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Abstract Background The integration of anaerobic digestion into bio-based industries can create synergies that help render anaerobic digestion self-sustaining. Two-stage digesters with separate acidification stages allow for the production of green hydrogen and short-chain fatty acids, which are promising industrial products. Heat shocks can be used to foster the production of these products, the practical applicability of this treatment is often not addressed sufficiently, and the presented work therefore aims to close this gap. Methods Batch experiments were conducted in 5 L double-walled tank reactors incubated at 37 °C. Short microwave heat shocks of 25 min duration and exposure times of 5–10 min at 80 °C were performed and compared to oven heat shocks. Pairwise experimental group differences for gas production and chemical parameters were determined using ANOVA and post–hoc tests. High-throughput 16S rRNA gene amplicon sequencing was performed to analyse taxonomic profiles. Results After heat–shocking the entire seed sludge, the highest hydrogen productivity was observed at a substrate load of 50 g/l with 1.09 mol H2/mol hexose. With 1.01 mol H2/mol hexose, microwave-assisted treatment was not significantly different from oven-based treatments. This study emphasised the better repeatability of heat shocks with microwave-assisted experiments, revealing low variation coefficients averaging 29%. The pre-treatment with microwaves results in a high predictability and a stronger microbial community shift to Clostridia compared to the treatment with the oven. The pre-treatment of heat shocks supported the formation of butyric acid up to 10.8 g/l on average, with a peak of 24.01 g/l at a butyric/acetic acid ratio of 2.0. Conclusion The results support the suitability of using heat shock for the entire seed sludge rather than just a small inoculum, making the process more relevant for industrial applications. The performed microwave-based treatment has proven to be a promising alternative to oven-based treatments, which ultimately may facilitate their implementation into industrial systems. This approach becomes economically sustainable with high-temperature heat pumps with a coefficient of performance (COP) of 4.3. Graphical abstract

Abstract Background The holistic characterization of different microbiomes in anaerobic digestion (AD) systems can contribute to a better understanding of these systems and provide starting points for bioengineering. The present study investigates the microbiome of 80 European full-scale AD systems. Operational, chemical and taxonomic data were thoroughly collected, analysed and correlated to identify the main drivers of AD processes. Results The present study describes chemical and operational parameters for a broad spectrum of different AD systems. With this data, Spearman correlation and differential abundance analyses were applied to narrow down the role of the individual microorganisms detected. The authors succeeded in further limiting the number of microorganisms in the core microbiome for a broad range of AD systems. Based on 16S rRNA gene amplicon sequencing, MBA03, Proteiniphilum, a member of the family Dethiobacteraceae, the genus Caldicoprobacter and the methanogen Methanosarcina were the most prevalent and abundant organisms identified in all digesters analysed. High ratios for Methanoculleus are often described for agricultural co-digesters. Therefore, it is remarkable that Methanosarcina was surprisingly high in several digesters reaching ratios up to 47.2%. The various statistical analyses revealed that the microorganisms grouped according to different patterns. A purely taxonomic correlation enabled a distinction between an acetoclastic cluster and a hydrogenotrophic one. However, in the multivariate analysis with chemical parameters, the main clusters corresponded to hydrolytic and acidogenic microorganisms, with SAOB bacteria being particularly important in the second group. Including operational parameters resulted in digester-type specific grouping of microbes. Those with separate acidification stood out among the many reactor types due to their unexpected behaviour. Despite maximizing the organic loading rate in the hydrolytic pretreatments, these stages turned into extremely robust methane production units. Conclusions From 80 different AD systems, one of the most holistic data sets is provided. A very distinct formation of microbial clusters was discovered, depending on whether taxonomic, chemical or operational parameters were combined. The microorganisms in the individual clusters were strongly dependent on the respective reference parameters. Graphical Abstract

Abstract Background Recent research articles indicate that direct interspecies electron transfer (DIET) is an alternative metabolic route for methanogenic archaea that improves microbial methane productivity. It has been shown that multiple conductive materials such as biochar can be supplemented to anaerobic digesters to increase the rate of DIET. However, the industrial applicability, as well as the impact of such supplements on taxonomic profiles, has not been sufficiently assessed to date. Results Seven industrial biogas plants were upgraded with a shock charge of 1.8 kg biochar per ton of reactor content and then 1.8 kg per ton were added to the substrate for one year. A joint analysis for all seven systems showed a decreasing trend for the concentration of acetic acid (p

Abstract Background Separating acidification and methanogenic steps in anaerobic digestion processes can help to optimize the process and contribute to producing valuable sub-products such as methane, hydrogen and organic acids. However, the full potential of this technology has not been fully explored yet. To assess the underlying fermentation process in more detail, a combination of high-throughput sequencing and proteomics on the acidification step of plant material (grass) at both mesophilic and thermophilic temperatures (37 and 55 °C, respectively) was applied for the first time. Results High-strength liquor from acidified grass biomass exhibited a low biodiversity, which differed greatly depending on temperature. It was dominated by Bacteroidetes and Firmicutes at 37 °C, and by Firmicutes and Proteobacteria at 55 °C. At the methane stage, Methanosaeta, Methanomicrobium and Methanosarcina proved to be highly sensitive to environmental changes as their abundance in the seed sludges dropped dramatically after transferring the seed sludges from the respective reactors into the experimental setup. Further, an increase in Actinobacteria coincided with reduced biogas production at the end of the experiment. Over 1700 proteins were quantified from the first cycle of acidification samples using label-free quantitative proteome analysis and searching protein databases. The most abundant proteins included an almost complete set of glycolytic enzymes indicating that the microbial population is basically engaged in the degradation and catabolism of sugars. Differences in protein abundances clearly separated samples into two clusters corresponding to culture temperature. More differentially expressed proteins were found under mesophilic (120) than thermophilic (5) conditions. Conclusion Our results are the first multi-omics characterisation of a two-stage biogas production system with separated acidification and suggest that screening approaches targeting specific taxa such as Methanosaeta, Methanomicrobium and Methanosarcina could be useful diagnostic tools as indicators of environmental changes such as temperature or oxidative stress or, as in the case of Actinobacteria, they could be used as a proxy of the gas production potential of anaerobic digesters. Metaproteome analyses only detected significant expression differences in mesophilic samples, whereas thermophilic samples showed more stable protein composition with an abundance of chaperones suggesting a role in protein stability under thermal stress.