils for aerobic biodegradation [39]. Air may be added to the soil either by injection or vacuum withdrawal; Brown and Crosbie highlight that withdrawal is more common because volatile organic compounds will also be removed [39]. However, a requirement to treat volatiles prior to releasing withdrawn air to the atmosphere will potentially increase the treatment costs significantly. Despite this, Brown and Crosbie identified bioventing as the most cost-effective means of providing an oxygen source for in situ bioremediation [39]. Bioventing also provides the added opportunity to pump warm and/or humidified air into the subsurface to extend the season that bioremediation is effective in colder or drier climates.
Soil permeability is the most significant factor in evaluating the applicability of bioventing at a contaminated site [32]. Cost is driven primarily by the extent of the contamination and soil permeability, as both parameters impact the number of injection/extraction wells that are required [44]. Bioventing is considered ineffective for clays and silts [32].

12.6. Ex Situ Bioremediation Strategies

Ex situ bioremediation strategies require that contaminated soil be excavated, and relocated to where it can then be treated either on- or off-site. Ex situ techniques facilitate greater control on environmental conditions, enabling biodegradation rate optimization [27]. Due to the ability to homogenize contaminated soil, treatment is typically more uniform and requires less time than in situ treatment techniques. However, ex situ technologies are more costly due to excavation, site preparation, and operation. In addition, excavation of soil increases exposure to, and mobility of, contaminants.
Site preparation for most ex situ bioremediation technologies requires the construction of a liner system in the treatment area to prevent the movement of contaminants into the subsurface, and surface water runoff control systems to prevent off-site transport. Other requirements include measures to maintain optimal environmental conditions. This could include nutrient and moisture application, or aeration via blower systems or mechanical agitation. Ex situ treatment technologies include landfarming, windrows and biopiles, which are described in detail in Sections 12.6.1 and 12.6.2.
Results from field application of ex situ treatment technologies are given in Table 12.3.

Table 12.3

Field Results From Select Ex Situ Bioremediation Programs

Site DescriptionContaminantTreatment Technique% ReductionReferences
(Analytical Method)
Kuwait desertCrude oilLandfarmingTPHa 82.5%[47]
Windrow composting pilesTPH 74.2%[47]
Static biopilesTPH 64.2%[47]
(Infrared)
Kuwait desertCrude oilWindrow pilesTPH ∼60%[48]
(GC-FIDb)
Oil field treatment facilityOily waste from crude oil productionLandfarmingOrganic fraction 78%[49]
(Soxhlet, TLCc)
Agricultural soil contaminated from leaking pipelineCrude oilBiopilesUp to 57%[16]
∗No aeration(Gravimetric)
Contaminated soil at refineryHydrocarbonsLandfarmingTPH 67–75%[50]
(Infrared)
Heavy vehicle maintenance yardOily wasteLandfarmingTPH 74%[51]
(GC)

image

∗ The analysis was completed on groundwater.

a Total petroleum hydrocarbons.

b Gas chromatography–flame ionization detector.

c Thin layer chromatography.

12.6.1. Landfarming

Landfarming involves application of contaminated soil in a 12–18 inch layer with nutrient and moisture addition, and tillage to increase aeration, stimulate, and enhance biodegradation [27,32]. A diagram of a typical landfarming system is shown in Fig. 12.4 [32].
Landfarming may also refer to the placement of thin lifts of soil in lined beds, versus applying directly to the land [44]. If contaminated soil is applied directly to the land without a barrier in place, on- and off-site impacts on ground and surface water, air, and the food chain must be prevented. As with biopiles, a liner is often utilized to prevent this contamination from occurring.
image
Figure 12.4 Typical landfarming system [32].
Bleckmann et al. highlighted that the petroleum industry has been successfully using landfarming for treatment and disposal for decades [49]. Landfarming is well suited to accommodate frequent additions of new waste, and is therefore effective for applications producing continuous waste streams [51]. This method is particularly effective in areas with low rainfall (275 mm), high evaporative climates (annual evaporation of 2700 mm), and large areas of available land [51]. Shallow contamination may be treated with this technique without the need for excavation [32].
A key limitation of land treatment is the loss of volatile organic contaminants to the atmosphere [51]. Legislation may prevent the use of landfarming as a remediation strategy, if air emissions of volatile organic compounds must be controlled.
During a one-year assessment of three ex situ bioremediation technologies (landfarming, windrows, and biopiles), landfarming resulted in the greatest reduction of oil contamination [47]. About 91% of total alkanes and 82.5% of TPH were degraded, where the soil was amended with nitrogen, phosphorus, and wood chips [47]. Field-scale landfarming successfully reduced soil concentrations of diesel contamination to target levels, but it is unknown to what degree volatilization contributed to these losses [27].
While the basic principles of landfarming, as described above, are consistent between field studies, every contaminated site is different, and a single remediation technique is not necessarily appropriate in every situation. For this reason, each remediation scheme should be tailored specifically for the environmental conditions and contaminants present. For example, a laboratory study investigating the optimization of environmental conditions for landfarming of oily sludges indicated that the addition of micronutrients supplied via dilution of Hoagland trace element solution and organic supplements (yeast extract and dried domestic sewage sludge) was not beneficial [52], demonstrating that the addition of external nutrients is not always necessary. The authors also found that the optimal conditions include a soil water-holding capacity of 30–90%, a pH of 7.5–7.8, C:N and C:P ratios of 60:1 and 800:1, respectively, and a temperature of 20°C or greater. Biodegradation of saturated hydrocarbons was greatest at low oil sludge application rates, but the opposite was true for aromatic hydrocarbons which were degraded more readily when oil sludge was added to the system more frequently. Thus, the authors concluded that an application rate of 5% w/w oil sludge was a good compromise between biodegradation rates of all hydrocarbon compounds and efficient land use.
Similarly, Chagas et al. demonstrated that biostimulation and bioaugmentation did little to enhance landfarming bioremediation of soil contaminated with diesel oil, but a difference in the proportions of the degraded hydrocarbons was observed. PAHs with fewer aromatic rings were more easily degraded using bioaugmented and/or biostimulated remediation, while compounds with four or more rings were more readily degraded using unamended land farming [53].

12.6.2. Biopiles

The biopile technique involves mixing excavated soils with amendments and placing the material in multiple lifts into a “pile” on a treatment area that encompasses both a leachate collection system and some form of aeration, usually accompanied by a system to deliver moisture and possibly nutrients to the soil [44]. A typical system is shown in Fig. 12.5.
Aeration may be achieved by placing soil in static piles with aeration pipes placed below or within the pile (referred to as a “biopile” [54]), or the piles are turned regularly with heavy equipment (referred to as a “windrow” [54]). In-vessel (contained) systems are uncommon due to high costs; windrows are considered to be the most cost-effective biopile method, and mechanical agitation facilitates the addition of moisture or nutrients [44]. Soil piles may be covered, enabling more favorable temperature and moisture conditions for effective bioremediation.
Typically, piles are placed on an impermeable liner to prevent seepage of contamination into the subsurface soils. Often front-end loaders are utilized for periodic turning and windrow construction, but dedicated windrow-turning machinery can be used.
Contaminated soil is often amended with bulking agents such as sawdust and straw, which function to increase air space in the matrix, and organic amendments such as fertilizer to ensure the required carbon to nitrogen ratio exists [32].
Al-Daher et al. evaluated the use of turned windrows at pilot scale following its selection as the most appropriate technology for remediation of oil-contaminated soil in the Kuwait desert [48]. On average, 60% and 55% reductions in TPH and total PAHs were observed, respectively. Wood chips, dried sewage sludge, and mature compost were assessed as soil amendments, but none significantly impacted hydrocarbon degradation. Covering the windrow during the summer months increased the moisture content from 3% to 12% and resulted in a 19.3% increase in total petroleum hydrocarbon degradation over 3 months, indicating that maintaining adequate moisture within the system is an important consideration when optimizing the remediation process. Static biopiles were found to reduce total petroleum hydrocarbon concentrations less than both windrow piles and landfarming, but resulted in significantly lower operation and maintenance costs, smaller operating area, and required less water for irrigation [47].
image
Figure 12.5 Typical biopile system [32].
Eszenyiova et al. determined during a pilot-scale experiment that turned windrows reduced the time required to decontaminate soil by two and a half to three times, compared to land treatment [50].

12.7. Enhanced Bioremediation

12.7.1. Biostimulation Strategies

Biostimulation improves the conditions for biodegradation to occur, either by indigenous microorganisms or inoculants. Apart from improving environmental conditions for microorganisms via the addition of nutrients, moisture, or an electron acceptor, additional biostimulation strategies exist to improve biodegradation.

12.7.1.1. Organic and Nutrient Amendments

Amending excavated contaminated soil with organic amendments is common, especially for biopile treatment technologies. Amendments may serve to increase air space within the soil matrix, thereby improving conditions for aerobic degradation, supplying nutrients for microbial growth, increasing the water-holding capacity of the soil, and supplying viable microbial populations [55]. Examples of organic amendments include, but are not limited to, agricultural crop residues such as straw, animal manure, wastewater biosolids, and commercial waste products such as sawdust.
Pometto et al. demonstrated in a laboratory study that biodegradation of petroleum hydrocarbons was increased by 20% when soil was mixed with soybean hulls [55]. A comparative study assessed peat or sawdust as a spill adsorbent and subsequent organic nutrient provider, and established that while peat increased alkane degradation by only 1.5% over sawdust, it also provided a soluble carbon source for sustaining biomass [56]. Although biosolids have been found to stimulate biodegradation of hydrocarbons in soil, Rivera-Espinoza and Dendooven concluded that the addition of biosolids or maize to clayey soil contaminated with polycyclic aromatic hydrocarbons did not accelerate biodegradation [57].
Altering the concentrations and ratios of essential nutrients, such as nitrogen and phosphorus, or the addition of fertilizers can also be utilized to stimulate biodegradation processes. This was discussed in detail in Section 12.4.4.
The addition of amendments may be dictated more by what is locally available versus what has been found to be most effective in improving biodegradation from literature studies.

12.7.1.2. Biosurfactants

Biodegradation of petroleum hydrocarbons may be limited by the availability of the contaminants to microorganisms in the soil [58]. The addition of synthetic or biogenic biosurfactants (amphiphilic, low molecular weight, biologically produced compounds which reduce surface and interfacial tension between two phases at their interface, and can increase the dispersion and bioavailability contaminants such as oil [59]) or biosurfactants (amphiphilic, higher molecular weight biopolymers or exopolysaccharide compounds which are responsible for solubilizing nonsoluble substrates, as well as stabilizing emulsions to prevent mixing of compounds, but are not surface-active like biosurfactants [59]) has been considered as a means for increasing accessibility of hydrocarbon compounds to microorganisms for biodegradation [2,60]. Since microorganisms are only able to degrade hydrocarbons at the oil–water interface, increasing the surface area of this interface will greatly increase the biodegradation potential.
The release of biosurfactants by bacteria and fungi represents an important process for the uptake of hydrocarbons [1]. There is a wide diversity of microorganisms able to produce biosurfactants, as well a variety of chemical structures [60]. The function of biosurfactants/bioemulsifiers is dependent both on the composition of the compound, as well as the environmental conditions in which it is used [60] Additionally, those microorganisms that are able to produce biosurfactants or emulsifiers may have a selective advantage to biodegrade hydrophobic hydrocarbon compounds, especially at lower temperatures where the effects of viscosity and water solubility are more intense [27]. For example, some Rhodococci have been found to produce cell-associated biosurfactants for alkanes that are solid at low temperatures [27]. Banat et al. isolated numerous bacterial species that produced biosurfactants [10]. A disadvantage of the use of biosurfactants is the high cost of production, which limits the feasibility of their use in large-scale applications such as remediation of polluted sites [61]. However, recent studies have shown promise in using alternative, low-cost sources to feed biosurfactant-producing organisms. If optimized, agricultural and food wastes could provide an economical solution to this bottleneck [61].
Martins et al. utilized the filamentous fungus Aspergillus fumigatus to produce a biosurfactant using solid state fermentation [62]. A 99% reduction in both aliphatic and polycyclic aromatic hydrocarbons was observed when the biosurfactant was utilized, versus 90% for a chemical disperser. Similar results were obtained by comparing the TPH removal capacity of two biosurfactants (rhamnolipids, surfactins) and two synthetic surfactants (Tween-80, Triton X-100) on heavy oil-contaminated soil. The addition of biosurfactants resulted in the biodegradation of 62–63% TPH, while the synthetic compounds resulted in only 35–40% degradation [63]. Martienssen and Schirmer emphasize that the use of industrial surfactants for bioremediation purposes is not advisable, as these compounds may possess inadequate biocompatibility with microorganisms, sometimes even causing toxic effects [58,61]. Thus, they conclude that biosurfactants, or “close to nature” synthetic surfactants, will effectively improve bioavailability of substrates to microorganisms without negative impacts. Additionally, as a result of their natural origin, biosurfactants are generally more stable than their synthetic counterparts when subjected to the harsh environmental conditions often encountered at sites undergoing remediation activities [61].
Recent studies have also demonstrated the effectiveness of combining surfactants with biostimulation and bioaugmentation technologies. Jeong et al. investigated the use of surfactants to improve landfarming treatment at low temperatures. The researchers sprayed a surfactant foam compound containing psychrophilic (cold-loving) hydrocarbon-degrading microorganisms, and nutrients on the surface of diesel-contaminated soil [64]. Application of the foam served to enhance degradation of hydrocarbons by insulating the soil and keeping it ∼2.2°C warmer than the ambient temperature, and introduced the necessary nutrients and microorganisms, leading to a 74% reduction in TPH, compared to the 46% observed for landfarming alone [64].

12.7.2. Bioaugmentation Strategies

12.7.2.1. Microorganisms

Terrestrial environments tend to contain a larger number of microorganisms compared to aquatic environments, due to higher concentrations of organic and inorganic matter. Indigenous microbial populations that are adapted to the particular soil environment are expected to negatively impact seeded microorganisms' ability to survive and thrive due to competition [1]. Studies have shown, however, that the addition of microorganisms already acclimated to hydrocarbons to a contaminated soil system can enhance the biodegradation rate [2]. If no increase in the biodegradation rate is observed following bioaugmentation, either sufficient indigenous microbial communities capable of biodegrading petroleum hydrocarbons already exist [2] or the introduced microorganisms could not compete with existing populations. For this reason, bioaugmentation with indigenous microbes grown to high concentrations is sometimes favorable. This is called autochthonous bioaugmentation, and has proven to be successful in some studies [65,66]. Łebkowska et al. isolated hydrocarbon-degrading microorganisms from their contaminated site using diesel oil, grew them to a high concentration, and performed a sequential injection of these indigenous microorganisms into a diesel-contaminated soil and demonstrated that this technique is significantly more effective than a single injection event [66]. Despite the successes described, Aislabie et al. concluded that, in general, concentrating efforts on alleviating inhibitory environmental conditions is more beneficial than bioaugmentation, especially in cold climates, since acclimated microorganisms capable of biodegrading petroleum hydrocarbons are prolific in the subsurface [27].
Another option for harnessing and potentially enhancing natural degradation processes is through the reutilization of previously remediated soils [67]. These soils already have an enriched hydrocarbon-degrading microbial community, and therefore are capable of degrading contaminants relatively quickly. This process was not enhanced by either additional bioaugmentation or biostimulation [67].
It is essential when selecting strains of microorganisms for bioaugmentation that not only their ability to metabolize the contaminants of concern is considered, but that they must also be able to survive and thrive in the environment into which they are being introduced.

12.7.2.2. Macroorganisms

Organisms other than microbes, such as earthworms, may improve biodegradation via a number of mechanisms. Earthworms maintain a complex relationship with microorganisms, whereby earthworms fragment organic matter and microorganisms serve as a major source of nutrients for earthworms [22]. The presence of three species of earthworm (Lumbricus terrestris, Allolobophora chlorotica and Eisenia fetida) significantly improved the biodegradation of TPH, reducing concentrations by up to 40%, versus 9–17% without worms [22]. Californian red worms contributed to bioremediation of oil polluted soils in 5 months [68]. An indirect benefit of the presence of earthworms is aeration of the soil due to their movement. It is, however, important to note that different hydrocarbon compounds have different levels of toxicity toward worms, with a 14-day LC50 ranging from 1079 to 15,609 mg/kg [69]. It is therefore possible that the use of worms may not be feasible in highly contaminated areas, or in areas impacted by a particularly toxic compound.
Also of interest is the use of plants for the remediation of hydrocarbons in soil. Phytoremediation is a commonly used remediation method, but its effectiveness for hydrocarbons is not well studied [70,71] and can be limited due to the finite physical area covered by the root system, as well as sensitivity to the toxic effects of the contaminants, and the limited bioavailability of compounds in the soil [70]. Recent studies suggest that hydrocarbon removal by plants can be enhanced by inoculating the rhizosphere (the soil region immediately surrounding the root of a plant that is impacted by root [72]) with hydrocarbon-degrading bacteria. This lends the plants an increased hydrocarbon tolerance, as well as enhanced biomass [70]. This phenomenon of rhizosphere-enhanced bioremediation has been noted elsewhere as well [72]. Preliminary studies suggest that the success of plant–bacteria partnerships for remediation is dependent on the environmental conditions present (including nutrient concentrations and soil type) [73,74], as well as the physiological condition of the microorganisms used to inoculate the rhizosphere [73]. Khan et al. demonstrated that the highest rates of bacterial colonization and hydrocarbon degradation occurred with Pantoea sp. grown in complex media prior to inoculation [73]. Under these optimized conditions, up to 87% of hydrocarbons were removed from diesel-contaminated soils [73]. Ribeiro et al. also showed that high nutrient concentrations and fine sediments inhibited hydrocarbon degradation, indicating that the interaction between the microorganisms and soil particles is a key element of the plant–bacteria association. This also demonstrates that the concentration of soil organic matter is also an essential consideration for the success of a given remediation project [74].

12.7.3. Combined Approaches to Bioremediation

Many recent laboratory studies have focused on comparing various remediation techniques on their own, and in conjunction with other approaches. This is often seen in regards to bioaugmentation and biostimulation.
Amendment with waste products such as compost materials [75], kitchen waste [76], and brewery effluent [77] have been explored and demonstrated to enhance remediation when complemented with other technologies. Most of these studies combine the use of these amendments with the use of bioaugmented microbial consortia, but in some the biostimulation is used in conjunction with bioventing [77], and the addition of other nutrients [76,78]. Biostimulation and bioaugmentation have also been used together successfully to enhance the effectiveness of one another [79,80], land farming [81,82], and biosurfactants [81].

12.8. Case Study: Kuwait Oil Spill

During the Gulf War, retreating Iraqi forces planted explosives in over 600 of Kuwait's oil wells, releasing an estimated 250 million gallons of oil. This in turn created over 300 “lakes” of oil covering an area of over 49 km2, resulting in one of the worst man-made disasters ever documented [47,83,84]. The Kuwait desert poses a number of remediation challenges. Kuwait's soil can reach high temperatures in the summer and mild temperatures in the winter, ranging between 5 and 39°C [85]. The soil is calcareous and predominantly sandy, alkaline, saline, low in organic matter, and has a low water-holding capacity [48,85,86]. Unlike water-based oil spills, there is no water movement in the desert to break up the oil, and weathering of the residual oil further inhibits biodegradation [83]. As a result of these unfavorable conditions, additional nutrients, moisture, and oxygen are likely required to provide an environment conducive to the degradation of oil by indigenous bacteria and fungi [48].
As part of the remediation efforts to reclaim affected areas in Kuwait, several bioremediation techniques have been assessed. These primarily included excavation of contaminated soil and treatment via ex situ techniques including landfarming [47], windrow composting piles [47,48], and static aerated soil piles [47], as well as in situ methods using rhizoremediation techniques [85,87–90]. Bioaugmentation, even with indigenous microbes isolated from the oil fields, was shown to be ineffective or even inhibitory to the remediation process [48,91,92].
Landfarming has been demonstrated to be one of the most effective methods for the treatment of oily soils in Kuwait [47,93]. Al-Daher et al. demonstrated that treatment of light (2–3% TPH) to moderately (3–5% TPH) contaminated soils from the Burgan Oil Field (lake #102, which has been drained of recoverable oil and weathered) could be treated to remove up to 83% TPH within 12 months with the addition of inorganic fertilizer, a mix of compost and woodchips, irrigation with fresh water via leaky pipes, and tilling twice per week [47]. In a similar set-up, Al-Awadhi also demonstrated over 80% removal of TPH, as well as a significant reduction in PAHs [93]. In both studies, degradation was significantly different from the control plots (<20% TPH removal) and all treatments saw an increase in the number of bacteria present [47].
The construction of windrow piles yielded similar results with a TPH reduction of 60–74.2% observed [47,48]. As with landfarming, fertilizer, compost, and woodchips were added, and the piles were irrigated with fresh water. Rather than tilling, the piles were turned over approximately once per month [48,85]. One study investigated the addition of other additives, including dried sewage sludge, and a culture of hydrocarbon-degrading organisms, but these treatments were not shown to have significant impact [48].
Treatment of oil waste in biopiles was also successful, although slightly less so than landfarming or windrows, with a reduction of 64.2% TPH [47]. The biopiles were similar in that they were supplemented with fertilizer, compost, and woodchips, and irrigated. However, instead of physically mixing the soils, aeration was facilitated via perforated pipes buried in the piles [47]. While less efficient than the other methods in treating TPH, this method has the added advantage of a smaller physical footprint, lower capital and labor costs, and a lower water requirement. Although less efficient, when taking all aspects into consideration, biopiles may be a more favorable remediation technique [47].
Rhizoremediation has also been heavily investigated for the Kuwait desert petroleum-contaminated soils. Rhizoremediation refers to the degradation of pollutants by the microorganisms inhabiting the rhizosphere of plants growing in the contaminated soil [85]. The harsh desert conditions pose a challenge for the growth of plants, in that they must be tolerant to extreme temperatures, high salinity, and other conditions common to the desert environment [86]. Some plants of interest include wild desert plants (such as Asthenatherum forsskalii, Cyperus conglomeratus, Launaea mucronata, Picris babylonica, Salsola imbricata, Senecio glaucus, and Stipagrostis plumosa) which quickly recolonized weakly to moderately contaminated areas following the oil spill [85,87,94]. Nonnative plants investigated include those known to have root systems associated with hydrocarbon-degrading microorganisms such as legume plants, grasses, and ornamental trees [85,87,94]. The idea of utilizing this plant–bacterial interaction in this context came from the observation that while desert plants grew in black, oily soils, their roots remained clean and white, and the soil in the immediate vicinity of the roots was also free of contaminants [94]. This observation led researchers to investigate the root-associated microbial community. It was demonstrated that these plants were densely associated with hydrocarbon-degrading bacteria, including those of the genuses Cellulomonas, Rhodococcus, Arthrobacter, Pseudomonas, and Bacillus, as well as the fungi Trichoderma, Penicilium, and Fusarium [87,94].
As a result of this work, the authors proposed that by cultivating the appropriate desert or crop plants in a high density, rhizoremediation could be a feasible approach for the reclamation of oil-contaminated soils [94]. Crop plants, including Vicia faba (broad beans) have been investigated for this purpose as well, as they tolerate up to 10% (w/w) oil, and have a significant hydrocarbon-degrading rhizosphere microbial community [88]. Both excised roots and whole plants were found to contribute to the attenuation of hydrocarbons in lab-based experiments. However, after growth on oily soils the plants are not recommended for human or animal consumption, so they have to be disposed of accordingly, and remediation in conjunction with growing a crop as a food source is not feasible [88].
Grasses, such as Bermuda Grass and American Grass, are also of interest for rhizoremediation. The roots of both were found to be associated with high levels of hydrocarbon-degrading, nitrogen-fixing bacteria, including Agrobacterium, Arthrobacter, Pseudomonas, Gordonia, and Rhodococcus. The ability of these organisms to fix nitrogen as well as degrade hydrocarbons make them ideal candidates for the remediation of soils low in nitrogen, such as those found in the Kuwait desert [89]. In microbial communities rich with nitrogen-fixing organisms, it may even be possible for remediation to proceed without the addition of fertilizers [95]. The growth of these grasses and their associated microbial communities were found to not only enhance degradation of hydrocarbons in the soil, but also served to reduce the volatilization of oil into the atmosphere, thereby minimizing pollution of the land and air [89]. Lastly, ornamental trees have also been recommended for rhizoremediation. A number of ornamental plants have been used to establish a bio-park in Kuwait in order to compare their growth on previously bioremediated soil to agricultural soil [48]. Bioremediated soils had no inhibitory effect on the growth of plants. Thus, it is suggested that the soils remaining after remediation (via landfarming, windrows, biopiles, etc.) can be utilized in this fashion [85,96].
Though vast quantities of pollution remain in the desert of Kuwait, progress has been made toward remediating the contaminated sites, and several promising methodologies have been developed.

12.9. Conclusion

In summary, a wide range of bioremediation techniques are available for the remediation of oil spills on land. These strategies, including monitored natural attenuation, have been used successfully in a variety of environments, and can be both a cost-effective and environmentally friendly remediation tactic. The approach utilized is largely dependent on the type and concentration/quantity of contaminant present, the environmental conditions of the site, the availability of bioaugmentation cultures and biostimulation amendments, the finances available to the operator, and the amount of physical space available for large-scale, ex situ remediation activities. Additionally, multiple techniques may need to be utilized in conjunction or succession to achieve optimal results.
Due to the complex nature of the bioremediation process, it is imperative that the environmental and biogeochemical characteristics of a contaminated site be well-understood prior to undertaking remediation activities. An assessment of the native microbial community is also useful and may help determine the most effective means to stimulate biodegradation. Additionally, knowledge of the contaminant(s) and the contaminated site can allow for the use of methods such as phyto or rhizoremediation with plants well suited to the environment.
Future research efforts should focus not only on the development of new technologies for remediation, but also on developing a more thorough understanding of the interaction of different environmental facts and their impact on the indigenous microbial community and its dynamics.
Readers are encouraged to refer to the following source for more information, especially with regard to designing engineered bioremediation systems:
Atlas, R. and Philip, J., Bioremediation of contaminated soils and aquifers, Bioremediation – Applied Microbial Solutions for Real-World Environmental Cleanup, ASM Press, 139, 2005.

References

[1] Leahy J.G, Colwell R.R. Microbial degradation of hydrocarbons in the environment. Microbiological Reviews. 1990;54.

[2] Mohammed N, Allayla R.I, Nakhla G.F, Farooq S, Husain T. State-of-the-art review of bioremediation studies. Journal of Environmental Science and Health. 1996;A31:1547.

[3] Horel A, Schiewer S. Investigation of the physical and chemical parameters affecting biodegradation of diesel and synthetic diesel fuel contaminating Alaskan soils. Cold Regions Science and Technology. 2009;58:113.

[4] Jorgensen K.S. In situ bioremediation. Advances in Applied Microbiology. 2007;61.

[5] Ahn J.H, Kim M.S, Kim M.C, Lim J.S, Lee G.T, et al. Analysis of bacterial diversity and community structure in forest soils contaminated with fuel hydrocarbons. Journal of Microbiology and Biotechnology. 2006;16:704.

[6] Benka-Coker M.O, Ekundayo J.A. Applicability of evaluating the ability of microbes isolated from an oil spill site to degrade oil. Environmental Monitoring and Assessment. 1997;45:259.

[7] Akoachere J.T.K, Akenji T.N, Yongabi F.N, Nkwelang G, Ndip R.N. Lubricating oil-degrading bacteria in soils from filling stations and auto- mechanic workshops in Buea, Cameroon: occurrence and characteristics of isolates. African Journal of Biotechnology. 2008;7.

[8] Biswas D, Dutta S, Saha R. Effect of Pseudomonas sp. on diesel and other petroleum fractions. Journal of the Indian Chemical Society. 2006;83:1185.

[9] Ijah U.J.J, Antai S.P. The potential use of chicken-drop micro-organisms for oil spill remediation. The Environmentalist. 2003;23:89.

[10] Banat I.M, Rahman K.S.M, Thahira-Rahman J. Bioremediation of hydrocarbon pollution using biosurfactant producing oil degrading bacteria. In: Brebbia C.A, ed. Oil and hydrocarbon spills III. 2002.

[11] Ellis B, Balba M.T, Theile P. Bioremediation of oil contaminated land. Environmental Technology. 1990;11:443.

[12] Nwachukwu S.U. Bioremediation of sterile agricultural soils polluted with crude petroleum by application of the soil bacterium, Pseudomonas putida, with inorganic nutrient supplementations. Current Microbiology. 2001;42.

[13] Rughavan P.U.M, Vivekanandan M. Bioremediation of oil-spilled sites through seeding of naturally adapted Pseudomonas putida. International Biodeterioration & Biodegradation. 1999;4:29.

[14] Rema S, Pandian M.R, Sudhakaran S, Devi R. Petroleum hydrocarbon degradation by native isolate Rhodococcus. Asian Journal of Microbiology, Biotechnology and Environmental Sciences. 2007;9:973.

[15] Lee M, Kim M.K, Singleton I, Goodfellow M, Lee S.T. Enhanced biodegradation of diesel oil by a newly identified Rhodococcus baikonurensis EN3 in the presence of mycolic acid. Journal of Applied Microbiology. 2006;100. .

[16] Christofi N, Ivshina I.B, Kuyukina M.S, Philp J.C. Biological treatment of crude oil contaminated soil in Russia. In: Lerner D.N, Walton N.R.G, eds. Contaminated land and groundwater: future directions. 1998.

[17] Banerjee M, Shrivastava S. Sensitivity of cyanobacterium Mastigocladus to petrol and its role in bioremediation. Journal of Industrial Pollution Control. 2005;21:77.

[18] Gallego J.L, Loredo J, Llamas J.F, Vazquez F, Sanchez J. Bioremediation of diesel-contaminated soils: evaluation of potential in situ techniques by study of bacterial degradation. Biodegradation. 2001;12.

[19] Hestbjerg H, Willumsen P.A, Christensen M, Andersen O, Jacobsen C.S. Field scale bioremediation of tar contaminated soil with commercial mushroom Pleurotus ostreatus refuse. In: Brebbia C.A, ed. Oil and hydrocarbon spills III. 2002.

[20] Yateem A, Balba M.T, Al-Awadhi N. White rot fungi and their role in remediating oil-contaminated soil. Environment International. 1998;24:181.

[21] Palittapongampim M, Pokethitiyook P, Upathm E.S, Tangbanluekal L. Biodegradation of crude oil by soil microorganisms in the tropic. Biodegradation. 1998;9:83.

[22] Schaefer M, Peterson S.O, Filser J. Effects of Lumbricus terrestris, Allolobophora chlorotica and Eisenia fetida on microbial community dynamics in oil-contaminated soil. Soil Biology and Biochemistry. 2005;37:2065.

[23] Dott W, Feidieker D, Steiof M, Becker P.M, Kämpfer P. Comparison of ex situ and in situ techniques for bioremediation of hydrocarbon-polluted soils. International Biodeterioration & Biodegradation. 1995;35.

[24] Foght J. Anaerobic biodegradation of aromatic hydrocarbons: pathways and prospects. Journal of Molecular Microbiology and Biotechnology. 2008;15.

[25] Widdel F, Rabus R. Anaerobic biodegradation of saturated and aromatic hydrocarbons. Current Opinion in Biotechnology. 2001;12.

[26] Ulrich A.C, Tappenden K, Armstrong J, Biggar K.W. Effect of cold temperature on the rate of natural attenuation of benzene, toluene, ethylbenzene, and the three isomers of xylene (BTEX). Canadian Geotechnical Journal. 2010;47.

[27] Aislabie J, Saul D.J, Foght J.M. Bioremediation of hydrocarbon-contaminated polar soils. Extremophiles. 2006;10.

[28] Margesin R, Schinner F. Biodegradation and bioremediation of hydrocarbons in extreme environments. Applied Microbiology and Biotechnology. 2001;56.

[29] Chang B.V, Shiung L.C, Yuan S.Y. Anaerobic biodegradation of polycyclic aromatic hydrocarbon in soil. Chemosphere. 2002;48.

[30] Ulrich A.C, Guigard S.E, Foght J.M, Semple K.M, Pooley K, et al. Effect of salt on aerobic biodegradation of petroleum hydrocarbons in contaminated groundwater. Biodegradation. 2009;20.

[31] Menendez-Vega D, Gallego J.L.R, Pelaez A.I, Fernandez de Cordoba G, Moreno J, et al. Engineered in situ bioremediation of soil and groundwater polluted with weathered hydrocarbons. European Journal of Soil Biology. 2007;43.

[32] US EPA. How to evaluate alternative cleanup technologies for underground storage tank sites: a guide for corrective action plan reviewers. United States Environmental Protection Agency; 2004.

[33] Agarry S.E, Aremu M.O, Aworanti O.A. Kinetic modelling and half-life study on bioremediation of soil co-contaminated with lubricating motor oil and lead using different bioremediation strategies. Soil and Sediment Contamination. 2013;22:800.

[34] Kauppi S, Sinkkonen A, Romantschuk M. Enhancing bioremediation of diesel-fuel-contaminated soil in a boreal climate: comparison of biostimulation and bioaugmentation. International Biodeterioration & Biodegradation. 2011;65. .

[35] Walworth J.L, Woolard C.R, Harris K.C. Nutrient amendments for contaminated peri-glacial soils: use of cod bone meal as a controlled release nutrient source. Cold Regions Science and Technology. 2003;37.

[36] Walworth J.L, Woolard C.R, Braddock J.F, Reynolds C.M, Battelle Mem I. The role of soil nitrogen concentration in bioremediation. Battelle Press; 1997.

[37] Boopathy R. Anaerobic biodegradation of no. 2 diesel fuel in soil: a soil column study. Bioresource Technology. 2004;94.

[38] Gogoi B.K, Dutta N.N, Goswami P, Krishna Mohan T.R. A case study of bioremediation of petroleum-hydrocarbon contaminated soil at a crude oil spill site. Advances in Environmental Research. 2003;7:767.

[39] Brown R.A, Crosbie J.R. Oxygen sources for bioremediation. In: Flathman P.E, Jerger D.E, Exner J.H, eds. Bioremediation: field experience. 1994.

[40] Hunkeler D, Meckenstock R.U, Lollar B.S, Schmidt T.S, Wilson J.T. A guide for assessing biodegradation and source identification of organic ground water contaminants using Compound Specific Isotope Analysis (CSIA) EPA 600/R-08/148. 2008.

[41] Aleer S, Adetutu E, Makadia T, Patil S, Ball A. Harnessing the hydrocarbon-degrading potential of contaminated soils for the bioremediation of waste engine oil. Water, Air, and Soil Pollution. 2011;218.

[42] Couto M.N, Monteiro E, Vasconcelos M.T. Mesocosm trials of bioremediation of contaminated soil of a petroleum refinery: comparison of natural attenuation, biostimulation and bioaugmentation. Environmental Science and Pollution Research International. 2010;17.

[43] Wiedemeier T.H, Haas P.E. Designing monitoring programs to effectively evaluate the performance of natural attenuation. Ground Water Monitoring and Remediation. 2002;22.

[44] Van Deuren J, Lloyd T, Chhetry S, Liou R, Peck J. Remediation technologies screening matrix and reference guide. Federal Remediation Technologies Roundtable; 2002.

[45] Nelson C.H, Hicks R.J, Andrews S.D. An integrated approach for in situ bioremediation of petroleum hydrocarbon-contaminated soil and groundwater. In: Flathman P.E, Jerger D.E, Exner J.H, eds. Bioremediation: field experience. 1994.

[46] Margesin R, Schinner F. Bioremediation (natural attenuation and biostimulation) of diesel-oil-contaminated soil in an alpine glacier skiing area. Applied and Environmental Microbiology. 2001;67.

[47] Balba M.T, Al-Daher R, Al-Awadhi N, Chino H, Tsuji H. Bioremediation of oil-contaminated desert soil: the Kuwaiti experience. Environment International. 1998;24.

[48] Al-Daher R, Al-Awadhi N, El-Nawawy A. Bioremediation of damaged desert environment using the windrow soil pile system in Kuwait. Environment International. 1998;24.

[49] Bleckmann C.A, Oxley M.E, Wilson E.J, Hayes K.W, Hercyk N.L. Land treatment of produced oily sand: field results. Waste Management and Research. 1997;15.

[50] Eszenyiova A, Polakovicova G, Bilska V, Rajnohova H. Biological clean-up of hydrocarbon pollution. PTQ. 2000;133(Autumn).

[51] Guerin T.F. Long-term performance of a land treatment facility for the bioremediation of non-volatile oily wastes. Resource, Conservation and Recycling. 2000;28.

[52] Dibble J.T, Bartha R. Effect of environmental parameters on the biodegradation of oil sludge. Applied and Environmental Microbiology. 1979;37.

[53] Chagas-Spinelli A.C, Kato M.T, de Lima E.S, Gavazza S. Bioremediation of a tropical clay soil contaminated with diesel oil. Journal of Environmental Management. 2012;113.

[54] Coulon F, Al Awadi M, Cowie W, Mardlin D, Pollard S, et al. When is a soil remediated? Comparison of biopiled and windrowed soils contaminated with bunker-fuel in a full-scale trial. Environmental Pollution. 2010;158. .

[55] Pometto A.L, Oulman C.S, DiSpirito A.A, Johnson K.E, Baranow S. Potential of agricultural by-products in the bioremediation of fuel spills. Journal of Industrial Microbiology and Biotechnology. 1998;20:369.

[56] Saez-Navarrete C, Gelmi C.A, Reyes-Bozo L, Godoy-Faundez A. An exploratory study of peat and sawdust as enhancers in the (bio)degradation of n-dodecane. Biodegradation. 2008;19.

[57] Rivera-Espinoza Y, Dendooven L. Dynamics of carbon and nitrogen in a mixture of polycyclic aromatic hydrocarbons contaminated soil amended with organic residues. Environmental Technology. 2007;28.

[58] Martienssen M, Schirmer M. Use of surfactants to improve the biological degradation of petroleum hydrocarbons in a field site study. Environmental Technology. 2007;28.

[59] Uzoigwe C, Burgess J.G, Ennis C.J, Rahman P.K.S.M. Bioemulsifiers are not biosurfactants and require different screening approaches. Frontiers in Microbiology. 2015;6.

[60] Calvo C, Manzanera M, Silva-Castro G.A, Uad I, Gonzalez-Lopez J. Application of bioemulsifiers in soil oil bioremediation processes. Future prospects. Science of Total Environment. 2009;407.

[61] Singh P, Saini H. Exploitation of agro-industrial wastes to produce low-cost microbial surfactants. In: Brar S.K, Dhillon G.S, Soccol C.R, eds. Biotransformation of waste biomass into high value biochemicals. vol. 18. 2014.

[62] Martins V, Kalil S, Costa J. In situ bioremediation using biosurfactant produced by solid state fermentation. World Journal of Microbiology and Biotechnology. 2009;25.

[63] Lai C.C, Huang Y.C, Wei Y.H, Chang J.S. Biosurfactant-enhanced removal of total petroleum hydrocarbons from contaminated soil. Journal of Hazardous Materials. 2009;167.

[64] Jeong S.W, Jeong J, Kim J. Simple surface foam application enhances bioremediation of oil-contaminated soil in cold conditions. Journal of Hazardous Materials. 2015;286.

[65] Ma J, Yan G, Ma W, Cheng C, Wang Q, et al. Isolation and characterization of oil-degradaing microorganisms for bench-scale evaluations of autochthonous bioaugmentation for soil remediation. Water, Air, and Soil Pollution. 2015;226:272.

[66] Łebkowska M, Zborowska E, Karwowska E, Miaśkiewicz-Pęska E, Muszyński A, et al. Bioremediation of soil polluted with fuels by sequential multiple injection of native microorganisms: field-scale processes in Poland. Ecological Engineering. 2011;37.

[67] Makadia T.H, Adetutu E.M, Simons K.L, Jardine D, Sheppard P.J, et al. Re-use of remediated soils for the bioremediation of waste oil sludge. Journal of Environmental Management. 2011;92.

[68] Ilarionov S.A, Kalachnikova I.G, Sergeev V.A. Rehabilitation of oil-polluted soils using products obtained in the process of organic wastes bioconversion. In: Brebbia C.A, ed. Oil and hydrocarbon spills III: modelling, analysis, and control. 2002.

[69] Moon Y, Yim U.H, Kim H.S, Kim Y.J, Shin W.S, et al. Toxicity and bioaccumulation of petroleum mixtures with alkyl PAHs in earthworms. Human and Ecological Risk Assessment. 2013;19.

[70] Khan S, Afzal M, Iqbal S, Khan Q.M. Plant–bacteria partnerships for the remediation of hydrocarbon contaminated soils. Chemosphere. 2013;90.

[71] Yavari S, Malakahmad A, Sapari N. A review on phytoremediation of crude oil spills. Water, Air, and Soil Pollution. 2015;226.

[72] Kirkpatrick W.D, White P.M, Wolf D.C, Thoma G.J, Reynolds C.M. Petroleum-degrading microbial numbers in rhizosphere and non-rhizosphere crude oil-contaminated soil. International Journal of Phytoremediation. 2008;10. .

[73] Khan S, Afzal M, Iqbal S, Mirza M.S, Khan Q.M. Inoculum pretreatment affects bacterial survival, activity and catabolic gene expression during phytoremediation of diesel contaminated soil. Chemosphere. 2013;91.

[74] Ribeiro H, Almeida C.M, Magalhaes C, Bordalo A.A, Mucha A.P. Salt marsh sediment characteristics as key regulators on the efficiency of hydrocarbons bioremediation by Juncus maritimus rhizospheric bacterial community. Environmental Science and Pollution Research International. 2015;22.

[75] Gomez F, Sartaj M. Field scale ex-situ bioremediation of petroleum contaminated soil under cold climate conditions. International Biodeterioration & Biodegradation. 2013;85.

[76] Grace Liu P.-W, Chang T.C, Whang L.-M, Kao C.-H, Pan P.-T, et al. Bioremediation of petroleum hydrocarbon contaminated soil: effects of strategies and microbial community shift. International Biodeterioration & Biodegradation. 2011;65.

[77] Agarry S.E, Latinwo G.K. Biodegradation of diesel oil in soil and its enhancement by application of bioventing and amendment with brewery waste effluents as biostimulation-bioaugmentation agents. Journal of Ecological Engineering. 2015;16:82.

[78] Llado S, Solanas A.M, de Lapuente J, Borras M, Vinas M. A diversified approach to evaluate biostimulation and bioaugmentation strategies for heavy-oil-contaminated soil. The Science of the Total Environment. 2012:435–436.

[79] Pontes J, Mucha A.P, Santos H, Reis I, Bordalo A, et al. Potential of bioremediation for buried oil removal in beaches after an oil spill. Marine Pollution Bulletin. 2013;76.

[80] Suja F, Rahim F, Taha M.R, Hambali N, Rizal Razali M, et al. Effects of local microbial bioaugmentation and biostimulation on the bioremediation of total petroleum hydrocarbons (TPH) in crude oil contaminated soil based on laboratory and field observations. International Biodeterioration & Biodegradation. 2014;90.

[81] Lin T.C, Pan P.T, Young C.C, Chang J.S, Chang T.C, et al. Evaluation of the optimal strategy for ex situ bioremediation of diesel oil-contaminated soil. Environmental Science and Pollution Research International. 2011;18.

[82] Sun G.D, Xu Y, Jin J.H, Zhong Z.P, Liu Y, et al. Pilot scale ex-situ bioremediation of heavily PAHs-contaminated soil by indigenous microorganisms and bioaugmentation by a PAHs-degrading and bioemulsifier-producing strain. Journal of Hazardous Materials. 2012:233–234.

[83] Shouse B. The Gulf War's aftermath: Kuwait unveils plan to treat festering desert wound. Science. 2001;293.

[84] Al-Attar M.H. An integrated approach for overcoming the adverse environmental impacts inflicted on Kuwait during the Iraqi occupation. European Centre Pollution Research; 1996.

[85] Yateem A. Rhizoremediation of oil-contaminated sites: a perspective on the Gulf War environmental catastrophe on the State of Kuwait. Environmental Science and Pollution Research International. 2013;20.

[86] Obuekwe C.O, Badrudeen A.M, Al-Saleh E, Mulder J.L. Growth and hydrocarbon degradation by three desert fungi under conditions of simultaneous temperature and salt stress. International Biodeterioration & Biodegradation. 2005;56.

[87] Radwan S.S, Al-Awadhi H, Sorkhoh N.A, El-Nemr I.M. Rhizospheric hydrocarbon-utilizing microorganisms as potential contributors to phytoremediation for the oily Kuwaiti desert. Microbiological Research. 1998;153.

[88] Radwan S.S, Al-Awadhi H, El-Nemr I.M. Cropping as a phytoremediation practice for oily desert soil with reference to crop safety as food. International Journal of Phytoremediation. 2000;2. .

[89] Mahmoud H.M, Suleman P, Sorkhoh N.A, Salamah S, Radwan S.S. The potential of established turf cover for cleaning oily desert soil using rhizosphere technology. International Journal of Phytoremediation. 2011;13.

[90] Radwan S.S, Sorkhoh N.A, Fardoun F, Al-Hasan R.H. Soil management enhancing hydrocarbon biodegradation in the polluted Kuwaiti desert. Applied Microbiology and Biotechnology. 1995;44.

[91] Radwan S.S, Sorkhoh N.A, ElNemr I.M, ElDesouky A.F. A feasibility study on seeding as a bioremediation practice for the oily Kuwaiti desert. Journal of Applied Microbiology. 1997;83.

[92] Cho B.H, Chino H, Tsuji H, Kunito T, Nagaoka K, et al. Laboratory-scale bioremediation of oil-contaminated soil of Kuwait with soil amendment materials. Chemosphere. 1997;35.

[93] Al-Awadhi N, Al-Daher R, El-Nawawy A, Balba M.T. Bioremediation of oil-contaminated soil in Kuwait 1. Landfarming to remediate oil-contaminated soil. Journal of Soil Contamination. 1996;5.

[94] Radwan S, Sorkhoh N, El-Nemr I. Oil biodegradation around roots. Nature. 1995;376.

[95] Al-Awadhi H, Al-Mailem D, Dashti N, Khanafer M, Radwan S. Indigenous hydrocarbon-utilizing bacterioflora in oil-polluted habitats in Kuwait, two decades after the greatest man-made oil spill. Archives of Microbiology. 2012;194.

[96] Al-Zalzaleh H, Ghulam S. Effect of bioremediated soil on growth of different plant species. Kuwait Journal of Science and Engineering. 2004;31.

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