Étude de stratégies visant à stimuler la production de protéase alcalines par Bacillus licheniformis en utilisation des boues d'épuration municipales comme substrat.

The present work is part of a research project which is aimed at developing an effective and economic protease production process using municipal wastewater sludge as raw material. Alkaline proteases produced by microorganism such as Bacillus licheniformis are among the most used industrial enzymes. However, the use of enzymes is oftenlimited by their high production costs. Thus, it is indispensible for industry to find cheaper ways to produce enzymes. As a great part of production costs originate from the culture medium, the use of cheaper raw materials is an interesting avenue. In this vein, municipal wastewater sludge, whose disposaI is an increasing challenge which many societies have to face, constitutes a promising raw material. Previous works have showed that wastewater sludge represents a great potential for protease production. Neverthe1ess, many elements still have to be studied and optimized. First, the composition of the fermentation media is having a great influence on protease production. The wastewater sludge composition depends on wastewater composition and on the type of wastewater treatment applied. AIso, various compounds (flocculants, coagulants) are often added into wastewaters or sludge in order to increase the purification yie1ds of the treatment processes, which can modify composition and characteristics of sludge. So, a part of this work consisted in the evaluation of the effect of theses parameters on protease production. Also, a significant portion of sludge potential nutrients is the parts of which are difficult to biodegrade, and sorne sludge inconvenient physical characteristics (presence of flocks, high viscosity) restrained the potential of sludge as a fermentation media. Thus, another part of this work was to evaluate the potential of a pre-treatment (thermal-alkaline hydrolysis) to improve the sludge physical and nutritional characteristics for protease production. Moreover, the operational parameters (pH, aeration, agitation) in fermentation processes also have a considerable effect on protease production. The impact of theses parameters was also studied in order to increase protease production yields using activated sludge. In order to attain the different fixed objectives, many fermentation essays were realized in 7.5 and 15 L bioreactors, using sludge from Québec and Victoriaville municipal wastewater treatment plants. With the Sludge from Victoriaville, the results demonstrated that the presence of ferric chloride, added during the treatment process in order to favor phosphorus removal, did not affect protease production. On the other hand, the addition of alum and polymers in Québec wastewaters in order to favor the partic1es flocculation and sedimentation had a beneficial effect (+ 16%) on protease production. The protease activities obtained in Québec sludge were fairly constant (-6% to +9%) from one batch to another, whereas the protease activities obtained in the different sludge batches from Victoriaville presented more considerable variations (-22% to +32%). The results obtained in Québec sludge demonstrated that a fermentation medium consisting of thermal-alkaline (hydrolyzed) pre-treated sludge only is not suitable for protease production. On the other hand, the use of a mixture (MIX sludge) of hydrolyzed sludge (25%) and nonhydrolyzed sludge (75%) permitted a significant increase (78%) in protease activities, in comparison with non-pretreated sludge (RAW sludge) only. The proteases produced in RAW and MIX sludge are active under wide pH and temperature ranges and generated maximum protease activity at pH 10 and T 55°C. Low cell concentration and protease activities were obtained under uncontrolled pH conditions. In comparison, the protease activities obtained under controlled pH conditions were considerably higher. In RA W sludge, the proteases activities were 65% higher when NHtOH was employed in replacement of NaOH as pH-control agent. On the other hand, the use of CH3COOH in replacement of H2S04 significantly inhibited protease production. Therefore, NHtOHlH2S04 was found the most suitable combination of pH-control agents. In MIX sludge, the use of NHtOH had an inhibition effect on cell growth and protease production and the NaOHlH2S04 combination permitted to obtain the highest protease activities. Fermentations were carried under different combinations of aeration and agitation rates, c1assified as low, medium and high oxygen transfer conditions. The results demonstrated that protease production was highly stimulated (high protease activity/cells) under high oxygen transfer conditions (high aeration and agitation rates). However, when such conditions were maintained throughout the whole fermentation, low cell concentrations and low protease activities were obtained. Also, the abundant foam formation associated with high aeration and agitation rates was a major concem, more particularly during the first hours of the fermentation. On the other hand, the drop of dissolved oxygen concentrations below 10% when lower agitation and aeration rates were employed strongly inhibited protease production. Thus, the use of variable oxygen transfer