2000 ApplMicroBiotech

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O R I G I N A L P A P E R

A. C. Singer E. S. Gilbert E. LuepromchaiD. E. Crowley

Bioremediation of polychlorinated biphenyl-contaminated soilusing carvone and surfactant-grown bacteria

Received: 9 March 2000 / Received revision: 27 June 2000 / Accepted: 16 July 2000

Abstract Partial bioremediation of polychlorinatedbiphenyl (PCB)-contaminated soil was achieved by

repeated applications of PCB-degrading bacteria and asurfactant applied 34 times over an 18-week period. Twobacterial species, Arthrobacter sp. strain B1B andRalstonia eutrophus H850, were induced for PCBdegradation by carvone and salicylic acid, respectively,and were complementary for the removal of dierentPCB congeners. A variety of application strategies wasexamined utilizing a surfactant, sorbitan trioleate, whichserved both as a carbon substrate for the inoculum andas a detergent for the mobilization of PCBs. In soilcontaining 100 lg Aroclor 1242 g)1 soil, bioaugmenta-tion resulted in 5559% PCB removal after 34 ap-plications. However, most PCB removal occurred within

the rst 9 weeks. In contrast, repeated addition ofsurfactant and carvone to non-inoculated soil resulted in3036% PCB removal by the indigenous soil bacteria.The results suggest that bioaugmentation with surf-actant-grown, carvone-induced, PCB-degrading bacteriamay provide an eective treatment for partial deconta-mination of PCB-contaminated soils.

Introduction

Ten million kilograms of polychlorinated biphenyls(PCBs) are estimated to persist in the environment, most

of which has become dispersed on large land areas insoils and sediments. There are no proven PCB bio-remediation technologies currently available, largely dueto the low bioavailability of PCBs, the requirement forinducing substrates, and the need for a high populationdensity of degrader organisms. These obstacles may be

alleviated by incorporating surfactants, adding inducingsubstrates, and repeated inoculation with PCB-degrad-

ing microorganisms, respectively.Previously, Gilbert and Crowley (1998) tested the

ecacy of repeated applications of a PCB-degradingmicroorganism induced by the non-toxic inducing agent,carvone, for removing Aroclor 1242 from contaminatedsoil. Carvone is a plant-derived monoterpene, commonlyfound in spearmint. Carvone-induced, fructose-growncultures of Arthrobacter sp. strain B1B were repeatedlyadded to PCB-contaminated soil, resulting in theremoval of 27 6% of the PCB after 17 applications(9 weeks). The more highly chlorinated congeners of theAroclor 1242 mixture, which had been shown to bedegraded in liquid culture (Gilbert and Crowley 1997;

Kohler et al. 1988), were not substantially removed fromthe treated soil in this study. This suggested that thebioavailability of higher-chlorinated congeners mayhave been limiting the extent of PCB removal. Incor-poration of surfactants may enhance PCB removal ratesby increasing their bioavailability (Fava and Di Gioia1998; Viney and Bewley 1990). Surfactant-enhancedbioaugmentation studies of PCB-contaminated soilalmost exclusively employ an aqueous or soil slurrysystem (Fava and Di Gioia 1998; Ghosh et al. 1995;Lajoie et al. 1994); and they utilize either biphenyl as theinducing substrate, or a recombinant microorganism(s)engineered to constitutively express the genes for

degrading PCBs (Lajoie et al. 1997). However, theapplication of surfactants and environmentally comp-atible inducing substrates for in situ soil remediation isstill poorly understood.

In the study reported here, we examined a bioreme-diation approach in which sorbitan trioleate, a non-toxicsurfactant, was used to increase the bioavailability ofPCBs for the carvone-induced, PCB-degrading inocu-lum. The inoculum consisted of two bacterial species,Arthrobacter sp. strain B1B and Ralstonia eutrophusH850. This allowed us to examine the extent of PCBremoval by using two complementary PCB-degradingbacteria having dierent congener specicities.

Appl Microbiol Biotechnol (2000) 54: 838843 Springer-Verlag 2000

A. C. Singer E. S. Gilbert E. Luepromchai D. E. Crowley (&)Department of Environmental Sciences,University of California Riverside,Riverside, CA 92521, USAe-mail: [email protected].:+1-909-7873785;Fax: +1-909-7873993


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Materials and methods

Chemicals

L-Carvone was obtained from Aldrich Chemical Co. (Milwaukee,Wis.). Aroclor 1242 was purchased from AccuStandard (NewHaven, Conn.). Sodium dodecyl sulfate (SDS) and sorbitan triol-

eate (ST) were purchased from Sigma (Saint Louis, Mo.). Salicylicacid (SA), biphenyl, and Bacto agar were obtained from FisherScientic (Pittsburgh, Pa.). All solvents were reagent grade orbetter. Glycerol monooleate, sodium stearate, triethanolaminestearate, triethylene glycol monostearate, and potassium laurylsulfate were purchased from Chem Service (West Chester, Pa.).

Culture medium

Ralstonia eutrophus H850, a microorganism isolated from theHudson River, N.Y., by the General Electric Corporate Researchand Development Group (Bedard et al. 1987; Unterman et al.1985), and Arthrobacter sp. strain B1B (Kohler et al. 1988), wereboth cultured on inverted 1.5% Bacto agarmineral salts medium(MSM) in petri-plates with biphenyl crystals on the lid, as previ-

ously described (Gilbert and Crowley 1997). Arthrobacter sp. strainB1B is available through the American Type Culture Collection(ATCC 700595). R. eutrophus H850 is available through the U.S.Department of Agriculture patent collection (NRRLB 15940).

Soil-washing assay

The ability of glycerol monooleate and sorbitan trioleate to desorbsoil-bound PCBs was evaluated. Samples (10 g) of Aroclor 1242-contaminated soil (100 lg g)1) were sealed in 40-ml glass vialscontaining either 15 ml of an aqueous sorbitan trioleate solution(180 lg ml)1), aqueous glycerol monooleate solution (100 lgml)1), or deionized water. The surfactants were tested at doubletheir critical micelle concentration (CMC). Surfactant surfacetension measurements were made with a Surface Tensiomat tensi-ometer (Fisher Scientic, Pittsburgh, Pa.). After shaking the vials

for 48 h, the supernatant was extracted with hexane, and analyzedby ame ionization gas chromatography (GC-FID; see Extractionof PCBs below).

Preparation of PCB-contaminated soil

The soil was the same as that used in our recent study (Gilbert andCrowley 1998) (Dello loamy sand; 15% silt, 0.5% clay; pH 7.7;C:N ratio 11:1; total C 0.9%). A sample (2.5 kg) of soil wascollected, sieved (2-mm mesh) and contaminated with Aroclor 1242as previously described (Gilbert and Crowley 1998). A dry soilsample (20 g) was distributed into 28 mm 100 mm borosilicateglass vials (40 ml capacity) and capped with polyurethane foam(PUF) plugs. The microcosms were then allowed to age for3 months prior to the start of the experiment. Ten vials were pre-

pared for each treatment, permitting four vials to be extracted intheir entirety after 17 and 34 applications of each amendment(9 weeks and 18 weeks).

Treatments

A series of treatments was designed to identify the variablesresponsible for enhanced PCB removal (Table 1). Three bioaug-mentation treatments were incorporated into the study design. Inthe rst treatment, Arthrobacter sp. strain B1B was grown on

1,000 lg sorbitan trioleate (ST) ml)1 in MSM which also contained100 lg carvone ml)1 (abbreviated as treatment `B1B' hereafter). Ina second treatment, a dual culture amendment was applied. TheB1B treatment was co-amended with a R. eutrophus H850 culture(abbreviated as treatment `B1B-H850' hereafter). R. eutrophus

H850 cells were grown in 500 lg SA ml)1 in a minimal salts solu-tions, with the pH adjusted to 7.2 with NaOH. SA was determinedto be a growth substrate and inducer of PCB co-metabolism forR. eutrophus H850 (data not shown). Half of the total volume ofeach amendment consisted of each of the two cultures (approxi-mately 325 ll of each culture application)1). The third treatmentalso consisted of a co-amendment containing a culture ofArthrobacter sp. strain B1B grown on 1000 lg ml)1 fructose in a

minimal salts solution, containing 100 lg ml)1

carvone and aminimal salts solution of 1,000 lg ST ml)1 and 100 lg carvoneml)1 (abbreviated `B1B-Fr' hereafter). As in the co-culture treat-ment, half of the total volume of each amendment consisted of eachof the two solutions (approximately 325 ll of each solutionapplication)1). To determine the potential for biostimulating in-digenous soil microorganisms for PCB removal, two additionaltreatments were included: (1) ltered (0.22-lm nylon lter; MicronSeparations, Westborough Mass.) spent medium from the ``B1B''treatment (abbreviated `Filt-B1B' hereafter), and (2) an amend-ment containing 1,000 lg ST ml)1 and 100 lg carvone-MSM ml)1

(abbreviated `ST-C' hereafter).

Application of amendments to soil microcosms

Each soil microcosm contained 12.5% moisture content at the start

ofthe study, atwhich time the weight ofeachvial wasnoted and usedthroughout the study to determine the evaporative water loss. Toensure that each vial received the same number of organisms, anaverage evaporative water loss from all vials was determined at eachapplication period; and the appropriate solution/cell culture wasadded to compensate for the loss. Cultures ofArthrobacter sp. strainB1B and Ralstonia eutrophus H850 were grown for 24 h and 16 h,respectively, until mid- to late-log phase (108 colony forming units(cfu) ml)1) before addition to the soil microcosms at a nal soilconcentrations of 5 106 cfu g)1 soil. Application volumes averaged650 150 ll microcosm)1 (32.5 ll g)1 soil), allowingfor a nal soilinoculum concentration of 3.25 106 cfu g)1 soil inoculation)1.Gilbert and Crowley (1998) used an average application volume of

567 ll microcosm)1 (28.4 ll g)1 soil). The study conducted byGilbert and Crowley (1998) will be used throughout the paper as abasis for comparison, because of the similarities in the soil, PCB,

microorganisms, and methodologies used.After 34 amendments, approximately 2.21 mg ST and carvone

were added to the B1B treatment. The B1B culture initially con-tained 1,000 lg ST, but this diminished during cell culture toapproximately 100 lg ST ml)1. Carvone was not signicantly me-tabolized during cell culture and remained approximately100 lg ml)1. The B1B-H850 treatment receivedhalfas much ST andcarvone as the B1B treatment, due to co-amendment with the H850culture. The Fr-B1B treatment was amended with 11.1 mg ST and2.21 mg carvone. The ST-C treatment, having not been metabolizedby a microbial culture nor diluted by a co-amendment, received22.1 mg sorbitan trioleate and 2.21 mg carvone after 34 applica-tions. The amendments were pipetted into each microcosm andstirred using a spatula after each augmentation to ensure homoge-neous delivery of the inoculum. The frequency of applications (every4 1 days) and the time-points chosen for sampling microcosms

for PCB recovery (17 and 34 applications; 9 weeks and 18 weeks,respectively), were identical to Gilbert and Crowley (1998). All mi-crocosms were maintained at room temperature (approximately25 C) in a fume hood having a constant air ow; and they wererotated in location after each inoculum or treatment application.

Extraction of PCBs

Aroclor 1242 was extracted from soil by adding 5 ml of a 3% SDSsolution, 5 ml acetone, and 10 ml hexane directly to the 40-mlmicrocosm vials. The microcosms were sealed with PTFE-linedcaps and shaken for 24 h on a horizontal platform shaker. Theresulting emulsion was broken by centrifuging at 1,000 g for 5 min.An aliquot of the hexane phase was transferred to a second vialwhere anhydrous sodium sulfate was added to dewater the sample.

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PCBs dissolved in the hexane fraction were transferred to GCautosampler vials for analysis by a Hewlett-Packard 5890 GCequipped with an FID (Hewlett-Packard Co., Palo Alto, Calif.), as

described previously (Gilbert and Crowley 1997). Percentagerecovery of each congener was based on the corresponding con-gener peak from an Aroclor 1242 standard (Schulz et al. 1989). PCBextraction eciency from contaminated soil was 85 percent, com-mensurate with other extraction techniques (Brunner et al. 1985;Lajoie et al. 1994). The PUF plugs that had capped the microcosmswere stored at )20 C until analysis, when they were extracted into40-ml vials using 20 ml double-deionized water, 5 ml acetone, and10 ml hexane. The PCBhexane solution was dewatered and ana-lyzed as described above. Total PCB removed from each samplewas calculated by summing the percentage of PCB recovered fromsoil and PUF plug and subtracting this from the initial soil PCBcontamination. Statistical signicances were determined for recov-ered PCB from soil by ANOVA and by StudentNewmanKeulsanalysis using SAS 8.0 for Windows (Cary, N.C.).

Results

Surfactant screening

Six commercially available surfactants, glycerol mo-nooleate, ST, sodium stearate, triethanolamine stearate,triethylene glycol monostearate, and potassium laurylsulfate were tested for their ability to serve as a solecarbon source for Arthrobacter sp. strain B1B. Twosurfactants, glycerol monooleate and ST, proved to befar superior growth substrates, as indicated by totalbiomass generated during growth studies (data not

shown). A single washing of soil spiked with 100 ppmAroclor 1242 (aged for 3 months) determined that STeectively desorbed more soil-bound PCB than glycerolmonooleate, removing 22% and 5%, respectively, of thetotal PCBs. In comparison, deionized water extractedonly 2 lg PC B g)1 soil (2%). ST was subsequentlychosen for use throughout the bioremediation study. STis a surface-active agent with a molecular weight of 956,containing three fatty acid groups, comprised primarilyof oleic acid (75%) and is attached to a sorbitol back-bone by ester linkages. The CMC was determined to be80 ml l)1, with a surface tension measuring 31.9dynes cm)2 at 25 C. ST's low risk to human health is

evident by its commercial use in emulsiers, cosmetics,pharmaceuticals, and food applications.

PCB recovery

The eectiveness of the bioaugmentation and biosti-mulation treatments were evaluated on the basis ofrecoverable PCB (i.e., soil and PUF plug extractablePCB). The B1B and B1B-H850 treatments resulted inthe recovery of 23 7 lg PCB g)1 soil and 24 3 lg PCB g)1 soil, respectively, after 17 applications;32 9 lg PCB g)1 soil was recovered from the B1B-Frtreatment. Extraction of the Filt-B1B and ST-C treat-ments resulted in the recovery of 40 4 lg PCBg)1 soil and 49 7 lg PCB g)1 soil, respectively

(Table 2). PCB recoveries after 34 applications werelargely unchanged from the recoveries achieved after 17applications (P > 0.05). The B1B-Fr treatment and theB1B-H850 treatments were the only exceptions, resultingin an additional decline in recovered PCB of 8 lg PCBg)1 soil and 4 lg PCB g)1 soil, respectively (P 0.05).PCB recoveries from bioaugmented treatments were notsignicantly dierent after 34 applications (P > 0.05).PCB removal by congener type was analyzed following17 applications, by comparison with an Aroclor 1242

standard. Bioaugmented treatments were more eectivefor degradation of the di-, tri-, and tetra-chlorinatedcongeners than the biostimulated treatments (Table 3).The B1B and B1B-H850 treatments degraded 59 7%and 56 1% of the tetra-chlorinated congeners, re-spectively, as compared with the Filt-B1B and ST-Ctreatments, which degraded 32 5% and 22 11%,respectively. After 17 applications, the B1B treatmentand the B1B-H850 treatments nearly doubled the penta-chlorinated PCB removal achieved in biostimulatedtreatments.

Congener analysis following 34 applications of thedual-inoculum showed enhanced removal of the more

Amendment variables Bioaugmented treatments Biostimulated Treatments

B1B B1B-H850 B1B-Fr Filt-B1B ST-C

Initial/amended concentrationSorbitan trioleate (lg ml)1) 1000/100a 1000/50b 1000/500c 1000/100a 1000/1000Carvone (lg ml)1) 100/100 100/50 100/100 100/100 100/100Arthrobacter sp. strain B1B (cfu ml)1) 1 108 1 108/0.5 108 0.5 108

Ralstonia eutrophus H850 (cfu ml)1) 1 108/0.5 108

Fructose (lg ml)1) 1000/NMSalicylic acid (lg ml)1) 500/NM

a Amended concentration accounts for metabolized substratebAmended concentration accounts for metabolized substrate and a co-amendment treatmentc Amended concentration accounts for a co-amendment treatment

Table 1 Treatments and amendments. See text for detailed de-scription of each treatment. For initial/amended concentrations,initial values indicate starting concentration and amended values

give lg amendment ml)1. Note that the average amendment vo-lume was 650 ll. All amendments are in a minimal salts solution.NM Not measured

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recalcitrant PCBs with chlorine substitutions in the

ortho-position, particularly double ortho-positions onboth rings (Table 4). R. eutrophus has previously beenshown to be particularly competent in degrading theseparticular congeners (Bedard and Haberl 1990; Bedardet al. 1986) as compared with other PCB-degradingmicroorganisms, such as Arthrobacter sp. strain B1B(Gilbert and Crowley 1997; Kohler et al. 1988).

Discussion

Polychlorinated biphenyl bioremediation in unsaturatedsoils comprises a complex and multifaceted set of

physical, chemical, and biological problems. In the

physicochemical realm, the highly hydrophobic charac-ter of PCBs results in their sequestration on clay parti-cles and within the soil organic matrix, with acorresponding decrease in their bioavailability. Biologi-cal problems include the requirement for an inducingsubstrate that will promote the aerobic co-metabolism ofPCBs. As many indigenous biphenyl degraders are rel-atively poor PCB degraders, bioaugmentation withPCB-degrading microorganism(s) may be required toachieve the maximum extent of aerobic biologicaldegradation. Thus, a fully integrated treatment strategymust simultaneously address all of these interdependentproblems to achieve extensive cleanup.

Table 2 Polychlorinated biphenyl (PCB) recovery after 17 applications and 34 applications (9 weeks and 18 weeks) of the indicatedtreatments. Paired values were recorded after 17/34 applications

Bioaugmented treatments Biostimulated treatments

B1B B1B-H850 B1B-Fr Filt-B1B ST-C

Initial soil PCB contamination 2000 2000 2000 2000 2000

PCB recovered (lg PCB)Soil 463/443 484/407 644/482 807/790 973/895PUF plug 450/455 375/429 450/340 525/486 495/508Total recovered 913/898 859/836 1094/823 1332/1276 1468/1403

PCB recovered (% PCB)Soila 23aA/22aA 24aA/20aB 32bA/24aB 40cA/39bA 49dA/45cA

PUF plug 23/23 19/21 23/17 26/24 25/25Total recovered 46/45 43/42 55/41 67/64 73/70

Total PCB removedb 54/55 57/58 45/59 33/36 27/30

a Soil PCB recoveries at the same time point are signicantlydierent (P < 0.05) if marked with dierent lower case letters. SoilPCB recoveries within the same treatment at dierent timepointsare signicantly dierent (P < 0.05) if marked with dierent

uppercase letters. All analyses were conducted using SAS (Cary,N.C.) by one-way ANOVA with repeated measure usingStudentNewmanKeulsbPCB remediation: 100 (% total PCB recovered)

Congener Bioaugmented treatments Biostimulated treatments

B1B B1B-H850 B1B-FrNo surfactant

B1B-FrPlus surfactant

Filt-B1B Fructose-C ST-C

Di-CB 98 0a 95 4a 88 3 87 5a 4310b 34 25 23 21c

Tri-CB 79 6a 83 0a 41 7 65 7b 44 7c 13 8 18 4d

Tetra-CB 59 7a 56 1ab 11 5 42 10bc 32 4cd 6 3 22 11d

Penta-CB 54 16a 41 0a 3 8 28 12a 26 8a 5 7 30 12a

Table 3 Percentage PCB removal by congener type after 9 weeks(17 applications). The (B1B-Fr, no surfactant) treatments fromGilbert and Crowley (1998) were added for comparison but werenot considered in the statistical analysis. Values are based on a

comparison with autoclaved control soils and are calculated as100 (% recovered PCB). Treatments are signicantly dierentif marked with dierent letters (Fishers PLSD; P < 0.05)

Table 4 Eect of co-inocula-tion of Arthrobacter sp. strainB1B and Ralstonia eutrophusH850 on the removal ofmultiple ortho-substitutedcongeners in Aroclor 1242 after34 applications. All pairs ofdata are signicantly dierent(Student's t test, P < 0.05)

Multiple ortho-substitutedPCB congeners

Percentage degraded,from surfactant-grownB1B treatment

Percentage degraded,from surfactant-grownB1B plus H850 treatment

% Change inPCB congenerremovala

2,3,6,2 34 5 45 6 112,5,2,5 29 3 56 7 272,4,2,5 27 3 49 6 222,4,2,4, 2,4,5,2b 30 5 44 6 142,3,2,3 30 6 51 6 212,3,5,2,4, 2,4,5,2,5b 13 4 25 4 12

a Values indicate increased removal of specic congener(s) by the H850-B1B dual amendmentbCongeners coelute

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The carvone-based, bacterial-surfactant treatmentpresented here combined several features that addressthe aforementioned problems. In previous work (Gilbertand Crowley 1997), carvone was determined to be a non-toxic, water-soluble inducer of PCB co-metabolism inArthrobacter sp. strain B1B. In subsequent work, re-peated application of the degrader microorganism wasdemonstrated to partially remove PCB from contami-nated soil (Gilbert and Crowley 1998). In the presentwork, a surfactant was identied that both supportedthe growth of a PCB-degrading inoculum and in-creased the bioavailability of soil-bound PCBs. Incor-poration of the surfactant greatly increased PCBremoval as compared with the Gilbert and Crowley(1998) study. The bioaugmented treatments resulted inan average recovery of 22 lg PCB g)1 soil from soil thatwas originally contaminated with 100 lg PCB g)1 soil.In contrast, Gilbert and Crowley (1998), using identicalexperimental conditions (Aroclor 1242 mixture andconcentration, soil and sample size, and microorgan-

ism), but without the use of a surfactant, achieved re-covery of 41 3 lg PCB g)1 soil (Gilbert and Crowley1998). Under the conditions and time frame of this ex-periment, bioaugmentation proved to be a superiortreatment method for the removal of PCBs, as comparedwith biostimulation. Approximately twice as much PCBwas removed in treatments receiving repeated inocula-tion with degrader microorganisms as compared withbiostimulated microcosms (Table 2).

The B1B-Fr treatment was designed to circumventproblems with biofouling which could potentially occurdue to the cell-clumping that occurs with surfactant-grown cells. As shown here, there was no reduction in

the ecacy of the bioaugmentation treatment after 34applications when Arthrobacter sp. strain B1B wasgrown on a non-surfactant medium and inoculated in amixture with added surfactant. The results suggest thatthe addition of surfactant and the replenishment ofactive PCB-degrading bacteria after each amendmentwere important for increasing the rate and extent of PCBremoval. Particularly noteworthy was the increase in theremoval of the higher-chlorinated congeners in com-parison with the prior study (Gilbert and Crowley 1998)where no surfactant was added. A four-fold increase inthe removal of tetra-chlorobiphenyl and a greater thanten-fold increase in the removal of the penta-chlorobi-

phenyl were observed, as compared with earlier results(Gilbert and Crowley 1998).

Co-inoculation of R. eutrophus H850 and Arthro-bacter sp. strain B1B did not increase the removal oftotal PCBs over Arthrobacter sp. strain B1B alone, de-spite the presence of an expanded suite of PCB-degrading enzymes. This result may simply be theproduct of having used two ecient PCB-degradingmicroorganisms, each of which was capable of degrad-ing the majority of bioavailable PCBs. Nevertheless, thecombination of the two strains signicantly increasedthe removal of selected congeners, particularly those

with multiple ortho-substitutions, which are particularlyrecalcitrant (Table 4).

A survey of the literature nds the work presentedhere is the rst to investigate surfactant-enhanced PCBbioaugmentation in a non-slurry, soil-based systemusing only environmentally benign compounds. Priorstudies have, however, investigated the use of surfactantsin improving PCB bioremediation in slurry (Fava andDi Gioia 1998; Ghosh et al. 1995; Lajoie et al. 1994) andcompletely aqueous systems (Lajoie et al. 1997), utiliz-ing biphenyl as an inducing substrate, or a recombinantmicroorganism(s) engineered to constitutively expressthe genes for degrading PCBs. ST was chosen to beeasily biodegraded by indigenous soil microorganisms,thereby decreasing the potential for soil and watercontamination if left undegraded in the soil.

Incorporating environmentally friendly surfactantsand inducing substrates into a PCB-bioaugmentationmethodology succeeded in increasing the removal ofPCBs from soil, as compared with an identical bioaug-

mentation methodology without the use of a surfactant.However, despite the use of a surfactant, bioavailabilitystill appeared to be limiting complete PCB removal.These results suggest that surfactant-enhanced bioaug-mentation may result in a more rapid removal of PCBand may be capable of reaching a lower nal PCB con-centration than biostimulation. Consideration of theenvironmental and human risk associated with non-bio-available and non-solvent-extractable PCBs may enablethe application of this or a similarly designed bioaug-mentation treatment in the future (Alexander et al. 1997).

Acknowledgements The authors are thankful to Jennifer Clark for

her assistance throughout the study and to Katechan Jampachaisrifor her invaluable help with the statistical analysis. This researchwas partially supported by a grant from EcoSoils Systems Inc. (SanDiego, Calif.). The experiments comply with the current laws ofthe United States, the country in which the experiments wereperformed.

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2000 ApplMicroBiotech