effects of bacterial inoculation and nonionic surfactants on degradation of polycyclic aromatic...

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Environmental Toxicology and Chemistry, Vol. 16, No. 4, pp. 631–637, 1997q 1997 SETAC

Printed in the USA0730-7268/97 $6.00 1 .00

EFFECTS OF BACTERIAL INOCULATION AND NONIONIC SURFACTANTS ONDEGRADATION OF POLYCYCLIC AROMATIC HYDROCARBONS IN SOIL

TORBEN MADSEN* and PREBEN KRISTENSENVKI, Agern Alle 11, DK-2970 Hørsholm, Denmark

(Received 3 April 1996; Accepted 16 August 1996)

Abstract—The aim of the study was to examine the effects of introduced bacteria and nonionic surfactants on the degradation ofpolycyclic aromatic hydrocarbons (PAHs) in soil. Mineralization experiments were conducted with freshly added [14C]phenanthreneor [14C]pyrene, whereas other experiments focused on the degradation of selected PAHs present in a coal tar-contaminated soil.Inoculation of soil samples with phenanthrene-utilizing bacteria stimulated the mineralization of [14C]phenanthrene. This effect,however, was most notable in soil with a low indigenous potential for PAH degradation, and a large inoculum was apparentlyrequired to establish phenanthrene mineralization in the soil. Addition of alcohol ethoxylate and glycoside surfactants to soil samplesenhanced the mineralization of [14C]phenanthrene and [14C]pyrene. The nonionic surfactants also enhanced the degradation ofcontaminant PAHs that were present in the soil coal tar. As an example, pyrene, benzo[b,j,k]fluoranthene, and benzo[a]pyrene wereresistant to degradation in the absence of surfactants, whereas significant degradation of these PAHs was observed when surfactantswere added. The surfactant-related enhancement of the degradation of PAH contaminants was less convincing when a rapidlydegradable glycoside surfactant was used. This suggests that surfactants that are mineralized at moderate rates may be more applicablefor increasing the availability of PAHs in soil.

Keywords—Polycyclic aromatic hydrocarbons Biodegradation Inoculation Availability Surfactants

INTRODUCTION

Polycyclic aromatic hydrocarbons (PAHs) are hydrophobicchemicals that sorb strongly to clay minerals, soil humus, orother organic solids [1–4]. Microbial degradation is believedto be the most important process for removal of PAHs fromcontaminated soils although hydrocarbons with less than threefused rings are also susceptible to volatilization [5]. Duringrecent years, a number of bacteria and fungi that degrade PAHswith two to five fused rings have been isolated [6–12], andenhanced PAH degradation rates in soil after the addition ofPAH-utilizing microorganisms have been demonstrated in sev-eral studies [10,11,13]. These results indicate a potential useof microbial inocula in bioremediation strategies. Due to thesorption of PAHs in soil, the success of bioremediation may,however, depend on the actions taken in order to increase thebioavailability of the hydrophobic contaminants.

Surfactants may enhance the apparent solubility of hydro-phobic organic compounds, especially when present at con-centrations above the critical micelle concentration (CMC) atwhich surfactant monomers aggregate to micelles [14–17]. Ina system of soil and water, most of the surfactant moleculesare sorbed onto the soil solids [15,18,19], and addition ofsurfactants to soil usually results in aqueous surfactant con-centrations below the CMC. Although to a more minor extentthan in micellar surfactant solutions, the apparent solubility ofhydrophobic chemicals increases linearly with an increase ofsub-CMC nonionic surfactant concentrations. It has been dem-onstrated that the apparent solubility for pyrene in nonionicsurfactant solution at the CMC may increase by a factor ofabout three compared to the solubility in water [18].

Studies of the factors affecting the biodegradation of hy-

* To whom correspondence may be addressed.

drophobic chemicals have frequently used freshly contami-nated soil or aquifer samples [11–13]. However, the desorptionmay be very slow for hydrocarbon contaminants that havediffused into micropores of soil aggregates after being presentin the soil for long periods of time [4]. The objective of thepresent study was to examine the degradation of freshly addedPAHs and of selected PAH contaminants present in a coal tar-polluted soil. The study focused on the effects of bacterialinoculation and of addition of nonionic surfactants on the deg-radation of PAHs. The nonionic surfactants used consisted offatty alcohol ethoxylates that are common ingredients in de-tergents and of glycolipids that have structural similarities tobiosurfactants produced by hydrocarbon-utilizing microorgan-isms [20].

MATERIALS AND METHODS

Chemicals

Model PAHs. Reagent-grade [9-14C]phenanthrene (8.3 mCi/mmol, radiochemical purity .98%) and [4,5,9,10-14C]pyrene(32.4 mCi/mmol, radiochemical purity .98%) were purchasedfrom Sigma Chemical Co. (St. Louis, MO, USA). Analyticalgrade phenanthrene (purity: .98%) was purchased fromMerck (Darmstadt, Germany).

Nonionic surfactants. Five different nonionic surfactantswere used for the experiments: a fatty alcohol ethoxylate (AE)with an alkyl chain length of C9 to C11 and eight ethoxyleneoxide units, denoted here as C9–11 AE-8 (C10H21O(CH2CH2O)8H;average molecular weight [MW], 510 g/mol; CMC, 1.50 3 1023

M); an AE with an alkyl chain length of C12 to C18, 10 ethoxyleneoxide units, and end-capped with butyl, denoted here as C12–18

AE-10 (C13.6H28.2(CH2CH2O)9CH2CH2O(CH2)3CH3; MW, 704g/mol; CMC, 0.24 3 1023 M); an alkyl polyglycoside (APG)with an alkyl chain length of C12 to C14 and an average degree

632 Environ. Toxicol. Chem. 16, 1997 T. Madsen and P. Kristensen

of polymerization of 1.4, denoted here as C12–14 APG(C12.7H26.4O(C5H8O5)1.4C5H9O5; MW, 551 g/mol; CMC, 0.23 31023 M); an ethyl glycoside monoester (EGE) with an alkylchain length of C12, denoted here as C12 EGE (C12H25CO(C5H8O5)OCH2CH3; MW, 390 g/mol; CMC, 0.50 3 1023 M);and an EGE with an alkyl chain length of C18, denoted here asC18:1 EGE (C18H35CO(C5H8O5)OCH2CH3; MW, 472 g/mol;CMC, 0.40 3 1023 M). The surfactants were of technical purityand contained 100% surfactant with the exception of C12–14 APG,which contained 50% surfactant. The CMCs were determinedat 228C by the ketonic and enolic absorption spectra of ben-zoylacetoanilide [21] by using a Unicam UV/VIS spectrometer(Unicam Ltd., Cambridge, UK). Samples of the surfactants wereobtained from Colgate Palmolive (Glostrup, Denmark) (C9–11

AE-8), P. Brøste (Lyngby, Denmark) (C12–18 AE-10 and C12–14

APG), and Novo Nordisk (Bagsvard, Denmark) (C12 EGE andthe C18:1 EGE).

Soil samples

Two samples of coal tar-contaminated soil were obtainedfrom an abandoned gas works site in Esbjerg, Denmark. Onesample was collected just outside the borders of a polluted siteand was designated soil A1. The other sample was collectedfrom the upper layers of the polluted site (depth of sampling:0.3–1.0 m) and was designated soil A2. A third soil sample(soil B) with no history of coal tar contamination was collectedfrom the surface layers of an agricultural field in Flakkebjerg,Denmark. The soil samples had the following characteristics.Soil A1: coarse sand (0.25–2 mm), 15.9%; fine sand (0.02–0.25 mm), 34.9%; silt (0.002–0.02 mm), 45.7%; clay (,0.002mm), 1.9%; organic matter, 1.7%; and pH 7.9. Soil A2: coarsesand, 15.3%; fine sand, 34.4%; silt, 46.3%; clay, 2.2%; organicmatter, 2.0%; and pH 8.2. Soil B: coarse sand, 17.5%; finesand, 37.0%; silt, 41.7%; clay, 1.2%; organic matter, 2.8%;and pH 6.5. The extractable amounts of coal tar were 0.24mg/g (dry weight) for soil A1 and 2.9 mg/g for soil A2 (dryweight) as determined by gas chromatography with flame ion-ization detection after extraction with 2-propanol : cyclohexane(1:2 v/v). The soils were air-dried at room temperature for 20h, sieved through a 2-mm mesh, and stored at 48C until use.

Microorganisms

Bacterial strains capable of utilizing phenanthrene as thesole source of carbon and energy were isolated from soil sam-ples. Agar plates were prepared by using a mineral medium[22] with 1.5% purified agar (Noble agar, Difco Laboratories,Detroit, MI, USA). The surface of the agar was sprayed with6% (w/v) phenanthrene in hexane : acetone (1:1 v/v) so that awhite layer remained [23]. Soil samples were diluted in themineral medium, and 0.1-ml aliquots were distributed onto theagar plates. Following incubation at 278C for up to 5 weeks,transparent zones surrounded some of the colonies. The growthof these bacteria was examined in liquid mineral medium withphenanthrene, and pure cultures were obtained after subcul-turing and transfers of three generations of colonies from solidto liquid medium [24]. One of the isolated bacteria was ob-tained from a fuel-oil contaminated soil. This bacterium wasdesignated strain PheAH. It was gram-positive and showed adimorph rod–coccus growth cyclus. These characteristics andthe range of carbon substrates utilized (API 20 NE; Biomer-ieux, France) indicate a relationship with Arthrobacter. Twoother bacteria were isolated from soil A1. One of these isolatesstained gram-negative and was tentatively identified as a Pseu-

domonas fluorescens by using API 20 NE (strain 17PHE128).The other bacterium was a gram-positive rod that was notidentified (strain 1PHE128). The pure cultures were grown inthe mineral medium with crystaline phenanthrene (5 mg/L).Before the experiments, the bacteria were harvested by cen-trifugation at 10,000 g for 10 min and concentrated aboutfivefold in the mineral medium.

Biodegradation experiments with radiolabelled compounds

Radiolabeled phenanthrene (approx. 0.75 mCi/ml) or pyrene(approx. 2.0 mCi/ml) were added from acetone solutions to10-g samples of air-dried soil A1 and B. [14C]Phenanthrene(approx. 0.15 mCi per bottle; 0.32 mg/g soil) and [14C]pyrene(approx. 0.40 mCi per bottle; 0.25 mg/g soil) were used in theexperiments with nonionic surfactants. The other experimentswith [14C]phenanthrene were performed by using a mixture inacetone of labeled and nonlabeled compound (approx. 0.30mCi per bottle; 1.00 mg/g soil). The mineralization of[14C]phenanthrene was examined after inoculation with strainPheAH, 17PHE128, or 1PHE128. The bacteria were added tosamples of soil A1 and B from aqueous suspensions (approx.1.0 ml) to achieve an initial inoculum density of 106 cells/gof air-dried soil. The number of bacteria in the inocula wasdetermined by acridine orange direct counting (AODC) [25].The influence of the inoculum density was examined by addingserial dilutions (1.0 ml) of strain 1PHE128 (101–106 cells/gsoil) to soil B samples. Effects of nonionic surfactants on themineralization of [14C]phenanthrene and [14C]pyrene werestudied by using the low level contaminated soil A1. The sur-factants were added to the soil from aqueous stock solutions(1.0 ml) to initial concentrations of 100 mg/g of air-dried soil.

Inoculated and surfactant-amended soil samples were sup-plemented with deionized water to initial soil wet weights of12.5 g/bottle (soil A1) or 12.0 g/bottle (soil B) correspondingto approximately 70% of the water-holding capacity. The par-allel controls received mineral medium (1.0 ml) and deionizedwater (inoculation experiments) or deionized water only. Allexperiments were performed in 100-ml respirometric bottleswith ground glass stoppers. A vial containing 2 ml of 0.5 NKOH was placed inside the bottles for trapping 14CO2. Incu-bation took place in the dark at 208C.

Recovery of added 14C

The contents of the vials were replaced with fresh KOH atspecific intervals, and the absorbed 14CO2 was quantified byliquid scintillation counting. The total 14C in the soil afterincubation ended was determined by combustion of 0.1-g sub-samples in excess of oxygen. In one experiment, the distri-bution of the residual 14C in the soil was examined in detail.The 14C present in the biomass was estimated by a modificationof the chloroform fumigation method of Jenkinson and Powl-son [26]. The soil was covered with alcohol-free CHCl3 andafter fumigation overnight, CHCl3 was evaporated by a streamof air. The fumigated soil was reinoculated with 1 g of freshsoil and incubated at 208C for 18 d with vials for absorptionof 14CO2, which is assumed to represent 14C assimilated bysoil microorganisms. The amount of 14C present as hydrolyz-able compounds (e.g., polysaccharides, proteins, and peptides)was estimated by extraction of the soil with HCl [27]. First,the samples were shaken with 15 ml 6 N HCl for 20 h, thenthey were transferred to 100-ml centrifuge tubes and, finally,centrifuged for 10 min at 2,380 g. The supernatants were re-moved, the soil pellets were washed twice with 15 ml of deion-

PAH degradation by addition of bacteria and surfactants Environ. Toxicol. Chem. 16, 1997 633

ized water, and the acidic extracts were combined and adjustedto 50 ml. The 14C associated with the soil humus (fulvic andhumic acids) was estimated by extraction of the hydrolyzedsoil with 15 ml of 1 N NaOH [28] using the procedure de-scribed for the acidic extraction. Eventually, subsamples ofthe soil were combusted in excess of oxygen to determine theamount of 14C that was not released by the extractions withHCl and NaOH.

Biodegradation experiments—coal tar contaminants

The effects of nonionic surfactants on the removal of PAHsin the coal tar were examined in 117-ml serum bottles con-taining 10 g of air-dried soil A2. Aqueous solutions of C12–18

AE-10, C12–14 APG, or C18:1 EGE (1.0 ml) were added toachieve initial surfactant concentrations of 100 mg/g of air-dried soil, and deionized water (1.5 ml) was used to adjust themoisture content to approximately 70% of the water-holdingcapacity. The serum bottles were sealed with teflon-linedrubber stoppers and were incubated in the dark at 208C. After28 d of incubation, the soil samples received a second 100-mg/g addition of the surfactants (0.5 ml from aqueous solutioncontaining 2.0 g surfactant/L). Triplicate series of bottles wereharvested after 0, 14, 28, and 42 d for chemical analyses.

Substrate competition between phenanthrene andsurfactants

The ability of strain 1PHE128 to utilize C12–18 AE-10 andC18:1 EGE as growth substrates was examined in 250-ml bottleswith 50 ml of mineral medium [22] and an initial cell densityof 3.0 6 0.02 3 103 cells/ml. The medium was supplementedwith phenanthrene at 0.94 mg/ml, and C12–18 AE-10 or C18:1

EGE was added to triplicate series of bottles at 10, 20, 40, or80 mg/ml. The bottles were incubated at 288C with shaking.Growth was evaluated after 70 h by AODC [25], and theconcentration of phenanthrene was determined in pentane ex-tracts by gas chromatography with flame ionization detection.

Analytical methods

The 14C activity was quantified by mixing 2 ml of the KOHfrom the 14CO2 absorbers or 4 ml of the extracts with 10 mlof scintillation cocktail (Insta-Gel II Plus, Packard, Groningen,The Netherlands) in 20 ml vials. The vials were allowed torest for 20 h and shaken for 20 min before the 14C-activitywas determined in a liquid scintillation counter (LKB 81000,Wallac OY, Turku, Finland). The PAHs contained in the soilcoal tar were extracted twice by adding 5 ml of deionizedwater and 8 ml of 2-propanol : cyclohexane (1:2 v/v) to the10-g soil samples. The soil was treated with ultrasonicationfor 5 min and shaken for 30 min. The extracts were mixedand washed with two 15-ml volumes of deionized water. Aque-ous and organic phases were separated by using a filter withNa2SO4, and the anhydrous extracts were evaporated to a finalvolume of 1 ml.

The gas chromatograph used to determine the coal tar con-tents of soil A1 and A2, as well as phenanthrene in the liquidmedium, was an HP 5890 Series II (Hewlett Packard, Am-sterdam, The Netherlands) equipped with a flame ionizationdetector and an HP-5 capillary column (film thickness, 0.11mm; inner diameter, 0.20 mm; length, 25 m). Injection tem-perature was 358C, the detector temperature was 2808C, andthe flow rate was 1.0 ml of H2 per min; the column temperaturewas 758C for 2 min, was then increased at 68C/min to 2758C,and was held for 10 min. Specific PAHs in the coal tar were

identified by gas chromatography–mass spectrometry (GC-MS). The extracts (1 ml) were injected on column in an HP5890 Series II gas chromatograph connected to an HP 5971Amass spectrometry unit (Hewlett Packard, Amsterdam, TheNetherlands). The system was equipped with an HP-1 capillarycolumn (film thickness, 0.11 mm; inner diameter, 0.20 mm;length, 25 m). The injection temperature was 358C, the detectortemperature was 2808C, and the flow rate was 0.3 ml of Heper min; the column temperature was 608C for 2 min, wasthen increased at 68C/min to 2858C, and was held for 15 min.The analyses were performed by using single ion monitoringwith deuterated internal standards (biphenyl-d10 and pyrene-d10). The detection limit for the GC-MS analyses of extractedPAHs was 0.01 mg/g of soil.

RESULTS

The addition of phenanthrene-utilizing bacteria to the lowlevel contaminated soil A1 only enhanced the mineralizationof [14C]phenanthrene when strain 1PHE128 was used as theinoculum. In this case, the maximum 14CO2 evolution rate fromthe labeled phenanthrene was 0.87 6 0.03 nmol/g/d comparedto 0.57 6 0.02 nmol/g/d without bacterial inoculation (linearregression correlation, r2 $ 0.98). Both the rates of 14CO2

evolution and the extent of phenanthrene mineralization weremarkedly increased when the bacteria were added to soil B,which had no history of coal tar contamination (Fig. 1). Themaximum 14CO2 evolution rate was 0.10 6 0.05 nmol/g/d insoil B without inoculation. Inoculation increased the phenan-threne mineralization rates to 0.24 6 0.01 nmol/g/d for strainPheAH, 0.54 6 0.08 nmol/g/d for strain 17PHE128, and 0.956 0.01 nmol/g/d for strain 1PHE128 (r2 $ 0.98). The influenceof the inoculum density was examined only with strain1PHE128. A large inoculum of 1PHE128 cells was requiredin order to establish phenanthrene mineralization in the soil.When this bacterium was introduced at an initial concentrationbelow 106 cells/g of soil, the mineralization of phenanthrenedid not exceed the level observed in noninoculated samples(data not shown).

The distribution of the residual 14C in the soil was examinedby use of CHCl3 fumigation, HCl and NaOH extraction, and,eventually, combustion of the soil samples. Fumigation of thesoil with CHCl3 and measurements of the released 14CO2 afterreinoculation showed that only 2 to 4% of the labeled carbonwas present in the biomass at the termination of the experiment(day 28). The acidic extraction of the fumigated soil indicatedthat a similar small amount of the residual 14C was associatedwith hydrolyzable compounds. The major part of the 14C wasrelated to NaOH-extractable fulvic and humic acids, or to hu-min and other soil materials that were not extracted with acidand alkali (Table 1).

Addition of nonionic surfactants to soil A1 enhanced themaximum mineralization rates (r2 $ 0.95) for [14C]phenanthreneand [14C]pyrene compared to the rates in the controls (Table2). The differences between the 14CO2 evolution rates in sur-factant-amended and control samples were significant accord-ing to the Student’s t test with p . 0.95 (5 and 11 d.f. forphenanthrene and pyrene, respectively). The surfactants alsoincreased the extent of hydrocarbon degradation. For example,in soil samples amended with C12–18 AE-10, the total evolutionof 14CO2 from mineralization of phenanthrene and pyrene at-tained 67 and 54% of the labeled compound after 35 d com-pared to 60 and 43% in the absence of surfactant (Fig. 2A andB).


Fig. 1. Effect of bacterial inoculation on the mineralization of [14C]phenanthrene in a noncontaminated soil (soil B). Inoculated soil samplesreceived 106 cells/g of air-dried soil. Standard deviations of three replicates are indicated by vertical bars.

Table 1. Effects of bacterial inoculation on mineralization of [14C]phenanthrene in a noncontaminated soil (soil B). Distribution and recoveryof 14C after 28 d of incubation. SD, standard deviations of three replicates

Treatment

Recovered 14C activity (% of added 14C 6 SD)

CO2 Microbial biomass HCl extractable NaOH extractable Not extractableTotal

recovery

None1 Strain PheAH1 Strain 1PHE1281 Strain 17PHE128

31.4 6 0.3239.0 6 0.5749.3 6 0.5048.6 6 3.5

3.7 6 0.923.0 6 0.363.9 6 0.192.4 6 0.22

1.9 6 0.222.7 6 0.302.9 6 0.083.3 6 0.47

12.9 6 2.610.3 6 1.68.0 6 0.66

12.0 6 1.2

34.8 6 10.025.6 6 2.814.3 6 0.6213.2 6 1.5

84.780.678.479.5

The recovery of 14C in the experiments with labeled phe-nanthrene or pyrene ranged between 78 and 85% of the addedradioactivity when the distribution of residual 14C was ex-amined (Table 1), whereas 85 to 90% was recovered by com-bustion of soil samples immediately after incubation ended.Small amounts of 14C were apparently lost during the 14C frac-tionation, for example, when the HCl-extracted soil sampleswere transferred to centrifuge tubes.

Three of the nonionic surfactants (C12–18 AE-10, C12–14 APG,and C18:1 EGE) were added to samples of soil A2 at day 0 andday 28, and the concentrations of PAHs contained in the coaltar were monitored during aerobic incubation for 42 d. Forhydrocarbons with two and three fused rings, enhanced deg-radation or abiotic loss in the presence of surfactants was notedafter 14 d, whereas a surfactant-related increase of the deg-radation of four- and five-ring PAHs was observed during thelast 2 weeks of the incubation period (data not shown). Two-and three-ring PAHs were still present in the soil after 42 d,especially in the control samples that did not receive nonionicsurfactants. The major part of the four-ring compounds per-sisted in the control samples throughout the experiment, andpyrene, benzo[b,j,k]fluoranthene, and benzo[a]pyrene were notdegraded in the absence of surfactants. Degradation of PAHsin soil samples that received surfactants was significantly moreextensive than PAH degradation in the controls according tothe Student’s t test (Table 3). The C18:1 EGE did not stimulatePAH degradation to the same extent as the other nonionicsurfactants; the hydrocarbon concentrations remaining after 42d in soil samples amended with C18:1 EGE were generallyhigher than concentrations in the samples treated with C12–18

AE-10 or C12–14 APG (Table 3).

One of the phenanthrene-degrading isolates, strain1PHE128, was grown in a defined medium with phenanthrene(0.94 mg/ml) and either C12–18 AE-10 or C18:1 EGE. The C12–18

AE-10 stimulated the growth of strain 1PHE128 when addedat 80 mg/ml. The cell density increased from the initial 3.0 60.02 3 103 cells/ml to 6.7 6 0.42 3 106 cells/ml in the presenceof C12–18 AE-10 compared to 4.0 6 0.53 3 106 cells/ml in thecontrols without surfactant. Addition of C18:1 EGE, at 10 to 80mg/ml, resulted in a doubling of the number of 1PHE128 cellswhen the concentration of the surfactant was increased by afactor of two, and 7.9 6 0.79 3 108 cells/ml were reachedduring growth with C18:1 EGE at 80 mg/ml. The degradationof phenanthrene was repressed when C12–18 AE-10 or C18:1 EGEwere used for growth by strain 1PHE128 (data not shown).

DISCUSSION

The inoculation experiment with [14C]phenanthrene showedthat a higher percentage of the added 14C was firmly sorbedto the soil in the control samples and in the samples inoculatedwith strain PheAH, where the mineralization of phenanthrenewas slow (Fig. 1 and Table 1). The [14C]phenanthrene residuesthat were not extracted by HCl or NaOH were probably boundto NaOH-insoluble humin, which is not extracted with alkali[28]. It is possible that the mineralization of phenanthrene afterinoculation with strain 1PHE128 or 17PHE128 was so rapidthat the time was limited for the slow diffusion of PAH intomicropores in clay and organic matter. Relatively low amountsof sorbed and recalcitrant residues have also been observedfor pesticides and other chemicals that were rapidly eliminatedfrom soil by degradation and abiotic loss mechanisms [28,29].A recent study by Harms and Zehnder [30] indicated that the


Table 2. Effects of nonionic surfactants on the rates ofmineralization of [14C]phenanthrene and [14C]pyrene in a coaltar-contaminated soil (soil A1). Differences between surfactant-

amended soil samples and the controls were significant accordingto the Student’s t test (p . 0.95). SD, standard deviations of three

replicates

Treatmenta

Maximum rates of mineralization(nmol/kg soil/d 6 SD)

[14C]Phenanthrene [14C]Pyrene

NoneC9–11 AE-8C12–18 AE-10C12 EGEC18:1 EGE

136 6 18206 6 18220 6 11218 6 22236 6 37

42 6 2.651 6 2.460 6 1.759 6 1.861 6 2.9

a C9–11 AE-8, a fatty alcohol ethoxylate (AE) with an alkyl chain lengthof C9 to C11 and eight ethoxylene oxide units; C12–18 AE-10, an AEwith an alkyl chain length of C12 to C18, 10 ethoxylene oxide units,and end-capped with butyl; C12 EGE, an ethyl glycoside monoester(EGE) with an alkyl chain length of C12; C18:1 EGE, an EGE withan alkyl chain length of C18.

Fig. 2. Effect of the addition of an alcohol ethoxylate with an alkyl chain length of C12 to C18, 10 ethoxylene units, and end-capped with butyl(C12–18 AE-10) (100 mg/g) on the mineralization of [14C]phenanthrene (A) and [14C]pyrene (B) in a coal tar-contaminated soil (soil A1). Standarddeviations of three replicates are indicated by vertical bars.

diffusion of 3-chlorodibenzofuran into the interior of a solidsorbent was dependent on the time of contact between thesorbent and the sorbate. The desorption and degradation ratesfor 3-chlorodibenzofuran declined markedly with increasingtime for sorption to porous granules [30].

Desorption of hydrophobic organic compounds from soilaggregates is often slow, and contaminants that have persistedin soil for years may not be readily accessible to the micro-organisms [4,29]. Despite the possible limitations on hydro-carbon degradation that may be the consequence of a dimin-ished availability in soil, it has been demonstrated that theaddition of competent microorganisms may enhance the deg-radation of PAHs in field-scale experiments [10]. In our lab-oratory experiments, two bacterial strains, 1PHE128 and17PHE128, clearly enhanced the mineralization of phenan-threne when introduced in the noncontaminated soil B with alow indigenous potential for degradation of phenanthrene.Strain PheAH, which originated from a fuel oil-contaminatedsoil, degraded phenanthrene more slowly than did strain1PHE128 and 17PHE128, which were both isolated from thecoal tar-contaminated soil A1 (Fig. 1). When the bacteria wereintroduced into soil A1, only strain 1PHE128 increased therate of phenanthrene degradation compared to the mineraliza-


Table 3. Effects of nonionic surfactants (100 mg/g; additions at day 0 and day 28) on theconcentrations of polycyclic aromatic hydrocarbons (PAHs)a in a coal tar-contaminated soil (soil A2).The PAH concentrations in surfactant-amended soil samples differed significantly from the controls

according to the Student’s t test when indicated by the following symbols: n, p . 0.975; ●, p .0.95; and C, p . 0.90. SD, standard deviations of three replicates. BDL, below detection limit

(0.01 mg/g)

PAHInitial concn.(mg/g 6 SD)

Concn. remaining after 42 d (mg/g 6 SD)

Control C12–18 AE-10 C12–14 APG C18:1 EGE

Naphthalene1-Methylnaphthalene2-MethylnaphthaleneBiphenylAcenaphthyleneAcenaphtheneFluoreneAnthracenePhenanthrene1-Methylphenanthrene2-Methylphenanthrene

15.4 6 2.210.2 6 3.321.4 6 3.3

2.5 6 0.6211.5 6 3.5

4.1 6 1.016.7 6 1.814.7 6 1.152.5 6 4.7

5.9 6 1.55.1 6 0.29

0.17 6 0.150.70 6 0.400.60 6 0.490.70 6 0.621.8 6 0.061.4 6 0.462.4 6 0.958.4 6 3.4

18.3 6 2.54.0 6 0.604.6 6 1.5

BDL0.13 6 0.06 C0.07 6 0.05

BDL0.53 6 0.23 n0.33 6 0.06 ●1.2 6 0.10 C3.0 6 0.30 C4.3 6 0.10 n1.4 6 0.53 n1.1 6 0.15 ●

0.10 6 0.100.20 6 0.10 C

BDLBDL

0.50 6 0.30 n1.6 6 0.211.3 6 0.453.6 6 0.20 C4.8 6 0.15 n1.7 6 0.12 n1.3 6 0.47 ●

0.07 6 0.060.27 6 0.210.13 6 0.05

BDL2.3 6 0.700.40 6 0.35 ●1.9 6 0.603.9 6 1.3 C6.1 6 1.7 n0.97 6 0.29 n1.3 6 0.40 ●

FluoranthenePyreneBenzo[a]fluoreneBenz[a]anthraceneChryseneBenzo[b,j,k]fluorantheneBenzo[a]pyreneBenzo[ghi]peryleneIndeno[1,2,3-cd]pyrene

30.4 6 3.023.6 6 2.7

8.6 6 1.011.6 6 1.713.5 6 1.614.3 6 1.7

7.1 6 0.973.0 6 1.15.1 6 1.1

17.5 6 1.826.4 6 4.7

7.0 6 0.998.5 6 1.28.7 6 2.1

13.0 6 0.306.2 6 0.953.4 6 0.704.8 6 1.5

12.5 6 2.0 ●13.5 6 2.1 n

3.9 6 0.65 n5.1 6 0.75 ●6.4 6 1.18.6 6 1.1 n4.3 6 0.87 C2.7 6 1.35.4 6 0.97

14.0 6 0.65 ●12.7 6 1.7 n

4.0 6 0.20 n5.1 6 0.25 n6.4 6 0.507.2 6 0.46 n3.5 6 0.30 n2.7 6 1.44.7 6 0.11

16.5 6 4.516.8 6 4.2 C4.7 6 1.7 C6.7 6 2.38.3 6 2.69.9 6 3.05.1 6 1.43.4 6 0.215.3 6 0.70

a C12–18 AE-10, an alcohol ethoxylate with an alkyl chain length of C12 to C18, 10 ethoxylene oxide units,and end-capped with butyl; C12–14 APG, an alkyl polyglycoside with a chain length of C12 to C14; C18:1 EGE,an ethyl glycoside monoester with an alkyl chain length of C18.

tion by the microorganisms in the native soil. In the experi-ments with strain 1PHE128, an inoculum density of at least106 cells/g of soil was necessary for the introduction of aphenanthrene degradation potential in soil B. Considering thepossible use of seed organisms in a field-scale treatment, alarge inoculum may be required in order to establish the xe-nobiotic degrading activity of the introduced organisms. Pre-vious laboratory studies of bacterial inoculation of soil samples[11,13] have also used a high concentration of the added or-ganisms (107 cells/g soil), and treatment of creosote contam-inated soil in a field study involved an inoculum (wet weight) :soil (dry weight) ratio of 1:10 [10].

The addition of nonionic surfactants significantly enhancedthe degradation of freshly added PAHs and of PAHs in thecoal tar. Compared to controls without surfactant, the maxi-mum mineralization rates for [14C]phenanthrene and[14C]pyrene in soil A1 increased by a factor of 1.24 to 1.69in the presence of AEs and EGEs (Table 2). The surfactant-related stimulation of PAH degradation (Table 2 and Fig. 2)is in accordance with a previous study by Aronstein and Al-exander [13]. They observed that low concentrations of non-ionic surfactants (10 and 100 mg/g of air-dried soil or aquifermaterial) enhanced the desorption and mineralization of phe-nanthrene and biphenyl. Additions of surfactants (100 mg/g)at the start of the experiment and after 28 d also enhanced thedegradation of PAH contaminants in soil A2. The persistenceof pyrene, benzo[b,j,k]fluoranthene, and benzo[a]pyrene in thecontrol samples indicates that these PAHs were scarcely avail-able to the soil microorganisms in the absence of surfactants.The low levels of naphthalenes and biphenyl that persisted inthe control samples indicate that a fraction of the two-ringcompounds was firmly sorbed and that the availability of thesecompounds could be influenced by nonionic surfactant solu-

tions (Table 3). The effect of the surfactants may be relatedto the enhancement of the apparent solubility of PAHs, whichoccurs at surfactant concentrations below the CMC [18]. Insoil, however, sorption of surfactant increases the soil organiccarbon content, which also increases the potential for hydro-carbon sorption. The net change in PAH availability in soiltreated with surfactant, therefore, depends on the relative con-tribution of solubility and sorption-enhancement effects [15].

The C12–18 AE-10 and C12–14 APG were both more beneficialto the degradation of the PAH contaminants than was C18:1

EGE. The limited effect of the EGE on the degradation of thecontaminant hydrocarbons contrasts with the results of theexperiments with the labeled compounds. The relative effectsof C12–18 AE-10, C12–14 APG, and C18:1 EGE may be explainedby differences in the rate of surfactant degradation in the soil.Addition of C18:1 EGE to liquid medium markedly increasedthe growth of strain 1PHE128 and simultaneously repressedthe degradation of phenanthrene. Previous studies have shownthat surfactants of the EGE type are more rapidly degradedthan is C12–14 APG under aerobic as well as methanogenicconditions [31,32]. The C12–18 AE-10 is expected to biodegrademore slowly than C18:1 EGE and C12–14 APG. The ethoxylatemoiety of C12–18 AE-10 is ended by a butyl group, and thisstructure may retard the oxidation of the terminal ethoxylate,which is assumed as a possible initial step in the aerobic bio-degradation of AEs [33]. Although the concentrations of thesurfactants were not measured during the experiment, it seemspossible that degradation of C18:1 EGE in the soil resulted ina rapid decrease in the aqueous surfactant concentration and,hence, in a less effective solubilization of PAHs.

This study has indicated that easily degradable PAHs suchas naphthalenes may resist desorption and persist at low levelsafter biotreatment of contaminated soil (Table 3). Although


mineralized as the 14C-labeled compound, pyrene was not de-graded as a soil contaminant, unless nonionic surfactants wereadded to enhance the bioavailability of the PAHs (Fig. 2B andTable 3). Degradation of hydrophobic substances in freshlycontaminated samples should, therefore, be interpreted withcaution as such experiments do not address the time-dependentinteractions between the chemicals and the components of soilsand sediments. One of the surfactants, C18:1 EGE, which de-grades rapidly under aerobic conditions [31], was less effectivein enhancing the degradation of the coal tar contaminants. Thissuggests that nonionic surfactants that are mineralized to harm-less products at moderate rates should receive attention if thesesubstances are considered for biotreatment purposes.

Acknowledgement—This study was financed equally by EuropeanCommunity contract EVWA-CT92-0004 and by the Danish Environ-mental Research Programme. We thank Jens Aamand and ClausJørgensen for providing the bacterial strains and Connie Seierø, HanneBeck Rasmussen, Ulla Steen Pedersen, and Steffen Nielsen for as-sistance in the laboratory. We also thank Susan Bennetzen and LisK. Søndergaard for making the chemical analyses, and ClausJørgensen for comments on the manuscript.

REFERENCES

1. Chin, Y.-P., W.J. Weber, Jr. and B.J. Eadle. 1990. Estimatingthe effects of dispersed organic polymers on the sorption of con-taminants by natural solids. 2. Sorption in the presence of humicand other natural macromolecules. Environ. Sci. Technol. 24:837–842.

2. Mahro, B. and M. Kastner. 1993. Mechanisms of microbialdegradation of polycyclic aromatic hydrocarbon (PAH) in soil-compost mixtures. In F. Arendt, G.J. Annokkee, R. Bosman andW.J. van den Brink, eds., Contaminated Soil 993, Vol. 2. Kluwer,Dordrecht, The Netherlands, pp. 1249–1256.

3. Mihelcic, J.R., D.R. Lueking, R.J. Mitzell and J.M. Stableton.1993. Bioavailability of sorbed- and separate-phase chemicals.Biodegradation 4:141–153.

4. Hatzinger, P.B. and M. Alexander. 1995. Effect of aging ofchemicals in soil on their biodegradability and extractability. En-viron. Sci. Technol. 29:537–545.

5. Park, K.S., R.C. Sims, R.R. Dupont, W.J. Doucette and J.E.Matthews. 1990. Fate of PAH compounds in two soil types:Influence of volatilization, abiotic loss and biological activity.Environ. Toxicol. Chem. 9:187–195.

6. Heitkamp, M.A., W. Franklin and C.E. Cerniglia. 1988. Mi-crobial metabolism of polycyclic aromatic hydrocarbons: Isola-tion and characterization of a pyrene-degrading bacterium. Appl.Environ. Microbiol. 54:2549–2555.

7. Weissenfels, W.D., M. Beyer and J. Klein. 1990. Degradationof phenanthrene, fluorene and fluoranthene by pure bacterial cul-tures. Appl. Microbiol. Biotechnol. 32:479–484.

8. Walter, U., M. Beyer, J. Klein and H.-J. Rehm. 1991. Deg-radation of pyrene by Rhodococcus sp. UW1. Appl. Microbiol.Biotechnol. 34:671–676.

9. Cerniglia, C.E. 1992. Biodegradation of polycyclic aromatic hy-drocarbons. Biodegradation 3:351–368.

10. Davis, M.W., J.A. Glaser, J.W. Evans and R.T. Lamar. 1993.Field evaluation of the lignin-degrading fungus Phanerochaetesordida to treat creosote-contaminated soil. Environ. Sci. Tech-nol. 27:2572–2576.

11. Grosser, R.J., D. Warshawsky and J.R. Vestal. 1995. Miner-alization of polycyclic and N-heterocyclic aromatic compoundsin hydrocarbon-contaminated soils. Environ. Toxicol. Chem. 14:375–382.

12. Schneider, J., R. Grosser, K. Jayasimhulu, W. Xue and D.Warshawsky. 1996. Degradation of pyrene, benz[a]anthracene,

and benzo[a]pyrene by Mycobacterium sp. strain RJGII-135, iso-lated from a former coal gasification site. Appl. Environ. Micro-biol. 62:13–19.

13. Aronstein, B.N. and M. Alexander. 1992. Surfactants at lowconcentrations stimulate biodegradation of sorbed hydrocarbonsin samples of aquifer sands and soil slurries. Environ. Toxicol.Chem. 11:1227–1233.

14. Edwards, D.A., R.G. Luthy and Z. Liu. 1991. Solubilization ofpolycyclic aromatic hydrocarbons in micellar nonionic surfactantsolutions. Environ. Sci. Technol. 25:127–133.

15. Edwards, D.A., Z. Liu and R.G. Luthy. 1994. Surfactant sol-ubilization of organic compounds in soil/aqueous systems. J. En-viron. Eng. 120:5–22.

16. Edwards, D.A., Z. Liu and R.G. Luthy. 1994. Experimentaldata and modeling for surfactant micelles, HOCs, and soil. J.Environ. Eng. 120:23–41.

17. Grimberg, S.J., J. Nagel and M.D. Aitken. 1995. Kinetics ofphenanthrene dissolution into water in the presence of nonionicsurfactants. Environ. Sci. Technol. 29:1480–1487.

18. Edwards, D.A., Z. Liu and R.G. Luthy. 1992. Interactions be-tween nonionic surfactant monomers, hydrophobic organic com-pounds and soil. Water Sci. Technol. 26:147–158.

19. Liu, Z., D.A. Edwards and R.G. Luthy. 1992. Sorption of non-ionic surfactants onto soil. Water Res. 26:1337–1345.

20. Hommel, R.K. 1990. Formation and physiological role of bio-surfactants produced by hydrocarbon-utilizing microorganisms.Biodegradation 1:107–119.

21. Shoji, N., M. Ueno and K. Meguro. 1978. Determination ofcritical micelle concentrations of some nonionic surfactants byketo-enol tautomerism of benzoylacetoanilide. J. Am. Oil Chem.Soc. 55:297–299.

22. Ridgway, H.F., J. Safarik, D. Phipps, P. Carl and D. Clark.1990. Identification and catabolic activity of well-derived gaso-line-degrading bacteria from a contaminated aquifer. Appl. En-viron. Microbiol. 56:3565–3575.

23. Kiyohara, H., K. Nagao and K. Yana. 1982. Rapid screen forbacteria degrading water-insoluble, solid hydrocarbons on agarplates. Appl. Environ. Microbiol. 43:454–457.

24. Aamand, J., G. Bruntse, M. Jepsen, C. Jørgensen and B.K.Jensen. 1995. Degradation of PAHs in soil by indigenous andinoculated bacteria. In R.E. Hinchee, J. Fredrickson and B.C.Alleman, eds., Bioaugmentation for Site Remediation, Vol. 3.Battelle, Columbus, OH, USA, pp. 121–127.

25. Hobbie, J.E., R.J. Daley and S. Jasper. 1977. Use of nucleoporefilters for counting bacteria by flourescence microscopy. Appl.Environ. Microbiol. 33:1225–1228.

26. Jenkinson, D.S. and D.S. Powlson. 1976. The effects of biocidaltreatments on metabolism in soil—V. A method for measuringsoil biomass. Soil Biol. Biochem. 8:209–213.

27. Stott, D.E., J.P. Martin, D.D. Focht and K. Haider. 1983. Bio-degradation, stabilization in humus, and incorporation into soilbiomass of 2,4-D and chlorocatechol carbons. Soil Sci. Soc. Am.J. 47:66–70.

28. McCall, P.J., S.A. Vrona and S.S. Kelly. 1981. Fate of uniformlycarbon-14 ring labeled 2,4,5-trichlorophenoxyacetic acid and 2,4-dichlorophenoxyacetic acid. J. Agric. Food Chem. 29:100–107.

29. Alexander, M. 1995. How toxic are toxic chemicals in soil?Environ. Sci. Technol. 29:2713–2717.

30. Harms, H. and A.J.B. Zehnder. 1995. Bioavailability of sorbed3-chlorodibenzofuran. Appl. Environ. Microbiol. 61:27–33.

31. Madsen, T., G. Petersen, C. Seierø and J. Tørsløv. 1996. Bio-degradability and aquatic toxicity of glycoside surfactants and anonionic alcohol ethoxylate. J. Am. Oil Chem. Soc. 73:929–933.

32. Madsen, T., H.B. Rasmussen and L. Nilsson. 1995. Anaerobicbiodegradation potentials in digested sludge, a freshwater swampand a marine sediment. Chemosphere 31:4243–4258.

33. Holt, M.S., G.C. Mitchell and R.J. Watkinson. 1992. The en-vironmental chemistry, fate and effects of nonionic surfactants.In N.T. de Oude, ed., Detergents. Springer-Verlag, Berlin, Ger-many, pp. 89–144.

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