Thesis of Delphine Riviere

2008 nov.

" Comparison of microbial populations in anaerobic digesters treating wastewater sludge : molecular analysis of the diversity and activity "
  Delphine Rivière (mail) supported his thesis on November the 14th in Evry. His research was carried out in the “Metagenomics of prokaryotes” team led by Denis Le Paslier (mail) and Abdelghani Sghir (mail).

Anaerobic digestion is a natural process of degradation of organic matter realised by a microbial consortium. With the development of molecular techniques, our knowledge of the diversity of this microbial ecosystem has been extended in the last years. In this study, the molecular study of prokaryotic diversity was performed on 30 digesters with various operating conditions. In this first phase, the most common phyla were identified. We were able to measure the extend of the diversity of this complex ecosystem and distinguish the most diversified phyla. Different types of structures were observed: some phyla are constituted of frequently observed phylotypes while others are mainly composed of endemic phylotypes. In a second phase, 7 digesters were selected to pursue the exploration of the diversity at a deeper level with a large number of clones sequenced for Archaea and Bacteria domain. A selection of statistic tools was used to compare the populations encountered and determine their level of similarity/difference. For Archaea domain, seven OTUs were found to be dominant and are in equilibrium. Depending on the availability and the concentration of the substrates, some OTUs of the seven may become more abundant. These dominant OTUs are affiliated with Methanosarcinales, Methanomicrobiales and Arc I phylogenetic groups. We highlighted the abundance of the lineage Arc I which is probably in competition with Methanosaeta species. This implies that Arc I could be an acetate consumer and therefore a major actor in acetoclastic methanogenesis. New cultivation experiments are being conducted to explore the metabolic ability of Arc I that are still unclear. Moreover, statistical analysis revealed that the Bacteria community can be described as a three component model: one third making up a core group of phylotypes defined as OTUs commonly found in most of the digesters, one third are phylotypes shared between a few digesters, and another third are endemic phylotypes. The core group is composed of 6 OTUs affiliated with the less diversified phyla: Chloroflexi, Betaproteobacteria, Bacteroidetes and Synergistetes. Other phyla such as Firmicutes, Alpha and Deltaproteobacteria are composed of a majority of endemic sequences. Among these OTUs 2 are commonly found in other environments while the other four seem to be specific of anaerobic environments. These populations commonly found in anaerobic digesters could be major actors of the degradation of the organic matter.

Based on the large database of 16S rDNA sequences specific probes were designed to target the dominant groups and quantify their activity inside the reactors. This quantitative analysis underlined the most active groups and especially the importance of the candidate division WWE1. These hybridization experiments also revealed a diversity that is not yet targeted by the existent probes and the limits in the use of primers 0008F and 1390R for the clones libraries construction. The comparison of qualitative and quantitative approach showed that some groups seem to be over-represented in clones libraries compared to their real activity. We followed a full-cycle RNA approach from a molecular inventory to quantification with specific probes. This comparison of anaerobic digester populations is a first step toward a future understanding on microbial resource management in order to manage complex microbial system. Ultimately, the relationship between biodiversity, operating conditions and digester efficiency could be established for a better process engineering in anaerobic digestion.

Keywords : Anaerobic digestion, Archaea, Bacteria, prokaryotic, biodiversity, 16S rRNA gene libraries, biostatistics, dot-blot, quantification, activity