This study demonstrates the power of SIP-viromics for resolving virus-host associations in complex anaerobic communities, linking viral diversity directly to metabolically active formatotrophic and methanogenic guilds.
Abstract
Viruses play a major role in regulating microbial community composition and activity, yet their diversity and host associations in anaerobic digestion (AD)—a complex biotechnological process converting organic waste into biogas—remain poorly characterized. Linking viral sequences to metabolically active hosts in such complex communities is particularly challenging. Here, we applied an integrative approach combining
13
C-stable isotope probing (SIP) with shotgun sequencing of viromes and microbiomes to identify and characterize viruses infecting the active formatotrophic guilds in batch AD microcosms.
Microcosms fed with
13
C-labeled formate as sole carbon source selectively enriched two primary formate-consuming guilds: hydrogenotrophic methanogens (Methanobacteriales, predominantly
Methanobacterium subterraneum
) and acetogenic bacteria (family Natronincolaceae, genus
Andreesenella
). From cross-assembly of six viromes on the one hand, and six
13
C-SIP microbiome fractions on the other, we assembled a catalogue of 2,368 vOTUs, of which 261 were selected for detailed analysis. The striking enrichment of vOTUs associated with methanogenic archaeal hosts, specifically in the heavy (
13
C-enriched) SIP fractions, contrasted with their near-absence in total viromes—demonstrates that DNA-SIP successfully resolved viruses of active formatotrophs at the viral community level. Novel viruses were identified for both primary guilds, including archaeal viruses of Methanobacteriales (family Anaerodiviridae and a possible novel family) and two
Andreesenella
viruses carrying diversity-generating retroelements. A provirus predicted to infect
Methanothrix
—a strictly acetoclastic methanogen—further illustrates the capacity of SIP-viromics to capture viral associations across trophic levels. Analysis of auxiliary viral genes (AVGs) revealed numerous defense-associated genes (
dcm
,
metK
,
queC
) and genuine metabolic AVG candidates, such as
cysH
(assimilatory sulfate reduction) and, a contiguous cluster including
uxe, galU
and
serB
(surface polysaccharide biosynthesis), which may influence on host metabolism.
This study demonstrates the power of SIP-viromics for resolving virus-host associations in complex anaerobic communities, linking viral diversity directly to metabolically active formatotrophic and methanogenic guilds. The identification of novel viral lineages infecting key AD microorganisms, combined with AVGs with potential consequences for carbon and sulfur cycling, provides a foundation for understanding the functional role of viruses in AD process performance.
The coexistence of autotrophic, methanotrophic, and heterotrophic metabolisms underscores the ecological versatility of these communities and their significant contributions to carbon and iron cycling in these deep-sea iron-rich ecosystems.
Elsa Mendes, Aina Astorch-Cardona, Carine Audiffrin et al.· Environmental Microbiome· 0 citations
Microbial interactions strongly influence carbon and nitrogen flows in mushroom compost, yet their functional roles during Agaricus bisporus colonization remain unresolved. We combined PLFA-SIP and nanoSIMS imaging with ITS amplicon sequencing to follow resource flows and microbial activity across spatial scales. Stable-isotope tracers (¹³C- glucose and ¹⁵N-ammonium) revealed that A. bisporus simultaneously facilitates and suppresses bacterial populations: fungal activity increased glucose assimilation by bacteria yet reduced overall bacterial biomass. NanoSIMS visualized nutrient-rich microenvironments along hyphae where bacterial ¹³C and ¹⁵N assimilation was elevated. Sequencing showed the fungal community to comprise essentially two organisms, A. bisporus and Mycothermus thermophilus, which differ approximately elevenfold in their content of the fungal biomarker C18:2ω6,9c. Total fungal PLFA therefore tracks which of the two dominates as much as it tracks fungal biomass. Together these findings reveal coupled fungal–bacterial nutrient processing and show that biomarker-based estimates of fungal biomass require community composition to be known. Multi-scale isotope probing provides a framework for resolving microbial interactions in complex detrital systems. Key Points A. bisporus raises bacterial glucose uptake but lowers bacterial biomass Bacteria near hyphae show elevated 13C and 15N uptake at single-cell scale Fungal PLFA reflects the combined signal of fungus identity and biomass Graphic abstract
Mădălina M. Vîță, F. van Dam, M. Kienhuis et al.· bioRxiv· 0 citations
Inoculating cyanobacteria is a widely adopted strategy to accelerate biocrust formation and restore dryland soil functions, yet the in situ mechanisms sustaining microbial biomass carbon (MBC) accumulation under seasonal stress remain poorly understood. Here, we combined a field inoculation experiment with multi-omic profiling (amplicon sequencing, metagenomics, metatranscriptomics, and metaproteomics) and genome-scale metabolic modeling to track the circannual development of induced cyanobacterial biocrusts in Hopq Desert, China. Although the inoculated Konicacronema strain (formerly Microcoleus) maintained significant taxonomic dominance, metatranscriptomic and metaproteomic data revealed a striking functional handoff. Microcoleus-like populations drove rapid initial biocrust formation via the oxidative pentose-phosphate pathway and enzymatic antioxidant systems, whereas Oscillatoria assumed transcriptional dominance during harsher seasonal transitions through enhanced light harvesting, cyclic electron transport, and non-enzymatic photoprotection. Genome-scale metabolic modeling predicted that dominant cyanobacteria acted primarily as net importers, receiving amino acids, vitamins, cell-wall precursors, and phosphate from heterotrophic partners, while supplying inorganic sulfur and iron-sulfur clusters. Co-occurrence networks and life-history analyses further revealed that MBC maintenance was associated with a shift from growth-oriented resource mobilization toward acquisition-oriented strategies mediated by ABC transporters. Structural equation modeling confirmed that solar radiation and soil salinity govern MBC indirectly by influencing cyanobacterial composition and the balance between phototrophic assimilation and resource-acquisition strategies. These findings demonstrate that biocrust carbon accumulation is sustained by temporal functional division among cyanobacterial guilds and metabolic subsidies from heterotrophic partners, rather than by the persistent activity of a single pioneer strain, and provide a trait-based roadmap for designing resilient, multifunctional synthetic inocula for dryland restoration.
Xian Zhang, Hua Li, Ying-Chun Han et al.· ISME Communications· 0 citations
Analysis of microbiome samples collected from hadal seawaters via in situ filtration during 12 human-occupied vehicle dives identifies 135,073 non-redundant active proteins, with over 95% being hadal-specific.
Wei-Jia Zhang, Aoran Hu, Ziheng Wu et al.· Cell Host and Microbe· 0 citations
Granular activated carbon (GAC) enhances anaerobic digestion (AD) performance, potentially via biofilm promotion, microbial enrichment, and facilitating direct interspecies electron transfer (DIET). In this work, to investigate the role of GAC, DNA was extracted from sludge in lab-scale digesters (with/without GAC) and GAC biofilm. Genome-resolved analysis revealed GAC established distinct niches enriching Methanothrix and Methanosarcina vacuolata. DIET potential would be indicated by strictly filtered pilA genes with the presence of aromatic amino acids, but not the overall pilA genes. While capable of DIET, Methanothrix primarily utilized the acetoclastic pathway. Methanosarcina vacuolata actively partnered with electroactive bacteria, likely via conductive pili. Enhanced performance with GAC primarily stemmed from increased glucose transport activity and microbial enrichment, not DIET. Mature biofilms during prolonged operation may amplify DIET contributions. Thus, the immediate value of GAC lies in optimizing substrate metabolism and spatial community structuring, with biofilm maturation potentially unlocking greater DIET-mediated efficiency.
Hongyu Dang, Ying-Di Zhang, Yang Liu· Bioresource Technology· 0 citations
Commonly used mcrA primer sets differ substantially in their ability to capture methanogenic diversity, with some showing broad representation of reactor-associated methanogens and others exhibiting strong lineage-specific biases.
C. Callejas, P. Bovio-Winkler, C. Etchebehere· Journal of Applied Microbiol...· 0 citations
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