Important conventional and genetic factors affecting bioethanol from Saccharomyces cerevisiae strains: A review article

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Introduction

Bioethanol is a versatile substance with a wide range of applications that may be produced spontaneously by yeast that ferments sugar, such as Saccharomyces cerevisiae. The ethanol produced during yeast fermentation is one of the main sources of stress for brewer's yeast because it prevents both cell growth and metabolism [1]. The high cost of the enzymes required for the saccharification of lignocellulosic biomass into fermentable sugars is a significant barrier to the production of bioethanol with second-generation feedstocks [2]. Because Saccharomyces cerevisiae has a substantially higher ethanol tolerance than most other species, it is often used in the commercial production of ethanol and alcoholic beverages. This issue might be solved by consolidated bioprocessing with saccharification and fermentation simultaneously using the lignocellulolytic enzyme-secreting Saccharomyces cerevisiae yeast [3,4]. Yeasts of the Saccharomyces genus are employed in biorefineries to ferment the carbohydrates produced during the breakdown of starch. They can also ferment galactose and mannose in addition to glucose. Xylose and arabinose can only be fermented by recombinant strains [5].

The most widely used yeast in the world for alcoholic fermentation is Saccharomyces cerevisiae's wild strains are typically unable to metabolize the pentose sugars (xylose and arabinose) that are generated during the hydrolysis of hemicelluloses [6]. Some other yeast genera, can also ferment these sugars to ethanol such as, Pichia [7] Candida, [8] and Pachysolen, [9] have been suggested as the most promising strains for producing bioethanol from different lignocellulosic feedstocks, either in monoculture or in coculture with conventional strains. The first-generation technique used in Brazil to manufacture ethanol is optimized and has a low cost of operation. But, given the volume of ethanol produced and exchanged annually, even a tiny process improvement might result in billions of dollars in cost savings. The enhancement of yeast strains that now take part in the first-generation process is receiving little attention from Brazilian research programs that are participating in sugarcane development. Sugarcane is used as a carbon source for the yeast Saccharomyces cerevisiae in the Brazilian ethanol manufacturing process. After that, yeast is cultivated in big, open tanks with cell recycling at high temperatures and cellular densities [10]. The yeast must be able to adapt to various circumstances. Two of the main stressors during the synthesis of alcohol are high temperatures and high ethanol concentrations inside the fermentation tanks. The struggle between the desired yeast strains, which are injected at the beginning of the process, and contaminants like wild-type yeasts and bacteria makes acid treatment required to successfully recycling the cells [10].

Literature Review

Important classical factors affecting ethanol production by Saccharomyces cerevisiae

The transformation processes for producing bioethanol from starches' disaccharides are looked at. Improvements through five potential interventions are discussed, namely: I an integrated energy-pinch of condensers and reboilers in the bio-ethanol distillation train; (ii) the use of Very High Gravity (VHG) fermentation; (iii) the development of hybrid processes using pervaporation membranes; (iv) the replacement of current ethanol dewatering processes to >99.5 wt.% pure ethanol by membrane technology; and (v) new distillation ideas or other advancements to enhance plant operation, like employing microfiltration of fermenter broth to prevent fouling of heat exchangers and distillation columns [11]. By adopting Very High Gravity (VHG) ethanol fermentation, energy is saved for ethanol distillation. The system's high ethanol yield, low waste generation, and low running costs may make it more efficient than existing ethanol production techniques on an industrial scale. The production of ethanol in a 1.5 L fed-batch bioreactor was subjected [12]. The primary benefit of this method is the up to 15% (v/v) rise in ethanol concentration, which results in a 5.4 L vinasse/L ethanol vinasse volume reduction [13]. Due to physiologic alterations in the microbial cells, ethanol is seldom produced in full in VHG fermentations. It has been common practice to produce industrial ethanol using Saccharomyces cerevisiae. It is recognized as a harmless microorganism that, in VHG conditions, may generate up to 20% (v/v) of ethanol [14]. Samples of S. cerevisiae cells Ultra-high temperature (UHT-6 and UHT-7) formed more moderately complex protein aggregates when the ethanol level was further increased, and the pH decreased, but the network became porous and weaker when the water capacity decreased [15-17]. An innovative element of some studies was the investigation of the effect of wort color modulated by dark special malts on the process and efficiency of fermentation, the characterization of yeast biomass after fermentation of strains with different characteristics [18,19]. The deacetylation pretreatment method during acetic acid was specifically eliminated by mild alkaline before acid pretreatment can improve the yield of ethanol production. It was demonstrated that ideal conditions would reduce the loss of fermentable sugar. Some studies suggested that the removal of acetic acid from hydrolysates through the deacetylation process can increase ethanol yield as well as efficiency. They also suggest that an acetic acid bypass strategy is necessary to effectively utilize cellulosic biomass as a bio-based energy source [20]. Throughout the 2000s, the rate of ethanol production and consumption in the US increased quickly along with the adoption of several ethanol laws (both state and federal) [21-23]. The Renewable Fuel Standard (RFS) program was the main factor in the rise of the ethanol industry in the US. The RFS program was implemented during the main growth phase and was specially created to enhance ethanol production and consumption which contributed to the ten-fold increase in ethanol production and consumption which modified gasoline and ethanol rating in the US from 2019 to 2002, according to several investigations [24-26]. An ongoing challenge for the profitable development of renewable transportation fuels has been the economical manufacture of ethanol from cellulosic biomass [27-29]. Since it can reduce investment costs, attenuate end-product inhibition on enzymatic hydrolysis of cellulosic biomass and minimize sugar loss while the separation and purification process, simultaneous Saccharification and Fermentation (SSF), which a potential method for turning lignocellulosic resources into ethanol all these factors has been recommended for the synthesis of cellulosic ethanol [30-32].

But still the ultimate ethanol concentration in the fermentation broth must be high, as well as the overall ethanol production. Thus, practically total conversion of both hexoses and pentoses at a high solid content is required in SSF was studied [33]. The engineered strain without supplementation of β-glucosidase showed almost the same or even better ethanol productivity than the parental strain with supplementation of β-glucosidase when initial cell mass concentrations were elevated. Ethanol production by SSF could be achieved by engineered yeast capable of fermenting cellobiose without addition of extracellular β-glucosidase, leading to economic production of cellulosic ethanol [34]. Simultaneous Saccharification and Fermentation (SSF) is an efficient process for producing cellulosic ethanol because it avoids end-product inhibition of cellulases through the immediate consumption of glucose by the fermenting microorganism [30].

One of the most significant biotechnological chemicals, ethanol is utilized extensively in a variety of industries including cosmetics, food and fuel, medicine, and pharmacology. Using baker's yeast Saccharomyces cerevisiae alcohol is fermented to produce ethanol as the primary process [35]. Glucose is converted into ethanol by S. cerevisiae very well; the amount of ethanol produced is greater than 90% of the theoretical maximum. But a little boost in ethanol output during an industrial-scale alcoholic fermentation can result in an extra 100 million t of ethanol being produced annually [36]. Yeast cells were subjected to long-term culture on the medium with high concentrations of glucose and ethanol in order to boost the production of ethanol with industrial S. cerevisiae strains. In compared to the original strains, most of the modified strains were distinguished by higher ethanol production during alcoholic fermentation [37,38]. The direct production of ethanol utilizing enzyme saccharification and fermentation without chemical liquefaction (pretreatment) of agarose is described for the first time as a single process [39,40]. Simultaneous saccharification and fermentation with Saccharomyces cerevisiae yeast that secretes a range of lignocellulolytic enzymes might addressed, ideally leading to consolidated bioprocessing. However, it has been unclear how many enzymes can be secreted simultaneously with the sugar fermentation performance and robustness of the second-generation yeast strain. Neither glucose nor the engineered xylose fermentation was significantly affected by the heterologous enzyme secretion [41]. The process of saccharification, followed by microbial fermentation and product recovery, is used to create second-generation bioethanol from lignocellulosic feedstock. One such renewable and abundant source of lignocellulose-rich biomass for the synthesis of bioethanol is agricultural leftovers produced as wastes during or after the processing of agricultural products [42-44].

Pretreatment processes are generally grouped into four categories, namely chemical, physical, biological, and physicochemical pretreatment [45,46]. Each pretreatment technique affects different properties, including chemical and physical properties. The effects of these pretreatments are demonstrated in various lignocellulosic biomasses, which could be helpful for their application in the biorefining process [47]. Among the various pretreatment processes, the most widely used process is chemical pretreatment because of its ability to alter the biopolymeric conformation of the biomass and its uncomplicated application compared to other pretreatment methods [47]. Chemical pretreatment helps in removing the chemical linkages as associated with three biomolecules together that it is facilitating further processes [48]. Various existing chemical pretreatment methods, including alkaline, acid, deep eutectic solvent extraction, and ionic liquid, was previously investigated to enhance sugar production and bioethanol production [49]. However, their application on an industrial scale is limited due to the investment cost. Pretreatment and hydrolysis processes are considered a high cost and time investment from an industrial point. Hence, it is preferable to select a potential pretreatment method, which can reduce the total cost for its industrial application [50,51]. Among the various types of chemical pretreatment techniques, a widely advanced technique is ionic liquid pretreatment, which has the potential to be recycled and reused [52-55].

Important genetics factors on ethanol production by Saccharomyces cerevisiae

The first insight into the unique genomic background of an industrial yeast-type strain regarding to chemical stress tolerance in comparison to a model reference genome. The development of next-generation biocatalysts for the manufacture of advanced biofuels has two significant technical challenges: enhanced stress tolerance, particularly inhibitor tolerance, and effective and balanced utilization of C-5 and C-6 biomass sugars employing lignocellulosic hydrolyzes. Due to their dissimilar genetic backgrounds, model lab strains and industrial yeast strains provide inconsistent phenotypic outcomes, which makes it difficult to address these technical issues efficiently [56]. Using global gene transcriptional analysis and functional analysis, new research has recently been conducted on the biochemical and molecular pathways underpinning yeast ethanol tolerance. Novel genetic and metabolic factors linked to ethanol tolerance have also been discovered. By overexpressing or disrupting the necessary genes, some researches have successfully produced new yeast strains with improved ethanol tolerance genetic engineering methods to improve yeast ethanol tolerance were discussed together with recent developments in the study of the molecular basis of yeast ethanol tolerance [57]. By increasing the production of heat shock-like proteins, decreasing the rate of RNA and protein accumulation, raising the incidence of small mutations, altering metabolism, denaturing intracellular proteins, and decreasing the activity of glycolytic enzymes, ethanol also affects cell metabolism and macromolecular biosynthetic pathway [58].

Brewer's yeast Saccharomyces cerevisiae is frequently employed in the commercial manufacture of ethanol and alcoholic drinks because it has a significantly greater ethanol tolerance than most other species. It has been difficult to comprehend the genetic mechanisms driving the high ethanol tolerance of this yeast. The multiple methods and challenges involved in creating improved industrial yeasts with higher ethanol tolerance are outlined in the next section [59].

Some studies the many strains enhancement and screening methods available for both common and uncommon yeasts. Superior industrial yeasts have been chosen by utilizing the natural diversity already present, creating artificial diversity through methods like mutagenesis, protoplast fusion, breeding, genome shuffling, and directed evolution, or by using genetic modification techniques to modify traits more specifically. The discovery of high-throughput methods to generate genetic variation, such as "global Transcription Machinery Engineering" (gTME), made it possible to create new sources of genetic diversity in yeast [60]. Recently, recombinant cellulases were genetically altered in Saccharomyces cerevisiae and numerous other yeasts to express them in media or show them on the cell surface. They change that may be made to a particular protein or to specific pathways that increase Saccharomyces cerevisiae's tolerance to ethanol and its ability to ferment alcohol more effectively [61,62]. An ecologically and industrially important characteristic of microorganisms is tolerance to high ethanol concentrations, but the molecular processes behind this complicated feature are still largely understood. They investigate adaptability to rising ethanol concentrations using long-term experimental evolution of isogenic yeast populations of various beginning ploidies. Whole-genome sequencing of these populations and clones showed how a complex interaction of clonal interference, copy number variation, ploidy changes, mutator phenotypes, and de novo single nucleotide mutations resulted in a significant increase in ethanol tolerance [63,64].

Increased production of ethanol by mutation: Physical and chemical mutagens methods is classical genetics methods used for yeast strain improvement [65]. Gene modification by mutations can generate new genetic modified characters important in industry after detecting and screening of desired important productivity [66]. Physical mutations in yeast can be induced by exposure to physical agents, such as heat or electrical shock. These mutations can result in changes to the DNA sequence or chromosomal rearrangements. Physical mutagens that are commonly used to induce mutations in yeast include X-rays and gamma rays [67].

Both chemical and physical mutations in yeast can be beneficial or detrimental to the organism, depending on the specific mutation and its effect on gene expression and phenotype. Researchers use yeast as a model organism to study the effects of mutations on gene expression, protein function, and cellular processes [68]. Yeast is a single-celled organism that is commonly used as a model organism in genetics research. Yeast can undergo both chemical and physical mutations, which can lead to changes in its genetic information and phenotype [69]. The development of Very High-Gravity (VHG) fermentation (fermentation at high sugar levels) for the manufacture of ethanol was made possible with the help of S. cerevisiae Ethidium Bromide (EtB) mutagenesis. According to random amplified polymorphic DNA analysis, this study discovered two evolved mutants of S. cerevisiae (EtB20a and EtB20b) with varying capacities for ethanol synthesis utilizing EtB [70]. Chemical mutations in yeast can be induced by exposure to mutagenic agents, such as chemicals or radiation. These mutations can result in changes to the DNA sequence, including point mutations (changes to a single nucleotide), deletions, and insertions. Chemical mutagens that are commonly used to induce mutations in yeast include Ethyl Methane Sulfonate (EMS), nitrogen mustard, and UV light [71]. In addition to being a valuable tool for genetic research, yeast is also widely used in the biotechnology industry for the production of various products, such as bread, beer, and biofuels. Understanding the mechanisms and consequences of chemical and physical mutations in yeast is important for both basic research and applied biotechnology [36]. Ethanol is a versatile substance with a wide range of applications that may be produced spontaneously by yeast that ferments sugar, such as Saccharomyces cerevisiae [3]. However, because ethanol is poisonous to yeast, we sought to increase yeast ethanol tolerance in some work. This might lead to higher production efficiency for alcohol [72]. Some other studies investigate the impact of using Ethyl Methane Sulfonate (EMS) to intentionally induce population-level variation on improving S. cerevisiae's tolerance to ethanol. The two treatment populations (AS-Artificial Selection only; and EMS-Exposure to EMS plus Artificial Selection) were compared to the initial parental population after numerous rounds of selection and increasing ethanol concentrations, ranging from 9-27% [73]. The parental strain, the selection strain, and the EMS-exposed strain were each independently plated on ten 27% ethanol plates and ten 0% ethanol plates, and growth was observed after 24 hours to test for strain differences. Only the EMS-exposed strain grew at 27% ethanol; all other strains grew at 0% ethanol. These findings suggest that EMS, artificial selection, and ethanol as a stressor may be useful in creating S. cerevisiae strains that can generate more ethanol before toxicity sets in Kadeba and Wilgers [73].

The application of a modern computational pipeline, phenetic, demonstrated that many mutations target functional modules involved in stress response, cell cycle control, DNA repair and respiration, even though the mutations vary throughout different evolutionary lineages. In non-evolved ethanol-sensitive cells, measuring the fitness impacts of chosen mutations led to the discovery of other adaptive mutations that were previously unrelated to ethanol tolerance, including mutations in PRT1, VPS70, and MEX67 [63,74].

The outcomes demonstrated that the advanced oxidative processes AOPs could eliminate aromatic substances) without having an impact on the hydrolysate's sugar content. The most efficient methods for hydrolysate detoxification were ozonation in an alkaline medium (pH 8) using H₂O₂ (treatment A3) or UV radiation (treatment A5). These methods also had a favourable impact on the hydrolysate's capacity to be fermented by yeast. Under these circumstances, it was shown that the removal of total phenols (above 40%), low molecular weight phenolic compounds (above 95%), and furans (above 52%) was greater. Additionally, compared to the untreated hydrolysate, P. stipites' ethanol volumetric productivity rose by around two-fold. These results demonstrate that AOPs are promising methods to reduce toxicity and improve the fermentability of lignocellulosic hydrolysates [75].

The effects of genome shuffling and gene cloning in ethanol production: Ethanol is produced during the fermentation of sugars by Kluyveromyces fragilis, Candida species, and S. cerevisiae in the case of hexoses and lactose or pentose. It is possible to produce ethanol at a rate of about 120 g/L under ideal circumstances. The fermentation stops when the concentration is this high because it slows down growth. S. cerevisiae fusion library formed by genome shuffling was examined for growth at 35, 40, 45, 50 and 55°C on agar plates with varied concentrations of ethanol [76]. After three rounds of genome shuffles, a strain was produced that could grow on plates up to 55°C, fully use 20% (w/v) glucose at 45-48°C, produce 99 g/L ethanol and tolerate 25% (v/v) ethanol stress. In silico metabolic models have been used to address the redox imbalance in S. cerevisiae that has the Xyl1 and Xyl2 genes from Pichia stripes [77,78]. Both genes were overexpressed, resulting in an overabundance of NADH and a deficiency in NADPH. Using xylose as the fermentation substrate, ethanol production increased with NADP+-dependent Glutamate Dehydrogenase (GGH1) deletion and the overexpression of NAD+-dependent GDH2. The byproducts glycerol and xylitol generation were reduced while ethanol production was increased using an in-silico genome-scale gene insertion method [79]. When glyceraldehyde-3-phosphate dehydrogenase was added to S. cerevisiae, glycerol synthesis dropped by 58%, xylitol production dropped by 33%, and ethanol production rose by 24%. Acetic acid is released when biomass is broken down and utilized as a carbon source for the production of ethanol. The generation of ethanol is hampered by the acid. Genome shuffles increased Candida krusei GL560's acetic acid tolerance [80]. After four cycles, a mutant was identified and chosen because it was more viable in acetic acid-containing medium than the parent strain. The mutant also increased its resistance to many stresses, including heat, alcohol, H2O2 and freeze-thaw. By cloning the alcohol dehydrogenase II and pyruvate decarboxylase genes from Zygomas mobiles, E. coli was transformed into an ethanol producer (43 g/L) [81]. The same two genes were cloned and expressed in Klebsiella oxytocic, and the recombinant efficiently converted crystalline cellulose to ethanol when fungal cellulases were introduced [82]. The maximum theoretical yield was between 81 and 86%, and from 100 g/L of cellulose, 47 g/L of ethanol was generated. With yields of 0.41-0.50 ethanol per gram of ingested sugar, recombinant strains of Saccharomyces, Zymomonas and E. coli can convert maize fibre hydrolysate to 21-35 g/L ethanol [83], it took 55 hours and 0.46 g of ethanol per gram of accessible sugar for a recombinant E. coli strain to produce 35 g/L, which is 90% of the theoretical limit [84-86].

Conclusion

The ability of ethanol production by yeast strain has been identified all over the world. Some wild-type yeast strains produce ethanol from different types of feedstocks depending on the fermentation process. Genetic methods also give several advantages in ethanol production by yeast strain. This review article concluded several types of ethanol fuel production using wild-type and engineered Saccharomyces cerevisiae strains, which are the most common microorganisms in bioethanol production, and it, can be achieved through microbial breeding. By using the low-cost lignocellulosic biomass resource that is used as raw materials which include cellulose, hemicellulose and lignin structural elements. Second-generation feedstocks have several benefits over first-generation substrates derived from food crops, which are often employed as food sources and as a result, suffer from the food versus fuel debate as well as from relatively high prices. Yeast can undergo both chemical and physical mutations, which can lead to changes in its genetic information and phenotype. In the case of yeast fermentation, it was possible to make mutagenesis with increased ethanol tolerance. The mutants will be tested by chemical and physical methods. Both chemical and physical mutations in yeast can be beneficial or detrimental to the organism, depending on the specific mutation and its effect on gene expression and phenotype. Researchers use yeast as a model organism to study the effects of mutations on gene expression, protein function, and cellular processes.

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