Bioethanol Production Research Articles

Phosphate-type zwitterionic liquid.

The imidazolium/phosphate zwitterionic liquid synthesised in this study dissolved cellulose and satisfies all other properties required for efficient cellulosic bioethanol production such as liquid at room temperature and low toxicity.

Enhancing bioethanol conversion from straw by a novel circulation promotion method

Crop straw, an inedible and abundant agricultural by-product, can be used for bioethanol production. However, ethanol conversion from straw is a series of complex processes and has encountered issues, which cause considerable restriction to the production of straw ethanol. Herein, the bioethanol conversion process was innovated. Ethanol as a reverse osmosis membrane accelerant was investigated to enhance the removal of inhibitors to increase ethanol production during conversion process. The results indicated that membrane accelerant used here could reduce the inhibitor retentions by 39.3 %–69.2 %. Then, an original innovation integrated method for the whole process of straw ethanol conversion was developed. The integrated method could increase 26.8 % the production of straw ethanol compared to the membrane process without an accelerant. A preliminary cost accounting indicated that the integrated method was economically feasible when total ethanol output exceeded 14000 kg. This study represents for the first time a circulation promotion method based on the whole process of straw ethanol conversion. The results showed that the integrated method was capable of effective conversion of straw ethanol.

Sustainable waste management: a comprehensive life cycle assessment of bioethanol production from agricultural and municipal waste.

Biofuels have emerged as a promising and eco-friendly alternative to conventional fossil fuels. Biofuel sourced from rice straw (RS) and municipal solid waste (MSW), which are abundant residues from agricultural and municipal activities, present a sustainable solution to address waste management challenges. Utilizing life cycle assessment, this study quantifies the environmental advantages by assessing the reduction in greenhouse gas emissions, energy consumption, and other environmental impacts linked with employing these waste materials for biofuel production. Employing a cradle-to-gate approach as the system boundary for bioethanol production, with the functional unit set as per liter of bioethanol produced, the analysis reveals that the global warming potential (GWP) for ethanol from MSW is 4.4 kg CO2 eq., whereas for RS, it is 2.1 kg CO2 eq. per functional unit. The total environmental impacts were primarily due to enzymatic hydrolysis and electricity consumption for ethanol production from MSW and RS. Despite advancements, fossil fuel consumption remains a potential energy source for biofuel production. The cumulative energy demand stands at 18.6 MJ for RS and 71.5 MJ for MSW per functional unit, underscoring the potential to significantly reduce overall impacts by transitioning to a more environmentally sustainable energy source. The uncertainty analysis acknowledges the inherent uncertainties associated with data, assumptions, and methodologies, highlighting the crucial need for ongoing research and updates to enhance the accuracy of future assessments. This analysis forms the foundation for well-informed decision-making, providing valuable insights for policymakers, industry stakeholders, and consumers.

Construction of an amylolytic Saccharomyces cerevisiae strain with high copies of α-amylase and glucoamylase genes integration for bioethanol production from sweet potato residue.

Sweet potato residue (SPR) is the by-product of starch extraction from fresh sweet potatoes and is rich in carbohydrates, making it a suitable substrate for bioethanol production. An amylolytic industrial yeast strain with co-expressing α-amylase and glucoamylase genes would combine enzyme production, SPR hydrolysis, and glucose fermentation into a one-step process. This consolidated bioprocessing (CBP) shows great application potential in the economic production of bioethanol. In this study, a convenient heterologous gene integration method was developed. Eight copies of a Talaromyces emersonii α-amylase expression cassette and eight copies of a Saccharomycopsis fibuligera glucoamylase expression cassette were integrated into the genome of industrial diploid Saccharomyces cerevisiae strain 1974. The resulting recombinant strains exhibited clear transparent zones in the iodine starch plates, and SDS-PAGE analysis indicated that α-amylase and glucoamylase were secreted into the culture medium. Enzymatic activity analysis demonstrated that the optimal temperature for α-amylase and glucoamylase was 60-70°C, and the pH optima for α-amylase and glucoamylase was 4.0 and 5.0, respectively. Initially, soluble corn starch with a concentration of 100 g/L was initially used to evaluate the ethanol production capability of recombinant amylolytic S. cerevisiae strains. After 7 days of CBP fermentation, the α-amylase-expressing strain 1974-temA and the glucoamylase-expressing strain 1974-GA produced 33.03 and 28.37 g/L ethanol, respectively. However, the 1974-GA-temA strain, which expressed α-amylase and glucoamylase, produced 42.22 g/L ethanol, corresponding to 70.59% of the theoretical yield. Subsequently, fermentation was conducted using the amylolytic strain 1974-GA-temA without the addition of exogenous α-amylase and glucoamylase, which resulted in the production of 32.15 g/L ethanol with an ethanol yield of 0.30 g/g. The addition of 20% glucoamylase (60 U/g SPR) increased ethanol concentration to 50.55 g/L, corresponding to a theoretical yield of 93.23%, which was comparable to the ethanol production observed with the addition of 100% α-amylase and glucoamylase. The recombinant amylolytic strains constructed in this study will facilitate the advancement of CBP fermentation of SPR for the production of bioethanol.

Integrated choline chloride/citric acid-microwave pretreatment for efficient nanolignin extraction and bioethanol production from cocoa pod husk waste

The necessity to address environmental issues has driven efforts toward exploring sustainable bio-based materials as a viable alternative to conventional energy sources. The current study explores the utilization of cocoa pod husk (CPH) biomass for lignin extraction and cellulose production, aiming to contribute to the eco-friendly production of lignin nanoparticles and bioethanol. A synergistic green deep eutectic solvent (DES) (choline chloride/citric acid)-microwave method was employed to effectively fractionate CPH biomass, resulting in an impressive 77.58% lignin removal at 600 W microwave power. The extracted lignin (211.56 mg/g biomass) was utilized to synthesize lignin nanoparticles that were subsequently characterized. Enzyme-driven hydrolysis of the residual cellulose yielded a reducing sugar content of 198.34 mg/g biomass, demonstrating a saccharification efficiency of 70.78%. Fermentation of monomeric sugars by Saccharomyces cerevisiae and Scheffersomyces stipitis, respectively, gave a maximum ethanol yield of 130 mg/g biomass with a high fermentation efficiency (67.17%). The alterations in the CPH's surface structure and morphology following sequential pretreatment were assessed through FT-IR, BET, and SEM analyses, facilitating effective enzymatic hydrolysis. The current investigation adds to the increasing recognition of sustainable approaches in leveraging waste biomass resources toward a more environmentally conscious future.

Application of In-House Xylanases as an Addition to a Commercial Cellulase Cocktail for the Sustainable Saccharification of Pretreated Blue Agave Bagasse Used for Bioethanol Production

The study involves the use of commercial cellulase Cellic CTec2 in combination with two in-house xylanases, PxXyn10A (XynA), a recombinant purified enzyme from Paenibacillus xylanivorans A59, and a xylanase enzymatic extract from native Moesziomyces aphidis PYCC 5535T (MaPYCC 5535T), for the enzymatic hydrolysis of pretreated blue agave bagasse (BAB) at the high solids load of 20% (w/v). Three different combinations of cellulase and xylanases were evaluated. When Cellic® CTec2 was used at a dosage of 10 FPU/g oven-dried solids (ODS) supplemented with XynA or MaPYCC 5535T at an endo-xylanase dosage of 100 U/g ODS, increases in the xylose yield of 30% and 33%, respectively, were obtained. When applying in-house xylanases alone (at an endo-xylanase dosage of 100 U/g ODS), xylan in BAB was selectively hydrolyzed into xylose with 5% yield with MaPYCC 5535T, while no xylose was detected with XynA. Interestingly, a synergic effect of Cellic® CTec 2 with both xylanases was observed when using a low dosage of 1 FPU/g ODS (allowing for some liquefaction of the reaction mixture), promoting xylose and glucose release by either xylanase. A higher concentration of monomeric sugars was obtained with 10 FPU/g ODS of Cellic® Ctec 2 supplemented with 100 U/g ODS of MaPYCC 5535T, followed by XynA. The improvement in saccharification through the synergistic combination of in-house xylanases and commercial cellulases allows for the obtention of sugar-rich hydrolysates, which enhances the technical sustainability of the process. Hydrolysates were then fermented using recombinant Cellux 4TM yeast to yield 45 g/L ethanol, representing an increase of about 30% with respect to the control obtained with only the commercial cellulase cocktail. The surface modification of agave biomass with the different combinations of enzymes was evidenced by scanning electron microscopy (SEM).

Upgraded cellulose and xylan digestions for synergistic enhancements of biomass enzymatic saccharification and bioethanol conversion using engineered Trichoderma reesei strains overproducing mushroom LeGH7 enzyme

Crop straws provide enormous lignocellulose resources transformable for sustainable biofuels and valuable bioproducts. However, lignocellulose recalcitrance basically restricts essential biomass enzymatic saccharification at large scale. In this study, the mushroom-derived cellobiohydrolase (LeGH7) was introduced into Trichoderma reesei (Rut-C30) to generate two desirable strains, namely GH7–5 and GH7–6. Compared to the Rut-C30 strain, both engineered strains exhibited significantly enhanced enzymatic activities, with β-glucosidases, endocellulases, cellobiohydrolases, and xylanase activities increasing by 113 %, 140 %, 241 %, and 196 %, respectively. By performing steam explosion and mild alkali pretreatments with mature straws of five bioenergy crops, diverse lignocellulose substrates were effectively digested by the crude enzymes secreted from the engineered strains, leading to the high-yield hexoses released for bioethanol production. Notably, the LeGH7 enzyme purified from engineered strain enabled to act as multiple cellulases and xylanase at higher activities, interpreting how synergistic enhancement of enzymatic saccharification was achieved for distinct lignocellulose substrates in major bioenergy crops. Therefore, this study has identified a novel enzyme that is active for simultaneous hydrolyses of cellulose and xylan, providing an applicable strategy for high biomass enzymatic saccharification and bioethanol conversion in bioenergy crops.

Optimizing bioethanol production from sweet sorghum stem juice under very high gravity fermentation and temperature stress conditions

This study optimized ethanol production from sweet sorghum stem juice (SSJ) by Saccharomyces cerevisiae NP01 under very high gravity (VHG) fermentation in 500-mL air–locked flasks at 30 °C. Response surface methodology based on a Box-Behnken design was employed to optimize initial sugar (267 g/L), urea (3.24 g/L), and cell concentration (1.32 × 108cells/mL) for maximization of ethanol concentration (PE), productivity (QP), and sugar consumption (%SC). The experimental values (PE, 119.29 g/L; QP, 2.49 g/L.h and %SC,91.83 %) under optimal conditions were close to the predicted values, verifying the optimization process. Aeration (2.5 vvm for 4 h) increased viable cell counts and decreased glycerol production (a by-product), but not fermentation efficiency. An osmoprotectant (40 mM potassium chloride combined with 10 mM potassium hydroxide, KCl/KOH) at 30 °C had no positive effect on ethanol fermentation efficiency. However, at 25 °C, the osmoprotectant increased PE from 106 to 116 g/L and ethanol yield from 0.46 to 0.49 g/g. At 35–37 °C, it prolonged cell viability, increasing PE by 5–12 g/L and %SC by 3–8 % without affecting ethanol yield. However, at 39 °C, no positive impact occurred on ethanol fermentation efficiency. The findings from this study, particularly the optimized fermentation conditions and stress tolerance strategies, could guide the scale-up to an industrial level of bioethanol production from sweet sorghum stem juice or other feedstocks using VHG fermentation, contributing to the development of more efficient and sustainable biofuel production processes.

Preparation of Bioethanol from Pineapple Peel Waste for Blending Pertalite into Alternative Fuel (Gasohol)

This study aims to obtain bioethanol according to the Indonesian National Standard (SNI) 7390:2012, obtain Gasohol according to the RON (Research Octane Number) standard in Pertalite, and produce alternative fuels that are more environmentally friendly. The bioethanol production process includes hydrolysis, fermentation, distillation, and adsorption, with Saccharomyces cerevisiae to ferment sugar in pineapple skin into ethanol with a content of 59.62% from a 5-day fermentation process with 4% Saccharomyces cerevisiae, 0.5% urea, 0.5% NPK. Bioethanol is then mixed with Pertalite in the composition of E5 (5 ml of bioethanol mixed with 95 ml of Pertalite) to E25 (25 ml of bioethanol mixed with 75 ml of Pertalite), lowering the flash point of the mixture from 29.8°C (E5) to 28.0°C (E25), increasing the density from 0.7239 gr.(cm3)-1 (E5) to 0.7250 gr.(cm3)-1 (E25) and the viscosity from 0.41 cSt (E5) to 0.49 cSt (E25). Still, the octane number (RON) tends to be stable at 91.4-95.6. As a result, the bioethanol content is close to SNI 99.5%, the bioethanol-Pertalite mixture improves several parameters but lowers the flash point, and the E25 mixture meets the RON standard of 95.6 for Pertalite.

Bioethanol derived from rice straw agricultural waste as alternative energy towards a pollution-free era

Abstract Rice production occupies the first position as raw material for bioethanol production, with a total output throughout Indonesia of 56.63 million tons in 2023 (BPS, 2023). The purpose of this innovation is to create bioethanol derived from new raw materials, namely rice straw, by applying the simultaneous saccharification and fermentation (SSF) method for the conversion of lignocellulose into alcohol, which will produce bioethanol with a purity level of about 99% with a more efficient process. This method goes through several processes, including breaking the size of the rice straw to 0.1 mm and mixing it with several bacteria for fermentation and hydrolysis. The saccharification process is also carried out until the distillation process to make high levels of bioethanol. Bioethanol from the simultaneous saccharification and fermentation (SSF) method is tested using an Octane Number Analyzer to determine the octane number content. The test included mixing bioethanol with Pertamax 92 to prove the effect of adding bioethanol to Pertamax, and the result was 93.1. Therefore, the innovation of making bioethanol from rice straw with the SSF method is expected to be a solution for Indonesia towards a pollution-free era.

Use of spent yeasts from bioethanol production plant as low-cost nitrogen sources for ethanol fermentation from sweet sorghum stem juice in low-cost bioreactors

Two spent yeasts from an ethanol production plant, spent yeast after distillation (SY-AD) and spent yeast after fermentation (SY-AF), were used as low-cost nitrogen sources for ethanol fermentation from sweet sorghum stem juice (SSJ) by a commercial dry yeast (Saccharomyces cerevisiae) in air-locked flasks. SY-AF was the more effective nitrogen source for ethanol fermentation, giving ethanol concentration (PE) and ethanol productivity (QE) values of 95.22 g/L and 1.98 g/L·h, respectively. When SY-AF was disrupted by autolysis, and the spent yeast hydrolysate (SYH) obtained was used as a nitrogen supplement. It was found that ethanol production in terms of PE and QE values increased to 102.20 g/L and 2.83 g/L·h, respectively. When three bioreactors, a stirred-tank bioreactor (STR, a typical bioreactor), a column bioreactor with stirrer (CS-R, a tower bioreactor) and an external loop bioreactor (ELR, a low-cost bioreactor with no agitation), were used for ethanol production from the SSJ supplemented with SYH, the fermentation efficiencies of all bioreactors were not different. Appropriate aeration during fermentation (0.31 vvm for 12 h) in the three bioreactors could enhance the QE value, reaching 3.36 g/L·h. Both the CR-S and ELR could be successfully used for ethanol production from SSJ supplemented with SYH.

Design and fabrication of a 20 kilogram capacity cassava-based bioethanol distillation apparatus with 2.4 liter bioethanol output

Background: The depletion of fossil fuel resources and growing environmental concerns have sparked interest in renewable energy sources like bioethanol. Cassava, an abundant crop in many tropical regions, shows promise as a feedstock for bioethanol production. This study aimed to design and fabricate an efficient small-scale distillation apparatus for producing bioethanol from cassava. Method: A distillation apparatus with 20 kg cassava capacity was designed and constructed using locally available materials. Key components included a distillation tank, condenser, cooling tower system, and burner. The apparatus was tested using fermented cassava mash to evaluate its performance in bioethanol production. Findings: The fabricated apparatus successfully produced 2.4 liters of bioethanol with 65% purity from 20 kg of cassava feedstock. Optimal distillation temperature was found to be 70°C, balancing ethanol yield and purity. Heat transfer calculations indicated 576 kW of cooling capacity was required in the condenser. The cooling tower system achieved 63% thermal efficiency. Conclusion: The designed distillation apparatus demonstrates the feasibility of small-scale bioethanol production from cassava. Further optimization of the distillation process and heat recovery systems could improve efficiency. This technology shows potential for decentralized biofuel production to meet local energy needs in cassava-producing regions. Novelty/Originality of this study: The study on designing small-scale distillation equipment for bioethanol production from cassava successfully demonstrated practical and affordable applications for decentralized biofuel production in cassava-producing areas.

Lignocellulosic Biomass for Bioethanol Recovery: A way towards Environmental Sustainability

The increasing population and industrialization have increased the day to day demand of energy. Global warming and other climatic disasters caused due to burning of conventional fossil fuels have laid the need for an alternative and renewable fuel. Concurrently, the worldwide demand and production of bio-ethanol, a bio-based sustainable fuel is increasing continuously. Fungal saccharification of lignocellulosic biomass takes place simultaneously with the secretion of various metabolites, which function as a catalytic system to liberate soluble sugars from insoluble composite biomass. Cellulase holds a major role for converting lignocellulosic feedstock into fermentable sugar. Fungi, especially filamentous fungi preferably the Aspergillus species are known to produce cellulase. Amongst the lignocellulosic biomass, rice straw is considered as one of the most attractive materials for producing bioethanol because of its high cellulosic and hemicellulosic content, which can be hydrolyzed into fermentable sugars. However, the recalcitrant nature of the substrate imposes considerable challenges and limitations to bioethanol production. To combat these challenges, various pretreatment techniques such as chemical, physical, and physico-chemical processes are known to be effective enough to enhance the efficiency of enzymatic saccharification to make the complete process economically feasible. The present work deals with the isolation and selection of hypercellulase-producing fungal species. These species were used along with the pretreatment techniques either alone or in combination for efficient hydrolysis of rice straw to obtain the maximum amount of the released sugar.

Valorization of Cheese Whey through Bioethanol Production and Probiotic Drink Development: A Sustainable Approach for Organic Waste Management

Cheese whey, a significant byproduct of the dairy industry, poses a substantial environmental challenge due to its organic pollutant load and large size demands for effective and affordable valorization methods. The abundance of lactose and other organic compounds in whey contributes to its elevated Biological Oxygen Demand (BOD) and Chemical Oxygen Demand (COD), further straining natural ecosystems. This study aimed to investigate the nutritional potential of whey and its conversion into value-added products: bioethanol and probiotic drinks. To achieve this, Bacillus megaterium and Bacillus subtilis strains, isolated from soil samples in Nepal, were co-cultured with Lactobacillus sp. and Saccharomyces cerevisiae in the whey broth for fermentation. Ethanol distillation was done using a rotary evaporator to maintain the viability of the fermenting organisms in the broth. Moreover, the probiotic criteria of the fermenting strains were extensively examined. Experimental observations revealed a remarkable concentration of 9.2 g/L of ethanol, resulting in an ethanol yield of 0.23 g/L (42% compared to theoretical yield). Extrapolating this rate, it is theoretically possible to produce an annual global bioethanol output of 5 million tons using whey as a sustainable and abundant resource. Furthermore, the study explores novel advancements in the distillation process, demonstrating the successful extraction of ethanol at room temperature. Additionally, the fermentation organisms employed in this research exhibit robust viability, surviving various in-vitro stresstolerance tests, including temperature, bile salt, pH fluctuations, and gastric juice. These resilient strains also demonstrated long-term stability in fermented whey broth, making them promising candidates for the development of probiotic beverages. This dual approach enables sustainable management of organic waste and presents an opportunity to create value-added products with positive implications for the environment and human health.

Bioethanol: A Sustainable Liquid Fuel as Substitute to Gasoline

Abstract: Fossil fuel dependence is a growing concern due to its contribution to greenhouse gas emission, climatic change and environmental pollution. This highlights the urgency for alternative source of energy that is renewable, environmental friendly, stability in price, and attractive for sustainable development. Bioethanol, a biofuel has emerged as the most acceptable liquid fuel and as a promising alternative to gasoline. Bioethanol, derived from sugars and starch, has raised sustainability concern as it can lead to competition for land use and potentially driven-up food prices especially in developing countries. Meanwhile, Lignocellulosic biomass, a non-food resources, abundant in cellulose and hemicellulose, present a more sustainable feedstock for bioethanol production. This approach could offer advantages like affordability, environmental friendliness, reduce reliance on traditional fuels and compensate for fuel scarcity. Furthermore, bioconversion technology of lignocellulosic biomass to bioethanol is required to improve its efficiency and cost effectiveness, making it a highly attractive option for a greener energy in the future.

Cellular damage and response mechanisms of Candida tropicalis SHC-03 induced by toxic byproducts in corn stover hydrolysate

This study advances the understanding of cellular damage and response mechanisms in Candida tropicalis (SHC-03) when exposed to toxic byproducts in corn stover hydrolysate, which is used for optimizing the industrial production of bioethanol and bio-based products. We found that the hydrolysate's toxic byproducts led to 84.61% accumulation of reactive oxygen species and considerable mitochondrial damage, thus inhibiting SHC-03 cell growth by 40%. The yeast combated these effects by enhancing the glutathione and thioredoxin systems, and increased their activity by 60% and 70%, respectively, to maintain intracellular redox balance. The ubiquitin–proteasome pathway was involved and endoplasmic reticulum stress was alleviated, which increased membrane thickness through ergosterol biosynthesis and improved inhibitor tolerance via upregulated expression of transporters and aldehyde reductases. These adaptations, along with the overexpression of genes related to the biosynthesis of impaired proteins and fatty acid degradation, promote SHC-03's resilience to hydrolysate toxic byproducts. Our findings could be useful for genetic modifications to increase the tolerance of fermentation strains, which could accelerate the industrial production of bioethanol and bio-based products.

Harnessing artificial intelligence for enhanced bioethanol productions: a cutting-edge approach towards sustainable energy solution

Abstract The adoption of biofuels as an energy source has experienced a substantial increase, exceeding the consumption of fossil fuels. The shift can be ascribed to the availability of renewable resources for energy production and the ecological advantages linked to their utilisation. Nevertheless, due to its intricate characteristics, the process of producing ethanol fuel from biomass poses difficulties in terms of administration, enhancement, and forecasting future results. To tackle these difficulties, it is crucial to utilise modelling techniques like artificial intelligence (AI) to create, oversee, and improve bioethanol production procedures. Artificial Neural Networks (ANN) is a prominent AI technique that offers significant advantages for modelling bioethanol production systems’ pretreatment, fermentation, and conversion stages. They are highly flexible and accurate, making them particularly well-suited. This study thoroughly examines several artificial intelligence techniques used in bioethanol production, specifically focusing on research published in the past ten years. The analysis emphasises the importance of using AI methods to address the complexities of bioethanol production and shows their role in enhancing efficiency and sustainability in the biofuel industry.

Utilization of local corn (Zea Mays) wastes for bioethanol production by separate hydrolysis and fermentation

Maize is the second most-common crop globally. Thus, enormous maize cobs are generated annually from maize processing activities which could serve as a potential and non-edible source for biofuel production. The primary aim of this research was to assess the practicality of producing bioethanol from discarded maize cob through a distinct two-step process referred to as separate hydrolysis and co-fermentation. After a 72-hour fermentation period, the greatest ethanol yield of 66.23 ± 8.35 mL/kg was obtained. This was followed by 54.33 ± 7.27 35 mL/kg after 48 h, and 21.68 ± 2.97 35 mL/kg after 24 h. Importantly, all ethanol yields at different time points exhibited statistical significance at p < 0.05. Moreover, the study revealed a robust positive correlation (r = 0.99, p < 0.01) between glucose and Total reducing Sugars (TRS) yields, and negative correlations were observed between ethanol yield and glucose (r = -0.97, p < 0.05) as well as ethanol and TRS (r = -0.98, p < 0.05). The results indicate the potential of maize cob waste as a valuable resource for bioethanol production. Significant enhancements in operational processes are necessary to enhance the economic feasibility of producing ethanol from maize cobs. Nigeria's utilization of waste for biofuel production is bolstered by substantial policy and financial backing for renewable fuels. The economic viability of ethanol production from maize cobs relies heavily on its competitiveness relative to other waste treatment methods and the effectiveness of policy measures.

Harnessing Ulva ohnoi for eco-friendly bioethanol production via hydrothermal pretreatment

BackgroundThe increasing demand for renewable energy sources has sparked interest in biofuels derived from green seaweeds, owing to their abundance and high carbohydrate content. Ulva ohnoi, harvested from Taiwan's shores, presents a promising candidate for bioethanol production due to its favorable characteristics. MethodsIn this study, Ulva ohnoi underwent hydrothermal pretreatment at temperatures of 120 °C, 150 °C, and 180 °C for 20 min. The pretreated slurry obtained at 180 °C was subjected to enzymatic saccharification at pH 4.8 and 50 °C for 72 h. This process resulted in the production of 5.8 g/L of glucose, which was subsequently concentrated to 18.86 g/L. Significant FindingsFermentation of the concentrated sugar fractions using Saccharomyces cerevisiae WLP 300 at 30 °C for 24 h yielded remarkable ethanol yields of 0.39 and 0.43 g/g of sugar. These yields approached the maximum theoretical values of 76.1% and 84.58%, respectively. Additionally, the study reported a maximum ethanol productivity of 0.54 g/L/h in the 12th hour in the concentrated fraction. These findings underscore the potential of Ulva ohnoi as a sustainable biofuel and clean energy source, aligning with Sustainable Development Goal 7 (SDG 7) of ensuring access to affordable, reliable, sustainable, and modern energy for mankind.

Development of a Robust Saccharomyces cerevisiae Strain for Efficient Co-Fermentation of Mixed Sugars and Enhanced Inhibitor Tolerance through Protoplast Fusion.

The economical and efficient commercial production of second-generation bioethanol requires fermentation microorganisms capable of entirely and rapidly utilizing all sugars in lignocellulosic hydrolysates. In this study, we developed a recombinant Saccharomyces cerevisiae strain, BLH510, through protoplast fusion and metabolic engineering to enhance its ability to co-ferment glucose, xylose, cellobiose, and xylooligosaccharides while tolerating various inhibitors commonly found in lignocellulosic hydrolysates. The parental strains, LF1 and BLN26, were selected for their superior glucose/xylose co-fermentation capabilities and inhibitor tolerance, respectively. The fusion strain BLH510 demonstrated efficient utilization of mixed sugars and high ethanol yield under oxygen-limited conditions. Under low inoculum conditions, strain BLH510 could completely consume all four kinds of sugars in the medium within 84 h. The fermentation produced 33.96 g/L ethanol, achieving 84.3% of the theoretical ethanol yield. Despite the challenging presence of mixed inhibitors, BLH510 successfully metabolized all four sugars above after 120 h of fermentation, producing approximately 30 g/L ethanol and reaching 83% of the theoretical yield. Also, strain BLH510 exhibited increased intracellular trehalose content, particularly under conditions with mixed inhibitors, where the intracellular trehalose reached 239.3 mg/g yeast biomass. This elevated trehalose content contributes to the enhanced stress tolerance of BLH510. The study also optimized conditions for protoplast preparation and fusion, balancing high preparation efficiency and satisfactory regeneration efficiency. The results indicate that BLH510 is a promising candidate for industrial second-generation bioethanol production from lignocellulosic biomass, offering improved performance under challenging fermentation conditions. Our work demonstrates the potential of combining protoplast fusion and metabolic engineering to develop superior S. cerevisiae strains for lignocellulosic bioethanol production. This approach can also be extended to develop robust microbial platforms for producing a wide array of lignocellulosic biomass-based biochemicals.