Cloning and expression of an endoglucanase gene from the thermotolerant fungus Aspergillus fumigatus DBiNU-1 in Kluyveromyces lactis

Abstract An intronless endoglucanase from thermotolerant Aspergillus fumigatus DBINU-1 was cloned, characterized and expressed in the yeast Kluyveromyces lactis. The full-length open reading frame of the endoglucanase gene from A. fumigatus DBiNU-1, designated Cel7, was 1383 nucleotides in length and encoded a protein of 460 amino acid residues. The predicted molecular weight and the isoelectric point of the A. fumigatus Cel7 gene product were 48.19 kDa and 5.03, respectively. A catalytic domain in the N-terminal region and a fungal type cellulose-binding domain/module in the C-terminal region were detected in the predicted polypeptide sequences. Furthermore, a signal peptide with 20 amino acid residues at the N-terminus was also detected in the deduced amino acid sequences of the endoglucanase from A. fumigatus DBiNU-1. The endoglucanase from A. fumigatus DBiNU-1 was successfully expressed in K. lactis, and the purified recombinant enzyme exhibited its maximum activity at pH 5.0 and 60 °C. The enzyme was very stable in a pH range from 4.0 to 8.0 and a temperature range from 30 to 60 °C. These features make it suitable for application in the paper, biofuel, and other chemical production industries that use cellulosic materials.

The potential of the newly isolated thermotolerant yeast Pichia kudriavzevii RZ8-1 for high-temperature ethanol production

Abstract High potential, thermotolerant, ethanol-producing yeasts were successfully isolated in this study. Based on molecular identification and phylogenetic analysis, the isolated thermotolerant yeasts were clustered in the genera of Pichia kudriavzevii, Candida tropicalis, Candida orthopsilosis, Candida glabrata and Kodamea ohmeri. A comparative study of ethanol production using 160 g/L glucose as a substrate revealed several yeast strains that could produce high ethanol concentrations at high temperatures. When sugarcane bagasse (SCB) hydrolysate containing 85 g/L glucose was used as a substrate, the yeast strain designated P. kudriavzevii RZ8-1 exhibited the highest ethanol concentrations of 35.51 g/L and 33.84 g/L at 37 °C and 40 °C, respectively. It also exhibited multi-stress tolerance, such as heat, ethanol and acetic acid tolerance. During ethanol fermentation at high temperature (42 °C), genes encoding heat shock proteins (ssq1 and hsp90), alcohol dehydrogenases (adh1, adh2, adh3 and adh4) and glyceraldehyde-3-phosphate dehydrogenase (tdh2) were up-regulated, suggesting that these genes might play a crucial role in the thermotolerance ability of P. kudriavzevii RZ8-1 under heat stress. These findings suggest that the growth and ethanol fermentation activities of this organism under heat stress were restricted to the expression of genes involved not only in heat shock response but also in the ethanol production pathway.

Ethanol production from sweet sorghum by Saccharomyces cerevisiae DBKKUY-53 immobilized on alginate-loofah matrices

Abstract Ethanol production from sweet sorghum juice (SSJ) using the thermotolerant Saccharomyces cerevisiae strain DBKKUY-53 immobilized in an alginate-loofah matrix (ALM) was successfully developed. As found in this study, an ALM with dimensions of 20 × 20 × 5 mm3 is effective for cell immobilization due to its compact structure and long-term stability. The ALM-immobilized cell system exhibited greater ethanol production efficiency than the freely suspended cell system. By using a central composite design (CCD), the optimum conditions for ethanol production from SSJ by ALM-immobilized cells were determined. The maximum ethanol concentration and volumetric ethanol productivity obtained using ALM-immobilized cells under the optimal conditions were 97.54 g/L and 1.36 g/L h, respectively. The use of the ALM-immobilized cells was successful for at least six consecutive batches (360 h) without any loss of ethanol production efficiency, suggesting their potential application in industrial ethanol production.

Ethanol production from sweet sorghum by Saccharomyces cerevisiae DBKKUY-53 immobilized on alginate-loofah matrices

Abstract Ethanol production from sweet sorghum juice (SSJ) using the thermotolerant Saccharomyces cerevisiae strain DBKKUY-53 immobilized in an alginate-loofah matrix (ALM) was successfully developed. As found in this study, an ALM with dimensions of 20 × 20 × 5 mm3 is effective for cell immobilization due to its compact structure and long-term stability. The ALM-immobilized cell system exhibited greater ethanol production efficiency than the freely suspended cell system. By using a central composite design (CCD), the optimum conditions for ethanol production from SSJ by ALM-immobilized cells were determined. The maximum ethanol concentration and volumetric ethanol productivity obtained using ALM-immobilized cells under the optimal conditions were 97.54 g/L and 1.36 g/L h, respectively. The use of the ALM-immobilized cells was successful for at least six consecutive batches (360 h) without any loss of ethanol production efficiency, suggesting their potential application in industrial ethanol production.

High-temperature ethanol production using thermotolerant yeast newly isolated from Greater Mekong Subregion

Abstract The application of high-potential thermotolerant yeasts is a key factor for successful ethanol production at high temperatures. Two hundred and thirty-four yeast isolates from Greater Mekong Subregion (GMS) countries, i.e., Thailand, The Lao People's Democratic Republic (Lao PDR) and Vietnam were obtained. Five thermotolerant yeasts, designated Saccharomyces cerevisiae KKU-VN8, KKU-VN20, and KKU-VN27, Pichia kudriavzevii KKU-TH33 and P. kudriavzevii KKU-TH43, demonstrated high temperature and ethanol tolerance levels up to 45 °C and 13% (v/v), respectively. All five strains produced higher ethanol concentrations and exhibited greater productivities and yields than the industrial strain S. cerevisiae TISTR5606 during high-temperature fermentation at 40 °C and 43 °C. S. cerevisiae KKU-VN8 demonstrated the best performance for ethanol production from glucose at 37 °C with an ethanol concentration of 72.69 g/L, a productivity of 1.59 g/L/h and a theoretical ethanol yield of 86.27%. The optimal conditions for ethanol production of S. cerevisiae KKU-VN8 from sweet sorghum juice (SSJ) at 40 °C were achieved using the Box-Behnken experimental design (BBD). The maximal ethanol concentration obtained during fermentation was 89.32 g/L, with a productivity of 2.48 g/L/h and a theoretical ethanol yield of 96.32%. Thus, the newly isolated thermotolerant S. cerevisiae KKU-VN8 exhibits a great potential for commercial-scale ethanol production in the future.

Fermentation of rice bran hydrolysate to ethanol using Zymomonas mobilis biofilm immobilization on DEAE-cellulose

Background The major challenges associated with the fermentation of lignocellulosic hydrolysates are the reduction in the operating cost and minimizing the complexity of the process. Zymomonas mobilis biofilm has been emerged to resolve these complexities. Biofilm has been reported to tolerate to the toxic inhibitors and easily manipulated toward the cell recycle through the cell immobilization. Results Z. mobilis ZM4 and TISTR 551 were able to develop biofilms on DEAE cellulose under the differences in the morphologies. Z. mobilis ZM4 developed homogeneous biofilm that brought DEAE fiber to be crosslinking, while Z. mobilis TISTR 551 developed heterogeneous biofilm in which crosslinking was not observed. Ethanol production under batch and repeated batch fermentation of rice bran hydrolysate containing toxic inhibitors were compared between these two biofilms. TISTR 551 biofilm produced the maximum yield (Y P/S) of 0.43 ± 0.09 g ethanol/g glucose (83.89% theoretical yield). However the repeated batch could not be proceeded due to the bacterial detachment. Z. mobilis ZM4 biofilm produced the maximum yield (Y P/S) of 0.177 ± 0.05 g ethanol/g glucose (34.74% theoretical yield) in the batch culture and the biofilm remained intact to proceed along the repeated batch. The highest ethanol yield (Y P/S) in the repeated batch of Z. mobilis ZM4 was 0.354 ± 0.07 g ethanol/g glucose (69.51% theoretical yield). Conclusions Homogeneous biofilm structure of Z. mobilis provided more recycle beneficial over the heterogeneous biofilm structure for the ethanol production from lignocellulosic hydrolysate.