In situ characterization of the genomic content of viruses of methanogenic archaea in organic waste fermentation bioprocesses – VIRAME

Establishing the link between a virus and its host within ecosystems is challenging since they do not systematically share common genes. To overcome this bottleneck, the link between activated methanogenesis pathways, archaea catalyzing methanogenesis and the genomic content of viruses infecting these archaea will be established thanks to an original integrated approach coupling state-of-the-art molecular ecology tools (meta-omics, isotopy, especially stable isotope probing SIP) and classical in silico analyses (comparative genomics) as well as specific ones (analyses of CRISPR spacers, proviruses and kmer compositions). The genomic content of viruses of methanogens in several replicated anaerobic digestion microcosms will be analyzed and compared with those from organic waste anaerobic digestion plants. The diversity of represented viral families and the presence of genes relevant to virus life cycle and to the ecophysiology of the methanogenic hosts will be especially examined.Regarding the experimental design, specific conditions and distinct carbon substrates, unlabeled or labeled with 13C, will be used to selectively activate distinct methanogenesis pathways and identify viruses and cells having incorporated 13C.

At this stage, the proof of concept of the application of SIP to viral DNA has been successfully established on a pure strain culture model.
Indeed, some T4 bacteriophage DNA was obtained by infecting bacterial cultures of Escherichia coli cells grown on minimal medium containing 13C-glucose. SIP analyses of this viral DNA revealed that it had a higher mass density compared to the case where 12C-glucose (unlabeled) had been employed.
It shows the incorporation of 13C in viral DNA thanks to the use of a 13C-labeled substrate consumed by the cellular host of the virus. It opens the possibility to track, within ecosystems, viruses infecting cellular hosts which grow on specific substrates.

The original integrated approach including SIP applied to viral nucleic acids will contribute to overcome an important bottleneck in microbial ecology, establishing the link between a virus and its host. By providing important insights into the genomic diversity of viruses of methanogenic archaea, VIRAME will moreover fill important knowledge gaps and contribute to a better understanding of the evolutionary history of viruses and their hosts. The results will be of general interest for the optimization of biogas production from organic waste, a sector presently enjoying a boom, since viruses of methanogens likely have important effects on the matter fluxes and on the dynamics of methanogens, a key functional group. Regarding biorefinery applications, VIRAME aims at suggesting innovative strategies for the biocontrol of methanogens by viruses or their constituants, by drawing inspiration from the most advanced application fields (medicine) and imagining, thanks to the project’s results, a transposition to bioprocesses. Methane being a potent greenhouse gas, biocontrol of methanogens also offers broader application perspectives (e.g. mitigation of methane emissions by ruminants) and could have a strong environmental impact.

Oral communication on k-mer profiles in genomes of cell, plasmids and viruses in the domain Archaea. Ariane Bize, Viruses of Microbes, 2018 (Poland, July 2018, meetings.embo.org/event/18-virus-microbe)

Organic solid waste is emerging as an attractive resource for the production of biofuels and synthons through anaerobic bioprocesses. However, waste matrix is complex, heterogeneous, and temporally variable and such features contribute to the complexity of their valorisation: organic waste biorefinery is currently in the exploratory research phase. To orientate fermentation pathways and establish stable processes, new sensitive operational levers are required. Viruses could thus serve as basis for the development of biocontrol tools specifically targeting certain functional microbial groups. In other sectors, including the medical field, agro-food-sector and waste water treatment, viruses and their components are indeed already used or considered for biocontrol applications. Regarding waste biorefinery, methanogens are particularly detrimental to the production of molecules more valuable than methane (e.g. ethanol, butanol) but viruses of methanogenic archaea are poorly characterized so that the development of such strategies is currently not possible.
Project VIRAME thus aims at characterizing in situ the genomic content of viruses infecting methanogens within anaerobic bioprocesses for organic waste valorisation. Establishing the link between a virus and its host within ecosystems is challenging since they do not systematically share common genes. To overcome this bottleneck, the link between activated methanogenesis pathways, archaea catalysing methanogenesis and the genomic content of viruses infecting these archaea will be established thanks to an original integrated approach coupling state-of-the-art molecular ecology tools (meta-omics, isotopy, especially stable isotope probing SIP) and classical in silico analyses (comparative genomics) as well as specific ones (analyses of CRISPR spacers, proviruses and kmer compositions). The genomic content of viruses of methanogens in several replicated methanization microcosms will be analysed and compared with those from organic waste methanization plants. The diversity of represented viral families and the presence of genes relevant to virus life cycle and to the ecophysiology of the methanogenic hosts will be especially examined.
Regarding the experimental design, specific conditions and distinct carbon substrates, unlabeled or labeled with 13C, will be used to selectively activate distinct methanogenesis pathways and identify viruses and cells having incorporated 13C. Inocula originating from industrial methanization plants will be used and the coordinator’s team has already accumulated and published evidence of the incorporation of 13C labeled substrates by methanogenic archaea in microcosms inoculated with such biomass and similar to those which will be established for VIRAME.
The original integrated approach including SIP applied to viral nucleic acids will contribute to overcome an important bottleneck in microbial ecology, establishing the link between a virus and its host. By providing important insights into the genomic diversity of viruses of methanogenic archaea, VIRAME will moreover fill important knowledge gaps and contribute to a better understanding of the evolutionary history of viruses and their hosts. The results will be of general interest for the optimisation of biogas production from organic waste, a sector presently enjoying a boom, since viruses of methanogens likely have important effects on the matter fluxes and on the dynamics of methanogens, a key functional group. Regarding biorefinery applications, VIRAME aims at suggesting innovative strategies for the biocontrol of methanogens by viruses, by drawing inspiration from the most advanced application fields (medicine) and imagining, thanks to the project’s results, a transposition to bioprocesses. Methane being a potent greenhouse gas, biocontrol of methanogens also offers broader application perspectives (e.g. mitigation of methane emissions by ruminants) and could have a strong environmental impact.