Contribution à l'étude de la diversité des bactéries thermophiles de quelques environnements chauds algériens et essai de de production et de caractérisation de leurs protéases extracellulaires.

Abstract

Natural hot environments can represent an important source of microbial biodiversity and biomolecules with great added value. Among these biomolecules, extracellular hydrolases and in particular proteases are of great interest for the food industry because of their single reactional properties at high temperatures and their stability in various environmental conditions. The primary objective of this thesis is the isolation of thermophilic aerobic bacteria with extracellular proteolytic activities from 7 sites representing different Algerian hot environments: three geothermal sources, two Saharan soils, a sebkha and an oil field. On the 138 isolates selected on the basis of their macroscopic and microscopic morphological characters, 53 isolates able to degrade casein and/or gelatin on solid culture media were characterized by amplified ribosomal DNA restriction analysis. After the elimination of clones, 28 strains were identified by sequencing and analyzing their 16S rRNA genes. These strains belong to 5 genera of aerobic thermophilic endospore-forming bacteria: Anoxybacillus, Bacillus, Geobacillus, Brevibacillus and Thermoactinomyces. The physiological and biochemical characterization of these strains confirm the relationship with these genera. The genomes of two strains, Geobacillus Sah69 and Thermoactinomyces AS95, were then characterized. The production of putative extracellular proteases of industrial interest was revealed. In addition, the biodiversity of the geothermal sources of Debagh and Ouled Ali was studied by metagenomic analysis of the 16S rRNA hypervariable V4 region sequences of environmental DNA collected from sediment samples. The thermophilic aerobic bacterial communities dominate the two sites with a minor presence of the phylum of Firmicutes and the genera isolated by cultural methods. Lastly, the production of extracellular proteases was estimated quantitatively for the 28 selected strains. Study of production kinetics and effect of fermentation conditions on three selected strains, Geobacillus Sah69, Thermoactinomyces AS95 and Brevibacillus OA30, were then carried out. After the extraction of the supernatant, two proteases, metalloprotease 32- F38 and serine-protease 16-F39, were produced and purified from strain Brevibacillus OA30. These proteases were acid, thermoactive and had a great stability in presence of SDS, solvents and other chemical agents tested. Thus revealing a good potential for industrial application.