الخلاصة:
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.
