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.

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. 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. 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.

Lignocellulosic residues, such as straw, are currently considered as candidates for biogas production. Therefore, straw fermentations were performed to quantitatively estimate methane yields and cell counts, as well as to qualitatively determine the microbiome. Six fully automated, continuously stirred biogas reactors were used: three mesophilic (41 °C) and three thermophilic (58 °C). They were fed every 8 h with milled wheat straw suspension in a defined, buffered salt solution, called ‘synthetic manure’. Total reflection X-ray fluorescence spectrometry analyses showed nickel and tungsten deficiency in the straw suspension. Supplementation of nickel and subsequently tungsten, or with an increasing combined dosage of both elements, resulted in a final concentration of approximately 0.1 mg/L active, dissolved tungsten ions, which caused an increase of the specific methane production, up to 63% under mesophilic and 31% under thermophilic conditions. That is the same optimal range for pure cultures of methanogens or bacteria found in literature. A simultaneous decrease of volatile fatty acids occurred. The Ni/W effect occurred with all three organic loading rates, being 4.5, 7.5, and 9.0 g volatile solids per litre and day, with a concomitant hydraulic retention time of 18, 10, or 8 days, respectively. A maximum specific methane production of 0.254 m³ CH₄, under standard temperature and pressure per kg volatile solids (almost 90% degradation), was obtained. After the final supplementation of tungsten, the cell counts of methanogens increased by 300%, while the total microbial cell counts increased by only 3–62%. The mesophilic methanogenic microflora was shifted from the acetotrophic Methanosaeta to the hydrogenotrophic Methanoculleus (85%) by tungsten, whereas the H₂-CO₂-converter, Methanothermobacter, always dominated in the thermophilic fermenters.

The anaerobic digestion of swine manure was performed for more than 2 years in a biogas pilot plant with cereal residues as a mono-input, either by a simple intermittent substrate feeding or by feeding with an automated “autopilot” system under the direction of a Fuzzy logic control (FLC) system, working with a closed-loop feedback control. The pilot plant of the University of Applied Sciences in Nordhausen consisted of a 2.5 m³ dosage tank, a 2.5 m³ digestate tank, and a 1 m³ biogas reactor. Only three control parameters were used for FLC: pH, methane %, and the specific gas production rate (GPR) related to the organic loading rate (OLR), that is GPR/OLR m³ biogas/(kg_VS d), vs = volatile solids. The specific GPR was referred to the OLR of the last feeding every 8 h in terms of kg_VS/(m³ d). In test period I without an FLC system, a safe process with just an OLR of 4 kg_VS/(m³ d) was reached, followed by an overloading and reactor disturbance at ≤6.3 kg_VS/(m³ d) as indicated by acidification with volatile fatty acids up to 25,000 mg/L. However, test period II (585 trial days) with an integrated FLC system allowed a safe OLR up to 11 kg_VS/(m³ d). Apparently, the microbes themselves directed the speed of substrate feeding by the dynamics of their substrate turnover and by the closed loop feedback control, while the three FLC parameters prevented acidification. Therefore, the application of FLC enabled a doubling of the throughput for a biogas reactor in the same time with a ‘turbo speed’. The concomitant hydraulic residence time (HRT) of only 10 days reduced the stirring and heating costs. The usage of an FLC system should open the door for networked biogas production to enable flexible biogas production on demand.