Three novel, facultatively anaerobic bacteria of the family Porphyromonadaceae (phylum Bacteroidetes) were isolated from mesophilic laboratory-scale biogas reactors. The strains were Gram-negative rods. Optimal growth occurred between 35 and 45 °C and at pH 7.1–7.8. The main fermentation products were acetic and propionic acids. The predominant fatty acid in all strains was anteiso-C15 : 0, and the only respiratory quinone detected was menaquinone MK-8. 16S rRNA gene sequence comparison indicated that strains M3/6T and ING2-E5BT were most closely related to the type strain of Proteiniphilum acetatigenes, with sequence similarities of 97.3 and 94.5 %. Strain ING2-E5AT showed the closest affiliation to the type strain of Petrimonas sulfuriphila, with 97 % sequence identity. DNA–DNA hybridization of strain M3/6T and ING2-E5AT with the most closely related type strains showed 43.3–45.6 and 23.8–25.7 % relatedness, respectively, which supports the conclusion that both isolates represent novel species. Phylogenetic analysis and comparison of cellular fatty acid patterns indicated that strain ING2-E5BT cannot be classified as a member of any previously described genus. Therefore, because of the physiological, genotypic and chemotaxonomic differences, it is proposed to designate novel species within the genera Proteiniphilum and Petrimonas, Proteiniphilum saccharofermentans sp. nov. (type strain M3/6T = DSM 28694T = CECT 8610T = LMG 28299T) and Petrimonas mucosa sp. nov. (type strain ING2-E5AT = DSM 28695T = CECT 8611T), and a novel species of a new genus, Fermentimonas caenicola gen. nov., sp. nov. (type strain of Fermentimonas caenicola is ING2-E5BT = DSM 28696T = CECT 8609T = LMG 28429T). In addition, an emended description of the genus Proteiniphilum is provided.

Volatile fatty acid intoxication (acidosis), a common process failure recorded in anaerobic reactors, leads to drastic losses in methane production. Unfortunately, little is known about the microbial mechanisms underlining acidosis and the potential to recover the process. In this study, triplicate mesophilic anaerobic reactors of 100 L were exposed to acidosis resulting from an excessive feeding with sugar beet pulp and were compared to a steady-state reactor. Stable operational conditions at the beginning of the experiment initially led to similar microbial populations in the four reactors, as revealed by 16S rRNA gene T-RFLP and high-throughput amplicon sequencing. Bacteroidetes and Firmicutes were the two dominant phyla, and although they were represented by a high number of operational taxonomic units, only a few were dominant. Once the environment became deterministic (selective pressure from an increased substrate feeding), microbial populations started to diverge between the overfed reactors. Interestingly, most of bacteria and archaea showed redundant functional adaptation to the changing environmental conditions. However, the dominant Bacteroidales were resistant to high volatile fatty acids content and low pH. The severe acidosis did not eradicate archaea and a clear shift in archaeal populations from acetotrophic to hydrogenotrophic methanogenesis occurred in the overfed reactors. After 11 days of severe acidosis (pH 5.2 ± 0.4), the process was quickly recovered (restoration of the biogas production with methane content above 50 %) in the overfed reactors, by adjusting the pH to around 7 using NaOH and NaHCO3. In this study we show that once the replicate reactors are confronted with sub-optimal conditions, their microbial populations start to evolve differentially. Furthermore the alterations of commonly used microbial parameters to monitor the process, such as richness, evenness and diversity indices were unsuccessful to predict the process failure. At the same time, we tentatively propose the replacement of the dominant Methanosaeta sp. in this case by Methanoculleus sp., to be a potential warning indicator of acidosis.

Knowledge of the microbial consortia participating in the generation of biogas, especially in methane formation, is still limited. To overcome this limitation, the methanogenic archaeal communities in six full-scale biogas plants supplied with different liquid manures and renewable raw materials as substrates were analyzed by a polyphasic approach. Fluorescencein situhybridization (FISH) was carried out to quantify the methanogenicArchaeain the reactor samples. In addition, quantitative real-time PCR (Q-PCR) was used to support and complete the FISH analysis. Five of the six biogas reactors were dominated by hydrogenotrophicMethanomicrobiales. The average values were between 60 to 63% of archaeal cell counts (FISH) and 61 to 99% of archaeal 16S rRNA gene copies (Q-PCR). Within this order,Methanoculleuswas found to be the predominant genus as determined by amplified rRNA gene restriction analysis. The aceticlastic familyMethanosaetaceaewas determined to be the dominant methanogenic group in only one biogas reactor, with average values for Q-PCR and FISH between 64% and 72%. Additionally, in three biogas reactors hitherto uncharacterized but potentially methanogenic species were detected. They showed closest accordance with nucleotide sequences of the hitherto unclassified CA-11 (85%) and ARC-I (98%) clusters. These results point to hydrogenotrophic methanogenesis as a predominant pathway for methane synthesis in five of the six analyzed biogas plants. In addition, a correlation between the absence ofMethanosaetaceaein the biogas reactors and high concentrations of total ammonia (sum of NH₃and NH₄⁺) was observed.

One of the most promising technologies to sustainably produce energy and to mitigate greenhouse gas emissions from combustion of fossil energy carriers is the anaerobic digestion and biomethanation of organic raw material and waste towards biogas by highly diverse microbial consortia. In this context, the microbial systems ecology of thermophilic industrial-scale biogas plants is poorly understood. The microbial community structure of an exemplary thermophilic biogas plant was analyzed by a comprehensive approach comprising the analysis of the microbial metagenome and metatranscriptome complemented by the cultivation of hydrolytic and acido-/acetogenic Bacteria as well as methanogenic Archaea. Analysis of metagenome-derived 16S rRNA gene sequences revealed that the bacterial genera Defluviitoga (5.5 %), Halocella (3.5 %), Clostridium sensu stricto (1.9 %), Clostridium cluster III (1.5 %), and Tepidimicrobium (0.7 %) were most abundant. Among the Archaea, Methanoculleus (2.8 %) and Methanothermobacter (0.8 %) were predominant. As revealed by a metatranscriptomic 16S rRNA analysis, Defluviitoga (9.2 %), Clostridium cluster III (4.8 %), and Tepidanaerobacter (1.1 %) as well as Methanoculleus (5.7 %) mainly contributed to these sequence tags indicating their metabolic activity, whereas Hallocella (1.8 %), Tepidimicrobium (0.5 %), and Methanothermobacter (<0.1 %) were transcriptionally less active. By applying 11 different cultivation strategies, 52 taxonomically different microbial isolates representing the classes Clostridia, Bacilli, Thermotogae, Methanomicrobia and Methanobacteria were obtained. Genome analyses of isolates support the finding that, besides Clostridium thermocellum and Clostridium stercorarium, Defluviitoga tunisiensis participated in the hydrolysis of hemicellulose producing ethanol, acetate, and H2/CO2. The latter three metabolites are substrates for hydrogentrophic and acetoclastic archaeal methanogenesis. Obtained results showed that high abundance of microorganisms as deduced from metagenome analysis does not necessarily indicate high transcriptional or metabolic activity, and vice versa. Additionally, it appeared that the microbiome of the investigated thermophilic biogas plant comprised a huge number of up to now unknown and insufficiently characterized species.