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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.

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)).

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.

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 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.