High rate treatment of terephthalic acid production wastewater in a two-stage anaerobic bioreactor

The feasibility was studied of anaerobic treatment of wastewater generated during purified terephthalic acid (PTA) production in two-stage upflow anaerobic sludge blanket (UASB) reactor system. The artificial influent of the system contained the main organic substrates of PTA-wastewater: acetate, benzoate, and terephthalate. Three parallel operated reactors were used for the second stage, and seeded with a suspended terephthalate degrading culture, with and without additional methanogenic granular sludge (two different types). The first stage UASB-reactor was seeded with methanogenic granular sludge. Reactors were operated at 37 degrees C and pH 7. During the first 300 days of operation a clear distinction between the biomass grown in both reactor stages was obtained. In the first stage, acetate and benzoate were degraded at a volumetric loading rate of 40 g-COD/L . day at a COD-removal efficiency of 95% within the first 25 days of operation. No degradation of terephthalate was obtained in the first stage during the first 300 days of operation despite operation of the reactor at a decreased volumetric loading rate with acetate and benzoate of 9 g-COD/L . day from day 150. Batch incubation of biomass from the reactor with terephthalate showed that the lag-phase prior to terephthalate degradation remained largely unchanged, indicating that no net growth of terephthalate degrading biomass occurred in the first stage reactor. From day 300, however, terephthalate degradation was observed in the first stage, and the biomass in this reactor could successfully be enriched with terephthalate degrading biomass, resulting in terephthalate removal capacities of 15 g-COD/L . day. Even though no single reason could be identified why (suddenly) terephthalate degradation was obtained after such a long period of operation, it is suggested that the solid retention time as well the prevailing reactor concentrations acetate and benzoate may have played an important role. From day 1 of operation, terephthalate was degraded in the second stage. In presence of methanogenic granular biomass, high terephthalate removal capacities were obtained in these reactors (15 g-COD/L . day) after approximately 125 days of operation. From the results obtained it is concluded that terephthalate degradation is the bottleneck during anaerobic treatment of PTA-wastewater. Pre-removal of acetate and benzoate in staged bioreactor reduces the lag-phase prior to terephthalate degradation in latter stages, and enables high rate treatment of PTA-wastewater.     

Identification and isolation of anaerobic, syntrophic phthalate isomer-degrading microbes from methanogenic sludges treating wastewater from terephthalate manufacturing

The microbial populations responsible for anaerobic degradation of phthalate isomers were investigated by enrichment and isolation of those microbes from anaerobic sludge treating wastewater from the manufacturing of terephthalic acid. Primary enrichments were made with each of three phthalate isomers (ortho-, iso-, and terephthalate) as the sole energy source at 37 degrees C with two sources of anaerobic sludge (both had been used to treat wastewater containing high concentrations of phthalate isomers) as the inoculum. Six methanogenic enrichment cultures were obtained which not only degraded the isomer used for the enrichment but also had the potential to degrade part of other phthalate isomers as well as benzoate with concomitant production of methane, presumably involving strictly syntrophic substrate degradation. Our 16S rRNA gene-cloning analysis combined with fluorescence in situ hybridization revealed that the predominant bacteria in the enrichment cultures were affiliated with a recently recognized non-sulfate-reducing subcluster (subcluster Ih) in the group 'Desulfotomaculum lineage I' or a clone cluster (group TA) in the class delta-PROTEOBACTERIA: Several attempts were made to isolate these microbes, resulting in the isolation of a terephthalate-degrading bacterium, designated strain JT, in pure culture. A coculture of the strain with the hydrogenotrophic methanogen Methanospirillum hungatei converted terephthalate to acetate and methane within 3 months of incubation, whereas strain JT could not degrade terephthalate in pure culture. During the degradation of terephthalate, a small amount of benzoate was transiently accumulated as an intermediate, indicative of decarboxylation of terephthalate to benzoate as the initial step of the degradation. 16S rRNA gene sequence analysis revealed that the strain was a member of subcluster Ih of the group 'Desulfotomaculum lineage I', but it was only distantly related to other known species.     

Interspecies Acetate Transfer Influences the Extent of Anaerobic Benzoate Degradation by Syntrophic Consortia

Benzoate degradation by an anaerobic, syntrophic bacterium, strain SB, in coculture with Desulfovibrio sp. strain G-11 reached a threshold value which depended on the amount of acetate added and ranged from about 2.5 to 29.9 (mu)M. Increasing acetate concentrations also uncompetitively inhibited benzoate degradation. The apparent V(infmax) and apparent K(infm) for benzoate degradation decreased with increasing acetate concentration, but the benzoate degradation capacities (V(infmax)/K(infm)) of cell suspensions remained comparable. The addition of an acetate-using bacterium to cocultures after the threshold was reached resulted in the degradation of benzoate to below the detection limit. Mathematical simulations showed that the benzoate threshold was not predicted by the inhibitory effect of acetate on benzoate degradation kinetics. With nitrate instead of sulfate as the terminal electron acceptor, no benzoate threshold was observed in the presence of 20 mM acetate even though the kinetics of benzoate degradation were slower with nitrate rather than sulfate as the electron acceptor. When strain SB was grown with Desulfovibrio sp. strain DG2 that had a fourfold-lower V(infmax) for hydrogen use than strain G-11, the V(infmax) for benzoate degradation was 37-fold lower than that of strain SB-G-11 cocultures. The Gibb's free energy for benzoate degradation was less negative in cell suspensions with a threshold than in suspensions without a threshold. These studies showed that the threshold was not a function of the inhibition of benzoate degradation by acetate or the toxicity of the undissociated form of acetate. Rather, a critical or minimal Gibb's free energy may exist where thermodynamic constraints preclude further benzoate degradation.     

Evidence for anaerobic syntrophic benzoate degradation threshold and isolation of the syntrophic benzoate degrader.

An anaerobic, motile, gram-negative, rod-shaped, syntrophic, benzoate-degrading bacterium, strain SB, was isolated in pure culture with crotonate as the energy source. Benzoate was degraded only in association with an H2-using bacterium. The kinetics of benzoate degradation by cell suspensions of strain SB in coculture with Desulfovibrio strain G-11 was studied by using progress curve analysis. The coculture degraded benzoate to a threshold concentration of 214 nM to 6.5 microM, with no further benzoate degradation observed even after extended incubation times. The value of the threshold depended on the amount of benzoate added and, consequently, the amount of acetate produced. The addition of sodium acetate, but not that of sodium chloride, affected the threshold value; higher acetate concentrations resulted in higher threshold values for benzoate. When a cell suspension that had reached a threshold benzoate concentration was reamended with benzoate, benzoate was used without a lag. The hydrogen partial pressure was very low and formate was not detected in cell suspensions that had degraded benzoate to a threshold value. The Gibbs free energy change calculations showed that the degradation of benzoate was favorable when the threshold was reached. These studies showed that the threshold for benzoate degradation was not caused by nutritional limitations, the loss of metabolic activity, or inhibition by hydrogen or formate. The data are consistent with a thermodynamic explanation for the existence of a threshold, but a kinetic explanation based on acetate inhibition may also account for the existence of a threshold.     

Influences of organic loading disturbances on the performance of anaerobic filter process to treat purified terephthalic acid wastewater

A lab-scale anaerobic filter process was operated for the treatment of purified terephthalic acid (PTA) wastewater, and the influences of organic loading disturbances on the process performance were investigated. After about 15 month operation, the COD removal efficiency was maintained at 79% under the volumetric loading rate of 5.05 kg-COD/m³/d and the hydraulic retention time (HRT) of 50 h. Interestingly, this performance could be further enhanced over 85% by applying a step-increase/decrease of the HRT, which was mainly due to the increased p-toluate degradation. In the shock loading tests of four major pollutants (benzoate, acetate, terephthalate and p-toluate), it was found that the overall process performance was adversely affected by all the shock loadings, indicating that the syntrophic microbial consortium involved in the PTA wastewater treatment is highly sensitive to the organic loading disturbances. The complex inhibition effects of the benzoate and acetate on the terephthalate and p-toluate degradations were mainly responsible for this sensitivity.     

Benzoate fermentation by the anaerobic bacterium Syntrophus aciditrophicus in the absence of hydrogen-using microorganisms.

The anaerobic bacterium Syntrophus aciditrophicus metabolized benzoate in pure culture in the absence of hydrogen-utilizing partners or terminal electron acceptors. The pure culture of S. aciditrophicus produced approximately 0.5 mol of cyclohexane carboxylate and 1.5 mol of acetate per mol of benzoate, while a coculture of S. aciditrophicus with the hydrogen-using methanogen Methanospirillum hungatei produced 3 mol of acetate and 0.75 mol of methane per mol of benzoate. The growth yield of the S. aciditrophicus pure culture was 6.9 g (dry weight) per mol of benzoate metabolized, whereas the growth yield of the S. aciditrophicus-M. hungatei coculture was 11.8 g (dry weight) per mol of benzoate. Cyclohexane carboxylate was metabolized by S. aciditrophicus only in a coculture with a hydrogen user and was not metabolized by S. aciditrophicus pure cultures. Cyclohex-1-ene carboxylate was incompletely degraded by S. aciditrophicus pure cultures until a free energy change (DeltaG') of -9.2 kJ/mol was reached (-4.7 kJ/mol for the hydrogen-producing reaction). Cyclohex-1-ene carboxylate, pimelate, and glutarate transiently accumulated at micromolar levels during growth of an S. aciditrophicus pure culture with benzoate. High hydrogen (10.1 kPa) and acetate (60 mM) levels inhibited benzoate metabolism by S. aciditrophicus pure cultures. These results suggest that benzoate fermentation by S. aciditrophicus in the absence of hydrogen users proceeds via a dismutation reaction in which the reducing equivalents produced during oxidation of one benzoate molecule to acetate and carbon dioxide are used to reduce another benzoate molecule to cyclohexane carboxylate, which is not metabolized further. Benzoate fermentation to acetate, CO(2), and cyclohexane carboxylate is thermodynamically favorable and can proceed at free energy values more positive than -20 kJ/mol, the postulated minimum free energy value for substrate metabolism.     

Production of aromatic compounds during methanogenic degradation of straw in rice field soil

Production of CH(4) in anoxic rice field soil is stimulated by the addition of rice straw. Previous experiments showed that acetate and propionate are the most important intermediates of the carbon flow to CH(4), and accumulate if CH(4) production is inhibited by 2-bromoethanesulfonate (BES). However, some unidentified compounds were found to accumulate in addition. We now identified them as benzoate, phenylpropionate, and phenylacetate by comparison of the retention times in HPLC chromatograms with authentic standards and by mass spectrometry. These aromatic compounds accumulated only to concentrations <100 microM, especially in soil amended with rice straw (stem, sheath or blade straw). Phenylpropionate and benzoate were the most abundant aromatic intermediates contributing up to 4% to total CH(4) production. Phenylacetate, on the other hand, contributed very little (<0.3%). Gibbs free energies (DeltaG) were calculated for different anaerobic degradation pathways of the aromatic compounds at the actual incubation conditions. Conversion of benzoate to acetate, CO(2) and H(2) was strongly exergonic (DeltaG = -86 kJ mol(-1)) under methanogenic conditions, but became less exergonic (DeltaG = -30 kJ mol(-1)) when CH(4) production was inhibited. The primary oxidation of phenylpropionate was only exergonic for alpha-oxidation (i.e. phenylacetate as product) but not for beta-oxidation (i.e. benzoate as product). However, the DeltaG values for the complete degradation of phenylpropionate to acetate, CO(2) and H(2) were similar for both pathways and were also similar to those of benzoate degradation. Collectively, the results suggest that aromatic compounds are minor intermediates of anaerobic degradation of organic matter in rice field soil, and are syntrophically degraded by coupling to methanogenesis.     

Kinetics of chlorophenol degradation by benzoate-induced culture of Rhodococcus erythropolis M1

A pure culture of Rhodococcus erythropolis was isolated with the ability to degrade 2-chlorophenol, 4-chlorophenol and 2,4-dichlorophenol. Degradation of 2-chlorophenol by the uninduced culture of Rhodococcus erythropolis began after a prolonged lag period and complete mineralization of the substrates took 45 days. With the aim of reducing the lag period and subsequently improving the rate of degradation, the cells of the isolate were induced with benzoate, phenol, toluene and catechol individually. Benzoate-induced cells showed the highest rate of degradation and were thus used for the study of the degradation kinetics of 2-chlorophenol, 4-chlorophenol and 2,4-dichlorophenol. Complete mineralization of these substrates was achieved up to a concentration of 300, 100 and 50 mg l^–1 respectively. Degradation of the chlorophenols was initiated without any significant lag and took the remarkably short time periods of 84, 64 and 144 h for the highest concentrations of the substrate. Evaluation of kinetic parameters showed chlorophenol degradation to follow substrate inhibition kinetics. This is evident from the decrease in specific growth rate, growth yield and substrate uptake rate with increase in the initial substrate concentrations. Toxicity of the chlorophenols was observed to depend on the position of chlorine on the benzene ring and the degree of chlorination.     

Inhibition of benzene,toluene, phenol and benzoate in single and combination on Anammox activity: implication to the denitrification–Anammox synergy

A previous study demonstrated that denitrification synergized with Anammox could accelerate the anaerobic degradation of benzene. The inhibitory effects of benzene, toluene, phenol and benzoate in single and combination on Anammox activity were investigated by short-term batch tests. The results indicated that the inhibition of single compounds on Anammox could be well fitted with the extended non-competitive and Luong inhibition kinetic models. The inhibitions of the individual compound were in order as follows: benzene?>?toluene?>?phenol?>?benzoate. The joint inhibitions of bi-component mixtures of benzene with toluene, benzene with phenol and benzene with benzoate on Anammox activity were additive; the joint inhibition of a tri-component mixture (benzene, toluene and phenol) was partly additive; and the joint inhibition of a multicomponent mixture (benzene, toluene, phenol and benzoate) was synergistic. The effect of benzoate on the denitrification–Anammox synergy for benzene degradation was evaluated using a long-term test. Although the average rate of benzene degradation decreased by 13% with the addition of 10 mg L^?1 benzoate, the average rate of NO₃^? and NH₄⁺ increased by approximately 1- and 0.56-fold, respectively, suggesting that benzoate favors the stability of the denitrification–Anammox synergy. The carboxylation of benzene would be a more favorable pathway for the anaerobic degradation of benzene under denitrification synergized with Anammox.     

Degradation of cinnamate via beta-oxidation to benzoate by a defined,syntrophic consortium of anaerobic bacteria

A syntrophic consortium was enriched in a basal medium containing cinnamate as the carbon and energy source. It was found to consist of three morphologically distinct microbes, viz., a short, rod-shaped, non-motile bacterium with distinctly pointed ends, Papillibacter cinnamivorans; a rod-shaped, motile bacterium with rounded ends, Syntrophus sp.; and a methanoarchaeon, Methanobacterium sp. This methanogen was then replaced by a collection strain of Methanobacterium formicicum. A syntrophic interdependency of the three partners of the consortium was observed during growth on cinnamate. In the presence of bromoethanesulfonic acid (BESA), cinnamate was transformed to benzoate, whereas under methanogenic conditions without BESA, cinnamate was first transformed to benzoate via beta-oxidation and subsequently completely degraded into acetate, CH(4), and CO(2). Papillibacter cinnamivorans was responsible for benzoate production from cinnamate, whereas a syntrophic association between Syntrophus sp. and the methanogen degraded benzoate to acetate, CH(4), and CO(2). A new anaerobic degradation pathway of cinnamate into benzoate via beta-oxidation by a pure culture of P. cinnamivorans is proposed.     

Two distinct pathways for anaerobic degradation of aromatic compounds in the denitrifying bacterium Thauera aromatica strain AR-1

Denitrifying bacteria degrade many different aromatic compounds anaerobically via the well-described benzoyl-CoA pathway. We have shown recently that the denitrifiers Azoarcus anaerobius and Thauera aromatica strain AR-1 use a different pathway for anaerobic degradation of resorcinol (1,3-dihydroxybenzene) and 3,5-dihydroxybenzoate, respectively. Both substrates are converted to hydroxyhydroquinone (1,2,4-trihydroxybenzene). In the membrane fraction of T. aromatica strain AR-1 cells grown with 3,5-dihydroxybenzoate, a hydroxyhydroquinone-dehydrogenating activity of 74 nmol min(-1)(mg protein)-1 was found. This activity was significantly lower in benzoate-grown cells. Benzoate-grown cells were not induced for degradation of 3,5-dihydroxybenzoate, and cells grown with 3,5-dihydroxybenzoate degraded benzoate only at a very low rate. With a substrate mixture of benzoate plus 3,5-dihydroxybenzoate, the cells showed diauxic growth. Benzoate was degraded first, while complete degradation of 3,5-dihydroxybenzoate occurred only after a long lag phase. The 3,5-dihydroxybenzoate-oxidizing and the hydroxyhydroquinone-dehydrogenating activities were fully induced only during 3,5-dihydroxybenzoate degradation. Synthesis of benzoyl-CoA reductase appeared to be significantly lower in 3,5-dihydroxybenzoate-grown cells as shown by immunoblotting. These results confirm that T. aromatica strain AR-1 harbors, in addition to the benzoyl-CoA pathway, a second, mechanistically distinct pathway for anaerobic degradation of aromatic compounds. This pathway is inducible and subject to catabolite repression by benzoate.     

Methane Fermentation of Ferulate and Benzoate: Anaerobic Degradation Pathways

The anaerobic biodegradation of ferulate and benzoate in stabilized methanogenic consortia was examined in detail. Up to 99% of the ferulate and 98% of the benzoate were converted to carbon dioxide and methane. Methanogenesis was inhibited with 2-bromoethanesulfonic acid, which reduced the gas production and enhanced the buildup of intermediates. Use of high-performance liquid chromatography and two gas chromatographic procedures yielded identification of the following compounds: caffeate, p-hydroxycinnamate, cinnamate, phenylpropionate, phenylacetate, benzoate, and toluene during ferulate degradation; and benzene, cyclohexane, methylcyclohexane, cyclohexanecarboxylate, cyclohexanone, 1-methylcyclohexanone, pimelate, adipate, succinate, lactate, heptanoate, caproate, isocaproate, valerate, butyrate, isobutyrate, propionate, and acetate during the degradation of either benzoate or ferulate. Based on the identification of the above compounds, more complete reductive pathways for ferulate and benzoate are proposed.     

Anaerobic degradation of benzoate to methane by a microbial consortium

A stabilized consortium of microbes which anaerobically degraded benzoate and produced CH₄ was established by inoculation of a benzoate-mineral salts medium with sewage sludge; the consortium was routinely subcultured anaerobically in this medium for 3 years. Acetate, formate, H₂ and CO₂ were identified as intermediates in the overall conversion of benzoate to CH₄ by the culture. Radioactivity was equally divided between the CH₄ and CO₂ from the degradation of uniformly ring-labeled [¹⁴C]benzoate. The methyl group of acetate was stoichiometrically converted to CH₄. Acetate, cyclohexanecarboxylate, 2-hydroxycyclohexanecarboxylate, o-hydroxybenzoic acid and pimelic acid were converted to CH₄ without a lag suggesting that benzoate was degraded by a reductive pathway. Addition of o-chlorobenzoate inhibited benzoate degradation but not acetate degradation or methane formation. Two methanogenic organisms were isolated from the mixed culture, neither organism was able to degrade benzoate, showing that the methanogenic bacteria served as terminal organisms of a metabolic food chain composed of several organisms. Removal of intermediates by the methanogenic bacteria provided thermodynamically favorable conditions for benzoate degradation.     

Acetoclastic methanogenic activity measurement by a titration bioassay.

A titration bioassay, designed to accurately determine the activity of acetoclastic methanogens, is described that also allows evaluation of inhibition due to potential toxicants on the active biomass. The instrument is made of a pH-stat connected to an anaerobic batch reactor. Acetate is blended and mixed with anaerobic sludge in the reactor where a 1:1 N2 and CO2 mixture is sparged at the beginning of each test. As the acetoclastic methanogens consume acetate, the pH increase, and the titration unit adds acetic acid and keeps the pH constant. The rate of titrant addition is directly proportional to the methanogenic activity. A very useful feature of the system is its potential to operate for long periods (days) at constant pH and substrate (acetate) concentration. The theoretical background and principle of operation are described as well as some of the practical problems encountered with the use of the instrument. Estimation of kinetic constants for an anaerobic culture according to the Michaelis-Menten model is presented. Examples of inhibition by inorganics (NaCl) and chlorinated solvents (chloroform) are also given.     

Identification of intermediates formed during anaerobic benzene degradation by an iron-reducing enrichment culture

Anaerobic benzene degradation is an important process in contaminated aquifers but is poorly understood due to the scarcity of microbial cultures for study. We have enriched a ferric iron-reducing culture that completely mineralizes benzene to CO₂. With ¹³C₆-labelled benzene as the growth substrate, ring-labelled benzoate was identified as a major intermediate by liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis of culture supernatants. With increasing incubation time, ¹³C₇-labelled benzoate appeared, indicating that the carboxyl group of benzoate derived from CO₂ that was produced from mineralization of labelled benzene. This was confirmed by growing the culture in ¹³C-bicarbonate-buffered medium with unlabelled benzene as the substrate, as the label appeared in the carboxyl group of benzoate produced. Phenol was also identified as an intermediate at high concentration. However, it was clearly shown that phenol was formed abiotically by autoxidation of benzene during the sampling and analysis procedure as a result of exposure to air. The results suggest that, in our culture, anaerobic benzene degradation proceeds via carboxylation and that caution should be exercised in interpreting hydroxylated benzene derivatives as metabolic intermediates of anaerobic benzene degradation.     

Tetrachloroethylene as electron acceptor for the anaerobic degradation of benzoate

Abstract Teltrachloroethylene (PCE) was biotransformed by reductive dehalgenation under anoxic conditions with benzoate as the electron donor. The experiments were carried out under batch culture conditions with biomass from an anoxic fixed bed reactor fed with benzoate and PCE. Inhibition of methanogenesis by bromoethane-sulfonic acid (BES) resulted in a complete inhibition of benzoate degradation. Benzoate, however, was decomposed in the presence of BES if PCE was added to the cultures. With 2.8 mmol/1 PCE, that was transformed to 1.4 mmol/1 cis-1,2-dichloroethylene (DCE) and 3.8 mmol/1 chloride, 2 mmol/1 benzoate were degraded to about 3.2 mmol/1 acetate. The elimination of benzoate was directly proportional to DCE accumulation, ranging between 1:0.5 and 1:1.     

Effects of acetate, propionate, and butyrate on the thermophilic anaerobic degradation of propionate by methanogenic sludge and defined cultures.

The effects of acetate, propionate, and butyrate on the anaerobic thermophilic conversion of propionate by methanogenic sludge and by enriched propionate-oxidizing bacteria in syntrophy with Methanobacterium thermoautotrophicum delta H were studied. The methanogenic sludge was cultivated in an upflow anaerobic sludge bed (UASB) reactor fed with propionate (35 mM) as the sole substrate for a period of 80 days. Propionate degradation was shown to be severely inhibited by the addition of 50 mM acetate to the influent of the UASB reactor. The inhibitory effect remained even when the acetate concentration in the effluent was below the level of detection. Recovery of propionate oxidation occurred only when acetate was omitted from the influent medium. Propionate degradation by the methanogenic sludge in the UASB reactor was not affected by the addition of an equimolar concentration (35 mM) of butyrate to the influent. However, butyrate had a strong inhibitory effect on the growth of the propionate-oxidizing enrichment culture. In that case, the conversion of propionate was almost completely inhibited at a butyrate concentration of 10 mM. However, addition of a butyrate-oxidizing enrichment culture abolished the inhibitory effect, and propionate oxidation was even stimulated. All experiments were conducted at pH 7.0 to 7.7. The thermophilic syntrophic culture showed a sensitivity to acetate and propionate similar to that of mesophilic cultures described in the literature. Additions of butyrate or acetate to the propionate medium had no effect on the hydrogen partial pressure in the biogas of an UASB reactor, nor was the hydrogen partial pressure in propionate-degrading cultures affected by the two acids.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acetate Inhibition of Methanogenic, Syntrophic Benzoate Degradation

Acetate inhibited benzoate degradation by a syntrophic coculture of an anaerobic benzoate degrader (strain BZ-2) and Methanospirillum strain PM-1; the apparent K_i for acetate was approximately 40 mM. The addition of acetate resulted in a decrease in the hydrogen concentration in the coculture, indicating that phenomena related to interspecies hydrogen transfer affected this value and that the effect of acetate on the benzoate-degrading partner was probably greater than the apparent K_i for the coculture suggests.     

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.