Increase In Ethanol Yield Research Articles

Fermentation of Biomass and Residues from Brazilian Agriculture for 2G Bioethanol Production.

Brazil is one of the world's leading producers of staple foods and bioethanol. Lignocellulosic residual sources have been proposed as a promising feedstock for 2G bioethanol and to reduce competition between food and fuels. This work aims to discuss residual biomass from Brazilian agriculture as lignocellulosic feedstock for 2G bioethanol production as bagasse, stalk, stem, and peels, using biorefining concepts to increase ethanol yields. Herein, we focused on biomass chemical characteristics, pretreatment, microorganisms, and optimization of process parameters that define ethanol yields for bench-scale fermentation. Although several techniques, such as carbon capture, linking enzymes to supports, and a consortium of microorganisms, emerge as future alternatives in bioethanol synthesis, these technologies entail necessary optimization efforts before commercial availability. Overcoming these challenges is essential to linking technological innovation to synthesizing environmentally friendly fuels and searching other biomass wastes for 2G bioethanol to increase the biofuel industry's potential. Thus, this work is the first to discuss underutilized lignocellulosic feedstock from other agrifoods beyond sugar cane or corn, such as babassu, tobacco, cassava, orange, cotton, soybean, potatoes, and rice. Residual biomasses combined with optimized pretreatment and mixed fermentation increase hydrolysis efficiency, fermentation, and purification. Therefore, more than a product with a high added value, bioethanol synthesis from Brazilian residual biomass prevents waste production.

Adaptive evolution of Clostridium thermocellum ATCC 27405 on alternate carbon sources leads to altered fermentation profiles.

Consolidated bioprocessing candidate, Clostridium thermocellum, is a cellulose hydrolysis specialist, with the ability to ferment the released sugars to produce bioethanol. C. thermocellum is generally studied with model substrates Avicel and cellobiose to understand the metabolic pathway leading to ethanol. In the present study, adaptive laboratory evolution, allowing C.thermocellum DSM 1237 to adapt to growth on glucose, fructose, and sorbitol, with the prospect that some strains will adapt their metabolism to yield more ethanol. Adaptive growth on glucose and sorbitol resulted in an approximately 1mM and 2mM increase in ethanol yield per millimolar glucose equivalent, respectively, accompanied by a shift in the production of the other expected fermentation end products. The increase in ethanol yield observed for sorbitol adapted cells was due to the carbon source being more reduced compared to cellobiose. Glucose and cellobiose have similar oxidation states thus the increase in ethanol yield is due to the rerouting of electrons from other reduced metabolic products excluding H2 which did not decrease in yield. There was no increase in ethanol yield observed for fructose adapted cells, but there was an unanticipated elimination of formate production, also observed in sorbitol adapted cells suggesting that fructose has regulatory implications on formate production either at the transcription or protein level.

Bioethanol production from sugarcane molasses by co-fermentation of Saccharomyces cerevisiae isolate TA2 and Wickerhamomyces anomalus isolate HCJ2F-19

PurposeCo-culturing is a widely used method to improve bioethanol production from biomass enriched in fermentable sugars. This study aims to produce bioethanol from sugarcane molasses by simultaneous co-fermentation of S. cerevisiae isolate TA2 and W. anomalus isolate HCJ2F-19.MethodsResponse surface methodology (RSM) based on the central composite design (CCD) was employed to optimize fermentation conditions, including mixing rate (110–150 rpm), temperature (25–35 °C), molasses concentration (25–35 obrix), and incubation time (36–72 h). The ethanol concentration was analyzed using HPLC equipped with a UV detector.ResultsThe monocultureS. cerevisiae isolate TA2 produced 17.2 g.L−1 of ethanol, 0.33 g.g−1 of ethanol yield, and 0.36 g.L−1.h−1 of productivity compared to W. anomalus isolate HCJ2F that produced 14.5 g.L−1, 0.30 g.g−1 and 0.28 g.L−1.h−1 ethanol, ethanol yield, and productivity under laboratory conditions, respectively. In comparison to single cultures of S. cerevisiae TA2 and W. anomalus HCJ2F, the co-fermentation using both isolates showed an increased ethanol yield of 29% and 53% compared to the single species fermentations, respectively. The results showed that the growth of W. anomalus HCJ2F-19 and S. cerevisiae TA2 was not influenced by each other during the co-fermentation process. The one variable at a time optimization (OVAT) analysis resulted in an ethanol concentration of 26.5 g.L−1 with a specific yield and productivity of 0.46 g.g−1, 0.55 g.L−1.h−1, respectively, at pH 5.5, 25 obrix, 48 h, 150 rpm, 30 °C, 60:40 inoculum ratio, and 10% overall inoculum size. The maximum ethanol concentration of 35.5 g.L−1 was obtained by co-fermentation using the RSM-CCD tool at 30 obrix, 30 °C, 54 h, and 130 rpm.ConclusionThe results suggested that the co-fermentation of S. cerevisiae isolate TA2 and W. anomalus isolate HCJ2F improves bioethanol production from sugar cane molasses under optimum fermentation conditions.

Specific activity and utility of recombinant cellobiohydrolase II (Cel6A) produced in maize endosperm.

Cellobiohydrolase II (CBH II) is an exo-glucanase that is part of a fungal mixture of enzymes from a wood-rot fungus, Trichoderma reesei. It is therefore difficult to purify and to establish a specific activity assay. The gene for this enzyme, driven by the rice Os glutelin promoter, was transformed into High II tissue culture competent corn, and the enzyme accumulated in the endosperm of the seed. The transgenic line recovered from tissue culture was bred into male and female elite Stine inbred corn lines, stiff stalk 16083-025 (female) and Lancaster MSO411 (male), for future production in their hybrid. The enzyme increases its accumulation throughout its 6 generations of back crosses, 27-266-fold between T1 and T2, and 2-10-fold between T2 and T3 generations with lesser increases in T4-T6. The germplasm of the inbred lines replaces the tissue culture corn variety germplasm with each generation, with the ultimate goal of producing a high-yielding hybrid with the transgene. The CBH II enzyme was purified from T5 inbred male grain 10-fold to homogeneity with 47.5% recovery. The specific activity was determined to be 1.544 units per µg protein. The corn-derived CBH II works in biopolishing of cotton by removing surface fibers to improve dyeability and increasing glucose from corn flour for increasing ethanol yield from starch-based first-generation processes.

The promoting effect of electron beam irradiation on enzymatic saccharification and alcohol fermentation of sorghum meal: Related mechanisms

In this study, sorghum meal underwent 2–10 kGy electron beam irradiation (EBI) to investigate alterations in its elemental composition, relevant physicochemical characteristics, and enzymatic hydrolysis fermentation properties. The results indicated a decrease in amylose content and an increase in reducing sugar content in sorghum meal after EBI, along with severe damage to the internal structure of starch granules, leading to increased solubility in water. Furthermore, irradiation enhanced the shear resistance and gelatinization stability of sorghum meal paste. Most importantly, EBI-induced degradation of sorghum starch molecules enhanced their susceptibility to interact with α-amylase, significantly improving the enzymatic hydrolysis and alcohol fermentation efficiency of sorghum meal. At an irradiation dose of 8 kGy, raw and cooked sorghum meal exhibited a 43.40% and 33.33% increase in ethanol concentration and a 43.44% and 30.29% increase in ethanol yield, respectively. This finding indicated that irradiation is an effective pretreatment method for starch-based ethanol fermentation.

Comparative Assessment of Ethanol Production from Six Typical German Waste Baked Products

AbstractThis study investigates the potential for bioethanol production of six types of typical German leftover baked products: bread rolls, pretzel rolls, fine rye bread, white bread, pastry, and cream cakes. The experimental setup consisted of two experiments—one as a control and another with the addition of diammonium phosphate (DAP) to the mash. In terms of monosaccharide concentration at 30% dry matter (DM), white bread mash exhibited the highest level at 251.5 g/L, while cream cakes mash had the lowest at 186 g/L. The highest ethanol production occurred after 96 h of fermentation with rye bread, yielding 78.4 g/L. In contrast, despite having the highest monosaccharide levels, white bread produced only 21.5 g/L of ethanol after 96 h. The addition of DAP accelerated monosaccharide consumption in all baked products, with cream cakes completing the process in just 24 h. Bread rolls, pretzel rolls, pastry, and white bread fermentations finished within 72 h. Ethanol yields significantly increased in three DAP samples, with pretzel rolls yielding the highest ethanol concentration at 98.5 g/L, followed by white bread with 90.6 g/L, and bread rolls with 87.7 g/L. DAP had a substantial impact on all samples, reducing fermentation time and/or increasing ethanol yield. This effect was particularly pronounced with white bread, where it improved conversion efficiency from 17 to 72%, resulting in 90.6 g/L of ethanol. These results demonstrate that waste baked products hold substantial potential for bioethanol production, and this potential can be further enhanced through the addition of DAP. Graphical Abstract

Towards a hybrid model-driven platform based on flux balance analysis and a machine learning pipeline for biosystem design

Metabolic modeling and machine learning (ML) are crucial components of the evolving next-generation tools in systems and synthetic biology, aiming to unravel the intricate relationship between genotype, phenotype, and the environment. Nonetheless, the comprehensive exploration of integrating these two frameworks, and fully harnessing the potential of fluxomic data, remains an unexplored territory. In this study, we present, rigorously evaluate, and compare ML-based techniques for data integration. The hybrid model revealed that the overexpression of six target genes and the knockout of seven target genes contribute to enhanced ethanol production. Specifically, we investigated the influence of succinate dehydrogenase (SDH) on ethanol biosynthesis in Saccharomyces cerevisiae through shake flask experiments. The findings indicate a noticeable increase in ethanol yield, ranging from 6 % to 10 %, in SDH subunit gene knockout strains compared to the wild-type strain. Moreover, in pursuit of a high-yielding strain for ethanol production, dual-gene deletion experiments were conducted targeting glycerol-3-phosphate dehydrogenase (GPD) and SDH. The results unequivocally demonstrate significant enhancements in ethanol production for the engineered strains Δsdh4Δgpd1, Δsdh5Δgpd1, Δsdh6Δgpd1, Δsdh4Δgpd2, Δsdh5Δgpd2, and Δsdh6Δgpd2, with improvements of 21.6 %, 27.9 %, and 22.7 %, respectively. Overall, the results highlighted that integrating mechanistic flux features substantially improves the prediction of gene knockout strains not accounted for in metabolic reconstructions. In addition, the finding in this study delivers valuable tools for comprehending and manipulating intricate phenotypes, thereby enhancing prediction accuracy and facilitating deeper insights into mechanistic aspects within the field of synthetic biology.

Ethanol tolerance in engineered strains of Clostridium thermocellum

Clostridium thermocellum is a natively cellulolytic bacterium that is promising candidate for cellulosic biofuel production, and can produce ethanol at high yields (75–80% of theoretical) but the ethanol titers produced thus far are too low for commercial application. In several strains of C. thermocellum engineered for increased ethanol yield, ethanol titer seems to be limited by ethanol tolerance. Previous work to improve ethanol tolerance has focused on the WT organism. In this work, we focused on understanding ethanol tolerance in several engineered strains of C. thermocellum. We observed a tradeoff between ethanol tolerance and production. Adaptation for increased ethanol tolerance decreases ethanol production. Second, we observed a consistent genetic response to ethanol stress involving mutations at the AdhE locus. These mutations typically reduced NADH-linked ADH activity. About half of the ethanol tolerance phenotype could be attributed to the elimination of NADH-linked activity based on a targeted deletion of adhE. Finally, we observed that rich growth medium increases ethanol tolerance, but this effect is eliminated in an adhE deletion strain. Together, these suggest that ethanol inhibits growth and metabolism via a redox-imbalance mechanism. The improved understanding of mechanisms of ethanol tolerance described here lays a foundation for developing strains of C. thermocellum with improved ethanol production.

Integrating surfactants with low enzyme loading to increase the glucan conversion and ethanol concentration of reed after combined pretreatment

In this study, in order to obtain high concentration bioethanol, reed pretreated with liquid hot water and ammonia oxygen (LHW-NH3·H2O/O2) was used as the substrate for fermentation. Enzymatic hydrolysis becomes difficult by the ineffective lignin adsorption and the decline in cellulase activity. It is worth noting that even under low enzyme loading (9 FPU/g pretreated solid), this study was able to maintain a higher glucan conversion rate (97.60%). In addition, under high solid loading conditions, the combined effect of PEG 6000 and rhamnolipids increased ethanol yield (78.40%) and ethanol concentration (71.5 g/L). The combined LHW-NH3·H2O/O2 pretreatment with fed-batch semi-simultaneous saccharification and fermentation technique may be utilized to industrialize the production of bioethanol from reed since we concentrated on the maximum exploitation of biomass resources and reduction in energy consumption.

Consolidated bioprocessing for bioethanol production by metabolically engineered cellulolytic fungus Myceliophthora thermophila

Using cellulosic ethanol as fuel is one way to help achieve the world's decarbonization goals. However, the economics of the present technology are unfavorable, especially the cost of cellulose degradation. Here, we reprogram the thermophilic cellulosic fungus Myceliophthora thermophila to directly ferment cellulose into ethanol by mimicking the aerobic ethanol fermentation of yeast (the Crabtree effect), including optimizing the synthetic pathway, enhancing the glycolytic rate, inhibiting mitochondrial NADH shuttles, and knocking out ethanol consumption pathway. The final engineered strain produced 52.8 g/L ethanol directly from cellulose, and 39.8 g/L from corncob, without the need for any added cellulase, while the starting strain produced almost no ethanol. We also demonstrate that as the ethanol fermentation by engineered M. thermophila increases, the composition and expression of cellulases that facilitate the degradation of cellulose, especially cellobiohydrolases, changes. The simplified production process and significantly increased ethanol yield indicate that the fungal consolidated bioprocessing technology that we develop here (one-step, one-strain ethanol production) is promising for fueling sustainable carbon-neutral biomanufacturing in the future.

Urea addition during hydrolysis for increased ethanol yield from white sweet potato: a promising strategy

Bioethanol is mainly produced from corn worldwide, but many countries face challenges in producing sufficient corn for this purpose. White sweet potato (Ipomoea batatas Linneaus) has a lower nutritional value and less carotene pigment compared to other sweet potatoes, which may make it less desirable as a food source in some regions. However, the affordability of white sweet potato could make it an attractive option for bioethanol production in areas where corn is not readily available. This study investigated the use of white sweet potatoes powder (40 mesh sieve) as a feedstock for ethanol production using Saccharomyces cerevisiae yeast. Optimization of the fermentation process was carried out using response surface methodology (RSM). Results showed that an ethanol yield of 18% could be achieved by applying pH at 4.5 and urea concentration of 60 mg/L. These findings suggest that the addition of urea during hydrolysis of white sweet potato is a promising alternative method for bioethanol production which contained higher etnaol yield compated to the previous finding that utilized amyloglucosidase enzyme during hydrolysis.

The Roles of Nicotinamide Adenine Dinucleotide Phosphate Reoxidation and Ammonium Assimilation in the Secretion of Amino Acids as Byproducts of Clostridium thermocellum.

Clostridium thermocellum is a cellulolytic thermophile that is considered for the consolidated bioprocessing of lignocellulose to ethanol. Improvements in ethanol yield are required for industrial implementation, but the incompletely understood causes of amino acid secretion impede progress. In this study, amino acid secretion was investigated via gene deletions in ammonium-regulated, nicotinamide adenine dinucleotide phosphate (NADPH)-supplying and NADPH-consuming pathways as well as via physiological characterization in cellobiose-limited or ammonium-limited chemostats. First, the contribution of the NADPH-supplying malate shunt was studied with strains using either the NADPH-yielding malate shunt (Δppdk) or a redox-independent conversion of PEP to pyruvate (Δppdk ΔmalE::Peno-pyk). In the latter, branched-chain amino acids, especially valine, were significantly reduced, whereas the ethanol yield increased from 46 to 60%, suggesting that the secretion of these amino acids balances the NADPH surplus from the malate shunt. The unchanged amino acid secretion in Δppdk falsified a previous hypothesis on an ammonium-regulated PEP-to-pyruvate flux redistribution. The possible involvement of another NADPH-supplier, namely, NADH-dependent reduced ferredoxin:NADP+ oxidoreductase (nfnAB), was also excluded. Finally, the deletion of glutamate synthase (gogat) in ammonium assimilation resulted in the upregulation of NADPH-linked glutamate dehydrogenase activity and decreased amino acid yields. Since gogat in C. thermocellum is putatively annotated as ferredoxin-linked, a claim which is supported by the product redistribution observed in this study, this deletion likely replaced ferredoxin with NADPH in ammonium assimilation. Overall, these findings indicate that a need to reoxidize NADPH is driving the observed amino acid secretion, likely at the expense of the NADH needed for ethanol formation. This suggests that metabolic engineering strategies that simplify the redox metabolism and ammonium assimilation can contribute to increased ethanol yields. IMPORTANCE Improving the ethanol yield of C. thermocellum is important for the industrial implementation of this microorganism in consolidated bioprocessing. A central role of NADPH in driving amino acid byproduct formation was demonstrated by eliminating the NADPH-supplying malate shunt and separately by changing the cofactor specificity in ammonium assimilation. With amino acid secretion diverting carbon and electrons away from ethanol, these insights are important for further metabolic engineering to reach industrial requirements on ethanol yield. This study also provides chemostat data that are relevant for training genome-scale metabolic models and for improving the validity of their predictions, especially considering the reduced degree-of-freedom in the redox metabolism of the strains generated here. In addition, this study advances the fundamental understanding on the mechanisms underlying amino acid secretion in cellulolytic Clostridia as well as on the regulation and cofactor specificity in ammonium assimilation. Together, these efforts aid in the development of C. thermocellum for the sustainable consolidated bioprocessing of lignocellulose to ethanol with minimal pretreatment.

Multi-omics network model reveals key genes associated with p-coumaric acid stress response in an industrial yeast strain

The production of ethanol from lignocellulosic sources presents increasingly difficult issues for the global biofuel scenario, leading to increased production costs of current second-generation (2G) ethanol when compared to first-generation (1G) plants. Among the setbacks encountered in industrial processes, the presence of chemical inhibitors from pre-treatment processes severely hinders the potential of yeasts in producing ethanol at peak efficiency. However, some industrial yeast strains have, either naturally or artificially, higher tolerance levels to these compounds. Such is the case of S. cerevisiae SA-1, a Brazilian fuel ethanol industrial strain that has shown high resistance to inhibitors produced by the pre-treatment of cellulosic complexes. Our study focuses on the characterization of the transcriptomic and physiological impact of an inhibitor of this type, p-coumaric acid (pCA), on this strain under chemostat cultivation via RNAseq and quantitative physiological data. It was found that strain SA-1 tend to increase ethanol yield and production rate while decreasing biomass yield when exposed to pCA, in contrast to pCA-susceptible strains, which tend to decrease their ethanol yield and fermentation efficiency when exposed to this substance. This suggests increased metabolic activity linked to mitochondrial and peroxisomal processes. The transcriptomic analysis also revealed a plethora of differentially expressed genes located in co-expressed clusters that are associated with changes in biological pathways linked to biosynthetic and energetical processes. Furthermore, it was also identified 20 genes that act as interaction hubs for these clusters, while also having association with altered pathways and changes in metabolic outputs, potentially leading to the discovery of novel targets for metabolic engineering toward a more robust industrial yeast strain.

The Relationship between Structural Features of Lignocellulosic Materials and Ethanol Production Yield

Lignocellulosic materials are a mixture of natural polymers which can be considered a great alternative source of chemical products and energy. Hence, pinewood, poplar wood, and rice straw, as representatives of different types of lignocelluloses, were subjected to several pretreatment types in order to increase ethanol production yield. All pretreatments increased enzymatic hydrolysis and ethanol yield, specifically pretreatment with phosphoric acid. This pretreatment increased ethanol yields by 304.6% and 273.61% for poplar wood and pinewood, respectively, compared to untreated substrates. In addition, a number of analyses, including a BET test, buffering capacity, crystallinity, accessible surface area, and composition measurement, were conducted on the pretreated substrates to investigate their structural modifications in detail. Accessible surface area, as one of the most important parameters for performance of enzymes and microorganisms in the fermentation process, was examined by the water retention value test. The results of this method (using centrifuge) showed that the maximum accessible surface area was related to the pretreated samples with phosphoric acid so that it increased WRV to 132.19%, 149.41%, and 68.44% for poplar wood, pinewood, and rice straw, respectively, as compared to untreated substrates. On the whole, pretreatments restructured and opened up the tangled structure of lignocelluloses, resulting in a considerable increase in ethanol yields. Moreover, in this study, for the first time, a new correlation was presented for each substrate which indicates the relationship between ethanol yield and structural features of the lignocellulosic substrate.

Techno-economic analysis of the production of 2G ethanol and technical lignin via a protic ionic liquid pretreatment of sugarcane bagasse

The increasing demand for renewable energy, fuels, and chemicals encourages the development of new biorefinery processes. This work presents a techno-economic analysis of the pretreatment of sugarcane bagasse using the protic ionic liquid 2-hydroxyethylammonium acetate ([MEA][OAc]) considering four washing methods to remove it from the pretreated pulp. In the best scenario, the increased enzymatic hydrolysis yield and the more efficient counter-current washing method contributed to an increased ethanol yield of 33 %. Despite the lower increment in ethanol yield compared with other second-generation processes, the production of technical lignin increased the reaction mass efficiency of the biorefinery by 5.6 % and led to an internal rate of return two percentage points higher. However, wastewater and vinasse production increased by 26 % and 50 %, respectively. These results indicate that protic ionic liquids are potentially good pretreatment agents to produce fuels and chemicals from biomass, thus underpinning the establishment of new biorefinery processes. • Process development to produce ethanol and technical lignin from bagasse. • Different washing steps yielded different hydrolysis and fermentation yields. • Techno-economic analysis indicates the importance of using protic ionic liquids. • Protic ionic liquid pretreatment has better economic results because of lignin. • Recycling of ionic liquid plays an important role in process economic feasibility.

Saccharomyces cerevisiae employs complex regulation strategies to tolerate low pH stress during ethanol production

BackgroundIndustrial bioethanol production may involve a low pH environment caused by inorganic acids, improving the tolerance of Saccharomyces cerevisiae to a low pH environment is of industrial importance to increase ethanol yield, control bacterial contamination, and reduce production cost. In our previous study, acid tolerance of a diploid industrial Saccharomyces cerevisiae strain KF-7 was chronically acclimatized by continuous ethanol fermentation under gradually increasing low-pH stress conditions. Two haploid strains B3 and C3 having excellent low pH tolerance were derived through the sporulation of an isolated mutant. Diploid strain BC3 was obtained by mating these two haploids. In this study, B3, C3, BC3, and the original strain KF-7 were subjected to comparison transcriptome analysis to investigate the molecular mechanism of the enhanced phenotype.ResultThe comparison transcriptome analysis results suggested that the upregulated vitamin B1 and B6 biosynthesis contributed to the low pH tolerance. Amino acid metabolism, DNA repairment, and general stress response might also alleviate low pH stress.ConclusionSaccharomyces cerevisiae seems to employ complex regulation strategies to tolerate low pH during ethanol production. The findings provide guides for the construction of low pH-tolerant industrial strains that can be used in industrial fermentation processes.

RNA-seq based transcriptomic analysis of the non-conventional yeast Spathaspora passalidarum during Melle-boinot cell recycle in xylose-glucose mixtures

The Melle-Boinot is a promising process for second-generation ethanol production by xylose-fermenting yeasts. However, the impact of this process on the physiology of the non-conventional yeast Spathaspora passalidarum during second-generation ethanol production remains unclear. Therefore, we performed a transcriptomic analysis of S. passalidarum to determine the differences and global responses of differentially expressed genes (DEGs) during five fed-batch fermentations with cell recycle. A cycle-to-cycle metabolic reprogramming was observed resulting in an increase in ethanol yield (32%), volumetric productivity (33%), and titer (33%); and an expressive decrease of 94% of xylitol production. A broad set of pathways operated synergically such as fatty acid metabolism, N-glycan biosynthesis, glyoxylate and dicarboxylate metabolism, oxidative phosphorylation, glutathione metabolism and sulfur metabolism, indicating those as important mechanisms for cell recycling adaptations due to increased ethanol concentration, long-term ethanol exposure and osmotic stress. Together these results suggest that cellular energy was redirected towards the production of cell wall components enabling S. passalidarum cells to thrive on consecutive recycles. Furthermore, these results are instrumental to guide genetic-engineering efforts of xylose-fermenting yeasts to improve the productiveness and feasibility of renewable energy production from lignocellulosic biomass and hemicellulosic hydrolysates.

Effect of phytase corn addition on ethanol yield and distillers' dried grains with soluble profile in corn dry‐grind process

AbstractBackground and ObjectivesPhytase as a feed additive improves the bioavailable phosphorus content of the poultry diet, thereby decreasing dependence on externally added phosphorus. The objective of this study was to determine the effect of adding phytase corn on ethanol yield and distillers' dried grains with soluble (DDGS) composition during a corn dry‐grind process.FindingsPhytase corn at 2.5%, 5%, 7.5%, and 10% w/w of fermentable corn was added during the dry‐grind process. Upon fermentation, the ethanol yield was observed to be in the range of 17.9%–18.9% v/v compared to control (17.7% v/v), representing an 1%–6.5% increase in ethanol yield. Relative to the corn that was introduced at the beginning of the fermentation, the DDGS obtained from the control corn or phytase corn addition all showed a threefold increase in oil, protein, and neutral detergent fiber content. However, the phytic acid content of the DDGS was reduced from 0.45% w/w (control) to 0.13% w/w (phytase treatments).ConclusionAdding phytase corn in a dry‐grind process improves the ethanol yield and reduces phytate‐bound phosphorus content.Significance and NoveltyPhytase corn addition will improve the ethanol yield, DDGS nutritional quality in a corn dry‐grind process, and reduce dependence on phosphorus supplementation.

Evasion of Cas9 toxicity to develop an efficient genome editing system and its application to increase ethanol yield in Fusarium venenatum TB01.

The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas9) system is a powerful genome editing tool that has been successfully established in some filamentous fungi due to its high flexibility and efficiency. However, the potential toxicity of Cas9 restricts the further popularization and application of this system to some degree. The AMA1 element is a self-replicator derived from Aspergillus nidulans, and its derived vectors can be readily lost without selection. In this study, we eliminated Cas9 toxicity to Fusarium venenatum TB01 based on 100% AMA1-based Cas9 expression vector loss. Meanwhile, two available endogenous Pol III promoters (FvU6374 and Fv5SrRNA) used for sgRNA expression of the CRISPR/Cas9 system were excavated. Compared to FvU6374 (40-50%), Fv5SrRNA exhibited higher single-gene editing efficiency (> 85%), and the efficiency of simultaneous editing of the two genes using Fv5SrRNA was over 75%. Based on this system, a butanediol dehydrogenase encoding gene FvBDH was deleted, and the ethanol yield in variants increased by 52% compared with that of the wild-type. The highly efficient CRISPR/Cas9 system developed here lays the technical foundation for advancing the development of F. venenatum TB01 through metabolic engineering, and the obtained FvBDH gene-edited variants have the potential to simultaneously produce mycoprotein and ethanol by further gene modification and fermentation process optimization in the future.Key points• Cas9 toxicity disappeared and DNA-free gene-edited strains obtained after vector loss• Promoter Fv5SrRNA conferred TB01 higher gene editing efficiency than FvU6374•Deletion of the FvBDH gene resulted in a 52% increase in ethanol yield.

Dextran-coated Gd-based ultrasmall nanoparticles as phosphatase-like nanozyme to increase ethanol yield via reduction of yeast intracellular ATP level

Nanozymes—functional materials that possess intrinsic enzyme-like characteristics—have gained tremendous attention in recent years owing to their unique advantages; however, further research is required to understand their scope in biological applications. In this study, dextran-coated nanogadolinia (DCNG) was synthesised, and its phosphatase mimetic activity was demonstrated. Specifically, the dephosphorylation of adenosine triphosphate (ATP), an important biomolecule, by DCNG was investigated. The results showed that DCNG could selectively catalyse the hydrolysis of the terminal high-energy phosphate bonds of ATP under physiological conditions. Furthermore, the biocompatible DCNG, with remarkable phosphatase mimicking activity, decreased the intracellular ATP content by dephosphorylation and increased ethanol yield during glucose fermentation by S. cerevisiae. These results indicate potential alternatives for improving ethanol yields and exploring novel biological applications of nanozymes.