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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.

The effects of the addition of an acetate-degrading enrichment culture to an anaerobic digester with a stagnating biogas production were investigated. Initially, a thermophilic batch-operated lab-scale digester was inoculated with the diluted fermenter sludge of a biogas plant, and process parameters including the concentration of volatile fatty acids (VFAs) and gases in the headspace were measured. After a phase of high gas production, a stagnation of biogas production followed for a further 30 days. An acetate enrichment culture was added 34 days after the commencement of the experiment and this resulted in a sharp decrease in the concentrations of accumulated VFAs and an increase in total biogas and CH₄ production. An archaeon with a sequence similarity of 98% to Methanosarcina sp. and the ability to degrade acetic acid was introduced with the enrichment culture and is proposed to have been the driving factor for the changes that occurred within a few days to the process.

Five differently developed soils aged 6, 35, 80, 150, and >5000 years with the same bedrock and the same (current) climate conditions were chosen to assess abiotic and enzymatic properties as well as methanogenic and methanotrophic activities. Most abiotic properties (dry weight, pH, soil organic matter, and ammonium content), enzyme activities (dehydrogenase [DH] activity, ammonification [AM] rate, dimethylsulfoxide reduction), and potential methane oxidation (PoMO) per gram of dry weight (DW) increased with soil age. In contrast, potential methane production (PoMP) as well as the nitrate content per gram of DW and most enzymatic properties per gram of soil organic matter (SOM) did not increase with soil age but reached its maximum in the middle-aged soils (80–150 years). Our results show that (i) microbial activity does not consequently increase with SOM content/soil age; (ii) methane production can be measured in undeveloped soils, whereas methane oxidation is more restricted to fully developed soils; and (iii) certain soil modifications (change in water content, ammonium addition) could influence potential methane production/oxidation. When considering the concurrent release of raw soil because of the melting of perpetual ice, these data could help to better understand and assess the consequences of global change.

Although Methanosarcinales are versatile concerning their methanogenic substrates, the ability of Methanosarcina thermophila to use carbon dioxide (CO2) for catabolic and anabolic metabolism was not proven until now. Here, we show that M. thermophila used CO2 to 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 CO2 consecutively, resulting in a biphasic methane production. Growth exclusively from CO2 occurred 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 from 13CO2 into 13CH4 molecules was measured to validate the obtained data. New insights into the physiology of M. thermophila can serve as reference for genomic analyses to link genes with metabolic features in uncultured organisms.

Background Proteinaceous wastes exhibit high theoretical methane yields and their residues are considered valuable fertilisers. The routine anaerobic degradation of proteins often raises problems like high aromatic compound concentrations caused by the entry of aromatic amino acids into the system. A profound investigation of the consequences of aromatic compound exposure on various microorganisms, which cascade-like and interdependently degrade complex molecules to biogas, is still pending. Results In mesophilic samples, methane was predominantly produced via acetoclastic methanogenesis. The highest positive correlation was observed between phenylacetate (PAA) and Psychrobacter spp. and between phenylpropionate (PPA) and Haloimpatiens spp. Moreover, Syntrophus spp. negatively correlated with PAA (Spearman’s rank correlations coefficient (rs) = − 0.46, p < 0.05) and PPA concentrations (rs = − 0.44, p < 0.05) and was also associated with anaerobic benzene ring cleavage. In thermophilic samples, acetate was predominantly oxidised by Tepidanaerobacter spp. or Syntrophaceticus spp. in syntrophic association with a hydrogenotrophic methanogen. The genera Sedimentibacter and Syntrophaceticus correlated positively with both PAA and PPA concentrations. Moreover, Sedimentibacter spp., Tepidanaerobacter spp., Acetomicrobium spp., and Sporanaerobacter spp. were significant LEfSe (linear discriminant analysis effect size) biomarkers for high meso- as well as thermophilic phenyl acid concentrations. Direct negative effects of phenyl acids on methanogenic properties could not be proven. Conclusions Anaerobic phenyl acid formation is not restricted to specific microbial taxa, but rather done by various meso- and thermophilic bacteria. The cleavage of the highly inert benzene ring is possible in methanogenic batch reactors—at least in mesophilic fermentation processes. The results indicated that phenyl acids rather affect microorganisms engaged in preceding degradation steps than the ones involved in methanogenesis.

Background Lignin intermediates resulting from lignocellulose degradation have been suspected to hinder anaerobic mineralisation of organic materials to biogas. Phenyl acids like phenylacetate (PAA) are early detectable intermediates during anaerobic digestion (AD) of aromatic compounds. Studying the phenyl acid formation dynamics and concomitant microbial community shifts can help to understand the microbial interdependencies during AD of aromatic compounds and may be beneficial to counteract disturbances. Results The length of the aliphatic side chain and chemical structure of the benzene side group(s) had an influence on the methanogenic system. PAA, phenylpropionate (PPA), and phenylbutyrate (PBA) accumulations showed that the respective lignin intermediate was degraded but that there were metabolic restrictions as the phenyl acids were not effectively processed. Metagenomic analyses confirmed that mesophilic genera like Fastidiosipila or Syntrophomonas and thermophilic genera like Lactobacillus, Bacillus, Geobacillus, and Tissierella are associated with phenyl acid formation. Acetoclastic methanogenesis was prevalent in mesophilic samples at low and medium overload conditions, whereas Methanoculleus spp. dominated at high overload conditions when methane production was restricted. In medium carbon load reactors under thermophilic conditions, syntrophic acetate oxidation (SAO)-induced hydrogenotrophic methanogenesis was the most important process despite the fact that acetoclastic methanogenesis would thermodynamically be more favourable. As acetoclastic methanogens were restricted at medium and high overload conditions, syntrophic acetate oxidising bacteria and their hydrogenotrophic partners could step in for acetate consumption. Conclusions PAA, PPA, and PBA were early indicators for upcoming process failures. Acetoclastic methanogens were one of the first microorganisms to be impaired by aromatic compounds, and shifts to syntrophic acetate oxidation coupled to hydrogenotrophic methanogenesis occurred in thermophilic reactors. Previously assumed associations of specific meso- and thermophilic genera with anaerobic phenyl acid formation could be confirmed.

Since the start of the 2019 pandemic, wastewater-based epidemiology (WBE) has proven to be a valuable tool for monitoring the prevalence of SARS-CoV-2. With methods and infrastructure being settled, it is time to expand the potential of this tool to a wider range of pathogens. We used over 500 archived RNA extracts from a WBE program for SARS-CoV-2 surveillance to monitor wastewater from 11 treatment plants for the presence of influenza and norovirus twice a week during the winter season of 2021/2022. Extracts were analyzed via digital PCR for influenza A, influenza B, norovirus GI, and norovirus GII. Resulting viral loads were normalized on the basis of NH4-N. Our results show a good applicability of ammonia-normalization to compare different wastewater treatment plants. Extracts originally prepared for SARS-CoV-2 surveillance contained sufficient genomic material to monitor influenza A, norovirus GI, and GII. Viral loads of influenza A and norovirus GII in wastewater correlated with numbers from infected inpatients. Further, SARS-CoV-2 related non-pharmaceutical interventions affected subsequent changes in viral loads of both pathogens. In conclusion, the expansion of existing WBE surveillance programs to include additional pathogens besides SARS-CoV-2 offers a valuable and cost-efficient possibility to gain public health information.

Background Substrate spectra for anaerobic digestion have been broadened in the past decade, inter alia, due to the application of different pretreatment strategies and now include materials rich in lignocellulose, protein, and/or fat. The application of these substrates, however, also entails risks regarding the formation of undesired by-products, among which phenolic compounds are known to accumulate under unfavorable digestion conditions. Methods Different states of overload were simulated in batch experiments while reviewing the generation of phenyl acids out of different lab-use substrates in order to evaluate the impact on biogas and methane production as well as some additional process performance parameters under defined laboratory conditions. Investigations were conducted under both mesophilic and thermophilic conditions. Results It could be shown that the tested input materials led to the formation of phenyl acids in a substrate-dependent manner with the formation itself being less temperature driven. Once formed, the formation of phenyl acids turned out to be a reversible process. Conclusions Although a mandatory negative impact of phenyl acids per se on the anaerobic digestion process in general and the methanogenesis process in particular could not be proven, phenyl acids, however, seem to play an important role in the microbial response to overloaded biogas systems.

The importance and need of renewable-based, sustainable feedstocks increased in recent years. Lignin-derived monomers have high potential, energetic and economic value in the microbial bioconversion to valuable biomolecules. The bacterium Paraburkholderia aromaticivorans AR20-38 produces a remarkable yield of vanillic acid from ferulic acid at moderate and low temperatures and is therefore a good candidate for biotechnological applications. To understand this bioconversion process on a molecular level, a transcriptomic study during the bioconversion process was conducted to elucidate gene expression patterns. Differentially expressed genes, cellular transporters as well as transcriptional factors involved in the bioconversion process could be described. Additional enzymes known for xenobiotic degradation were differentially expressed and a potential membrane vesicle mechanism was detected. The bioconversion mechanism on a transcriptional level of P. aromaticivorans could be elucidated and results can be used for strain optimization. Additionally, the transcriptome study showed the high potential of the strain for other degradation applications.KeypointsP. aromaticivorans AR20-38 differentially regulates three main enzymes for the bioconversion of ferulic acid to vanillic acid.ATP binding cassette transporters, major facilitator superfamily transporters and outer membrane vesicles are putative transport routes for catabolic enzymes.Regulated degradation enzymes indicate xenobiotic degradation potential

The contribution of cold-adapted yeasts to the emerging field of lignin biovalorization has not yet been studied. The red-pigmented basidiomycetous yeast strain Rhodosporidiobolus colostri DBVPG 10655 was examined for its potential to degrade five selected lignin-derived aromatic monomers (syringic acid, p-coumaric acid, 4-hydroxybenzoic acid, ferulic acid, and vanillic acid). The strain utilized p-coumaric acid, 4-hydroxybenzoic acid, and ferulic acid not only as the sole carbon source; full biodegradation occurred also in mixtures of multiple monomers. Vanillic acid was not utilized as the sole carbon source, but was degraded in the presence of p-coumaric acid, 4-hydroxybenzoic acid, and ferulic acid. Syringic acid was utilized neither as the sole carbon source nor in mixtures of compounds. Biodegradation of lignin-derived aromatic monomers was detected over a broad temperature range (1–25 °C), which is of ecological significance and of biotechnological relevance.