The biomethane formation from 4 H₂ + CO₂ by pure cultures of two methanogens, Methanocaldococcus fervens and Methanobacterium thermophilum, has been studied. The goal of the study was to understand the regulation of the enzymatic steps associated with biomethane biosynthesis by H₂, using metagenomic, pan-genomic, and transcriptomic approaches. Methanogenesis in the autotrophic methanogen M. fervens could be easily “switched off” and “switched on” by H₂/CO₂ within about an hour. In contrast, the heterotrophic methanogen M. thermophilum was practically insensitive to the addition of the H₂/CO₂ trigger although this methanogen also converted H₂/CO₂ to CH₄. From practical points of view, the regulatory function of H₂/CO₂ suggests that in the power-to-gas (P2G) renewable excess electricity conversion and storage systems, the composition of the biomethane-generating methanogenic community is essential for sustainable operation. In addition to managing the specific hydrogenotrophic methanogenesis biochemistry, H₂/CO₂ affected several, apparently unrelated, metabolic pathways. The redox-regulated overall biochemistry and symbiotic relationships in the methanogenic communities should be explored in order to make the P2G technology more efficient. Key points: • Hydrogenotrophic methanogens may respond distinctly to H₂/CO₂ in bio-CH₄ formation. • H₂/CO₂ can also activate metabolic routes, which are apparently unrelated to methanogenesis. • Sustainable conversion of the fluctuating renewable electricity to bio-CH₄ is an option.

The recent human Monkeypox outbreak underlined the importance of studying basic biology of orthopoxviruses. However, the transcriptome of its causative agent has not been investigated before neither with short-, nor with long-read sequencing approaches. This Oxford Nanopore long-read RNA-Sequencing dataset fills this gap. It will enable the in-depth characterization of the transcriptomic architecture of the monkeypox virus, and may even make possible to annotate novel host transcripts. Moreover, our direct cDNA and native RNA sequencing reads will allow the estimation of gene expression changes of both the virus and the host cells during the infection. Overall, our study will lead to a deeper understanding of the alterations caused by the viral infection on a transcriptome level.

Biogas production through co-digestion of second and third generation substrates is an environmentally sustainable approach. Green willow biomass, chicken manure waste and microalgae biomass substrates were combined in the anaerobic digestion experiments. Biochemical methane potential test showed that biogas yields of co-digestions were significantly higher compared to the yield when energy willow was the sole substrate. To scale up the experiment continuous stirred-tank reactors (CSRTs) are employed, digestion parameters are monitored. Furthermore, genome-centric metagenomics approach was employed to gain functional insight into the complex anaerobic decomposing process. This revealed the importance of Firmicutes, Actinobacteria, Proteobacteria and Bacteroidetes phyla as major bacterial participants, while Methanomicrobia and Methanobacteria represented the archaeal constituents of the communities. The bacterial phyla were shown to perform the carbohydrate hydrolysis. Among the representatives of long-chain carbohydrate hydrolysing microbes Bin_61: Clostridia is newly identified metagenome assembled genome (MAG) and Bin_13: DTU010 sp900018335 is common and abundant in all CSTRs. Methanogenesis was linked to the slow-growing members of the community, where hydrogenotrophic methanogen species Methanoculleus (Bin_10) and Methanobacterium (Bin_4) predominate. A sensitive balance between H₂ producers and consumers was shown to be critical for stable biomethane production and efficient waste biodegradation.

This study employed both short-read sequencing (SRS, Illumina) and long-read sequencing (LRS Oxford Nanopore Technologies) platforms to conduct a comprehensive analysis of the equid alphaherpesvirus 1 (EHV-1) transcriptome. The study involved the annotation of canonical mRNAs and their transcript variants, encompassing transcription start site (TSS) and transcription end site (TES) isoforms, in addition to alternative splicing forms. Furthermore, the study revealed the presence of numerous non-coding RNA (ncRNA) molecules, including intergenic and antisense transcripts, produced by EHV-1. An intriguing finding was the abundant production of chimeric transcripts, some of which potentially encode fusion polypeptides. Moreover, EHV-1 exhibited a greater incidence of transcriptional overlaps and splicing compared to related viruses. It is noteworthy that many genes have their unique TESs along with the co-terminal transcription ends, a characteristic scarcely seen in other alphaherpesviruses. The study also identified transcripts that overlap the replication origins of the virus. Moreover, a novel ncRNA, referred to as NOIR, was found to intersect with the 5′-ends of longer transcript isoform specified by the major transactivator genes ORF64 and ORF65, surrounding the OriL. These findings together imply the existence of a key regulatory mechanism that governs both transcription and replication through, among others, a process that involves interference between the DNA and RNA synthesis machineries.

Short rotation plantations of willow genotypes, harvested in vegetative growth phases, were tested as an alternative biomass for methane production. The substrate characteristics, maximal methane yields (K) and highest methane production rates (µmax) were determined. Leaves and stems from diploid Energo (EN) and tetraploid (PP) plants, harvested in June were superior methane sources to woody tissue. This could be related to the lower lignin contents in green willow. Fermentation of pooled biomasses from tetraploid genotypes harvested in June-August was more efficient than methane production from diploid tissues. Microbial community analyses by 16S rRNA genes showed a dominance of the order Clostridiales. In field study, based on Energo plantation, the maximum in green biomass accumulation was in early month 9 of the vegetation period. A theoretical calculation showed similar or better energy potential per unit area for willow than in the case of maize silage. This study encourages the use of green willow biomass as feedstock in biomethanation processes due to its relatively low production costs and uncomplicated agricultural practice.

In this work, a long-read sequencing (LRS) technique based on the Oxford Nanopore Technology MinION platform was used for quantifying and kinetic characterization of the poly(A) fraction of bovine alphaherpesvirus type 1 (BoHV-1) lytic transcriptome across a 12-h infection period. Amplification-based LRS techniques frequently generate artefactual transcription reads and are biased towards the production of shorter amplicons. To avoid these undesired effects, we applied direct cDNA sequencing, an amplification-free technique. Here, we show that a single promoter can produce multiple transcription start sites whose distribution patterns differ among the viral genes but are similar in the same gene at different timepoints. Our investigations revealed that the circ gene is expressed with immediate–early (IE) kinetics by utilizing a special mechanism based on the use of the promoter of another IE gene (bicp4) for the transcriptional control. Furthermore, we detected an overlap between the initiation of DNA replication and the transcription from the bicp22 gene, which suggests an interaction between the two molecular machineries. This study developed a generally applicable LRS-based method for the time-course characterization of transcriptomes of any organism.