Preekamol Klanrit

Ethanol production from sweet sorghum juice using very high gravity technology: Effects of carbon and nitrogen supplementations

Ethanol production from sweet sorghum juice by Saccharomyces cerevisiae NP01 was investigated under very high gravity (VHG) fermentation and various carbon adjuncts and nitrogen sources. When sucrose was used as an adjunct, the sweet sorghum juice containing total sugar of 280 g l(-1), 3 g yeast extract l(-1) and 5 g peptone l(-1) gave the maximum ethanol production efficiency with concentration, productivity and yield of 120.68+/-0.54 g l(-1), 2.01+/-0.01 g l(-1) h(-1) and 0.51+/-0.00 g g(-1), respectively. When sugarcane molasses was used as an adjunct, the juice under the same conditions gave the maximum ethanol concentration, productivity and yield with the values of 109.34+/-0.78 g l(-1), 1.52+/-0.01 g l(-1) h(-1) and 0.45+/-0.01 g g(-1), respectively. In addition, ammonium sulphate was not suitable for use as a nitrogen supplement in the sweet sorghum juice for ethanol production since it caused the reduction in ethanol concentration and yield for approximately 14% when compared to those of the unsupplemented juices.

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

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 Peoples 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.

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

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

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

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.

Agrobacterium-mediated transformation of Dendrobium orchid with the flavanone 3-hydroxylase gene

The Ascocenda flavanone 3-hydroxylase (AcF3H) gene was successfully transformed into Dendrobium 5N white orchid plants using Agrobacterium-mediated gene transformation. In this study, for the first time, we report the construction of a plant expression vector harboring the AcF3H gene using the Gateway cloning system. Protocorm-like bodies (PLBs) were cocultivated with the Agrobacterium tumefaciens strain AGL1 harboring the plant expression vector pGWB5-AcF3H, which contained the hygromycin phosphotransferase (hpt) gene as a selectable marker. The highest transformation efficiency (10.13%) was achieved when PLBs were cocultivated with Agrobacterium cells for 15 min. Three months after the transformation, the plantlets were regenerated, and the transgenic plants were confirmed by polymerase chain reaction (PCR) analysis using specific primers for the hpt gene and 35S promoter region. PCR products of approximately 400 and 500 bp, corresponding to the hpt gene and the 35S promoter, respectively, were detected in the transgenic plants, while no such product was observed in nontransgenic plants, indicating that the AcF3H gene was integrated into the genome of Dendrobium 5N white orchid plants. The transient expression of the AcF3H gene in Dendrobium 5N white and Dendrobium Anna petals was performed using the agroinfiltration technique, and the results demonstrated that no cyanidin content was detected in the Dendrobium 5N white petals after AcF3H infiltration. In contrast, the cyanidin content was increased by approximately 6% in the Dendrobium Anna petals, suggesting that the AcF3H gene was transiently expressed in this orchid.