Limitation of syntrophic coculture growth by the acetogen.

The syntrophic cooperation between hydrogen-producing acetogens and hydrogenotrophic methanogens relies on a critical balance between both partners. A recent study, provided several indications for the dependence of the biomass-specific growth rate of a methanogenic coculture on the acetogen. Nevertheless, final experimental proof was lacking since biomass-specific rates were obtained from a descriptive model, and not from direct measurement of individual biomass concentrations. In this study, a recently developed quantitative PCR approach was used to measure the individual biomass concentrations in the coculture of Desulfovibrio sp. G11 and Methanospirillum hungatei JF1 on lactate, formate or both. The model-derived growth yields and biomass-specific rates were successfully validated. Experimental findings identified the acetogen as the growth-limiting partner in the coculture on lactate. While the acetogen was operating at its maximum biomass-specific lactate consumption rate, the hydrogenotrophic methanogen showed a significant overcapacity. Furthermore, this study provides experimental evidence for different growth strategies followed by the syntrophic partners in order to maintain a common biomass-specific growth rate. During syntrophic lactate conversion, the biomass-specific electron transfer rate of Methanospirillum hungatei JF1 was three-fold higher compared to Desulfovibrio sp. G11. This is to compensate for the lower methanogenic biomass yield per electron-mole of substrate, which is dictated by the thermodynamics of the underlying reaction.

Archaeal community in a human-disturbed watershed in southeast China: diversity, distribution, and responses to environmental changes.

The response of freshwater bacterial community to anthropogenic disturbance has been well documented, yet the studies of freshwater archaeal community are rare, especially in lotic environments. Here, we investigated planktonic and benthic archaeal communities in a human-perturbed watershed (Jiulong River Watershed, JRW) of southeast China by using Illumina 16S ribosomal RNA gene amplicon sequencing. The results of taxonomic assignments indicated that SAGMGC-1, Methanobacteriaceae, Methanospirillaceae, and Methanoregulaceae were the four most abundant families in surface waters, accounting for 12.65, 23.21, 18.58 and 10.97 % of planktonic communities, whereas Nitrososphaeraceae and Miscellaneous Crenarchaeotic Group occupied more than 49 % of benthic communities. The compositions of archaeal communities and populations in waters and sediments were significantly different from each other. Remarkably, the detection frequencies of families Methanobacteriaceae and Methanospirillaceae, and genera Methanobrevibacter and Methanosphaera in planktonic communities correlated strongly with bacterial fecal indicator, suggesting some parts of methanogenic Archaea may come from fecal contamination. Because soluble reactive phosphorus (SRP) and the ratio of dissolved inorganic nitrogen to SRP instead of nitrogen nutrients showed significant correlation with several planktonic Nitrosopumilus- and Nitrosotalea-like OTUs, Thaumarchaeota may play an unexplored role in biogeochemical cycling of river phosphorus. Multivariate statistical analyses revealed that the variation of α-diversity of planktonic archaeal community was best explained by water temperature, whereas nutrient concentrations and stoichiometry were the significant drivers of ß-diversity of planktonic and benthic communities. Taken together, these results demonstrate that the structure of archaeal communities in the JRW is sensitive to anthropogenic disturbances caused by riparian human activities.

Methanospirillum stamsii sp. nov., a psychrotolerant, hydrogenotrophic, methanogenic archaeon isolated from an anaerobic expanded granular sludge bed bioreactor operated at low temperature.

A psychrotolerant hydrogenotrophic methanogen, strain Pt1, was isolated from a syntrophic propionate-oxidizing methanogenic consortium obtained from granulated biomass of a two-stage low-temperature (3-8 °C) anaerobic expanded granular sludge bed (EGSB) bioreactor, fed with a mixture of volatile fatty acids (VFAs) (acetate, propionate and butyrate). The strain was strictly anaerobic, and cells were curved rods, 0.4-0.5×7.5-25 µm, that sometimes formed wavy filaments from 25 to several hundred micrometres in length. Cells stained Gram-negative and were non-sporulating. They were gently motile by means of tufted flagella. The strain grew at 5-37 °C (optimum at 20-30 °C), at pH 6.0-10 (optimum 7.0-7.5) and with 0-0.3 M NaCl (optimum 0 M NaCl). Growth and methane production was found with H2/CO2 and very weak growth with formate. Acetate and yeast extract stimulated growth, but were not essential. The G+C content of the DNA of strain Pt1 was 40 mol%. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain Pt1 was a member of the genus Methanospirillum and showed 97.5 % sequence similarity to Methanospirillum hungatei JF1(T) and 94 % sequence similarity to Methanospirillum lacunae Ki8-1(T). DNA-DNA hybridization of strain Pt1 with Methanospirillum hungatei JF1(T) revealed 39 % relatedness. On the basis of its phenotypic characteristics and phylogenetic position, strain Pt1 is a representative of a novel species of the genus Methanospirillum, for which the name Methanospirillum stamsii sp. nov. is proposed. The type strain is Pt1(T) ( = DSM 26304(T) = VKM B-2808(T)).

Growth- and substrate-dependent transcription of formate dehydrogenase and hydrogenase coding genes in Syntrophobacter fumaroxidans and Methanospirillum hungatei.

Transcription of genes coding for formate dehydrogenases (fdh genes) and hydrogenases (hyd genes) in Syntrophobacter fumaroxidans and Methanospirillum hungatei was studied following growth under different conditions. Under all conditions tested, all fdh and hyd genes were transcribed. However, transcription levels of the individual genes varied depending on the substrate and growth conditions. Our results strongly suggest that in syntrophically grown S. fumaroxidans cells, the [FeFe]-hydrogenase (encoded by Sfum_844-46), FDH1 (Sfum_2703-06) and Hox (Sfum_2713-16) may confurcate electrons from NADH and ferredoxin to protons and carbon dioxide to produce hydrogen and formate, respectively. Based on bioinformatic analysis, a membrane-integrated energy-converting [NiFe]-hydrogenase (Mhun_1741-46) of M. hungatei might be involved in the energy-dependent reduction of CO(2) to formylmethanofuran. The best candidates for F(420)-dependent N(5),N(10)-methyl-H(4) MPT and N(5),N(10),-methylene-H(4)MPT reduction are the cytoplasmic [NiFe]-hydrogenase and FDH1. 16S rRNA ratios indicate that in one of the triplicate co-cultures of S. fumaroxidans and M. hungatei, less energy was available for S. fumaroxidans. This led to enhanced transcription of genes coding for the Rnf-complex (Sfum_2694-99) and of several fdh and hyd genes. The Rnf-complex probably reoxidized NADH with ferredoxin reduction, followed by ferredoxin oxidation by the induced formate dehydrogenases and hydrogenases.

Effect of tungsten and molybdenum on growth of a syntrophic coculture of Syntrophobacter fumaroxidans and Methanospirillum hungatei.

The effect of tungsten (W) and molybdenum (Mo) on the growth of Syntrophobacter fumaroxidans and Methanospirillum hungatei was studied in syntrophic cultures and the pure cultures of both the organisms. Cells that were grown syntropically were separated by Percoll density centrifugation. Measurement of hydrogenase and formate dehydrogenase levels in cell extracts of syntrophically grown cells correlated with the methane formation rates in the co-cultures. The effect of W and Mo on the activity of formate dehydrogenase was considerable in both the organisms, whereas hydrogenase activity remained relatively constant. Depletion of tungsten and/or molybdenum, however, did not affect the growth of the pure culture of S. fumaroxidans on propionate plus fumarate significantly, although the specific activities of hydrogenase and especially formate dehydrogenase were influenced by the absence of Mo and W. This indicates that the organism has a low W or Mo requirement under these conditions. Growth of M. hungatei on either formate or H2/CO2 required tungsten, and molybdenum could replace tungsten to some extent. Our results suggest a more prominent role for H2 as electron carrier in the syntrophic conversion of propionate, when the essential trace metals W and Mo for the functioning of formate dehydrogenase are depleted.

Methane production in an UASB reactor operated under periodic mesophilic-thermophilic conditions.

Methane production was studied in a laboratory-scale 10 L anaerobic upflow sludge bed (UASB) reactor with periodic variations of the reactor temperature. On a daily basis the temperature was varied between 35 and 45 degrees C or 35 and 55 degrees C with a heating period of 6 h. Each temperature increase was accompanied by an increase in methane production and a decrease in the concentration of soluble organic matter in the effluent. In comparison to a reactor operated at 35 degrees C, a net increase in methane production of up to 22% was observed. Batch activity tests demonstrated a tolerance of mesophilic methanogenic populations to short-term, 2-6 h, temperature increases, although activity of acetoclastic methanogens decreased after 6 h exposure to a temperature of 55 degrees C. 16S sequencing of DGGE bands revealed proliferation of temperature-tolerant Methanospirillum hungatii sp. in the reactor.

Isolation and characterization of a motile hydrogenotrophic methanogen from rice paddy field soil in Japan.

A hydrogenotrophic motile methanogen was isolated from flooded Japanese paddy field soil. Anaerobic incubation of the paddy soil on H(2)-CO(2) at 20 degrees C led to the enrichment of symmetrically curved motile autofluorescent rods. The methanogenic strain TM20-1 isolated from the culture was halotolerant and utilized H(2)-CO(2), 2-propanol-CO(2), or formate as a sole methanogenic substrate. Based on the 16S rRNA gene sequence similarity (94.8%) with Methanospirillum hungateii, and on the physiological and phenotypic characteristics, TM20-1 was suggested to be a newly identified species belonging to the genus Methanospirillum. This is the first report of isolation of the genus Methanospirillum strain from a rice paddy field.

Degradation of acetaldehyde and its precursors by Pelobacter carbinolicus and P. acetylenicus.

Pelobacter carbinolicus and P. acetylenicus oxidize ethanol in syntrophic cooperation with methanogens. Cocultures with Methanospirillum hungatei served as model systems for the elucidation of syntrophic ethanol oxidation previously done with the lost "Methanobacillus omelianskii" coculture. During growth on ethanol, both Pelobacter species exhibited NAD+-dependent alcohol dehydrogenase activity. Two different acetaldehyde-oxidizing activities were found: a benzyl viologen-reducing enzyme forming acetate, and a NAD+-reducing enzyme forming acetyl-CoA. Both species synthesized ATP from acetyl-CoA via acetyl phosphate. Comparative 2D-PAGE of ethanol-grown P. carbinolicus revealed enhanced expression of tungsten-dependent acetaldehyde: ferredoxin oxidoreductases and formate dehydrogenase. Tungsten limitation resulted in slower growth and the expression of a molybdenum-dependent isoenzyme. Putative comproportionating hydrogenases and formate dehydrogenase were expressed constitutively and are probably involved in interspecies electron transfer. In ethanol-grown cocultures, the maximum hydrogen partial pressure was about 1,000 Pa (1 mM) while 2 mM formate was produced. The redox potentials of hydrogen and formate released during ethanol oxidation were calculated to be EH2 = -358±12 mV and EHCOOH = -366±19 mV, respectively. Hydrogen and formate formation and degradation further proved that both carriers contributed to interspecies electron transfer. The maximum Gibbs free energy that the Pelobacter species could exploit during growth on ethanol was -35 to -28 kJ per mol ethanol. Both species could be cultivated axenically on acetaldehyde, yielding energy from its disproportionation to ethanol and acetate. Syntrophic cocultures grown on acetoin revealed a two-phase degradation: first acetoin degradation to acetate and ethanol without involvement of the methanogenic partner, and subsequent syntrophic ethanol oxidation. Protein expression and activity patterns of both Pelobacter spp. grown with the named substrates were highly similar suggesting that both share the same steps in ethanol and acetalydehyde metabolism. The early assumption that acetaldehyde is a central intermediate in Pelobacter metabolism was now proven biochemically.

Changes in process performance and microbial characteristics of retained sludge during low-temperature operation of an EGSB reactor.

A lab-scale expanded granular sludge bed (EGSB) reactor was seeded with granular sludge and operated to investigate the influence of temperature decrease on both process performance and the microbial community structure of the granular sludge. Synthetic wastewater containing sucrose and volatile fatty acids was used as feed. The EGSB reactor was brought online at a starting temperature of 15 degrees C and was reduced stepwise to a final temperature of 5 degrees C. The reactor exhibited sufficient COD removal efficiency between 10 degrees C and 15 degrees C. However at 5 degrees C serious deterioration of process performance was observed. The methane-producing activity of the retained sludge increased when it was compared to the activity of the seed sludge (day 0) during 10 degrees C to 15 degrees C operation. When hydrogen fed, sludge showed much higher methanogenic activity as compared with seed sludge activity at test temperatures of 15 degrees C and 20 degrees C on day 196 of reactor operation. At this time, proliferation of the genus Methanospirillum in the retained sludge was observed and a decrease in Methanobacterium species was also measured. Throughout the experiment, the genus Methanosaeta was detected in abundance and the community structure of the Domain Bacteria was stably maintained. The sugar-degrading acid-forming bacteria, Lactococcus and Anaerovibrio were detected in the retained sludge throughout the experiment as well and the propionate-degrading acetogen Syntrophobacter fumaroxidans was also detected, although its population size decreased at 5 degrees C.

[Microbial community in granules from an EGSB reactor operated at 20 degrees C].

The microbial communities in 6 granular sludge samples taken from a lab-scale EGSB reactor operated under different organic loading rates (OLR) at 20 degrees C were studied using PCR-DGGE and RTQ-PCR technique. The results indicate that as the OLR increased from 1.5 kg/(m3 x d) to 10.0 kg/(m3 x d), the microbial communities of archaea and eubacteria in the 6 sludge samples are not changed greatly. The archaea population is highly similar among the 6 samples. The Shannon indexes of archaea population are 5.51, 5.88, 5.47, 5.25, 5.32 and 5.11, respectively. Except the seed sludge, the eubacteria population is similar among the other 5 samples. The Shannon indexes of eubacteria population are 2.97, 5.07, 5.44, 6.38, 6.66 and 5.21, respectively. The dominant archaea in the 6 granular samples are Methanobacterium, Methanocorpusculum, Methanosaeta and Methanospirillum. As the OLR of the reactor increased and the operation time elapsed, Methanocorpusculum parvum and Methanospirillum hungatei, both are hydrotrophic methanogens, become the dominant archaea. The archaea content in the samples decreases slightly at the beginning, but increases later and finally reaches to a level obviously higher than that in the seed sludge.

Pelotomaculum propionicicum sp. nov., an anaerobic, mesophilic, obligately syntrophic, propionate-oxidizing bacterium.

An anaerobic, mesophilic, syntrophic, propionate-oxidizing bacterium, strain MGP(T), was isolated as a defined co-culture with Methanospirillum hungatei from the methanogenic sludge of a mesophilic upflow anaerobic sludge blanket (UASB) reactor. The strain grew in the presence of propionate, but only in co-culture with methanogens, suggesting that it is an obligately syntrophic bacterium. The optimum temperature for growth was 37 degrees C, and the optimum pH was between 6.5 and 7.2. Based on comparative 16S rRNA gene sequence analysis, strain MGP(T) was affiliated with subcluster Ih of 'Desulfotomaculum cluster I', in which it was found to be moderately related to known species of the genera Pelotomaculum and Cryptanaerobacter. Similar to known species of the genus Pelotomaculum, strain MGP(T) could degrade propionate in syntrophy, but had no ability to reduce sulfate, sulfite and thiosulfate. Further phenotypic and genetic studies supported the affiliation of the strain as a novel species in this genus, for which the name Pelotomaculum propionicicum sp. nov. is proposed. The type strain is MGP(T) (=DSM 15578(T)=JCM 11929(T)). The strain has been deposited in the DSM and JCM culture collections as a defined co-culture with Methanospirillum hungatei.

Pelotomaculum terephthalicum sp. nov. and Pelotomaculum isophthalicum sp. nov.: two anaerobic bacteria that degrade phthalate isomers in syntrophic association with hydrogenotrophic methanogens.

An anaerobic phthalate isomer-degrading strain (JT(T)) that we previously isolated was characterized. In addition, a strictly anaerobic, mesophilic, syntrophic phthalate isomer-degrading bacterium, designated strain JI(T), was isolated and characterized in this study. Both were non-motile rods that formed spores. In both strains, the optimal growth was observed at temperatures around 37 degrees C and neutral pH. In syntrophic co-culture with the hydrogenotrophic methanogen Methanospirillum hungatei, both strains could utilize two or three phthalate isomers for growth, and produce acetate and methane as end products. Strain JT(T) was able to grow on isophthalate, terephthalate, and a number of low-molecular weight aromatic compounds, such as benzoate, hydroquinone, 2-hydroxybenzoate, 3-hydroxybenzoate, 2,5-dihydroxybenzoate, 3-phenylpropionate in co-culture with M. hungatei. It could also grow on crotonate, hydroquinone and 2,5-dihydroxybenzoate in pure culture. Strain JI(T) utilized all of the three phthalate isomers as well as benzoate and 3-hydroxybenzoate for growth in co-culture with M. hungatei. No substrates were, however, found to support the axenic growth of strain JI(T). Neither strain JT(T) nor strain JI(T) could utilize sulfate, sulfite, thiosulfate, nitrate, fumarate, Fe (III) or 4-hydroxybenzoate as electron acceptor. Phylogenetically, strains JT(T) and JI(T) were relatively close to the members of the genera Pelotomaculum and Cryptanaerobacter in 'Desulfotomaculum lineage I'. Physiological and chemotaxonomic characteristics indicated that the two isolates should be classified into the genus Pelotomaculum, creating two novel species for them. Here, we propose Pelotomaculum terephthalicum sp. nov. and Pelotomaculum isophthalicum sp. nov. for strain JT(T) and strain JI(T), respectively. The type strains are strains JT(T) (= DSM 16121(T )= JCM 11824(T )= NBRC 100523(T)) and JI(T) (= JCM 12282(T) = BAA-1053(T)) for P. terephthalicum and P. isophthalicum, respectively.

The first true obligately syntrophic propionate-oxidizing bacterium, Pelotomaculum schinkii sp. nov., co-cultured with Methanospirillum hungatei, and emended description of the genus Pelotomaculum.

A Gram-positive, spore-forming, syntrophic propionate-oxidizing bacterium, Pelotomaculum schinkii sp. nov. strain HH(T), was isolated as a co-culture with Methanospirillum hungatei JF-1(T) from anaerobic, freeze-dried granular sludge obtained from an upflow anaerobic sludge bed reactor treating sugar beet wastewater. The bacterium converted propionate to acetate in co-culture with Methanospirillum hungatei JF-1(T) or Methanobacterium formicicum MF(NT), but not in co-culture with Methanobrevibacter arboriphilus AZ. The organism could not be cultured axenically with any of the substrates tested and therefore can be considered as a (the first) true anaerobic syntrophic bacterium. The bacterium contained two distinct 16S rRNA gene sequences, with 96.8% sequence similarity, which were both expressed during syntrophic growth on propionate as revealed by fluorescent in situ hybridization. The most closely related organisms are Cryptanaerobacter phenolicus LR7.2(T), a bacterium that transforms phenol into benzoate, and Pelotomaculum thermopropionicum SI(T), a thermophilic, syntrophic propionate-oxidizing bacterium. Other related species belong to the Gram-positive, sulfate-reducing genus Desulfotomaculum. The type strain of Pelotomaculum schinkii is strain HH(T) (=ATCC BAA-615(T)=DSM 15200(T)).

Syntrophomonas erecta sp. nov., a novel anaerobe that syntrophically degrades short-chain fatty acids.

Two novel anaerobes, strains GB4-38(T) and SB9-1, were isolated from an upflow anaerobic sludge blanket reactor for treating bean-curd farm wastewater and lotus field mud, respectively. The strains degraded straight-chain fatty acids with 4-8 carbon atoms in syntrophic association with methanogens and converted 1 mol butyrate into about 2 mol acetate and presumably 2 mol H(2). None of the branched-chain fatty acids tested could be degraded. Benzoate was not degraded. Fumarate, sulfate, thiosulfate, sulfur and nitrate did not serve as electron acceptors for butyrate degradation. In the absence of a methanogen partner, strain GB4-38(T) grew on crotonate in pure culture; the generation time was about 5 h at 37 degrees C. However, strain SB9-1 grew on butyrate plus pentenoate, but not crotonate, in pure culture and the generation time was 18 h at 37 degrees C. Cells of GB4-38(T) and SB9-1 were straight rods and stained Gram-negative. The major cellular fatty acids of GB4-38(T) were C(14 : 0) (29.74 %), C(16 : 0) (17.00 %), C(16 : 1)omega5c (16.63 %) and isoC(17 : 1) I (15.34 %). ll-Diaminopimelic acid existed in the cellular peptidoglycan. The genomic DNA G+C content of strain GB4-38(T) was 43.2 mol%. Phylogenetic analysis based on 16S rRNA gene sequences supported clustering of the two strains with syntrophic bacterial species of the genus Syntrophomonas (89.6-92.4 % sequence similarity), but phenotypic, chemotaxonomic and genetic characters differentiated the two strains from members of this genus. Therefore, it is proposed that the two strains are representatives of a novel species, Syntrophomonas erecta sp. nov. The type strain is GB4-38(T) (=CGMCC 1.5013(T)=DSM 16215(T)).

Energetics of syntrophic propionate oxidation in defined batch and chemostat cocultures.

Propionate consumption was studied in syntrophic batch and chemostat cocultures of Syntrophobacter fumaroxidans and Methanospirillum hungatei. The Gibbs free energy available for the H(2)-consuming methanogens was <-20 kJ mol of CH(4)(-1) and thus allowed the synthesis of 1/3 mol of ATP per reaction. The Gibbs free energy available for the propionate oxidizer, on the other hand, was usually >-10 kJ mol of propionate(-1). Nevertheless, the syntrophic coculture grew in the chemostat at steady-state rates of 0.04 to 0. 07 day(-1) and produced maximum biomass yields of 2.6 g mol of propionate(-1) and 7.6 g mol of CH(4)(-1) for S. fumaroxidans and M. hungatei, respectively. The energy efficiency for syntrophic growth of S. fumaroxidans, i.e., the biomass produced per unit of available Gibbs free energy was comparable to a theoretical growth yield of 5 to 12 g mol of ATP(-1). However, a lower growth efficiency was observed when sulfate served as an additional electron acceptor, suggesting inefficient energy conservation in the presence of sulfate. The maintenance Gibbs free energy determined from the maintenance coefficient of syntrophically grown S. fumaroxidans was surprisingly low (0.14 kJ h(-1) mol of biomass C(-1)) compared to the theoretical value. On the other hand, the Gibbs free-energy dissipation per mole of biomass C produced was much higher than expected. We conclude that the small Gibbs free energy available in many methanogenic environments is sufficient for syntrophic propionate oxidizers to survive on a Gibbs free energy that is much lower than that theoretically predicted.

Metabolism of benzoate, cyclohex-1-ene carboxylate, and cyclohexane carboxylate by "Syntrophus aciditrophicus" strain SB in syntrophic association with H(2)-using microorganisms.

The metabolism of benzoate, cyclohex-1-ene carboxylate, and cyclohexane carboxylate by "Syntrophus aciditrophicus" in cocultures with hydrogen-using microorganisms was studied. Cyclohexane carboxylate, cyclohex-1-ene carboxylate, pimelate, and glutarate (or their coenzyme A [CoA] derivatives) transiently accumulated during growth with benzoate. Identification was based on comparison of retention times and mass spectra of trimethylsilyl derivatives to the retention times and mass spectra of authentic chemical standards. (13)C nuclear magnetic resonance spectroscopy confirmed that cyclohexane carboxylate and cyclohex-1-ene carboxylate were produced from [ring-(13)C(6)]benzoate. None of the metabolites mentioned above was detected in non-substrate-amended or heat-killed controls. Cyclohexane carboxylic acid accumulated to a concentration of 260 microM, accounting for about 18% of the initial benzoate added. This compound was not detected in culture extracts of Rhodopseudomonas palustris grown phototrophically or Thauera aromatica grown under nitrate-reducing conditions. Cocultures of "S. aciditrophicus" and Methanospirillum hungatei readily metabolized cyclohexane carboxylate and cyclohex-1-ene carboxylate at a rate slightly faster than the rate of benzoate metabolism. In addition to cyclohexane carboxylate, pimelate, and glutarate, 2-hydroxycyclohexane carboxylate was detected in trace amounts in cocultures grown with cyclohex-1-ene carboxylate. Cyclohex-1-ene carboxylate, pimelate, and glutarate were detected in cocultures grown with cyclohexane carboxylate at levels similar to those found in benzoate-grown cocultures. Cell extracts of "S. aciditrophicus" grown in a coculture with Desulfovibrio sp. strain G11 with benzoate or in a pure culture with crotonate contained the following enzyme activities: an ATP-dependent benzoyl-CoA ligase, cyclohex-1-ene carboxyl-CoA hydratase, and 2-hydroxycyclohexane carboxyl-CoA dehydrogenase, as well as pimelyl-CoA dehydrogenase, glutaryl-CoA dehydrogenase, and the enzymes required for conversion of crotonyl-CoA to acetate. 2-Ketocyclohexane carboxyl-CoA hydrolase activity was detected in cell extracts of "S. aciditrophicus"-Desulfovibrio sp. strain G11 benzoate-grown cocultures but not in crotonate-grown pure cultures of "S. aciditrophicus". These results are consistent with the hypothesis that ring reduction during syntrophic benzoate metabolism involves a four- or six-electron reduction step and that once cyclohex-1-ene carboxyl-CoA is made, it is metabolized in a manner similar to that in R. palustris.

Pathway of propionate oxidation by a syntrophic culture of Smithella propionica and Methanospirillum hungatei.

The pathway of propionate conversion in a syntrophic coculture of Smithella propionica and Methanospirillum hungatei JF1 was investigated by (13)C-NMR spectroscopy. Cocultures produced acetate and butyrate from propionate. [3-(13)C]propionate was converted to [2-(13)C]acetate, with no [1-(13)C]acetate formed. Butyrate from [3-(13)C]propionate was labeled at the C2 and C4 positions in a ratio of about 1:1.5. Double-labeled propionate (2,3-(13)C) yielded not only double-labeled acetate but also single-labeled acetate at the C1 or C2 position. Most butyrate formed from [2,3-(13)C]propionate was also double labeled in either the C1 and C2 atoms or the C3 and C4 atoms in a ratio of about 1:1.5. Smaller amounts of single-labeled butyrate and other combinations were also produced. 1-(13)C-labeled propionate yielded both [1-(13)C]acetate and [2-(13)C]acetate. When (13)C-labeled bicarbonate was present, label was not incorporated into acetate, propionate, or butyrate. In each of the incubations described above, (13)C was never recovered in bicarbonate or methane. These results indicate that S. propionica does not degrade propionate via the methyl-malonyl-coenzyme A (CoA) pathway or any other of the known pathways, such as the acryloyl-CoA pathway or the reductive carboxylation pathway. Our results strongly suggest that propionate is dismutated to acetate and butyrate via a six-carbon intermediate.

Propionate formation by Opitutus terrae in pure culture and in mixed culture with a hydrogenotrophic methanogen and implications for carbon fluxes in anoxic rice paddy soil.

Propionate-forming bacteria seem to be abundant in anoxic rice paddy soil, but biogeochemical investigations show that propionate is not a correspondingly important intermediate in carbon flux in this system. Mixed cultures of Opitutus terrae strain PB90-1, a representative propionate-producing bacterium from rice paddy soil, and the hydrogenotrophic Methanospirillum hungatei strain SK maintained hydrogen partial pressures similar to those in the soil. The associated shift away from propionate formation observed in these cultures helps to reconcile the disparity between microbiological and biogeochemical studies.

Syntrophus aciditrophicus sp. nov., a new anaerobic bacterium that degrades fatty acids and benzoate in syntrophic association with hydrogen-using microorganisms.

Strain SBT is a new, strictly anaerobic, gram-negative, nonmotile, non-sporeforming, rod-shaped bacterium that degrades benzoate and certain fatty acids in syntrophic association with hydrogen/formate-using microorganisms. Strain SBT produced approximately 3 mol of acetate and 0.6 mol of methane per mol of benzoate in coculture with Methanospirillum hungatei strain JF1. Saturated fatty acids, some unsaturated fatty acids, and methyl esters of butyrate and hexanoate also supported growth of strain SBT in coculture with Desulfovibrio strain G11. Strain SBT grew in pure culture with crotonate, producing acetate, butyrate, caproate, and hydrogen. The molar growth yield was 17 +/- 1 g cell dry mass per mol of crotonate. Strain SBT did not grow with fumarate, iron(III), polysulfide, or oxyanions of sulfur or nitrogen as electron acceptors with benzoate as the electron donor. The DNA base composition of strain SBT was 43.1 mol% G+C. Analysis of the 16 S rRNA gene sequence placed strain SBT in the delta-subdivision of the Proteobacteria, with sulfate-reducing bacteria. Strain SBT was most closely related to members of the genus Syntrophus. The clear phenotypic and genotypic differences between strain SBT and the two described species in the genus Syntrophus justify the formation of a new species, Syntrophus aciditrophicus.

Sporotomaculum syntrophicum sp. nov., a novel anaerobic, syntrophic benzoate-degrading bacterium isolated from methanogenic sludge treating wastewater from terephthalate manufacturing.

An anaerobic, mesophilic, syntrophic benzoate-degrading bacterium, designated strain FB(T), was isolated from methanogenic sludge which had been used to treat wastewater from the manufacture of terephthalic acid. Cells were non-motile gram-positive rods that formed spores. The optimum temperature for growth was 35-40 degrees C, and the optimum pH was 7.0-7.2. A co-culture with the hydrogenotrophic methanogen Methanospirillum hungatei converted benzoate to acetate, carbon dioxide, and methane. Butyrate transiently accumulated at a high concentration of 2.5 mM during degradation. Besides benzoate, no other compound tested supported growth of the co-culture. Crotonate supported growth of strain FB(T) in pure culture. Furthermore, the strain degraded benzoate in pure culture with crotonate as co-substrate to produce acetate and butyrate. The strain was not able to utilize sulfate, sulfite, thiosulfate, nitrate, fumarate, or Fe(III) as electron acceptor. The G+C content of the DNA was 46.8 mol%. Strain FB(T) contained MK-7 as the major quinone and C(16:1) as the major fatty acid. 16S rDNA sequence analysis revealed that the strain was a member of the genus Sporotomaculum, even though it exhibited significant differences, such as the capacity for syntrophic growth, to the known member of the genus. Hence, we propose the name Sporotomaculum syntrophicum sp. nov. for strain FB(T). The type strain is strain FB(T) (DSM 14795, JCM 11475).