Seleção de leveduras amazônicas capazes de metabolizar hidrolisado hemicelulósico e fermentar D-xilose

Abstract

Yeasts were isolated from different Amazon habitat: beetles gut (Carabidae), midgut and hindgut of Nasutitermes sp., soil samples from savanna (Boa Vista city, Roraima state) and Amazon rain forest. The yeasts were selected according to the capability of metabolize sugarcane bagasse hemicellulosic hydrolysate (HACA) as one carbon source, assimilate and fermenting D-xylose. To selective medium preparation, HACA was diluted until reducing sugar concentration was 1% and supplemented with Yeast Nitrogen Base (YNB 6,7 g/L) and Agar (20 g/L). Xylose assimilating ability was investigated using replica-plating, as described by Barnett et al. (1990), using D-xylose (50 mM), YNB (6,7 g/L) and Agar (20 g/L). Fermenting capability was evaluated using flasks with 10 mL of YUKX medium (Yeast extract 1,5 g/L; Urea 1,25 g/L; KH2PO4 1,1 g/L and D-xylose 50 g/L), containing a Durham tube. Another fermentation test was performed using Falcon® tubes (50 mL) with 10 mL of YUKX, sealed with rubber stopper. A valve allowed CO2 liberation as fermentative evidence. A number of 76 colonies were isolated (2 from savanna, 8 from rain forest, 12 from Carabidae and 54 from Nasutitermes sp.), among these were detected 28 that have ability to growth on D-xylose as unique carbon source and 3 were able to ferment that sugar. These results agree with Breznak (1982) and Blackwell et al. (2004), which consider that insect gut (as termites and beetle) is a rich source of microorganisms that metabolize hemicelluloses for biotechnology purpose. Three D-xylose fermenting strains isolated from Amazon insects (LC27, IB04 and IB09) were cultivated at HACA and YUKX medium. The cell growth and sugar consumption were evaluated using OD600 and DNS methods, respectively. Thermo tolerance and ethanol tolerance were investigated using heat-shock (52 ºC, 9 minutes) and ethanol-shock (YPD + 20% ethanol during one week), evaluating cell viability. A fermenting assay was performed, monitoring CO2 releasing to estimate ethanol production. Sugar consumption was 78,3% at HACA medium, while at YUKX was 55,9%. Both strains are able to do hemicellulosic hydrolysate saccharification, raising reducing sugar from 42,5 g/L up to 63 g/L. The cell viability after heat-shock was upper 85% for IB04 and IB09 strains, but LC27 had a cell-viability lower than 40%. The cultivation after ethanol shock shows tolerance by all tested strains. The fermenting assay allows estimate low ethanol yield, average about 3,34 g/L. These results indicate potential for biotechnological using of hemicellulosic derived.