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High-gravity fermentation, used for ethanol production from sugarcane, corn, and mixed substrates, offers several benefits. Yeast, a rapidly multiplying unicellular microorganism, can be adapted for high sugar and ethanol tolerance on a lab scale. However, different substrates can enhance fermentation efficiency. Our study consisted of two experiments. In the first, we compared simple batch feeding with a fed-batch system for yeast selection in high-gravity fermentation. We ran eight cycles with increasing initial sugar contents (50 to 300 g L−1). No significant differences were observed in the first seven cycles, but in the eighth, the fed-batch system showed lower glycerol and fructose contents and higher cell viability than the simple batch system. In the second experiment, we used the fed-batch system with 300 g L−1 from sugarcane, corn, and mixed wort. The results showed that mixed wort produced higher ethanol contents and greater fermentation efficiency compared to corn and sugarcane as substrates. In conclusion, our findings indicate that the fed-batch system is more suitable for high-gravity fermentation on a lab scale, and the combination of sugarcane juice and corn can enhance fermentation efficiency, paving the way for integrating these substrates in industrial ethanol production.

High-gravity fermentation, used for ethanol production from sugarcane, corn, and mixed substrates, offers several benefits. Yeast, a rapidly multiplying unicellular microorganism, can be adapted for high sugar and ethanol tolerance on a lab scale. However, different substrates can enhance fermentation efficiency. Our study consisted of two experiments. In the first, we compared simple batch feeding with a fed-batch system for yeast selection in high-gravity fermentation. We ran eight cycles with increasing initial sugar contents (50 to 300 g L−1). No significant differences were observed in the first seven cycles, but in the eighth, the fed-batch system showed lower glycerol and fructose contents and higher cell viability than the simple batch system. In the second experiment, we used the fed-batch system with 300 g L−1 from sugarcane, corn, and mixed wort. The results showed that mixed wort produced higher ethanol contents and greater fermentation efficiency compared to corn and sugarcane as substrates. In conclusion, our findings indicate that the fed-batch system is more suitable for high-gravity fermentation on a lab scale, and the combination of sugarcane juice and corn can enhance fermentation efficiency, paving the way for integrating these substrates in industrial ethanol production. [ABSTRACT FROM AUTHOR]

The stress imposed by ethanol to Saccharomyces cerevisiae cells are one of the most challenging limiting factors in industrial fuel ethanol production. Consequently, the toxicity and tolerance to high ethanol concentrations has been the subject of extensive research, allowing the identification of several genes important for increasing the tolerance to this stress factor. However, most studies were performed with well-characterized laboratory strains, and how the results obtained with these strains work in industrial strains remains unknown. In the present work, we have tested three different strategies known to increase ethanol tolerance by laboratory strains in an industrial fuel–ethanol producing strain: the overexpression of the TRP1 or MSN2 genes, or the overexpression of a truncated version of the MSN2 gene. Our results show that the industrial CAT-1 strain tolerates up to 14% ethanol, and indeed the three strategies increased its tolerance to ethanol. When these strains were subjected to fermentations with high sugar content and cell recycle, simulating the industrial conditions used in Brazilian distilleries, only the strain with overexpression of the truncated MSN2 gene showed improved fermentation performance, allowing the production of 16% ethanol from 33% of total reducing sugars present in sugarcane molasses. Our results highlight the importance of testing genetic modifications in industrial yeast strains under industrial conditions in order to improve the production of industrial fuel ethanol by S. cerevisiae. [ABSTRACT FROM AUTHOR]

Ethanol tolerant Saccharomyces cerevisiae is compulsory for ethanol production in starch based biorefinery, especially during high-gravity fermentation. In this study, adaptive evolution with increased initial ethanol concentrations as a driving force was harnessed for achieving ethanol tolerant S. cerevisiae. After evolution, an outstanding ethanol tolerant strain was screened, which contributed to significant improvements in glucose consumption and ethanol production in scenarios of 300 g/L initial glucose, high solid loadings (30 wt%, 33 wt%, 35 wt% and 40 wt%) of corn, and high solid loadings (30 wt% and 33 wt%) of cassava, compared with the original strain. Genome re-sequencing was applied for the evolved strain, and 504 sense mutations in 205 genes were detected, among which PAM1 gene was demonstrated related to the elevated ethanol tolerance. In sum, this study provided a practical approach for obtaining ethanol tolerant strain and the identified PAM1 gene enhanced our understanding on ethanol tolerant mechanism, as well as provided a target basis for rational metabolic engineering., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

While Saccharomyces cerevisiae is a popular organism to produce ethanol, its fermentation performance is affected at high sugar concentrations due to osmotic stress. We hypothesized that adaptation under ionic stress conditions will improve the fermentation performance at high sugar concentrations due to cross-stress adaptation. We, therefore, adapted a high-performance yeast strain, S. cerevisiae CEN.PK 122, to increasing salt concentrations in an industrial medium. Control cells were adapted in the medium without added salt. The cells adapted to 3.5% (w/v) salt concentration demonstrated a superior performance when fermenting 10–30% (w/v) glucose. When fermenting 30% (w/v) glucose, the ethanol yields of the adapted cells (0.49 ± 0.01 g g−1) were about 30% higher than the control cells (0.37 ± 0.01 g g−1) and are comparable with the best reported to date for any medium employed. Similar improvements were also observed when fermenting 10% (w/v) sucrose. However, little improvement in fermentation was observed at the higher temperature tested (40 °C), even though the growth of the adapted cells was greater when tested in YPD medium. The improvements in fermentation at 30 °C were primarily related to the faster growth of the adapted cells and not to an increase in specific intake rates. Additionally, a significantly reduced lag phase was also observed when fermenting 30% (w/v) glucose. Thus, our work shows the application of a simple strategy to significantly improve high-gravity fermentation (HGF) performance through adaptation. Key points: • Cell adapted on 3.5% NaCl made 28% more ethanol when fermenting 30% glucose. • The adapted cells had reduced lag phase, grew faster, and produced less glycerol. • The improvements were not related to increased specific rates of production. [ABSTRACT FROM AUTHOR]

The stress imposed by ethanol to Saccharomyces cerevisiae cells are one of the most challenging limiting factors in industrial fuel ethanol production. Consequently, the toxicity and tolerance to high ethanol concentrations has been the subject of extensive research, allowing the identification of several genes important for increasing the tolerance to this stress factor. However, most studies were performed with well-characterized laboratory strains, and how the results obtained with these strains work in industrial strains remains unknown. In the present work, we have tested three different strategies known to increase ethanol tolerance by laboratory strains in an industrial fuel–ethanol producing strain: the overexpression of the TRP1 or MSN2 genes, or the overexpression of a truncated version of the MSN2 gene. Our results show that the industrial CAT-1 strain tolerates up to 14% ethanol, and indeed the three strategies increased its tolerance to ethanol. When these strains were subjected to fermentations with high sugar content and cell recycle, simulating the industrial conditions used in Brazilian distilleries, only the strain with overexpression of the truncated MSN2 gene showed improved fermentation performance, allowing the production of 16% ethanol from 33% of total reducing sugars present in sugarcane molasses. Our results highlight the importance of testing genetic modifications in industrial yeast strains under industrial conditions in order to improve the production of industrial fuel ethanol by S. cerevisiae.

In modern wine-making technology, there is an increasing concern in relation to the preservation of the biodiversity, and the employment of “new”, “novel” and wild-type Saccharomyces cerevisiae strains as cell factories amenable for the production of wines that are not “homogenous”, expressing their terroir and presenting interesting and “local” sensory characteristics. Under this approach, in the current study, several wild-type Saccharomyces cerevisiae yeast strains (LMBF Y-10, Y-25, Y-35 and Y-54), priorly isolated from wine and grape origin, selected from the private culture collection of the Agricultural University of Athens, were tested regarding their biochemical behavior on glucose-based (initial concentrations ca 100 and 200 g/L) shake-flask experiments. The wild yeast strains were compared with commercial yeast strains (viz. Symphony, Cross X and Passion Fruit) in the same conditions. All selected strains rapidly assimilated glucose from the medium converting it into ethanol in good rates, despite the imposed aerobic conditions. Concerning the wild strains, the best results were achieved for the strain LMBF Y-54 in which maximum ethanol production (EtOHmax) up to 68 g/L, with simultaneous ethanol yield on sugar consumed = 0.38 g/g were recorded. Other wild strains tested (LMBF Y-10, Y-25 and Y-35) achieved lower ethanol production (up to ≈47 g/L). Regarding the commercial strains, the highest ethanol concentration was achieved by S. cerevisiae Passion Fruit (EtOHmax = 91.1 g/L, yield = 0.45 g/g). Subsequently, the “novel” strain that presented the best technological characteristics regards its sugar consumption and alcohol production properties (viz. LMBF Y-54) and the commercial strain that equally presented the best previously mentioned technological characteristics (viz. Passion Fruit) were further selected for the wine-making process. The selected must originated from red and white grapes (Assyrtiko and Mavrotragano, Santorini Island; Greece) and fermentation was performed under wine-making conditions showing high yields for both strains (EtOHmax = 98–106 g/L, ethanol yield = 0.47–0.50 g/g), demonstrating the production efficiency under microaerophilic/anaerobic conditions. Molecular identification by rep-PCR carried out throughout fermentations verified that each inoculated yeast was the one that dominated during the whole bioprocess. The aromatic compounds of the produced wines were qualitatively analyzed at the end of the processes. The results highlight the optimum technological characteristics of the selected “new” wild strain (S. cerevisiae LMBF Y-54), verifying its suitability for wine production while posing great potential for future industrial applications.

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Highlights • Wheat gluten hydrolysates (WGH) and soy protein hydrolysates (SPH) were prepared. • WGH and SPH exhibited significant yeast growth and fermentation promoting activity. • The bioactivity was not linear with the degree of hydrolysis and addition amount. • Bioactivity of hydrolysates was related to the molecular structure characteristics. • Glu, Leu, Lys and Arg in hydrolysates were responsible for their bioactivity. Abstract Wheat gluten hydrolysates (WGH) and soy protein hydrolysates (SPH) were used to examine their effects on the growth and fermentation performance of yeast during high-gravity fermentation. Results showed that both the kind and the addition amount of hydrolysates had significant impacts on the biomass, viability, fermentability and ethanol yield of yeast cells. Worts supplemented with WGH and SPH (with the hydrolysis time of 24 h and 12 h, and the addition amount of 1.0% and 0.1%, respectively) increased 5.5% and 12.6% of net cell growth, 0.3% and 1.1% of fermentability, and 19.3% (v/v) and 23.7% (v/v) of ethanol yields, respectively. All these results indicated that both hydrolysates were effective and preferential nitrogen sources for yeast growth and fermentation. The moderate molecular weight distribution, the complexity of nitrogen source and the higher hydrophilic and basic amino acids contents in hydrolysates might be responsible for their specific bioactivity. [ABSTRACT FROM AUTHOR]

Incompletely degraded corn starch particles often seriously inhibit wort filtration and decrease a brewery's beer productivity. Herein, the inhibiting factors of starch hydrolysis and the application of amylases to degrade residual starch were evaluated. The results showed that resistant starch and the amylopectin of corn starch were not the inhibiting factors. Almost all residual starch left in the spent grain layer was proved to be degradable by amylases. Mesophilic α-amylase was selected through a comparison of nine amylases, which increased the wort filtration rate by 44%. However, >6% of corn starch was still left after mashing when a high ratio of corn starch to water (>1:3.5) was used in liquefaction. The low water content in liquefaction was proved to be the key inhibiting factor. Considering the existing equipment and brewing technology, the application of mesophilic α-amylases should be a simple and effective method for enhancing the hydrolysis of corn starch and accelerating the wort lautering process during a high-adjunct-ratio beer brewing process. [ABSTRACT FROM AUTHOR]

A co-fermentation process involving Saccharomyces cerevisiae and Fusarium oxysporum was studied, using hydrothermally pretreated wheat straw as substrate. In the first step of the study, we examined liquefaction of the material in a free-fall reactor. Both the enzyme loading and the dry matter content affected severely the liquefaction efficiency. In the second step (simultaneous saccharification and fermentation (SSF) experiments), we found that the enzymatic system of F. oxysporum contributed significantly to substrate hydrolysis, while its metabolic system played a secondary role in fermentation. SSF in the presence of F. oxysporum cells and enzymes gave 62 g L −1 ethanol. In the third step of the study, a semi-consolidated bioprocess was designed in which F. oxysporum culture (submerged or solid-state) was added at the SSF stage along with S. cerevisiae . The addition of solid F. oxysporum culture increased ethanol production by 19%, leading to a final ethanol concentration of 58 g L −1 . The present study proposes a semi-consolidated process combining two microorganisms for the fermentation at high solids concentration of a liquefied material using an in house free fall mixing reactor. The semi-consolidated process proposed not only increased the ethanol yields significantly, but could also lead to lower overall cost of the process by incorporating in-situ enzyme production. [ABSTRACT FROM AUTHOR]

Significant positive correlations between wort fermentability and the assimilation of Lys and His under normal-gravity and high-gravity conditions indicated that Lys and His were the key amino acids for lager yeast during beer brewing. In order to obtain insight into the roles of Lys and His in nitrogen regulation, the influences of Lys, His and their mixture supplementations on the fermentation performance and nitrogen metabolism in lager yeast during high-gravity fermentation were further investigated in the present study. Results showed that Lys and His supplementations improved yeast growth, wort fermentability, ethanol yield and the formation of flavor volatiles. Lys supplementation up-regulated Ssy1p-Ptr3p-Ssy5p (SPS)-regulated genes ( LYP1, HIP1, BAP2 and AGP1) dramatically compared to nitrogen catabolite repression (NCR)-sensitive genes ( GAP1 and MEP2), whereas His supplementation activated SPS-regulated genes slightly in exponential phase, and repressed NCR-sensitive genes significantly throughout the fermentation. Lys and His supplementations increased the consumption of Glu and Phe, and decreased the consumption of Ser, Trp and Arg. Moreover, Lys and His supplementations exhibited similar effects on the fermentation performance, and were more effective than their mixture supplementation when the same dose was kept. These results demonstrate that both Lys and His are important amino acids for yeast nitrogen metabolism and fermentation performance. [ABSTRACT FROM AUTHOR]

Vacuolar H+-ATPase (V-ATPase) is thought to play a role in stress tolerance. In this study it was found that bottom-fermenting yeast strains, in which the V-ATPase-related genes DBF2, VMA41/CYS4/NHS5 and RAV2 were overexpressed, exhibited stronger ethanol tolerance than the parent strain and showed increased fermentation rates in a high-sugar medium simulating high-gravity fermentation. Among the strains examined, the DBF2-overexpressing bottom-fermenting yeast strain exhibited the highest ethanol tolerance and fermentation rate in YPM20 medium. Using this strain, high-gravity fermentation was performed by adding sugar to the wort, which led to increased fermentation rates and yeast viability compared with the parent strain. These findings indicate that V-ATPase is a stress target in high-gravity fermentation and suggests that enhancing the V-ATPase activity increases the ethanol tolerance of bottom-fermenting yeast, thereby improving the fermentation rate and cell viability under high-gravity conditions. Copyright © 2012 The Institute of Brewing & Distilling [ABSTRACT FROM AUTHOR]