Study of the microbial diversity of the deep anoxic hypersaline basins of the Eastern Mediterranean Sea

Since 1983, the existence of Deep Hypersaline anoxic basins (DHABs) has been revealed in the Eastern Mediterranean basin. These basins which represent extremes environments (anoxic, hypersalty, dark, high hydrostatic pressure)  have  been  under  investigation  and  increasing  interest  the  last  fifteen  years.  The  molecular  approaches revealed  the  presence  of  active  and  uncultivated  microbial  communities  in  the  DHABs  with  two  main  metabolic processes  such  as  methanogenesis  and  sulphate-reduction.  Until  now,  no  representative  strain  of  the  different uncultivated lineages thriving in the DHABs (ex : lineages MSBL, Mediteranean Sea Brine Lake) or involved in the two processes  have  been  yet  cultivated.  The  main  objectives  of  the  thesis  work  were  to  (i)  identify  the  microbial  key players in the processes of methanogenesis and sulphate-reduction, (ii) isolate new microbes adapted to hypersaline conditions and to study their physiology, (iii) to compare the genome and the physiology of the strains belonging to the genus  Methanohalophilus,  isolated  from  three  different  basins  (Thetis,  Kryos,  Tyro),  (iv)  attempt  to  cultivate  and isolate  strains  belonging  to  the  uncultivated  lineages  described  in  the  DHABs.  Our  cultivation  approach  allow  to cultivate and isolate three methanogenic methylotrophic moderately halophile strains belonging to the genus Methanohalophilus from the basins Thetis, Kryos and Tyro. A chemo-taxonomic and genomic characterization of the isolates  revealed  the  capacity  of  the  strains  to  grow  under  in  situ  conditions.  Genome  analysis  revealed  the streamlining reduction (by 10%) of the 3 genomes of our deep sea isolates compared to the terrestrial species of the genus  Methanohalophilus  and  also  an  adaptation  of  the  isolates  to  the  in  situ  conditions.  The  isolate  SLHTYRO represent a new species of the genus for which we propose the name M. profundus strain SLHTYRO. Other microbial isolates belonging to genus Marinobacter, Halomonas, and Halanaerobium obtained could be involved in a syntrophic relationship with archaeal partners of the Methanohalophilus genus in order to produce methylated compounds from betaine which in turn is used as catabolic substrates in the methanogenesis process. A new strain belonging to a new genus affiliated the the Bacteroidetes phylum and phylogenetically close to clones identified in the DHABs has been cultivated and isolated in pure culture. This hard to grow isolate represents the first cultivated members of the diverse uncultivated lineages discovered in the DHABs.