A syntrophic acetate-oxidizing bacterium, strain BST (T = type strain), was isolated from a previously described mesophilic triculture that was able to syntrophically oxidize acetate and form methane in stoichiometric amounts. Strain BST was isolated with substrates typically utilized by homoacetogenic bacteria. Strain BST was a spore-forming, gram-positive, rod-shaped organism which utilized formate, glucose, ethylene glycol, cysteine, betaine, and pyruvate. Acetate and sometimes formate were the main fermentation products. Small amounts of alanine were also produced from glucose, betaine, and cysteine. Strain BST grew optimally at 37°C and pH 7. The G+C content of the DNA of strain BST was 32 mol%. A 16s rRNA sequence analysis revealed that strain BST was a member of a new species of the genus Closfridium. We propose the name Clostridium ultunense for this organism; strain BS is the type strain of C. ultunense.Anaerobic oxidations of fatty acids to hydrogen and carbon dioxide by proton-reducing bacteria are, from a thermodynamic point of view, very unfavorable reactions (38). Under standard conditions, these hydrogen-producing reactions are all endergonic. However, standard conditions are not the conditions that occur in most natural anaerobic environments; at a very low partial pressure of hydrogen, like the partial pressure achieved in the presence of hydrogenotrophs, such as methanogenic or sulfate-reducing bacteria, the oxidation reaction becomes exergonic (9, 48). By consuming hydrogen, the hydrogenotrophs can create conditions under which the protonreducing bacteria can perform oxidations that otherwise could not yield energy; i.e., the latter organisms depend entirely on the former to fulfill their function.Several different species of bacteria are able to oxidize fatty acids (38). Syntrophomonas wolfei degrades straight-chain fatty acids up to octanoate, forming acetate and propionate together with H,, in cocultures with either a methanogen or a sulfate reducer (25). Propionate is degraded to acetate, carbon dioxide, and H, by Syntrophobacter wolinii in cocultures with members of the genus Desulfovibrio (5). Clostridium blyantii oxidizes fatty acids with 4 to 11 carbon atoms in associations with several different hydrogen-utilizing bacteria (43). Acetic acid is associated with the lowest levels of energy released from the fatty acids degraded in syntrophic cooperations. Thus, the partial pressure of hydrogen has to be very low for this reaction to proceed, and the energy available is influenced by the electron acceptor used. Under standard conditions, acetate oxidation coupled to methane formation releases less energy than acetate oxidation coupled to sulfate reduction (AGO' = -31.0 kJ/mol and AGO' = -47.7 kJ/mol, respectively). The changes in free energy are also slightly greater at higher temperatures. Syntrophic oxidation of acetate has been observed with thermophilic and mesophilic cultures, as well as at moderate tem- peratures with both methanogenic and sulfate-reducing bacteria as the hydrogen-c...

In biogas processes, methane production from acetate proceeds by either aceticlastic methanogenesis or through syntrophic acetate oxidation (SAO). In the present study, the pathway for methane production from acetate was analysed; i) during a gradual increase of the ammonia concentration (final concentration 7 g NH(4)(+) -N/L) in a semi-continuous lab-scale anaerobic digester (4.3 L), operating at mesophilic temperature (37 degrees C) or ii) in diluted enrichment cultures (100 ml) experiencing a gradual increase in ammonia, sodium, potassium and propionic acid. The pathway for methane formation was determined by calculating the (14)CO(2)/(14)CH(4) ratio after incubating samples with (14)C-2-acetate. In the anaerobic digester, as well as in the enrichment cultures, the (14)CO(2)/(14)CH4 ratio clearly increased with increasing ammonium-nitrogen concentration, i.e. as the ammonia concentration increased, a shift from the aceticlastic mechanism to the syntrophic pathway occurred. The shift was very distinct and occurred as the NH(4)(+) -N concentration rose above 3 g/l. No shift in pathway was seen during increasing concentrations of sodium, potassium or propionic acid. The shift to SAO in the biogas digester resulted in a twofold decrease in the specific gas and methane yield.

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A mesophilic, syntrophic acetate-oxidizing bacterium, designated strain Sp3(T), was isolated from sludge from a mesophilic methanogenic digestor operating at a high ammonium concentration (6.4 g L(-1) NH(4)(+)-N). The strain showed acetate-oxidizing ability in cocultivation with a hydrogen-consuming methanogen. Comparative 16S rRNA gene sequence analysis confirmed that strain Sp3(T) belonged to the Firmicutes-Clostridia class. The most closely related species was Thermacetogenium phaeum (16S rRNA gene sequence identity 92%). Strain Sp3(T) used ethanol, betaine and lactate as carbon and electron sources and showed growth between 25 and 40 degrees C and pH 6.0 and 8.0. Based on the phylogenetic position and the physiological characteristics of strain Sp3(T), this new syntrophic, acetate-oxidizing bacterium is proposed as the new genus and species Syntrophaceticus schinkii, with Sp3(T) (=JCM 16669(T)) as the type strain. An isolate (strain Esp=JCM 16670) with high 16S rRNA gene sequence identity (99%) to syntrophic acetate-oxidizing Clostridium ultunense was also retrieved from the methanogenic digestor.

Methane (CH4) is produced as an end product from feed fermentation in the rumen. Yield of CH4 varies between individuals despite identical feeding conditions. To get a better understanding of factors behind the individual variation, 73 dairy cows given the same feed but differing in CH4 emissions were investigated with focus on fiber digestion, fermentation end products and bacterial and archaeal composition. In total 21 cows (12 Holstein, 9 Swedish Red) identified as persistent low, medium or high CH4 emitters over a 3 month period were furthermore chosen for analysis of microbial community structure in rumen fluid. This was assessed by sequencing the V4 region of 16S rRNA gene and by quantitative qPCR of targeted Methanobrevibacter groups. The results showed a positive correlation between low CH4 emitters and higher abundance of Methanobrevibacter ruminantium clade. Principal coordinate analysis (PCoA) on operational taxonomic unit (OTU) level of bacteria showed two distinct clusters (P < 0.01) that were related to CH4 production. One cluster was associated with low CH4 production (referred to as cluster L) whereas the other cluster was associated with high CH4 production (cluster H) and the medium emitters occurred in both clusters. The differences between clusters were primarily linked to differential abundances of certain OTUs belonging to Prevotella. Moreover, several OTUs belonging to the family Succinivibrionaceae were dominant in samples belonging to cluster L. Fermentation pattern of volatile fatty acids showed that proportion of propionate was higher in cluster L, while proportion of butyrate was higher in cluster H. No difference was found in milk production or organic matter digestibility between cows. Cows in cluster L had lower CH4/kg energy corrected milk (ECM) compared to cows in cluster H, 8.3 compared to 9.7 g CH4/kg ECM, showing that low CH4 cows utilized the feed more efficient for milk production which might indicate a more efficient microbial population or host genetic differences that is reflected in bacterial and archaeal (or methanogens) populations.

BackgroundSyntrophic acetate oxidation (SAO) is the predominant pathway for methane production in high ammonia anaerobic digestion processes. The bacteria (SAOB) occupying this niche and the metabolic pathway are poorly understood. Phylogenetic diversity and strict cultivation requirements hinder comprehensive research and discovery of novel SAOB. Most SAOB characterised to date are affiliated to the physiological group of acetogens. Formyltetrahydrofolate synthetase is a key enzyme of both acetogenic and SAO metabolism. The encoding fhs gene has therefore been identified as a suitable functional marker, using a newly designed primer pair. In this comparative study, we used a combination of terminal restriction fragment length polymorphism profiling, clone-based comparison, qPCR and Illumina amplicon sequencing to assess the bacterial community and acetogenic sub-community prevailing in high- and low-ammonia laboratory-scale digesters in order to delineate potential SAOB communities. Potential candidates identified were further tracked in a number of low-ammonia and high-ammonia laboratory-scale and large-scale digesters in order to reveal a potential function in SAO.ResultsAll methodical approaches revealed significant changes in the bacterial community composition concurrently with increasing ammonia and predominance of SAO. The acetogenic community under high ammonia conditions was revealed to be generally heterogeneous, but formed distinct phylogenetic clusters. The clusters differed clearly from those found under low-ammonia conditions and represented an acetogenic assemblage unique for biogas processes and recurring in a number of high-ammonia processes, indicating potential involvement in SAO.ConclusionsThe phylogenetic affiliation and population dynamics observed point to a key community, belonging mainly to the Clostridia class, in particular to the orders Clostridiales and Thermoanaerobacterales, which appear to specialise in SAO rather than being metabolically versatile. Overall, the results reported here provide evidence of functional importance of the bacterial families identified in high-ammonia systems and extend existing knowledge of bacterial and acetogenic assemblages at low and high ammonia levels. This information will be of help in monitoring and assessing the impacts on the SAOB community in order to identify characteristics of robust and productive high ammonia biogas processes.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0454-9) contains supplementary material, which is available to authorized users.

The bacterial and archaeal community structure was examined in two methanogenic anaerobic digestion processes degrading organic household waste at mesophilic (37 degrees C) and thermophilic (55 degrees C) temperatures. Analysis of bacterial clone libraries revealed a predominance of Bacteroidetes (34% of total clones) and Chloroflexi (27%) at the mesophilic temperature. In contrast, in the thermophilic clone library, the major group of clones were affiliated with Thermotogae (61%). Within the domain Archaea, the phyla Euryarchaeota and Crenarchaeota were both represented, the latter only at the mesophilic temperature. The dominating archaeons grouped with Methanospirillum and Methanosarcina species at the mesophilic and thermophilic temperature, respectively. Generally, there was a higher frequency of different sequences at the lower temperature, suggesting a higher diversity compared to the community present at the thermophilic temperature. Furthermore, it was not only the species richness that was affected by temperature, but also the phylogenetic distribution of the microbial populations.

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