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The microbiomes involved in liquid anaerobic digestion process have been investigated extensively, but the microbiomes underpinning solid-state anaerobic digestion (SS-AD) are poorly understood. In this study, microbiome composition and temporal succession in batch SS-AD reactors, operated at mesophilic or thermophilic temperatures, were investigated using Illumina sequencing of 16S rRNA gene amplicons. A greater microbial richness and evenness were found in the mesophilic than in the thermophilic SS-AD reactors. Firmicutes accounted for 60 and 82 % of the total Bacteria in the mesophilic and in the thermophilic SS-AD reactors, respectively. The genus Methanothermobacter dominated the Archaea in the thermophilic SS-AD reactors, while Methanoculleus predominated in the mesophilic SS-AD reactors. Interestingly, the data suggest syntrophic acetate oxidation coupled with hydrogenotrophic methanogenesis as an important pathway for biogas production during the thermophilic SS-AD. Canonical correspondence analysis (CCA) showed that temperature was the most influential factor in shaping the microbiomes in the SS-AD reactors. Thermotogae showed strong positive correlation with operation temperature, while Fibrobacteres, Lentisphaerae, Spirochaetes, and Tenericutes were positively correlated with daily biogas yield. This study provided new insight into the microbiome that drives SS-AD process, and the findings may help advance understanding of the microbiome in SS-AD reactors and the design and operation of SS-AD systems.

Background Glacier ice archives information, including microbiology, that helps reveal paleoclimate histories and predict future climate change. Though glacier-ice microbes are studied using culture or amplicon approaches, more challenging metagenomic approaches, which provide access to functional, genome-resolved information and viruses, are under-utilized, partly due to low biomass and potential contamination. Results We expand existing clean sampling procedures using controlled artificial ice-core experiments and adapted previously established low-biomass metagenomic approaches to study glacier-ice viruses. Controlled sampling experiments drastically reduced mock contaminants including bacteria, viruses, and free DNA to background levels. Amplicon sequencing from eight depths of two Tibetan Plateau ice cores revealed common glacier-ice lineages including Janthinobacterium , Polaromonas , Herminiimonas , Flavobacterium , Sphingomonas , and Methylobacterium as the dominant genera, while microbial communities were significantly different between two ice cores, associating with different climate conditions during deposition. Separately, ~355- and ~14,400-year-old ice were subject to viral enrichment and low-input quantitative sequencing, yielding genomic sequences for 33 vOTUs. These were virtually all unique to this study, representing 28 novel genera and not a single species shared with 225 environmentally diverse viromes. Further, 42.4% of the vOTUs were identifiable temperate, which is significantly higher than that in gut, soil, and marine viromes, and indicates that temperate phages are possibly favored in glacier-ice environments before being frozen. In silico host predictions linked 18 vOTUs to co-occurring abundant bacteria ( Methylobacterium , Sphingomonas , and Janthinobacterium ), indicating that these phages infected ice-abundant bacterial groups before being archived. Functional genome annotation revealed four virus-encoded auxiliary metabolic genes, particularly two motility genes suggest viruses potentially facilitate nutrient acquisition for their hosts. Finally, given their possible importance to methane cycling in ice, we focused on Methylobacterium viruses by contextualizing our ice-observed viruses against 123 viromes and prophages extracted from 131 Methylobacterium genomes, revealing that the archived viruses might originate from soil or plants. Conclusions Together, these efforts further microbial and viral sampling procedures for glacier ice and provide a first window into viral communities and functions in ancient glacier environments. Such methods and datasets can potentially enable researchers to contextualize new discoveries and begin to incorporate glacier-ice microbes and their viruses relative to past and present climate change in geographically diverse regions globally. 8eoz6b1Gq7e8M2dM_SBXdy Video Abstract

Anthropogenic-driven climate change is accelerating permafrost thaw, threatening to release vast carbon stores through increased microbial activity. While microbial roles are increasingly studied, the contributions of viruses remain largely unexplored, in part due to soil-associated technical challenges that have hindered their detection and characterization. Here, we applied an optimized virion enrichment workflow along a permafrost thaw gradient, identifying 9,963 viral populations (vOTUs), including single- and double-stranded DNA viruses, with 99.9% novelty compared to other soils. Hosts were predicted for 38% of vOTUs, spanning nine archaeal, and 36 bacterial phyla, 22% of which were linked to metagenome-assembled genomes, including key carbon-cycling taxa. Genomic analyses revealed 811 putative auxiliary metabolic genes (AMGs) from 658 vOTUs, nearly half involved in carbon processing. These included 59 glycoside hydrolases (GH) across nine GH families, 45 for monosaccharide degradation, and seven involved in short-chain fatty acid and C1 metabolism, linking viruses to both early and late stages of carbon turnover. Additionally, six vOTUs carried racD , which may stabilize microbial necromass and promote long-term carbon storage. Viral and AMG functional diversity increased with thaw stage, indicating that viruses might participate in a broadening range of microbial metabolic processes as permafrost thaws. These findings expand our understanding of virus contributions in microbial carbon processing and suggest their important role in deciphering soil carbon fate under changing climate conditions. The potential roles of viruses in microbial-induced permafrost thaw, which is accelerated by climate change, are unclear. Here, Trubl et al. sample a permafrost thaw gradient and identify thousands of new viruses carrying genes with metabolic-related functions, which might influence the fate of stored carbon.

Sphagnum -dominated bogs are climatically impactful systems that exhibit two puzzling characteristics: CO 2 :CH 4 ratios are greater than those predicted by electron balance models and C decomposition rates are enigmatically slow. We hypothesized that Maillard reactions partially explain both phenomena by increasing apparent CO 2 production via eliminative decarboxylation and sequestering bioavailable nitrogen (N). We tested this hypothesis using incubations of sterilized Maillard reactants, and live and sterilized bog peat. Consistent with our hypotheses, CO 2 production in the sterilized peat was equivalent to 8–13% of CO 2 production in unsterilized peat, and the increased formation of aromatic N compounds decreased N-availability. Numerous sterility assessments rule out biological contamination or extracellular enzyme activity as significant sources of this CO 2 . These findings suggest a need for a reevaluation of the fixed CO 2 :CH 4 production ratios commonly used in wetland biogeochemical models, which could be improved by incorporating abiotic sources of CO 2 production and N sequestration.

Soils impact global carbon cycling and their resident microbes are critical to their biogeochemical processing and ecosystem outputs. Based on studies in marine systems, viruses infecting soil microbes likely modulate host activities via mortality, horizontal gene transfer, and metabolic control. However, their roles remain largely unexplored due to technical challenges with separating, isolating, and extracting DNA from viruses in soils. Some of these challenges have been overcome by using whole genome amplification methods and while these have allowed insights into the identities of soil viruses and their genomes, their inherit biases have prevented meaningful ecological interpretations. Here we experimentally optimized steps for generating quantitatively-amplified viral metagenomes to better capture both ssDNA and dsDNA viruses across three distinct soil habitats along a permafrost thaw gradient. First, we assessed differing DNA extraction methods (PowerSoil, Wizard mini columns, and cetyl trimethylammonium bromide) for quantity and quality of viral DNA. This established PowerSoil as best for yield and quality of DNA from our samples, though ∼1/3 of the viral populations captured by each extraction kit were unique, suggesting appreciable differential biases among DNA extraction kits. Second, we evaluated the impact of purifying viral particles after resuspension (by cesium chloride gradients; CsCl) and of viral lysis method (heat vs bead-beating) on the resultant viromes. DNA yields after CsCl particle-purification were largely non-detectable, while unpurified samples yielded 1–2-fold more DNA after lysis by heat than by bead-beating. Virome quality was assessed by the number and size of metagenome-assembled viral contigs, which showed no increase after CsCl-purification, but did from heat lysis relative to bead-beating. We also evaluated sample preparation protocols for ssDNA virus recovery. In both CsCl-purified and non-purified samples, ssDNA viruses were successfully recovered by using the Accel-NGS 1S Plus Library Kit. While ssDNA viruses were identified in all three soil types, none were identified in the samples that used bead-beating, suggesting this lysis method may impact recovery. Further, 13 ssDNA vOTUs were identified compared to 582 dsDNA vOTUs, and the ssDNA vOTUs only accounted for ∼4% of the assembled reads, implying dsDNA viruses were dominant in these samples. This optimized approach was combined with the previously published viral resuspension protocol into a sample-to-virome protocol for soils now available at protocols.io , where community feedback creates ‘living’ protocols. This collective approach will be particularly valuable given the high physicochemical variability of soils, which will may require considerable soil type-specific optimization. This optimized protocol provides a starting place for developing quantitatively-amplified viromic datasets and will help enable viral ecogenomic studies on organic-rich soils.

Summary Mixed plug‐flow loop reactor (MPFLR) has been widely adopted by the US dairy farms to convert cattle manure to biogas. However, the microbiome in MPFLR digesters remains unexplored. In this study, the microbiome in a MPFLR digester operated on a mega‐dairy farm was examined thrice over a 2 month period. Within 23 days of retention time, 55–70% of total manure solid was digested. Except for a few minor volatile fatty acids (VFAs), total VFA concentration and pH remained similar along the course of the digester and over time. Metagenomic analysis showed that although with some temporal variations, the bacterial community was rather stable spatially in the digester. The methanogenic community was also stable both spatially and temporally in the digester. Among methanogens, genus Methanosaeta dominated in the digester. Quantitative polymerase chain reaction (qPCR) analysis and metagenomic analysis yielded different relative abundance of individual genera of methanogens, especially for Methanobacterium, which was predominant based on qPCR analysis but undetectable by metagenomics. Collectively, the results showed that only small microbial and chemical gradients existed within the digester, and the digestion process occurred similarly throughout the MPFLR digester. The findings of this study may help improve the operation and design of this type of manure digesters. In this study, the microbiome in a MPFLR digester operated on a mega‐dairy farm was examined thrice over a two‐month period. The results showed that only small microbial and chemical gradients existed within the digester, and the digestion process occurred similarly throughout the MPFLR digester. The findings of this study may help improve operation and design of this type of manure digesters.

Recovered microbial community structure is known to be influenced by sample storage conditions and nucleic acid extraction methods, and the impact varies by sample type. Peat soils store a large portion of soil carbon and their microbiomes mediate climate feedbacks. Here, we tested three storage conditions and five extraction protocols on peat soils from three physicochemically distinct habitats in Stordalen Mire, Sweden, revealing significant methodological impacts on microbial (here, meaning bacteria and archaea) community structure. Initial preservation method impacted alpha but not beta diversity, with in-field storage in LifeGuard buffer yielding roughly two-thirds the richness of in-field flash-freezing or transport from the field on ice (all samples were stored at −80 °C after return from the field). Nucleic acid extraction method impacted both alpha and beta diversity; one method ( the PowerSoil Total RNA Isolation kit with DNA Elution Accessory ki t) diverged from the others ( PowerMax Soil DNA Isolation kit-High Humic Acid Protocol , and three variations of a modified PowerMax Soil DNA/RNA isolation kit) , capturing more diverse microbial taxa, with divergent community structures. Although habitat and sample depth still consistently dominated community variation, method-based biases in microbiome recovery for these climatologically-relevant soils are significant, and underscore the importance of methodological consistency for accurate inter-study comparisons, long-term monitoring, and consistent ecological interpretations.

Glaciers archive time-structured information on climates and ecosystems, including microorganisms. However, the long-term ecogenomic dynamics or biogeography of the preserved viruses and their palaeoclimatic connections remain uninvestigated. Here we use metagenomes to reconstruct viral genomes from nine time horizons, spanning three cold-to-warm cycles over the past >41,000 years, preserved in an ice core from Guliya Glacier, Tibetan Plateau. We recover genomes of 1,705 approximately species-level viral operational taxonomic units. Viral communities significantly differ during cold and warm climatic conditions, with the most distinct community observed ~11,500 years ago during the major climate transition from the Last Glacial Stage to the Holocene. In silico analyses of virus–host interactions reveal persistently high viral pressure on Flavobacterium (a common dominant glacier lineage) and historical enrichment in the metabolism of cofactors and vitamins that can contribute to host adaptation and virus fitness under extreme conditions. Biogeographic analyses show that approximately one-fourth of Guliya viral operational taxonomic units overlap with the global dataset, primarily with the Tibetan Plateau metagenomes, suggesting regional associations of a subset of the Guliya-preserved viruses over time. We posit that the cold-to-warm variations in viral communities might be attributed to distinct virus sources and/or environmental selections under different temperature regimes. Genomes recovered from a Tibetan Plateau ice core extending back 41,000 years show that preserved viral communities varied substantially with cold-to-warm climate cycles.

Hemolytic–uremic syndrome is a life-threating disease most often associated with Shiga toxin-producing microorganisms like Escherichia coli (STEC), including E. coli O157:H7. Shiga toxin is encoded by resident prophages present within this bacterium, and both its production and release depend on the induction of Shiga toxin-encoding prophages. Consequently, treatment of STEC infections tend to be largely supportive rather than antibacterial, in part due to concerns about exacerbating such prophage induction. Here we explore STEC O157:H7 prophage induction in vitro as it pertains to phage therapy—the application of bacteriophages as antibacterial agents to treat bacterial infections—to curtail prophage induction events, while also reducing STEC O157:H7 presence. We observed that cultures treated with strictly lytic phages, despite being lysed, produce substantially fewer Shiga toxin-encoding temperate-phage virions than untreated STEC controls. We therefore suggest that phage therapy could have utility as a prophylactic treatment of individuals suspected of having been recently exposed to STEC, especially if prophage induction and by extension Shiga toxin production is not exacerbated.

Peatlands are a large source of methane (CH4) to the atmosphere, yet the uncertainty around the estimates of CH4 flux from peatlands is large. To better understand the spatial heterogeneity in temperate peatland CH4 emissions and their response to physical and biological drivers, we studied CH4 dynamics throughout the growing seasons of 2017 and 2018 in Flatiron Lake Bog, a kettle-hole peat bog in Ohio. The site is composed of six different hydro-biological zones: an open water zone, four concentric vegetation zones surrounding the open water, and a restored zone connected to the main bog by a narrow channel. At each of these locations, we monitored water level (WL), CH4 pore-water concentration at different peat depths, CH4 fluxes from the ground and from representative plant species using chambers, and microbial community composition with a focus here on known methanogens and methanotrophs. Integrated CH4 emissions for the growing season were estimated as 315.4±166 mgCH4m-2d-1 in 2017 and 362.3±687 mgCH4m-2d-1 in 2018. Median CH4 emission was highest in the open water, then it decreased and became more variable through the concentric vegetation zones as the WL dropped, with extreme emission hotspots observed in the tamarack mixed woodlands (Tamarack) and low emissions in the restored zone (18.8–30.3 mgCH4m-2d-1). Generally, CH4 flux from above-ground vegetation was negligible compared to ground flux (<0.4 %), although blueberry plants were a small CH4 sink. Pore-water CH4 concentrations varied significantly among zones, with the highest values in the Tamarack zone, close to saturation, and the lowest values in the restored zone. While the CH4 fluxes and pore-water concentrations were not correlated with methanogen relative abundance, the ratio of methanogens to methanotrophs in the upper portion of the peat was significantly correlated to CH4 transfer velocity (the CH4 flux divided by the difference in CH4 pore-water concentration between the top of the peat profile and the concentration in equilibrium with the atmosphere). Since ebullition and plant-mediated transport were not important sources of CH4 and the peat structure and porosity were similar across the different zones in the bog, we conclude that the differences in CH4 transfer velocities, and thus the flux, are driven by the ratio of methanogen to methanotroph relative abundance close to the surface. This study illustrates the importance of the interactions between water-level and microbial composition to better understand CH4 fluxes from bogs and wetlands in general.