Recherche de micro-organismes producteurs d’hémi-cellulases à partir de la biomasse lignocellulosique ; valorisation des hémicelluloses dans les domaines des bio-polymères et les fermentations non agroalimentaires.

Abstract

The main objective of this work is to search for microorganisms capable of degrading lignocellulosic biomass by lignocellulosic enzymes and more particularly, hemicellulase. The exploration of soil samples from two forests and a former landfill in Constantine (Algeria), resulted in the selection of 16 microbial isolates. A single mold isolate (MGA) was selected for its high cellulase and hemicellulase activity. Molecular identification of MGA revealed it to be Aspergillus fumigatus ON226990. The selected strain was subsequently subjected to a scaling-up study to test its ability to grow and produce hemicellulase on a large scale. A. fumigatus showed an ability to grow in a liquid medium based on xylose as substrate, in 250 mL Erlenmeyer flasks, 2 and 20 liter bioreactors. In addition, three shaking speeds: 350, 500 and 650 rpm were tested in the 2 L bioreactor in order to follow the impact of the shear effect on the morphology of A. fumigatus. Indeed, this strain showed a certain ""robustness"" to the shear that can occur on an industrial scale; by increasing the agitation speed to 500 rpm, the mold forms free micro-pellets ensuring a good homogeneity in the culture medium. On the other hand, at agitation speeds lower than 300 rpm, the mycelium develops as macro-pellets. Furthermore, according to HPLC analyses, A. fumigatus showed a very high potential for production of endo-1,4-β-xylanase and xylan 1,4-β xylosidase during using corn xylan as substrate in a 2-L bioreactor fermentation. The production process in liquid medium, remains very expensive because of the high price of pure substrate. Therefore, the interest is redirected to the valorization of Stipa tenacissima (Alfa) as raw material for the production of hemicellulase by A. fumigatus. It is important to emphasize that this work is the first to have exploited Alfa as a raw material for the production of enzymes without pretreatment by A. fumigatus. For this purpose, the polymers of dried and ground esparto samples were analyzed by chemical methods (strong and dilute acid). Indeed, the results obtained by HPLC analysis show that this herb is mainly composed of 46-44% of cellulose and 12.5-13.33% of hemicellulose. On another aspect, experiments were statistically planned with the Box-Behnken design to optimize the production of xylanase and cellulase by A. fumigatus, using Alfa as the only carbon source, where the inputs are; pH, temperature, humidity and incubation time. The optimal conditions established by the response surface method for maximum xylanase production (841.78 U/gds) are: pH; 4.5, temperature; 30 °C, humidity; 89.19% and a fermentation time of 144 h, and for maximum cellulase production (74.41 U/gds): pH; 6, temperature; 31.61 °C, humidity; 90% for 144 h incubation. Subsequently, 11 factors, including organic and inorganic nitrogen sources, carbon and phosphorus sources, were screened using the Plackett-Burman design to investigate their influence on xylanase production. Malt extract, MgSO4, NaNO3 and (NH4)2SO4 were selected for further optimization using the Central-Composite design. The maximum xylanase production under the optimized conditions is 1659.41 U/gds, which represents a nearly 3-fold increase over the first xylanase production before optimization. Furthermore, the enzyme activity produced by A. fumigatus under the optimized conditions, is more efficient than that of commercial enzymes. This enzyme complex also showed its efficiency in the saccharification of Alfa, wheat straw, oat straw and wheat bran, compared to the saccharification of these substrates by commercial hemicellulase. A saccharification rate of 35.82% was observed with wheat bran, oat straw (25.91%), Alfa (21.29%) and wheat straw (2.29%) after 24 h of incubation. Finally, a cost-effectiveness study confirmed the value of xylanase production by A. fumigatus; enzyme production with Alfa is 3 times cheaper than with pure commercial xylan.