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Concept: Ethanol fermentation

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Yeast and cancer cells share the unusual characteristic of favoring fermentation of sugar over respiration. We now reveal an evolutionary conserved mechanism linking fermentation to activation of Ras, a major regulator of cell proliferation in yeast and mammalian cells, and prime proto-oncogene product. A yeast mutant (tps1∆) with overactive influx of glucose into glycolysis and hyperaccumulation of Fru1,6bisP, shows hyperactivation of Ras, which causes its glucose growth defect by triggering apoptosis. Fru1,6bisP is a potent activator of Ras in permeabilized yeast cells, likely acting through Cdc25. As in yeast, glucose triggers activation of Ras and its downstream targets MEK and ERK in mammalian cells. Biolayer interferometry measurements show that physiological concentrations of Fru1,6bisP stimulate dissociation of the pure Sos1/H-Ras complex. Thermal shift assay confirms direct binding to Sos1, the mammalian ortholog of Cdc25. Our results suggest that the Warburg effect creates a vicious cycle through Fru1,6bisP activation of Ras, by which enhanced fermentation stimulates oncogenic potency.Yeast and cancer cells both favor sugar fermentation in aerobic conditions. Here the authors describe a conserved mechanism from yeast to mammals where the glycolysis intermediate fructose-1,6-bisphosphate binds Cdc25/Sos1 and couples increased glycolytic flux to increased Ras proto-oncoprotein activity.

Concepts: Enzyme, Cell cycle, Bacteria, Yeast, Adenosine triphosphate, Ethanol fermentation, Glucose, Glycolysis

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BACKGROUND: Very high gravity (VHG) fermentation using medium in excess of 250 g/L sugars for more than 15 % (v) ethanol can save energy consumption, not only for ethanol distillation, but also for distillage treatment; however, stuck fermentation with prolonged fermentation time and more sugars unfermented is the biggest challenge. Controlling redox potential (ORP) during VHG fermentation benefits biomass accumulation and improvement of yeast cell viability that is affected by osmotic pressure and ethanol inhibition, enhancing ethanol productivity and yield, the most important techno-economic aspect of fuel ethanol production. RESULTS: Batch fermentation was performed under different ORP conditions using the flocculating yeast and media containing glucose of 201 [PLUS-MINUS SIGN] 3.1, 252 [PLUS-MINUS SIGN] 2.9 and 298 [PLUS-MINUS SIGN] 3.8 g/L. Compared with ethanol fermentation by non-flocculating yeast, different ORP profiles were observed with the flocculating yeast due to the morphological change associated with the flocculation of yeast cells. When ORP was controlled at [MINUS SIGN]100 mV, ethanol fermentation with the high gravity (HG) media containing glucose of 201 [PLUS-MINUS SIGN] 3.1 and 252 [PLUS-MINUS SIGN] 2.9 g/L was completed at 32 and 56 h, respectively, producing 93.0 [PLUS-MINUS SIGN] 1.3 and 120.0 [PLUS-MINUS SIGN] 1.8 g/L ethanol, correspondingly. In contrast, there were 24.0 [PLUS-MINUS SIGN] 0.4 and 17.0 [PLUS-MINUS SIGN] 0.3 g/L glucose remained unfermented without ORP control. As high as 131.0 [PLUS-MINUS SIGN] 1.8 g/L ethanol was produced at 72 h when ORP was controlled at [MINUS SIGN]150 mV for the VHG fermentation with medium containing 298 [PLUS-MINUS SIGN] 3.8 g/L glucose, since yeast cell viability was improved more significantly. CONCLUSIONS: No lag phase was observed during ethanol fermentation with the flocculating yeast, and the implementation of ORP control improved ethanol productivity and yield. When ORP was controlled at [MINUS SIGN]150 mV, more reducing power was available for yeast cells to survive, which in turn improved their viability and VHG ethanol fermentation performance. On the other hand, controlling ORP at [MINUS SIGN]100 mV stimulated yeast growth and enhanced ethanol production under the HG conditions. Moreover, the ORP profile detected during ethanol fermentation with the flocculating yeast was less fluctuated, indicating that yeast flocculation could attenuate the ORP fluctuation observed during ethanol fermentation with non-flocculating yeast.

Concepts: Enzyme, Ethanol fermentation, Carbon dioxide, Fermentation, Ethanol fuel, Ethanol, Yeast

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The thermotolerant methylotrophic yeast Hansenula polymorpha is able to grow at elevated temperature up to 48 °C as one of a few yeast strains which are naturally capable of alcoholic fermentation of xylose, a pentose sugar abundant in lignocellulosic biomass. However, the current level of ethanol production from xylose by H. polymorpha is still very low compared to those of other xylose-fermenting strains. Therefore, it is necessary to analyze and remodel the xylose metabolism in H. polymorpha at the whole genome level to identify and overcome these limits. In the present study, the transcriptomes of H. polymorpha grown on xylose were compared with those of glucose-grown cells under both aerobic and microaerobic conditions. Approximately, two percent of H. polymorpha genes were either up- or down-regulated by more than two-fold during the growth on xylose. The majority of the up-regulated genes were involved in metabolism. Some genes involved in xylose metabolism, such as XYL1, XYL2, and TAL1 were also up-regulated, despite the fact that the differences in their induction level were only about three-fold. On the other hand, the majority of the down-regulated genes were involved in metabolism and cellular transport. Interestingly, some genes involved in glycolysis and ethanol fermentation were also repressed during growth on xylose, suggesting that these genes are good targets for engineering H. polymorpha to improve xylose fermentation.

Concepts: Glycolysis, Alcohol, Ethanol fuel, Alcoholic beverage, Fermentation, Ethanol, Ethanol fermentation, Yeast

2

Dekkera yeasts have often been considered as alternative sources of ethanol production that could compete with S. cerevisiae. The two lineages of yeasts independently evolved traits that include high glucose and ethanol tolerance, aerobic fermentation, and a rapid ethanol fermentation rate. The Saccharomyces yeasts attained these traits mainly through whole genome duplication approximately 100 million years ago (Mya). However, the Dekkera yeasts, which were separated from S. cerevisiae approximately 200 Mya, did not undergo whole genome duplication (WGD) but still occupy a niche similar to S. cerevisiae. Upon analysis of two Dekkera yeasts and five closely related non-WGD yeasts, we found that a massive loss of cis-regulatory elements occurred in an ancestor of the Dekkera yeasts, which led to improved mitochondrial functions similar to the S. cerevisiae yeasts. The evolutionary analysis indicated that genes involved in the transcription and translation process exhibited faster evolution in the Dekkera yeasts. We detected 90 positively selected genes, suggesting that the Dekkera yeasts evolved an efficient translation system to facilitate adaptive evolution. Moreover, we identified that 12 vacuolar H+-ATPase (V-ATPase) function genes that were under positive selection, which assists in developing tolerance to high alcohol and high sugar stress. We also revealed that the enzyme PGK1 is responsible for the increased rate of glycolysis in the Dekkera yeasts. These results provide important insights to understand the independent adaptive evolution of the Dekkera yeasts and provide tools for genetic modification promoting industrial usage.

Concepts: Ethanol fermentation, Fungus, Evolution, Genetics, Genome, DNA, Saccharomyces cerevisiae, Yeast

2

Contamination of corn mash by lactic acid bacteria (LAB) reduces the efficiency of the ethanol fermentation process. The industry relies heavily on antibiotics for contamination control and there is a need to develop alternative methods. The goals of this study were to determine the diversity and abundance of bacteria contaminating commercial ethanol fermentations, and to evaluate the potential of anti-LAB bacteriophages in controlling production losses.

Concepts: Yeast, Ethanol fermentation, Lactic acid, Microbiology, Lactobacillus, Fermentation, Bacteria

2

Genetic variation in chemosensory genes can explain variability in individual’s perception of and preference for many foods and beverages. To gain insight into variable preference and intake of alcoholic beverages, we explored individual variability in the responses to sampled ethanol (EtOH). In humans, EtOH elicits sweet, bitter, and burning sensations. Here, we explore the relationship between variation in EtOH sensations and polymorphisms in genes encoding bitter taste receptors (TAS2Rs) and a polymodal nociceptor (TRPV1).

Concepts: DNA, Ethanol fermentation, Yeast, Genetics, Ethanol, Taste, Alcohol, Alcoholic beverage

1

Efficient ethanol production from waste paper requires the addition of expensive nutrients. To reduce the production cost of ethanol from waste paper, a study on how to produce ethanol efficiently by adding kitchen waste (potentially as a carbon source, nutrient source, and acidity regulator) to waste paper was performed and a process of successive liquefaction, presaccharification, and simultaneous saccharification and fermentation (L+PSSF) was developed. The individual saccharification performances of waste paper and kitchen waste were not influenced by their mixture. Liquefaction of kitchen waste at 90°C prior to presaccharification and simultaneous saccharification and fermentation (PSSF) was essential for efficient ethanol fermentation. Ethanol at concentrations of 46.6 or 43.6g/l was obtained at the laboratory scale after fermentation for 96h, even without pH adjustment and/or the addition of extra nutrients. Similarly, ethanol at a concentration of 45.5g/l was obtained at the pilot scale after fermentation for 48h. The ethanol concentration of L+PSSF of the mixture of waste paper and kitchen waste was comparable to that of PSSF of waste paper with added nutrients (yeast extract and peptone) and pH adjustment using H2SO4, indicating that kitchen waste is not only a carbon source but also an excellent nutrient source and acidity regulator for fermentation of the mixture of waste paper and kitchen waste.

Concepts: Ethanol fermentation, Fermentation, Acetic acid, Ethanol fuel, Alcohol, Ethanol, Yeast, Carbon dioxide

1

Incidence of methanol contamination of traditionally fermented beverages is increasing globally resulting in the death of several persons. The source of methanol contamination has not been clearly established in most countries. While there were speculations that unscrupulous vendors might have deliberately spiked the beverages with methanol, it is more likely that the methanol might have been produced by contaminating microbes during traditional ethanol fermentation, which is often inoculated spontaneously by mixed microbes, with a potential to produce mixed alcohols. Methanol production in traditionally fermented beverages can be linked to the activities of pectinase producing yeast, fungi and bacteria. This study assessed some traditional fermented beverages and found that some beverages are prone to methanol contamination including cachaca, cholai, agave, arak, plum and grape wines. Possible microbial role in the production of methanol and other volatile congeners in these fermented beverages were discussed. The study concluded by suggesting that contaminated alcoholic beverages be converted for fuel use rather than out rightly banning the age-long traditional alcohol fermentation.

Concepts: Carbon dioxide, Fermentation, Ethanol fermentation, Wine, Alcohol, Yeast, Ethanol, Alcoholic beverage

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The growing demand to replace fossil fuels with renewable alternatives has generated an urgent and imminent global need to find new non-fossil sources. Sweet sorghum is widely recognized as a highly promising biomass energy crop with particular potential to complement sugarcane for ethanol production. Our aim in this study was to evaluate the influence of pH during the clarification method on the composition of essential nutrients in the sorghum juice and how this affects the efficiency of the ethanol fermentation process. We found that a higher pH directly affected residual concentrations of key nutrients (P, Ca, Zn, Mn) and as a consequence the efficiency of ethanol fermentation. In conclusion we recommend a clarification procedure at pH 6-6.5 in order not to significantly affect nutritional parameters important for the yeast fermentation process.

Concepts: Ethanol fermentation, Fermentation, Alcoholic beverage, Carbon dioxide, Ethanol fuel, Biofuel, Ethanol, Yeast

0

There is a worldwide interest for sustainable and environmentally-friendly ways to produce fuels and chemicals from renewable resources. Among them, the production of acetone, butanol and ethanol (ABE) or Isopropanol, Butanol and Ethanol (IBE) by anaerobic fermentation has already a long industrial history. Isopropanol has recently received a specific interest and the best studied natural isopropanol producer is C. beijerinckii DSM 6423 (NRRL B-593). This strain metabolizes sugars into a mix of IBE with only low concentrations of ethanol produced (< 1 g/L). However, despite its relative ancient discovery, few genomic details have been described for this strain. Research efforts including omics and genetic engineering approaches are therefore needed to enable the use of C. beijerinckii as a microbial cell factory for production of isopropanol.

Concepts: Ethanol fermentation, Bacteria, Microbiology, Metabolism, Yeast, Fermentation, Alcohol, Ethanol