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Enhanced Biodegradation of Petroleum Hydrocarbons in Contaminated Soil 37

1058-8337/03/$.50© 2003 by CRC PressBioremediation Journal 7(1):37–51 (2003)

Enhanced Biodegradation of PetroleumHydrocarbons in Contaminated Soil

Laleh Yerushalmi,1,4 Sylvie Rocheleau,1 Ruxandra Cimpoia,1 ManonSarrazin,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 H4Y1G7; 3Environment Canada, 105 McGill Street, Montreal, Quebec, Canada H2Y 2E7; 4Presentaddress: Atara Corporation, 390 Guy Street, Montreal, Quebec, H3J 1S6

ABSTRACT: Soil samples taken from a contaminated site in Northern Quebec, Canada, exhibited a low capacityfor biodegradation of total petroleum hydrocarbons (TPH), despite a high capacity for the mineralization ofaromatic hydrocarbons and a low toxicity of soil leachates as measured by Microtox assay. Toxicity assays directlyperformed on surface soil, including earthworm mortality and barley seedling emergence, indicated moderate tohigh levels of toxicity. Soil biostimulation did not improve the removal of petroleum hydrocarbons, whilebioaugmentation of soil with a developed enrichment culture increased the efficiency of hydrocarbon removal from20.4% to 49.2%. A considerable increase in the removal of TPH was obtained in a bioslurry process, enhancingthe mass transfer of hydrocarbons from soil to the aqueous phase and increasing the efficiency of hydrocarbonremoval to over 70% after 45 days of incubation. The addition of ionic or nonionic surfactants did not have asignificant impact on biodegradation of hydrocarbons. The extent of hydrocarbon mineralization during thebioslurry process after 45 days of incubation ranged from 41.3% to 58.9%, indicating that 62.7% to 83.1% of theeliminated TPH were transformed into CO2 and water.

Keywords: soil contamination, petroleum hydrocarbons, biostimulation, bioaugmentation, bioslurry.

* Corresponding author

IntroductionAn extensive soil contamination with petroleum hy-drocarbons was found in a former meteorological andradio station located in Northern Quebec, Canada. Theconcentration of total petroleum hydrocarbons (TPH,C10-C50) in soil ranged from 1700 to 10,000 mg/kg (drysoil), exceeding the MENV B (Quebec Ministry ofEnvironment) criteria for residential sites (700 mg/kgdry soil) and the MENV C criteria for commercial/industrial sites (3500 mg/kg dry soil). The monitoringactivities conducted as part of an environmental siteassessment showed a lack of natural attenuation ofTPH in soil after more than a decade.

The contamination of soil and groundwater withpetroleum hydrocarbon-based fuels as a result of acci-dental spills or improper storage has been reportedfrequently (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 theUnited States Environmental Protection Agency (USEPA), Quebec Ministry of Environment (MENV), andmany other environment and health organizations inthe world. These chemicals pose serious health andecological threats due to their toxicity and mutagenic-ity.

There are several physical-chemical technologiesfor the treatment of soil contaminated with organic andhazardous material such as petroleum hydrocarbons.They include vapor extraction, stabilization, solidifi-cation, soil flushing, soil washing, thermal desorption,vitrification, and incineration (Balba et al., 1998; Zappiet al., 1996). However, most of these techniques areexpensive to implement at full scale and require con-tinuous monitoring and control for optimum perfor-mance. In addition, they do not usually result in acomplete destruction of the contaminants. Biological

38 Yerushalmi et al.

treatment or bioremediation techniques are alternativemethods for the treatment of contaminated soil. Thesetechniques are economically and politically attractiveand have shown promising results in the treatment ofsoil contaminated with organic compounds, particu-larly 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 site-specific process, and its efficiency may be limited bymicrobiological and physical-chemical conditions ofthe soil. The limiting factors for soil bioremediationinclude the type and concentration of contaminantsand the indigenous microbial population, availabilityof 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 projectwith Transport Canada and Environment Canada. Thestudy investigates the potential for reduction of TPHconcentration in contaminated soil by bioremediationtechniques and identifies the limiting factors, leadingto the recommendation of possible processes to im-prove soil biotreatability.

Materials and MethodsSoil CharacteristicsPrevious assessments of the site had shown that thecontaminated soil contained medium to coarse sand inthe upper layers and a mixture of sand and silt in thelower layers. Soil samples used in the present studywere collected from various locations and depths (0 to15 cm or 1 to 1.5 m) on the contaminated site. Thepercentage of sand in soil samples extracted from thedepth of 1 to 1.5 m was 91.6%, while the soil densitywas 2.2 g/mL. The soil had an average organic contentof 4.3 ± 0.3 g/kg (dry soil).

The chemical analysis of samples showed the pres-ence of metals, including aluminum (1900 to 3000 mg/kg soil), calcium (1100 to 1700 mg/kg soil), iron (6100to 7100 mg/kg soil), magnesium (880 to 1300 mg/kgsoil), and titanium (520 to 600 mg/kg soil). Theseanalyses also showed that the concentrations of poly-cyclic aromatic hydrocarbons (PAHs), volatile organiccompounds

(VOCs), ethylene glycol, and PCBs were belowthe MENV B criteria. The surface soil (0 to 15 cm) hadan average moisture content of 6%, whereas the deepersoil (1 to 1.5 m) had an average moisture content of17%. The pH of soil samples ranged from 5.8 to 6.7.The temperature of the contaminated site changed fromnear zero in the winter to 10°C in the summer.

Preparation of Soil LeachateSoil leachates, used for COD analysis and Microtoxassays, were prepared according to the US EPA method#1312 (United States Environmental ProtectionAgency, 1997). The soil was extracted with deionizedwater, in which the pH was adjusted to 4.5 with asolution of 60% H2SO4 and 40% HNO3. The extractionwas performed by mixing 7 g dry soil with 140 mLextraction liquid for 18 ± 2 h, at 22°C ± 3°C andshaking at 30 ± 2 rpm. Prior to the toxicity test, theresulting soil leachate was filtered through a 0.7-µmborosilicate filter under nitrogen pressure using aMillipore Teflon®-coated filtration system.

Microbial CountThe presence of microbial activity in soil was deter-mined by enumeration of total heterotrophic bacteria,growing on YTS 250 solid medium (yeast extract, 250mg/L; tryptone, 250 mg/L and starch, 250 mg/L), fol-lowed by colony count after a 14 day-incubation at10°C.

Analytical TechniquesTotal Petroleum Hydrocarbons (TPH). The total hy-drocarbons in soil were extracted with hexane accord-ing to the Method 410-HYD 1.0 of Quebec Ministry ofEnvironment. A high-performance liquid chromatog-raphy (HPLC) was used for the analysis of extractedsamples. The system included a pump (Model W600,Waters, Milford, MA, USA), an autosampler (ModelW717, Waters, Milford, MA, USA), a column heater(Model TCM, Waters, Milford, MA, USA), a fluores-cence detector (Model Spectroflow 980, ABI Analyti-cal, Ramsey, NJ, USA), and an ultraviolet detector(Model W490, Waters, Milford, MA, USA). A 20 µLor 100 µL sample was separated on a Supelcosil LC-PAH C18 column (15 cm × 4.6 mm, with a particlesize of 5 µm, Supelco, Bellafonte, PA, USA) with a40:60 (v/v) acetonitrile:water mobile phase for 5 min.The acetonitrile fraction was subsequently increasedto 100%, at a flow rate of 1.5 mL/min, for a period of25 min. Quantification was done by a UV detector at254 nm and a fluorescence detector at 280 nm.

Ion Analysis. The concentrations of sulfate, nitrate,nitrite, phosphate, and chloride in the liquid phasewere monitored by high-performance liquid chroma-tography (HPLC, Model Spectra-Physics, SP8800 andSP8760) using a Hamilton PRP-X100 polymer-basedchromatography column (250 × 41 mm O.D.). A Wa-ters Millipore detector (Model 431) was used to obtainconductivity data. The mobile phase was 4.0 mMp-hydroxybenzoic acid with a flow rate of 2 mL/min.


The injection volume was 100 µL and the injectionport temperature was 40°C.

Chemical Oxygen Demand (COD). The COD of soilleachates was estimated colorimetrically using a HachCOD incubator (Model 45600) and a Hach spectro-photometer (Model DR/3000). The reaction proceededfor 2 at 150°C.

Toxicity AssaysThe toxicity assays were performed in order to deter-mine whether the observed lack of natural attenuationof hydrocarbons at the contaminated site was due tothe high toxicity of soil. The soil samples used fortoxicity tests were taken from depths of 0 to 15 cmfrom three different locations on the contaminated site,referred to as locations #1, #2, and #3. The toxicity ofcontaminated soil samples was compared to that of areference soil taken from a noncontaminated area ofthe site under investigation.

Microtox. Microtox is a standard toxicity assay (Envi-ronment Canada, 1992), measuring the reduction inbioluminescence of the marine bacterium, Vibriofischeri after 15 min or 30 min exposure to the targetcontaminants. The bioluminescence reduction is ex-pressed as the percentage inhibition of light emissionwhen compared with a control containing only 2%NaCl. The test was performed on soil leachates at thetime of their reception (t = 0) as well as on thoseincubated at 10°C for a period of 5 or 10 weeks. Thetests were performed in triplicate and phenol was usedas the reference toxicant. The phenol EC50 value forthe Microtox assay was between 18 and 26 mg/L. TheEC50 value (effective concentration) refers to the con-centration that reduces the average response of the testorganisms by 50% within the test period.

Barley Seedling Emergence and Growth Inhibition.This toxicity test is based on the protocol developed bythe US-EPA (United States Environmental ProtectionAgency, 1989). It measures the seedling emergence(germination) inhibition of barley Hordeum vulgareseeds exposed to contaminated soil. Forty barley seedsof homogeneous size were placed in Petri dishes (150× 20 mm) containing 100 g of the test soil. Deionizedwater was added to the soil to obtain an 85% waterholding capacity. The seeds were covered with sand,and each Petri dish was enclosed in a polyethylene bagcontaining air. The seeds were incubated in the dark at24 ± 2°C for 48 h and were then exposed to a dailyphoto-period, including 16 h of light. The emergedseedlings were counted after 5 and 14 days, and the

percentage of seedling emergence inhibition was esti-mated in comparison with the control, which con-tained deionized water. After 14 days of exposure, thegrowth inhibition was calculated by measuring theplant dry weight when compared with results of thecontrol soil. The test was done in triplicate and thereference toxicant was mercuric chloride. The EC50value for barley seedling emergence was between 130and 330 mg/kg HgCl2 at 5 days, and between 215 and375 mg/kg HgCl2 at 14 days. The EC50 value forbarley growth was between 140 and 360 mg/kg HgCl2

after 14 days.

Earthworm Mortality. This toxicity assay uses Eiseniafoetida, a small (<10 cm long) red worm found incompost and manure. E. foetida has been widely usedfor toxicity evaluation of various toxicants, providingan extensive toxicity databank. The acute toxicity testwas performed directly on soil samples (United StatesEnvironmental Protection Agency, 1989). For eachreplicate, 10 worms (Carolina Biological Supply,Burlington, NC, USA) weighing between 300 and 600mg were exposed to 200 g of test soil in containerswith perforated lids. Deionized water was added to soilin order to obtain a 75% water-holding capacity. After14 days of exposure at 22 ± 2°C with a daily photo-period of 16 h of light, the percentage of worm sur-vival was estimated. The test was performed in tripli-cate and the reference toxicant was potassium chlo-ride. The EC50 value for earthworm was between5000 and 7000 mg/kg KCl. The OECD soil (Organiza-tion for Economic Cooperation and Development,1993) was used as a quality control of the bioassay.

Development of Enrichment CultureThe soil samples taken from depths of 1 to 1.5 m wereused in the development of enrichment culture. Afterthorough mixing of all samples, 100 g of soil wasadded to 100 mL minimal salts medium (MSM) andshaken overnight at 200 rpm. After the soil was al-lowed to settle, the supernatant was removed and dis-tributed in 1000-mL Erlenmeyer flasks, where dieselat a concentration of 90 mg/L was used as the sub-strate. The culture was enriched by regular transfers of10% (v/v) inoculum every 3 weeks into a fresh MSMmedium containing 90 mg/L diesel. The flasks wereshaken at 150 rpm and incubated at 20°C in the dark.The incubation temperature of 20°C was used for thedevelopment of enrichment culture in order to enhancethe rate of microbial growth and to increase biomassproduction.

The efficiency of hydrocarbon removal improvedgradually with the increasing number of transfers, reach-


ing 92.7% after the fourth transfer. The high hydrocar-bon removal efficiency was maintained after 10 suc-cessive transfers.

The minimal salts medium (MSM) had the fol-lowing composition, in g/L: KH2 PO4, 0.87; K2H PO4,2.26; (NH4)2 SO4, 1.1; and Mg SO4.7H2O, 0.097. Tothis solution was added 1 mL (per liter) of a tracemetals solution, in g/L composed of: Co (NO3)2. 6H2O,0.291; AlK (SO4)2. 12H2O, 0.474; Cu SO4, 0.16; ZnSO4. 7H2O, 0.288; Fe SO4. 7H2O, 2.78; Mn SO4. H2O,1.69; Na2 MoO4. 2H2O, 0.482; and Ca (NO3)2. 4H2O,2.36. The final pH of the medium was in the range of6.9 to 7.1. The medium was sterilized by autoclavingat 120°C for 20 min. Distilled water was used in thepreparation of medium throughout this study.

Biodegradation Activity TestsTwo series of activity tests were carried out using soilstaken from depths of 0 to 15 cm and 1 to 1.5 m,respectively. The dynamics of hydrocarbon biodegra-dation by the indigenous microbial culture in soil un-der aerobic condition was determined in 250-mLµ–Carrier Spinner flasks (Belco Biotechnology,Ontario, Canada) containing 220 g of soil. The experi-mental flasks had two side arms, tightly closed withTeflon®-lined caps in order to prevent evaporation ofhydrocarbons and to minimize their loss through ad-sorption onto the caps. The experiments were per-formed at 10°C in order to simulate summer condi-tions at the contaminated site.

Two sets of experiments were performed usingnutrient-amended (biostimulated) and non-amended soilsamples. The biostimulated soil (220 g) received 1 mLof a concentrated nutrient solution, containing 0.96 gfertilizer 20-20-20 (Plantco Inc., Brampton, Ontario,Canada) in 100 mL water. The activity tests wereperformed in duplicate. No bioaugmentation of soilwas performed during these tests. The experimentalflasks were incubated unshaken in the dark for 53days. Soil samples were withdrawn at predeterminedintervals and were analyzed for their content of TPHand COD. The content of flasks was mixed prior tosampling.

Radioactive Mineralization TestsMicrocosm tests measured the extent of mineralizationof 14C-labeled compounds by the microbial culture.The tests were carried out at 10°C in 120-mL serumbottles equipped with a KOH vial, which trapped theproduced carbon dioxide. Soil samples taken from thedepths of 1 to 1.5 m were used in these tests. The CO2

produced by dissimilation of the labeled substrate wassubsequently measured by scintillation counting. Val-

ues in disintegrations per minute (dpm) were thenconverted to mg or percent (%) of substrate mineral-ized over a given period of time. Hexadecane, analiphatic hydrocarbon, and naphthalene, an aromatichydrocarbon, were used in separate bottles as repre-sentative hydrocarbons during the mineralization tests.The initial concentration of hexadecane or naphtha-lene in each bottle was about 0.05 µCi total radioactiv-ity and 10 mg/kg soil. The bottles contained 20 g ofsoil.

Two sets of experiments were performed usingnon-amended and nutrient-amended (biostimulated) soilsamples. All experiments were performed in a closedenvironment to prevent the evaporation of hydrocar-bons or the produced volatile intermediate metabo-lites. The atmospheric oxygen in the serum bottles wassufficient to support aerobic growth. However, in or-der to prevent oxygen deficiency during the later stagesof mineralization, 0.5 mL air was injected into thebottles by a sterile syringe once a week after 30 daysof incubation. No pressure buildup was observed. Themineralization experiments were performed in tripli-cate and lasted for 90 days. Control bottles were auto-claved 3 times with 24-h intervals to eliminate biologi-cal activities.

Bioaugmentation and BioslurryTreatment SystemsTwo processes were investigated in an effort to createfavorable conditions for biological degradation of hy-drocarbon contaminants of soil. They includedbioaugmentation, using the enrichment culture devel-oped from soil indigenous microbial population, andbioslurry process, to increase the mass transfer of soil-bound hydrocarbons to the aqueous phase. Soil samplestaken from the depths of 1 to 1.5 m were used in thesestudies.

Bioaugmentation. Soil bioaugmentation is a solid-phase process where specific seed microorganisms areadded to soil in order to enhance its biological activity.The seed microorganisms are often developed throughan enrichment process. This procedure results in theselection of the most efficient microorganism(s) thatpossess the necessary metabolic pathways and enzy-matic system for degradation of contaminants(Cookson, 1995). Soil bioaugmentation is most effec-tive when the soil is not nutrient deficient, but theindigenous microbial population lacks the requiredactivity or metabolic capability. However, this tech-nology has a limited capacity if the bioavailability ofcontaminants, controlled by their desorption from soil,is the rate-limiting step in bioremediation.


During the present study, the contaminated soilwas amended with the developed enrichment cultureand was stimulated by the addition of nutritional supple-ments (1 mL fertilizer solution added to 220 g soil) andpure oxygen. The experiments were performed at 10°Cin 500-mL Erlenmeyer flasks, shaken in a wrist-actionshaker (Model 75, Burrell Corporation, PA, USA) forthe entire duration of tests, which lasted for 60 days.The soil inside the flasks was manually mixed daily inorder to ensure homogeneity and to maximize oxygen-ation of soil. The soil used in the control system wasirradiated by gamma ray (Nordion, Montreal, Canada)to eliminate its biological activities. All experimentswere performed in triplicate. Soil samples were with-drawn at predetermined intervals and were analyzedfor their content of TPH.

Bioslurry Process. The bioslurry treatment systemconsists of a mixture of soil in water maintained in astirred reactor. Bioslurry treatment is an establishedand accepted site remediation method for decontami-nation of soils, sediments, and sludges (Cookson, 1995;Fava et al., 2000; Zappi et al., 1996), offering analternative technique to solid-phase processes for theenhancement of soil biotreatability. It enhances theavailability of contaminants, electron acceptors, andnutrients to the microbial population, and offers themost suitable conditions for the treatment of contami-nated soil, especially when mass transfer limitationscontrol the rate of contaminant biodegradation by themicrobial biomass (Zappi et al., 1996). The reductionof contaminant concentration in soil is considerablyhigher in bioslurry reactors compared with that ob-served in solid-phase reactors because of the increasedsolid-liquid mass transfer. The improved homogeneityand increased flexibility of operation, as well as anenhanced control over the operation of system, makebioslurry the most efficient treatment technology forcontaminated soils. The application of bioslurry reac-tors in the treatment of soil contaminated with petro-leum hydrocarbons has been reported before (Castaldiand Ford, 1992; Cassidy et al., 2000; Zappi et al.,1996).

Nonradioactive, aerobic microcosm tests were usedto simulate various soil bioslurry conditions and todefine the most effective biotreatment system for theremoval of TPH from soil. These tests were carried outat a higher temperature of 20°C in order to eliminatethe contribution of temperature as a limiting factor inorder to determine the potential for the biodegradationof hydrocarbons in the contaminated soil. All of thestudied treatment conditions used 5% soil slurry inphosphate buffer solution. 120-mL gas tight serumbottles with a working volume of 45 mL, used as the

bioslurry reactors, were shaken at 100 rpm on an or-bital shaker in the dark for 45 days. The headspace ofthe bottles was sparged with pure oxygen in order toensure a sufficient supply of oxygen during the entirecourse of the process. Several treatment conditionswere evaluated to obtain information on the rate andextent of hydrocarbon removal and to determine themost favorable condition for biodegradation of hydro-carbons. They included:

• Slurry + Tergitol NP-10: using contaminated soilwith the addition of nonionic surfactant TergitolNP-10 (Sigma, Oakville, Ontario, Canada), nutri-tional supplement, and enriched biomass.

• Slurry + SDS: using contaminated soil with theaddition of anionic surfactant sodium dodecyl sul-fate (SDS) (Sigma, Oakville, Ontario, Canada),nutritional supplement, and enriched biomass.

• Slurry: using contaminated soil with the addition ofnutritional supplement and enriched biomass, butwithout any external surfactants.

• Control system: using irradiated soil to eliminatebiological activities, without the addition of surfac-tants, enriched biomass, or nutritional supplement.

The concentration of surfactants was 1 mg/g soil.The nutrient solution, prepared by dissolving 0.96 gfertilizer 20-20-20 in 100 mL distilled water, was addedin sufficient quantities to provide C:N:P ratios of50:10:1. One mL concentrated enriched biomass, pre-pared by centrifuging the entire content of an enrich-ment flask (100 mL), was added to 220 g soil in eachbottle.

Three experimental bottles were opened and sac-rificed at each sampling time for the analysis of TPHin the aqueous and soil phases. Gas samples werealso taken from the headspace of separate bottlesoperating under similar conditions to determine theproduction of carbon dioxide as an indicator of hy-drocarbon mineralization. The slurry pH was main-tained at 6.8 ± 0.3.

The total extent of hydrocarbon mineralizationwas estimated from the amount of carbon dioxideproduced as a result of the complete mineralization ofhydrocarbons. The theoretical ratio of mg CO2 pro-duced to mg hydrocarbons mineralized was calculatedfrom stoichiometric relationships for oxidation of pe-troleum hydrocarbons with the following composition(Potter and Simmons, 1998): alkanes, 20%; alkenes,2.0%; mono-aromatics, 27%; naphthalenes, 3.5%;cycloalkanes, 40%; and polyaromatics, 7.5%. The es-timated value of the above ratio was 3.2. The changeof fuel composition had only a minor impact on theestimated value of this ratio.


Results

Toxicity AssaysThe Microtox assays showed a low level of toxicity inthe contaminated soil leachates. The maximal inhibi-tion of soil leachates was < 39.0% during the Microtoxassay (Table 1), representing a low level of toxicityunder all of the examined conditions. The reportedvalues of maximal inhibition represent the percentagereduction of bioluminescence in a diluted (50%) soilleachate. Similar trends were observed after 15 and 30min of exposure.

A moderate inhibition of barley seedling emer-gence was observed after both 5 and 14 days of expo-sure to soil samples. The toxicity slightly increased insamples incubated for 10 weeks (t= 10w) comparedwith those tested at t=0 (Figure 1). Seedling

emergence inhibition greater than 50% was obtainedin soil samples incubated for 10 weeks, except for thereference soil and the contaminated soil of location #1.Growth inhibition of barley seedlings was observedafter 14 days of exposure to the soil samples in spite ofan initial stimulation of seedling growth.

A relatively low 14-day earthworm mortality of< 10% was observed in the contaminated soil samplestaken from locations #1 and #2, whereas soil samplestaken from location #3 exhibited a high toxicity toearthworms (Figure 2). Incubation of soil samples for10 weeks did not have a significant impact on thesurvival of earthworms.

Radioactive Mineralization TestsThe presence of biological activity in soil was indi-cated by the total heterotrophic bacterial count that

Table 1. Results of Micrtox toxicity of soil leachates using 15 and 30 min bacterial exposrue time.


Figure 1. Barley seedling emergence and growth inhibition assay in surface soil (0-15 cm) at the reception of the soil samples (t=0)and after 10 weeks of incubation (t=10 w) at 10°C. The emergence of barley seeds was assessed after 5 days (5 d) and 14 days(14 d) of exposure to soil. Error bars represent the corresponding standard deviations.

Figure 2. Earthworm mortality assay in surface soil (0 to 15 cm) at the reception of the soil samples (t=0) and after 10 weeks ofincubation (t=10 weeks) at 10°C. Error bars represent the corresponding standard deviations.


ranged from 4.28 × 105 to 8.07 × 105 colony formingunits (CFUs/g soil). The indigenous microbial consor-tium exhibited a high capacity to mineralize aromaticcompounds, while presenting a lower capacity formineralization of aliphatic compounds. The extent ofmineralization of naphthalene used as the representa-tive aromatic compound was 62.4% ± 2.6% after 90days of incubation (Figure 3). The efficiency of naph-thalene mineralization increased to 93.2% ± 7.4% afterbiostimulation of soil, representing a considerable in-crease in the overall efficiency of mineralization ofaromatic compounds. The mineralization of hexadecaneused as the representative aliphatic compound was1.8% ± 0.8% after 90 days of incubation, increasing to8.4% ± 3.4% after the addition of nutritional supple-ments (Figure 4).

First-order kinetics adequately described the mineral-ization trends of hexadecane and naphthalene with or with-out soil biostimulation. The rate constants for hexadecanemineralization were 0.002 d–1 and 0.0002 d–1 (R2 > 0.99)for the nutrient-amended (biostimulated) andnonamended soil, respectively. For naphthalene, thesevalues changed to 0.05 d–1 and 0.01 d–1 under the tworespective conditions (R2 >0.93). The model predic-tions are presented as solid lines in Figures 3 and 4.These results indicated that biostimulation of soil hada positive effect and increased the efficiencies and

rates of mineralization of aliphatic and aromatic com-pounds.

Biodegradation Activity TestsSimilar results were obtained in the two series of tests usingsoils taken from depths of 0 to 15 cm and 1 to 1.5 m, respec-tively. Only the results of experiments with soil samplestaken 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 contaminatedsoil had a low capacity for biodegradation of petro-leum hydrocarbons. The concentration of TPH de-creased from 4400 mg/kg dry soil to 3500 mg/kg drysoil after 53 days of incubation, representing a removalefficiency of 20.4% (Figure 5). The supply of nutrientsdid not increase the removal efficiency. During thesetests, the COD concentration of soil leachates decreasedfrom 267 mg/L to 177 mg/L, representing a removalefficiency of 33.7%. The activity tests were carried outwithout any bioaugmentation and the biodegradationactivities were attributed to the indigenous microbialpopulation in the contaminated soil. The abiotic re-moval of hydrocarbons was 6.8%, leaving 13.6% elimi-nation due to biological degradation. The first-orderrate constants for the removal of hydrocarbons fromsoil were 0.007 d–1 and 0.009 d–1 in the absence andpresence of biostimulation, respectively.

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


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

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


The low removal efficiency of hydrocarbons, de-spite a high mineralization capacity for naphthaleneand a low toxicity of soil leachates, suggested thefollowing possibilities:

1. The microbial consortium does not have theenzymatic diversity required to degrade all ofthe contaminating hydrocarbons in soil.

2. The hydrocarbons in soil are recalcitrant andresist further biotransformation.

3. The hydrocarbons are strongly sorbed to thesoil matrix, rendering them inaccessible formicrobial degradation.

Bioaugmentation and bioslurry studies were car-ried out in order to verify the above hypotheses and todevelop possible methods to enhance the biodegrada-tion of hydrocarbon contaminants of soil.

Soil BioaugmentationSoil bioaugmentation using the developed enrichmentculture resulted in an overall TPH removal efficiencyof 49.2% after 60 days of operation (Figure 6), ofwhich more than 79% of the overall removal wasobtained during the first 15 days of operation. Theabiotic removal of TPH was 23.1%, as indicated in thenonbioaugmented control system, leaving 26.1% elimi-nation as a result of biological degradation. The abiotic

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

loss of hydrocarbons during the soil bioaugmentationtests was higher than that obtained during the activitytests due to the nature of experimental set up and thefrequency of sampling that affected the contribution ofphysical-chemical processes to the removal of hydro-carbons.

Compared with the previously mentioned soil bio-degradation activity results where the biological elimi-nation of hydrocarbons was equal to 13.6%, thebioaugmentation of soil increased the biological deg-radation of contaminants by 92%. However, it shouldbe noted that the initial removal rate of hydrocarbonsdid not increase as a result of soil bioaugmentation,because values of 153.3 mg TPH/kg dry soil.day and150.0 mg TPH/kg dry soil.day were obtained for soilwith or without bioaugmentation, respectively.

Despite an increase in biodegradation of contami-nants as a result of soil bioaugmentation, the overallextent of contaminant removal was still below 50%,suggesting that bioaugmentation alone would not besufficient to decontaminate soil according to the envi-ronmental regulatory limits.

Diesel fuel is known to have a low bioavailabilitydue to sorption to the minerals and humic fraction ofsoil (Cookson, 1995). It has also been established thatsolid-phase technologies, such as bioaugmentation,suffer from mass transfer limitations, controlling thetransfer of contaminants from soil to the aqueous phase.


Accordingly, soil bioslurry experiments were performedin order to increase solid-liquid mass transfer, thusenhancing the biodegradation rates of the contami-nants.

Bioslurry TreatmentThe efficiency of TPH removal from soil increased toover 70% during the bioslurry experiments (Figure 7).The control bioslurry system exhibited an overall re-moval efficiency of 35.6% ± 2.6%, while the TPHremoval efficiency in the bioslurry reactors after 45days of incubation ranged from 65.8% ± 6.3% to 70.9%± 6.8%. This increase was possibly due to the in-creased transfer of contaminants from the soil surfaceto the aqueous phase and their enhanced dissolutionrate that increased the bioavailability of contaminantsto the microbial culture. An increased biological activ-ity was apparent as evaluated by an increase in theproduction of carbon dioxide in the headspace of theexperimental bottles, demonstrating that petroleumhydrocarbons were mineralized during the process.While no carbon dioxide was detected in the controlsystem, its production ranged from 7.8 to 9.3 g/kg drysoil in the slurry flasks. Based on the theoretical pro-duction of 3.2 mg CO2/mg hydrocarbons mineralized,these values represent 41.3% to 58.9% hydrocarbonmineralization during the bioslurry process based onthe initial TPH concentration in the system. Given the

total extent of hydrocarbon removal, these values indi-cate that 62.7% to 83.1% of the eliminated hydrocar-bons were transformed into CO2 and water. The trans-formation of volatile TPH to the gas phase ofexperimental flasks (evaporation), adsorption to theexperimental system, and chemical transformation inthe presence of oxygen all account for the abiotic lossof TPH in the control system.

High efficiencies of TPH removal were obtainedin the presence as well as the absence of selectedsurfactants (Figure 7). This implies that the addition ofexternal surfactants was not necessary to enhance TPHbiodegradation in soil. Foaming was observed underall of the applied conditions, suggesting the productionof biosurfactants by the microbial culture even whenexternal surfactants were not added. In fact, prelimi-nary experiments using the groundwater sampled fromthe contaminated site under investigation showed thereduction of surface tension in water from 59 dynes/cm in the control system to 27 dynes/cm in the biologi-cally active system, indicating the production ofbiosurfactants by the indigenous microbial populationas reported before (Cassidy et al., 2000). These resultssuggest that a biological surfactant was produced at aconcentration sufficient to promote the desorption ofsorbed contaminants from the soil matrix, thus in-creasing their contact with the microorganisms andimproving their biodegradation.

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


The mechanism underlying the enhanced biodeg-radation of TPH was evaluated through independentanalysis of TPH concentrations in the liquid and soilphases of the bioslurry flasks (Figure 8 ). The presenceof surfactants, both ionic and nonionic, promoted thedesorption of TPH from soil and increased their disso-lution rate in the aqueous phase as indicated by theincreased initial liquid-phase concentration of TPHwhen compared with the control system. The nonionicsurfactant Tergitol NP-10 had a more pronounced ef-fect compared with the anionic surfactant SDS, result-ing in a higher initial TPH concentration in the liquidphase (Figure 8). The percentage of TPH initially trans-ferred to the liquid phase was 9.0% in the controlsystem, indicating the very low solubility of the sorbedTPH in the aqueous phase. This value increased to38.1% in the presence of SDS and further to 72.1% inthe presence of Tergitol NP-10, emphasizing the highdissolution rate of contaminants. In the absence ofexternal surfactant (slurry system), the initial increasein the liquid-phase concentration of TPH was 12.6%,which is slightly more than that observed in the controlsystem, suggesting the low initial rate of biosurfactantproduction that depressed the rate of contaminant de-sorption from the soil matrix. However, the high re-moval of TPH in the slurry system implies that thegradual production of biosurfactants was sufficient toensure an overall high removal efficiency of contami-nants. Correspondingly, the remaining TPH concen-tration in soil phase was considerably lower in thepresence of surfactants (Figure 8). The total initialTPH in the experimental flasks that did not receive anysurfactants (slurry) were slightly lower than those inthe other bottles, possibly due to the heterogeneity ofsoil samples, despite a thorough mixing of soil beforethe start of experiments.

DiscussionThe results obtained from the bioaugmentation andbioslurry treatment studies indicated that the contami-nated soil from a site in Northern Quebec, Canada, isbiotreatable, and its contaminants may be removedwith >70% efficiency. The slight increase of removalefficiency in the solid phase bioaugmentation treat-ment system with the addition of seed microorganismsand nutrients showed the possible deficiency of theunsupplemented (original) soil with respect to theseparameters. However, the remarkable increase of thebiodegradation rate in the slurry reactors, comparedwith that observed in the bioaugmented andbiostimulated soil, indicated that sorption of TPH tosoil was the limiting factor, controlling the biodegra-dation of contaminants. The considerably higher water

to solid ratio and thorough mixing in the slurry system,combined with the presence of surface active com-pounds, increased hydrocarbon dissolution rate. Thesefactors thus increased the rate of mass transfer to theliquid phase and improved the bioavailability of con-taminants to microorganisms. Increasing the tempera-ture to 20°C may have increased the biodegradationrate as a result of higher solubility of hydrocarbons andan increased degree of distribution. However, the effi-ciency of cold-adapted hydrocarbon degraders at lowtemperature has been shown to be comparable to thatof mesophiles at higher temperatures (Margesin andSchinner, 1997). Of course, the low temperature of thecontaminated site reduced biological activities andresulted in a low rate of hydrocarbon biodegradation.It is plausible that a higher extent of biodegradationwould result after a considerably longer period of time.

The biological availability of contaminants to soilbacteria is recognized as a potential limiting factorduring bioremediation studies (Autry and Ellis, 1992).The hydrophobicity of compounds, sorption onto soilmatrix, or volatilization of compounds are the majorfactors limiting the bioavailability of compounds. Li etal. (1995) measured biodegradation rates, oxygen trans-fer rates, and oil transfer rates during bioremediationof oil-contaminated soil and observed that the rate-controlling step was the mass transfer of oil into aque-ous solution.

The addition of surfactants has been reported toimprove the efficiency of bioslurry processes by in-creasing the rate of mass transfer of contaminants tothe aqueous phase. Autry and Ellis (1992) employed atechnology known as SafesoilTM to increase the bio-degradation of gasoline–derived TPH in soil. In thistechnology, the amendment of soil with surfactantsincreased the mass transfer of contaminants to theaqueous phase, increasing the contact of contaminantswith microbial population in soil and enhancing theirbiodegradation. It is believed that the addition of sur-factants reduces the initial adaptation period of theprocess by making the contaminants more available tothe microbial population. The enhanced biodegrada-tion of crude oil by the addition of chemical surfac-tants has been reported by Van Hamme and Ward(1999). However, in the present study the use of exter-nal surfactants was not necessary because the micro-bial culture may have produced biosurfactants, and theaddition of external surfactants to the experimentalsystem did not increase the overall efficiency of con-taminant biodegradation.

The addition of adapted bacterial culture to soil(bioaugmentation) has demonstrated a limited increasein the overall efficiency of hydrocarbon removal. Autryand Ellis (1992) observed a minor increase in biodeg-


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


radation of petroleum hydrocarbons as a result ofsoil bioaugmentation, implying that the limitedbiotreatability of soil was not due to the absence ofhydrocarbon-degrading bacteria. As reported byMargesin and Schinner (1997), the addition of micro-organisms can accelerate the initial phase of biodegra-dation and can be advantageous when thecontaminants have a toxic effect on the indigenousmicroorganisms. Demque et al. (1997) also reportedthat bioaugmentation with adapted indigenous micro-organisms had little or possibly negative effects onbiodegradation of diesel fuel in a contaminated sand.The inoculation of soil seems to have a more pro-nounced effect on the rate of hydrocarbon biodegrada-tion (or mineralization) at low temperatures. Whyte etal. (1999) and Mohn and Stewart (2000) reported areduced lag time and an increased rate of hydrocarbonmineralization as a result of soil bioaugmentation attemperatures below 10°C.

First-order kinetics for the removal of petroleumhydrocarbons from soil were previously observed byTaylor and Viraraghavan (1999), who reported rateconstants of 0.01 d–1 and 0.03 d–1 during the biodegra-dation of hydrocarbons in nonamended and amendedsoil, respectively. These values are higher than thoseobtained in the present study by the original andbiostimulated soil (0.007 and 0.009 d–1), indicating thelow capacity of soil for biodegradation of petroleumhydrocarbons.

The abiotic removal of hydrocarbons ranged from6.8% during the activity tests to 35.6% in the bioslurrytreatment. These values are in the range of the reportedvalues for abiotic elimination of hydrocarbons.Margesin and Schinner (1997) observed 30% removalof diesel oil from alpine soils at 10°C as a result ofabiotic processes. Bragg et al. (1994) also noticed aloss of 30% of hydrocarbons due to physical pro-cesses. The elimination of contaminating hydrocar-bons by abiotic processes constitutes a significant frac-tion of oil removal and must be taken into considerationin order to avoid over estimation of hydrocarbon bio-degradation.

Although the soil bioslurry process exhibited anenhanced removal of contaminating hydrocarbons,further studies are still needed in order to optimize theprocess and assess its applicability for treatment of thecontaminated site. These studies should investigate theeffect of environmental parameters on the productionof biosurfactants by the indigenous microbial popula-tion since the formation of biological surfactants wasshown to improve hydrocarbon biodegradation duringthe bioslurry treatment process. The overall efficiencyof hydrocarbon removal and the kinetics of bioslurryprocess at the conditions of the site must be deter-

mined for a proper design of full-scale treatment tech-nology.

AcknowledgmentsThe authors thank Chantale Beaulieu and the technicalofficers in the Analytical Chemistry Lab of Dr. JalalHawari for their technical support. Thanks are also dueto Serge Delisle from the Environmental Microbiol-ogy Group for his advice and support during ground-water and soil sampling procedures.

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