microbial transformation of styrene by anaerobic consortia

Journal of Applied Bacferiology 1990.69, 247-260 3 IR9/09/89

Microbial transformation of styrene by anaerobic consortia

D U N J A G R B I ~ - G A L I C * , N I N A C H U R C H M A N - E I S E L t & I S K R A

M R A K o v I C Environmental Engineering and Science, Department of Civil Engineering, Stanford University, Stanford, Calfornia 943054020, USA

Accepted I3 January 1990

G R B I ~ - G A L I ~ . , D., CHURCHMAN-EISEL. N . & M R A K O V I ~ , I . 1990. Micro- bial transformation of styrene by anaerobic consortia. Journal of Applied Bacre- riology 69. 247-260

Methanogenic microbial consortia, originally enriched from anaerobic sewage sludge with ferulic acid or styrene (vinylbenzene) as sole organic carbon and energy sources, were used to study transformation of styrene under strictly anaerobic conditions. Styrene. which was added as the substrate in a range of concentrations from 0.1 to 10 mmol/l. was extensively degraded but no methane production was observed during incubation for eight months. The addition of yeast extract during the enrichment stage completely inhibited degradation of styrene. Gas chromatography (GC), gas chromatography/mass spectrometry (GC/MS). high performance liquid chromatography (HPLC) analyses of the culture fluid, and GC analyses of the anaerobic headspace, indicated that the transformation of this arylalkene was initiated through an oxidation-reduction reaction and that the favoured mechanism was most likely the addition of water across the double bond in the alkenyl side- chain. The degradation proceeded through to carbon dioxide, the final product. Benzoic acid and phenol were transient compounds found in highest concentrations in the spent culture fluid and are suggested as the key intermediates of the transformation process. The tentative routes of anaerobic transformation partially overlap with those previously proposed for aromatic hydrocarbons such as toluene. Several pure cultures, which were tentatively identified as Clostridium spp. and Enterobacfer spp., were isolated from the styrene-degrading consortia. Two of these cultures were demonstrated to grow on styrene as sole carbon and energy source. Additionally, a pure culture of Enterohacfer cloacae DG-6 (ATCC 35929) which had been isolated previously from the ferulate-degrading consortium, was shown to degrade styrene through to carbon dioxide.

Although investigations on anaerobic transformation of unsubstituted aromatic hydrocarbons (such as benzene or naphthalene) and their alkylated counterparts (toluene, ethylbenzene, xylenes, etc.) started only in the early 1980s (Ward et a/. 1980), there is now a substantial literature on these phenomena (Vogel & Grbic- Galic 1986; Kuhn et al. 1988). The anaerobic degradation of mononuclear and binuclear aromatic hydrocarbons has been demonstrated under denitrifying (Kuhn et a/ . 1985, 1988;

Corresponding author. t Present address: Environmental Protection

Agency, Denver, Colorado 80208. USA.

Zeyer et a/. 1986; Major et a/ . 1988; Mihelcic & Luthy 1988a, b), methanogenic (Vogel & Grbic- Galic 1986; Wilson et a/ . 1986, 1987; Grbic- Galic & Vogel 1987; Grbic-Cali6 1989). and iron-reducing conditions (Lovley et al. 1989). In all cases examined so far, the anaerobic transformation of these compounds is initiated by an oxidative reaction, resulting in formation of a phenol- or aromatic alcohol-type intermediate. The oxygen necessary for this reaction is most likely derived from water (Vogel & Grbic-Galic 1986).

As opposed to unsubstituted or alkylated aromatic hydrocarbons, microbial degradation of arylalkenes (e.g. styrene or methylstyrenes) has

248 Dunja Grbit-Galit et al. been studied only under aerobic conditions (Omori et al. 1974, 1975; Sielicki et a/. 1978; Shirai & Hisatsuka 1979). Aerobic micro- organisms oxidize styrene to phenylethanol and phenylacetic acid which can be subsequently completely mineralized through an oxygenative pathway. Styrene is a very important synthetic monomer in the manufacture of plastics and may therefore also be a significant environmental pollutant. It is moderately toxic and has not been shown to act as a carcinogen; however, in mammals it is rapidly transformed to an epoxide (Liebman 1975). This can act as a carcinogenic substance by alkylating proteins and nucleic acids (van Duuren et al. 1963). It must be stressed that styrene can be produced naturally by micro-organisms which transform aromatic plant derivatives. Fungi have been shown to produce styrene and its derivatives from cinnamic acid (Jaminet 1950; Chen & Pepler 1956; Clifford et al. 1969) and p- coumaric acid (Harada & Mino 1973). Bacterial formation of hydroxystyrene from p- hydroxylated cinnamic acid has been observed under aerobic conditions (Finkle et al. 1962) and under anaerobic (fermentative) conditions (Scheline 1968). Consequently, whilst a significant portion of styrenes which reach the environment are man-made, micro-organisms in soil, sediments, or sewage (aerobic or anaerobic) may have been exposed to such compounds from natural sources as well and may theoretically harbour considerable capabilities for further transformation of these compounds. The competing reaction, which may scavenge styrene, is its spontaneous polymerization into polystyrene because styrene undergoes free- radical formation very easily (Bolker 1974); this polymerization occurs under oxidative conditions, and may be significant in formation of humic acids in soil (Finkle 1965). Commercial styrene, however, contains an anti-oxidant polymerization inhibitor, p-tertiary butyl catechol @-TCB), which has so far not been shown to biodegrade.

In this paper we report anaerobic microbial transformation of styrene amended with p-TCB by anaerobic consortia which were originally methanogenic and enriched from anaerobic sewage sludge, and by pure cultures isolated from these consortia. Degradation routes, intermediates, and products of transformation are discussed. This is the first report on transform-

ation of this arylalkene in the absence of molecular oxygen which elucidates some details of the transformation sequence. The only available ref- erence suggesting disappearance of styrene under anaerobic conditions is that of Wilson ef a/. (1986) but these authors did not analyze for intermediates and products of degradation.

Materials a d Metbods

MICROBIAL C O N S O R T I A A N D C U L T U R E S

The original methanogenic consortia (suspended mixed cultures) were enriched from anaerobic municipal sludge using a lignin derivative, ferulic acid (1.5 mmol/l), or styrene (1.2 mmol/l) amended with p-TCB (15 mgp styrene), as sole organic carbon and energy sources. The ferulic acid consortia (GrbiC-Galii: & Young 1985) had been maintained on their original substrate for a year before they were used for the styrene degradation experiments. Subsequently, the original ferulate consortia were also used to develop toluene ( 1 5-30 mrnol/l) and benzene ( 15 mmol/l) degrading consortia (GrbiC-GaliC & Vogel 1987); these were used in later styrene degradation experiments. The ferulate, toluene, and benzene consortia were degrading their respective substrates stoichiometrically to carbon dioxide and methane; the incubation times varied from two weeks (ferulate) to two months (toluene) for complete degradation. The ‘ferulate’, ‘toluene’. and ‘benzene’ inocula to be used for styrene degradation experiments were with- drawn from the respective consortia at the end of an incubation period when their substrates were completely degraded to CO, and CH, (as demonstrated by GC and GC/MS analyses and carbon mass balance calculations). In this manner, no additional organic substrates were transferred to the new cultures, and styrene was the sole organic carbon and energy source (except in the cases when yeast extract was deliberately added, as described later in this text).

In addition to the consortia, a pure culture of a facultatively anaerobic fermentative bacterium, Enterobacter cloacae DG-6 (ATCC 35929) was also used in the styrene degradation experiments. This bacterium had been isolated from the original ferulate consortium (GrbiC-Galii: 1985) and shown to degrade ferulic acid under anaerobic (fermentative) conditions (GrbiC-

Anaerobic degradation of styrene 249

Cali6 & La Pat-Polasko 1985; Grbic-Galic 1986). DG-6 was maintained aerobically on nutrient agar slants; the aerobically grown inoculum was used in the experiments of anaerobic styrene degradation.

CHEMICALS

Styrene (Aldrich Chemical Co., Inc., Milwaukee, WI) was at least 99% pure and was amended with 15 ppm p-TCB to inhibit polymerization. All the other chemicals used in media prep- aration or analytical work were of reagent grade, and were purchased from Aldrich, or from Baker Chemical Co. (Phillipsburg, NJ). Yeast extract was obtained from Difco.

MEDIA A N D C U L T I V A T I O N PROCEDURES

A prereduced, defined anaerobic mineral medium with vitamins (Healy & Young 1979) was used in these experiments. The medium contained 2 mmol/l of each ferrous chloride and sodium sulphide as reducing agents, and 1 mgJ resazurin as the redox indicator, The medium was buffered at pH 7.0 with a bicarbonate-CO, system under a gas atmosphere of 30% CO, : 70% N,. No exogenous electron acceptors other than CO, were added. In the first series of experiments, where ferulate, toluene, and styrene-enriched consortia and the pure culture DG-6 were used, styrene was added to the autoclaved medium in nominal concentrations of 3, 5, and 10 mmol/l. In the second series of experiments, the consortia were refed styrene, and additionally, new enrichments were obtained from anaerobic sludge, using styrene as sole organic carbon and energy source. The concentrations of styrene which were tested this time were 0.1, I , 3, 5, and 10 mmol/l. In the third series of experiments, benzene-degrading consortia were examined for their ability to degrade styrene and the original ferulate- degrading consortia were used in a new enrichment procedure which included both yeast extract and styrene as the enrichment substrates. This experiment was performed to test for the effect of additional carbon sources on the induction of the styrene-transforming capability. The concentration of styrene used in the third series of experiments was 3 mmol/l. Since the highest styrene concentrations ( 5 and 10 mmol/l) exceeded the solubility limit for styrene

in the aqueous medium, which had been esti- mated to be 2.7 mmol/l (Yalkowsky el a/. 1983). two liquid phases were present in the respective experiments; the dissolution of styrene in the medium was controlled by microbial removal of styrene from aqueous solution. At least three replicate cultures were established for each com- bination of inocula, styrene concentration. and yeast extract addition (where applicable); additionally, duplicate autoclaved biological controls and sterile abiotic (chemical) controls were monitored in parallel with the cultures. The purpose of the controls was to account for the background, non-microbiological disappearance (such as sorption) or changes of the substrate. The culture containers were 150-1111 volume serum bottles stoppered with teflon-coated butyl rubber stoppers, fixed in place with alu- minium crimps. Each bottle contained 100 ml of fluid ( 1 5% v/v inoculum, 85% v/v of prereduced defined medium) and 50 ml headspace (30% CO, : 7oYO N,). The bottles were incubated at 35°C in the dark, for a total of eight months. During this time, they were all sampled with disposable syringes for culture fluid and headspace at least biweekly and the samples were analysed by GC, HPLC, and GC/MS for substrate removal, transient intermediates appearance, and end product formation. Each culture was vigorously shaken for several seconds before sampling. Three different procedures (GC, HPLC. GC/MS) were used independently to follow the fate of styrene and/or its transformation intermediates in order to obtain more conclusive results and better proofs for the Occurrence of degradation. Total gas production was continuously measured throughout the incubation period with a glass syringe as described by Healy & Young (1979).

Isolation of pure cultures from the styrene- transforming enrichments was attempted with the same defined mineral medium, but with the addition of 1.5% agar, a lower concentration of reducing agents (half the concentration used in liquid medium) and 5 mmol/l styrene as sole carbon and energy source. The isolation and incubation were performed in an anaerobic chamber (Coy Laboratories, Inc., Ann Arbor, MI) in a 5% H,: 10% CO,: 85% N, atmosphere. The organisms were tested for their abil- ities to transform styrene in liquid medium as described above. Two of the strains were studied for growth on styrene (0.1 mmol/l) and,

250 Dunja Grbit-Galit et al. comparatively, gloucose (0. I mmol/l) as sole carbon and energy sources under anaerobic conditions. The cells were enumerated using acridin orange epiflourescence procedure (Hobbie et a/ . 1977).

A N A L Y T I C A L P R O C E D U R E S

Gas chromatography (GC)

The composition of gas produced by the microbial cultures was determined on a Fisher- Hamilton gas partitioner, Model 25 V, with helium as the carrier gas. The instrument was calibrated using a commercial gas mixture (Liquid Carbonic, Chicago, ILL) consisting of 9.94% CO,. 0.04% CH,, and balanced with N,.

The amount of methane in the sample gas was determined with a Hewlett-Packard 5730 A gas chromatograph equipped with a 60/80 Car- bosieve G packed column (1.7 m x 3 mm internal diameter), and a flame ionization detector. The G C was connected to a Spectra-Physics 4OOO data system. The oven and injector tem- peratures were 120°C and 200°C respectively. Helium was the carrier gas. An external standard procedure was used for quantification.

Styrene determinations and detection of potential transformation intermediates were performed on a Carlo Erba Fractovap Model 2900 G C (Carlo Erba Strumentazione, Milano, Italy) with a Spectra-Physics 4OOO data system. The GC was equipped with a fused silica capillary column DB-5 30 m x 0.32 mm i.d., 1.0 pm film thickness (J&W Scientific, Rancho Cordova, CA), and a photoionization detector (HNU Systems, Inc., Newton, MA). Helium was used as the carrier gas. Samples of the culture fluid were extracted with diethylether; o- dichlorobenzene was added as a n internal standard. The initial oven temperature was 60°C (for 5 min), and was subsequently increased to 160°C at a rate of I"C/min. The injector temperature was set a t 230°C.

H i g h performance liquid chromatography ( H P LC)

Transient appearance of water-soluble intermediates of styrene transformation was also fol- lowed by HPLC, with a Spectra-Physics S P 3500 B HPLC system with a Spherisorb ODS

10 pm reverse-phase column, a variable wave- length UV detector and a computing integrator (Spectra-Physics Autolab System I). The mobile phase consisted of acetonitrile and 0.01 N per- chloric acid in a linear gradient of 10 to 50% acetonitrile. The flow rate was 1.2 ml/min. The detector was set at five different wavelengths (190,210, 222, 254, and 284 nm), and calibration curves for standards of each suspected transformation intermediate were determined at all five wavelengths. Centrifuged samples of culture fluid were injected directly on the column without extraction. A minimum of five replicate injections were made for each sample taken. The lower sensitivity limit of the procedure was 0.1 mgJ for each compound.

Gas chromatography/mass spectrometry (GCIMS)

Final quantification, detection, and identification of styrene and its transformation intermediates and products (except gaseous products, which were independently determined by GC) was performed on Finnigan MAT 4OOO GC and 4500 MS with an INCOS data system, by using selective masses. Samples of culture fluid were extracted with diethyl ether under acidic conditions; one microlitre of the extract was injected splitless (for 30 s) on a 60-m DB-5 fused silica capillary column with a 0.32 mm i.d. and 1.0 pm film thickness. The carrier gas was helium. The initial column temperature was 60°C and was increased to 250°C at a rate of 4"C/min. The injector temperature was 250"C, and the ionizer temperature was 140°C. The fore- pressure of the column was adjusted to 7.5 x lo4 Pa. The scanning rate was approx- imately one scan per second with an ionization voltage of about 1500 V. Identifications of the unknowns were based on comparison with a library of known spectra and by comparison with retention times and mass spectra of the known standards. o-Fluorophenol was used as an internal standard for quantitation.

Results

The initial series of experiments, in which various cultures were fed styrene as sole carbon and energy source for the first time, indicated that the initial lag time before the onset of substrate degradation was short. The first sampling

Anaerobic degradation of styrene 25 1

of the culture fluid was performed 12 d after the feeding, and numerous potential transformation intermediates, including ethylbenzene, toluene, benzene, phenylacetic acid, benzyl alcohol, benzaldehyde, benzoic acid, 2-ethylphenol, p- cresol, a-cresol, phenol, methylcyclohexane, acetic acid, and formic acid, were already present in the culture fluid. No CO, was formed in the same time period. Subsequent samplings of the active consortia resulted in detection and identification of more compounds. All are sum- marized in Table 1 and may be divided into several structural groups: aromatic and aliphatic hydrocarbons, phenols, aromatic alcohols, aromatic aldehydes, aromatic acids, alicyclic compounds, aliphatic alcohols, branched aliphatic acids, straight-chain aliphatic acids, and one-carbon compounds (CO,). Neither of the controls formed any of these transformation intermediates or products. In cultures which received higher initial concentrations of styrene ( 5 or 10 mmol/l) many of the compounds transiently accumulated in the culture fluid and were therefore easier to detect and identify than in the cultures fed lower (3 mmol/l) concentration of styrene. However, the formation of two separate liquid phases in the bottles receiving high concentrations of styrene, facilitated the sorption of styrene into rubber stoppers after the repeated sampling damaged the teflon coating on the stoppers and exposed the rubber; this occurred both in the cultures

and the controls. It is probably for this reason that only about 50% of the original styrene carbon could be accounted for at the end of the experiments with high styrene concentrations, whereas most of the substrate carbon could be accounted for in cultures and controls when the initial styrene concentrations did not exceed the solubility limit (see Table 2). Some of the postu- lated transformation intermediates (those marked by an asterisk in Table 1) were detected in all the consortia irrespective of the inoculum source. In addition to the compounds shown in Table 1, some other compounds (not shown) which were most likely products of secondary methylation reactions, were found : p-xylene, a- xylene, 4-methylbenzaldehyde, and methoxycyclohexane. Although the inocula had been initially methanogenic when fed their original substrates, and although precursors of methane, such as acetate, formate, and CO, were produced from styrene (Table I), no methane was formed by any of the styrene-degrading consortia during eight months of incubation.

In the second series of experiments, where the original styrene enrichments were refed styrene, there was no lag period before the onset of styrene transformation. In the new anaerobic sludge enrichments on styrene, however, a 7-day long lag was observed. With the lowest styrene concentrations tested (0.1 and 1 mmol/l), a good carbon mass balance could be established, but the intermediates were transformed faster and

Table 1. A complete list of intermediates and products detected in the culture fluid of styrene-degrading anaerobic cultures. The compounds labelled by an asterisk were found in all the cultures tested, irrespective

of the inoculum source?

hydrocarbons compounds compounds compounds Gases Aromatic Oxygenated aromatic Alicyclic Aliphatic

Ethylbenzene Phenylethanol 2-Methylcyclohexanol 2-Ethyl-I-hexanol CO,* Toluene* Phenylacetaldeh yde Cyclohexanol 2-Ethyl- I-hexanoic Benzene Phenylacetic acid* 2-Ethylcycl0- acid

2-Hydroxyphenylacetic hexanone 2-Methylpentanoic acid Cyclohexanone acid

I-Phenylethanone 2-Methyl- 1 - 2-Hexenoic acid Benzyl alcohol* cyclohexene Butanoic acid Benzaldehyde* Methylcyclohexane' Propionic acid 4Hydroxybenzaldehyde Acetic acid* 2-Hydroxybenzaldehyde* Formic acid Benzoic acid* 2-Ethylphenol* Heptane pCresol 2-Heptene o-Cresol Phenol*

t Chemical structures for the compounds are shown in Figures 1.2, and 3.

252 Dunja Grbi&Galk et al. fewer of them could be detected. The compounds which were still appearing in relatively high concentrations were phenol, 2-ethylphenol, and benzoic acid. Table 2 shows carbon mass balances for some of the cultures (originally ferulate, toluene, and styrene consortia) receiving 1 mmol/l styrene. A greater than 100% recovery of carbon in styrene enrichments (Table 2) may be explained by more pronounced cell decay and subsequent degradation of dead cells to CO, in these cultures. A lower than lOO% recovery of the substrate carbon in the other two types of cultures (ferulate and toluene-degrading inocula) may be explained by the possibility that not only carbon dioxide and the reduced compounds listed in Table 2. but also other reduced compounds (which could not be detected with the analytical techniques used), were still present at the end of the incubation period. Moreover, some of the reduced compounds (benzene, heptane, 2-heptene) were

found in low concentrations and are not included in Table 2. Again, no methane production was observed in any of the consortia.

In the third series of experiments, benzene consortia were tested for their ability to transform styrene and the original ferulate-degrading consortia were enriched on styrene in the presence of yeast extract to study the influence of additional organic substrates on the induction of styrene transformation. Both sets of experiments yielded negative results. Benzene consortia were incapable of degrading styrene. Yeast extract completely inhibited styrene transformation in the ferulate consortia, whereas previous experiments had shown that the styrene- transforming capability could be easily enriched in these consortia. Methane was produced from yeast extract in the presence of styrene (which was not degraded). Upon depletion of yeast extract, the cultures were subcultured into fresh styrene-amended media which did not contain

Table 2. Carbon mass balances for anaerobic (fermentative) cultures fed 1 mmol/l styrene as sole organic carbon and energy source and incubated for 8 months at 35°C

pmol/l carbon in cultures derived from:'

Ferulatedegrading Toluene-degrading St yrene-enriched inoculum inoculum sludge consortium

Compounds Months Months Months detected 0 4 8 0 4 8 0 4 8

Styrene

Ethylbenzene

Toluene

Phenol

2-Ethylphenol

p-Cresol

Benzoic acid

Methylcyclohexane

CO,

Total carbon 8000t

150 f 9.4

720 & 7.3

2340 f 12.5

1260 f 18.3

210 f 7.5

2950 f 15.5

7630

-1

44 f 2.6

98 f 4.7

560 f 14.4 5530 & 10.2

6232

300 f 7.8

50 f 0.8

70 f 0.4 420 & 4.6 -

440 f 7.8 840 f 12.4 -

4800 17.3

6920

75 f 6.2

35 f 0.6

340

6230 6.3

f 9.5

6680

80 f 2.3 -

60 f 0.7 -

140 f 2.8 -

8300 & 12.4

8580

80 f 3.2

8470 9.6

8550 ~~

The results (except for the initial concentration of styrene) are expressed as mean f standard deviation, and have been obtained from measurements on triplicate cultures of each type. Only the compounds which were detected in concentrations higher than 10 pmol/l carbon, are taken into account and shown in this table. The duplicate autoclaved controls and duplicate abiotic (chemical) controls did not produce any intermediates, or carbon dioxide.

t The initial concentration of styrene is expressed as the amount added to the cultures (8000 pmol/l carbon, i.e. 1.0 mmol/l styrene), and not actually measured in the culture fluid.

1 Compound not detected in the culture fluid (or headspace) during this particular measurement.

Anaerobic degradation of styrene 253 yeast extract; however, the styrene-degrading capability was not induced during a four- month incubation period.

Enferohacter cloacae DG-6 successfully transformed styrene in concentration range from 0. I to 3 mmol/l. The intermediates and products from styrene which were formed by DG-6 include benzyl alcohol, benzaldehyde, 2- hydroxybenzaldehyde, benzoic acid, p-cresol, o- cresol. phenol, methylcyclohexane, and carbon dioxide. Some of these compounds, notably benzyl alcohol and benzaldehyde, were detected in the culture fluid already in the tirst week of incubation. N o aliphatic compounds were detected. Carbon mass balances were calculated for DG-6 growing on 1 mmol/l styrene. After 4.5 months of incubation, 28 k 3.4 pmol/l styrene carbon were left of the original 8OOO pmol/l; additionally, 39 f 1.2 pmol/l of phenol carbon, 7 f 0.4 /cmol/l of benzyl alcohol carbon, 16 k 2.6 pmol/l of methylcyclohexane carbon, and 7400 14.4 pmol/l CO, were detected, for a total of 7490 /cmol/l carbon accounted for.

Fifteen strains were isolated from styrene- degrading consortia --six from the original styrene enrichments, six from the original toluene consortia, and three from the original ferulate consortia. All of the strains were Gram- negative rods, which were tentatively assigned to the genus Enferohacrrr, except two of them which were sporogenous rods resembling members of the genus Clostridium. Six of these strains were tested for their ability to grow on styrene as sole carbon and energy source. Two of these six, both Gram-negative asporogenous rods of small size ( 0 . 2 0 3 by 0.5-1.5 pm) which were isolated from 'toluene' consortia, grew on styrene in a concentration of 0.1 mmol/l. These bacteria had generation times of 24 hours on styrene, and 20 hours on glucose.

Discussion

Anaerobic removal of styrene as sole organic carbon and energy source, its degradation to a range of aromatic, alicyclic, and aliphatic intermediates and ultimately carbon dioxide and a small amount of reduced organic compounds, was observed with all the inocula tested except with the benzene-degrading methanogenic consortium. N o degradation was observed in sterile controls, which excludes the possibility of

abiotic transformations. The microbial degradation can be described by the term 'primary substrate utilization'. The assumption of styrene utilization rather than cometabolism or for- tuitous transformation is supported by the tinding that an additional, easily biodegradable substrate (yeast extract) completely inhibited styrene degradation in consortia simultaneously grown on yeast extract plus styrene. Futhermore, the styrene-degrading ability was not induced after subculturing the yeast extract/ styrene-fed cultures into the medium with styrene as the sole substrate, because the addition of yeast extract possibily selected for different subpopulations within the ferulate- degrading community. The faster-growing yeast extract utilizers could have outcompeted the micro-organisms harbouring the styrene- transforming capability, and they may have been gradually eliminated from the consortium. Finally, a conclusive proof of styrene utilization is the fact that pure cultures of fermentative bacteria isolated from the consortia grew on styrene as sole carbon and energy source.

The degradation process was a typical fermentation, which resulted in formation of carbon dioxide and reduced hydrocarbon compounds (such as ethylbenzene, toluene, benzene, methylcyclohexane, heptane, and 2-heptene). In fermentations, the substrate plays the role of an electron donor as well as an electron acceptor, since there are no exogenous electron acceptors; some of the products become more oxidized than the substrate, whereas others become more reduced. Although the original consortia were all methanogenic, no methane production was observed during styrene degradation. This may be due to the toxicity of either styrene, or some of its degradation products to methanogens. The observation that the yeast extract- degrading consortia produced methane in the presence of styrene indicates that it was some of the products, rather than styrene itself, which adversely affected methanogenic bacteria. Schink (1985) found that aromatic hydrocarbons, such as toluene and benzene, and alicy- clics, such as cyclopentadiene and cyclo- heptatriene, inhibited methanogenesis in slurries from sewage sludge and sediments. However, some of our methanogenic inocula. especially those that degrade ferulate and toluene, had been shown previously not to be affected by aromatic hydrocarbons (Grbic-Galic & Young

254

H,O-

Dunja Grbit-Galit et a].

+HCOOH @

. . Phenylet honol 0

I- Phenyl -ethonone

Pheny locetoldehyde

0 ------) @CH,

'O2 Toluene

4 C H l 8 -L- O C H ,

2 -Methyl - I - cyclohexene

Benzyl olcohol Met hylcyclohexone

- 4 H l

4 @C"O

co2 f Benzoldehyde Benzene

I

J. /' 63 '@CO 0 H

Benrolc acid '2 / 2-Hexenoic acid

CH, (CH, )& H=CHCOOH ;I

@ Q - CH,(CHz)2COOH CH,COOH

8

Q OH OH Butyric ocid Acetic ocid

Phenol Cyclohexonol

Fig. 1. The 'phenylacetate route', a tentative transformation sequence which appears to occur most frequently in the anaerobic consortia transforming styrene. Reductive reactions which create some of the reduced electron and proton sink compounds (ethylbenzene, toluene, benzene) are also shown. All compounds marked by an asterisk were detected in culture fluids.

Anaerobic degradation of styrene 255 1985; Vogel & Grbic-Galic 1986; Grbic-Galic & Vogel 1987). Another possibility is the toxic effect of both phenol and 2-ethylphenol, which were produced in relatively high concentrations during styrene breakdown. We believe that the effect was toxic (damaging) to methanogenic cells rather than only inhibitory, because methanogenic activity was not revived after the elimination of phenols through degradation (Table 2).

Some of the intermediates and products of styrene degradation were found in all the cultures tested, regardless of the inoculum (Table I). Some of these compounds, especially phenylacetic acid, benzyl alcohol, benzaldehyde, benzoic acid, and phenol, point at the most likely initial transformation reactions which could happen in anaerobic microbial communities degrading styrene. These reactions are depicted in Fig. 1 which represents a tentative transformation route which we named 'the phenylacetate route'. The route is not completely shown in Fig. I , because only the compounds which were actually detected in the culture fluid, are represented. Here, styrene is initially transformed through addition of water across the double bond in the side chain, with the simultaneous oxidation of one carbon atom and reduction of another carbon atom. I t has been shown previously (Thomas 1970) that compounds which contain unsaturated double bonds, such as oleic acid, easily undergo this type of transformation (hydration) under anaerobic conditions. The hydration of styrene results in formation of phenylethanol, which is subsequently oxidized to phenylacetaldehyde and phenylacetic acid. These initial transformation reactions correspond to those that occur under aerobic conditions (Sielicki et a / . 1978; Shirai & Hisatsuka 1979). However, whereas the anaerobic reactions probably utilize water as an oxygen source, it is not clear in the aerobic reactions whether the initial conversion of styrene to phenylethanol occurs through an oxygenation, or through a hydration. After the formation of phenylacetic acid, the anaerobic routes become entirely different from those that are aerobic. Phenylacetic acid was previously shown to be easily biodegraded by methanogenic consortia degrading aromatic lignin derivatives (GrbiC- Galic & Young 1985), and to be possibly converted (through oxidative decarboxylation) to benzyl alcohol and subsequently oxidized to

benzaldehyde and benzoic acid. In support of this finding, it is worth mentioning that Barik et a/. (1983) observed that their methanogenic phenylacetate enrichments were simultaneously adapted to benzoate. Another decarboxylation of benzoic acid to yield phenol, rather than its reduction to cyclohexanecarboxylic acid as suggested by Evans (1977) is hypothesized because alicyclic acids were never found in our styrene- degrading consortia, whereas cyclohexanol, a likely product of phenol reduction (Evans 1977; Balba & Evans 1980; Barik et a / . 1985). was detected (Fig. I ) . Decarboxylation of 4- hydroxybenzoate to phenol and CO, has been demonstrated under anaerobic conditions (Scheline 1966; Curtius et a/ . 1976; Tschech & Schink 1986); but whether the decarboxylation of benzoic acid to phenol in styrene-degrading cultures occurs directly, or through a hydroxylated intermediate, is presently unknown. Both benzoic acid and cyclohexanol are precursors for ring cleavage and formation of long and short chain aliphatic acids (Evans 1977; Young 1984). In Fig. I , this is partially shown only for phenol, and not for benzoic acid, for sake of clarity of the figure.

The addition of water across the double bond in the styrene side chain could result theoretically in two products: phenylethanol (if the terminal carbon in the side chain is oxidized) and alternately, a secondary alcohol (if the subterminal carbon atom is oxidized), which can be subsequently converted to 1 -phenylethanone (Fig. I ) . Indeed, both these compounds were formed in some of the tested consortia (ferulate consortia and styrene enrichments from sewage sludge). However, the concentration of 1- phenylethanone measured in the culture fluid was always very low, suggesting that the oxidation of the terminal, rather than subterminal carbon in the styrene side chain, is the preferred route of anaerobic oxidation. The fate of 1- phenylethanone in our consortia is unknown.

Another early transformation intermediate found in all the cultures tested was 2- ethylphenol. Figure 2 ('2-ethylphenol route') shows that 2-ethylphenol can be derived from styrene through a reaction with water, which results in hydroxylation of the ring and a reduction of the double bond in the side chain (detailed mechanisms, the number of steps, and all the reactants involved in this reaction are presently unknown and therefore not shown in

256 Dunja Grbii.-Gulii. et al.

@CH=CH, f &CHz CH, /

STYRENE , Ethyl benzene

H.0 0

w OH

2- Ethylphenol J

@ J . CH,(CH,),CHCH,OH

I CH,CH,

2-Ethyl-I-hexanol J d 2 C H l

2' -i-'2 C H 7 @CH,(CH, ),CHCOOH

I CHZCH,

2-Hydroxy- phenylacet ic acid

2-Ethylhexonoic acid Fig. 2. The '2-ethylphenol route', a tentative transformation sequence which appears to be important in the anaerobic consortia transforming styrene. All compounds marked by an asterisk were detected in culture fluids.

Fig. 2). Judging from the concentration of 2- ethylphenol (Table 2). ring oxidation during the initial redox transformation seems to be almost as important a mechanism of styrene transformation as the terminal side chain carbon oxidation. If a parallel is made with the anaerobic toluene transformation by methanogenic consortia (Grbic-Galic & Vogel 1987). i t becomes obvious that in the toluene-degrading cultures, the two possible mechanisms (the initial side chain oxidation and the initial ring oxidation) were also occurring simultaneously, due to the presence of a number of different types of fermentative bacteria. These observations point at the great complexity of anaerobic consortia degrading aromatic hydrocarbons and arylalkenes.

2-Ethylphenol could theoretically undergo two degradation routes, firstly by further oxidation to o-hydroxylated phenylethanol, phenylacetaldehyde, and phenylacetic acid and secondly, by ring reduction and cleavage without previous oxidation of the ethyl side chain, to yield ethylated aliphatic products. Indications of both routes were found in our cultures; some of the compounds which were

detected (2-hydroxyphenylacetic acid, 2-ethyl- hexanol, 2-ethylhexanoic acid) are shown in Fig. 2. Further degradation of 2-hydroxyphenylacetic acid could occur either through phenylacetate route (Fig. I) , if the 2- hydroxyphenylacetic acid is dehydroxylated to phenylacetic acid, or through another sequence of reactions, some of which are shown in Figure 3. This figure (tentatively termed 'cresol route') attempts to establish connections between various hydroxylated intermediates, acids, aldehydes, and cresols, which were detected in the tested cultures. The conversion of toluene to cresols, and cresols to hydroxylated benz- aldehydes, had been shown previously (GrbiC- Galit & Vogel 1987). Since these compounds (with the exception of 2-hydroxybenzaldehyde) were not occurring as ubiquitously in different styrene-degrading cultures as the compounds discussed above, the routes depicted in Fig. 3 possibly d o not have as great a significance in styrene degradation as the routes shown in Figs 1 and 2.

It needs to be emphasized here that reduction reaction-resulting in formation of ethylbenzene, toluene, benzene, methylcyclohexane,

Anaerobic degradation of styrene 257

Q H O @ @CH=CH, HO@CH, -5. --• L HO@CHO

STYRENE p-Cresol 2CH’ 2CH3 4 -Hydroxy- benzoldehyde

Q + @ I 4 C H I 63 @CH,COOH - - T -- @ - I , +\+ O C H 3

Phenylacetic acid Toluene 2-Methyl-l-

I I

/

I

/ 2-Hydroxyphenyl- , acetic acid /

c yclohexene

I

Meth ylcyclohexane

o-Cresol

q2.4

2- Hydroxy- benzaldehyde

2- Methylcyclohexonol 2- Methylpentanoic acid

Fig. 3. The ‘cresol route’, a tentative transformation sequence involving hydroxylated aromatic aldehydes, acids and substituted phenols. Formation and transformation of toluene, one of the important intermediates and electron sinks in the styrene transformation, is also shown (modified after GrbiC-Galic & Vogel 1987). All compounds marked by an asterisk were detected in culture fluids or headspaces.

heptane, and 2-heptene, were occurring simultaneously with oxidation reactions, which is typical for fermentation processes; some of these compounds and the possible reactions of their formation (including reductive decarboxylation) are shown in Figs 1, 2, and 3. Ward et al. (1987) observed anaerobic reductive decarboxylation of p-hydroxyphenylacetic acid to p-cresol by Lactobacillus spp.; the reaction of decarboxylation of phenylacetic acid to toluene which we propose (Figs 1 and 3) is novel, and has not been suggested before. Reductive, electron sink- forming reactions similar t o the ones we propose had earlier been observed in the original methanogenic ferulate consortia degrading ferulate, when the methanogens were inhibited by 2-bromoethanesulfonic acid (GrbiC-GaliC &

Young 1985). Obviously, the inactivation of methanogens in anaerobic communities results in change of the fermentative metabolism and formation of more reduced organic compounds. In addition to the reduction reactions, various methylation reactions were observed in the styrene-degrading consortia (not shown in the figures). These reactions were either 0- methylations (e.g., formation of O-methylcyclohexane [methoxycyclohexane], possibly from cyclohexanol), or C-methylations (e.g., formation of 0- or p-xylene, possibly from toluene or cresols, or of 4-methylbenzaldehyde, possibly from benzaldehyde or p-hydroxybenzaldehyde). Such reactions are common in anaerobic consortia degrading aromatic compounds as suggested by Pereira et al. (1987) and they may play

258 Dunja Grbii-Galii et a].

a role either in formation of proton and electron sinks during the fermentative oxidation, or in detoxification processes.

The short initial lag period before the onset of degradation of styrene in various enrichments (toluene degraders, ferulate degraders, anaerobic sludge) and the immediate response of the cultures upon refeeding, indicates that the enzymic activity catalyzing styrene degradation can be easily induced without previous exposure to this compound. This supports the hypothesis that natural microbial communities may have an ability to degrade styrene derivatives (many of which may be of natural origin), especially if these communities are feeding on aromatic plant derivatives, such as ferulate (GrbiC-GaliC & Young 1985), or if they are remarkably diverse feeders, such as the communities in anaerobic municipal sludge. Of all the inocula tested, only benzene-degrading methanogenic consortia did not show any activity towards styrene. I t is probable that the initial enrichment procedure, with benzene as sole carbon and energy source, selected for micro-organisms which were unable to transform aromatic hydrocarbons carrying a side chain. This hypothesis is supported by our unpublished observations that benzene consortia could not develop the capability to degrade toluene either. Conversely, however, they did develop the ability to degrade naphthalene, which is another unsubstituted aromatic hydrocarbon (GrbiC- GaliC 1989).

Whether the natural anaerobic degradation of styrene will occur or not depends on numerous factors such as temperature, the presence of the necessary micro-organisms, availability of nutri- ents, elimination of other organic substrates which would otherwise interfere with styrene transformation, presence or absence of toxic or inhibitory compounds, and many others. If methanogens in styrene-degrading communities are inhibited, some of the products of styrene degradation may be more hazardous than styrene itself (e.g. benzene). However, these compounds are known to be degraded under anaerobic conditions (Vogel & GrbiC-GaliC 1986; Wilson et 01. 1986; Major et a/. 1988). Further- more, it is conceivable that, in natural habitats, the toxic effects of styrene transformation intermediates on methanogens would not be nearly as pronounced as in closed laboratory containers, because these intermediates would be

considerably more diluted. Therefore, the fermentative degradation of styrene in natural habitats may be carried out to completion, without production of reduced hydrocarbon compounds.

It is important to point out that there are obvious similarities and overlaps between the toluene and benzene anaerobic transformation routes (GrbiC-GaliC & Vogel 1987; Kuhn et a/. 1988) and the styrene transformation route suggested in this paper, although the initiating transformation reactions are different. The initial reaction with styrene is a simultaneous oxidation and reduction of the styrene molecule, whereby one of the carbon atoms in styrene becomes more reduced, and another, more oxidized (hydration). With toluene and benzene, the initial transformation is an oxidation with water-derived oxygen (hydroxylation), resulting in the production of a cresol, benzyl alcohol, or phenol, respectively and in reduction of an oxidized proton- and electron-carrier. The reason for this difference resides in the structure of styrene, whose unsaturated double bond facilitates water addition. After this initial transformation reaction, however, the transformation routes for aromatic hydrocarbons and arylalkenes start overlapping. For example, phenol, substituted phenols, and benzoic acid seem to be the key transformation intermediates; the oxygen source for oxidative reactions is most likely water and the degradation sequence (after the initial transformation reaction) includes side chain transformations, ring reduction, ring cleavage, and degradation of aliphatic intermediates to simple final products. It could be con- cluded that unsubstituted and substituted aromatic hydrocarbons, as well as arylalkenes, probably experience very similar fates in anaerobic natural environments. The most pronounced difference that we observed between the styrene degradation in these experiments and toluene (or benzene) degradation in our previous experiments was a remarkably longer incubation period needed to complete the styrene degradation process (eight months, against two months for toluene), although there was no difference in the duration of the initial lag before the onset of degradation. This can be explained by the fact that methanogens were obviously non-functional in the styrene- degrading cultures; consequently, there were no active hydrogen utilizers which could pull the

Anaerobic degradation of styrene 259 overall fermentation process forward. Since no molecular hydrogen could be produced, various electron and proton sinks (reduced compounds) were formed instead and the fermentative degradation was considerably slowed down. There- fore, provided with an ellicient hydrogen- scavenging mechanism, styrene degradation might be conceivably accelerated.

E n t e r o b u c t r r cloacue DG-6, previously isolated from the original ferulate-degrading consortium, elliciently transformed styrene through a fermentative route which is probably the closest to the suggested 'phenylacetate route' (Fig. 1). Phenylacetate was not detected in the culture fluid of DG-6 cultures grown on styrene, but some of the further transformation products, such as benzyl alcohol, benzaldehyde, and benzoic acid, were. DG-6 performed an almost stoichiometric conversion of styrene to carbon dioxide and low concentrations of aromatic and alicyclic compounds in 4.5 months of incubation. Still, a t this point, we feel it is premature to claim that DG-6 is capable of complete degradation of styrene; in order to prove this hypothesis, it would be necessary to perform the experiments with ''C-labelled styrene.

The performance of DG-6, as well as the growth of two styrene isolates on styrene as sole carbon and energy source, indicate that fermentative bacteria (and especially Enterobacte- riaceae which easily switch from the aerobic to anaerobic metabolism) may be quite significant in degradation of arylalkenes in nature. This significance is even pronounced by the fact that these bacteria are quite ubiquitous in the environment. Our work with the pure cultures isolated from styrene-degrading consortia is by no means complete ; these micro-organisms still need to be studied and characterized, and the mechanisms they utilize to transform styrene need to be elucidated in order to better under- stand the anaerobic pathways of arylalkene transformation. This research is currently in progress.

This work was funded by the U.S. Environ- mental Protection Agency under contract R811610-01-0 to D. Grbic-Calk.

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microbial transformation of styrene by anaerobic consortia

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