ORIGINAL PAPER

B. P. Ressler á H. Kneifel á J. Winter

Bioavailability of polycyclic aromatic hydrocarbons and formationof humic acid-like residues during bacterial PAH degradation

Received: 1 June 1999 / Received revision: 16 July 1999 /Accepted: 1 August 1999

Abstract The degradation of single polycyclic aromatichydrocarbons (PAHs: naphthalene, acenaphthene,¯uorene, phenanthrene, anthracene, ¯uoranthene andpyrene) and a mixture of all seven PAHs by a bacterialculture enriched from contaminated soil resulted in theformation of a dark-coloured residual fraction of dis-solved (DOM) and particulate organic matter (POM).This fraction was highly resistant to bacterial degrada-tion. Analysis of the DOM revealed a molecular-size-distribution similar to that of natural humic acids. Acomplete degradation of PAHs was apparently pre-vented by an irreversible incorporation of about 10% ofthe carbon from single PAHs or 20% of the carbon fromthe mixture of seven PAHs into the DOM- and POM-fraction. Some metabolites excreted during bacterialPAH-degradation were identi®ed as known precursorsfor humi®cation.

Introduction

The use of microorganisms for soil remediation aftercontamination with hydrophobic organic pollutants likepolycyclic aromatic hydrocarbons (PAHs) is oftenlimited by the bioavailability of these compounds. Thefate of organic pollutants during bioremediation is in-¯uenced by the competing processes of adsorption andbiodegradation (Guerin and Boyd 1992). Whereas thehydrophobic PAHs adsorb tightly to the organic frac-tion of soil and sediments (Kordel et al. 1997), sorptionto the mineral fraction of soil is usually much weaker

and is of importance for bioavailability, mainly by in-traparticle di�usion (Steinberg et al. 1987; Chiou et al.1998). Within the fraction of organic soil compounds,humic substances (humic acids, fulvic acids and hum-ins) play an important role for binding PAHs or PAHmetabolites (Richnow et al. 1997; Rebhuhn et al. 1998).Humic substances are considered to be a sink for in-termediates of aromatic and non-aromatic compounds(Gauthier et al. 1987). Humi®cation during microbialdegradation of organic material in soil and watermainly procedes with PAH transformation products.Humic substances consist of dark-coloured, inhomo-geneous macromolecules with a molecular weightranging from 800 Da to more than 100,000 Da and arecomposed almost entirely of the elements C, O and H,with an average ratio of 5.5:4.5:0.5 (Ziechmann 1994).The formation of humic acid-like substances duringmicrobial degradation of hazardous organic pollutantslike PAHs has been an important matter of concern inthe ®eld of bioremediation of contaminated soil in re-cent years. Humi®cation has been observed and wasinvestigated to study the interaction of soil fungi andthe complex reaction of soil-substrate mixtures (KaÈ st-ner and Mahro 1996). Walter (1990; Walter et al. 1991)observed the formation of recalcitrant humic acid-likesubstances during the bacterial degradation of anthra-cene oil, a complex mixture of PAHs and other sub-stances.

The mechanism of humi®cation can be illustrated bytwo models: the Maillard model, which describes theformation of melanoides from sugars and amino acids;and the polyphenol model, which describes the irre-versible polymerization of aromatic compounds viaphenolic radicals. In the case of microbial degradationof PAHs, it is likely that the formation of humic acid-like molecules starts with an oxidative coupling of partlyoxidized, phenolic PAH-metabolites that are releasedinto the medium by the cells. An extracellular accumu-lation of PAH-metabolites has been observed duringmicrobial metabolism of di�erent PAHs (Bouchez et al.1996). Whereas in most of the available literature the

Appl Microbiol Biotechnol (1999) 53: 85±91 Ó Springer-Verlag 1999

B. P. Ressler á J. Winter (&)Institut fuÈ r Ingenieurbiologie und Biotechnologie des Abwassers,UniversitaÈ t Karlsruhe, Am Fasanengarten,D-76131 Karlsruhe, Germany

H. KneifelInstitut fuÈ r Biotechnologie III, Forschungszentrum JuÈ lich,D-52425 JuÈ lich, Germany

formation of humic substances was attributed to com-plex reactions in microcosms with the participation ofsoil fungi, in this study evidence is presented that theformation of organic residues with humic acid-like be-haviour is mediated mainly by soil bacteria.

Materials and methods

Chemicals were obtained from Riedel de Haen (Seelze, Germany),Sigma-Aldrich (Steinheim, Germany), Sigma (Deisenhofen, Ger-many) and Merck (Darmstadt, Germany) and were of pure ana-lytical grade. The PAHs listed by the Environmental ProtectionAgency (EPA-PAHs, consisting of naphthalene, acenaphtene,¯uorene, phenanthrene, anthracene, ¯uoranthene and pyrene) wereused for experiments (Table 1). The PAHs were applied singly or asa mixture as the sole source of carbon for bacterial growth.

A mixed bacterial population from the PAH-contaminated soilof a former coal gas plant was enriched in a medium containing theseven EPA-PAHs as a carbon source. A subsequent screening re-vealed that the enriched mixed culture could grow with either oneof the PAHs or a combination of all seven PAHs. The culturemedium for enrichment and further experiments was prepared ac-cording to Lockhead and Chase (1973) and contained per liter:0.8 g K2HPO4, 0.2 g KH2PO4, 1.0 g KNO3, 0.2 g MgSO4 � 7H2O,0.1 g CaCl2 � 2H2O, 0.1 g NaCl, 0.01 g FeCl3 � 6H2O, 1 ml traceelement solution. The trace elemtent solution contained per liter:500 mg EDTA-sodium salt, 10 mg ZnSO4 � 7H2O, 200 mg FeSO3 �7H2O, 3 mg MnCl2 � 4H2O, 30 mg H3BO3, 20 mg CoCl2 � 6H2O,10 mg CuSO4 � 2H2O, 6 mg NiCl2 � 6H2O, 3 mg Na2MoO4 �2H2O. CaCl2 was dissolved separately in 100 ml distilled water.Culture media and trace element solutions were autoclaved(20 min, 121 °C, 105 Pa) separately. After sterilization the traceelement and CaCl2 solutions were slowly poured intothe stirred media. The pH of the media was 7.0±7.2. Crystals of theseven PAH compounds were sieved through a 0.5 mm sieve and25 mg of each fraction <0.25 mm were added to 250 ml of culturemedium, resulting in a ®nal concentration of 100 mg/l. To obtainmedia with the PAH mixture, 14.3 mg of each PAH was added to1 l medium. At a PAH concentration of 100 mg/l no agglomerationof PAH crystals occurred, although this was observed at PAHconcentrations of 700±1,000 mg/l. For preparation of solid medi-um, 20 g agar were added per liter and 20-ml portions were pouredinto Petri dishes.

For the degradation experiments, 500-ml Schott ¯asks (Schott,Mainz, Germany) were ®lled with 250 ml culture media containing100 mg/l single PAH or PAH mixture. The ¯asks were inoculatedwith 250 ll concentrated enrichment culture (optical densityE630 nm � 1.0) by scratching colonies from the surface of agarplates and preparing a suspension in 0.1 M phosphate-bu�er, pH 7,to give an optical density of E630 nm � 1.0. The ¯asks were shakenat 180 rpm on rotary shakers in the dark at 22 °C. For measure-ment of microbial oxygen consumption, the ¯asks were incubated

in a Voigt-Sapromat (Voigt, Sulzbach, Germany). PAH-analyseswere performed on a Hewlett Packard HPLC with ¯uorescencedetector as described previously (Ressler and Winter 1995). Anal-ysis of metabolites was performed according to Soeder et al. (1996)and organic residues were analyzed by gel permeation chroma-tography (GPC) as described by Gremm et al. (1991). Dissolvedorganic carbon (DOC) was measured with a Dohrmann DOC-Analyser (Dohrmann, Germany). CO2-evolution was measured asdescribed by JaÈ ggi (1976). Protein-measurement was performedusing the method of Bradford (1976). To determine the relativetoxicity of the PAHs in culture media, a standard bioluminescencetest (DIN 38412) with Photobacterium phosphoreum was used(DEV, 1995). In this test, toxic e�ects on the metabolism of P.phosphoreum can be measured through a reduced bacterial lumi-nescence. The organic carbon content of particular organic matter(POM) was measured with a LECO Multiphase carbon determi-nator RC-412 (LECO, St. Joseph, Mich., USA). One g driedsample (105 °C, 24 h) was oxidized in an oxygen atmosphere at550 °C within 4 min and the CO2 produced was measured by infra-red spectroscopy.

Adsorption isotherms were determined with phenanthrene asthe adsorptive. The fraction of POM residues from the bacterialdegradation of the PAH mixture (PAH1±7) and the bacterial cells ofthe mixed population were used as adsorbent. Prior to adsorption,the POM fraction containing the bacteria was hydrolysed in 1.0 MNaOH at 95 °C for 20 min to lyse the bacteria. The sample waspassed through a 0.45 lm ®lter to separate cell wall fragments. Theadsorbed PAHs on the POM fraction were removed by twowashing steps with acetone at 60 °C, followed by three washingsteps with distilled water to remove the acetone. The PAH-freePOM fraction was dried at 105 °C for 24 h, powdered with a pestleand used as adsorbent. For the determination of adsorption iso-therms of cells from the mixed culture, fresh bacterial cells wereused. The biomass dry weight was measured in parallel. The bio-mass was cultivated on mineral-agar plates covered with a thincrystalline layer of an equimolar mixture of PAH1±7. The plateswere prepared as described by Kiyohara et al. (1982). The bacterialcolonies were harvested carefully to avoid contact with the sur-rounding surface of the agar-plates and washed three times in0.1 M phosphate bu�er, pH 7. Cells were separated by centrifu-gation and used as adsorbent.

For the determination of adsorption isotherms, 100 ml phena-nthrene-saturated water containing 0.05% (w/v) of NaN3 for in-activation of the bacteria was pipetted into 100-ml glass vials. Toachieve homo-ionic conditions NaN3 was also added to controlvials for measuring the adsorption onto the glass walls. Followingequilibration (phenanthrene concentration, ceq � 0.9±1.1 mg/l), thesorbents (washed POM or bacterial cells) were added to the vials.The vials were shaken 24 h on a rotary shaker at 120 rpm until anew equilibrium was reached. As a criterion for the equilibriumconcentration, a constant PAH concentration over 12 h was taken.After a total equilibration time of 24 h the solution in the vials wascentrifuged at 8,000 g for 30 min and the residual PAH concen-tration in the liquid phase was measured. After measurement of thepartition equilibrium the adsorbent concentration was increasedand the procedure was repeated until a new partition equilibriumwas reached. Taking into account the adsorption onto the glasswalls in the controls, the amount of PAH that was sorbed onto thesorbent could be calculated. For documentation of the desorptionisotherms, an analog procedure was used. The adsorbent, saturatedwith the adsorptive, was centrifuged and half of the supernatantwas replaced by distilled water. After the equilibrium for re-solubilization was reached the procedure was repeated until theconcentration of the respective PAH in the aqueous phase was nolonger detectable. For a mathematical description of the adsorptionisotherms the Freundlich and the Langmuir equations were applied(see below).

To investigate the e�ect of POM on the bioavailability ofphenanthrene, di�erent amounts of POM, prepared in the sameway as for adsorption isotherms, were suspended in the culturemedium before it was inoculated with the mixed culture. Allexperiments were conducted at room temperature (21 � 2 °C).

Table 1 Physical and chemical parameters of the seven polyaro-matic hydrocarbons listed by the Environmental Protection Agency(EPA-PAHs) (Sims and Overcash 1983). KOW Octanol/water par-titition coe�cient

EPA-PAHs Molecularweight

Solubilityin water (mg/l)

Log KOW

Naphthalene 128.19 30 3.37Acenaphthene 154.2 3.47 4.33Fluorene 166.2 1.98 4.18Phenanthrene 178.24 1.29 4.46Anthracene 178.24 0.07 4.45Fluoranthene 202.26 0.26 5.33Pyrene 202.26 0.14 5.32

86

Results

PAH degradation and formation of DOM

The degradation of phenanthrene alone and the mixtureof seven EPA-PAHs by the enrichment culture was an-alysed. In the assay with phenanthrene 10% of the PAHand in the assay with the PAH1±7 mixture 19% of theamount of the original PAHs were found as DOM after30±80 h of incubation (Fig. 1a, b). If each of the sevenPAHs was separately degraded by the mixed culture 10±12% of the supplied carbon entered a non-biodegrad-able or non-bioavailable soluble state (DOM) (Fig. 2).Not included in either case is the POM already formedduring degradation. A detailed carbon balance wascarried out using the mixed culture fed with 100 mg/lEPA-PAHs mixture (Table 2). It was found that 60.5%of the PAH carbon was respired to carbon dioxide and13.8% ®xed into bacterial biomass. The rest was eitherDOM (19.1%) or POM (6.2%). It could not be furthermetabolized by the bacterial population upon prolongedincubation.

In the course of the bacterial degradation of PAHmixtures, the colour of the culture medium turned fromyellow to brown and ®nally to black. The culture

medium was then passed through a 0.45 lm ®lter. Byacidifying the dark-brown ®ltrate to a pH of <2, blackparticles precipitated, which later changed colour viadark brown to light brown. This behaviour is typical forhumic acids, which are water-insoluble at low pH. Tofurther substantiate humic acid formation from PAH bythe enrichment culture, gel permeation chromatography(GPC) was performed on the dissolved organic com-pounds in the medium. The results revealed a molecularsize distribution similar to that of natural humic acids(Fig. 3).

The particulate organic residues consisted of biomassand presumably polymerized organic residues fromPAH degradation. The biomass in the dried POMfraction was estimated from the protein content, whichamounted to 12±15% (w/w). The protein content ofdried cells of the mixed culture, grown and harvestedfrom agar plates, was determined to be 50% (w/w).Thus, about 30% of the POM fraction consisted ofbiomass and 70% was formed of organic residues fromthe PAH degradation.

Fig. 1 Degradation of phenanthrene (a) and of a mixture of the sevenpolyaromatic hydrocarbons listed by the Environmental ProtectionAgency (EPA-PAHs) (b) and formation of dissolved organiccompounds (DOC) by the enrichment culture. (a) 100 mg phenanth-rene/l, (b) 100 mg PAH mixture/l (=14.3 mg/l for each single PAH)

Fig. 2 Residual DOC after degradation of the seven single PAHs(100 mg/l) and a mixture of PAH1±7 (SSPAK1±7 � 100 mg/l) by theenrichment culture. Cultures were prepared as described in Materialsand methods and incubated for 80 h at 22 °C

Table 2 Carbon balance for the degradation of PAH mixtures(100 mg/l = 100%) by the enrichment culture from soil. Incuba-tion time 80 h. DOC dissolved organic compound; DOM DOmatter; POM particulate OM

Fraction Amount(mg/l)

Proportionof carbon (%)

CO2 209 60.5Biomass 32.8 13.8DOM (DOC) 18 19.1POM 10 6.2

Recovery 99.6

87

Metabolites of PAH degradation

In order to determine some of the possible reactants forDOM formation, the water soluble metabolites excretedduring degradation of single PAHs were determined. Inthe exponential growth phase of the bacterial population1-naphthol was excreted during the degradation ofphenanthrene (Fig. 4), 1-hydroxy-indene during the de-gradation of ¯uorene, 7-hydroxy-acenaphthylene duringthe degradation of ¯uoranthene (not shown) and 4-hy-droxy-tetralone during the degradation of ¯uorene(Fig. 5). They were either taken up later for further de-gradation or reacted with DOM and ®nally were ad-sorbed onto or reacted with POM.

Bioavailability of polyaromatic hydrocarbons

The e�ect of POM on the bioavailability of PAHs forthe bacterial population was investigated with phena-nthrene as a single PAH in cultures supplemented with100±400 mg/l POM. Increasing concentrations of POMin the cultures reduced microbial CO2 formation sig-

ni®cantly (Fig. 6). In cultures without POM, respirationof 100 mg phenanthrene resulted in the production of261 mg CO2 and utilization of 296 mg oxygen, whereasin cultures with increasing amounts of up to 400 mgPOM/l the CO2 evolution and oxygen consumption werereduced (Table 3). Protein production as a measure ofbacterial growth was 22 lg/ml in cultures without POMand was reduced to 15.9 lg/ml in cultures containing400 mg POM/l. Thus it seemed that the presence ofPOM reduced the proportion of phenanthrene availablefor bacterial growth and respiration. To exclude possibletoxic e�ects of the POM fraction on the bacterial pop-ulation, toxicity tests were conducted with P. phosphor-eum. Results of these experiments revealed that thePOM fraction did not inhibit luminescence of P. phos-phoreum and therefore POM had no toxic e�ect on theenrichment culture (data not shown).

Adsorption of phenanthrene to particulateorganic matter

Experiments of bacterial PAH degradation in the pres-ence of increasing amounts of POM implied that this

Fig. 3 Comparison of the elution behaviour (analyzed by gelpermeation chromatography) of natural humic acids and the dissolvedorganic matter fraction (DOM) formed during degradation of amixture of PAH1±7

Fig. 4 UV absorption spectrum of metabolite Phe1 from phenanth-rene degradation by the mixed culture (solid line) compared to thespectrum of 1-naphthol (dotted line)

Fig. 5 UV absorption spectrum of metabolite Flu1 from ¯uorenedegradation by the mixed culture (solid line) compared to thespectrum of 4-hydroxy-tetralone (dotted line)

Fig. 6 CO2-production by the mixed culture during degradation ofphenanthrene (100 mg/l) in the presence of di�erent amounts ofparticulate organic matter (POM)

88

fraction had a high a�nity either to PAH molecules insolution and/or to water soluble microbial degradationproducts. Therefore, adsorption of phenanthrene as areference PAH to the POM fraction, separated from theculture liquid, was investigated by determining adsorp-tion isotherms. As a control, adsorption isotherms ofphenanthrene to the mixed bacterial PAH-degradingpopulation were also determined. For this purpose, cellsuspensions were inactivated with 0.2% (w/v) NaN3 toavoid degradation of the sorptive. Figure 7 illustratesadsorption isotherms of phenanthrene to POM, mixedbacterial cells and mixtures of both in di�erent pro-portions. It can be seen that the POM fraction had ahigher a�nity to adsorb dissolved phenanthrene mole-cules than the bacterial cells. At increasing phenanthreneconcentrations, the a�nity of phenanthrene to the POMfraction increased to a higher degree than it did for thebacterial cells. In the presence of both sorbents, theamount of phenanthrene on the sorbents was dependenton the relative concentration of POM to bacterial cells.

As shown in Fig. 8, the adsorption of phenanthreneto POM was not completely reversible. A complete

desorption of phenanthrene from the POM fractioncould only be achieved if the adsorption was mediatedby weak physical forces, such as van der Waals anddipole-dipole forces.

Discussion

Laboratory results on the degradation of PAHs in soilsuspensions by enrichment cultures from contaminatedsoil indicated that as much as 20% of the organic carbonof a mixture of PAHs remained in the medium, either asDOM or adsorbed to/forming POM. This could be dueto a tight binding of the PAHs to DOM or POM, eitherdirectly or via reaction with metabolites. The role ofmetabolites in the formation of humic acid-like sub-stances might be deduced from the fact that, duringfermentation of a mixture of PAHs, more metaboliteswere formed and more humic acid-like substances re-leased than during fermentation of single compounds.The metabolites were only excreted in measurableamounts during the exponential phase of degradation,indicating that the subsequent reactions were rate-lim-iting. PAHs and/or metabolites presumably reacted withDOM and formed the humic acid- or humic substance-like precursors that were ®nally bound into the POMfraction as humic residues. It seems likely that an irre-versible covalent binding of PAH-metabolites to thePOM fraction was the prevailing mechanism that pre-vented total bacterial mineralization of the PAHs.

The metabolites detected during PAH degradationhad functional groups such as hydroxy- and carboxy-moieties, typical substituents necessary for biologicaldegradation. If the subsequent reactions were rate-lim-iting, these intermediate substances were excreted intothe medium and there served for the genesis of humicacids (Hedges 1988; Hatcher and Spiker 1988). 1-Nap-hthol was a typical metabolite of the degradation ofnaphthalene by fungi and yeasts (Cerniglia and Crow1981; Cerniglia et al. 1983), preferentially formed by a

Fig. 7 Adsorption isotherms of phenanthrene on POM and onbacterial cells (BC)

Fig. 8 Adsorption and desorption isotherms of phenanthrene ontoand from POM

Table 3 Respiratory activity of the mixed bacterial populationduring degradation of phenanthrene (100 mg/l) in culture mediacontaining di�erent concentrations of POM, derived from the de-gradation of PAH mixtures

POM(mg/l)

CO2 productionrate (mg/l � h)

CO2 production(mg total)

Decline of CO2

production (%)

0 7.1 261 100100 6.2 246 94.1200 5.5 237 90.7400 4.5 220 84.2

POM(mg/l)

O2 consumptionrate (mg/l � h)

ConsumedO2 (mg)

Decline of oxygenconsumption (%)

0 11.1 296 100100 9.6 280 94.7200 7.4 266 90.3400 6.3 251 84.9

89

monoxygenase reaction, whereas dioxygenase reactionswere involved in the bacterial degradation of phena-nthrene (Cerniglia et al. 1984). The formation of1-naphthol from phenanthrene by our mixed culturemight indicate that a monooxygenase was also involvedin the bacterial metabolism of phenanthrene. 1-Nap-hthol is a reactant for the formation of higher molecularhumic acid complexes via the polyphenol model ofhumic matter genesis (Martin and Haider 1980; Bollaget al. 1983). The principal mechanism of polymerizationof 1-naphthol to polynaphthol under catalysis of per-oxidases has been described for white rot fungi (Su¯ita1981).

The metabolite 4-hydroxy-1-tetralone, which was ex-creted during the degradation of ¯uorene (Fig. 5), isknown to be a degradation metabolite of naphthaleneproduced by eucaryotic microorganisms. It has also beendetected during naphthalene degradation by cyanobac-teria (Cerniglia et al. 1980). One of the known pathwaysfor the formation of 4-hydroxy-1-tetralone is via naph-thalene-1,2-oxide, 1- or 2-naphthylol and 1,4-naphtho-chinone, involving a nonenzymatic shift of the hydroxysubstituent during the hydroxylation of the arenoxide to1- or 2-naphthol (Gibson 1984).

For the formation of humic substances the impor-tance of chinoid structures, which originate from phe-nolic precursors, was outlined by Stevenson (1982) andZiechmann (1994). Since 4-hydroxy-1-tetralone and itsprecursor 1,4-naphthochinone are chinoidic molecules,they may also play a role as precursors in humi®cationprocesses and therefore are likely to be involved in theformation of DOM and POM during bacterial PAHdegradation.

Compared with data from the literature, the KF andKL values for the adsorption of phenanthrene to thePOM fraction were relatively small, although they werein the same order of magnitude as the values from otherinvestigations for the adsorption of three- and four-ringPAHs to humic substances (Gremm et al. 1991; Gaut-hier et al. 1987). Maxin and KoÈ gel-Knabner (1995)found adsorption to soil organic matter only for PAHswith more than three rings. No signi®cant adsorption ofphenanthrene could be found. The broad range of valuesfor partition coe�cients in the literature might be ex-plained by the fact that adsorption was investigatedusing di�erent natural humic substances with very het-erogeneous structures and di�erent adsorptive qualities.

For the adsorption of aromatic hydrocarbons tobacterial cells, data from the literature indicated thatpartition coe�cients were dependent on the status of theadsorbent biomass and the way the biomass was inac-tivated to prevent degradation of the sorbed material. Inthe review of Baughman and Paris (1981), a partitioncoe�cient of 6.3 1/g was reported for the adsorption ofphenanthrene to bacterial cells. Flieder (1991) found apartition coe�cient of 13 l/g for the adsorption ofphenanthrene to a bio®lm and Whitman et al. (1995)reported a KF value of 0.4 l/g for the adsorption ofnaphthalene to cells of P. ¯uorescens.

Adsorption isotherms for phenanthrene on POM hada sigmoid shape with quasi-linear parts between con-centrations of 0±0.1 mg/l and 0.1±0.4 mg/l, respectively.At higher concentrations an over-proportional adsorp-tion of phenanthrene to POM or bacterial cells wasobserved. To decribe adsorption for concentrations be-tween 0±0.1 mg/l, the Langmuir equation (Eq. 1) couldbe applied with su�cient accuracy:

qS �KL � qmax � ceq1�KL � ceq �1�

qS � loading of the adsorbent with the sorptiveceq � equilibrium concentration in the liquid phaseKL � Langmuir-sorption-coe�cientqmax � maximun sorption capacity of the sorbent

The Langmuir constants KL and qm were obtained bythe linearized plot of the measured data (Eq. 2):

1

qS� 1

qmax

� 1

qmaxKL� 1ceq

�2�

At higher concentrations (>0.2 mg phenanthrene/l), theLangmuir model was not applicable since there was nomaximum for the sorption (qm), which is de®ned in theLangmuir model by a monomolecular layer that com-pletely covers the surface of the sorbent. Therefore, theFreundlich equation (Eq. 3) was applied to describe theconcentration range up to 0.4 mg/l:

qS � KF � c1=neq �3�qS � loading of the adsorbent with the sorptiveceq � equilibrium concentration in the liquid phaseKF � Freundlich-coe�cient1/n � Freundlich-exponent

The Freundlich parameters KF and 1/n were obtainedfrom the following regression (Eq. 4):

log qS � logKF � 1=n � log c �4�Langmuir and Freundlich constants were calculated andare listed in Table 4.

In summary, the formation of DOM and POMduring bacterial degradation of PAHs reduced thebioavailability of PAHs. The DOM and POM fraction

Table 4 Langmuir and Freundlich Isotherm constants for the ad-sorption of phenanthrene to POM and bacterial cells. The Lang-muir model was applied at very low concentration of dissolvedphenanthrene (<0.1 mg/l) and the Freundlich model was appliedfor the concentration range <0.4 mg phenanthrene/l. KF Freun-dlich partitition; KL Langmuir partitition coe�cient; qm maximumloading of the sorbent with a monomolecular layer of phenan-threne molecules; ceq concentration at equilibrium

Fraction ceq < 0.4 mgphenanthrene/l

ceq < 0.1 mgphenanthrene/l

KF

(l/g)1/n r2 KL

(l/g)qm(mg/g)

r2

POM 7.02 0.93 0.98 6.05 1.93 0.994Bacterial cells 4.21 1.18 0.97 3.43 0.92 0.997

90

in contaminated soil can therefore be seen as an in-creasing sorptive matrix that competes with microor-ganisms for dissolved PAHs and microbial metabolites.Binding and incorporating those molecules into thehumic-acid-like matrix removes them from the pool ofbioavailable compounds and keeps them from re-utili-zation by the bacterial population. The polymerizationof transformation products of the bacterial PAH me-tabolism to humic-acid-like complexes is one conditionto render biodegradable metabolites into a recalcitrantstate. Speci®c functional structures like the phenolic andchinoidic structures of metabolites are known to beamong the most important for initiating this process.Therefore it seems probable that, in the course of bac-terial PAH degradation, mechanisms are involved thatare similar to those which take place in the naturalhumi®cation of other aromatic compounds.

Acknowledgement We thank the Deutsche Forschungsgemeinsc-haft, Bonn for supporting this study as a part of the ``OÈ kologischeWasserwirtschaft'' (``Ecological Water Management'').

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