Recherche et isolement de microorganismes des milieux extrêmes algériens producteurs de métabolites d’intérêt sur différents substrats agro-industriels pertinents.

Abstract

In this study, the cellulolytic activity of bacteria isolated from various locations in an extreme algerian ecosystme was investigated. The salt lake of El M'Ghair was specifically examined (along with its nearby soil and water, as well as a nearby palmeraie). In fact, 9 of the 43 bacterial isolates were able to degrade carboxymethyl-cellulose (CMC) with varying cellulolytic index values. The analysis has shown that the bacterial isolate H1, which was isolated from the soil of a palmeraie near a lake, has the best cellulolytic potential, with a value of 5.5 of CI. The isolate's molecular analysis revealed that it belongs to the species Bacillus velezensis, whose accession numbers to GenBank (NCBI) are OM510337 for the 16S ARN and OM523097 for the gyrA. This strain exhibits optimal growth at 37 °C in 863 broth without NaCl, however, it resisted at 55 °C in the same medium with 10% NaCl, giving rise to its thermotolérance and halotolérance characteristics. The strain was inoculated on a medium containing 5% olive mill wastes (GO), 0.5% yeast extract, and 150 rpm stirring to test its capacity to hydrolyse lignocellulosic material and produce lipopeptides with biosurfactant activity. Indeed, during the 72 hours of fermentation, the olive mill wastes was hydrolyzed by the release of various oligosaccharides due to strain ability to produce a cellulase (CelH1). This degradation also made it possible to produce lipopeptides with surfactant and emulsifying activity. A significant positive correlation of the cellulase and biosurfactants production was observed (77%). The CelH1 enzyme was, then, purified 44.9 folds and recovered with a yield of 80% and a specific activity of 453.04 U/mg of protein, using only two successive steps: ultrafiltration and ion exchange chromatography. Indeed, the molecular mass of the enzyme was estimated at 26 kDa by SDS-PAGE, and its identification by LC-ESI-MS/MS analysis, showed that it covers 40% of the sequence of an endo-1,3-1,4-β-glucanase from family 16 of glycosyl hydrolases. After 48 hours of incubation under test conditions, the enzyme remained stable and displayed optimal enzymatic activity at pH 6.0-6.5 and at 60-65°C. The highest CelH1 activity was measured on barley β-glucan substrate (604.5 U/mL) followed by lichenan and CMC, confirming that the studied enzyme is an endo-1,4-β-glucanase . CelH1 is highly thermotolerant and halotolerant retaining 77% of its activity at 70°C and 70% in the presence of 4 M NaCl. The enzyme has a Vmax of 625 U/min/mL and a high affinity with barley β-glucan with a Km of 0.69 mg/mL. It has also shown a significant ability to release cello-oligosaccharides by hydrolyzing cellopentaose. Lipopeptides produced by B. velezensis H1 are partially purified using a C18 column. HPLC analysis of the lipopeptide extract showed that the B. velezensis H1 strain has the ability to produce at least two lipopeptides: surfactin and fengycin, on the GO-based medium. The biosurfactant and tensioactif potential of these products was demonstrated by a Drop Collapse test using olive oil and paraffin as hydrophobic substrates, a complete collapse of the lipopeptides drop was observed, due to the reduction of surface tension of the hydrophobic substrates under the effect of the lipopeptide extract.