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Summary A new thermophilic, coccoid methanogen from a potato waste digester, Methanogenium thermophilicum strain Ratisbona is described. Single cells possess one flagellum and several pili. The optimum growth temperature is 58 °C and the minimum doubling time 3.4 h. Methane is formed from H 2 : CO 2 or formate. The G+C content of the DNA is 57 mol %. The new isolate is closely related to Methanogenium thermophilicum DSM 2373 and the H 2 -oxidizing methanogen of Ferguson and Mah (1983) , which we assign to Methanogenium thermophilicum as strain Los Angeles. The species description of Methanogenium thermophilicum is emended.

Summary A new rod-shaped, Gram-positive, autotrophic, thermophilic, tungsten-requiring methanogen was isolated from enrichment cultures prepared from a mixture of sewage sludge and river sediment. The organism uses H2 + C02 for growth and methane production, but not formate, acetate, methanol, dimethylamine and trimethylamine. Its DNA contains 61 ± 1 mol % G+C. Due to the presence of pseudomurein, it is grouped into the order Methanobacteriales. With respect to its temperature optimum and its ability to grow autotrophically on H2+C02 it resembles Methanobacterium tbermoautotropbicum. However, the G+C content of its DNA is much higher, and it requires tungsten for growth. Therefore, it is considered as a new species of the genus Methanobacterium and is named Methanobacterium wolfei sp. nov., in honour of R.S. Wolfe, who pioneered the research on methanogenesis.

The present knowledge of the microbiology, physiology and regulation of anaerobic digestion in conventional or advanced processes is reviewed. In all systems the carbon flow from biopolymers to biogas is determined by syntrophic interactions of fermentative or acetogenic bacteria with methanogens at the level of interspecies hydrogen transfer. Inhibitors or heavy metal ions may interfere at different levels. The stabilization of waste at mesophilic and thermophilic temperatures is compared and the process stability as well as the inactivation of pathogens is discussed. Characteristics of conventional digestion systems and of recently developed advanced processes with solids and liquids uncoupling are compared and selection criteria with respect to the type of sludge are outlined. Areas of future research for a better understanding of the biochemistry, the physiology and the regulation of the degradation of pollutants are suggested.

Out of 22 methanogens Methanobacterium formicicum, Methanobacterium bryantii M.o.H., Methanogenium marisnigri, Methanomicrobium paynteri, Methanocorpusculum parvum and the new coccoid methanogenic isolates GKZPZ and SZSXXZ were found to grow at the expense of 2-propanol and 2-butanol + CO2. 2-Propanol was oxidized to acetone and 2-butanol was converted to 2-butanone during CO2-reduction to methane. Growth was poor compared to that on H2/CO2, and in the presence of both, 2-propanol and H2, molecular hydrogen was the preferred reductant. Acetone, formed during oxidation of 2-propanol in the absence of hydrogen, was reduced again to 2-propanol, when the culture was supplied with H2/CO2. Ethanol, 1-propanol, 1-butanol, 2-pentanol and cyclohexanol could not serve as hydrogen donors for methanogenesis.

Summary The effect of aphidicolin on growing cultures of Methanococcus vannielii, Methanogenium tatii, Methanogenium marisnigri, Methanobacterium formicicum, Methanobacterium wolfei, Methanosarcina barken strains MS and Naples and Methanospirillum hungatei was tested. While growth of Mc. vannielii, M. wolfei, Mg. tatii, Mg. marisnigri and Ms. barkeri strain Naples (coccoid form) was completely inhibited by ≦ 20 μg aphidicolin per ml, as was the eucaryote Soja mandarin, M. formicicum, Msp. hungatei and Ms. barkeri strain MS were less sensitive. In Mc. vannielii, incorporation of 3H-dTTP into DNA is prevented by aphidicolin, suggesting a similar mode of action as in eucaryotes. In cell-free crude extracts of methanogenic bacteria and of the eucaryote Physarum polycephalum, inhibition of DNA-synthesis was found in the presence of aphidicolin and ara-CTP, while E. coli extracts were insensitive. N-ethylmaleimide inhibited DNA-synthesis in Mc. vannielii extracts, but not in extracts of M. formicicum or M. wolfei. The DNA-polymerase of Mc. vannielii was purified to homogeneity. A protein and activity band with a molecular weight of 108 kD was detected in polyacrylamide gels. In the absence of salt the enzyme had a tendency to form complexes or aggregates of up to 1600 kD. Its activity was stimulated by NaCl (20–200 mmol/l), KCl (60–200 mmol/l) and spermidine (2–20 mmol/l); maximum activity was obtained at a pH around 8.0. Two well-known inhibitors of the α-polymerase of eucaryotes, aphidicolin and butylphenyl-dGTP, inhibit the DNA-polymerase of Mc. vannielii in pure enzyme preparations. This is regarded as evidence for the presence of DNA-polymerases type α in methanogens and is another example of an eucaryotic feature within this group of archaebacteria.

The mode of action of aphidicolin on DNA synthesis catalysed by the DNA polymerase of Methanococcus vannielii is competitive for dCTP, noncompetitive for dATP, dGTP and dTTP and uncompetitive for activated DNA. The kinetic data are accounted for by a mechanism in which dCTP and aphidicolin compete for the dCTPspecific binding site on the DNA polymerase. The dissociation constant for the aphidicolin - DNA-polymerase complex is 0.04-0.07 pM. Similar modes of inhibition of DNA synthesis exist for DNA polymerase a of higher eucaryotes but not for eubacteria or viruses and suggests a close functional relationship between the DNA polymerase of eucaryotes and of the archaebacterium M. vannielii. Aphidicolin is a specific inhibitor of animal a DNA polymerases, dike DNA polymerases of plants and yeasts, some animal virus-encoded DNA polymerases [l, 21 and of DNA polymerases of some archaebacteria [3 - 61, but not of eubacteria [2]. The mode of inhibition of DNA synthesis by aphidicolin was investigated with the DNA polymerase of sea-urchin [7] and herpes simplex virus [8, 9). In sea-urchin aphidicolin inhibits the incorporation of dCTP competitively and of dATP, dGTP and dTTP noncompetitively [7], while the mode of action on the DNA polymerase of herpes simplex virus is competitive with dATP, dCTP and dTTP and mixed with dGTP [8], which conflicts with results published earlier [9]. This is the first report on the mode of action of aphidicolin on the DNA polymerase of an archaebacterium, the methanogen Methanococcus vannielii.

The fermentation of isoleucine, arginine and isoleucine + arginine by pure and syntrophic cultures of Clostridium sporogenes was investigated. Growth of C. sporogenes on isoleucine, if any, was poor, but some isoleucine was fermented to 2-methylbutyrate and hydrogen. In syntrophic cultures with Methanobacterium formicicum or Methanosarcina barkeri growth was better, and isoleucine was completely fermented, the hydrogen being used for methane production. Pure cultures of C. sporogenes grew on arginine and produced 5-aminovalerate, ornithine and acetate. The reducing equivalents for 5-aminovalerate production from intermediarily formed proline were provided by oxidative conversion of arginine to acetate and by oxidative metabolism of some amino acids present in the yeast extract. However, when isoleucine was available together with arginine in syntrophic cultures of C. sporogenes and M. formicicum, the reducing equivalents for arginine fermentation came mainly from the oxidation of isoleucine (Stickland reaction), and the hydrogen produced in excess served for the reduction of CO2 to methane.

A new coccoid methanogen, Methanogenium tatii, was isolated and characterized. The mesophilic isolate can grow on and produce methane from H2:CO2 and formate. For growth acetate is strictly required. The cell shape, the G+C content of 54 mol% and DNA-DNA homology data suggest it to be a Methanogenium species.

Summary A new, Gram-negative, strictly anaerobic, non-sporeforming, thermophilic, motile rod-shaped organism was enriched from sewage sludge and isolated from a continuous culture. The new isolate grew best at temperatures between 70–73°C and at a pH of 6.5–7.0. It required yeast extract for growth and fermented a variety of hexoses and pentoses. Glucose was fermented via the Emden-Meyerhof-Parnas pathway with the formation of lactate, acetate, ethanol, C02 and H2. Pentoses were fermented to acetate, ethanol, CO 2 and little lactate. DNA-DNA hybridization revealed 64% homology with Acetomicrobium flavidum . Thus, the isolate represents a new, although closely related species and is named Acetomicrobium faecalis .

The soluble hydrogenase of Acetomicrobium flavidum was purified by chromatography of crude extracts on phenyl-Sepharose, DEAE-cellulose and hydroxylapatite. The final purification was 28-fold with a yield of 11% and a specific activity of 6.0 μmol H2 released from reduced methylviologen per mg protein and min. The Km value for methylviologen was 2.65 mM. The enzyme had a molecular weight of 52 000 and consisted of only one polypeptide. It contained seven or eight atoms of iron and eight or nine atoms of acid labile sulfur per molecule, but no nickel. Its temperature optimum of 58°C was correlated to the optimal growth temperature, its pH optimum of 7.5 was little higher than the optimal pH for growth. The isoelectric point was 5.2. The membrane-associated hydrogenase activity was solubilized with Triton X-100 and was purified 18-fold. It crossreacted with antibodies against the soluble hydrogenase and differed only in vm. Both hydrogenases could be purified and were reasonably stable in the presence of oxygen. However, for long-term storage they had to be kept under hydrogen at 4°C to conserve activity.

The effect of monensin and 2-bromoethanesulfonic acid (BESA) on methane production from cattle manure and on volatile fatty acids metabolism was tested. At 10 days retention time 0.81 biogas per liter cattle manure and day were produced. Methanogenesis was inhibited 20% by 3 mM BESA per liter and 45% by 2–5 mg monensin per liter. When the digestion was inhibited with either of the both drugs, the acetate pool increased drastically. Like in untreated fermentations the propionate pool increased in BESA-inhibited fermentations for several hours after substrate addition. After 24 h however it did not decrease to the low level reached in non-inhibited fermentations. When monensin was the inhibitor, the propionate pool did not change for 15 h, but then decreased with the same rate as in the control experiment. Adaptation processes or detoxification may be responsible for the delayed degradation.

The isolation and characterization of a new methanogen from a peat bog, Methanobacterium palustre spec. nov., strain F, is described. Strain F grew on H2/CO2 and formate in complex medium. It also grew autotrophically on H2/CO2. Furthermore, growth on 2-propanol/CO2 was observed. Methane was formed from CO2 by oxidation of 2-propanol to acetone or 2-butanol to 2-butanone, but growth on 2-butanol plus CO2 apparently was too little to be measurable. Similarly, Methanobacterium bryantii M. o. H. and M. o. H. G formed acetone and 2-butanone from 2-propanol and 2-butanol, but no growth was measurable. On the basis of morphological and biochemical features strain F could be excluded from the genus Methanobrevibacter. Due to its cell morphology, lipid composition and polyamine pattern it belonged to the genus Methanobacterium. From known members of this genus strain F could be distinguished either by a different G+C content of the DNA, low DNA-DNA homology with reference strains, lacking serological reactions with anti-S probes and differences in the substrate spectrum. An alcohol dehydrogenase activity, specific for secondary alcohols and its substrate specificity was determined in crude extracts of strain F. NADP+ was the only electron carrier that was utilized. No reaction was found with NAD+, F420, FMN and FAD.

Growth of 5 strains of secondary alcohol-utilizing methanogens on cyclopentanol/CO2 was demonstrated. Cultures reached only low optical densities, comparable to those observed during growth on 2-propanol/CO2 or 2-butanol/CO2. The conversion of cyclopentanol to cyclopentanone was reversible in the presence of hydrogen. The specificity of ADHs of nine secondary alcohol-utilizing methanogens for coenzyme requirement and substrates was tested using crude enzyme preparations. The ADH of Methanobacterium bryantii M.o.H.G, Methabobacterium palustre, Methanocorpusculum parvum and Methanocorpusculum bavaricum was only active with NADP+, the ADH of Methanomicrobium paynteri, Methanogenium marisnigri, Methanogenium bourgense, strain GKZPZ and strain INSLUZ with F420 as electron carrier. FAD, FMN, methylene blue, benzyl viologen and tetrazolium, chloride could be used as electron carriers by the F420-dependent ADHs if endogenous F420 was introduced with the crude ADH preparation. No reaction with these electron carriers occurred if dialyzed extracts were used as a source of ADH. The ADH of all tested strains oxidized 2-propanol, 2-butanol, 2-pentanol and cyclopentanol, while the ADH of Mg. marisnigri and Mm. paynteri oxidized cyclohexanol, of Mb. palustre and Mcp. bavaricum cyclopentanol and 2,3-butanediol,and of Mcp. parvum cyclopentanol, 2,3-butanediol, ethanol and 1-propanol in addition.

On the basis of an extended investigation of morphological, biochemical and physiological properties and its phylogenetic affiliation it is proposed to place Methanomicrobium paynteri, validly published in 1984 within a new genus, Methanolacinia gen. nov. as Methanolacinia paynteri comb. nov.

Summary A new, Gram-negative, strictly anaerobic, non-sporeforming, thermophilic, non-motile rod-shaped bacterium, strain TN, was enriched and isolated from sewage sludge, digesting at 60°C. The optimum temperature for growth was 58°C., the optimum pH was between 7 and 8. Yeast extract and peptone (approximately 0.2% of each), as well as 1 μg/l of vitamin B12 were required for optimal growth. In the absence of vitamin B12 cells formed long filaments. From a variety of different substrates only glucose and fructose were utilized. Glucose was fermented via the Embden-Meyerhof-Parnas pathway, yielding 2 mol acetate, 2 mol CO2 and 4 mol H2. Increasing H2 concentrations in the gas phase inhibited growth of strain TN, while elevated acetate concentrations did not affect growth. Growth was also inhibited by kanamycin, streptomycin and cycloserin, while nisin, chloramphenicol, phosphomycin and penicillin were ineffective. The G+C content of the DNA was 62 mol%. DNA-DNA-hybridization with Acetomicrobium flavidum did not indicate a close relation between both strains (24%, spectrophotometric method). Thus, although both organisms had a similar metabolism, strain TN was considered a member of a new genus and was named Acetothermus paucivorans gen. nov., sp. nov.

A fixed film loop reactor was developed for the stabilization of undiluted sour whey. Porous clay beads were used to immobilize the population. The fermentation system was self-supporting with the aid of a pH-titrator. Within 2 months; the loading increased automatically to its maximum of 14 kg COD (chemical oxygen demand)/m3 per day. Parallel to this, the bacterial film was formed on the surface of the support material. For a pH of 6.7 the steady state was reached at a hydraulic retention time of 5 days equivalent to a loading of 14 kg COD/m3 per day. An amount of 5.6 m3 biogas was produced per m3 digester content and day and the COD-reduction was 95%. The pH-controlled whey addition led to only minor disturbances when overloading or oxygenation occured and a fast recovery of methanogenesis was observed. The economics of anaerobic whey digestion compared with conventional whey utilization is estimated by a simple cost/benefit calculation.

Summary A new, strictly anaerobic, non-sporeforming, non-motile, rigid, long filaments forming bacterium, strain MN, was isolated from a sewage sludge fermenter. Cells stained Gram-negative. Single cells were extremely thin, 0.18–0.3 × 2–3μm in size. Filaments of more than 100 μm in length were frequently formed. Optimum growth was between 35–38°C, optimum pH between 7.3–8.5. Growth was dependent on the presence of yeast extract. Acetate and H2 in a molar ratio of 1:2 as well as CO2 were formed from hexoses. Increasing hydrogen partial pressure inhibited growth, while elevated acetate concentration did not affect growth of strain MN. The G+C content of the DNA was 47 mol%. The new mesophilic isolate was named Acetofilamentum rigidum gen. nov., sp. nov. with isolate MN, deposited in the Deutsche Sammlung fur Mikroorganismen under DSM 20769, as the type strain.

Summary Ninety % of the bacterial population of an anaerobic whey digester were isolated and characterized. Fifty-five % of the isolates were fermentative, 5% acetogenic and 40% methanogenic organisms. Of the fermentative isolates, one strain of Lactobacillus , three strains of Eubacterium and 2 strains that could not be assigned to any known genus were predominant, accounting for 45% of the culturable population. The remaining 10% of the fermentative strains comprised Clostridia, Fusobacterium and Bacteroides . Each of these isolates represented less than 2% of the culturable population. The isolates probably belonging to the acetogenic stage could be assigned to Desulfovibrio and constituted approximately 5% of the culturable population. The methanogens consisted of 5 morphologically different groups. One group had the characteristic morphology of Methanospirilium , but no strains were isolated. Isolates of the other 4 groups were identified as strains of Methanobacterium formicicum, Methanobrevibacter arboriphilus, Methanothrix soehngenii and Methanocorpusculum parvum ( Zellner et al., 1987). On the basis of the metabolic capabilities of the isolates a microbial food chain for the degradation of the whey components to methane and CO 2 is proposed, fitting the theoretical model of Bryant (1979).

Summary Survival of several strains of methanogens during short-term and long-term conservation was tested. If oxygenation is prevented during storage, freezing of cultures in the growth medium conserves methanogens for at least 4 months, and organisms from colonies on agar slants were still viable after one year. Cell suspensions in glycerol stored at — 18°C survived for 20 months.

Cultures of the pseudomurein-containing archaebacterium Methanobacterium wolfei regularly lysed a short while after the energy source H2 was exhausted, or when H2 in growing cultures was replaced by N2. During lysis of cells, the DNA was released into the culture medium.

Summary A group of 11 facultatively heterofermentative rod-shaped bacteria were isolated at a late fermentation stage of pre-wilted grass. These bacteria were found to differ from currently described lactobacilli in the spectrum of fermented sugars, in particular in their ability to ferment xylose. The isolates were similar to Lactobacillus curvatus and Lactobacillus sake with respect to D(-)- and L(+)- lactate formation, R f -values of the D- and L-LDH, G+C content of the DNA and the L-Lys-D-Asp murein type. Teichoic acid was lacking. DNA-DNA homology among the isolates was high, while low homology values were obtained with the type strains of L. curvatus and L. sake , indicating only a distant relationship. It is proposed that the isolates represent a new species named Lactobacillus graminis sp. nov.

Summary Acetomicrobium flavidum gen. nov., sp. nov. was isolated from a thermophilic, anaerobic sewage sludge digester operated at 60 °c. The Gram-negative, highly motile, non-sporeforming rods form 2 mol acetic acid, 2 mol CO 2 and 4 mol H 2 from 1 mol hexose. They represent at least 25% of the total population (microscopic count) of the fermenting sludge.

Desulfovibrio simplex spec. nov. strain XVI was isolated from an anaerobic sour whey digester. Single cells had a vibrioid shape and were motile by a single, polar flagellum. The size of cells was 0.5–1.0 μ×1.5–3.0 μm. The G+C content of the DNA of D. simplex strain XVI was 47.5 mol%. The only other Desulfovibrio species with a similar G+C content of the DNA was Desulfovibrio salexigens (46.1 mol%. D. simplex grew on H2/CO2, formate, pyruvate, L(+)-lactate, fumarate, malate, ethanol, 1-propanol and 1-butanol as electron donors, while Desulfovibrio salexigens grew in addition on methanol, 2-propanol, 2-butanol, glycerol, succinate, citrate, choline and glucose. Electron acceptors for D. simplex were sulfate, thiosulfate and nitrate. L(+)-Lactate was incompletely oxidized to acetate and CO2 during sulfate reduction. Furthermore, both species could be distinguished by the ability of D. simplex but not of D. salexigens to grow on and to oxidize benzaldehyde derivatives to the respective acids, including vanillin, p-anisaldehyde and syringaldehyde. Moreover, D. simplex could grow in the presence of trace amounts of NaCl, while D. salexigens had an obligate requirement of 25 g/l NaCl. In addition, D. simplex can be distinguished from D. salexigens by its differing polyamine pattern. On the basis of the presented data the description of strain XVI as Desulfovibrio simplex spec. nov. is proposed.

A new mesophilic, monotrichously flagellated methane-producing coccus of ≦1μm in diameter was isolated from an anaerobic sour whey digester, originally inoculated with sewage sludge. Growth and methane production were observed with H2/CO2, formate and — less effectively — with 2-propanol/CO2. The isolate grew at temperatures between 15° C and 45° C with the optimum at around 37° C. Acetate, yeast extract and tungstate were required in the medium. Clarified rumen fluid stimulated growth.

Summary An extremely thermophilic, strictly anaerobic sulfate reducing bacterium, strain Z, was isolated from sediments of a hydrothermal vent located near Mount Vulcanello, Vulcano, Italy. On the basis of 16S rRNA cataloguing, strain Z was shown to belong to the archaebacterial kingdom, representing another strain of Archaeoglobus fulgidus . The weakly motile, irregular coccoid cells showed the fluorescence regarded as typical for methanogens, when viewed under the fluorescence microscope, although no relationship with methanogens was seen from comparative analysis of antigenic fingerprints with antibody S-probes. Furthermore, methane was not produced in significant amounts. Dissimilatory sulfate reduction was found with lactate, pyruvate and 2,3-butandiol as substrates. With thiosulfate as electron acceptor H 2 /CO 2 , formate, lactate, pyruvate or fumarate were utilized as electron donors. Sulfur bloom was not used as electron acceptor. Growth was inhibited by molybdate, a typical inhibitor of sulfate reducers. Lactate was decarboxylated to acetate and part of the acetate was oxidized to CO 2 with the reducing equivalents serving for sulfate reduction. The cell envelope consisted of a hexagonally arranged S-layer. The apparent molecular weight of its subunits, staining PAS-positive, was 132000. No significant amounts of polyamines were detected. The DNA polymerase was sensitive towards aphidicolin, a specific inhibitor of DNA polymerases type a of eucaryotes and of the DNA polymerase of Methanococcus vannielii . The DNA base composition was 45 mol% G+C. Archaeoglobus fulgidus strain Z has been deposited in the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH under number DSM 4139.

Anaerobic phenol degrading consortia were selected in sewage sludge and culture conditions were improved to allow maximum degradation rates of 0.9 g/l·d. Phenol had to be added in two portions of 0.45 g/l at intervals of 12 h to keep the fermentation at stable conditions. From U-14C-phenol little benzoate and acetate were formed as intermediates under a N2:CO2 gas phase. Final products were methane and CO2. When methanogenesis was inhibited by BESA, less labeled methane and CO2 were formed and labeled acetate remained undegraded. Turnover rates of phenol were significantly reduced in the presence of a H2:CO2 gas atmosphere and benzoate was formed from phenol and CO2. Acetate did not accumulate remarkably. After the H2:CO2 was converted to methane or was exchanged by N2:CO2 the accumulated benzoate was further degraded to methane and CO2. Elevated pools of acetate in sewage sludge led also to a reduction of the phenol degradation rates and presumably to an increased concentration of benzoate. In fresh sewage sludge benzoate degradation proceeds immediately, while the degradation of phenol starts only after a lag-phase of 3–10 days.

An anaerobic enrichment culture that degraded 0.4 mmol/l per day of o-phenylphenol was selected from sediment of a waste water pond of a sugar factory. From the consortium an o-phenylphenol-degrading bacterium, strain B10, was isolated. Strain B10 could not degrade other aromatic substances, including phenylacetic acid, benzoate, o-hydroxybenzoate, p-hydroxybenzoate and phenol. Best growth was observed with glucose, pyruvate, lactate, methanol and H2/CO2 as substrates. o-Phenylphenol was slowly degraded if supplied as the only carbon source and was cometabolized in the presence of >5 mmol/l glucose. Strain B10 has not yet been assigned to a known species or family.

Whey could be anaerobically digested at space loadings up to 36 kg COD/m3·d in an upflow digester containing porous clay beads for immobilization of microorganisms. In a parallel fermenter without immobilization a space loading of only 8 kg/m3·d was reached. The start-up time was very much reduced by the support material. The COD-reduction in both reactors was 95% and volatile fatty acids in the effluent were below 10 mmol/l. During the digestion of whey a thick layer of Methanothrix soehngenii and occasionally Methanobrevibacter arboriphilus was immobilized on the clay beads. The Methanothrix soehngenii layer disappeared, when whey permeate was fed. Methanosarcina spec. became the predominant acetotrophic methanogen, probably due to the lower pH resulting from digestion of whey permeate. Methanosarcina spec., however, was suspended and only occasionally trapped in the pores of the clay beads. No significant adhesion of other bacteria occurred.

A phenylacetic acid-degrading mixed culture was enriched from effluent of an anaerobic reactor for the treatment of waste water from cellulose bleaching. From this consortium a phenylacetic acid-degrading pure culture, strain DSU3, was isolated and, due to its typical morphology and substrate spectrum, tentatively classified as a Desulfosarcina sp. It could grow on and degrade phenylacetic acid, cyclohexane carboxylate, cyclohexylacetate, benzoate, fumaric acid and several volatile fatty acids, while phenol, o-hydroxybenzoate, p-hydroxybenzoate and glucose were not utilized. Production of mandelic acid from phenylacetic acid by the enrichment culture and utilization of benzoate, an intermediate of the mandelic acid pathway, by strain DSU3 may presumably indicate degradation of phenylacetic acid via the mandelic acid pathway.