influence of peanut oil on microbial degradation of polycyclic aromatic hydrocarbons

Influence of peanut oil on microbial degradation ofpolycyclic aromatic hydrocarbons

Jasvir K. Pannu, Ajay Singh, and Owen P. Ward

Abstract: Peanut oil amendment (0.1%–0.2% (v/v)) increased the biodegradation of various polycyclic aromatic hydro-carbons (PAHs) by 15%–80% with a mixed bacterial culture and a pure culture of Comamonas testosteroni in aqueousmedia and in PAH-contaminated weathered soil slurry systems. The stimulatory effect on biodegradation was more pro-nounced with the high molecular weight PAHs (e.g., >3 rings). The presence of peanut oil also accelerated thebiodegradation of PAHs sorbed onto activated carbon, indicating its potential application in the bioregeneration of acti-vated carbon.

Key words: polycyclic aromatic hydrocarbons, microorganisms, peanut oil, biodegradation.

Résumé : Un ajout d’huile d’arachide (0,1 % – 0,2 % (v/v)) a augmenté la biodégradation de divers hydrocarburesaromatiques polycycliques (HAP) de 15 % – 80 % par une culture bactérienne mixte et une culture pure de Comamo-nas testosteroni dans un milieu aqueux et des systèmes de boues provenant de sols altérés et contaminés aux HAP.L’effet de stimulation de la biodégradation fut davantage prononcé avec des HAP de poids moléculaires élevés (p. ex.>3 cycles). La présence d’huile d’arachide a accéléré la biodégradation des HAP retenus sur du charbon actif, ce quisignale une application possible pour la biorégénération du charbon actif.

Mots clés : hydrocarbures aromatiques polycycliques, micro-organismes, huile d’arachide, biodégradation.

[Traduit par la Rédaction] Pannu et al. 513

Introduction

Although biological treatment of contaminated soil is awell-established technology, a disadvantage of the techniqueis that the rates of degradation of hydrophobic compoundscan be slow because of their limited bioavailability (reviewedby Providenti et al. 1993). Strategies to increase the bio-availability of compounds promoting biodegradation includethe use of (bio)surfactants (Rouse et al. 1994; Banat et al.2000), oxidative enzymes (Alexander 2000), various chemi-cal pretreatment methods (Scott and Ollis 1995), and parti-tioning into organic solvents (Efroymson and Alexander 1991).The polycyclic aromatic hydrocarbons (PAHs), containingfused aromatic rings, constitute a family of hazardous com-pounds that are widely present as contaminants in the envi-ronment. They are found naturally in petroleum oil and coaland are major products of incomplete combustion of organicmaterials. The higher molecular weight PAHs, such as chry-sene, pyrene, and benzo[a]pyrene, especially, exhibit lowbioavailability characteristics because of their very low wa-ter solubility (Stucki and Alexander 1987; Boldrin et al.1993; Tiehm 1994). PAH bioavailability in contaminated soilis further reduced by strong PAH sorption to soil particles(Stapleton et al. 1994). Furthermore, while the biodegradationof smaller PAHs is well recognized, no strains have yet beenfound to use PAHs with greater than four rings, such as

benzo[a]pyrene, as sole carbon source. However, their co-metabolic transformations have been demonstrated (reviewedby Kanaly and Harayama 2000).

The use of synthetic and biological surfactants, which candesorb and solubilize nonpolar substrates in soil, have beenshown to have beneficial, inhibitory, or neutral effects on thebiodegradation of hydrophobic contaminants (Desai and Banat1997). Chemical surfactants, such as Tween 80, promotedegradation of PAHs (Mueller et al. 1991), and nonylphenolethoxylate accelerates degradation of the various hydrocar-bon components in crude oil (Van Hamme and Ward 1999).The disadvantages of using surfactants are related to incon-sistencies in their performance, their relatively high costs,and in some cases their toxicity and persistence in the envi-ronment. Furthermore, the concentrations of surfactants re-quired to solubilize hydrophobic compounds in soil slurriesare much higher than the corresponding concentrationsneeded in an aqueous solution because a large portion of thesurfactant is removed from the aqueous phase by beingbound to soil (Rouse et al. 1994).

Solvent extraction is an effective method for the removalof organic hydrophobic contaminants from soil (Richter etal. 1996). However, the solvents used, for example, alkanes,alcohols, ketones, and alkylamines, are typically flammable,volatile, and toxic. Their use involves significant safety andenvironmental risks, and their application requires stringent

Can. J. Microbiol. 49: 508–513 (2003) doi: 10.1139/W03-068 © 2003 NRC Canada

508

Received 24 February 2003. Revision received 2 September 2003. Accepted 4 September 2003. Published on the NRC ResearchPress Web site at http://cjm.nrc.ca on 13 October 2003.

J.K. Pannu, A. Singh, and O.P. Ward.1 Department of Biology, University of Waterloo, Waterloo, ON N2L 3G1, Canada.

1Corresponding author (e-mail: [email protected]).

I:\cjm\cjm4908\W03-068.vpOctober 6, 2003 9:41:21 AM

Color profile: DisabledComposite Default screen

and costly measures to minimize those risks. In contrast,vegetable oil is a natural, nontoxic, cost-effective, biode-gradable extractant and is capable of solubilizing PAHs. Inaddition, any oil remaining in soil after extraction has thepotential to enhance the biodegradation of residual contami-nants by acting as a growth substrate for biodegrading mi-croorganisms and by enhancing bioavailability. We havepreviously shown that peanut oil is effective in extractinghydrophobic contaminants from aqueous and soil systems(Pannu et al. 2003). In this study, the influence of peanut oilon the biodegradation of PAHs in aqueous and soil slurrymedia is evaluated.

Materials and methods

PAHs of highest purity (92%–99%), peanut oil and pow-dered activated carbon (Darco G-60, 100 mesh), were ob-tained from Aldrich (St. Louis, Mo., U.S.A.). WeatheredPAH-contaminated soil, obtained from Biorem TechnologiesInc., Guelph, Ont., was partially ground with a pestle andmortar and sieved (2 mm) for uniform consistency.

The mixed bacterial culture used in the present in-vestigation was isolated in our laboratory from a PAH-contaminated site. The culture was streaked on Difco nutri-ent agar plates containing 0.2 µg/mL of anthracene and0.05% of glucose. Plates were incubated at 30 °C for up to7 days. Morphologically distinct colonies were purified byserial transfer onto the nutrient agar plates. The identifica-tion of pure isolates was performed by the Agriculture andFood Laboratory Services, Guelph, Ont., and based on afatty acid profile. The major species found in this mixed cul-ture were Enterobacter agglomerans, Erwinia herbicola,Comamonas testosteroni, Pseudomonas syringae, and Pseu-domonas fluorescens. The medium contained the following(per litre): K2HPO4, 0.5 g; NH4Cl, 1.0 g; Na2SO4, 2.0 g;KNO3, 2.0 g; MgSO4·7H2O, 0.2 g; yeast extract, 1.0 g;bactopeptone, 1.0 g; and trace metal solution, 3 mL. ThepH of the medium was adjusted to 7.0. The trace metals so-lution contained the following (per litre): FeCl3·6H2O,0.162 g; ZnCl3·4H2O, 0.014 g; CoCl2· 6H2O, 0.012 g;Na2MoO4·2H2O, 0.012 g; CuSO4·5H2O, 1.9 g; and H3BO3,0.05 g.

For the preparation of pure inoculum, microbial colonieswere inoculated into 250-mL Erlenmeyer flasks containing50 mL of medium and 50 ppm of anthracene and incubatedfor 24 h at 30 °C on an orbital shaker (200 r/min). The cellpellet was washed twice with 0.025 mol/L potassium phos-phate buffer (pH 7.0) and resuspended to an optical densityof 0.5 at 600 nm (1-cm light path) in the same buffer. Thisinoculum was used in biodegradation studies at 1% (v/v)level. The mixed bacterial inoculum was prepared by com-bining equal volumes (0.2% for a total inoculum size of 1%(v/v)) of inoculum from the five bacterial species describedabove.

Biodegradation experiments were performed in aqueousand soil slurry systems with 50-mL glass serum bottles(10 mL of the medium) and 250-mL Erlenmeyer flasks (50 gof 1:1, soil/medium slurry), respectively. For aqueous cul-ture, the appropriate amount of anthracene from the stocksolution prepared in dichloromethane (DCM) was added tothe sterile serum bottle, and the DCM was allowed to evapo-

rate before adding medium. After inoculation, cultures wereincubated with orbital shaking (200 r/min) for up to 24 daysat 30 °C. Heat-killed controls were included to detect anyabiotic losses. No significant abiotic losses were observed inany experiment. All the experiments were carried out in du-plicate, and variability between replicate samples within ex-periments was less than 5%. Variability in reproducibilitybetween experiments was less than 10%.

For the extraction of PAHs from aqueous cultures, includ-ing the medium containing activated carbon, 25 µg/mL ofmethyl anthracene was added as surrogate extraction stan-dard to the whole sample (10 mL) and then was extractedwith 20 mL of DCM for 20 min on an orbital shaker. DCMextracts, recovered with a Pasteur pipette, were passedthrough a sodium sulfate column and concentrated to 1 mLunder nitrogen. For extractions from soil slurries, 50 µg/mLof methyl anthracene and 5 g anhydrous sodium sulfate wereadded to 10 g samples. Samples were extracted for 20 minwith 1:1 (v/w) DCM, centrifuged at 3000g for 5 min, andthe DCM extract was passed through a Florisil (Sigma)(60/100 mesh, methanol treated) column. The efficiency ofPAH extraction with DCM was greater than 99%.

PAH samples were analyzed with a gas chromatograph(Shimadzu, Kyoto, Japan) connected to a data integrator andequipped with a split injector, a flame ionization detector,and an AOC-17 auto injector. The column was a fused silicaRtx-5MS W/Integra-Guard (30 m length, 0.32 mm diameter,and 0.25 µm film thickness; Chromatographic Specialties,Brockville, Ont.,). Sample split ratio was 25:1. Helium(10 mL/min) and nitrogen (40 mL/min) were the carrier andmake up gases, respectively. Injector and detector tempera-tures were 280 and 300 °C, respectively. PAH analysis con-ditions were set at an initial temperature of 100 °C for 3 min,a temperature program of 10 °C/min to the final columntemperature of 300 °C for 7 min.

Results and discussion

In preliminary experiments, the effect of peanut oil on thedegradation of 100 µg/mL anthracene by C. testosteroni wasevaluated in the medium over a 24-day incubation period. Aremarkably positive effect of oil supplementation on anthra-cene degradation was observed. Anthracene concentrationwas reduced by 21.5, 25.7, and 40.3 µg/mL in cultures con-taining 0%, 0.01%, and 0.1% peanut oil, respectively, after24 days. Thus, supplementation with 0.1% oil increased theextent of anthracene degradation by 87%. Peanut oil addi-tion had little effect on microbial counts after 24 days, indi-cating that peanut oil did not support any significant bacterialgrowth. Log colony-forming units per millilitre ranged from7.4 in the no peanut oil control to 7.85 in the 0.1% peanutoil supplemented culture. When the pure culture inoculumwas replaced by a mixed culture the extent of degradationafter 24 days was 20.6 µg/mL and 45.4 µg/mL with no oiland 0.1% oil, respectively.

The effect of anthracene concentration on its biodegra-dation by the mixed culture in the presence of 0.1% peanutoil is presented in Fig. 1. Over the first 6 days the extent ofanthracene biodegradation increased with increasing sub-strate concentration in the range 0–3000 µg/mL. Over a 24-day incubation period the extent of anthracene degradation

© 2003 NRC Canada

Pannu et al. 509



increased with increasing substrate concentration in the range0–1000 µg/mL. However, relative to the initial amount, thepercentage of anthracene degraded was lower at 3000 µg/mL,possibly due to an accumulation of metabolites causing cyto-toxicity (Stringfellow and Aitken 1995; Shuttleworth andCerniglia 1996; Juhasz et al. 2002). Microbial counts inthese cultures remained fairly constant (log CFU/mL range7.0–7.5), indicating no significant changes in microbialgrowth. The increase in biodegradation with increased sub-strate concentration followed saturation kinetics (Sherill andSayler 1980; Herbs 1981).

The effect of 0.1% peanut oil on the biodegradation of amixture of five PAHs by C. testosteroni and a mixed bacte-rial culture in an aqueous culture is presented in Table 1.Peanut oil increased the extent of degradation of all PAHsafter 24 days. The stimulatory effect on biodegradation wasmore pronounced with the more difficult-to-degrade highmolecular weight PAHs. For example, after 24 days, the ex-tent of degradation of benzo[a]pyrene by C. testosteroni in-creased from 32.0% in the absence of oil to 49.6% with0.1% peanut oil. The same patterns were observed when theexperiment was repeated with the mixed culture inoculum.In this case after 24 days, the extent of degradation ofbenzo[a]pyrene increased from 41.6% in the absence ofpeanut oil to 63.5% with 0.1% peanut oil. The better perfor-

© 2003 NRC Canada

510 Can. J. Microbiol. Vol. 49, 2003

Fig. 1. Biodegradation of anthracene in aqueous culture byComamonas testosteroni. The experiment was carried out in 50-mLcrimp-closed serum bottles with different anthracene concentrations(10–3000 mg/mL) in the presence of 0.1% peanut oil. The bottleswere incubated with shaking (200 r/min) for 24 days at 30 °C. Thedata presented are the means of duplicate samples. The experimentwas repeated at least two times and trends were identical in the in-dependent experiments.

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mance of the mixed culture, as compared with the bestPAH-degrading pure culture, C. testosteroni, is consistentwith other PAH biodegradation studies (Keck et al. 1989;Bhatnagar and Fathepure 1991; Trzesicka-Mlynarz and Ward1995) and is likely due to the wider range of enzymes andgreater diversity of metabolic pathways present in the mixedculture.

The effect of peanut oil amendment on the biodegradationof PAHs by the mixed culture in weathered soil (50%, w/vslurry in water) was also evaluated. Given the previouslynoted observation that higher concentrations of surfactantsare required to solubilize hydrophobic contaminants in soilthan in aqueous systems (see Introduction), the effect of pea-nut oil supplementation was evaluated in the range 0%–0.3%and up to 24 days of incubation. The results are presented inTable 2. Supplementation of 0.1% peanut oil only slightlyincreased the percentage of degradation. Significantly in-creased degradation was observed with oil supplementationup to 0.2% after 24 days. Further peanut oil supplementationto 0.3% inhibited PAH degradation (relative to 0.2% peanutoil). When the extents of PAH degradation with and without0.2% peanut oil supplementation were compared, similar de-gradation patterns relative to the aqueous system were ob-served as in aqueous systems. The beneficial effect of peanutoil supplementation on degradation of all the PAH com-pounds is evident, except for naphthalene, the degradation ofwhich was not affected by peanut oil addition. Again themost profound beneficial effects on degradation occurredwith the higher molecular weight PAHs. For example, after24 days, the extents of benzo[a]pyrene degradation with andwithout 0.2% oil were 42.8% and 70.2%, respectively, repre-senting a 64% increase in degradation as a result of the oilamendment.

We demonstrated in our laboratory a process for extract-ing different PAHs from soil with peanut oil (Pannu et al.2003). Depending on the type of soil and PAH concentra-tion, up to 98% removal of PAHs was observed within 3 h.We further showed that activated carbon can be used toremove the extracted PAHs from the peanut oil, facilitatingre-use of the oil. We investigated the capacity of activatedcarbon to bind anthracene by passing anthracene-contaminatedoil through a column of activated carbon. We found that 1 gof activated carbon adsorbs up to 4200 µg of anthracene(Pannu et al. 2003). In this study, we investigated the poten-tial of biodegrading the PAHs recovered on the activated car-bon. Activated carbon (1% (w/v)), suspended in the mediumcontaining 1000 µg/mL anthracene and 0.1% peanut oil, wasinoculated with appropriate controls with the mixed cultureand incubated for up to 24 days. Percentages of degradationof anthracene in each culture are presented in Table 3. Thepeanut oil control and activated carbon controls confirmedthat both oil and activated carbon promote PAH degradationover the no oil and (or) no activated carbon control. It wasinteresting to observe that the combination of oil and acti-vated carbon increased anthracene degradation compared withthe oil or activated carbon supplementation alone. Our re-sults in the presence of activated carbon are consistent withstudies reported on the bioregeneration of activated carbonwhere higher biodegradation was observed in the presenceof activated carbon (Sigurdson and Robson 1978; Rice andRobson 1982). De Jonge et al. (1996) observed 15%–85%

© 2003 NRC Canada

Pannu et al. 511

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bioregeneration of activated carbon containing aromatics,such as o-cresol and 3-chlorobenzoic acid. We believe theobservation that peanut oil can accelerate the activated car-bon bioregeneration process is a novel finding.

In the context of biodegradation of petroleum hydrocar-bons, two of the more widely recognized modes of hydrocar-bon accession are direct adherence of bacteria to oil dropletsand interaction of bacteria with pseudosolubilized oil(Bouchez-Naïtali et al. 1999). Van Hamme and Ward (2001)described a Rhodococcus strain that associated directly oncrude oil droplets, while a Pseudomonas species requiredsurfactant-solubilized oil to efficiently access hydrocarbons.Hence the vegetable oil may provide a hydrophobic mediumlike crude oil in which the PAHs may be solubilized suchthat they can pass to bacteria associated with the lipid drop-let surface. Encapsulation of straight chain alkanes in lipo-somes increased their biodegradation and utilization as agrowth substrate by a Pseudomonas strain, indicating possi-ble delivery of the encapsulated hydrocarbons to membrane-bound enzymes (Miller and Bartha 1989). Alternatively, thepeanut oil may lead to the production of molecular productswith surfactant-like properties, which facilitates the uptakeof PAHs after their pseudsolubilization. For example, vege-table oils, such as canola, corn, olive, and soy, have beenshown to promote the production of biosurfactants by bacte-ria (Sim and Ward 1997), which can enhance the removal ofhydrocarbons from soils and accelerate their biodegradation(Lang and Wullbrandt 1999; Rosenberg and Ron 1999).

We have demonstrated the beneficial effects of supple-menting peanut oil to an aqueous culture as well as toweathered contaminated soil in PAH biodegradation. Themultiple beneficial effects may include increased desorptionof contaminant from soil, increased bioavailability of PAHs,increased co-metabolic transformation, and promotion ofbiosurfactant production by the microorganisms.

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512 Can. J. Microbiol. Vol. 49, 2003

Amendment % anthracene removal after:

Activated carbon Peanut oil 6 days 12 days 24 days

+ + 15.8 30.6 45.2+ – 9.8 26.8 35.1– + 10.5 23.2 33.8– – 8.3 17.2 20.5

Note: Conditions: crimp-closed serum bottles (50 mL) containing1000 mg/mL anthracene with (+) or without (–) 1% activated carbon and0.1% peanut oil in an aqueous culture were incubated with shaking(200 r/min) for 24 days at 30 °C. The data presented are the means ofduplicate samples. The experiment was repeated at least two times andtrends were identical in the independent experiments.

Table 3. Biodegradation of anthracene in the presence of acti-vated carbon and peanut oil by the mixed bacterial culture.



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influence of peanut oil on microbial degradation of polycyclic aromatic hydrocarbons

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