Estratégias de melhoria do bioprocesso de produção de enzimas celulolíticas por Aspergillus niger a partir de bagaço de cana-de-açúcar para a produção de etanol de segunda geração
Cunha, Fernanda Marisa da
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The second generation ethanol (2G) production from sugarcane bagasse is a promising strategy to increase ethanol production per hectare. The 2G ethanol production comprises the steps of biomass pretreatment, hydrolysis of lignocellulose polymers into simple sugars, fermentation and purification. Aiming to reduce 2G ethanol production costs, the development of more efficient bioprocesses for the production of cellulase with high specificity to lignocellulose hydrolysis is needed. Therefore, the goal of this work was finding the cultivation condition more favorable to the production of enzymatic cocktails with higher performance on sugarcane bagasse hydrolysis and 2G ethanol production. In order to achieve this, the implementation of strategies to improve the bioprocess for cellulase production by Aspergillus niger in a stirred tank bioreactor was carried out. The enzymatic production under a new fermentation method, referred to as sequential fermentation (FSeq), was compared to the conventional submerged fermentation method in both shake flasks and stirred tank bioreactor. Experiments were performed to evaluate the effect of the fermentation method, agitation speed, pH control, sugarcane bagasse pretreatment and particle size, and impeller type on enzyme production using sugarcane bagasse as substrate. The highest enzymes production in cultivations using Rushton turbine impeller were 1,599 and 4,212 IU.L-1 for endoglucanase and xylanase, respectively, in cultivations under the new FSeq method, using steam explosion pretreated sugarcane bagasse of particle sizes smaller than 0.5 mm, agitation speed of 700 rpm, aeration of 0,5 vvm and pH 5.0. Cultivations carried out using the Elephant ear up-pumping flow impeller allowed higher enzymes production of 2,712 and 5,837 IU.L-1 for endoglucanase and xylanase, respectively. Given the superior performance of FSeq method in comparison to the FSm method, the decision was made to evaluate the FSeq method in cultivations employing different Aspergillus sp strains and with different lignocellulosic substrates, such as wheat bran, soybean meal, and mixtures of wheat bran and sugarcane bagasse, in shake flasks cultivations. Besides the steam-explosion pretreatment of the sugarcane biomass, which had shown to be favorable to endoglucanase production, the liquid hot water pretreatment was also evaluated, which was found to be favorable to xylanase induction. The enzymatic cocktails produced were employed on sugarcane bagasse hydrolysis, combined with a commercial enzyme, and the extracts produced under the FSeq method resulted in a percentual hydrolysis gain about 10% higher than FSm cocktails. Moreover, the 2G ethanol production from sugarcane bagasse hydrolysates by FSeq cocktails were up to 7% more efficient than the ethanol production using FSm sugarcane bagasse hydrolysates. The results obtained here validate the new sequential fermentation method as a promising strategy for the production of enzymatic cocktails with higher enzymatic activities as well as with higher performance on sugarcane bagasse hydrolysis and 2G ethanol production in comparison to the FSm method. The findings concerning the effects of different operational variables and cultivation methods on (hemi)cellulolytic enzymes production in a stirred tank bioreactor will contribute to the development of three-phasic cultivation systems employing different types of lignocellulosic biomass in bioreactors.