Removal of phenolic compounds from a petrochemical effluent with a methanogenic consortium.

A methanogenic consortium was used to degrade phenol and ortho- (o-) cresol from a specific effluent of a petrochemical refinery. This effluent did not meet the local environmental regulations for phenolic compounds (178 mg/L), oils and greases (61 mg/L), ammoniacal nitrogen (75 mg/L) or sulfides (3.2 mg/L). The consortium, which degrades phenol via its carboxylation to benzoic acid, was progressively adapted to the effluent. Despite the very high effluent toxicity (EC50 of 2% with Microtox), the adapted consortium degraded 97% of 156 mg/L phenol in the supplemented effluent after 13 days in batch cultures (serum bottle). The addition of proteose peptone to the effluent is essential for phenol degradation. o-cresol was also transformed but not meta- or para-cresols. A continuous flow fixed-film anaerobic bioreactor was developed with the consortium. Treating the effluent with the bioreactor reduced phenol and phenolic compounds concentrations by 97 and 83%, respectively, for a hydraulic residence time of 6 h. This treatment also reduced by about half the effluent toxicity. Oils and greases and ammoniacal nitrogen were not affected. Similar microbiological forms were observed in serum bottles and in the bioreactors with or without the petrochemical effluent. These results indicate that this methanogenic consortium can treat efficiently the phenolic compounds in this specific petrochemical effluent.     

Anaerobic degradation of 2,4-dichlorophenoxyacetic acid by <Emphasis Type="Italic">Thauera</Emphasis> sp. DKT

Thauera sp. strain DKT isolated from sediment utilized 2,4-dichlorophenoxyacetic acid (2,4D) and its relative compounds as sole carbon and energy sources under anaerobic conditions and used nitrate as an electron acceptor. The determination of 2,4D utilization at different concentrations showed that the utilization curve fitted well with the Edward model with the maximum degradation rate as 0.017?±?0.002 mM/day. The supplementation of cosubstrates (glucose, acetate, sucrose, humate and succinate) increased the degradation rates of all tested chemical substrates in both liquid and sediment slurry media. Thauera sp. strain DKT transformed 2,4D to 2,4-dichlorophenol (2,4DCP) through reductive side-chain removal then dechlorinated 2,4DCP to 2-chlorophenol (2CP), 4-chlorophenol (4CP) and phenol before complete degradation. The relative degradation rates by the isolate in liquid media were: phenol?>?2,4DCP?>?2CP?>?4CP?>?2,4D?≈?3CP. DKT augmentation in sediment slurry enhanced the degradation rates of 2,4D and chlorophenols. The anaerobic degradation rates in the slurry were significantly slower compared to the rates in liquid media.     

Studies on composition and stability of a large membered bacterial consortium degrading phenol

A ten member microbial consortium (AS) consisting of eight phenol-degrading and two non-phenol-degrading strains of bacteria was developed and maintained in a fed-batch reactor by feeding 500 mg l⁻¹ phenol for four years at 28 ± 3 °C. The consortium could degrade 99% of 500 mg l⁻¹ phenol after 24 hours incubation with a biomass increase of 2.6 × 10⁷ to 4 × 10¹² CFU ml⁻¹. Characterization of the members revealed that it consisted of 4 principal genera, Bacillus, Pseudomonas, Rhodococcus, Streptomyces and an unidentified bacterium. Phenol degradation by the mixed culture and Bacillus subtilis, an isolate from the consortium was compared using a range of phenol concentrations (400 to 700 mg l⁻¹) and by mixing with either 160 mg l⁻¹ glucose or 50 mg l⁻¹ of 2,4-dichlorophenol in the medium. Simultaneous utilization of unrelated mixed substrates (glucose/2,4-dichlorophenol) by the consortium and Bacillus subtilis, indicated the diauxic growth pattern of the organisms. A unique characteristic of the members of the consortia was their ability to oxidize chloro aromatic compounds via meta pathway and methyl aromatic compounds via ortho cleavage pathway. The ability of a large membered microbial consortia to maintain its stability with respect to its composition and effectiveness in phenol degradation indicated its suitability for bioremediation applications.     

Degradation of phenol under meso- and thermophilic, anaerobic conditions

Based on the results of preliminary studies on phenol degradation under mesophilic conditions with a mixed methanogenic culture, we proposed a degradation pathway in which phenol is fermented to acetate: Part of the phenol is reductively transformed to benzoate while the rest is oxidised, forming acetate as end product. According to our calculations, this should result in three moles of phenol being converted to two moles of benzoate and three moles of acetate (3 phenol + 2 CO2 + 3 H2O --> 3 acetate + 2 benzoate): To assess the validity of our hypothesis concerning the metabolic pathway, we studied the transformation of phenol under mesophilic and thermophilic conditions in relation to the availability of hydrogen. Hence, methanogenic meso- and thermophilic cultures amended with phenol were run with or without an added over-pressure of hydrogen under methanogenic and non-methanogenic conditions. Bromoethanesulfonic acid (BES) was used to inhibit methanogenic activity. In the mesophilic treatments amended with only BES, about 70% of the carbon in the products found was benzoate. During the course of phenol transformation in these BES-amended cultures, the formation pattern of the degradation products changed: Initially nearly 90% of the carbon from phenol degradation was recovered as benzoate, whereas later in the incubation, in addition to benzoate formation, the aromatic nucleus degraded completely to acetate. Thus, the initial reduction of phenol to benzoate resulted in a lowering of H2 levels, giving rise to conditions allowing the degradation of phenol to acetate as the end product. Product formation in bottles amended with BES and phenol occurred in accordance with the hypothesised pathway; however, the overall results indicate that the degradation of phenol in this system is more complex. During phenol transformation under thermophilic conditions, no benzoate was observed and no phenol was transformed in the BES-amended cultures. This suggests that the sensitivity of phenol transformation to an elevated partial pressure of H2 is higher under thermophilic conditions than under mesophilic ones. The lack of benzoate formation could have been due to a high turnover of benzoate or to a difference in the phenol degradation pathway between the thermophilic and mesophilic cultures.     

Microbial transformation of styrene by anaerobic consortia

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 Enterobacter 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 Enterobacter cloacae DG-6 (ATCC 35929) which had been isolated previously from the ferulate-degrading consortium, was shown to degrade styrene through to carbon dioxide.     

para-hydroxybenzoate as an intermediate in the anaerobic transformation of phenol to benzoate

Anaerobic phenol transformation was studied using a consortium which transformed phenol to benzoate without complete mineralization of benzoate. Products of monofluorophenol transformation indicated para-carboxylation. Phenol and benzoate were detected during para-hydroxybenzoate (p-OHB) degradation. p-OHB was detected in phenol-transforming cultures containing 6-hydroxynicotinic acid (6-OHNA), a structural analogue of p-OHB, or at elevated initial concentrations of phenol (greater than or equal to 5 mM), or benzoate (greater than or equal to 10 mM).     

Water purification through bioconversion of phenol compounds by tyrosinase and chemical adsorption by chitosan beads

Enzymatic removal of various phenol compounds from artificial wastewater was undertaken by the combined use of mushroom tyrosinase (EC 1.14.18.1) and chitosan beads as function of pH value, temperature, tyrosinase dose, and hydrogen peroxide-to-substrate ratio. Chitosan film incubated in a p-crersol+tyrosinase mixture had the main peaks at 400-470 nm assigned to chemically adsorbed quinone derivatives, which increased over the immersion time. These results indicate that removal of phenol compounds is caused by their tyrosinase-catalyzed oxidation to the corresponding quinone derivatives and the subsequent chemical adsorption on the chitosan film. The optimum conditions for quinone adsorption were determined to be pH 7 and 45 degrees C for p-cresol. Some alkyl-substituted phenol compounds were removed by adsorption of quinone derivatives enzymatically generated on the chitosan beads, and the % removal for p-cresol, 4-ethylphenol, 4-n-propylphenol, 4-n-butylphenol, and p-chlorophenol went up to 93%. In addition, 4-tert-butylphenol underwent tyrosinase-catalyzed oxidation in the presence of hydrogen peroxide. This procedure was applicable to removal of chlorophenols and alkyl-substituted phenols.     

Carboxylation of o-cresol by an anaerobic consortium under methanogenic conditions.

The metabolism of o-cresol under methanogenic conditions by an anaerobic consortium known to carboxylate phenol to benzoate was investigated. After incubation with the consortium at 29 degrees C for 59 days, o-cresol was transformed to 3-methylbenzoic acid, which was not further metabolized by the consortium. Proteose peptone in the culture medium was essential for the transformation of o-cresol. In addition, a transient compound detected in the culture was identified as 4-hydroxy-3-methylbenzoic acid. o-Cresol-6d was transformed by the consortium to deuterated hydroxy-methylbenzoic acid and deuterated methylbenzoic acid. These results demonstrate that o-cresol is carboxylated in the para position relative to the phenolic hydroxyl group and dehydroxylated by the anaerobic consortium.     

Carboxylation of o-cresol by an anaerobic consortium under methanogenic conditions.

The metabolism of o-cresol under methanogenic conditions by an anaerobic consortium known to carboxylate phenol to benzoate was investigated. After incubation with the consortium at 29 degrees C for 59 days, o-cresol was transformed to 3-methylbenzoic acid, which was not further metabolized by the consortium. Proteose peptone in the culture medium was essential for the transformation of o-cresol. In addition, a transient compound detected in the culture was identified as 4-hydroxy-3-methylbenzoic acid. o-Cresol-6d was transformed by the consortium to deuterated hydroxy-methylbenzoic acid and deuterated methylbenzoic acid. These results demonstrate that o-cresol is carboxylated in the para position relative to the phenolic hydroxyl group and dehydroxylated by the anaerobic consortium.     

Microbial transformation of styrene by anaerobic consortia

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 chromatog-raphy (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 Enterobacter 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 Enterobacter cloacae DG-6 (ATCC 35929) which had been isolated previously from the ferulate-degrading consortium, was shown to degrade styrene through to carbon dioxide.     

Removal of chlorophenols from synthetic solutions using Phanerochaete chrysosporium

The potential use of the fungus Phanerochaete chrysosporium to remove chlorophenols (phenol, o-chlorophenol, p-chlorophenol and 2,4,6-trichlorophenol) from aqueous solutions was evaluated. The kinetics of both adsorption and desorption of phenolic compounds was rapid for all adsorbates. The maximum adsorptions of phenol and chlorophenols onto the Phanerochaete chrysosporium were 1.23 mmol/g for phenol, 1.49 mmol/g for o-chlorophenol, 1.78 mmol/g for p-chlorophenol and 2.14 mmol/g for 2,4,6-trichlorophenol. The affinity order was as follows: 2,4,6-trichlorophenol > p-chlorophenol > o-chlorophenol > phenol. Phenol and chlorophenols binding with Phanerochaete chrysosporium were clearly pH dependent. The adsorption of phenol and chlorophenols increased as pH increased. Desorption of phenol or chlorophenols was achieved using methanol solution (30% (v/v)). Phanerochaete chrysosporium is suitable for reuse for more than ten cycles without noticeable loss of adsorption capacity.     

CO2-dependent fermentation of phenol to acetate, butyrate and benzoate by an anaerobic, pasteurised culture

Fermentative degradation of phenol was studied using a non-methanogenic, pasteurised enrichment culture containing two morphologically different bacteria. Phenol was fermented to benzoate, acetate and butyrate and their relative occurrence depended on the concentration of hydrogen. Proportionately more benzoate was formed with high initial levels of H2. The influence of PH2 on the fermentation pattern was studied both in dense cell suspensions and in growing cultures by addition of hydrogen. An increase in growth yield (OD578) was observed, compared to controls, as a consequence of phenol degradation; however, the increase was less in H2-amended treatments, in which most of the phenol ended up as benzoate. The degradation of phenol in the dense cell suspension experiments was dependent on CO2. Benzoate was not degraded when added as a substrate to the growing culture. This is, to our knowledge, the first report concerning the fermentative degradation of phenol to nonaromatic products.     

氯酚类化合物的微生物降解研究进展

综述了近年在具有降解氯酚类化合物能力的微生物的筛选、氯酚类化合物的好氧和厌氧降解机制以及现代生物技术的开发利用研究．阐述了氯酚类化合物在不同条件下的降解路径．在好氧条件下,单氯酚和二氯酚在氧化酶的攻击下形成氯代邻二酚,邻二酚开环生成相应的氯代粘康酸或半醛,粘康酸内酯化过程中释放氯离子;高度氯代的化合物则是在氢氧化酶作用下生成氯代醌,并逐步脱去所有的氯原子生成苯酚后才开环．在厌氧或缺氧条件下,氯酚进行还原脱氯,在得到电子的同时去掉一个氯取代基．     

Thermophilic anaerobic degradation of butyrate by a butyrate-utilizing bacterium in coculture and triculture with methanogenic bacteria

We studied syntrophic butyrate degradation in thermophilic mixed cultures containing a butyrate-degrading bacterium isolated in coculture with Methanobacterium thermoautotrophicum or in triculture with M. thermoautotrophicum and the TAM organism, a thermophilic acetate-utilizing methanogenic bacterium. Butyrate was beta-oxidized to acetate with protons as the electron acceptors. Acetate was used concurrently with its production in the triculture. We found a higher butyrate degradation rate in the triculture, in which both hydrogen and acetate were utilized, than in the coculture, in which acetate accumulated. Yeast extract, rumen fluid, and clarified digestor fluid stimulated butyrate degradation, while the effect of Trypticase was less pronounced. Penicillin G, d-cycloserine, and vancomycin caused complete inhibition of butyrate utilization by the cultures. No growth or degradation of butyrate occurred when 2-bromoethanesulfonic acid or chloroform, specific inhibitors of methanogenic bacteria, was added to the cultures and common electron acceptors such as sulfate, nitrate, and fumarate were not used with butyrate as the electron donor. Addition of hydrogen or oxygen to the gas phase immediately stopped growth and butyrate degradation by the cultures. Butyrate was, however, metabolized at approximately the same rate when hydrogen was removed from the cultures and was metabolized at a reduced rate in the cultures previously exposed to hydrogen.     

Comparison of ligninase-I and peroxidase-M2 from the white-rot fungus Phanerochaete chrysosporium

Ligninase-I (Mr 42,000-43,000; carbohydrate, 21%) and peroxidase-M2 (Mr 45,000-47,000; carbohydrate, 17%), two representative, hydrogen peroxide-dependent extracellular enzymes produced by ligninolytic cultures of the white-rot fungus Phanerochaete chrysosporium BKM-F-1767, were purified and their properties compared. Spectroscopic studies showed that both native enzymes are heme proteins containing protoporphyrin IX. EPR spectroscopy indicated that iron ions are coordinated with the enzymes' prosthetic groups as high-spin ferriheme complexes. We confirmed reports of others that the ligninase-hydrogen peroxide complex (activated enzyme) reverts to its native state on addition of dithionite or one of the enzyme's substrates (e.g., veratryl alcohol); however, we found that the peroxidase-M2-hydrogen peroxide complex required Mn2+ ions to accomplish a similar cycle. The peroxidase oxidized Mn2+ to a higher oxidation state, and the oxidized Mn acted as a diffusible catalyst able to oxidize numerous organic substrates. Unlike ligninase-I which is found free extracellularly, peroxidase-M2 appears to be associated closely with the fungal mycelium. In its peroxidatic reactions, ligninase-I oxidizes a variety of nonphenolic and phenolic lignin model compounds. In the presence of Mn2+, peroxidase-M2 oxidizes numerous phenolic compounds, especially syringyl (3,5-dimethoxy-4-hydroxyphenyl) and vinyl side-chain substituted substrates. Also, the peroxidase-Mn2+ system (without hydrogen peroxide) expresses oxidase activity against NADPH, GSH, dithiothreitol, and dihydroxymaleic acid, forming hydrogen peroxide at the expense of oxygen. Both enzymes were believed to play roles in lignin degradation, and these are discussed.     

Microbial metabolism of 2-chlorophenol, phenol and rho-cresol by Rhodococcus erythropolis M1 in co-culture with Pseudomonas fluorescens P1

Chlorophenolic waste most often contains phenol and rho-cresol along with chlorophenols. A Rhodococcus erythropolis strain M1 was isolated with the ability to degrade 2-chlorophenol, phenol and p-cresol (100 mgl(-1), each) in 18, 24 and 20 h, respectively, with negligible lag. However, Rhodococcus sp. characterized by low growth rate, pose a threat to be outgrown by bacteria occurring in natural habitats. In the present study, interaction of R. erythropolis M1 with another isolated bacteria generally encountered in activated sludge for water treatment like Pseudomonas fluorescens P1 was studied. 2-chlorophenol, phenol and p-cresol were selected as the substrates for the study. Viable cell counts showed competitive interaction between the species on 2-chlorophenol and phenol. Specific growth rate of pure culture of R. erythropolis M1 was higher than P. fluorescens P1 on 2-chlorophenol. However, in mixed culture, P. fluorescens P1 showed higher growth rate. Degradation of phenol showed higher growth rate of R. erythropolis M1 both in pure and in mixed culture form. Degradation of p-cresol had shown similar counts for both populations indicating neutral type of interaction. This observation was substantiated by detecting the growth rate, where both cultures had similar growth rate in pure and in the mixed culture form. Rate of 2-chlorophenol degradation was higher when R. erythropolis M1 was used as the pure culture as compared to the degradation rates observed with the P. fluorescens P1 or with the mixed culture. However, in case of phenol and p-cresol, degradation by the mixed culture had resulted in higher degradation rates as compared to the degradation of the substrates by both the axenic cultures.     

Enhanced biodegradation of phenol by a microbial consortium in a solid–liquid two phase partitioning bioreactor

Two phase partitioning bioreactors (TPPBs) operate by partitioning toxic substrates to or from an aqueous, cell-containing phase by means of second immiscible phase. Uptake of toxic substrates by the second phase effectively reduces their concentration within the aqueous phase to sub-inhibitory levels, and transfer of molecules between the phases to maintain equilibrium results in the continual feeding of substrate based on the metabolic demand of the microorganisms. Conventionally, a single pure species of microorganism, and a pure organic solvent, have been used in TPPBs. The present work has demonstrated the benefits of using a mixed microbial population for the degradation of phenol in a TPPB that uses solid polymer beads (comprised of ethylene vinyl acetate, or EVA) as the second phase. Polymer modification via an increase in vinyl acetate concentration was also shown to increase phenol uptake. Microbial consortia were isolated from three biological sources and, based on an evaluation of their kinetic performance, a superior consortium was chosen that offered improved degradation when compared to a pure strain of Pseudomonas putida ATCC 11172. The new microbial consortium used within a TPPB was capable of degrading high concentrations of phenol (2000mgl^–1), with decreased lag time (10h) and increased specific rate of phenol degradation (0.71g phenolg^–1cellh). Investigation of the four-member consortium showed that it consisted of two Pseudomonas sp., and two Acinetobacter sp., and tests conducted upon the individual isolates, as well as paired organisms, confirmed the synergistic benefit of their existence within the consortium. The enhanced effects of the use of a microbial consortium now offer improved degradation of phenol, and open the possibility of the degradation of multiple toxic substrates via a polymer-mediated TPPB system.     

Pentachlorophenol Dechlorination in Fluidized-Bed Reactors under Methanogenic Conditions

The reductive dechlorination of chlorophenols was studied in three fluidized-bed reactors (FBRs) with respect to enrichment, pathways, complete dechlorination, and overall performance. The methanogenic consortia, developed by previous researchers in our laboratory, have been further enriched by reducing the ratio of substrate to pentachlorophenol (PCP) and increasing the PCP loading. The performance of the consortia was improved, and complete dechlorination at high PCP loading rates was observed, reaching a PCP loading of 1227 µmol/L d with 99% chlorophenol removal. The dechlorination rates in the reactors for chlorophenol (CP) congeners were obtained and were used to evaluate the performance of the three consortia and to quantitatively estimate the fates of these chlorophenols in the reactors. The consortium with the best performance was further investigated in bottle tests by treatment with heat and metabolic inhibitors to examine chlorophenol degradation and to characterize the CP degraders. The degradation of all monochlorophenols was completely inhibited after heat treatment, but the degradation of all other tested chlorophenols was hardly affected by heat treatment, indicating that spore-forming bacteria likely were involved in dechlorination. Addition of sulfate negatively affected CP degradation, but addition of molybdate reduced the effect of sulfate. Tests with 2-bromoethanesulfonic acid and vancomycin indicated that bacteria were responsible for chlorophenol degradation in the consortium.     

Degradation of Monochlorinated and Nonchlorinated Aromatic Compounds under Iron-Reducing Conditions

The capacity for Fe(sup3+) to serve as an electron acceptor in the microbial degradation of monochlorinated and nonchlorinated aromatic compounds was investigated in anoxic sediment enrichments. The substrates tested included phenol, benzoate, aniline, their respective monochlorinated isomers, o-, m-, and p-cresol, and all six dimethylphenol isomers. Phenol and 2-, 3-, and 4-chlorophenol were utilized by anaerobic microorganisms, with the concomitant reduction of Fe(sup3+) to Fe(sup2+). The amount of Fe(sup2+) produced in the enrichments was 89 to 138% of that expected for the stoichiometric degradation of these substrates to CO(inf2), suggesting complete mineralization at the expense of Fe reduction. Under Fe-reducing conditions, there was initial loss of benzoate and 3-chlorobenzoate but not of 2- or 4-chlorobenzoate. In addition, there was initial microbial utilization of aniline but not of the chloroaniline isomers. There was also initial loss of o-, m-, and p-cresol in our enrichments. None of the dimethylphenol isomers, however, was degraded within 300 days. Furthermore, we tested the capacity of an Fe-reducing, benzoate-grown culture of Geobacter metallireducens GS-15 to utilize monochlorinated benzoates and phenols. G. metallireducens was able to degrade benzoate and phenol but none of their chlorinated isomers, suggesting that the degradation of chlorophenols in our sediment enrichments may be due to novel Fe-reducing organisms that have yet to be isolated.     

Fluorophenols and 3-fluorobenzoate in phenol-degrading methanogenic cultures

3-Fluorobenzoate and all three isomers of fluorophenol were used as analogues and inhibitors of phenol degradation in a methanogenic consortium. 3-Fluorobenzoate was not transformed by phenol-degrading cultures, but it facilitated the detection of the formation of 4-hydroxybenzoate and benzoate from phenol. The effects of the fluorophenols depended on their concentration in the cultures. When added at 0.90 mM, all fluorophenols prevented phenol transformation. At concentrations of 0.45 to 1.8 mM, 2-fluorophenol was transformed to 3-fluoro-4-hydroxybenzoate which accumulated in the medium. When both 2-fluorophenol and phenol were added to cultures at concentrations of 1 mM each, 3-fluoro-4-hydroxybenzoate, 4-hydroxybenzoate, 3-fluorobenzoate and benzoate were detected. 4-Fluorophenol was never transformed, and when it was present at 0.22 mM, it had no effect on phenol degradation. At concentrations 0.09 mM, 2-fluorophenol was mineralized by the phenol-degrading cultures to methane, carbon dioxide, and fluoride. The release of fluoride was also observed from 3-fluorophenol when it was initially present at 0.09 mM. These results support the proposed pathway for phenol degradation involving an initial para-carboxylation to 4-hydroxybenzoate followed by dehydroxylation to benzoate and further metabolism to carbon dioxide and methane. They also demonstrate defluorination of 2- and 3-fluorophenols under methanogenic conditions.