Anaerobic digestion (AD) is a microbiological process of degradation of the organic matter which produces biogas rich in methane that can be converted into valuable electrical and thermal energy. It is commonly used to manage different types of organic waste at industrial scale using anaerobic digesters. However, this bioprocess is not fully mastered and still has an important potential for improvement. One of the major limitations of AD is the important susceptibility of the microbial communities to changes in operational conditions of the digesters. It can lead to unstable methane formation. Controlling AD microbial community stability, though, is not a trivial task. Knowledge on the determinants of anaerobic microbial process stability (i.e. the conditions and the succession of microbial events that allow maintaining a balance after a disruption or, on the contrary, that generate a domino effect leading to total failure) over time is still missing. Emerging omics high-throughput approaches can now lead to unprecedented data to portray AD microbiome. Metagenomics, metatranscriptomics, metaproteomics and metabolomics enable to describe a community at different levels (genes, gene expression, and metabolites production). Appropriate and efficient analytical methods are required to analyse these big and complex data and unravel the intricate networks of functional processes of AD. Novel computational and statistical methods are progressively becoming available to fully harvest and integrate these complex datasets. In this context, the aim of STABILICS is to conduct the first sets of high-throughput multi-omics longitudinal experiments, with an unprecedented sampling depth, in anaerobic digesters under constant environmental parameters or subject to different model perturbations created by the addition of NaCl. Experiments in lab-scale semi-continuous reactors will be set-up and monitored in the long run (more than one year). Two levels of analysis will be applied. 1) A high frequency monitoring of different descriptors of microbiota activity, where non-targeted metabolomics and isotopic analyses will characterise the degradation pathways and metabarcoding of RNA and DNA will target both active and present microorganisms. 2) An in-depth monitoring of microbiota functioning with both metagenomics and metatranscriptomics on selected samples and conditions. These unprecedented sets of data will be thoroughly analysed and integrated using cutting-edge statistical methods. For example, multivariate dimension reduction methods will be used for data mining, omics integration and feature selection; specific analytical framework for longitudinal data will be developed. The objectives of this interdisciplinary project will be 1) to evaluate at different omics levels the dynamics of AD microbiome in long term and replicated time course experiments, 2) to describe the succession of events that, under stress, leads to microbiota equilibrium unbalance and digester disruption or on the contrary microbiota equilibrium preservation and maintenance of stability, 3) to propose an original analytical framework of multi-omics longitudinal studies accounting for temporality, and 4) to deliver generic knowledge to understand the determinants of perturbations.

In urban areas, the amount of electrical energy expenditure for water treatment and supply could reach up to 18%. Within wastewater resource recovery facilities (WRRF), oxygen supply to microorganisms for carbon and nitrogen biological removal remains the main source of energy consumption (up to 75% of the overall power expenditure of the WRRF). Also, the development of alternatives to conventional processes is essential to reduce the environmental impact of these treatment units. Bio-electrochemical systems represent a technology in the making for the treatment and valorization of waste, based on catalysis of electrochemical reactions by microbial biofilms on electrode surfaces. Among these technologies, the bioelectrochemical snorkel (BIOTUBA) is original due to its simpler operation that allows to consider short-term implementation in existing WWTP bioreactors. This technology consists of a bio-anode and a (bio-)cathode connected in short-circuit. It ensures a maximal efficiency of the oxidation of organic matter and substantial energy savings can be considered. In addition, this technology could meet other issues of the water treatment (process control, reduction of the sludge production or metallic micro-pollutant treatment). Several scientific and technical challenges must be raised before considering the implementation of such technologies at industrial scale. The cost of electrodes, due to the expensive materials used for their design, is a constraint on scale-up. In addition, researches shall be conducted to allow fundamental understanding and to optimize the bioelectochemical snorkel specifying both microbial and electrochemical processes governing its performance. The BIOTUBA project aims to raise scientific and technical barriers to the implantation of the bioelectrochemical snorkel at industrial scale through a transdisciplinary and multiscale approach. The scientific program of the project is divided in different tasks: (i) Studies at laboratory scale to understand and optimize the operation of BIOTUBA by focusing on the use of low-cost recycled materials and application to real matrices (WP1); (ii) Development and use of models for electrode design and implementation in industrial bioreactors (WP2); (iii) Evaluation of energetic and environmental impacts of BIOTUBA on wastewater bioprocesses using an life cycle analysis coupled to experiments at semi-industrial scale (WP3). The development of the BIOTUBA will be based on the precise knowledge of the electrochemical and biological mechanisms to optimize its operation and to maintain its performance over time. Experiments at a semi-industrial scale will allow to characterize its behavior and induced impacts at a representative scale and to validate the technological choices. The economic development will be supported a partner of the project (6TMIC). The project involves five partners: an Institute for applied research (Irstea-HBAN), an academic laboratory (LGC), a public industrial enterprise for urban wastewater management and treatment of the Parisian area (SIAAP) and a private company SME specialized in the development of innovative processes and technology transfer (6TMIC). Coordination will be provided by Irstea-HBAN.

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.