With regard to social and environmental sustainability, second-generation biofuel and biogas production from lignocellulosic material provides considerable potential, since lignocellulose represents an inexhaustible, ubiquitous natural resource, and is therefore one important step towards independence from fossil fuel combustion. However, the highly heterogeneous structure and recalcitrant nature of lignocellulose restricts its commercial utilization in biogas plants. Improvements therefore rely on effective pretreatment methods to overcome structural impediments, thus facilitating the accessibility and digestibility of (ligno)cellulosic substrates during anaerobic digestion. While chemical and physical pretreatment strategies exhibit inherent drawbacks including the formation of inhibitory products, biological pretreatment is increasingly being advocated as an environmentally friendly process with low energy input, low disposal costs, and milder operating conditions. Nevertheless, the promising potential of biological pretreatment techniques is not yet fully exploited. Hence, we intended to provide a detailed insight into currently applied pretreatment techniques, with a special focus on biological ones for downstream processing of lignocellulosic biomass in anaerobic digestion.

Although Methanosarcinales are versatile concerning their methanogenic substrates, the ability ofMethanosarcina thermophilato use carbon dioxide (CO₂) for catabolic and anabolic metabolism was not proven until now. Here, we show thatM. thermophilaused CO₂to perform hydrogenotrophic methanogenesis in the presence as well as in the absence of methanol. During incubation with hydrogen, the methanogen utilized the substrates methanol and CO₂consecutively, resulting in a biphasic methane production. Growth exclusively from CO₂occurred slowly but reproducibly with concomitant production of biomass, verified by DNA quantification. Besides verification through multiple transfers into fresh medium, the identity of the culture was confirmed by 16s RNA sequencing, and the incorporation of carbon atoms from¹³CO₂into¹³CH₄molecules was measured to validate the obtained data. New insights into the physiology ofM. thermophilacan serve as reference for genomic analyses to link genes with metabolic features in uncultured organisms.

Substrates with high sulfate levels pose problems for biogas production as they allow sulfate reducing bacteria to compete with syntrophic and methanogenic members of the community. In addition, the end product of sulfate reduction, hydrogen sulfide, is toxic and corrosive. Here we show how sulfate addition affects physiological processes in a thermophilic methanogenic system by analyzing the carbon flow and the microbial community with quantitative PCR and amplicon sequencing of the 16s rRNA gene. A sulfate addition of 0.5 to 3 g/L caused a decline in methane production by 73–92%, while higher sulfate concentrations had no additional inhibitory effect. Generally, sulfate addition induced a shift in the composition of the microbial community towards a higher dominance ofFirmicutesand decreasing abundances ofBacteroidetesandEuryarchaeota. The abundance of methanogens (e.g.,MethanoculleusandMethanosarcina) was reduced, while sulfate reducing bacteria (especiallyCandidatus DesulforudisandDesulfotomaculum) increased significantly in presence of sulfate. The sulfate addition had a significant impact on the carbon flow within the system, shifting the end product from methane and carbon dioxide to acetate and carbon dioxide. Interestingly, methane production quickly resumed, when sulfate was no longer present in the system. Despite the strong impact of sulfate addition on the carbon flow and the microbial community structure during thermophilic biogas production, short-term process disturbances caused by unexpected introduction of sulfate may be overcome due to the high resilience of the engaged microorganisms.

PH is a central environmental factor influencing CH₄ production from organic substrates, as every member of the complex microbial community has specific pH requirements. Here, we show how varying pH conditions (5.0–8.5, phosphate buffered) and the application of a phosphate buffer per se induce shifts in the microbial community composition and the carbon flow during nine weeks of thermophilic batch digestion. Beside monitoring the methane production as well as volatile fatty acid concentrations, amplicon sequencing of the 16S rRNA gene was conducted. The presence of 100 mM phosphate resulted in reduced CH₄ production during the initial phase of the incubation, which was characterized by a shift in the dominant methanogenic genera from a mixed Methanosarcina and Methanoculleus to a pure Methanoculleus system. In buffered samples, acetate strongly accumulated in the beginning of the batch digestion and subsequently served as a substrate for methanogens. Methanogenesis was permanently inhibited at pH values ≤5.5, with the maximum CH₄ production occurring at pH 7.5. Adaptations of the microbial community to the pH variations included shifts in the archaeal and bacterial composition, as less competitive organisms with a broad pH range were able to occupy metabolic niches at unfavorable pH conditions.

Aromatic compounds like phenyl acids derived from lignocellulose degradation have been suspected to negatively influence biogas production processes. However, results on this topic are still inconclusive. To study phenyl acid formation in batch reactors during the start-up phase of anaerobic degradation, different amounts of straw from grain were mixed with mesophilic and thermophilic sludge, respectively. Molecular biological parameters were assessed using next-generation sequencing and qPCR analyses. Metagenomic predictions were done via the program, piphillin. Methane production, concentrations of phenylacetate, phenylpropionate, phenylbutyrate, and volatile fatty acids were monitored chromatographically. Methanosarcina spp. was the dominant methanogen when high straw loads were effectively degraded, and thus confirmed its robustness towards overload conditions. Several microorganisms correlated negatively with phenyl acids; however, a negative effect, specifically on methanogens, could not be proven. A cascade-like increase/decrease from phenylacetate to phenylpropionate, and then to phenylbutyrate could be observed when methanogenesis was highly active. Due to these results, phenylacetate was shown to be an early sign for overload conditions, whereas an increase in phenylbutyrate possibly indicated a switch from degradation of easily available to more complex substrates. These dynamics during the start-up phase might be relevant for biogas plant operators using complex organic wastes for energy exploitation.