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Abstract Background To elucidate biogas microbial communities and processes, the application of high-throughput DNA analysis approaches is becoming increasingly important. Unfortunately, generated data can only partialy be interpreted rudimentary since databases lack reference sequences. Results Novel cellulolytic, hydrolytic, and acidogenic/acetogenic Bacteria as well as methanogenic Archaea originating from different anaerobic digestion communities were analyzed on the genomic level to assess their role in biomass decomposition and biogas production. Some of the analyzed bacterial strains were recently described as new species and even genera, namely Herbinix hemicellulosilytica T3/55T, Herbinix luporum SD1DT, Clostridium bornimense M2/40T, Proteiniphilum saccharofermentans M3/6T, Fermentimonas caenicola ING2-E5BT, and Petrimonas mucosa ING2-E5AT. High-throughput genome sequencing of 22 anaerobic digestion isolates enabled functional genome interpretation, metabolic reconstruction, and prediction of microbial traits regarding their abilities to utilize complex bio-polymers and to perform specific fermentation pathways. To determine the prevalence of the isolates included in this study in different biogas systems, corresponding metagenome fragment mappings were done. Methanoculleus bourgensis was found to be abundant in three mesophilic biogas plants studied and slightly less abundant in a thermophilic biogas plant, whereas Defluviitoga tunisiensis was only prominent in the thermophilic system. Moreover, several of the analyzed species were clearly detectable in the mesophilic biogas plants, but appeared to be only moderately abundant. Among the species for which genome sequence information was publicly available prior to this study, only the species Amphibacillus xylanus, Clostridium clariflavum, and Lactobacillus acidophilus are of importance for the biogas microbiomes analyzed, but did not reach the level of abundance as determined for M. bourgensis and D. tunisiensis. Conclusions Isolation of key anaerobic digestion microorganisms and their functional interpretation was achieved by application of elaborated cultivation techniques and subsequent genome analyses. New isolates and their genome information extend the repository covering anaerobic digestion community members.

High ammonia levels inhibit anaerobic digestion (AD) process and bioaugmentation with ammonia tolerant methanogenic culture is proposed to alleviate ammonia inhibition. In the current study, hydrogenotrophic Methanoculleus bourgensis was bioaugmented in an ammonia-inhibited continuous reactor fed mainly with microalgae (a protein-rich biomass), at extreme ammonia levels (i.e. 11 g NH 4 +-N L−1). The results showed 28% increase in methane production immediately after bioaugmentation. Moreover, volatile fatty acids decreased rapidly from more than 5 g L−1 to around 1 g L−1, with a fast reduction in propionate concentration. High throughput 16s rRNA gene sequencing demonstrated that the bioaugmented M. bourgensis doubled its relative abundance after bioaugmentation. "Microbiological domino effect", triggered by the bioaugmented M. bourgensis establishing a newly efficient community, was proposed as the working mechanism of the successful bioaugmentation. Additionally, a strong aceticlastic methanogenesis was found at the end of the experiment evidenced by the dominant presence of Methanosarcina soligelidi and the low abundance of syntrophic acetate oxidising bacteria at the final period. Overall, for the first time, this study proved the positive effect of bioaugmentation on ammonia inhibition alleviation of the microalgae-dominating fed reactor, paving the way of efficient utilization of other protein-rich substrates in the future. Image 1 • Bioaugmentation alleviated ammonia inhibition of a reactor fed with microalgae. • Bioaugmented M. bourgensis increased methane yield by 28% and decreased VFA by 80%. • Reduction of hydrogen pressure and propionate is crucial in alleviating inhibition. • The bioaugmentation success lies on the triggered "microbiological domino effect". • M. soligelidi was the dominant methanogen three HRTs after bioaugmentation. [ABSTRACT FROM AUTHOR]

Objective: To isolate and identify new methanogens from the rumen of Holstein steers in Korea. Methods: Representative rumen contents were obtained from three ruminally cannulated Holstein steers (793±8 kg). Pre-reduced media were used for the growth and isolation of methanogens. Optimum growth temperature, pH, and sodium chloride (NaCl) concentration as well as substrate utilization and antibiotic tolerance were investigated to determine the physiological characteristics of the isolated strain. Furthermore, the isolate was microscopically studied for its morphology. Polymerase chain reaction of 16S rRNA and mcrA gene-based amplicons was used for identification. Results: One strain designated as KOR-2 was isolated and found to be a non-motile irregular coccus with a diameter of 0.2 to 0.5 µm. KOR-2 utilized H2+CO2 and formate but was unable to metabolize acetate, methanol, trimethylamine, 2-propanol, and isobutanol for growth and methane production. The optimum temperature and pH for the growth of KOR-2 were 38°C and 6.8 to 7.0, respectively, while the optimum NaCl concentration essential for KOR-2 growth was 1.0% (w/v). KOR-2 tolerated ampicillin, penicillin G, kanamycin, spectromycin, and tetracycline. In contrast, the cell growth was inhibited by chloramphenicol. Phylogenetic analysis of 16S rRNA and mcrA genes revealed the relatedness between KOR-2 and Methanoculleus bourgensis. Conclusion: Based on the physiological and phylogenetic characteristics, KOR-2 was thought to be a new strain within the genus Methanoculleus and named Methanoculleus bourgensis KOR-2. [ABSTRACT FROM AUTHOR]

Background: To elucidate biogas microbial communities and processes, the application of high-throughput DNA analysis approaches is becoming increasingly important. Unfortunately, generated data can only partialy be interpreted rudimentary since databases lack reference sequences. Results: Novel cellulolytic, hydrolytic, and acidogenic/acetogenic Bacteria as well as methanogenic Archaea originating from different anaerobic digestion communities were analyzed on the genomic level to assess their role in biomass decomposition and biogas production. Some of the analyzed bacterial strains were recently described as new species and even genera, namely Herbinix hemicellulosilytica T3/55T, Herbinix luporum SD1DT, Clostridium bornimense M2/40T, Proteiniphilum saccharofermentans M3/6T, Fermentimonas caenicola ING2-E5BT, and Petrimonas mucosa ING2-E5AT. High-throughput genome sequencing of 22 anaerobic digestion isolates enabled functional genome interpretation, metabolic reconstruction, and prediction of microbial traits regarding their abilities to utilize complex bio-polymers and to perform specific fermentation pathways. To determine the prevalence of the isolates included in this study in different biogas systems, corresponding metagenome fragment mappings were done. Methanoculleus bourgensis was found to be abundant in three mesophilic biogas plants studied and slightly less abundant in a thermophilic biogas plant, whereas Defluviitoga tunisiensis was only prominent in the thermophilic system. Moreover, several of the analyzed species were clearly detectable in the mesophilic biogas plants, but appeared to be only moderately abundant. Among the species for which genome sequence information was publicly available prior to this study, only the species Amphibacillus xylanus, Clostridium clariflavum, and Lactobacillus acidophilus are of importance for the biogas microbiomes analyzed, but did not reach the level of abundance as determined for M. bourgensis and D. tunisiensis. Conclusions: Isolation of key anaerobic digestion microorganisms and their functional interpretation was achieved by application of elaborated cultivation techniques and subsequent genome analyses. New isolates and their genome information extend the repository covering anaerobic digestion community members. [ABSTRACT FROM AUTHOR]

Background: Decomposition of biomass for biogas production can be practiced under wet and dry fermentation conditions. In contrast to the dry fermentation technology, wet fermentation is characterized by a high liquid content and a relatively low total solid content. In this study, the composition and functional potential of a biogas-producing microbial community in an agricultural biogas reactor operating under wet fermentation conditions was analyzed by a metagenomic approach applying 454-pyrosequencing. The obtained metagenomic dataset and corresponding 16S rRNA gene amplicon sequences were compared to the previously sequenced comparable metagenome from a dry fermentation process, meeting explicitly identical boundary conditions regarding sample and community DNA preparation, sequencing technology, processing of sequence reads and data analyses by bioinformatics tools. Results: High-throughput metagenome sequencing of community DNA from the wet fermentation process applying the pyrosequencing approach resulted in 1,532,780 reads, with an average read length of 397 bp, accounting for approximately 594 million bases of sequence information in total. Taxonomic comparison of the communities from wet and dry fermentation revealed similar microbial profiles with Bacteria being the predominant superkingdom, while the superkingdom Archaea was less abundant. In both biogas plants, the bacterial phyla Firmicutes, Bacteroidetes, Spirochaetes and Proteobacteria were identified with descending frequencies. Within the archaeal superkingdom, the phylum Euryarchaeota was most abundant with the dominant class Methanomicrobia. Functional profiles of the communities revealed that environmental gene tags representing methanogenesis enzymes were present in both biogas plants in comparable frequencies. 16S rRNA gene amplicon high-throughput sequencing disclosed differences in the sub-communities comprising methanogenic Archaea between both processes. Fragment recruitments of metagenomic reads to the reference genome of the archaeon Methanoculleus bourgensis MS2T revealed that dominant methanogens within the dry fermentation process were highly related to the reference. Conclusions: Although process parameters, substrates and technology differ between the wet and dry biogas fermentations analyzed in this study, community profiles are very similar at least at higher taxonomic ranks, illustrating that core community taxa perform key functions in biomass decomposition and methane synthesis. Regarding methanogenesis, Archaea highly related to the type strain M. bourgensis MS2T dominate the dry fermentation process, suggesting the adaptation of members belonging to this species to specific fermentation process parameters. [ABSTRACT FROM AUTHOR]