scholarly journals ATPase-based implementation of enforced ATP wasting in Saccharomyces cerevisiae for improved ethanol production

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Ahmed Zahoor ◽  
Katrin Messerschmidt ◽  
Simon Boecker ◽  
Steffen Klamt
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Atpase Activity ◽  
Ethanol Production ◽  
Ethanol Yield ◽  
Volumetric Productivity ◽  
Anaerobic Growth ◽  
Control Strain ◽  
Genomic Integration ◽  
Expression Of Genes ◽  
Atp Generation

Abstract Background Enforced ATP wasting has been recognized as a promising metabolic engineering strategy to enhance the microbial production of metabolites that are coupled to ATP generation. It also appears to be a suitable approach to improve production of ethanol by Saccharomyces cerevisiae. In the present study, we constructed different S. cerevisiae strains with heterologous expression of genes of the ATP-hydrolyzing F1-part of the ATPase enzyme to induce enforced ATP wasting and quantify the resulting effect on biomass and ethanol formation. Results In contrast to genomic integration, we found that episomal expression of the αβγ subunits of the F1-ATPase genes of Escherichia coli in S. cerevisiae resulted in significantly increased ATPase activity, while neither genomic integration nor episomal expression of the β subunit from Trichoderma reesei could enhance ATPase activity. When grown in minimal medium under anaerobic growth-coupled conditions, the strains expressing E. coli’s F1-ATPase genes showed significantly improved ethanol yield (increase of 10% compared to the control strain). However, elevated product formation reduces biomass formation and, therefore, volumetric productivity. We demonstrate that this negative effect can be overcome under growth-decoupled (nitrogen-starved) operation with high and constant biomass concentration. Under these conditions, which mimic the second (production) phase of a two-stage fermentation process, the ATPase-expressing strains showed significant improvement in volumetric productivity (up to 111%) compared to the control strain. Conclusions Our study shows that expression of genes of the F1-portion of E. coli’s ATPase induces ATPase activity in S. cerevisiae and can be a promising way to improve ethanol production. This ATP-wasting strategy can be easily applied to other metabolites of interest, whose formation is coupled to ATP generation.

scholarly journals Engineering of Saccharomyces cerevisiae for Efficient Anaerobic Alcoholic Fermentation of l-Arabinose

2007 ◽  
Vol 73 (15) ◽  
pp. 4881-4891 ◽  
Author(s):  
H. Wouter Wisselink ◽  
Maurice J. Toirkens ◽  
M. del Rosario Franco Berriel ◽  
Aaron A. Winkler ◽  
Johannes P. van Dijken ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ethanol Production ◽  
Alcoholic Fermentation ◽  
Plant Biomass ◽  
Ethanol Yield ◽  
Dry Weight ◽  
Pentose Phosphate ◽  
Anaerobic Growth ◽  
Evolutionary Engineering ◽  
First Case

ABSTRACT For cost-effective and efficient ethanol production from lignocellulosic fractions of plant biomass, the conversion of not only major constituents, such as glucose and xylose, but also less predominant sugars, such as l-arabinose, is required. Wild-type strains of Saccharomyces cerevisiae, the organism used in industrial ethanol production, cannot ferment xylose and arabinose. Although metabolic and evolutionary engineering has enabled the efficient alcoholic fermentation of xylose under anaerobic conditions, the conversion of l-arabinose into ethanol by engineered S. cerevisiae strains has previously been demonstrated only under oxygen-limited conditions. This study reports the first case of fast and efficient anaerobic alcoholic fermentation of l-arabinose by an engineered S. cerevisiae strain. This fermentation was achieved by combining the expression of the structural genes for the l-arabinose utilization pathway of Lactobacillus plantarum, the overexpression of the S. cerevisiae genes encoding the enzymes of the nonoxidative pentose phosphate pathway, and extensive evolutionary engineering. The resulting S. cerevisiae strain exhibited high rates of arabinose consumption (0.70 g h−1 g [dry weight]−1) and ethanol production (0.29 g h−1 g [dry weight]−1) and a high ethanol yield (0.43 g g−1) during anaerobic growth on l-arabinose as the sole carbon source. In addition, efficient ethanol production from sugar mixtures containing glucose and arabinose, which is crucial for application in industrial ethanol production, was achieved.

scholarly journals Effects of Ultrasound on Fermentation of Glucose to Ethanol by Saccharomyces cerevisiae

Fermentation ◽  
2019 ◽  
Vol 5 (1) ◽  
pp. 16 ◽  
Author(s):  
Luis Huezo ◽  
Ajay Shah ◽  
Frederick Michel
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mass Transfer ◽  
Glucose Uptake ◽  
Ethanol Production ◽  
Ethanol Yield ◽  
Biofuel Production ◽  
Inhibitory Effects ◽  
Negative Effects ◽  
Intraparticle Mass Transfer ◽  
Improve Mass Transfer

Previous studies have shown that pretreatment of corn slurries using ultrasound improves starch release and ethanol yield during biofuel production. However, studies on its effects on the mass transfer of substrates and products during fermentation have shown that it can have both beneficial and inhibitory effects. In this study, the effects of ultrasound on mass transfer limitations during fermentation were examined. Calculation of the external and intraparticle observable moduli under a range of conditions indicate that no external or intraparticle mass transfer limitations should exist for the mass transfer of glucose, ethanol, or carbon dioxide. Fermentations of glucose to ethanol using Saccharomyces cerevisiae were conducted at different ultrasound intensities to examine its effects on glucose uptake, ethanol production, and yeast population and viability. Four treatments were compared: direct ultrasound at intensities of 23 and 32 W/L, indirect ultrasound (1.4 W/L), and no-ultrasound. Direct and indirect ultrasound had negative effects on yeast performance and viability, and reduced the rates of glucose uptake and ethanol production. These results indicate that ultrasound during fermentation, at the levels applied, is inhibitory and not expected to improve mass transfer limitations.

scholarly journals Ethanol Production from Nondetoxified Dilute-Acid Lignocellulosic Hydrolysate by Cocultures of Saccharomyces cerevisiae Y5 and Pichia stipitis CBS6054

2012 ◽  
Vol 2012 ◽  
pp. 1-6 ◽  
Author(s):  
Ping Wan ◽  
Dongmei Zhai ◽  
Zhen Wang ◽  
Xiushan Yang ◽  
Shen Tian
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ethanol Production ◽  
Ethanol Concentration ◽  
Ethanol Yield ◽  
Synthetic Medium ◽  
Pichia Stipitis ◽  
Dilute Acid ◽  
Acid Hydrolysate ◽  
Issatchenkia Orientalis ◽  
Final Ethanol Concentration

Saccharomyces cerevisiae Y5 (CGMCC no. 2660) and Issatchenkia orientalis Y4 (CGMCC no. 2159) were combined individually with Pichia stipitis CBS6054 to establish the cocultures of Y5 + CBS6054 and Y4 + CBS6054. The coculture Y5 + CBS6054 effectively metabolized furfural and HMF and converted xylose and glucose mixture to ethanol with ethanol concentration of 16.6 g/L and ethanol yield of 0.46 g ethanol/g sugar, corresponding to 91.2% of the maximal theoretical value in synthetic medium. Accordingly, the nondetoxified dilute-acid hydrolysate was used to produce ethanol by co-culture Y5 + CBS6054. The co-culture consumed glucose along with furfural and HMF completely in 12 h, and all xylose within 96 h, resulting in a final ethanol concentration of 27.4 g/L and ethanol yield of 0.43 g ethanol/g sugar, corresponding to 85.1% of the maximal theoretical value. The results indicated that the co-culture of Y5 + CBS6054 was a satisfying combination for ethanol production from non-detoxified dilute-acid lignocellulosic hydrolysates. This co-culture showed a promising prospect for industrial application.

Ethanol Production from Different Substrates by a Flocculent Saccharomyces cerevisiae Strain

2009 ◽  
Vol 7 (1) ◽  
Author(s):  
José Duarte ◽  
Vera Lourenço ◽  
Belina Ribeiro ◽  
Maria Céu Saagua ◽  
Joana Pereira ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ethanol Production ◽  
Yeast Strain ◽  
Fossil Fuels ◽  
Ethanol Yield ◽  
Corn Fiber ◽  
Production Costs ◽  
Raw Material ◽  
Continuous Reactor ◽  
Different Strains

During the last years there has been an increasing interest in using ethanol as a substitute for fossil fuels. The bioethanol used today is mainly produced from sugar cane and cereals, but reducing the production costs of ethanol is still crucial for a viable economic process. Cellulose from vegetable biomass will be the next cheap raw material for second generation fuel ethanol production and agricultural by-products with a low commercial value, as corn stover, corn fiber and cane bagasses would become an attractive feedstock for bioethanol production.In this study, different strains of Saccharomyces cerevisiae have been screened for the ability of bioethanol production. Yeasts were grown in a synthetic liquid medium containing sucrose in batch regime and the growth rates, ethanol and biomass productions were determined as well as their growth ability in cane molasses.The results indicate that a flocculent yeast, isolated in our lab and designated by strain F, was the most promising yeast strain among those tested for continuous ethanol production. This strain was isolated from corn hydrolysates, obtained from a Portuguese distillery facility (DVT, Torres Novas, Portugal) showing highest growth rate (0.49h-1), highest ethanol yield (0.35g/g) and high flocculation capacity.The study on ethanol production in continuous reactor process with the selected yeast strain (strain F) was made on sucrose and cane molasses at different dilution rates (0.05-0.42 h-1). A steady flocculating yeast fluidized bed reactor system was established allowing the functioning of the reactor for 1000 h. Data shows that when the dilution rate rose to 0.42h-1, the highest productivity (20g/Lh) was obtained attaining an ethanol concentration in the reactor of 47g/L for sucrose and molasses media.

scholarly journals Directed Evolution of Xylose Isomerase for Improved Xylose Catabolism and Fermentation in the Yeast Saccharomyces cerevisiae

2012 ◽  
Vol 78 (16) ◽  
pp. 5708-5716 ◽  
Author(s):  
Sun-Mi Lee ◽  
Taylor Jellison ◽  
Hal S. Alper
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ethanol Production ◽  
Directed Evolution ◽  
Ethanol Yield ◽  
Xylose Isomerase ◽  
Mutant Enzyme ◽  
Xylose Utilization ◽  
Xylose Consumption ◽  
Content Type ◽  
Consumption Rates

ABSTRACTThe heterologous expression of a highly functional xylose isomerase pathway inSaccharomyces cerevisiaewould have significant advantages for ethanol yield, since the pathway bypasses cofactor requirements found in the traditionally used oxidoreductase pathways. However, nearly all reported xylose isomerase-based pathways inS. cerevisiaesuffer from poor ethanol productivity, low xylose consumption rates, and poor cell growth compared with an oxidoreductase pathway and, additionally, often require adaptive strain evolution. Here, we report on the directed evolution of thePiromycessp. xylose isomerase (encoded byxylA) for use in yeast. After three rounds of mutagenesis and growth-based screening, we isolated a variant containing six mutations (E15D, E114G, E129D, T142S, A177T, and V433I) that exhibited a 77% increase in enzymatic activity. When expressed in a minimally engineered yeast host containing agre3knockout andtal1andXKS1overexpression, the strain expressing this mutant enzyme improved its aerobic growth rate by 61-fold and both ethanol production and xylose consumption rates by nearly 8-fold. Moreover, the mutant enzyme enabled ethanol production by these yeasts under oxygen-limited fermentation conditions, unlike the wild-type enzyme. Under microaerobic conditions, the ethanol production rates of the strain expressing the mutant xylose isomerase were considerably higher than previously reported values for yeast harboring a xylose isomerase pathway and were also comparable to those of the strains harboring an oxidoreductase pathway. Consequently, this study shows the potential to evolve a xylose isomerase pathway for more efficient xylose utilization.

scholarly journals Optimizing anaerobic growth rate and fermentation kinetics in Saccharomyces cerevisiae strains expressing Calvin-cycle enzymes for improved ethanol yield

2018 ◽  
Vol 11 (1) ◽  
Author(s):  
Ioannis Papapetridis ◽  
Maaike Goudriaan ◽  
María Vázquez Vitali ◽  
Nikita A. de Keijzer ◽  
Marcel van den Broek ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Growth Rate ◽  
Ethanol Yield ◽  
Calvin Cycle ◽  
Anaerobic Growth ◽  
Fermentation Kinetics ◽  
Calvin Cycle Enzymes

Improvement in Ethanol Yield from Lignocellulo-Starch Biomass using Saccharomyces cerevisiae alone or its Co-culture with Scheffersomyces stipitis

2020 ◽  
Vol 9 (1) ◽  
pp. 57-76
Author(s):  
Madhanamohanan G. Mithra ◽  
Gouri Padmaja
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ethanol Production ◽  
Ethanol Yield ◽  
Relative Advantage ◽  
Vegetable Crops ◽  
Fed Batch ◽  
Scheffersomyces Stipitis ◽  
Separate Hydrolysis And Fermentation ◽  
Ethanol Yields ◽  
Sugar Levels

Background: Literature on ethanol production from Lignocellulo-Starch Biomass (LCSB) containing starch besides cellulose and hemicellulose, is scanty. Fed-Batch Separate Hydrolysis And Fermentation (F-SHF) was earlier found more beneficial than Fed-Batch Simultaneous Saccharification and Fermentation (F-SSF). Objective: The study aimed at modification of the saccharification and fermentation strategies by including a prehydrolysis step prior to the SSF and compared the ethanol yields with co-culture fermentation using hexose-fermenting Saccharomyces cerevisiae and pentose-fermenting Scheffersomyces stipitis. Methods: Fed-batch hybrid-SSF and Fed-Batch Separate Hydrolysis and Co-culture Fermentation (F-SHCF) in improving ethanol yield from Steam (ST) or Dilute Sulfuric Acid (DSA) pretreated LCSBs (peels of root and vegetable crops) were studied. Results: There was a progressive build-up of ethanol during F-HSSF up to 72h and further production up to 120h was negligible, with no difference among pretreatments. Despite very high ethanol production in the initial 24h of fermentation by S.cerevisiae under F-SHCF, the further increase was negligible. A rapid hike in ethanol production was observed when S. stipitis was also supplemented because of xylose conversion to ethanol. Conclusion: While ST gave higher ethanol (296-323 ml/kg) than DSA under F-HSSF, the latter was advantageous under F-SHCF for certain residues. Prehydrolysis (24h; 50°C) enhanced initial sugar levels favouring fast fermentation and subsequent saccharification and fermentation occurred concurrently at 37°C for 120h, thus leading to energy saving and hence F-HSSF was advantageous. Owing to the low hemicellulose content in LCSBs, the relative advantage of co-culture fermentation over monoculture fermentation was not significant.

scholarly journals Assessment of the Effect of Nitrogen Concentration on Fermentation and Selection of a Highly Competitive Saccharomyces cerevisiae Strain for Efficient Ethanol Production

Energies ◽  
2019 ◽  
Vol 12 (13) ◽  
pp. 2614
Author(s):  
Barahona ◽  
Martín-Gil ◽  
Martín-Ramos ◽  
Pérez ◽  
Barriga
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ethanol Production ◽  
Ethanol Yield ◽  
Nitrogen Concentration ◽  
Glucose Consumption ◽  
Cellular Growth ◽  
Yeast Strains ◽  
Two Factors ◽  
Pilot Plant Scale ◽  
Reducing Costs

: The optimum nitrogen concentration for media supplementation and strain dominance are aspects of key importance to the industrial production of ethanol with a view to reducing costs and increasing yields. In this work, these two factors were investigated for four ethanologenic Saccharomyces cerevisiae strains (CLQCA-INT-001, CLQCA-INT-005, CLQCA-10-099, and UCLM 325), selected from the screening of 150 isolates, mostly from Ecuadorian yeast biodiversity. The effect of nitrogen concentration was assessed in terms of cellular growth, glucose consumption and ethanol production, and the yeast strains’ dominance was evaluated in continuous co-fermentation with cellular recycling by mitochondrial DNA analyses. Among the four selected yeast strains under study, CLQCA-INT-005 presented the highest glucose consumption at a nitrogen supplement concentration as low as 0.4 g·L−1, attaining an ethanol yield of up to 96.72% in 24 h. The same yeast strain was found to be highly competitive, showing a dominance of 80% after four cycles of fermentation in co-culture. Thus, CLQCA-INT-005 may be deemed as a very promising candidate to be used both at pilot-plant scale and at industrial scale cellulosic ethanol production.

scholarly journals An Innovative Biocatalyst for Continuous 2G Ethanol Production from Xylo-Oligomers by Saccharomyces cerevisiae through Simultaneous Hydrolysis, Isomerization, and Fermentation (SHIF)

Catalysts ◽  
2019 ◽  
Vol 9 (3) ◽  
pp. 225 ◽  
Author(s):  
Thais Milessi-Esteves ◽  
Felipe Corradini ◽  
Willian Kopp ◽  
Teresa Zangirolami ◽  
Paulo Tardioli ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ethanol Production ◽  
Ethanol Yield ◽  
Xylose Isomerase ◽  
Ethanol Productivity ◽  
Spherical Particles ◽  
Birchwood Xylan ◽  
Alginate Gel ◽  
Industrial Implementation ◽  
Ca Alginate

Many approaches have been considered aimed at ethanol production from the hemicellulosic fraction of biomass. However, the industrial implementation of this process has been hindered by some bottlenecks, one of the most important being the ease of contamination of the bioreactor by bacteria that metabolize xylose. This work focuses on overcoming this problem through the fermentation of xylulose (the xylose isomer) by native Saccharomyces cerevisiae using xylo-oligomers as substrate. A new concept of biocatalyst is proposed, containing xylanases and xylose isomerase (XI) covalently immobilized on chitosan, and co-encapsulated with industrial baker’s yeast in Ca-alginate gel spherical particles. Xylo-oligomers are hydrolyzed, xylose is isomerized, and finally xylulose is fermented to ethanol, all taking place simultaneously, in a process called simultaneous hydrolysis, isomerization, and fermentation (SHIF). Among several tested xylanases, Multifect CX XL A03139 was selected to compose the biocatalyst bead. Influences of pH, Ca2+, and Mg2+ concentrations on the isomerization step were assessed. Experiments of SHIF using birchwood xylan resulted in an ethanol yield of 0.39 g/g, (76% of the theoretical), selectivity of 3.12 gethanol/gxylitol, and ethanol productivity of 0.26 g/L/h.

scholarly journals Robustness and Ethanol Production of Industrial Strains of Saccharomyces cerevisiae Using Different Sugarcane Bagasse Hydrolysates

2019 ◽  
Vol 7 (1) ◽  
pp. 23 ◽  
Author(s):  
Vanessa S. Teixeira ◽  
Suéllen P. H. Azambuja ◽  
Priscila H. Carvalho ◽  
Fátima A. A. Costa ◽  
Patricia R. Kitaka ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ethanol Production ◽  
Sugarcane Bagasse ◽  
Ethanol Yield ◽  
Raw Materials ◽  
Fermentation Inhibitors ◽  
Severe Condition ◽  
Inhibitor Tolerance ◽  
Industrial Strains ◽  
Materials Used

Sugarcane bagasse is one of the main lignocellulosic raw materials used for the production of second-generation ethanol. Technological studies on fermentation processes have focused on the search for and development of more robust microorganisms that are able to produce bioethanol efficiently and are resistant to the main fermentation inhibitors. The purpose of this study was to evaluate the robustness and ethanol production of industrial strains of Saccharomyces cerevisiae using acid, alkaline, and enzymatic sugarcane bagasse hydrolysates. Hydrolysis was carried out to release fermentable sugars from sugarcane bagasse. Fermentations were performed in shake flasks containing sugarcane hydrolysates supplemented with 150 g L−1 glucose to evaluate the kinetic parameters of the reaction. Inhibitor tolerance was evaluated by incubating cells with different concentrations of inhibitors in 96-well plates. The biomass yield on substrate, ethanol yield on substrate, and ethanol productivity of the six strains were higher in 0.5% acid, 0.5% alkaline, and enzymatic hydrolysates (i.e., under milder conditions). The SA-1 (Santa Adélia-1) strain had a better performance in comparison with the other strains for its ability to produce ethanol in a very severe condition (7% acid hydrolysis) and for its robustness in growing at several inhibitor concentrations.

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