Produção de etanol 2g em reator de leito fixo e por bioprocessamento consolidado utilizando leveduras de alta performance

Abstract

In order to enable the industrial production of second generation (2G) ethanol, the development of processes with high productivity using hydrolysates of lignocellulosic material containing high concentration of inhibitors, with minimum enzymes concentration due to its impact on the total cost of the process, are some of the main challenges to be overcome. In this sense, researches in genetic engineering have been working towards obtaining yeast strains that are more tolerant to inhibitors present in hydrolysates and to the ethanol produced, as well as strains presenting capacity to produce and secrete enzymes, in addition to ferment pentoses and hexoses. The immobilization of cells in calcium alginate contributes to protect cells from the harmful effects of inhibitors, besides allowing the use of high cell densities, facilitating the recovery of products and the reuse of biocatalysts. Thus, the present work aimed to contribute to the development of a high productivity 2G ethanol production process using hemicellulose hydrolysates and superior recombinant yeast cells. At the first step of the research, after toxic and irreversible effects to the immobilized cells caused by the use of fermentative medium composed by concentrated hemicellulose hydrolysate during continuous fermentation were evidenced, experiments in mini-reactors unveiled the strong and synergistic effects between the high inhibitors load in fermentative medium and ethanol produced during fermentation, associated to the exposure time and the yeast strain characteristics. The results led the study to searching more robust and promising yeast strains, along with fermentative media presenting reduced inhibitors’ load. Three superior recombinant yeasts (S. cerevisae T18, HAA1 and MDS130) modified for xylose consumption, kindly donated by Prof. Johan Thevelein (KU Leuven and NovelYeast®, Belgium), were evaluated in different fermentative media formulated with concentrated crude hydrolysate (HC), detoxified hydrolysate (HD) and crude hydrolysate supplemented with molasses (MHB). The most encouraging results were achieved by the use of MDS130 strain and MHB medium, being this combination selected for further scale up in fixed bed bioreactors (BLF). Promising data regarding ethanol productivity (up to 20 g/L/h) and yield (up to 100% of the theoretical), besides high sugar conversion during the 20 recycles performed were reached with BLF. These results confirm the feasibility of the industrial application of the 2G ethanol production process from hemicellulose hydrolysate and molasses using recombinant yeast. In the second step of the present study, the superior recombinant yeast strain S. cerevisiae AC14, with capacity to secrete 7 hydrolytic enzymes, was evaluated for 2G ethanol production in a Consolidated Bioprocessing (BPC) system. Preliminary studies showed that free cells in high load enabled the hydrolysis and sequential fermentation of substrates such as cellobiose, corncob xylan and hydrothermal liquor (LH). Complementary experiments revealed that hydrolysis and fermentation steps from synthetic medium and solid (BTH) and liquid (LH) fractions obtained after hydrothermal pretreatment of sugarcane bagasse occurred in less than 10 hours in the presence of high cell load (optical density of 100), surpassing productivities from industrial enzymatic cocktails. Although the hydrolysis of the oligomers present in LH was not complete, the results here obtained are promising, indicating that the AC14 strain has potential of being used in BPC, enabling the 2G ethanol production from pretreated biomass.