Congratulations to Julie Hardy (MEB) who defended her thesis on October 28, 2021
On the following topic:
"Study of the metatranscriptome of anaerobic digestion: from bioinformatic development to the study of stability under inhibitory conditions"
Supervisor: Patricia Bonin, DR CNRS
Co-director: Sébastien Lacroix, DR Véolia
Abstract in French
Anaerobic digestion (AD) is a process of degradation of organic matter in the absence of oxygen which leads to the energetic valorisation of organic residues in the form of methane-rich biogas. The optimization of these processes requires a better understanding of the functioning of complex communities and their functional response to perturbations. One of the main objectives of this thesis was the construction and validation of a bioinformatics pipeline dedicated to the comparative analysis of metatranscriptomes from anaerobic digesters subjected to perturbations.
Meta-omics approaches based on high-throughput sequencing produce large amounts of data, challenging their processing. An efficient pipeline to analyze the functions of active microbial communities was developed during this thesis. The pipeline is designed to control sequence quality, remove rRNA, perform functional annotation of reads against functional databases, and process differential gene expression analysis. Its efficiency was validated by analyzing data from (i) synthetic communities, (ii) a previous metatranscriptomic study, and (iii) 7 triplicate samples from a continuous experimental methanizer (CSTR) in which we simulated different stress episodes.
To test the pipeline developed in this thesis, we chose to study the impact of ammonium inputs on the AD microbiome. Indeed, during anaerobic digestion (AD) of protein-rich wastewater, ammonium is released by the degradation of amino acids. High ammonium concentrations are well known to disrupt the equilibrium of the AD microbiome, and result in degradation of methanogenic activity.
In a preliminary study, we applied ammonium inputs of increasing concentration (from 1.7 to 15.2 g N-NH4+ L-1) to closed reaction systems consisting of synthetic wastewater, using two strategies: (i) independent inputs, and (ii) successive inputs. Functional performance and community composition of present and transcriptionally active microorganisms were analyzed by 16S rRNA amplicon sequencing. Regardless of the addition strategy, the active community of methanogenic archaea initially enriched in Methanosaeta undergoes changes to be enriched by Methanosarcina and finally by Methanoculleus during the increase of NH4+ content. A dynamic succession of CH4 precursor-producing bacteria (H2 and volatile fatty acids, VFAs) and bacteria syntrophically oxidizing VFAs was also observed, which was very different between the two ammonium addition strategies. From these metabarcoding data we can hypothesize a potential shift in metabolism from methanogenesis performed by the acetoclast pathway to syntrophic acetate oxidation coupled with hydrogenotrophic methanogenesis when ammonium content increases.
To validate the metatranscriptomic pipeline and test these hypotheses, different operative perturbations, including increasing ammonium inputs (between 3.95 and 5.71 g/L), were applied to 2 CH4-producing CSTRs. Process performance was evaluated by monitoring COD (chemical oxygen demand), CH4 and VFA during the 300 days of experimentation. Microbial community structure and functional response were routinely assessed by metabarcoding and metatranscriptomics. We applied our pipeline to study metatranscriptomes. Metabarcoding and metatranscriptomic analysis suggests that the anaerobic system responds to ammonium stress by reconstructing syntrophic associations between acetoclastic and hydrogenotrophic methanogens and acetate oxidants.