Enhanced Biodegradation of Petroleum Hydrocarbons in Contaminated Soil Laleh Yerushalmi,1,4 Sylvie Rocheleau,1 Ruxandra Cimpoia,1 Manon Sarrazin,1 Geoffrey Sunahara,1 Adriana Peisajovich,2 Gervais Leclair,3 and Serge R. Guiot1* 1Biotechnology Research Institute, National Research Council Canada, 6100 Royalmount Avenue, Montreal, Canada, H4P 2R2; 2Transport Canada, 700 Leigh Caperol, Dorval, Quebec, Canada H4Y 1G7; 3Environment Canada, 105 McGill Street, Montreal, Quebec, Canada H2Y 2E7; 4Present address: Atara Corporation, 390 Guy Street, Montreal, Quebec, H3J 1S6

ABSTRACT: Soil samples taken from a contaminated site in Northern Quebec, Canada, exhibited a low capacity for biodegradation of total petroleum hydrocarbons (TPH), despite a high capacity for the mineralization of aromatic hydrocarbons and a low toxicity of soil leachates as measured by Microtox assay. Toxicity assays directly performed on surface soil, including earthworm mortality and barley seedling emergence, indicated moderate to high levels of toxicity. Soil biostimulation did not improve the removal of petroleum hydrocarbons, while bioaugmentation of soil with a developed enrichment culture increased the efficiency of hydrocarbon removal from 20.4% to 49.2%. A considerable increase in the removal of TPH was obtained in a bioslurry process, enhancing the mass transfer of hydrocarbons from soil to the aqueous phase and increasing the efficiency of hydrocarbon removal to over 70% after 45 days of incubation. The addition of ionic or nonionic surfactants did not have a significant impact on biodegradation of hydrocarbons. The extent of hydrocarbon mineralization during the bioslurry process after 45 days of incubation ranged from 41.3% to 58.9%, indicating that 62.7% to 83.1% of the eliminated TPH were transformed into CO2 and water. Keywords: soil contamination, petroleum hydrocarbons, biostimulation, bioaugmentation, bioslurry.

Introduction An extensive soil contamination with petroleum hydrocarbons was found in a former meteorological and radio station located in Northern Quebec, Canada. The concentration of total petroleum hydrocarbons (TPH, C10-C50) in soil ranged from 1700 to 10,000 mg/kg (dry soil), exceeding the MENV B (Quebec Ministry of Environment) criteria for residential sites (700 mg/kg dry soil) and the MENV C criteria for commercial/ industrial sites (3500 mg/kg dry soil). The monitoring activities conducted as part of an environmental site assessment showed a lack of natural attenuation of TPH in soil after more than a decade. The contamination of soil and groundwater with petroleum hydrocarbon-based fuels as a result of accidental spills or improper storage has been reported frequently (Balba et al., 1998; Nadim et al., 2000; Rhykerd et al., 1999). Petroleum hydrocarbons, *

including polycyclic aromatic hydrocarbons (PAHs), have been categorized as priority pollutants by the United States Environmental Protection Agency (US EPA), Quebec Ministry of Environment (MENV), and many other environment and health organizations in the world. These chemicals pose serious health and ecological threats due to their toxicity and mutagenicity. There are several physical-chemical technologies for the treatment of soil contaminated with organic and hazardous material such as petroleum hydrocarbons. They include vapor extraction, stabilization, solidification, soil flushing, soil washing, thermal desorption, vitrification, and incineration (Balba et al., 1998; Zappi et al., 1996). However, most of these techniques are expensive to implement at full scale and require continuous monitoring and control for optimum performance. In addition, they do not usually result in a complete destruction of the contaminants. Biological

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treatment or bioremediation techniques are alternative methods for the treatment of contaminated soil. These techniques are economically and politically attractive and have shown promising results in the treatment of soil contaminated with organic compounds, particularly with petroleum hydrocarbons (Cho et al., 1997; Demque et al., 1997; Margesin and Schinner, 1997; Rhykerd et al., 1999). Despite its advantages, bioremediation is a sitespecific process, and its efficiency may be limited by microbiological and physical-chemical conditions of the soil. The limiting factors for soil bioremediation include the type and concentration of contaminants and the indigenous microbial population, availability of nutrients and electron acceptors, pH, temperature, moisture content of soil, and substrate bioavailability (Autry and Ellis, 1992; Balba et al., 1998). The present study was initiated as part of a project with Transport Canada and Environment Canada. The study investigates the potential for reduction of TPH concentration in contaminated soil by bioremediation techniques and identifies the limiting factors, leading to the recommendation of possible processes to improve soil biotreatability.

Materials and Methods Soil Characteristics Previous assessments of the site had shown that the contaminated soil contained medium to coarse sand in the upper layers and a mixture of sand and silt in the lower layers. Soil samples used in the present study were collected from various locations and depths (0 to 15 cm or 1 to 1.5 m) on the contaminated site. The percentage of sand in soil samples extracted from the depth of 1 to 1.5 m was 91.6%, while the soil density was 2.2 g/mL. The soil had an average organic content of 4.3 ± 0.3 g/kg (dry soil). The chemical analysis of samples showed the presence of metals, including aluminum (1900 to 3000 mg/ kg soil), calcium (1100 to 1700 mg/kg soil), iron (6100 to 7100 mg/kg soil), magnesium (880 to 1300 mg/kg soil), and titanium (520 to 600 mg/kg soil). These analyses also showed that the concentrations of polycyclic aromatic hydrocarbons (PAHs), volatile organic compounds (VOCs), ethylene glycol, and PCBs were below the MENV B criteria. The surface soil (0 to 15 cm) had an average moisture content of 6%, whereas the deeper soil (1 to 1.5 m) had an average moisture content of 17%. The pH of soil samples ranged from 5.8 to 6.7. The temperature of the contaminated site changed from near zero in the winter to 10°C in the summer.

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Preparation of Soil Leachate Soil leachates, used for COD analysis and Microtox assays, were prepared according to the US EPA method #1312 (United States Environmental Protection Agency, 1997). The soil was extracted with deionized water, in which the pH was adjusted to 4.5 with a solution of 60% H2SO4 and 40% HNO3. The extraction was performed by mixing 7 g dry soil with 140 mL extraction liquid for 18 ± 2 h, at 22°C ± 3°C and shaking at 30 ± 2 rpm. Prior to the toxicity test, the resulting soil leachate was filtered through a 0.7-µm borosilicate filter under nitrogen pressure using a Millipore Teflon®-coated filtration system.

Microbial Count The presence of microbial activity in soil was determined by enumeration of total heterotrophic bacteria, growing on YTS 250 solid medium (yeast extract, 250 mg/L; tryptone, 250 mg/L and starch, 250 mg/L), followed by colony count after a 14 day-incubation at 10°C.

Analytical Techniques Total Petroleum Hydrocarbons (TPH). The total hydrocarbons in soil were extracted with hexane according to the Method 410-HYD 1.0 of Quebec Ministry of Environment. A high-performance liquid chromatography (HPLC) was used for the analysis of extracted samples. The system included a pump (Model W600, Waters, Milford, MA, USA), an autosampler (Model W717, Waters, Milford, MA, USA), a column heater (Model TCM, Waters, Milford, MA, USA), a fluorescence detector (Model Spectroflow 980, ABI Analytical, Ramsey, NJ, USA), and an ultraviolet detector (Model W490, Waters, Milford, MA, USA). A 20 µL or 100 µL sample was separated on a Supelcosil LCPAH C18 column (15 cm × 4.6 mm, with a particle size of 5 µm, Supelco, Bellafonte, PA, USA) with a 40:60 (v/v) acetonitrile:water mobile phase for 5 min. The acetonitrile fraction was subsequently increased to 100%, at a flow rate of 1.5 mL/min, for a period of 25 min. Quantification was done by a UV detector at 254 nm and a fluorescence detector at 280 nm. Ion Analysis. The concentrations of sulfate, nitrate, nitrite, phosphate, and chloride in the liquid phase were monitored by high-performance liquid chromatography (HPLC, Model Spectra-Physics, SP8800 and SP8760) using a Hamilton PRP-X100 polymer-based chromatography column (250 × 41 mm O.D.). A Waters Millipore detector (Model 431) was used to obtain conductivity data. The mobile phase was 4.0 mM p-hydroxybenzoic acid with a flow rate of 2 mL/min.

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The injection volume was 100 µL and the injection port temperature was 40°C. Chemical Oxygen Demand (COD). The COD of soil leachates was estimated colorimetrically using a Hach COD incubator (Model 45600) and a Hach spectrophotometer (Model DR/3000). The reaction proceeded for 2 at 150°C.

Toxicity Assays The toxicity assays were performed in order to determine whether the observed lack of natural attenuation of hydrocarbons at the contaminated site was due to the high toxicity of soil. The soil samples used for toxicity tests were taken from depths of 0 to 15 cm from three different locations on the contaminated site, referred to as locations #1, #2, and #3. The toxicity of contaminated soil samples was compared to that of a reference soil taken from a noncontaminated area of the site under investigation. Microtox. Microtox is a standard toxicity assay (Environment Canada, 1992), measuring the reduction in bioluminescence of the marine bacterium, Vibrio fischeri after 15 min or 30 min exposure to the target contaminants. The bioluminescence reduction is expressed as the percentage inhibition of light emission when compared with a control containing only 2% NaCl. The test was performed on soil leachates at the time of their reception (t = 0) as well as on those incubated at 10°C for a period of 5 or 10 weeks. The tests were performed in triplicate and phenol was used as the reference toxicant. The phenol EC50 value for the Microtox assay was between 18 and 26 mg/L. The EC50 value (effective concentration) refers to the concentration that reduces the average response of the test organisms by 50% within the test period. Barley Seedling Emergence and Growth Inhibition. This toxicity test is based on the protocol developed by the US-EPA (United States Environmental Protection Agency, 1989). It measures the seedling emergence (germination) inhibition of barley Hordeum vulgare seeds exposed to contaminated soil. Forty barley seeds of homogeneous size were placed in Petri dishes (150 × 20 mm) containing 100 g of the test soil. Deionized water was added to the soil to obtain an 85% water holding capacity. The seeds were covered with sand, and each Petri dish was enclosed in a polyethylene bag containing air. The seeds were incubated in the dark at 24 ± 2°C for 48 h and were then exposed to a daily photo-period, including 16 h of light. The emerged seedlings were counted after 5 and 14 days, and the

percentage of seedling emergence inhibition was estimated in comparison with the control, which contained deionized water. After 14 days of exposure, the growth inhibition was calculated by measuring the plant dry weight when compared with results of the control soil. The test was done in triplicate and the reference toxicant was mercuric chloride. The EC50 value for barley seedling emergence was between 130 and 330 mg/kg HgCl2 at 5 days, and between 215 and 375 mg/kg HgCl2 at 14 days. The EC50 value for barley growth was between 140 and 360 mg/kg HgCl2 after 14 days. Earthworm Mortality. This toxicity assay uses Eisenia foetida, a small ( 0.99) for the nutrient-amended (biostimulated) and nonamended soil, respectively. For naphthalene, these values changed to 0.05 d–1 and 0.01 d–1 under the two respective conditions (R2 >0.93). The model predictions are presented as solid lines in Figures 3 and 4. These results indicated that biostimulation of soil had a positive effect and increased the efficiencies and

rates of mineralization of aliphatic and aromatic compounds.

Biodegradation Activity Tests Similar results were obtained in the two series of tests using soils taken from depths of 0 to 15 cm and 1 to 1.5 m, respectively. Only the results of experiments with soil samples taken from depths of 1 to 1.5 m are presented in this article, because these samples were investigated in more detail. The activity tests showed that the contaminated soil had a low capacity for biodegradation of petroleum hydrocarbons. The concentration of TPH decreased from 4400 mg/kg dry soil to 3500 mg/kg dry soil after 53 days of incubation, representing a removal efficiency of 20.4% (Figure 5). The supply of nutrients did not increase the removal efficiency. During these tests, the COD concentration of soil leachates decreased from 267 mg/L to 177 mg/L, representing a removal efficiency of 33.7%. The activity tests were carried out without any bioaugmentation and the biodegradation activities were attributed to the indigenous microbial population in the contaminated soil. The abiotic removal of hydrocarbons was 6.8%, leaving 13.6% elimination due to biological degradation. The first-order rate constants for the removal of hydrocarbons from soil were 0.007 d–1 and 0.009 d–1 in the absence and presence of biostimulation, respectively.

Figure 3. Mineralization of naphthalene by the indigenous microbial population in soil at 10°C. Solid lines represent model predictions.

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Figure 4. Mineralization of hexadecane by the indigenous microbial population in soil at 10°C. Solid lines represent model predictions.

Figure 5. Removal of total petroleum hydrocarbons (TPH) during the biodegradation activity tests by the indigenous microbial population in soil at 10°C. Solid lines represent model predictions.

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The low removal efficiency of hydrocarbons, despite a high mineralization capacity for naphthalene and a low toxicity of soil leachates, suggested the following possibilities: 1. The microbial consortium does not have the enzymatic diversity required to degrade all of the contaminating hydrocarbons in soil. 2. The hydrocarbons in soil are recalcitrant and resist further biotransformation. 3. The hydrocarbons are strongly sorbed to the soil matrix, rendering them inaccessible for microbial degradation. Bioaugmentation and bioslurry studies were carried out in order to verify the above hypotheses and to develop possible methods to enhance the biodegradation of hydrocarbon contaminants of soil.

Soil Bioaugmentation Soil bioaugmentation using the developed enrichment culture resulted in an overall TPH removal efficiency of 49.2% after 60 days of operation (Figure 6), of which more than 79% of the overall removal was obtained during the first 15 days of operation. The abiotic removal of TPH was 23.1%, as indicated in the nonbioaugmented control system, leaving 26.1% elimination as a result of biological degradation. The abiotic

loss of hydrocarbons during the soil bioaugmentation tests was higher than that obtained during the activity tests due to the nature of experimental set up and the frequency of sampling that affected the contribution of physical-chemical processes to the removal of hydrocarbons. Compared with the previously mentioned soil biodegradation activity results where the biological elimination of hydrocarbons was equal to 13.6%, the bioaugmentation of soil increased the biological degradation of contaminants by 92%. However, it should be noted that the initial removal rate of hydrocarbons did not increase as a result of soil bioaugmentation, because values of 153.3 mg TPH/kg dry soil.day and 150.0 mg TPH/kg dry soil.day were obtained for soil with or without bioaugmentation, respectively. Despite an increase in biodegradation of contaminants as a result of soil bioaugmentation, the overall extent of contaminant removal was still below 50%, suggesting that bioaugmentation alone would not be sufficient to decontaminate soil according to the environmental regulatory limits. Diesel fuel is known to have a low bioavailability due to sorption to the minerals and humic fraction of soil (Cookson, 1995). It has also been established that solid-phase technologies, such as bioaugmentation, suffer from mass transfer limitations, controlling the transfer of contaminants from soil to the aqueous phase.

Figure 6. Removal of total petroleum hydrocarbons (TPH) from soil during bioaugmentation treatment at 10°C. Error bars represent the corresponding standard deviations.

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Accordingly, soil bioslurry experiments were performed in order to increase solid-liquid mass transfer, thus enhancing the biodegradation rates of the contaminants.

Bioslurry Treatment The efficiency of TPH removal from soil increased to over 70% during the bioslurry experiments (Figure 7). The control bioslurry system exhibited an overall removal efficiency of 35.6% ± 2.6%, while the TPH removal efficiency in the bioslurry reactors after 45 days of incubation ranged from 65.8% ± 6.3% to 70.9% ± 6.8%. This increase was possibly due to the increased transfer of contaminants from the soil surface to the aqueous phase and their enhanced dissolution rate that increased the bioavailability of contaminants to the microbial culture. An increased biological activity was apparent as evaluated by an increase in the production of carbon dioxide in the headspace of the experimental bottles, demonstrating that petroleum hydrocarbons were mineralized during the process. While no carbon dioxide was detected in the control system, its production ranged from 7.8 to 9.3 g/kg dry soil in the slurry flasks. Based on the theoretical production of 3.2 mg CO2/mg hydrocarbons mineralized, these values represent 41.3% to 58.9% hydrocarbon mineralization during the bioslurry process based on the initial TPH concentration in the system. Given the

total extent of hydrocarbon removal, these values indicate that 62.7% to 83.1% of the eliminated hydrocarbons were transformed into CO2 and water. The transformation of volatile TPH to the gas phase of experimental flasks (evaporation), adsorption to the experimental system, and chemical transformation in the presence of oxygen all account for the abiotic loss of TPH in the control system. High efficiencies of TPH removal were obtained in the presence as well as the absence of selected surfactants (Figure 7). This implies that the addition of external surfactants was not necessary to enhance TPH biodegradation in soil. Foaming was observed under all of the applied conditions, suggesting the production of biosurfactants by the microbial culture even when external surfactants were not added. In fact, preliminary experiments using the groundwater sampled from the contaminated site under investigation showed the reduction of surface tension in water from 59 dynes/ cm in the control system to 27 dynes/cm in the biologically active system, indicating the production of biosurfactants by the indigenous microbial population as reported before (Cassidy et al., 2000). These results suggest that a biological surfactant was produced at a concentration sufficient to promote the desorption of sorbed contaminants from the soil matrix, thus increasing their contact with the microorganisms and improving their biodegradation.

Figure 7. Removal of total petroleum hydrocarbons (TPH) from soil during bioslurry treatment at 20°C. Error bars represent the corresponding standard deviations.

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The mechanism underlying the enhanced biodegradation of TPH was evaluated through independent analysis of TPH concentrations in the liquid and soil phases of the bioslurry flasks (Figure 8 ). The presence of surfactants, both ionic and nonionic, promoted the desorption of TPH from soil and increased their dissolution rate in the aqueous phase as indicated by the increased initial liquid-phase concentration of TPH when compared with the control system. The nonionic surfactant Tergitol NP-10 had a more pronounced effect compared with the anionic surfactant SDS, resulting in a higher initial TPH concentration in the liquid phase (Figure 8). The percentage of TPH initially transferred to the liquid phase was 9.0% in the control system, indicating the very low solubility of the sorbed TPH in the aqueous phase. This value increased to 38.1% in the presence of SDS and further to 72.1% in the presence of Tergitol NP-10, emphasizing the high dissolution rate of contaminants. In the absence of external surfactant (slurry system), the initial increase in the liquid-phase concentration of TPH was 12.6%, which is slightly more than that observed in the control system, suggesting the low initial rate of biosurfactant production that depressed the rate of contaminant desorption from the soil matrix. However, the high removal of TPH in the slurry system implies that the gradual production of biosurfactants was sufficient to ensure an overall high removal efficiency of contaminants. Correspondingly, the remaining TPH concentration in soil phase was considerably lower in the presence of surfactants (Figure 8). The total initial TPH in the experimental flasks that did not receive any surfactants (slurry) were slightly lower than those in the other bottles, possibly due to the heterogeneity of soil samples, despite a thorough mixing of soil before the start of experiments.

Discussion The results obtained from the bioaugmentation and bioslurry treatment studies indicated that the contaminated soil from a site in Northern Quebec, Canada, is biotreatable, and its contaminants may be removed with >70% efficiency. The slight increase of removal efficiency in the solid phase bioaugmentation treatment system with the addition of seed microorganisms and nutrients showed the possible deficiency of the unsupplemented (original) soil with respect to these parameters. However, the remarkable increase of the biodegradation rate in the slurry reactors, compared with that observed in the bioaugmented and biostimulated soil, indicated that sorption of TPH to soil was the limiting factor, controlling the biodegradation of contaminants. The considerably higher water 48

to solid ratio and thorough mixing in the slurry system, combined with the presence of surface active compounds, increased hydrocarbon dissolution rate. These factors thus increased the rate of mass transfer to the liquid phase and improved the bioavailability of contaminants to microorganisms. Increasing the temperature to 20°C may have increased the biodegradation rate as a result of higher solubility of hydrocarbons and an increased degree of distribution. However, the efficiency of cold-adapted hydrocarbon degraders at low temperature has been shown to be comparable to that of mesophiles at higher temperatures (Margesin and Schinner, 1997). Of course, the low temperature of the contaminated site reduced biological activities and resulted in a low rate of hydrocarbon biodegradation. It is plausible that a higher extent of biodegradation would result after a considerably longer period of time. The biological availability of contaminants to soil bacteria is recognized as a potential limiting factor during bioremediation studies (Autry and Ellis, 1992). The hydrophobicity of compounds, sorption onto soil matrix, or volatilization of compounds are the major factors limiting the bioavailability of compounds. Li et al. (1995) measured biodegradation rates, oxygen transfer rates, and oil transfer rates during bioremediation of oil-contaminated soil and observed that the ratecontrolling step was the mass transfer of oil into aqueous solution. The addition of surfactants has been reported to improve the efficiency of bioslurry processes by increasing the rate of mass transfer of contaminants to the aqueous phase. Autry and Ellis (1992) employed a technology known as SafesoilTM to increase the biodegradation of gasoline–derived TPH in soil. In this technology, the amendment of soil with surfactants increased the mass transfer of contaminants to the aqueous phase, increasing the contact of contaminants with microbial population in soil and enhancing their biodegradation. It is believed that the addition of surfactants reduces the initial adaptation period of the process by making the contaminants more available to the microbial population. The enhanced biodegradation of crude oil by the addition of chemical surfactants has been reported by Van Hamme and Ward (1999). However, in the present study the use of external surfactants was not necessary because the microbial culture may have produced biosurfactants, and the addition of external surfactants to the experimental system did not increase the overall efficiency of contaminant biodegradation. The addition of adapted bacterial culture to soil (bioaugmentation) has demonstrated a limited increase in the overall efficiency of hydrocarbon removal. Autry and Ellis (1992) observed a minor increase in biodegYerushalmi et al.

Figure 8. Concentration profile of total petroleum hydrocarbons (TPH) in the soil phase and aqueous phase during bioslurry treatment at 20°C. Error bars represent the corresponding standard deviations.

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radation of petroleum hydrocarbons as a result of soil bioaugmentation, implying that the limited biotreatability of soil was not due to the absence of hydrocarbon-degrading bacteria. As reported by Margesin and Schinner (1997), the addition of microorganisms can accelerate the initial phase of biodegradation and can be advantageous when the contaminants have a toxic effect on the indigenous microorganisms. Demque et al. (1997) also reported that bioaugmentation with adapted indigenous microorganisms had little or possibly negative effects on biodegradation of diesel fuel in a contaminated sand. The inoculation of soil seems to have a more pronounced effect on the rate of hydrocarbon biodegradation (or mineralization) at low temperatures. Whyte et al. (1999) and Mohn and Stewart (2000) reported a reduced lag time and an increased rate of hydrocarbon mineralization as a result of soil bioaugmentation at temperatures below 10°C. First-order kinetics for the removal of petroleum hydrocarbons from soil were previously observed by Taylor and Viraraghavan (1999), who reported rate constants of 0.01 d–1 and 0.03 d–1 during the biodegradation of hydrocarbons in nonamended and amended soil, respectively. These values are higher than those obtained in the present study by the original and biostimulated soil (0.007 and 0.009 d–1), indicating the low capacity of soil for biodegradation of petroleum hydrocarbons. The abiotic removal of hydrocarbons ranged from 6.8% during the activity tests to 35.6% in the bioslurry treatment. These values are in the range of the reported values for abiotic elimination of hydrocarbons. Margesin and Schinner (1997) observed 30% removal of diesel oil from alpine soils at 10°C as a result of abiotic processes. Bragg et al. (1994) also noticed a loss of 30% of hydrocarbons due to physical processes. The elimination of contaminating hydrocarbons by abiotic processes constitutes a significant fraction of oil removal and must be taken into consideration in order to avoid over estimation of hydrocarbon biodegradation. Although the soil bioslurry process exhibited an enhanced removal of contaminating hydrocarbons, further studies are still needed in order to optimize the process and assess its applicability for treatment of the contaminated site. These studies should investigate the effect of environmental parameters on the production of biosurfactants by the indigenous microbial population since the formation of biological surfactants was shown to improve hydrocarbon biodegradation during the bioslurry treatment process. The overall efficiency of hydrocarbon removal and the kinetics of bioslurry process at the conditions of the site must be deter50

mined for a proper design of full-scale treatment technology.

Acknowledgments The authors thank Chantale Beaulieu and the technical officers in the Analytical Chemistry Lab of Dr. Jalal Hawari for their technical support. Thanks are also due to Serge Delisle from the Environmental Microbiology Group for his advice and support during groundwater and soil sampling procedures.

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