On the following subject:
"Study of the metatranscriptome of anaerobic digestion: from bioinformatics development to the study of stability under inhibitory conditions".
Thesis supervisor: Patricia Bonin, DR CNRS
Co-director: Sébastien Lacroix, DR Véolia
In the photo from left to right: Eric Trably, Patricia Bonin, Julie Hardy, Valérie Michotey, Sébastien Lacroix, Marion Leclerc and Eric Pelletier.
Léa Cabrol was on video from Chile
Summary in French
Anaerobic digestion (AD) is a process for degrading organic matter in the absence of oxygen, leading to energy recovery from organic residues in the form of methane-rich biogas. Optimising these processes requires a better understanding of the functioning of complex communities and their functional response to disturbances. One of the main objectives of this thesis was to build and validate 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 quantities of data, making their processing a challenge. An efficient pipeline for analysing 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 effectiveness was validated by analysing data from (i) synthetic communities, (ii) a previous metatranscriptomic study and (iii) 7 triplicate samples from a continuous experimental methaniser (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. During anaerobic digestion (AD) of protein-rich wastewater, ammonium is released by the degradation of amino acids. High ammonium concentrations are well known to upset the balance of the AD microbiome, leading to a deterioration in methanogenic activity.
In a preliminary study, we applied ammonium inputs of increasing concentration (from 1.7 to 15.2 g N-NH4+ L-1) in closed reaction systems consisting of synthetic wastewater, using two strategies: (i) independent inputs, and (ii) successive inputs. Functional performance and the composition of the communities of microorganisms present and transcriptionally active were analysed by sequencing 16S rRNA amplicons. Regardless of the addition strategy, the active community of methanogenic archaea initially enriched with Methanosaeta underwent changes to become enriched with Methanosarcina and finally with Methanoculleus as the NH4+ content increased. A dynamic succession of bacteria producing CH4 precursors (H2 and volatile fatty acids, VFAs) and bacteria syntrophically oxidising the VFAs was also observed, very different between the two ammonium addition strategies. Based on these metabarcoding data, we can hypothesise a potential change in metabolism from methanogenesis via the acetoclast pathway to syntrophic oxidation of acetate coupled with hydrogenotrophic methanogenesis when the ammonium content increases.
To validate the metatranscriptomic pipeline and verify these hypotheses, various operational perturbations, including increasing ammonium inputs (between 3.95 and 5.71 g/L), were applied to 2 CSTRs producing CH4. The performance of the process was assessed by monitoring COD (chemical oxygen demand), CH4 and VFA over the 300 days of experimentation. Microbial community structure and functional response were regularly assessed using 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.