BackgroundInteractions between microorganisms during specific steps of anaerobic digestion determine metabolic pathways in bioreactors and consequently the efficiency of fermentation processes. This study focuses on conversion of lactate and acetate to butyrate by bacteria of dark fermentation. The recently recognized flavin-based electron bifurcation as a mode of energy coupling by anaerobes increases our knowledge of anaerobic lactate oxidation and butyrate formation.ResultsMicrobial communities from dark fermentation bioreactors or pure culture of Clostridium butyricum are able to convert lactate and acetate to butyrate in batch experiments. The ability of C. butyricum to transform lactate and acetate to butyrate was shown for the first time, with ethanol identified as an additional end product of this process. A search for genes encoding EtfAB complexes and their gene neighbourhood in C. butyricum and other bacteria capable of lactate and acetate conversion to butyrate as well as butyrate-producers only and the lactate oxidiser Acetobacterium woodii, revealed that the Etf complexes involved in (i) lactate oxidation and (ii) butyrate synthesis, form separate clusters. There is a more extent similarity between Etf subunits that are involved in lactate oxidation in various species (e.g. A. woodii and C. butyricum) than between the different etf gene products within the same species of butyrate producers. A scheme for the metabolic pathway of lactate and acetate transformation to butyrate in C. butyricum was constructed.ConclusionsStudies on the conversion of lactate and acetate to butyrate by microbial communities from dark fermentation bioreactors or Clostridium butyricum suggest that a phenomenon analogous to cross-feeding of lactate in gastrointestinal tract also occurs in hydrogen-yielding reactors. A scheme of lactate and acetate transformation pathway is proposed, based on the example of C. butyricum, which employs flavin-based electron bifurcation. This process utilizes electron-transferring flavoprotein (Etf) complexes specific for (i) lactate oxidation and (ii) butyrate formation. Phylogenetic analysis revealed that such complexes are encoded in the genomes of other bacteria capable of lactate and acetate conversion to butyrate. These findings contribute significantly to our understanding of the metabolic pathways and symbiotic interactions between bacteria during the acidogenic step of anaerobic digestion.

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A system for biohydrogen production was developed based on long-term continuous cultures grown on sugar beet molasses in packed bed reactors. In two separate cultures, consortia of fermentative bacteria developed as biofilms on granitic stones. In one of the cultures, a granular sludge was also formed. Metagenomic analysis of the microbial communities by 454-pyrosequencing of amplified 16S rDNA fragments revealed that the overall biodiversity of the hydrogen-producing cultures was quite small. The stone biofilm from the culture without granular sludge was dominated by Clostridiaceae and heterolactic fermentation bacteria, mainly Leuconostocaeae. Representatives of the Leuconostocaeae and Enterobacteriaceae were dominant in both the granules and the stone biofilm formed in the granular sludge culture. The culture containing granular sludge produced hydrogen significantly more effectively than that containing only the stone biofilm: 5.43 vs. 2.8 mol H(2)/mol sucrose from molasses, respectively. The speculations that lactic acid bacteria may favor hydrogen production are discussed.

Anaerobic digestion is a complex process involving hydrolysis, acidogenesis, acetogenesis and methanogenesis. The separation of the hydrogen-yielding (dark fermentation) and methane-yielding steps under controlled conditions permits the production of hydrogen and methane from biomass. The characterization of microbial communities developed in bioreactors is crucial for the understanding and optimization of fermentation processes. Previously we developed an effective system for hydrogen production based on long-term continuous microbial cultures grown on sugar beet molasses. Here, the acidic effluent from molasses fermentation was used as the substrate for methanogenesis in an upflow anaerobic sludge blanket bioreactor. This study focused on the molecular analysis of the methane-yielding community processing the non-gaseous products of molasses fermentation. The substrate for methanogenesis produces conditions that favor the hydrogenotrophic pathway of methane synthesis. Methane production results from syntrophic metabolism whose key process is hydrogen transfer between bacteria and methanogenic Archaea. High-throughput 454 pyrosequencing of total DNA isolated from the methanogenic microbial community and bioinformatic sequence analysis revealed that the domain Bacteria was dominated by Firmicutes (mainly Clostridia), Bacteroidetes, δ- and γ-Proteobacteria, Cloacimonetes and Spirochaetes. In the domain Archaea, the order Methanomicrobiales was predominant, with Methanoculleus as the most abundant genus. The second and third most abundant members of the Archaeal community were representatives of the Methanomassiliicoccales and the Methanosarcinales. Analysis of the methanogenic sludge by scanning electron microscopy with Energy Dispersive X-ray Spectroscopy and X-ray diffraction showed that it was composed of small highly heterogeneous mineral-rich granules. Mineral components of methanogenic granules probably modulate syntrophic metabolism and methanogenic pathways. A rough functional analysis from shotgun data of the metagenome demonstrated that our knowledge of methanogenesis is poor and/or the enzymes responsible for methane production are highly effective, since despite reasonably good sequencing coverage, the details of the functional potential of the microbial community appeared to be incomplete.

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