Ethanol Tolerance Research Articles

Characterization of malolactic fermentation by Lactiplantibacillus plantarum in red grape must

Malolactic fermentation (MLF) is a desirable biotransformation in red and some high-acidity white wines. This process involves the decarboxylation of L-malic acid into L-lactic acid by lactic acid bacteria (LAB). Among them, Oenococcus oeni and Lactiplantibacillus plantarum are the most common, being the last usually used as starter to perform MLF in wine. Compared with O. oeni, L. plantarum is less tolerant to wine conditions such as pH and ethanol; thus, it is preferable to inoculate it in must. The current literature has not yet addressed the impact of some parameters on the inoculation of this bacterium in grape must. Therefore, the aim of this work was to characterize the effect of the inoculum concentration, pH and ethanol tolerance, as well as the inoculation strategy, on the MLF performance of L. plantarum in grape must. We observed that a population < 108 CFU/mL could compromise MLF performance. Additionally, L. plantarum was able to survive at low pH values, such as 2.50, but in the presence of ethanol or a fermenting yeast, it could tolerate pH values in the range of 3.00–3.25. Finally, coinoculation or sequential inoculation with S. cerevisiae allows for the completion of MLF before the end of alcoholic fermentation.

APOE and Alzheimer's disease: Pathologic clues from transgenic Drosophila melanogaster

Alzheimer's disease (AD) is one of the most common forms of neurodegenerative diseases. Apolipoprotein E4 (ApoE4) is the main genetic risk factor in the development of late-onset AD. However, the exact mechanism underlying ApoE4-mediated neurodegeneration remains unclear. We utilized Drosophila melanogaster to examine the neurotoxic effects of various human APOE isoforms when expressed specifically in glial and neural cells. We assessed impacts on mitochondrial dynamics, ER stress, lipid metabolism, and bio-metal ion concentrations in the central nervous system (CNS) of the transgenic flies. Dachshund antibody staining revealed a reduction in the number of Kenyon cells. Behavioral investigations including ethanol tolerance and learning and memory performance demonstrated neuronal dysfunction in APOE4-expressing larvae and adult flies. Transcription level of marf and drp-1 were found to be elevated in APOE4 flies, while atf4, atf6, and xbp-1 s showed down regulation. Enhanced concentrations of triglyceride and cholesterol in the CNS were observed in APOE4 transgenic flies, with especially pronounced effects upon glial-specific expression of the gene. Spectrophotometry of brain homogenate revealed enhanced Fe++ and Zn++ ion levels in conjunction with diminished Cu++ levels upon APOE4 expression. To explore therapeutic strategies, we subjected the flies to heat-shock treatment, aiming to activate heat-shock proteins (HSPs) and assess their potential to mitigate the neurotoxic effects of APOE isoforms. The results showed potential therapeutic benefits for APOE4-expressing flies, hinting at an ability to attenuate memory deterioration. Overall, our findings suggest that APOE4 can alter lipid metabolism, bio metal ion homeostasis, and disrupt the harmonious fission-fusion balance of neuronal and glial mitochondria, ultimately inducing ER stress. These alterations mirror the main clinical manifestations of AD in patients. Therefore, our work underscores the suitability of Drosophila as a fertile model for probing the pathological roles of APOE and furthering our understanding of diverse isoform-specific functions.

Engineering diaryl alcohol dehydrogenase KpADH reveals importance of retaining hydration shell in organic solvent tolerance.

Alcohol dehydrogenases (ADHs) are synthetically important biocatalysts for the asymmetric synthesis of chiral alcohols. The catalytic performance of ADHs in the presence of organic solvents is often important since most prochiral ketones are highly hydrophobic. Here, the organic solvent tolerance of KpADH from Kluyveromyces polyspora was semi-rationally evolved. Using tolerant variants obtained, meticulous experiments and computational studies were conducted to explore properties including stability, activity and kinetics in the presence of various organic solvents. Compared with WT, variant V231D exhibited 1.9-fold improvement in ethanol tolerance, while S237G showed a 6-fold increase in catalytic efficiency, a higher , as well as 15% higher tolerance in 7.5% (v/v) ethanol. Based on 3 × 100 ns MD simulations, the increased tolerance of V231D and S237G against ethanol may be ascribed to their enhanced ability in retaining water molecules and repelling ethanol molecules. Moreover, 6.3-fold decreased KM value of V231D toward hydrophilic ketone substrate confirmed its capability of retaining hydration shell. Our results suggest that retaining hydration shell surrounding KpADH is critical for its tolerance to organic solvents, as well as catalytic performance. This study provides useful guidance for engineering organic solvent tolerance of KpADH and other ADHs.

Improving furfural tolerance in a xylose-fermenting yeast Spathaspora passalidarum CMUWF1–2 via adaptive laboratory evolution

BackgroundSpathaspora passalidarum is a yeast with the highly effective capability of fermenting several monosaccharides in lignocellulosic hydrolysates, especially xylose. However, this yeast was shown to be sensitive to furfural released during pretreatment and hydrolysis processes of lignocellulose biomass. We aimed to improve furfural tolerance in a previously isolated S. passalidarum CMUWF1−2, which presented thermotolerance and no detectable glucose repression, via adaptive laboratory evolution (ALE).ResultsAn adapted strain, AF2.5, was obtained from 17 sequential transfers of CMUWF1−2 in YPD broth with gradually increasing furfural concentration. Strain AF2.5 could tolerate higher concentrations of furfural, ethanol and 5-hydroxymethyl furfuraldehyde (HMF) compared with CMUWF1−2 while maintaining the ability to utilize glucose and other sugars simultaneously. Notably, the lag phase of AF2.5 was 2 times shorter than that of CMUWF1−2 in the presence of 2.0 g/l furfural, which allowed the highest ethanol titers to be reached in a shorter period. To investigate more in-depth effects of furfural, intracellular reactive oxygen species (ROS) accumulation was observed and, in the presence of 2.0 g/l furfural, AF2.5 exhibited 3.41 times less ROS accumulation than CMUWF1−2 consistent with the result from nuclear chromatins diffusion, which the cells number of AF2.5 with diffuse chromatins was also 1.41 and 1.24 times less than CMUWF1−2 at 24 and 36 h, respectively.ConclusionsAn enhanced furfural tolerant strain of S. passalidarum was achieved via ALE techniques, which shows faster and higher ethanol productivity than that of the wild type. Not only furfural tolerance but also ethanol and HMF tolerances were improved.

Characterization of a Novel Hyperthermophilic GH1 β-Glucosidase from Acidilobus sp. and Its Application in the Hydrolysis of Soybean Isoflavone Glycosides.

A putative β-glucosidase gene, BglAc, was amplified from Acidilobus sp. through metagenome database sampling from a hot spring in Yellowstone National Park. BglAc is composed of 485 amino acid residues and bioinformatics analysis showed that it belongs to the GH1 family of β-glucosidases. The gene was successfully expressed in Escherichia coli with a molecular weight of approximately 55.3 kDa. The purified recombinant enzyme showed the maximum activity using p-nitrophenyl-β-D-glucopyranoside (pNPG) as the substrate at optimal pH 5.0 and 100 °C. BglAc exhibited extraordinary thermostability, and its half-life at 90 °C was 6 h. The specific activity, Km, Vmax, and Kcat/Km of BglAc toward pNPG were 357.62 U mg-1, 3.41 mM, 474.0 μmol min-1·mg-1, and 122.7 s-1mM-1. BglAc exhibited the characteristic of glucose tolerance, and the inhibition constant Ki was 180.0 mM. Furthermore, a significant ethanol tolerance was observed, retaining 96% relative activity at 10% ethanol, and even 78% at 20% ethanol, suggesting BglAc as a promising enzyme for cellulose saccharification. BglAc also had a strong ability to convert the major soybean isoflavone glycosides (daidzin, genistin, and glycitin) into their corresponding aglycones. Overall, BglAc was actually a new β-glucosidase with excellent thermostability, ethanol tolerance, and glycoside hydrolysis ability, indicating its wide prospects for applications in the food industry, animal feed, and lignocellulosic biomass degradation.

A novel GH1 β-glucosidase from an Arctic bacterium: Characterization and secretory expression in Bacillus subtilis

β-Glucosidases are widely applied to biofuel production, food industry, and improvement in flavors. The novel β-glucosidase gene debgl was identified from the Arctic bacterium Devosia sp. Arc12 and expressed in Escherichia coli BL21 (DE3). The putative gene, belonging to the glycoside hydrolase family 1, consists of 1311 nucleotides and encodes 437 amino acid residues. The optimal temperature and pH of the recombinant DeBgl were 55 °C and 7, respectively. The recombinant DeBgl displayed salt and ethanol tolerance, and the Vmax, Km, and kcat for the substrate cellobiose were 754 μmol min−1 mg−1, 12.8 mM, and 661.4 s−1, respectively. The concentration of glucose was reached 51.6 g L−1 after incubation at 40 °C for 8 h in 100 g L−1 cellobiose solution. It suggests that the recombinant DeBgl is a good candidate enzyme to degrade complex celluloses. The signal peptide (SP) citH has been proven to be the most suitable for the secretory expression of β-glucosidase by screening the signal peptide library in Bacillus subtilis. The secretory activity of the SP citH fused DeBgl in the vector pMA5 was 9-fold higher than that of the DeBgl without signal peptide. This secretory expression system of β-glucosidase may benefit its industrial application.

Differences in the Volatile Profile of Apple Cider Fermented with Schizosaccharomyces pombe and Schizosaccharomyces japonicus

In this study, two strains of Schizosaccharomyces pombe (NCAIM Y01474T and SBPS) and two strains of Schizosaccharomyces japonicus (DBVPG 6274T, M23B) were investigated for their capacity to ferment apple juice and influence the volatile compounds of cider compared to Saccharomyces cerevisiae EC1118. The ethanol tolerance and deacidification capacity of Schizosaccharomyces yeasts could make them potential substitutes for the commonly used S. cerevisiae starter cultures. Despite different time courses (10–30 d), all strains could complete the fermentation process, and Schizosaccharomyces strains reduced the concentration of malic acid in the apple juice. Results indicated that each yeast exerted a distinctive impact on the volatile profile of the apple cider, giving final products separated using a principal component analysis. The volatile composition of the cider exhibited significant differences in the concentration of alcohols, esters, and fatty acids. Particularly, the flocculant strain S. japonicus M23B increased the levels of ethyl acetate (315.44 ± 73.07 mg/L), isoamyl acetate (5.99 ± 0.13 mg/L), and isoamyl alcohol (24.77 ± 15.19 mg/L), while DBVPG 6274T incremented the levels of phenyl ethyl alcohol and methionol up to 6.19 ± 0.51 mg/L and 3.72 ± 0.71 mg/L, respectively. A large production of terpenes and ethyl esters (e.g., ethyl octanoate) was detected in the cider fermented by S. cerevisiae EC1118. This study demonstrates, for the first time, the possible application of S. japonicus in cider-making to provide products with distinctive aromatic notes”.

Selection of ethanol tolerant strains of Candida albicans by repeated ethanol exposure results in strains with reduced susceptibility to fluconazole.

Candida albicans is a commensal yeast that has important impacts on host metabolism and immune function, and can establish life-threatening infections in immunocompromised individuals. Previously, C. albicans colonization has been shown to contribute to the progression and severity of alcoholic liver disease. However, relatively little is known about how C. albicans responds to changing environmental conditions in the GI tract of individuals with alcohol use disorder, namely repeated exposure to ethanol. In this study, we repeatedly exposed C. albicans to high concentrations (10% vol/vol) of ethanol-a concentration that can be observed in the upper GI tract of humans following consumption of alcohol. Following this repeated exposure protocol, ethanol small colony (Esc) variants of C. albicans isolated from these populations exhibited increased ethanol tolerance, altered transcriptional responses to ethanol, and cross-resistance/tolerance to the frontline antifungal fluconazole. These Esc strains exhibited chromosomal copy number variations and carried polymorphisms in genes previously associated with the acquisition of fluconazole resistance during human infection. This study identifies a selective pressure that can result in evolution of fluconazole tolerance and resistance without previous exposure to the drug.

Isolation, selection and characterisation of microorganisms with potential to form unique consortium for wine production

This paper focuses on a selection of microorganisms that should eventually form a Defined Consortium of Wine Microorganisms. The consortium might serve as a sophisticated oenological product for the production of wine with attractive organoleptic features. The Defined Consortium of Wine Microorganisms was obtained from non-saccharomyces and saccharomyces yeasts and lactic acid bacteria isolated from places where autochthonous microflora can be expected to occur (grape berries, wine lees or cellar spaces). A total of 42 microorganisms were obtained by surface smears, fallouts from the air or isolation from fermented must. All isolates were then tested and the best strains were selected on the basis of technological and phenotypic characterization using standard microbiological techniques. The main criteria for the selection of strains were the ability to ferment and the production of organoleptically active compounds. Sulphur dioxide or ethanol tolerance, β-glucosidase activity, a taxonomic identification, the tendency to produce sulphane or the ability of lactic acid bacteria to perform malolactic fermentation were also considered. According to these results, the non-saccharomyces yeast Starmerella bacillaris, the saccharomyces yeast Saccharomyces cerevisiae and the lactic acid bacterium Levilactobacillus brevis were selected as part of the Defined Consortium of Wine Microorganisms.

Heterologous expression of small heat shock protein by Oenococcus oeni improves its ethanol tolerance

Small heat shock proteins (sHsps) promote microbial resistance to harsh environmental conditions. Three sHsp genes (lphsp) from Lactobacillus plantarum and one (lohsp) from Oenococcus oeni were cloned and transformed into Lactococcus lactis MG1363, which does not contain sHSPs for heterologous expression. Comparing the tolerance of different recombinant strains to ethanol, the lphsp19.3 gene with the highest tolerance was selected and inserted into the expression vector pHH40 and transformed into the new host O. oeni. The results showed that when compared with the ethanol tolerance of the original O. oeni strain, i.e., 12% v/v, the recombinant strain could tolerate approximately 14% v/v. Moreover, the recombinant strain shortened the malo-lactic fermentation time of wine by 2 days without any adverse effects on aroma. Therefore, the recombinant O. oeni strain showed better application prospects.

Laboratory domestication of Lactiplantibacillus plantarum alters some phenotypic traits but causes non-novel genomic impact.

Laboratory domestication has been negligibly examined in lactic acid bacteria (LAB). Lactiplantibacillus plantarum is a highly studied and industrially relevant LAB. Here, we passaged L. plantarum JGR2 in a complex medium to study the effects of domestication on the phenotypic properties and the acquisition of mutations. Lactiplantibacillus plantarum JGR2 was passaged in mMRS medium (deMan Rogossa Sharpe supplemented with 0.05%w/v L-cysteine) in three parallel populations for 70 days. One pure culture from each population was studied for various phenotypic properties and genomic alterations. Auto-aggregation of the evolved strains was significantly reduced, and lactic acid production and ethanol tolerance were increased. Other probiotic properties and antibiotic sensitivity were not altered. Conserved synonymous and non-synonymous mutations were observed in mobile element proteins (transposases), β-galactosidase, and phosphoketolases in all three isolates. The evolved strains lost all the repeat regions and some of the functions associated with them. Most of the conserved mutations were found in the genomes of other wild-type strains available in a public database, indicating the non-novel genomic impact of laboratory passaging. Laboratory domestication can affect the phenotypic and genotypic traits of L. plantarum and similar studies are necessary for other important species of LAB.

Screening and characterization of potential bioethanol production yeast from tropical fruits

This study aims to separate and characterise indigenous yeast (IY) from tropical fruit waste. The techniques include isolating and characterising yeast from different kinds of fruit waste, testing yeast for ethanol and glucose tolerance, and producing bioethanol in vitro. Using a microscope and visual inspection, the yeast’s morphological identification is done. Using a spectrophotometer to measure optical density, the tolerance tests for glucose and ethanol are used to select yeast biochemically. With the Gas Chromatography-Flame Ionisation Detector (GC-FID), one can measure the amount of ethanol present. Yeast was isolated using selective media to yield six isolates: code A1 from grapes, codes NG1, NG2 from jackfruit, and codes N1, N2, and N3 from pineapple; mango produced no results. Three isolates with the codes A1, NG1, and NG2 were chosen based on test results for resistance to glucose and ethanol. The Saccharomyces cerevisae bioethanol production test yielded 6.60%, 3.30%, 4.5%, and.4.85% of ethanol for the yeast species coded A1, NG1, and NG2, respectively, in terms of ethanol. According to the study’s findings, yeast bearing the NG2 code may be used in the fermentation process to produce bioethanol.

Large analysis of genetic manipulations reveals an inverse correlation between initial alcohol resistance and rapid tolerance phenotypes

AbstractTolerance occurs when, following an initial experience with a substance, more of the substance is required subsequently to induce identical behavioral effects. Tolerance is not well‐understood, and numerous researchers have turned to model organisms, particularly Drosophila melanogaster, to unravel its mechanisms. Flies have high translational relevance for human alcohol responses, and there is substantial overlap in disease‐causing genes between flies and humans, including those associated with Alcohol Use Disorder. Numerous Drosophila tolerance mutants have been described; however, approaches used to identify and characterize these mutants have varied across time and labs and have mostly disregarded any impact of initial resistance/sensitivity to ethanol on subsequent tolerance development. Here, we analyzed our own, as well as data published by other labs to uncover an inverse correlation between initial ethanol resistance and tolerance phenotypes. This inverse correlation suggests that initial resistance phenotypes can explain many ‘perceived’ tolerance phenotypes, thus classifying such mutants as ‘secondary’ tolerance mutants. Additionally, we show that tolerance should be measured as a relative increase in time to sedation between an initial and second exposure rather than an absolute change in time to sedation. Finally, based on our analysis, we provide a method for using a linear regression equation to assess the residuals of potential tolerance mutants. These residuals provide predictive insight into the likelihood of a mutant being a ‘primary’ tolerance mutant, where a tolerance phenotype is not solely a consequence of initial resistance, and we offer a framework for understanding the relationship between initial resistance and tolerance.

Domestication signatures in the non-conventional yeast Lachancea cidri.

Evaluating domestication signatures beyond model organisms is essential for a thorough understanding of the genotype-phenotype relationship in wild and human-related environments. Structural variations (SVs) can significantly impact phenotypes playing an important role in the physiological adaptation of species to different niches, including during domestication. A detailed characterization of the fitness consequences of these genomic rearrangements, however, is still limited in non-model systems, largely due to the paucity of direct comparisons between domesticated and wild isolates. Here, we used a combination of sequencing strategies to explore major genomic rearrangements in a Lachancea cidri yeast strain isolated from cider (CBS2950) and compared them to those in eight wild isolates from primary forests. Genomic analysis revealed dozens of SVs, including a large reciprocal translocation (~16 kb and 500 kb) present in the cider strain, but absent from all wild strains. Interestingly, the number of SVs was higher relative to single-nucleotide polymorphisms in the cider strain, suggesting a significant role in the strain's phenotypic variation. The set of SVs identified directly impacts dozens of genes and likely underpins the greater fermentation performance in the L. cidri CBS2950. In addition, the large reciprocal translocation affects a proline permease (PUT4) regulatory region, resulting in higher PUT4 transcript levels, which agrees with higher ethanol tolerance, improved cell growth when using proline, and higher amino acid consumption during fermentation. These results suggest that SVs are responsible for the rapid physiological adaptation of yeast to a human-related environment and demonstrate the key contribution of SVs in adaptive fermentative traits in non-model species.IMPORTANCEThe exploration of domestication signatures associated with human-related environments has predominantly focused on studies conducted on model organisms, such as Saccharomyces cerevisiae, overlooking the potential for comparisons across other non-Saccharomyces species. In our research, employing a combination of long- and short-read data, we found domestication signatures in Lachancea cidri, a non-model species recently isolated from fermentative environments in cider in France. The significance of our study lies in the identification of large array of major genomic rearrangements in a cider strain compared to wild isolates, which underly several fermentative traits. These domestication signatures result from structural variants, which are likely responsible for the phenotypic differences between strains, providing a rapid path of adaptation to human-related environments.

Oral administration of Alcohol-Tolerant Lactic Acid Bacteria Alleviates Blood Alcohol Levels and Ethanol-Induced Liver Damage in Rodents.

Excessive alcohol consumption can have serious negative consequences on health, including addiction, liver damage, and other long-term effects. The causes of hangovers include dehydration, alcohol and alcohol metabolite toxicity, and nutrient deficiency due to absorption disorders. Additionally, alcohol consumption can slow reaction times, making it more difficult to rapidly respond to situations that require quick thinking. When consuming a large amount of alcohol, it takes a longer time to recover the righting reflex and balance following ethanol exposure. This study aimed to evaluate the potential of lactic acid bacteria (LAB) to alleviate alcohol-induced effects and behavioral responses. The two LAB strains isolated from kimchi, Levilactobacillus brevis WiKim0168 and Leuconostoc mesenteroides WiKim0172, were selected for their ethanol tolerance and potential to alleviate hangover symptoms. Enzyme activity assays for alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) were conducted to evaluate the role of these bacteria in alcohol metabolism. Through in vitro and in vivo studies, these strains were assessed for their ability to reduce blood alcohol concentrations and to protect against alcohol-induced liver damage. Results indicated that these LAB strains exhibited significant ethanol tolerance and elevate ADH and ALDH activities. LAB administration remarkedly reduced blood alcohol levels in rats with excessive alcohol consumption. Moreover, the LAB strains showed hepatoprotective effects and enhanced behavioral outcomes, highlighting their potential as probiotics for counteracting the adverse effects of alcohol consumption. These findings support the development of functional foods incorporating LAB strains that can mediate behavioral improvements following alcohol intake.

Perspectives and Progress in Bioethanol Processing and Social Economic Impacts

The liquid biofuel bioethanol is widely produced worldwide via fermenting sugars extracted from a variety of raw materials, including lignocellulose biomass, one of the world’s most abundant renewable resources. Due to its recalcitrant character, lignocellulose is usually pretreated by mechanical, chemical, and biological methods to maximize sugar recovery. Pretreated lignocellulose biomass undergoes a fermentation process performed sequentially or simultaneously to saccharification. The different fermentation strategies (e.g., separate or simultaneous hydrolysis and fermentation or co-fermentation) and conditions (e.g., inoculum type load, agitation, temperature, and pH) affect ethanol yield. Genetic modification of the inoculum has been focused recently to improve ethanol tolerance and as well as to use different sugars to enhance the performance of the microorganisms involved in fermentation. Nonetheless, these improvements result in a substantial increase in costs and have certain environmental costs. This review offers an overview of advancements in bioethanol production, with a primary focus on lignocellulosic feedstock, while also considering other feedstocks. Furthermore, it provides insights into the economic, social, and environmental impacts associated with bioethanol production.

Isolation, genetic identification of Amazonian yeasts and analysis of thermotolerance and alcohol tolerance of &lt;i&gt;Saccharomyces cerevisiae&lt;/i&gt; from &lt;i&gt;Theobroma grandiflorum&lt;/i&gt; and &lt;i&gt;Eugenia stipitata&lt;/i&gt;

&lt;abstract&gt; &lt;p&gt;Although yeasts of the &lt;italic&gt;Saccharomyces cerevisiae&lt;/italic&gt; species are industrially significant, few studies have investigated their presence in environmental samples from the Amazon rainforest. This study aimed to isolate &lt;italic&gt;S. cerevisiae&lt;/italic&gt; yeasts associated with trees of the Amazon Forest and investigate their thermotolerance, alcohol tolerance, and single nucleotide polymorphism (SNP) characteristics, along with those of regional strains from previous research and reference strains from the industry. We collected fruits, bark and decaying plant material from &lt;italic&gt;Theobroma grandiflorum&lt;/italic&gt;, &lt;italic&gt;Spondias mombin&lt;/italic&gt; L., &lt;italic&gt;Mangifera indica&lt;/italic&gt; L., and &lt;italic&gt;Eugenia stipitata&lt;/italic&gt;, and isolated yeasts using the culture media. To identify the yeasts, we conducted morphological and biochemical analyses, including sugar assimilation and fermentation, and sequencing analyses of the rDNA (ITS and LSU (D1 and D2)). We also performed fermentation tests to determine the optimum temperature, thermotolerance and ethanol tolerance. Finally, we subjected the selected strains to SNP analysis to study the reported genes that are important for alcohol tolerance in &lt;italic&gt;S. cerevisiae&lt;/italic&gt;: FPS1 (farnesyl diphosphate synthase1) and ASR1/YPR093 (alcohol sensitive RING/PHD finger1) genes. As a result, we isolated 53 yeasts, and 10 of which exhibited a sugar assimilation and fermentation profile that was similar to that of &lt;italic&gt;S. cerevisiae&lt;/italic&gt;. These ten isolates were identified using sequencing of the ITS and LSU regions, which revealed the species to be &lt;italic&gt;Wickerhamomyces anomalus&lt;/italic&gt; (n = 4), &lt;italic&gt;Torulaspora pretoriensis&lt;/italic&gt; (n = 3), &lt;italic&gt;Debaryomyces hansenni&lt;/italic&gt; (n = 1), and &lt;italic&gt;Saccharomyces cerevisiae&lt;/italic&gt; (n = 2). Through the analysis of the ASR1 and FPS1 regions, we found an SNP at nucleotide 1552 A &amp;gt; G (FPS1), which was associated with ethanol tolerance under our experimental conditions. This work is significant because it is one of the first studies to focus specifically on the isolation of &lt;italic&gt;S. cerevisiae&lt;/italic&gt; from samples in the Amazon region. Furthermore, the SNP analysis allowed us to differentiate isolates that showed greater tolerance to ethanol.&lt;/p&gt; &lt;/abstract&gt;

Selection of Starter from Decaying Oranges and Pineapple for Bioethanol Production

Screening of yeasts for bioethanol production is an important aspect of industrial microbiology and biotechnology. Optimal yeast nutrition also requires availability of nutrients allowing for growth and ethanol production and ability to withstand the increasing concentration of the medium during fermentation. This study seeks to use appropriate methods to isolate, characterize and identify yeasts isolates with essential attributes for bioethanol production. Microbiological and Physicochemical assessment of the isolated strains was carried out on yeast maintenance media for the determination of ethanol, temperature and salt tolerance ability, growth at different pH and temperature, chloramphenicol and nalidixic acid test, determination of killer toxins production capacity of yeast, nitrate and carbon assimilation and sugar fermentation tests were carried out to select the best starter for production.&#x0D; Fifteen yeasts were isolated belonging to the genera Saccharomyces, Candida, Rhodotorula, Kluvyreomyces, Trichosporon, Pichia. S. cerevisae and K. marxianus showed efficient physico-chemical attributes. Proximate analysis of fruits juice showed a moisture content between 81.83 – 86.37%, crude protein 1.33 – 2.00%, Ash 0.73 – 0.83%, carbohydrate 11.87 – 15.67% and Ether 0.1 – 0.5%. The total sugar ranged from 1.83 – 13.17, equal mixture of oranges and pineapple juice showed a sugar content of 15 which reduced during fermentation while the alcohol content increased. Organisms were able to tolerate a percentage of 4-30% ethanol content, tolerating 15% sodium chloride which is an index of osmotolerance. At higher concentration, growth reduced, organisms produced catalase, reduced nitrate and showed a variation in utilization of different sugars.&#x0D; At the end of the screening, S. cerevisiae and K. marxianus showed the best attribute essential for bioethanol production and were chosen as starters. The ability of the selected yeast isolates to produce Killer toxins against E. coli showed a negative result. Antibiotic sensitivity test was carried out on the selected yeast isolates using chloramphenicol and nalidixic and the two organisms were resistant to the antibiotics tested.&#x0D; The ability of the organisms to grow in changing environmental conditions and ethanol tolerance are attributes essential for production while Saccharomyces cerevisiae showed the highest attribute followed by Kluveromyces marxinus. This study concluded that Saccharomyces cerevisiae can be employed as starter in the industry for the production of bioethanol and in the conversion of agricultural waste to wealth.

Osmotic Stress Alleviation in Saccharomyces cerevisiae for High Ethanol Fermentations with Different Wort Substrates

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.

Bioethanol production from deproteinized cheese whey powder by local strain isolated from soured milk: influence of operating parameters

ABSTRACT In this paper, a local isolated yeast strain was studied for bioethanol production using protein-free cheese whey powder (CWP) in batch alcoholic fermentation experiments. Ethanol tolerance tests were performed with different ethanol concentrations (from 0% to 20%). The maximum ethanol tolerance obtained for our strain was 5% (v/v). The influence of initial lactose concentration, temperature and pH on fermentation kinetic was studied. For initial lactose concentration ranging from 57.5 to 260 gL−1, the highest ethanol production 34.8 gL−1 was obtained at 109 gL−1 initial lactose concentration corresponding to 0.413 gg−1 ethanol yield (YP/S); (While it was noticed less ethanol production, namely 0.178 g g−1) at a higher initial whey sugar concentration of 260 gL−1. The effect of the initial pH on the fermentation was studied at three different values: 3, 4 and 5. The ethanol production reached a maximum of 22 g L−1 with a lactose consumption of 77.62% at pH 4. However, an observed decrease occurred in ethanol production and lactose consumption at pH 3, with values of 10.34 gL-1 and 24.46%, respectively. For the fermentation conducted at the three temperatures of 37°C, 40°C and 43°C, the maximum ethanol production and lactose consumption were 34.82 gL-1 and 80.75%, respectively, achieved at 37°C. The experimental data was analyzed using the Gompertz model to obtain the following kinetic parameters: the maximum ethanol concentration (Pm) was found to be 36.97 gL-1, the maximum ethanol production rate (rp,m) was 0.402 gL−1h−1 and the lag phase (tl) was 10.8 h. The comparison of the isolated Kluyveromyces marxianus TXID4911 strain with the reference strain Kluyveromyces marxianus DSM 5421, showed a relatively close ethanol yield (YP/S) 0.415 and 0.505 gg,−1 respectively.