Ethanol Production Rate Research Articles

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.

Transcriptomic response to prolonged ethanol production in the cyanobacterium Synechocystis sp. PCC6803

BackgroundThe production of biofuels in photosynthetic microalgae and cyanobacteria is a promising alternative to the generation of fuels from fossil resources. To be economically competitive, producer strains need to be established that synthesize the targeted product at high yield and over a long time. Engineering cyanobacteria into forced fuel producers should considerably interfere with overall cell homeostasis, which in turn might counteract productivity and sustainability of the process. Therefore, in-depth characterization of the cellular response upon long-term production is of high interest for the targeted improvement of a desired strain.ResultsThe transcriptome-wide response to continuous ethanol production was examined in Synechocystis sp. PCC6803 using high resolution microarrays. In two independent experiments, ethanol production rates of 0.0338% (v/v) ethanol d-1 and 0.0303% (v/v) ethanol d-1 were obtained over 18 consecutive days, measuring two sets of biological triplicates in fully automated photobioreactors. Ethanol production caused a significant (~40%) delay in biomass accumulation, the development of a bleaching phenotype and a down-regulation of light harvesting capacity. However, microarray analyses performed at day 4, 7, 11 and 18 of the experiment revealed only three mRNAs with a strongly modified accumulation level throughout the course of the experiment. In addition to the overexpressed adhA (slr1192) gene, this was an approximately 4 fold reduction in cpcB (sll1577) and 3 to 6 fold increase in rps8 (sll1809) mRNA levels. Much weaker modifications of expression level or modifications restricted to day 18 of the experiment were observed for genes involved in carbon assimilation (Ribulose bisphosphate carboxylase and Glutamate decarboxylase). Molecular analysis of the reduced cpcB levels revealed a post-transcriptional processing of the cpcBA operon mRNA leaving a truncated mRNA cpcA* likely not competent for translation. Moreover, western blots and zinc-enhanced bilin fluorescence blots confirmed a severe reduction in the amounts of both phycocyanin subunits, explaining the cause of the bleaching phenotype.ConclusionsChanges in gene expression upon induction of long-term ethanol production in Synechocystis sp. PCC6803 are highly specific. In particular, we did not observe a comprehensive stress response as might have been expected.

Kinetics and Thermodynamics of Ethanol Production by Saccharomyces cerevisiae MLD10 Using Molasses

In this study, we have used ultraviolet (UV) and γ-ray induction to get a catabolite repression resistant and thermotolerant mutant with enhanced ethanol production along with optimization of sugar concentration and temperature of fermentation. Classical mutagenesis in two consecutive cycles of UV- and γ-ray-induced mutations evolved one best catabolite-resistant and thermotolerant mutant Saccharomyces cerevisiae MLD10 which showed improved ethanol yield (0.48 ± 0.02 g g(-1)), theoretical yield (93 ± 3%), and extracellular invertase productivity (1,430 ± 50 IU l(-1) h(-1)), respectively, when fermenting 180 g sugars l(-1) in molasses medium at 43 °C in 300 m(3) working volume fermenter. Ethanol production was highly dependent on invertase production. Enthalpy (ΔH*) (32.27 kJ M(-1)) and entropy (ΔS*) (-202.88 J M(-1) K(-1)) values at 43 °C by the mutant MLD10 were significantly lower than those of β-glucosidase production by a thermophilic mutant derivative of Thermomyces lanuginosus. These results confirmed the enhanced production of ethanol and invertase by this mutant derivative. These studies proved that mutant was significantly improved for ethanol production and was thermostable in nature. Lower fermentation time for ethanol production and maintenance of ethanol production rates (3.1 g l(-1) h(-1)) at higher temperature (43 °C) by this mutant could decrease the overall cost of fermentation process and increase the quality of ethanol production.

Systematic and evolutionary engineering of a xylose isomerase-based pathway in

Efficient xylose fermentation by yeast would improve the economical and sustainable nature of biofuels production from lignocellulosic biomass. However, the efficiency of xylose fermentation by the yeast Saccharomyces cerevisiae is suboptimal, especially in conversion yield, despite decades of research. Here, we present an improved performance of S. cerevisiae in xylose fermentation through systematic and evolutionary engineering approaches. The engineering of S. cerevisiae harboring xylose isomerase-based pathway significantly improved the xylose fermentation performance without the need for intensive downstream pathway engineering. This strain contained two integrated copies of a mutant xylose isomerase, gre3 and pho13 deletion and XKS1 and S. stipitis tal1 overexpression. This strain was subjected to rapid adaptive evolution to yield the final, evolved strain (SXA-R2P-E) which could efficiently convert xylose to ethanol with a yield of 0.45 g ethanol/g xylose, the highest yield reported to date. The xylose consumption and ethanol production rates, 0.98 g xylose g cell−1 h−1 and 0.44 g ethanol g cell−1 h−1, respectively, were also among the highest reported. During this process, the positive effect of a pho13 deletion was identified for a xylose isomerase-containing strain and resulted in up to an 8.2-fold increase in aerobic growth rate on xylose. Moreover, these results demonstrated that low inoculum size and the cell transfer at exponential phase was found to be the most effective adaptation strategy during a batch culture adaptation process. These results suggest that the xylose isomerase pathway should be the pathway of choice for efficient xylose fermentation in S. cerevisiae as it can outperform strains with the oxidoreductase pathway in terms of yield and ethanol production and xylose consumption rates. Consequently, the strain developed in this study could significantly improve the prospect of biofuels production from lignocellulosic biomass.

Transcription analysis of recombinant industrial and laboratory Saccharomyces cerevisiae strains reveals the molecular basis for fermentation of glucose and xylose

BackgroundThere has been much research on the bioconversion of xylose found in lignocellulosic biomass to ethanol by genetically engineered Saccharomyces cerevisiae. However, the rate of ethanol production from xylose in these xylose-utilizing yeast strains is quite low compared to their glucose fermentation. In this study, two diploid xylose-utilizing S. cerevisiae strains, the industrial strain MA-R4 and the laboratory strain MA-B4, were employed to investigate the differences between anaerobic fermentation of xylose and glucose, and general differences between recombinant yeast strains, through genome-wide transcription analysis.ResultsIn MA-R4, many genes related to ergosterol biosynthesis were expressed more highly with glucose than with xylose. Additionally, these ergosterol-related genes had higher transcript levels in MA-R4 than in MA-B4 during glucose fermentation. During xylose fermentation, several genes related to central metabolic pathways that typically increase during growth on non-fermentable carbon sources were expressed at higher levels in both strains. Xylose did not fully repress the genes encoding enzymes of the tricarboxylic acid and respiratory pathways, even under anaerobic conditions. In addition, several genes involved in spore wall metabolism and the uptake of ammonium, which are closely related to the starvation response, and many stress-responsive genes mediated by Msn2/4p, as well as trehalose synthase genes, increased in expression when fermenting with xylose, irrespective of the yeast strain. We further observed that transcript levels of genes involved in xylose metabolism, membrane transport functions, and ATP synthesis were higher in MA-R4 than in MA-B4 when strains were fermented with glucose or xylose.ConclusionsOur transcriptomic approach revealed the molecular events underlying the response to xylose or glucose and differences between MA-R4 and MA-B4. Xylose-utilizing S. cerevisiae strains may recognize xylose as a non-fermentable carbon source, which induces a starvation response and adaptation to oxidative stress, resulting in the increased expression of stress-response genes.

Production and characterization of algae extract from Chlamydomonas reinhardtii

Algae offer many advantages as biofuel sources including: high growth rates, high lipid content, the ability to grow on non-agricultural land, and the genetic versatility to improve strains rapidly and produce co-products. Research is ongoing to make algae biofuels a more financially attractive energy option; however, it is becoming evident that the economic viability of algae-based fuels may hinge upon high-value co-products. This work evaluated the feasibility of using a co-product, algae extract, as a nutrient source in cell culture media. Algae extract prepared from autolysed Chlamydomonas reinhardtii was found to contain 3.0% protein, 9.2% total carbohydrate, and 3.9% free α-amino acid which is similar to the nutrient content of commercially available yeast extract. The effects of algae extract on the growth and metabolism of laboratory strains of Escherichia coli and Saccharomyces cerevisiae were tested by substituting algae extract for yeast extract in LB and YPAD growth media recipes. Complex laboratory media supplemented with algae extract instead of yeast extract showed markedly improved effects on the growth and metabolism of common laboratory microorganisms in all cases except ethanol production rates in yeast. This study showed that algae extract derived from C. reinhardtii is similar, if not superior, to commercially available yeast extract in nutrient content and effects on the growth and metabolism of E. coli and S. cerevisiae. Bacto™ yeast extract is valued at USD $0.15–0.35 per gram, if algae extract was sold at similar prices, it would serve as a high-value co-product in algae-based fuel processes.

Kinetic Studies on Fermentative Production of Biofuel from Synthesis Gas UsingClostridium ljungdahlii

The intrinsic growth, substrate uptake, and product formation biokinetic parameters were obtained for the anaerobic bacterium, Clostridium ljungdahlii, grown on synthesis gas in various pressurized batch bioreactors. A dual-substrate growth kinetic model using Luong for CO and Monod for H2 was used to describe the growth kinetics of the bacterium on these substrates. The maximum specific growth rate (μ max = 0.195 h−1) and Monod constants for CO (K s,CO = 0.855 atm) and H2 (K s,H2 = 0.412 atm) were obtained. This model also accommodated the CO inhibitory effects on cell growth at high CO partial pressures, where no growth was apparent at high dissolved CO tensions (P CO ∗ > 0.743 atm). The Volterra model, Andrews, and modified Gompertz were, respectively, adopted to describe the cell growth, substrate uptake rate, and product formation. The maximum specific CO uptake rate (q max = 34.364 mmol/gcell/h), CO inhibition constant (K I = 0.601 atm), and maximum rate of ethanol (R max = 0.172 mmol/L/h at P CO = 0.598 atm) and acetate (R max = 0.096 mmol/L/h at P CO = 0.539 atm) production were determined from the applied models.

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.

Bioethanol Production from Glycerol by Mixed Culture System

Glycerol, a by-product from biodiesel industry, is a promising feedstock for subsequent bioconversion to higher-value products. Potential application of a mixed microbial consortium on the fermentative conversion of glycerol to ethanol was demonstrated in this study. Maximum ethanol concentration of 11.1 g l-1 was produced after 72 h fermentation from the initial pure glycerol concentration of 45 g l-1, at 30 ̊C, and pH 7 under anaerobic conditions, corresponding to the ethanol production rate and yield of 0.34 g l-1 h-1, and 0.81 mol ethanolmol-1 glycerol, respectively. The microbial consortium yielded lower ethanol concentration (6.5 g l-1) but similar ethanol yield (0.85 mol ethanol mol-1 glycerol) when crude glycerol of 45 g l-1 was fermented at the same condition. At the optimum fermentative condition of the pure glycerol, phylogenetic analysis of microbial consortium based on 16S rRNA gene sequences indicated that Gammaproteobacteria represented 95% of the microbial diversity in the consortium while the rest belonged to Betaproteobacteria. The consortium was dominated by bacteria closely related to genera Enterobacter and Klebsiella, which could play the role on conversion of glycerol to ethanol in this system.

Ethanol fermentation of very high gravity (VHG) maize mashes by Saccharomyces cerevisiae with spent brewer's yeast supplementation

The subject of this research project was ethanol productivity from very high gravity maize mashes supplemented with spent brewer's yeast as a nutrient source. Batch fermentation of maize mashes (360 g of maize per kilogram of mash) supplemented with 30 g of spent brewer's yeast's wet biomass, dried spent brewer's yeast and yeast extract per kilogram of mash was conducted for 96 h at 30 °C. In all supplemented mashes higher ethanol production rate was observed in comparison to the control sample, especially in the initial stages of fermentation. After 96 h fermentation the ethanol concentration ranged from 120.95 to 142.34 g dm−3 in supplemented mashes (120.74 g dm−3 in the control sample) which corresponded with practical ethanol yields of 78.05–91.85% (77.91% in the control sample). It was also important that maximum ethanol yield in supplemented mashes was achieved after ca. 48 h and 72 h of fermentation while in the control sample ethanol production continued until the end of the process.

Optimizing medium for producing ethanol from industrial crop Jerusalem artichoke by one-step fermentation and recombinant Saccharomyces cerevisiae

In order to obtain a high ethanol yield from the Jerusalem artichoke raw extract and reduce the fermentation cost, we have engineered a new recombinant Saccharomyces cerevisiae strain that could produce ex-inulinase. The response surface methodology based on Plackett–Burman and Box–Behnken design was used to optimize the medium for the ethanol production from the Jerusalem artichoke raw extracts by the recombinant strain. In the first optimization step, Plackett–Burman design was employed to select significant factors, including concentrations of yeast extract, inoculum, and MgSO4·7H2O. In the second step, the steepest ascent experiment was carried out to determine the center point with the three significant factors; the selected combinations were further optimized using the Box–Behnken design. The maximum ethanol production rate was predicted at 91.1 g/l, which was based on a medium consisting of yeast extract 9.24 g/l, inoculum 39.8 ml/l, and MgSO4·7H2O 0.45 g/l. In the validating experiment, the ethanol fermentation rate reached 102.1 g/l, closely matching the predicted rate.

Enzymatic hydrolysis and fermentation of crushed whole sugar beets

Pectinase and cellulase enzymes were used for hydrolysis of whole sugar beets and the hydrolyzates were fermented with Escherichia coli KO11 and Saccharomyces cerevisiae via simultaneous saccharification and fermentation (SSF). Ethanol production rate was significantly higher for S. cerevisiae than for E. coli KO11. The combined effect of pectinase and cellulase loadings on ethanol production as well as residual galacturonic acid and arabinose concentrations were modeled for fermentations with S. cerevisiae. Ethanol yields of more than 92% were reached with moderate to high cellulase and pectinase loadings at 0.51 FPU g−1 and 51 U g−1 of dry biomass, respectively. Ethanol yields of 85% were achieved without any enzyme addition. However, addition of cellulase and pectinase enzymes increased effluent arabinose and galacturonic acid concentrations and reduced total suspended solids. This study demonstrated the yield potential of fermentation of crushed, whole sugar beets with or without the addition of cellulase and pectinase enzymes.

Kinetic Modeling of Ethanol Production by Scheffersomyces stipitis from Xylose

This work focuses on the kinetics of ethanol production by Scheffersomyces stipitis on xylose with the development of a mathematical model considering the effect of substrate and product concentrations on growth rate. Experiments were carried out in batch and continuous modes, with substrate concentration varying from 7.2 to 145 g L(-1). Inhibitory effects on cell growth, substrate uptake, and ethanol production rates were found to be considerable. Kinetic parameters were obtained through linear and non-linear regression methods. Experiments in continuous mode were performed at different dilution rates to evaluate the inhibitory effect of ethanol. A mixed mathematical model which combined Andrews and Levenspiel's models, combining substrate and product inhibition, was used. A quasi-Newton routine was applied to obtain a more accurate fitting of kinetic parameters. The parameters such as cell to product factor (YP/X) and limiting cell yield (YX) were shown to be dependent on substrate concentration. The kinetic model fitted satisfactorily the experimental data.

Research on Ethanol Production by Biological Fermentation of Potato Pulp

Bioconversion of potato pulp to fuel ethanol, analysing the potato pulp chemical composition and determining the potato pulp in the role of microorganism produce ethanol under the best conditions is the major research. An analysis of the chemical composition of potato pulp showed that : the basic ingredients are Protein (9.72%), Starch (25.52%), Cellulose (17.90%). The effects of ethanol production rate of solid-liquid ratio, fermentation temperature, inoculumconcertration, fermentation time. The results showed that: the best conditions producting ethanol from potato pulp obtained by single factor experiments are: solid-liquid ratio: 1:15, fermentation temperature: 35°C, inoculumconcertration: 3mL, fermentation time: 20h. Under this occasion, the ethanol production rate was 0.183mL·g-1.

Growth and Fermentation Characteristics of Saccharomyces cerevisiae NK28 Isolated from Kiwi Fruit

The influences of glucose concentration, initial medium acidity (pH), and temperature on the growth and ethanol production of Saccharomyces cerevisiae NK28, which was isolated from kiwi fruit, were examined in shake flask cultures. The optimal glucose concentration, initial medium pH, and temperature for ethanol production were 200 g/l, pH 6.0, and 35oC, respectively. Under this growth condition, S. cerevisiae NK28 produced 98.9 ± 5.67 g/l ethanol in 24 h with a volumetric ethanol production rate of 4.12 ± 0.24 g/l·h. S. cerevisiae NK28 was more tolerant to heat and ethanol than laboratory strain S. cerevisiae BY4742, and its tolerance to ethanol and fermentation inhibitors was comparable to that of an ethanologen, S. cerevisiae D5A.

A Two-Stage Continuous Fermentation System for Conversion of Syngas into Ethanol

We have established a two-stage continuous fermentation process for production of ethanol from synthesis gas (syngas) with Clostridium ljungdahlii. The system consists of a 1-L continuously stirred tank reactor as a growth stage and a 4-L bubble column equipped with a cell recycle module as an ethanol production stage. Operating conditions in both stages were optimized for the respective purpose (growth in stage one and alcohol formation in stage two). The system was fed with an artificial syngas mixture, mimicking the composition of syngas derived from lignocellulosic biomass (60% CO, 35% H2, and 5% CO2). Gas recycling was used to increase the contact area and retention time of gas in the liquid phase, improving mass transfer and metabolic rates. In stage two, the biocatalyst was maintained at high cell densities of up to 10 g DW/L. Ethanol was continuously produced at concentrations of up to 450 mM (2.1%) and ethanol production rates of up to 0.37 g/(L·h). Foam control was essential to maintain reactor stability. A stoichiometric evaluation of the optimized process revealed that the recovery of carbon and hydrogen from the provided carbon monoxide and hydrogen in the produced ethanol was 28% and 74%, respectively.

Influence of addition of insoluble solids, different yeast strains and pectinesterase enzyme on the quality of apple wine

Rate of fermentation and physico-chemical characteristics of apple wines, owing to the addition of different yeast strains, insoluble solids and pectinesterase enzyme, were examined. The highest rates of fermentation and ethanol production were found in the wine fermented by yeast strain UCD 505, while strain UCD 595 gave the smallest amount of methanol. The addition of insoluble solids to the apple juice significantly increased the levels of methanol, vitamin C, amyl alcohol, total volatiles, rate of fermentation, tannins, colour units, Mn and Zn. Addition of insoluble solids decreased pH, titrable acidity, ethanol, total sugars, total esters, and K, Mg, Ca, Cu and Fe content of the wine. Addition of pectinesterase enzyme significantly increased all the parameters examined except for pH, vitamin C, total esters, Mn and Mg content. Application of cluster analysis to the results of rate of fermentation, reducing sugars, volatile acidity and ethanol showed that the influence of the yeast strains was more than the influence of insoluble solids or pectinesterase enzyme addition. Consideration of more parameters showed that there was a clear interaction between the yeast strain, insoluble solids and the pectinesterase enzyme. Addition of insoluble solids to the must led to the production of some undesirable quality characteristics. In contrast, specific yeast strains and enzyme addition improved various physico-chemical characteristics of the wine. Pre-settled or clarified juice was preferred to produce a quality apple wine. Copyright © 2013 The Institute of Brewing & Distilling

Short-term adaptation improves the fermentation performance of Saccharomyces cerevisiae in the presence of acetic acid at low pH.

The release of acetic acid due to deacetylation of the hemicellulose fraction during the treatment of lignocellulosic biomass contributes to the inhibitory character of the generated hydrolysates. In the present study, we identified a strain-independent adaptation protocol consisting of pre-cultivating the strain at pH 5.0 in the presence of at least 4 g L−1 acetic acid that enabled aerobic growth and improved fermentation performance of Saccharomyces cerevisiae cells at low pH (3.7) and in the presence of inhibitory levels of acetic acid (6 g L−1). During anaerobic cultivation with adapted cells of strain TMB3500, the specific ethanol production rate was increased, reducing the fermentation time to 48 %.

Mixed culture of Saccharomyces cerevisiae and Acetobacter pasteurianus for acetic acid production

Mixed culture of Saccharomyces cerevisiae and Acetobacter pasteurianus was carried out for high yield of acetic acid. Acetic acid production process was divided into three stages. The first stage was the growth of S. cerevisiae and ethanol production, fermentation temperature and aeration rate were controlled at 32°C and 0.2vvm, respectively. The second stage was the co-culture of S. cerevisiae and A. pasteurianus, fermentation temperature and aeration rate were maintained at 34°C and 0.4vvm, respectively. The third stage was the growth of A. pasteurianus and production of acetic acid, fermentation temperature and aeration rate were controlled at 32°C and 0.2vvm, respectively. Inoculation volume of A. pasteurianus and S. cerevisiae was 16% and 0.06%, respectively. The average acetic acid concentration was 52.51g/L under these optimum conditions. To enhance acetic acid production, a glucose feeding strategy was subsequently employed. When initial glucose concentration was 90g/L and 120g/L glucose was fed twice during fermentation, acetic acid concentration reached 66.0g/L.

Comparison between Theoretical Simulation and Experimental Result for Continues Fermenter Product the Baker’s Yeast

Baker’s yeast is an important additive to improve bread quality. Baker yeast produced in two methods (batch or continues fermenter). In this investigation the continuous operating conditions in reactors were studied to predict the best conditions of product. Experimental runs were implemented in a 12-liter pilot-scale fermenter. The simulation carried out between the experimental and the theoretical results and gives a good results presentation of growth rote, the rate of substrate additives, pH and rate of ethanol production. The process is modeled and par formed using a computer programming prepares for this purpose.