Ethanologenic Yeast Research Articles

Optimal trade-off between boosted tolerance and growth fitness during adaptive evolution of yeast to ethanol shocks

BackgroundThe selection of Saccharomyces cerevisiae strains with higher alcohol tolerance can potentially increase the industrial production of ethanol fuel. However, the design of selection protocols to obtain bioethanol yeasts with higher alcohol tolerance poses the challenge of improving industrial strains that are already robust to high ethanol levels. Furthermore, yeasts subjected to mutagenesis and selection, or laboratory evolution, often present adaptation trade-offs wherein higher stress tolerance is attained at the expense of growth and fermentation performance. Although these undesirable side effects are often associated with acute selection regimes, the utility of using harsh ethanol treatments to obtain robust ethanologenic yeasts still has not been fully investigated.ResultsWe conducted an adaptive laboratory evolution by challenging four populations (P1–P4) of the Brazilian bioethanol yeast, Saccharomyces cerevisiae PE-2_H4, through 68–82 cycles of 2-h ethanol shocks (19–30% v/v) and outgrowths. Colonies isolated from the final evolved populations (P1c–P4c) were subjected to whole-genome sequencing, revealing mutations in genes enriched for the cAMP/PKA and trehalose degradation pathways. Fitness analyses of the isolated clones P1c–P3c and reverse-engineered strains demonstrated that mutations were primarily selected for cell viability under ethanol stress, at the cost of decreased growth rates in cultures with or without ethanol. Under this selection regime for stress survival, the population P4 evolved a protective snowflake phenotype resulting from BUD3 disruption. Despite marked adaptation trade-offs, the combination of reverse-engineered mutations cyr1A1474T/usv1Δ conferred 5.46% higher fitness than the parental PE-2_H4 for propagation in 8% (v/v) ethanol, with only a 1.07% fitness cost in a culture medium without alcohol. The cyr1A1474T/usv1Δ strain and evolved P1c displayed robust fermentations of sugarcane molasses using cell recycling and sulfuric acid treatments, mimicking Brazilian bioethanol production.ConclusionsOur study combined genomic, mutational, and fitness analyses to understand the genetic underpinnings of yeast evolution to ethanol shocks. Although fitness analyses revealed that most evolved mutations impose a cost for cell propagation, combination of key mutations cyr1A1474T/usv1Δ endowed yeasts with higher tolerance for growth in the presence of ethanol. Moreover, alleles selected for acute stress survival comprising the P1c genotype conferred stress tolerance and optimal performance under conditions simulating the Brazilian industrial ethanol production.

Engineering Compositionally Uniform Yeast Whole-Cell Biocatalysts with Maximized Surface Enzyme Density for Cellulosic Biofuel Production.

In recent decades, whole-cell biocatalysis has played an increasingly important role in the food, pharmaceutical, and energy sector. One promising application is the use of ethanologenic yeast displaying minicellulosomes on the cell surface to combine cellulose hydrolysis and fermentation into a single step for consolidated bioprocessing. However, cellulosic ethanol production using existing yeast whole-cell biocatalysts (yWCBs) has not reached industrial feasibility due to their inefficient cellulose hydrolysis. As prior studies have demonstrated enzyme density on the yWCB surface to be one of the most important parameters for enhancing cellulose hydrolysis, we sought to maximize this parameter at both the population and single-cell levels in yWCBs displaying tetrafunctional minicellulosomes. At the population level, enzyme density is limited by the presence of a nondisplay population constituting 25-50% of all cells. In this study, we identified the cause to be plasmid loss and successfully eliminated the nondisplay population to generate compositionally uniform yWCBs. At the single-cell level, we demonstrate that enzyme density is limited by molecular crowding, which hinders minicellulosome assembly. By adjusting the integrated gene copy number, we obtained yWCBs of tunable enzyme display levels. This tunability allowed us to avoid the crowding-limited regime and achieve a maximum enzyme density per cell. As a result, the best strain showed a cellulose-to-ethanol yield of 4.92 g/g, corresponding to 96% of the theoretical maximum and near-complete conversion (∼96%) of the starting cellulose (1% PASC). Our holistic engineering strategy that combines a population and single-cell level approach is broadly applicable to enhance the WCB performance in other biocatalytic cascade schemes.

Ethanologenic yeasts from Ethiopian fermented beverages and optimization of fermentation conditions

The rising global demand of renewable energy sources such as bioethanol is pushing stakeholders to search for efficient ethanologenic microbes. This study aimed to isolate yeasts from Ethiopian traditional fermented beverages, evaluate their ethanol yield using banana peel as a substrate, and identify best-performing isolates. Moreover, this study attempted to statistically optimize fermentation conditions through Placket Burman Design and Central Composite Design based Response Surface Methodology (RSM). A progressive screening and selection approach was followed to select best performing isolates. Factors that significantly influence fermentation efficiency were selected using Plackett-Burman Design (PBD) and then optimized via Central Composite Design (CCD). A total of 11 isolates were obtained. Three isolates (DMTD2, GB1D5 and RTj3D3) were selected based on their tolerance to stress conditions, and carbohydrate fermentation and assimilation performances. GB1D5 and RTj3D3 were highly tolerant whereas DMTD2 was moderately tolerant to 10 % ethanol concentration with survival percentages ranging from 40.8 ± 5.6 to 65.7 ± 10.5 %. However, they were slightly tolerant to 15 and 20 %, with an insignificant survival percentage rate. They yield 11.6 ± 0.9 to 12.9 ± 0.3 g/L ethanol from 8 % (w/v) banana peel waste. D1/D2 genotyping identified them as Saccharomyces cerevisiae strains. Glucose concentration and incubation period significantly affect ethanol production. The maximum actual ethanol yield produced by isolates at optimum condition was the same i.e. 31.4 g/L, whereas the ethanol yield predicted by the model ranged between 31.8 and 32.5 g/L. Results demonstrate these strains are promising candidates for bioethanol production. Optimization of fermentation conditions using banana peel waste as substrate is recommended.

Utilization of pretreated oil palm empty fruit bunches and their hydrolysate for ethanol production by Indonesian ethanologenic yeast

Abstract. Anita SH, Oktaviani M, Hermiati E. 2023. Utilization of pretreated oil palm empty fruit bunches and their hydrolysate for ethanol production by Indonesian ethanologenic yeast. Biodiversitas 24: 5243-5252. Oil Palm Empty Fruit Bunches (OPEFB) represent a polysaccharide-rich raw material with promising potential for ethanol production. This study aimed to investigate the ethanologenic yeasts, specifically Saccharomyces cerevisiae InaCC Y93 and Kluyveromyces marxianus InaCC Y119, affect bioethanol production in three different systems: Separate Hydrolysis and Fermentation (SHF), Simultaneous Saccharification and Fermentation (SSF), and Prehydrolysis-Simultaneous Saccharification and Fermentation (PSSF). This work is distinguished by the use of indigenous Indonesian yeast strains, including a thermotolerant strain. In the pretreatment process, 1.13% oxalic acid was added to OPEFB and subjected to microwave treatment at 190°C for 3.01 min. Subsequently, cellulase enzymes (40 FPU/g) and a 10% (w/v) yeast inoculum were introduced into 5.27 g dry weight of pretreated OPEFB pulp. The OPEFB acid hydrolysate was also subjected to fermentation. Ethanol content was monitored at 24 h intervals for 72 h. The PSSF system employs K. marxianus InaCC Y119 at 48 h exhibited the highest ethanol concentration, yielding 0.290 g/g, equivalent to approximately 51.20% of the theoretical yield. Additionally, K. marxianus InaCC Y119 demonstrated its capability to ferment the OPEFB acid hydrolysate into ethanol. These findings underscore the considerable potential of K. marxianus for applications in fermenting both hexose and pentose sugars to produce ethanol within higher-temperature systems.

Isolation, screening and identification of ethanol producing yeasts from Ethiopian fermented beverages

The growing demand for renewable energy sources such as bioethanol is facing a lack of efficient ethanologenic microbes. This study aimed to isolate and screen ethanologenic yeasts from Ethiopian fermented beverages. A progressive screening and selection approach was employed. Selected isolates were evaluated for bioethanol production using banana peel waste as substrate. A total of 102 isolates were obtained. Sixteen isolates were selected based on their tolerance to stress conditions and carbohydrate fermentation and assimilation capacity. Most found moderately tolerant to 10 %, but slightly tolerant at 15 and 20 % (v/v) ethanol concentration. They yield 15.3 to 20.1 g/L and 9.1 ± 0.6 to 12.9 ± 1.3 g/L ethanol from 2 % (w/v) glucose and 80 g/L banana peel, respectively. Molecular characterization identified them as Saccharomyces cerevisiae strains. Results demonstrate insight about their potential role in the ethanol industry. Optimization of the fermentation conditions is recommended.

Optimized biotransformation of acid-treated water melon peel hydrolyzate into ethanol.

Today, global focus of research is to explore the solution of energy crisis and environmental pollution. Like other agricultural countries, bulk quantities of watermelon peels (WMP) are disposed-off in environment as waste in Pakistan and appropriate management of this waste is the need of hour to save environment from pollution. The work emphasizes the role of ethanologenic yeasts to utilize significant sugars present in WMP for low-cost bioethanol fermentation. Dilute hydrochloric acid hydrolysis of WMP was carried out on optimized conditions employing RSM (response surface methodology) following central composite design (CCD). This experimental design is based on optimization of ethanologenesis involving some key independent parameters such as WMP hydrolysate and synthetic media ratio (X1), incubation temperature (X2) and incubation temperature (X3) for maximal ethanol yield exploiting standard (Saccharomyces cerevisiae K7) as well as experimental (Metchnikowia cibodasensisY34) yeasts. The results revealed that maximal ethanol yields obtained from S. cerevisiae K7 was 0.36±0.02 g/g of reducing sugars whereas M. cibodasensisY34, yielded 0.40±0.01 g ethanol/g of reducing sugars. The yeast isolate M. cibodasensisY34 appeared as promising ethanologen and embodies prospective potential for fermentative valorization of WMP-to-bioethanol.

The potential of multistress tolerant yeast, Saccharomycodes ludwigii, for second-generation bioethanol production

Ethanol production at high temperatures using lignocellulosic biomass as feedstock requires a highly efficient thermo and lignocellulosic inhibitor-tolerant ethanologenic yeast. In this study, sixty-three yeast isolates were obtained from tropical acidic fruits using a selective acidified medium containing 80 mM glacial acetic acid. Twenty-nine of the yeast isolates exhibited significant thermo and acetic acid-tolerant fermentative abilities. All these isolates were classified into three major yeast species, namely Saccharomycodes ludwigii, Pichia kudriavzevii, and P. manshurica, based on molecular identification. Saccharomycodes ludwigii APRE2 displayed an ability to grow at high temperatures of up to 43 °C and exhibited significant multistress tolerance toward acetic acid, furfural, 5-hydroxymethyl furfural (5-HMF), and ethanol among the isolated yeast species. It can produce a maximum ethanol concentration of 63.07 g/L and productivity of 1.31 g/L.h in yeast extract malt extract (YM) medium containing 160 g/L glucose and supplemented with 80 mM acetic acid and 15 mM furfural as a cocktail inhibitor. When an acid-pretreated pineapple waste hydrolysate (PWH) containing approximately 106 g/L total sugars, 131 mM acetic acid, and 3.95 mM furfural was used as a feedstock, 38.02 g/L and 1.58 g/L.h of ethanol concentration and productivity, respectively, were achieved. Based on the results of the current study, the new thermo and acetic acid-tolerant yeast S. ludwigii APRE2 exhibited excellent potential for second-generation bioethanol production at high temperatures.

Exploring Natural Fermented Foods as a Source for New Efficient Thermotolerant Yeasts for the Production of Second-Generation Bioethanol

Considering the cost-effectiveness of bioethanol production at high temperatures, there is an enduring need to find new thermotolerant ethanologenic yeasts. In this study, a total of eighteen thermotolerant yeasts were isolated from various natural fermented products in Morocco. Ethanol production using 50 g/L glucose or 50 g/L xylose as the sole carbon source revealed potential yeasts with high productivities and volumetric ethanol productivities at high temperatures. Based on molecular identification, the selected thermotolerant fermentative isolates were affiliated with Pichia kudriavzevii, Kluyveromyces marxianus, and Kluyveromyces sp. During the simultaneous saccharification and fermentation of lignocellulosic biomass at a high temperature (42 °C), the designated yeast P. kudriavzevii YSR7 produced an ethanol concentration of 22.36 g/L, 18.2 g/L and 6.34 g/L from 100 g/L barley straw (BS), chickpea straw (CS), and olive tree pruning (OTP), respectively. It also exhibited multi-stress tolerance, such as ethanol, acetic acid, and osmotic tolerance. Therefore, the yeast P. kudriavzevii YSR7 showed promising attributes for biorefinery-scale ethanol production in the future.

Metabolomic profiling of Spathaspora passalidarum fermentations reveals mechanisms that overcome hemicellulose hydrolysate inhibitors.

Understanding the mechanisms involved in tolerance to inhibitors is the first step in developing robust yeasts for industrial second-generation ethanol (E2G) production. Here, we used ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) and MetaboAnalyst 4.0 for analysis of MS data to examine the changes in the metabolic profile of the yeast Spathaspora passalidarum during early fermentation of hemicellulosic hydrolysates containing high or low levels of inhibitors (referred to as control hydrolysate or CH and strategy hydrolysate or SH, respectively). During fermentation of SH, the maximum ethanol production was 16g L-1 with a yield of 0.28gg-1 and productivity of 0.22g L-1h-1, whereas maximum ethanol production in CH fermentation was 1.74g L-1 with a yield of 0.11gg-1 and productivity of 0.01g L-1h-1. The high level of inhibitors in CH induced complex physiological and biochemical responses related to stress tolerance in S. passalidarum. This yeast converted compounds with aldehyde groups (hydroxymethylfurfural, furfural, 4-hydroxybenzaldehyde, syringaldehyde, and vanillin) into less toxic compounds, and inhibitors were found to reduce cell viability and ethanol production. Intracellularly, high levels of inhibitors altered the energy homeostasis and redox balance, resulting in lower levels of ATP and NADPH, while that of glycolytic, pentose phosphate, and tricarboxylic acid (TCA) cycle pathways were the most affected, being the catabolism of glucogenic amino acids, the main cellular response to inhibitor-induced stress. This metabolomic investigation reveals interesting targets for metabolic engineering of ethanologenic yeast strains tolerant against multiple inhibitors for E2G production. KEY POINTS: • Inhibitors in the hydrolysates affected the yeast's redox balance and energy status. • Inhibitors altered the glycolytic, pentose phosphate, TCA cycle and amino acid pathways. • S. passalidarum converted aldehyde groups into less toxic compounds.

Cellulosic Ethanol Production Using a Dual Functional Novel Yeast.

Reducing the cost of cellulosic ethanol production, especially for cellulose hydrolytic enzymes, is vital to growing a sustainable and efficient cellulosic ethanol industry and bio-based economy. Using an ethanologenic yeast able to produce hydrolytic enzymes, such as Clavispora NRRL Y-50464, is one solution. NRRL Y-50464 is fast-growing and robust, and tolerates inhibitory compounds 2-furaldehyde (furfural) and 5-hydroxymethyl-2-furaldehyde (HMF) associated with lignocellulose-to-fuel conversion. It produces three forms of β-glucosidase isozymes, BGL1, BGL2, and BGL3, and ferment cellobiose as the sole carbon source. These β-glucosidases exhibited desirable enzyme kinetic parameters and high levels of enzyme-specific activity toward cellobiose and many oligosaccharide substrates. They tolerate the product inhibition of glucose and ethanol, and are stable to temperature and pH conditions. These characteristics are desirable for more efficient cellulosic ethanol production by simultaneous saccharification and fermentation. NRRL Y-50464 provided the highest cellulosic ethanol titers and conversion rates at lower cellulase loadings, using either pure cellulose or agricultural residues, as so far reported in the literature. This review summarizes NRRL Y-50464 performance on cellulosic ethanol production from refined cellulose, rice straw, and corn stover processed in various ways, in the presence or absence of furfural and HMF. This dual functional yeast has potential to serve as a prototype for the development of next-generation biocatalysts. Perspectives on continued strain development and process engineering improvements for more efficient cellulosic ethanol production from lignocellulosic materials are also discussed.

Revitalizing the ethanologenic bacterium Zymomonas mobilis for sugar reduction in high-sugar-content fruits and commercial products

The excessive consumption of sugars can cause health issues. Different strategies have been developed to reduce sugars in the diets. However, sugars in fruits and commercial products may be difficult to reduce, limiting their usage among certain populations of people. Zymomonas mobilis is a generally recognized as safe (GRAS) probiotic bacterium with the capability to produce levan-type prebiotics, and thrives in high-sugar environments with unique characteristics to be developed for lignocellulosic biofuel and biochemical production. In this study, the sugar reduction capabilities of Z. mobilis ZM4 were examined using two fruits of pear and persimmon and three high-sugar-content commercial products of two pear pastes (PPs) and one Chinese traditional wine (CTW). Our results demonstrated that Z. mobilis ZM4 can utilize sugars in fruits with about 20 g/L ethanol and less than 5 g/L sorbitol produced within 22 h using pears, and about 45 g/L ethanol and 30 g/L sorbitol produced within 34 h using persimmons. When PPs made from pears were used, Z. mobilis can utilize nearly all glucose (ca. 60 g/L) and most fructose (110 g/L) within 100 h with 40 ~ 60 g/L ethanol and more than 20 g/L sorbitol produced resulting in a final sorbitol concentration above 80 g/L. In the high-sugar-content alcoholic Chinese traditional wine, which contains mostly glucose and ethanol, Z. mobilis can reduce nearly all sugars with about 30 g/L ethanol produced, resulting in a final ethanol above 90 g/L. The ethanol yield and percentage yield of Z. mobilis in 50 ~ 60% CTW were 0.44 ~ 0.50 g/g and 86 ~ 97%, respectively, which are close to its theoretical yields—especially in 60% CTW. Although the ethanol yield and percentage yield in PPs were lower than those in CTW, they were similar to those in fruits of pears and persimmons with an ethanol yield around 0.30 ~ 0.37 g/g and ethanol percentage yield around 60 ~ 72%, which could be due to the formation of sorbitol and/or levan in the presence of both glucose and fructose. Our study also compared the fermentation performance of the classical ethanologenic yeast Saccharomyces cerevisiae BY4743 to Z. mobilis, with results suggesting that Z. mobilis ZM4 had better performance than that of yeast S. cerevisiae BY4743 given a higher sugar conversion rate and ethanol yield for sugar reduction. This work thus laid a foundation for utilizing the advantages of Z. mobilis in the food industry to reduce sugar concentrations or potentially produce alcoholic prebiotic beverages.Graphical

Compost as an untapped niche for thermotolerant yeasts capable of high-temperature ethanol production.

Efficient bioethanol production from lignocellulosic biomass requires thermotolerant yeasts capable of utilizing multiple sugars, tolerating inhibitors and fermenting at high temperatures. In this study, 98 thermotolerant yeasts were isolated from nine compost samples. We selected 37 yeasts that belonged to 11 species; 31 grew at 45°C; 6 strains grew at 47°C, while 9 yeasts could utilize multiple sugars. Many yeast isolates showed high ethanol production in the range of 12-24 g l-1 , with fermentation efficiencies of 47-94% at 40°C using 5% glucose. Kluyveromyces marxianus CSV3.1 and CSC4.1 (47°C), Pichia kudriavzevii CSUA9.3 (45°C) produced 21, 22 and 23 g l-1 of ethanol with efficiencies of 83, 87 and 90%, respectively, using 5% glucose. Among these yeasts, K. marxianus CSC4.1 and P. kudriavzevii CSUA9.3 exhibited high tolerance against furfural, 5-HMF, acetic acid and ethanol. These two strains produced high amounts of ethanol from alkali-treated RS, with 84 and 87% efficiency via separate hydrolysis and fermentation; 76 and 74% via simultaneous saccharification and fermentation at 47 and 45°C, respectively. Therefore, this study demonstrates compost as a potential anthropogenic niche for multiple sugar-utilizing, inhibitor-tolerant ethanologenic yeasts suitable for high-temperature ethanol production via SHF of rice straw.

Development of multiple inhibitor tolerant yeast via adaptive laboratory evolution for sustainable bioethanol production

The present research work aimed at developing robust yeast cell factory via adaptive laboratory evolution (ALE) for improved cellulosic bioethanol production. Kluyveromyces marxianus JKH5, a newly isolated thermotolerant ethanologenic yeast, was engineered by serial passaging for 60 generations in medium supplemented with gradually higher concentration of inhibitors (acetic acid, furfural, and vanillin) that are generated during dilute acid pretreatment. The improved strain K. marxianus JKH5 C60, showed 3.3-fold higher specific growth rate, 56% reduced lag phase and 80% enhanced fermentation efficiency at 42 °C in comparison to parent strain in inhibitor cocktail comprising medium. Bioethanol production by simultaneous saccharification and fermentation of sequential dilute acid-alkali pretreated sugarcane bagasse in presence of inhibitors, resulted in ethanol titre and yield, respectively, 54.8 ± 0.9 g/L and 0.40 g/g. The adapted yeast can be used to ferment unwashed pretreated biomass, thereby, reducing overall cost, time, and wastewater generation, hence making bioethanol production sustainable.

Proteomic analysis revealed the roles of YRR1 deletion in enhancing the vanillin resistance of Saccharomyces cerevisiae

BackgroundVanillin is one of the important phenolic inhibitors in Saccharomyces cerevisiae for bioconversion of lignocellulosic materials and has been reported to inhibit the translation process in cells. In our previous studies, it was confirmed that the deletion of the transcription factor gene YRR1 enhanced vanillin resistance by promoting some translation-related processes at the transcription level. In this work, we investigated the effects of proteomic changes upon induction of vanillin stress and deletion of YRR1 to provide unique perspectives from a transcriptome analysis for comprehending the mechanisms of YRR1 deletion in the protective response of yeast to vanillin.ResultsIn wild-type cells, vanillin reduced two dozens of ribosomal proteins contents while upregulated proteins involved in glycolysis, oxidative phosphorylation, and the pentose phosphate pathway in cells. The ratios of NADPH/NADP+ and NADH/NAD+ were increased when cells responded to vanillin stress. The differentially expressed proteins perturbed by YRR1 deletion were much more abundant than and showed no overlaps with transcriptome changes, indicating that Yrr1 affects the synthesis of certain proteins. Forty-eight of 112 upregulated proteins were involved in the stress response, translational and transcriptional regulation. YRR1 deletion increased the expression of HAA1-encoding transcriptional activator, TMA17-encoding proteasome assembly chaperone and MBF1-encoding coactivator at the protein level, as confirmed by ELISA. Cultivation data showed that the overexpression of HAA1 and TMA17 enhanced resistance to vanillin in S. cerevisiae.ConclusionsCells conserve energy by decreasing the content of ribosomal proteins, producing more energy and NAD(P)H for survival in response to vanillin stress. Yrr1 improved vanillin resistance by increasing the protein quantities of Haa1, Tma17 and Mbf1. These results showed the response of S. cerevisiae to vanillin and how YRR1 deletion increases vanillin resistance at the protein level. These findings may advance our knowledge of how YRR1 deletion protects yeast from vanillin stress and offer novel targets for genetic engineering of designing inhibitor-resistant ethanologenic yeast strains.

Bioprospecting of wild type ethanologenic yeast for ethanol fuel production from wastewater-grown microalgae

BackgroundWild-type yeasts have been successfully used to obtain food products, yet their full potential as fermenting microorganisms for large-scale ethanol fuel production has to be determined. In this study, wild-type ethanologenic yeasts isolated from a secondary effluent were assessed for their capability to ferment saccharified microalgae sugars.ResultsYeast species in wastewater were identified sequencing the Internal Transcribed Spacers 1 and 2 regions of the ribosomal cluster. Concurrently, microalgae biomass sugars were saccharified via acid hydrolysis, producing 5.0 ± 0.3 g L−1 of fermentable sugars. Glucose consumption and ethanol production of yeasts in hydrolyzed-microalgae liquor were tested at different initial sugar concentrations and fermentation time. The predominant ethanologenic yeast species was identified as Candida sp., and glucose consumption for this strain and S. cerevisiae achieved 75% and 87% of the initial concentration at optimal conditions, respectively. Relatively similar ethanol yields were determined for both species, achieving 0.45 ± 0.05 (S. cerevisiae) and 0.46 ± 0.05 g ethanol per g glucose (Candida sp.).ConclusionOverall, the results provide a first insight of the fermentation capacities of specific wild-type Candida species, and their potential role in ethanol industries seeking to improve their cost-efficiency.

Optimization of ethanol production using newly isolated ethanologenic yeasts

Yeasts are important microorganisms used for ethanol production; however, they are not equally efficient in the amount of ethanol production under different environmental conditions. It is, therefore, necessary to screen for elite strains to utilize them for commercial production of these commodities. In this study, yeasts were isolated from different Ethiopian traditional fermented alcoholic beverages (teji, tella, shamiata and areqe tinisis), milk and ergo, teff and maize dough, soil and compost, flowers, and fruits to evaluate their potential use for ethanol fermentation process. Isolates were screened for efficient ethanol production and the selected ones were identified using phenotypic and genetic characters using D1/D2 region of LSU rDNA sequence analysis. The yeast isolates were evaluated based on their growth and fermentation of different carbon sources. Response surface methodology (RSM) was applied to optimize temperature, pH and incubation time using central composite design (CCD) in Design-Expert 7.0.0. A total of 211 yeasts colonies were isolated of which 60% were ethanologenic yeasts (ethanol producers) and 40% were non-ethanol producers. The yeast population detected from various sources was in the range of 105CFU from traditional foods and beverages to that of 103CFU from fruits and soil samples. The data also showed that the number of colony types (diversity) did not correlate with population density. The highly fermentative isolates were taxonomically characterized into four genera, of which 65% of the isolates (ETP37, ETP50; ETP53, ETP89, ETP94) were categorized under Saccharomyces cerevisiae, and the remaining were Pichia fermentans ETP22, Kluyveromyces marxianus ETP87, and Candida humilis ETP122. The S. cerevisiae isolates produced ethanol (7.6-9.0 g/L) similar with K. marxianus ETP87 producing 7.97 g/L; comparable to the ethanol produced from commercial baker's yeast (8.43 g/L) from 20 g/L dextrose; whereas C. humilis ETP122 and P. fermentans ETP22 produced 5.37 g/L and 6.43 g/L ethanol, respectively. S. cerevisiae ETP53, K. marxianus ETP87, P. fermentans ETP22 and C. humilis ETP122 tolerated 10% extraneous ethanol but the percentage of ethanol tolerance considerably decreased upon 15%. S. cerevisiae ETP53 produced ethanol optimally at pH 5.0, 60 h, and 34oC. pH 4.8, temperature 36oC, and 65 h of time were optimal growth conditions of ethanol fermentation by K. marxianus ETP87. The ethanol fermentation conditions of P. fermentans ETP22 was similar to S. cerevisiae ETP53 though the ethanol titer of S. cerevisiae ETP53 was higher than P. fermentans ETP22. Therefore, S. cerevisiae ETP53, K. marxianus and P. fermentans ETP22 are good candidates for ethanol production.

Acid hydrolysis optimization of pomegranate peels waste using response surface methodology for ethanol production

Agro-industrial wastes can be processed into valuable products. Successively, current investigation is an effort to optimize the acid hydrolysis of pomegranate peels waste (PPW) using central composite design (CCD) of response surface methodology (RSM) for ethanol production. Concentration of sulfuric acid, temperature, and time of hydrolysis were used as dependent variables, whereas reducing sugars, total carbohydrates, extractives, weight loss, hemicellulose, cellulose, and lignin contents were recorded as responses for PPW decomposition. The highest glucose level of 0.56 ± 0.04 mg mL−1 (with 5% acid concentration at 100 °C for 30 min) and carbohydrate contents of 1.53 ± 0.07 mg mL−1 (with 3% acid concentration at 75 °C for 45 min) were obtained. Subsequently, detoxification of hydrolysate was conducted employing 2.5% activated charcoal that reduced 62% of phenolic compounds. Detoxified hydrolysate was subjected to fermentation by ethanologenic yeasts: Metschnikowia sp. Y31, Metschnikowia cibodasensis Y34, and Saccharomyces cerevisiae K7 for 10 days experiment. Significant ethanol yield of 0.42 ± 0.08 g g−1 was noticed by Metschnikowia sp. Y31 on day 5 and 0.41 ± 0.07 g g−1 for Metschnikowia cibodasensis Y34 on day 2. The results demonstrated the hopeful prospect for bioethanologenesis using cellulosic wastes at marketable level.

Xylanolytic and Ethanologenic Potential of Gut Associated Yeasts from Different Species of Termites from India

Xylophagous termites are capable of degrading lignocellulose by symbiotic gut microorganisms along with the host’s indigenous enzymes. Therefore, the termite gut might be a potential niche to obtain natural yeasts with celluloytic, xylanolytic and ethanologenic traits required for bioethanol production from lignocellulosic biomass. In this study, we cultured 79 yeasts from three different termites viz. Coptotermes heimi, Odontotermes javanicus and Odontotermes obesus. After suitable screening methods, we identified 53 yeasts, which belonged to 10 genera and 16 different species of both ascomycetous and basidiomycetous yeasts. Most yeasts in the present study represent their first-ever isolation from the termite gut. Representative strains of identified yeasts were evaluated for their cellulolytic, xylanolytic, and ethanologenic abilities. None of the isolates showed cellulase activity; 22 showed xylanolytic activity, while six produced substantial quantities of ethanol. Among xylanolytic cultures, Pseudozyma hubeiensis STAG 1.7 and Hannaella pagnoccae STAG 1.14 produced 1.31 and 1.17 IU of xylanase. Among ethanologenic yeasts, the strains belonging to genera Candida and Kodamaea produced high amount of ethanol. Overall, highest ethanol level of 4.42 g/L was produced by Candida tropicalis TS32 using 1% glucose, which increased up to 22.92 g/L at 35 °C, pH 4.5 with 5% glucose. Fermentation of rice straw hydrolysate gave 8.95 g/l of ethanol with a yield of 0.42 g/g using the strain TS32. Our study highlights the gut of wood-feeding termites as a potential source of diverse yeasts that would be useful in the production of xylanase and bioethanol.

Substrate utilization of ethanologenic yeasts co-cultivation of Pichia kudriavzevii and Saccharomyces cerevisiae

Bioethanol is one of the renewable alternative energies and can be used as a substitute for fossil fuels. Bioethanol is produced from a fermentation process, which is assisted by microbial yeast groups (ethanologenic yeast). Yeast Pichia kudriavzevii is capable of converting both carbon source pentose and hexose sugars to ethanol. In the previous study, P. kudriavzevii ethanol stress - tolerant mutant strains T5 was successfully constructed via directed mutagenesis thus suggesting their potential as fermentation agent. One strategy to increase ethanol production is by applying co-cultivation techniques. Thus, this study aimed to examine the ability of sugar substrate utilization by ethanologenic yeast S. cerevisiae and P. kudriavzeviiat various inoculum ratio. The results of the study indicated that no antagonist interaction detected between S. cerevisiae with all of P. kudriavzevii, both mutant and wild type strains. Based on sugar consumption analysis, both yeast isolates could be used in ethanol production simultaneously to maximize mixed substrate utilization. Our study revealed that the best sugar consumption found on co-cultivation of S. cerevisiae with wild type P. kudriavzevii T (wt) in 1: 1 inoculum ratio.

Heterologous secretory expression of β-glucosidase from Thermoascus aurantiacus in industrial Saccharomyces cerevisiae strains.

The use of plant biomass for biofuel production will require efficient utilization of the sugars in lignocellulose, primarily cellobiose, because it is the major soluble by-product of cellulose and acts as a strong inhibitor, especially for cellobiohydrolase, which plays a key role in cellulose hydrolysis. Commonly used ethanologenic yeast Saccharomyces cerevisiae is unable to utilize cellobiose; accordingly, genetic engineering efforts have been made to transfer β-glucosidase genes enabling cellobiose utilization. Nonetheless, laboratory yeast strains have been employed for most of this research, and such strains may be difficult to use in industrial processes because of their generally weaker resistance to stressors and worse fermenting abilities. The purpose of this study was to engineer industrial yeast strains to ferment cellobiose after stable integration of tabgl1 gene that encodes a β-glucosidase from Thermoascus aurantiacus (TaBgl1). The recombinant S. cerevisiae strains obtained in this study secrete TaBgl1, which can hydrolyze cellobiose and produce ethanol. This study clearly indicates that the extent of glycosylation of secreted TaBgl1 depends from the yeast strains used and is greatly influenced by carbon sources (cellobiose or glucose). The recombinant yeast strains showed high osmotolerance and resistance to various concentrations of ethanol and furfural and to high temperatures. Therefore, these yeast strains are suitable for ethanol production processes with saccharified lignocellulose.