Pretreatment of extruded Napier grass byhydrothermal process with dilute sulfuric acid and fermentation using a cellulose-hydrolyzing and xylose-assimilating yeast for ethanol production.

One of the potential bioresources for bioethanol production is Napier grass, considering its high cellulose and hemicellulose content. However, the cost of pretreatment hinders the bioethanol produced from being economical. This study examines the effect of hydrothermal process with dilute acid on extruded Napier grass, followed by enzymatic saccharification prior to simultaneous saccharification and co-fermentation (SScF). Extrusion facilitated lignin removal by 30.2 % prior to dilute acid steam explosion. Optimum pretreatment condition was obtained by using 3% sulfuric acid, and 30-min retention time of steam explosion at 190 °C. Ethanol yield of 0.26 g ethanol/g biomass (60.5% fermentation efficiency) was attained by short-term liquefaction and fermentation using a cellulose-hydrolyzing and xylose-assimilating Saccharomyces cerevisiae NBRC1440/B-EC3-X ΔPHO13, despite the presence of inhibitors. This proposed method not only reduced over-degradation of cellulose and hemicellulose, but also eliminated detoxification process and reduced cellulase loading.

Zinc, magnesium, and calcium ion supplementation confers tolerance to acetic acid stress in industrial Saccharomyces cerevisiae utilizing xylose.

Lignocellulosic biomass is a potential substrate for ethanol production. However, pretreatment of lignocellulosic materials produces inhibitory compounds such as acetic acid, which negatively affect ethanol production by Saccharomyces cerevisiae. Supplementation of the medium with three metal ions (Zn(2+) , Mg(2+) , and Ca(2+) ) increased the tolerance of S. cerevisiae toward acetic acid compared to the absence of the ions. Ethanol production from xylose was most improved (by 34%) when the medium was supplemented with 2 mM Ca(2+) , followed by supplementation with 3.5 mM Mg(2+) (29% improvement), and 180 µM Zn(2+) (26% improvement). Higher ethanol production was linked to high cell viability in the presence of metal ions. Comparative transcriptomics between the supplemented cultures and the control suggested that improved cell viability resulted from the induction of genes controlling the cell wall and membrane. Only one gene, FIT2, was found to be up-regulated in common between the three metal ions. Also up-regulation of HXT1 and TKL1 might enhance xylose consumption in the presence of acetic acid. Thus, the addition of ionic nutrients is a simple and cost-effective method to improve the acetic acid tolerance of S. cerevisiae.

Co-expression of TAL1 and ADH1 in recombinant xylose-fermenting Saccharomyces cerevisiae improves ethanol production from lignocellulosic hydrolysates in the presence of furfural.

Lignocellulosic biomass dedicated to bioethanol production usually contains pentoses and inhibitory compounds such as furfural that are not well tolerated by Saccharomyces cerevisiae. Thus, S. cerevisiae strains with the capability of utilizing both glucose and xylose in the presence of inhibitors such as furfural are very important in industrial ethanol production. Under the synergistic conditions of transaldolase (TAL) and alcohol dehydrogenase (ADH) overexpression, S. cerevisiae MT8-1X/TAL-ADH was able to produce 1.3-fold and 2.3-fold more ethanol in the presence of 70 mM furfural than a TAL-expressing strain and a control strain, respectively. We also tested the strains' ability by mimicking industrial ethanol production from hemicellulosic hydrolysate containing fermentation inhibitors, and ethanol production was further improved by 16% when using MT8-1X/TAL-ADH compared to the control strain. Transcript analysis further revealed that besides the pentose phosphate pathway genes TKL1 and TAL1, ADH7 was also upregulated in response to furfural stress, which resulted in higher ethanol production compared to the TAL-expressing strain. The improved capability of our modified strain was based on its capacity to more quickly reduce furfural in situ resulting in higher ethanol production. The co-expression of TAL/ADH genes is one crucial strategy to fully utilize undetoxified lignocellulosic hydrolysate, leading to cost-competitive ethanol production.

Time-based comparative transcriptomics in engineered xylose-utilizing Saccharomyces cerevisiae identifies temperature-responsive genes during ethanol production.

Agricultural residues comprising lignocellulosic materials are excellent sources of pentose sugar, which can be converted to ethanol as fuel. Ethanol production via consolidated bioprocessing requires a suitable microorganism to withstand the harsh fermentation environment of high temperature, high ethanol concentration, and exposure to inhibitors. We genetically enhanced an industrial Saccharomyces cerevisiae strain, sun049, enabling it to uptake xylose as the sole carbon source at high fermentation temperature. This strain was able to produce 13.9 g/l ethanol from 50 g/l xylose at 38 °C. To better understand the xylose consumption ability during long-term, high-temperature conditions, we compared by transcriptomics two fermentation conditions: high temperature (38 °C) and control temperature (30 °C) during the first 12 h of fermentation. This is the first long-term, time-based transcriptomics approach, and it allowed us to discover the role of heat-responsive genes when xylose is the sole carbon source. The results suggest that genes related to amino acid, cell wall, and ribosomal protein synthesis are down-regulated under heat stress. To allow cell stability and continuous xylose uptake in order to produce ethanol, hexose transporter HXT5, heat shock proteins, ubiquitin proteins, and proteolysis were all induced at high temperature. We also speculate that the strong relationship between high temperature and increased xylitol accumulation represents the cell's mechanism to protect itself from heat degradation.

Gene expression cross-profiling in genetically modified industrial Saccharomyces cerevisiae strains during high-temperature ethanol production from xylose.

Production of ethanol from xylose at high temperature would be an economical approach since it reduces risk of contamination and allows both the saccharification and fermentation steps in SSF to be running at elevated temperature. Eight recombinant xylose-utilizing Saccharomyces cerevisiae strains developed from industrial strains were constructed and subjected to high-temperature fermentation at 38 °C. The best performing strain was sun049T, which produced up to 15.2 g/L ethanol (63% of the theoretical production), followed by sun048T and sun588T, both with 14.1 g/L ethanol produced. Via transcriptomic analysis, expression profiling of the top three best ethanol producing strains compared to a negative control strain, sun473T, led to the discovery of genes in common that were regulated in the same direction. Identification of the 20 most highly up-regulated and the 20 most highly down-regulated genes indicated that the cells regulate their central metabolism and maintain the integrity of the cell walls in response to high temperature. We also speculate that cross-protection in the cells occurs, allowing them to maintain ethanol production at higher concentration under heat stress than the negative controls. This report provides further transcriptomics information in the interest of producing a robust microorganism for high-temperature ethanol production utilizing xylose.