Specific Ethanol Production Rate Research Articles

Kinetic modeling and optimization of ethanol fermentation by the marine yeast Wickerhamomyces subpelliculosus ZE75

The study aims to enhance ethanol production by Wickerhamomyces subpelliculosus ZE75 isolated from marine sediment. In addition, analyzing the kinetic parameters of ethanol production and optimization of the fermentation conditions was performed. The marine yeast isolate ZE75 was selected as the front runner ethanol-producer, with an ethanol yield of 89.77 gL−1. ZE75 was identified relying on the phenotypic and genotypic characteristics of W. subpelliculosus. The genotypic characterization based on the Internal Transcribed Spacer (ITS) sequence was deposited in the GenBank database with the accession number OP715873. The maximum specific ethanol production rate (vmax) was 0.482 gg−1 h−1 at 175 gL−1 glucose concentration, with a high accuracy of R2 0.95. The maximum growth specific rates (μmax) were 0.141 h−1 obtained at 150 gL−1 glucose concentration with R2 0.91. Optimization of the fermentation parameters such as pH and salinity has been achieved. The highest ethanol yield 0.5637 gg−1 was achieved in a 100% natural seawater-based medium. The maximum ethanol production of 104.04 gL−1 was achieved at pH 4.5 with a specific ethanol rate of 0.1669 gg−1 h−1. The findings of the present study recommend the possibility of ethanol production from a seawater-based medium on a large scale using W. subpelliculosus ZE75.Graphical abstract

Induction of Chromosomal Aneuploids from Brewery Shochu Yeast with Novel Brewery Characteristics

The first development method of brewery shochu yeast focusing on chromosomal aneuploidy is reported in this study. Euploidy diploid shochu yeast S-3 was treated with a microtubule inhibitor, nocodazole, for the purpose of inducing aneuploidy. Next, 2,3,5-triphenyl tetrazolium chloride (TTC) staining and the growth rate were investigated to select aneuploids. Aneuploids were selected at a frequency of 8.2 × 10−4, which was significantly higher than that of the control group, mainly at chromosomes I, II, III, IX, XII, XIII, and XVI. The acquired aneuploids were evaluated for their metabolic and brewing characteristics. A hierarchical cluster analysis based on endogenous metabolite data discriminated euploid S-3 and aneuploids. In addition, principal-component analysis of the constituents of the broth brewed with the strains discriminated between euploid S-3 and aneuploids. Sensory evaluation of the broth brewed with euploid S-3 and aneuploids showed that it tended to differ in aroma and taste. Specific ethanol production rates of the aneuploids were not deteriorated. The method of this selection made it possible to efficiently obtain aneuploids with various brewing characteristics from brewer’s yeast, which do not correspond to genetically modified organisms. This novel breeding method focusing on chromosomal aneuploidy will facilitate the development of novel shochu yeast strains.

Assessment of ethanol tolerance of Kluyveromyces marxianus CCT 7735 selected by adaptive laboratory evolution.

Kluyveromyces marxianus CCT 7735 shows potential for producing ethanol from lactose; however, its low ethanol tolerance is a drawback for its industrial application. The first aim of this study was to obtain four ethanol-tolerant K. marxianus CCT 7735 strains (ETS1, ETS2, ETS3, and ETS4) by adaptive laboratory evolution. The second aim was to select among them the strain that stood out and to evaluate metabolic changes associated with the improved ethanol tolerance in this strain. The ETS4 was selected for displaying a specific growth rate higher than the parental strain under ethanol stress (122%) and specific ethanol production rate (0.26g/g/h) higher than those presented by the ETS1 (0.22g/g/h), ETS2 (0.17g/g/h), and ETS3 (0.17g/g/h) under non-stress condition. Further analyses were performed with the ETS4 in comparison with its parental strain in order to characterize metabolic changes. Accumulation of valine and metabolites of the citric acid cycle (isocitric acid, citric acid, and cis-aconitic acid) was observed only in the ETS4 subjected to ethanol stress. Their accumulation in this strain may have been important to increase ethanol tolerance. Furthermore, the contents of fatty acid methyl esters and ergosterol were higher in the ETS4 than in the parental strain. These differences likely contributed to enhance ethanol tolerance in the ETS4. KEY POINTS: • K. marxianus ethanol-tolerant strains were selected by adaptive laboratory evolution. • Valine and metabolites of the TCA cycle were accumulated in the ETS4. • High contents of fatty acids and ergosterol contributed to enhance ethanol tolerance.

Improve biotransformation of crude glycerol to ethanol of Enterobacter aerogenes by two-stage redox potential fed-batch process under microaerobic environment

Ethanol production from crude glycerol under microaerobic condition was optimized. The aeration rate in the reactor was controlled using redox potential (ORP) as to replace a conventional oxygen sensor that became insensitive at low oxygen concentration. The performance of Enterobacter aerogenes TISTR1468 in a batch reactor using a simple medium (crude glycerol and tuna condensate (GT) medium) was evaluated on various aeration strategies: continuous aeration at 0.5 vvm; controlled aeration at −350 mV and −400 mV ORP; and no aeration. The 72-h batch fermentation showed that a more reductive environment (lower aeration and ORP value) increased ethanol concentration and yield, but decreased the ethanol productivity. No-aeration fermentation with the lowest ORP value achieved the highest ethanol production (18.78 g L−1) and yield (0.94 g-ethanol.g-glycerol−1). A more oxidative environment (higher aeration and ORP value) fasten the specific growth rate (μ), but ethanol concentration and yield decreased. The optimum specific ethanol production rate (qp) and glycerol consumption rate occurred at ORP of −400 mV and −350 mV, respectively. When the ORP was very low, the μ decreased following substrate depletion, but this phenomenon was not observed at higher ORP value, an evident that adequate oxygen supply could maintain cell viability.Fed-batch with two-stage aeration strategy (−350 mV and −400 mV ORP) acquired higher ethanol production (30.31 g L−1) than fed-batch with single-stage aeration (25.95 g L−1) and batch process (12.33 g L−1).This study has revived the potential of crude glycerol biotransformation to ethanol and opened up opportunities for optimization of related microaerobic systems other than ethanol.

Transomics data-driven, ensemble kinetic modeling for system-level understanding and engineering of the cyanobacteria central metabolism

In silico kinetic modeling is an essential tool for rationally designing metabolically engineered organisms based on a system-level understanding of their regulatory mechanisms. However, an estimation of enzyme parameters has been a bottleneck in the computer simulation of metabolic dynamics. In this study, the ensemble-modeling approach was integrated with the transomics data to construct kinetic models. Kinetic metabolic models of a photosynthetic bacterium, Synechocystis sp. PCC 6803, were constructed to identify engineering targets for improving ethanol production based on an understanding of metabolic regulatory systems. A kinetic model ensemble was constructed by randomly sampling parameters, and the best 100 models were selected by comparing predicted metabolic state with a measured dataset, including metabolic flux, metabolite concentrations, and protein abundance data. Metabolic control analysis using the model ensemble revealed that a large pool size of 3-phosphoglycerate could be a metabolic buffer responsible for the stability of the Calvin–Benson cycle, and also identified that phosphoglycerate kinase (PGK) is a promising engineering target to improve a pyruvate supply such as for ethanol production. Overexpression of PGK in the metabolically engineered PCC 6803 strain showed that the specific ethanol production rate and ethanol titers at 48 h were 1.23- and 1.37-fold greater than that of the control strain. PGK is useful for future metabolic engineering since pyruvate is a common precursor for the biosynthesis of various chemicals.

Comparative analysis of fermentation and enzyme expression profiles among industrial Saccharomyces cerevisiae strains.

Industrial diploid strains of Saccharomyces cerevisiae are selected from natural populations and then domesticated by optimizing the preferred properties for producing products such as bread, wine, and sake. In this study, for comparing the fermentation performance of various industrial yeasts, seven diploid strains of S. cerevisiae, namely, BY4947 (laboratory yeast derived from S288C), Kyokai7 and Kyokai9 (sake yeasts), Red Star and NBRC0555 (bread yeasts), and QA23 and EC1118 (wine yeasts), were cultivated in a synthetic medium. The fermentation profiles of the seven yeast strains showed significant differences. The specific ethanol production rates of sake yeasts (Kyokai7 and Kyokai9) and wine strains (QA23 and EC1118) were higher and lower than those of laboratory strains, respectively. Targeted proteome analysis was also conducted to investigate the variation in the expression of metabolism-related enzymes. The expression profiles of central metabolism-related enzymes showed considerable variations among the industrial strains. Upregulation of the TCA cycle in wine strains was observed both in the synthetic and grape-juice media. These results suggested that these variations should be consequences of complex interactions between the domestication process, genetic polymorphism, and environmental factors such as the fermentation conditions.

Disruption of the transcription factors Thi2p and Nrm1p alleviates the post-glucose effect on xylose utilization in Saccharomyces cerevisiae

BackgroundThe recombinant Saccharomyces cerevisiae strains that acquired the ability to utilize xylose through metabolic and evolutionary engineering exhibit good performance when xylose is the sole carbon source in the medium (designated the X stage in the present work). However, the xylose consumption rate of strains is generally low after glucose depletion during glucose–xylose co-fermentation, despite the presence of xylose in the medium (designated the GX stage in the present work). Glucose fermentation appears to reduce the capacity of these strains to “recognize” xylose during the GX stage, a phenomenon termed the post-glucose effect on xylose metabolism.ResultsTwo independent xylose-fermenting S. cerevisiae strains derived from a haploid laboratory strain and a diploid industrial strain were used in the present study. Their common characteristics were investigated to reveal the mechanism underlying the post-glucose effect and to develop methods to alleviate this effect. Both strains showed lower growth and specific xylose consumption rates during the GX stage than during the X stage. Glycolysis, the pentose phosphate pathway, and translation-related gene expression were reduced; meanwhile, genes in the tricarboxylic acid cycle and glyoxylic acid cycle demonstrated higher expression during the GX stage than during the X stage. The effects of 11 transcription factors (TFs) whose expression levels significantly differed between the GX and X stages in both strains were investigated. Knockout of THI2 promoted ribosome synthesis, and the growth rate, specific xylose utilization rate, and specific ethanol production rate of the strain increased by 17.4, 26.8, and 32.4%, respectively, in the GX stage. Overexpression of the ribosome-related genes RPL9A, RPL7B, and RPL7A also enhanced xylose utilization in a corresponding manner. Furthermore, the overexpression of NRM1, which is related to the cell cycle, increased the growth rate by 8.7%, the xylose utilization rate by 30.0%, and the ethanol production rate by 76.6%.ConclusionsThe TFs Thi2p and Nrm1p exerted unexpected effects on the post-glucose effect, enhancing ribosome synthesis and altering the cell cycle, respectively. The results of this study will aid in maintaining highly efficient xylose metabolism during glucose–xylose co-fermentation, which is utilized for lignocellulosic bioethanol production.

Metabolic engineering and adaptive evolution ofEscherichia coliKO11 for ethanol production through the Entner-Doudoroff and the pentose phosphate pathways

BACKGROUND Ethanologenic Escherichia coli KO11 was modified to channel carbon flux from glucose through the Entner–Doudoroff (ED-P) and pentose phosphate (PP-P) pathways by using a phosphoglucose isomerase (pgi) knockout in the glycolytic pathway. This strain grows very slowly under non-aerated conditions with minimal media supplemented with 4% glucose. To improve the capacity to grow, KO11 Δpgi was evolved for 60 days; the resultant strain was named KO11 E35, which directs the carbon flux through the PP-P and ED-P to lactate and acetate production. RESULTS The activities of glucose-6-phosphate dehydrogenase and the ED-P enzymes increased 17-fold and 2-fold, respectively, in KO11 E35 in comparison with KO11. A homoethanologenic derivative was constructed from KO11 E35 by deleting the pta, ack and ldh genes, yielding the KO11 PPAL strain. This strain channels most of the carbon flux from pyruvate to ethanol and increased expression of heterologous pyruvate decarboxylase and alcohol dehydrogenase from Zymomonas mobilis allowed us to obtain specific ethanol production rates similar to those found in KO11, but with half the cell mass, i.e. larger ethanol/glucose and ethanol/biomass yields. CONCLUSIONS These results suggest that it is possible to obtain the same carbon flux using the PP-P and ED-P as when using the Embden–Meyerhof–Parnas pathway for glucose catabolism to ethanol. © 2016 Society of Chemical Industry

Improved glycerol to ethanol conversion by E. coli using a metagenomic fragment isolated from an anaerobic reactor.

Crude glycerol obtained as a by-product of biodiesel production is a reliable feedstock with the potential to be converted into reduced chemicals with high yields. It has been previously shown that ethanol is the primary product of glycerol fermentation by Escherichia coli. However, few efforts were made to enhance this conversion by means of the expression of heterologous genes with the potential to improve glycerol transport or metabolism. In this study, a fosmid-based metagenomic library constructed from an anaerobic reactor purge sludge was screened for genetic elements that promote the use and fermentation of crude glycerol by E. coli. One clone was selected based on its improved growth rate on this feedstock. The corresponding fosmid, named G1, was fully sequenced (41kbp long) and the gene responsible for the observed phenotype was pinpointed by in vitro insertion mutagenesis. Ethanol production from both pure and crude glycerol was evaluated using the parental G1 clone harboring the ethanologenic plasmid pLOI297 or the industrial strain LY180 complemented with G1. In mineral salts media containing 50% (v/v) pure glycerol, ethanol concentrations increased two-fold on average when G1 was present in the cells reaching up to 20g/L after 24h fermentation. Similar fermentation experiments were done using crude instead of pure glycerol. With an initial OD620 of 8.0, final ethanol concentrations after 24h were much higher reaching 67 and 75g/L with LY180 cells carrying the control fosmid or the G1 fosmid, respectively. This translates into a specific ethanol production rate of 0.39g h(-1) OD(-1) L(-1).

Purification, enzymatic activity and inhibitor discovery for recombinant human carbonic anhydrase XIV

In the important industrial yeast Saccharomyces cerevisiae, galactose metabolism requires energy production by respiration; therefore, this yeast cannot metabolize galactose under strict anaerobic conditions. While the respiratory dependence of galactose metabolism provides benefits in terms of cell growth and population stability, it is not advantageous for producing fuels and chemicals since a substantial fraction of consumed galactose is converted to carbon dioxide. In order to force S. cerevisiae to use galactose without respiration, a subunit (COX9) of a respiratory enzyme was deleted, but the resulting deletion mutant (Δcox9) was impaired in terms of galactose assimilation. Interestingly, after serial sub-cultures on galactose, the mutant evolved rapidly and was able to use galactose via fermentation only. The evolved strain (JQ-G1) produced ethanol from galactose with a 94% increase in yield and 6.9-fold improvement in specific productivity as compared to the wild-type strain. 13C-metabolic flux analysis demonstrated a three-fold reduction in carbon flux through the TCA cycle of the evolved mutant with redirection of flux toward the fermentation pathway. Genome sequencing of the JQ-G1 strain revealed a loss of function mutation in a master negative regulator of the Leloir pathway (Gal80p). The mutation (Glu348*) in Gal80p was found to act synergistically with deletion of COX9 for efficient galactose fermentation, and thus the double deletion mutant Δcox9Δgal80 produced ethanol 2.4 times faster and with 35% higher yield than a single knockout mutant with deletion of GAL80 alone. When we introduced a functional COX9 cassette back into the JQ-G1 strain, the JQ-G1-COX9 strain showed a 33% reduction in specific galactose uptake rate and a 49% reduction in specific ethanol production rate as compared to JQ-G1. The wild-type strain was also subjected to serial sub-cultures on galactose but we failed to isolate a mutant capable of utilizing galactose without respiration. We concluded that the metabolic “death valley” (i.e. no galactose utilization by the Δcox9 mutant) is a necessary intermediate phenotype to facilitate galactose utilization without respiration in yeast. The results in this study demonstrate a promising approach for directing adaptive evolution toward fermentative metabolism and for generating evolved yeast strains with improved phenotypes under anaerobic conditions.

Rapid and efficient galactose fermentation by engineered Saccharomyces cerevisiae

In the important industrial yeast Saccharomyces cerevisiae, galactose metabolism requires energy production by respiration; therefore, this yeast cannot metabolize galactose under strict anaerobic conditions. While the respiratory dependence of galactose metabolism provides benefits in terms of cell growth and population stability, it is not advantageous for producing fuels and chemicals since a substantial fraction of consumed galactose is converted to carbon dioxide. In order to force S. cerevisiae to use galactose without respiration, a subunit (COX9) of a respiratory enzyme was deleted, but the resulting deletion mutant (Δcox9) was impaired in terms of galactose assimilation. Interestingly, after serial sub-cultures on galactose, the mutant evolved rapidly and was able to use galactose via fermentation only. The evolved strain (JQ-G1) produced ethanol from galactose with a 94% increase in yield and 6.9-fold improvement in specific productivity as compared to the wild-type strain. 13C-metabolic flux analysis demonstrated a three-fold reduction in carbon flux through the TCA cycle of the evolved mutant with redirection of flux toward the fermentation pathway. Genome sequencing of the JQ-G1 strain revealed a loss of function mutation in a master negative regulator of the Leloir pathway (Gal80p). The mutation (Glu348*) in Gal80p was found to act synergistically with deletion of COX9 for efficient galactose fermentation, and thus the double deletion mutant Δcox9Δgal80 produced ethanol 2.4 times faster and with 35% higher yield than a single knockout mutant with deletion of GAL80 alone. When we introduced a functional COX9 cassette back into the JQ-G1 strain, the JQ-G1-COX9 strain showed a 33% reduction in specific galactose uptake rate and a 49% reduction in specific ethanol production rate as compared to JQ-G1. The wild-type strain was also subjected to serial sub-cultures on galactose but we failed to isolate a mutant capable of utilizing galactose without respiration. We concluded that the metabolic “death valley” (i.e. no galactose utilization by the Δcox9 mutant) is a necessary intermediate phenotype to facilitate galactose utilization without respiration in yeast. The results in this study demonstrate a promising approach for directing adaptive evolution toward fermentative metabolism and for generating evolved yeast strains with improved phenotypes under anaerobic conditions.

Deletion of JJJ1 improves acetic acid tolerance and bioethanol fermentation performance of Saccharomyces cerevisiae strains.

To improve tolerance to acetic acid that is present in lignocellulosic hydrolysates andaffects bioethanol production by Saccharomyces cerevisiae. Saccharomyces cerevisiae strains with improved tolerance to acetic acid were obtained through deletion of the JJJ1 gene. The lag phase of the JJJ1 deletion mutant BYΔJJJ1 was ~16 h shorter than that of the parent strain, BY4741, when the fermentation medium contained 4.5 g acetic acid/l. Additionally, the specific ethanol production rate of BYΔJJJ1 was increased (0.057 g/gh) compared to that of the parent strain (0.051 g/gh). Comparative transcription and physiological analyses revealed higher long chain fatty acid, trehalose, and catalase contents might be critical factors responsible for the acetic acid resistance of JJJ1 knockout strains. JJJ1 deletion improves acetic acid tolerance and ethanol fermentation performance of S. cerevisiae.

Microaerobic conversion of xylose to ethanol in recombinant Saccharomyces cerevisiae SX6MUT expressing cofactor-balanced xylose metabolic enzymes and deficient in ALD6

Xylose is a major monosugar in cellulosic biomass and should be utilized for cost-effective ethanol production. In this study, xylose-converting ability of recombinant Saccharomyces cerevisiae SX6MUT expressing NADH-preferring xylose reductase mutant (R276H) and other xylose-metabolic enzymes, and deficient in aldehyde dehydrogenase 6 (Ald6p) were characterized at microaerobic conditions using various sugar mixtures. The reduction of air supply from 0.5vvm to 0.1vvm increased specific ethanol production rate by 75% and did not affect specific xylose consumption rate. In batch fermentations using various concentrations of xylose (50–104g/L), higher xylose concentration enhanced xylose consumption rate and ethanol productivity but reduced ethanol yield, owing to the accumulation of xylitol and glycerol from xylose. SX6MUT consumed monosugars in pitch pine hydrolysates and produced 23.1g/L ethanol from 58.7g/L sugars with 0.39g/g ethanol yield, which was 14% higher than the host strain of S. cerevisiae D452-2 without the xylose assimilating enzymes. In conclusion, S. cerevisiae SX6MUT was characterized to possess high xylose-consuming ability in microaerobic conditions and a potential for ethanol production from cellulosic biomass.

Efficient butanol-ethanol (B-E) production from carbon monoxide fermentation by Clostridium carboxidivorans.

The fermentation of waste gases rich in carbon monoxide using acetogens is an efficient way to obtain valuable biofuels like ethanol and butanol. Different experiments were carried out with the bacterial species Clostridium carboxidivorans as biocatalyst. In batch assays with no pH regulation, after complete substrate exhaustion, acetic acid, butyric acid, and ethanol were detected while only negligible butanol production was observed. On the other side, in bioreactors, with continuous carbon monoxide supply and pH regulation, both C2 and C4 fatty acids were initially formed as well as ethanol and butanol at concentrations never reported before for this type of anaerobic bioconversion of gaseous C1 compounds, showing that the operating conditions significantly affect the metabolic fermentation profile and butanol accumulation. Maximum ethanol and butanol concentrations in the bioreactors were obtained at pH 5.75, reaching values of 5.55 and 2.66 g/L, respectively. The alcohols were produced both from CO fermentation as well as from the bioconversion of previously accumulated acetic and butyric acids, resulting in low residual concentrations of such acids at the end of the bioreactor experiments. CO consumption was often around 50% and reached up to more than 80%. Maximum specific rates of ethanol and butanol production were reached at pH 4.75, with values of 0.16 g/h*g of biomass and 0.07 g/h*g of biomass, respectively, demonstrating that a low pH was more favorable to solventogenesis in this process, although it negatively affects biomass growth which does also play a role in the final alcohol titer.

Continuous Ethanol Production from D-xylose: Using Free Cells of Clavispora opuntiae

The continuous fermentation of D-xylose to ethanol by free cells of Clavispora opuntiae was studied in a continuous stirred tank reactor at 25 ± 0.5°C and pH 4.2 ± 0.2. The maximum 89.8 ± 0.04% ethanol yield was obtained as compared to theoretical yield and about 96.00 ± 0.05% sugar was utilized at a dilution rate of 0.014 h–1. The maximum values of volumetric ethanol (Qp) and biomass (Qx) productivity were found to be 0.67 ± 0.04 gp l–1 h–1 and 0.18 ± 0.007 gx l–1 h–1, respectively, at a dilution rate of 0.09 h–1. The maintenance energy coefficient for C. opuntiae was found to be 0.21 ± 0.005 gs gx–1 h–1, whereas the values of specific ethanol production rate (qp) and specific sugar uptake rate (qs) were found to increase almost linearly with the increase of dilution rate. The maximum specific ethanol productivity (qp max) and maximum specific sugar uptake rate (qs max) were 0.50 ± 0.004 gp gx–1 h–1 and 4.00 ± 0.006 gs gx–1 h–1, respectively.

Kinetic Models and Effects of Initial Total Soluble Sugar Concentrations on Batch Ethanol Fermentation of Sweet Sorghum Stalk Juice by Saccharomyces cerevisiae Strain

Effects of initial total soluble sugar concentrations on batch ethanol fermentation of sweet sorghum stalk juice by Saccharomyces cerevisiae strain were investigated at initial total soluble sugar concentrations of 85–156 g/L. The result showed that cell growth and the ethanol yield were increased with the increase of initial total soluble sugar concentrations. The kinetic parameters at initial total soluble sugar concentrations of 85–156 g/L were obtained. With the increase of initial total soluble sugar concentration, an inhibition of initial total soluble sugar concentration on the maximum specific growth rate (μmax) was observed. However, the increase of initial total soluble sugar concentration favors in the increase of the maximum specific ethanol production rate (qmax). The models proposed by Hinshelwood were found to be appropriate for describing the batch growth and ethanol fermentation of sweet sorghum stalk juice by immobilized Saccharomyces cerevisiae at initial total soluble sugar concentrations of 85–156 g/L because the correlation coefficients for the cell growth kinetic model and the ethanol production kinetic model were greater than 0.99.

High temperature stimulates acetic acid accumulation and enhances the growth inhibition and ethanol production by Saccharomyces cerevisiae under fermenting conditions

Cellular responses of Saccharomyces cerevisiae to high temperatures of up to 42°C during ethanol fermentation at a high glucose concentration (i.e., 100g/L) were investigated. Increased temperature correlated with stimulated glucose uptake to produce not only the thermal protectant glycerol but also ethanol and acetic acid. Carbon flux into the tricarboxylic acid (TCA) cycle correlated positively with cultivation temperature. These results indicate that the increased demand for energy (in the form of ATP), most likely caused by multiple stressors, including heat, acetic acid, and ethanol, was matched by both the fermentation and respiration pathways. Notably, acetic acid production was substantially stimulated compared to that of other metabolites during growth at increased temperature. The acetic acid produced in addition to ethanol seemed to subsequently result in adverse effects, leading to increased production of reactive oxygen species. This, in turn, appeared to cause the specific growth rate, and glucose uptake rate reduced leading to a decrease of the specific ethanol production rate far before glucose depletion. These results suggest that adverse effects from heat, acetic acid, ethanol, and oxidative stressors are synergistic, resulting in a decrease of the specific growth rate and ethanol production rate and, hence, are major determinants of cell stability and ethanol fermentation performance of S. cerevisiae at high temperatures. The results are discussed in the context of possible applications.

Evaluation of the tolerance of acetic acid and 2-furaldehyde on the growth of Pichia stipitis and its respiratory deficient

The use of lignocellulosic residues for ethanol production is limited by toxic compounds in fermenting yeasts present in diluted acid hydrolysates like acetic acid and 2-furaldehyde. The respiratory deficient phenotype gives the cell the ability to resist several toxic compounds. So the aim of this work was to evaluate the tolerance to toxic compounds present in lignocellulosic hydrolysates like acetic acid and 2-furaldehyde in Pichia stipitis and its respiratory deficient strains. The respiratory deficient phenotype was induced by exposure to chemical agents such as acriflavine, acrylamide and rhodamine; 23 strains were obtained. The selection criterion was based on increasing specific ethanol yield (gethanolg(-1)biomass) with acetic acid and furaldehyde tolerance. The screening showed that P. stipitis NRRL Y-7124 ACL 2-1RD (lacking cytochrome c), obtained using acrylamide, presented the highest specific ethanol production rate (1.82gg(-1)h(-1)). Meanwhile, the ACF8-3RD strain showed the highest acetic acid tolerance (7.80gL(-1)) and the RHO2-3RD strain was able to tolerate up to 1.5gL(-1) 2-furaldehyde with a growth and ethanol production inhibition of 23 and 22%, respectively. The use of respiratory deficient yeast phenotype is a strategy for ethanol production improvement in a medium with toxic compounds such as hydrolysed sugarcane bagasse amongst others.

Technological properties of indigenous wine yeast strains isolated from wine production regions of Turkey

The purpose of this study was to evaluate the important technological and fermentative properties of wine yeast strains previously isolated from different wine producing regions of Turkey. The determination of the following important properties was made: growth at high temperatures; fermentative capability in the presence of high sugar concentration; fermentation rate; hydrogen sulfide production; killer activity; resistance to high ethanol and sulfur dioxide; foam production; and enzymatic profiles. Ten local wine yeast strains belonging to Saccharomyces, and one commercial active dry yeast as a reference strain were evaluated. Fermentation characteristics were evaluated in terms of kinetic parameters, including ethanol yield (YP/S), biomass yield (YX/S), theoretical ethanol yield (%), specific ethanol production rate (qp; g/gh), specific glucose uptake rate (qs; g/gh), and the substrate conversion (%). All tested strains were able to grow at 37°C and to start fermentation at 30° Brix, and were resistant to high concentrations of sulfur dioxide. 60% of the strains were weak H2S producers, while the others produced high levels. Foam production was high, and no strains had killer activity. Six of the tested strains had the ability to grow and ferment at concentrations of 14% ethanol. Except for one strain, all fermented most of the media sugars at a high rate, producing 11.0-12.4% (v/v) ethanol. Although all but one strain had suitable characteristics for wine production, they possessed poor activities of glycosidase, esterase and proteinase enzymes of oenological interest. Nine of the ten local yeast strains were selected for their good oenological properties and their suitability as a wine starter culture.

Enhanced expression of genes involved in initial xylose metabolism and the oxidative pentose phosphate pathway in the improved xylose-utilizing Saccharomyces cerevisiae through evolutionary engineering

Fermentation of xylose in lignocellulosic hydrolysates by Saccharomycescerevisiae has been achieved through heterologous expression of the xylose reductase (XR)-xylitol dehydrogenase (XDH) pathway. However, the fermentation efficiency is far from the requirement for industrial application due to high yield of the byproduct xylitol, low ethanol yield, and low xylose consumption rate. Through evolutionary engineering, an improved xylose-utilizing strain SyBE005 was obtained with 78.3% lower xylitol production and a 2.6-fold higher specific ethanol production rate than those of the parent strain SyBE004, which expressed an engineered NADP(+)-preferring XDH. The transcriptional differences between SyBE005 and SyBE004 were investigated by quantitative RT-PCR. Genes including XYL1, XYL2, and XKS1 in the initial xylose metabolic pathway showed the highest up-regulation in SyBE005. The increased expression of XYL1 and XYL2 correlated with enhanced enzymatic activities of XR and XDH. In addition, the expression level of ZWF1 in the oxidative pentose phosphate pathway increased significantly in SyBE005, indicating an elevated demand for NADPH from XR. Genes involved in the TCA cycle (LAT1, CIT1, CIT2, KGD1, KGD, SDH2) and gluconeogenesis (ICL1, PYC1) were also up-regulated in SyBE005. Genomic analysis revealed that point mutations in transcriptional regulators CYC8 and PHD1 might be responsible for the altered expression. In addition, a mutation (Y89S) in ZWF1 was identified which might improve NADPH production in SyBE005. Our results suggest that increasing the expression of XYL1, XYL2, XKS1, and enhancing NADPH supply are promising strategies to improve xylose fermentation in recombinant S. cerevisiae.