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.

Industrial biotechnology goes thermophilic: Thermoanaerobes as promising hosts in the circular carbon economy.

Transitioning away from fossil feedstocks is imperative to mitigate climate change, and necessitates the utilization of renewable, alternative carbon and energy sources to foster a circular carbon economy. In this context, lignocellulosic biomass and one-carbon compounds emerge as promising feedstocks that could be renewably upgraded by thermophilic anaerobes (thermoanaerobes) via gas fermentation or consolidated bioprocessing to value-added products. In this review, the potential of thermoanaerobes for cost-efficient, effective and sustainable bioproduction is discussed. Metabolic and bioprocess engineering approaches are reviewed to draw a comprehensive picture of current developments and future perspectives for the conversion of renewable feedstocks to chemicals and fuels of interest. Selected bioprocessing scenarios are outlined, offering practical insights into the applicability of thermoanaerobes at a large scale. Collectively, the potential advantages of thermoanaerobes regarding process economics could facilitate an easier transition towards sustainable bioprocesses with renewable feedstocks.

Highly Efficient Production of Cellulosic Ethanol from Poplar Using an Optimal C6/C5 Co-Fermentation Strain of Saccharomyces cerevisiae.

Cellulosic ethanol is the key technology to alleviate the pressure of energy supply and climate change. However, the ethanol production process, which is close to industrial production and has a high saccharification rate and ethanol yield, still needs to be developed. This study demonstrates the effective conversion of poplar wood waste into fuel-grade ethanol. By employing a two-step pretreatment using sodium chlorite (SC)-dilute sulfuric acid (DSA), the raw material achieved a sugar conversion rate exceeding 85% of the theoretical value. Under optimized conditions, brewing yeast co-utilizing C6/C5 enabled a yield of 35 g/L ethanol from 10% solid loading delignified poplar hydrolysate. We increased the solid loading to enhance the final ethanol concentration and optimized both the hydrolysis and fermentation stages. With 20% solid loading delignified poplar hydrolysate, the final ethanol concentration reached 60 g/L, a 71.4% increase from the 10% solid loading. Our work incorporates the pretreatment, enzymatic hydrolysis, and fermentation stages to establish a simple, crude poplar waste fuel ethanol process, expanding the range of feedstocks for second-generation fuel ethanol production.

A cellulosomal yeast reaction system of lignin-degrading enzymes for cellulosic ethanol fermentation.

Biofuel production from lignocellulose feedstocks is sustainable and environmentally friendly. However, the lignocellulosic pretreatment could produce fermentation inhibitors causing multiple stresses and low yield. Therefore, the engineering construction of highly resistant microorganisms is greatly significant. In this study, a composite functional chimeric cellulosome equipped with laccase, versatile peroxidase, and lytic polysaccharide monooxygenase was riveted on the surface of Saccharomyces cerevisiae to construct a novel yeast strain YI/LVP for synergistic lignin degradation and cellulosic ethanol production. The assembly of cellulosome was assayed by immunofluorescence microscopy and flow cytometry. During the whole process of fermentation, the maximum ethanol concentration and cellulose conversion of engineering strain YI/LVP reached 8.68 g/L and 83.41%, respectively. The results proved the availability of artificial chimeric cellulosome containing lignin-degradation enzymes for cellulosic ethanol production. The purpose of the study was to improve the inhibitor tolerance and fermentation performance of S. cerevisiae through the construction and optimization of a synergistic lignin-degrading enzyme system based on cellulosome.

Life cycle assessment and techno-economic analysis of wood-based biorefineries for cellulosic ethanol production.

Poplar is widely used in the paper industry and accompanied by abundant branches waste, which is potential feedstock for bioethanol production. Acid-chlorite pretreatment can selectively remove lignin, thereby significantly increasing enzymatic efficiency. Moreover, lignin residues valorization via gasification-syngas fermentation can achieve higher fuel yield. Herein, environmental and economic aspects were conducted to assess technological routes, which guides further process optimization. Life cycle assessment results show that wood-based biorefineries especially coupling scenarios have significant advantages in reducing global warming potential in contrast to fossil-based automotive fuels. Normalization results indicate that acidification potential surpasses other indicators as the primary impact category. In terms of economic feasibility, coupling scenarios present better investment prospects. Bioethanol yield is the most critical factor affecting market competitiveness. Minimum ethanol selling price below ethanol international market price is promising with higher-levels technology. Further work should be focused on technological breakthrough, consumable reduction or replacement.

Dual assistance of surfactants in glycerol organosolv pretreatment and enzymatic hydrolysis of lignocellulosic biomass for bioethanol production.

This study investigated an innovative strategy of incorporating surfactants into alkaline-catalyzed glycerol pretreatment and enzymatic hydrolysis to improve lignocellulosic biomass (LCB) conversion efficiency. Results revealed that adding 40 mg/g PEG 4000 to the pretreatment at 195 °C obtained the highest glucose yield (84.6%). This yield was comparable to that achieved without surfactants at a higher temperature (240 °C), indicating a reduction of 18.8% in the required heat input. Subsequently, Triton X-100 addition during enzymatic hydrolysis of PEG 4000-assisted pretreated substrate increased glucose yields to 92.1% at 6 FPU/g enzyme loading. High-solid fed-batch semi-simultaneous saccharification and co-fermentation using this dual surfactant strategy gave 56.4 g/L ethanol and a positive net energy gain of 1.4 MJ/kg. Significantly, dual assistance with surfactants rendered 56.3% enzyme cost savings compared to controls without surfactants. Therefore, the proposed surfactant dual-assisted promising approach opens the gateway to economically viable enzyme-mediated LCB biorefinery.

Efficient degradation of cellulosic ethanol wastewater by perovskite activation of Sr element A-site doped lanthanide copper chalcogenide materials.

The degradation of cellulosic ethanol wastewater by peroxymonosulfate (PMS) is one of the important methods to solve the environmental problems caused by it. In order to improve the degradation efficiency of cellulosic ethanol wastewater, the design of more catalytically active and stable chalcogenide catalysts has become a problem that needs to be solved nowadays. The application of foreign cations to replace the A- or B-site to increase the oxygen vacancy of the chalcocite catalyst to improve the efficiency of chalcocite catalytic degradation of wastewater has received much attention. In this work, the perovskite material LaCuO3 was synthesized using a citric acid-sol-gel method, and the novel material La1-xSrxCuO3 was prepared by doping of Sr element at the A position. In order to prepare catalytic materials with better performance, this study carried out performance-optimized degradation experiments on the prepared materials and determined that the catalytic efficiency of La0.5Sr0.5CuO3 prepared under the conditions of the complexing agent dosage of 1:2, the gel temperature of 80 °C, and the calcination temperature of 700 °C was better than that of the catalytic materials prepared under other conditions. The prepared material has good recycling function; after four times recycling, the removal rate of pollutant COD is still more than 85%. This work provides a new synthesis method of perovskite material with good recycling function and high catalytic efficiency for the degradation technology of cellulosic ethanol wastewater.

An atlas of rational genetic engineering strategies for improved xylose metabolism in Saccharomyces cerevisiae.

Xylose is the second most abundant carbohydrate in nature, mostly present in lignocellulosic material, and representing an appealing feedstock for molecule manufacturing through biotechnological routes. However, Saccharomyces cerevisiae-a microbial cell widely used industrially for ethanol production-is unable to assimilate this sugar. Hence, in a world with raising environmental awareness, the efficient fermentation of pentoses is a crucial bottleneck to producing biofuels from renewable biomass resources. In this context, advances in the genetic mapping of S. cerevisiae have contributed to noteworthy progress in the understanding of xylose metabolism in yeast, as well as the identification of gene targets that enable the development of tailored strains for cellulosic ethanol production. Accordingly, this review focuses on the main strategies employed to understand the network of genes that are directly or indirectly related to this phenotype, and their respective contributions to xylose consumption in S. cerevisiae, especially for ethanol production. Altogether, the information in this work summarizes the most recent and relevant results from scientific investigations that endowed S. cerevisiae with an outstanding capability for commercial ethanol production from xylose.

Life cycle assessment of calcium oxide pretreatment of corn stover with carbon dioxide neutralization for ethanol production.

This work used life-cycle assessment (LCA) to determine the environmental and human health impacts of four ethanol production scenarios (S1: CaO pretreatment + H2SO4 neutralization + C6 yeast fermentation; S2: CaO pretreatment + CO2 neutralization + C6 yeast fermentation; S3: CaO pretreatment + H2SO4 neutralization + C6/C5 yeast fermentation; and S4: CaO pretreatment + CO2 neutralization + C6/C5 yeast fermentation), with the functional unit being 1 kg of 95 % ethanol. TheLCA results showed that the total ozone depletion, global warming potential, smog, acidification, eutrophication, and ecotoxicity values were comparable when CO2 or H2SO4 were used to adjust the pH of CaO-pretreated slurry. However, using CO2 for neutralization and C6/C5 yeast for fermentation demonstrated significant benefits in terms of carcinogenicity, non-carcinogenicity, respiratory effect, ecotoxicity, and fossil fuel depletion. The findings indicate that the choice of chemicals and strains plays a key role in determining environmental and human health impacts.

Cellulosic Ethanol Production from Weed Biomass Hydrolysate of Vietnamosasa pusilla.

Lignocellulosic biomass can be used as a renewable and sustainable energy source to help reduce the consequences of global warming. In the new energy age, the bioconversion of lignocellulosic biomass into green and clean energy displays remarkable potential and makes efficient use of waste. Bioethanol is a biofuel that can diminish reliance on fossil fuels while minimizing carbon emissions and increasing energy efficiency. Various lignocellulosic materials and weed biomass species have been selected as potential alternative energy sources. Vietnamosasa pusilla, a weed belonging to the Poaceae family, contains more than 40% glucan. However, research on the applications of this material is limited. Thus, here we aimed to achieve maximum fermentable glucose recovery and bioethanol production from weed biomass (V. pusilla). To this end, V. pusilla feedstocks were treated with varying concentrations of H3PO4 and then subjected to enzymatic hydrolysis. The results indicated that after pretreatment with different concentrations of H3PO4, the glucose recovery and digestibility at each concentration were markedly enhanced. Moreover, 87.5% of cellulosic ethanol was obtained from V. pusilla biomass hydrolysate medium without detoxification. Overall, our findings reveal that V. pusilla biomass can be introduced into sugar-based biorefineries to produce biofuels and other valuable chemicals.

High bioethanol titer and yield from phosphoric acid plus hydrogen peroxide pretreated paper mulberry wood through optimization of simultaneous saccharification and fermentation.

The optimization of key simultaneous saccharification and fermentation (SSF) parameters for bioethanol production from phosphoric acid plus hydrogen peroxide pretreated paper mulberry wood was carried out under two isothermal scenarios; the yeast optimum and trade-off temperatures of 35 and 38 °C, respectively. The optimal conditions established for SSF at 35 °C (solid loading: 16%; enzyme dosage: 9.8 mg protein/g glucan; and yeast concentration: 6.5 g/L) achieved high ethanol titer and yield of 77.34 g/L and 84.60% (0.432 g/g), respectively. These corresponded to 1.2 and 1.3-folds increases, compared to the results of the optimal SSF at a relatively higher temperature of 38 °C. The information from this study would prove beneficial in reducing process energy demands to some extent, while also helping to achieve high levels of both ethanol concentration and yield that are desired in cellulosic ethanol production.

Challenges and perspectives of green-like lignocellulose pretreatments selectable for low-cost biofuels and high-value bioproduction.

Lignocellulose represents the most abundant carbon-capturing substance that is convertible for biofuels and bioproduction. Although biomass pretreatments have been broadly applied to reduce lignocellulose recalcitrance for enhanced enzymatic saccharification, they mostly require strong conditions with potential secondary waste release. By classifying all major types of pretreatments that have been recently conducted with different sources of lignocellulose substrates, this study sorted out their distinct roles for wall polymer extraction and destruction, leading to the optimal pretreatments evaluated for cost-effective biomass enzymatic saccharification to maximize biofuel production. Notably, all undigestible lignocellulose residues are also aimed for effective conversion into value-added bioproduction. Meanwhile, desired pretreatments were proposed for the generation of highly-valuable nanomaterials such as cellulose nanocrystals, lignin nanoparticles, functional wood, carbon dots, porous and graphitic nanocarbons. Therefore, this article has proposed a novel strategy that integrates cost-effective and green-like pretreatments with desirable lignocellulose substrates for a full lignocellulose utilization with zero-biomass-waste liberation.

Investigation of stress tolerance of Pichia kudriavzevii for high gravity bioethanol production from steam-exploded wheat straw hydrolysate.

This study investigated a newly isolated thermotolerant strain of Pichia kudriavzevii with respect to its stress tolerance and fermentation performance. Response surface methodology was applied to evaluate the combined effects of furfural, osmotic and thermal stress on ethanol yield. The proposed model shows that P. kudriavzevii has a natural resistance against multiple stress factors. Further evolutionary adaptation of the isolated strain in lignocellulosic hydrolysates improved the ethanol yield by ≥ 24 %. The adapted strain HYPK213_ELA was able to produce ethanol from wheat straw hydrolysates at a high solid loading of 37 %ww-1 at 40 °C and anaerobic conditions. The highest ethanol concentration of 56.8 ± 1.0 gL-1 was reached at 40°C with an inoculum size of 2.5 × 106cellsmL-1. The results show that Pichia kudriavzevii has the potential to enable high gravity bioethanol production under conditions where most yeast strains are unable to grow.

Characterization of Thermotoga neapolitana Alcohol Dehydrogenases in the Ethanol Fermentation Pathway.

Hyperthermophilic Thermotoga spp. are candidates for cellulosic ethanol fermentation. A bifunctional iron-acetaldehyde/alcohol dehydrogenase (Fe-AAdh) has been revealed to catalyze the acetyl-CoA (Ac-CoA) reduction to form ethanol via an acetaldehyde intermediate in Thermotoga neapolitana (T. neapolitana). In this organism, there are three additional alcohol dehydrogenases, Zn-Adh, Fe-Adh1, and Fe-Adh2, encoded by genes CTN_0257, CTN_1655, and CTN_1756, respectively. This paper reports the properties and functions of these enzymes in the fermentation pathway from Ac-CoA to ethanol. It was determined that Zn-Adh only exhibited activity when oxidizing ethanol to acetaldehyde, and no detectable activity for the reaction from acetaldehyde to ethanol. Fe-Adh1 had specific activities of approximately 0.7 and 0.4 U/mg for the forward and reverse reactions between acetaldehyde and ethanol at a pHopt of 8.5 and Topt of 95 °C. Catalyzing the reduction of acetaldehyde to produce ethanol, Fe-Adh2 exhibited the highest activity of approximately 3 U/mg at a pHopt of 7.0 and Topt of 85 °C, which were close to the optimal growth conditions. These results indicate that Fe-Adh2 and Zn-Adh are the main enzymes that catalyze ethanol formation and consumption in the hyperthermophilic bacterium, respectively.

Feasibility assessment of bioethanol production from humic acid-assisted alkaline pretreated Kentucky bluegrass (Poa pratensis L.) followed by downstream enrichment using direct contact membrane distillation.

The effective fractionation of structural components of abundantly available lignocellulosic biomass is essential to unlock its full biorefinery potential. In this study, the feasibility of humic acid on the pretreatment of Kentucky bluegrass biomass in alkaline condition was assessed to separate 70.1% lignin and hydrolyzable biocomponents. The humic acid-assisted delignification followed by enzymatic saccharification yielded 0.55 g/g of reducing sugars from 7.5% (w/v) pretreated biomass loading and 16 FPU/g of cellulase. Yeast fermentation of the biomass hydrolysate produced 76.6% (w/w) ethanol, which was subsequently separated and concentrated using direct contact membrane distillation. The hydrophobic microporous flat-sheet membrane housed in a rectangular-shaped crossflow module and counter-current mode of flow of the feed (hot) and distillate (cold) streams yielded a flux of 11.6 kg EtOH/m2/24 h. A modular, compact, flexible, and eco-friendly membrane-integrated hybrid approach is used for the first time to effectively valorize Kentucky bluegrass biomass for sustainable production of biofuel.

Rapid evolution and mechanism elucidation for efficient cellobiose-utilizing Saccharomyces cerevisiae through Synthetic Chromosome Rearrangement and Modification by LoxPsym-mediated Evolution.

Lack of cellobiose utilization capability for many microorganisms results in carbon source waste in lignocellulosic biorefinery. In this study, genes for cellobiose transport and hydrolysis were introduced to Saccharomyces cerevisiae synV, a semi-synthetic yeast with an inducible SCRaMbLE (Synthetic Chromosome Rearrangement and Modification by LoxPsym-mediated Evolution) system incorporated into its chromosome V, endowing cellobiose utilization capability to this strain. Thereafter, two evolved strains with 98.1% and 79.2% improvement, respectively, in cellobiose utilization rate were obtained through induced SCRaMbLE. Further studies suggested that the enhanced cellobiose utilization capability directly correlated with copy number increases of introduced genes and some chromosome structural variations. In particular, it was experimentally demonstrated for the first time that deletion of redox stress related gene MXR1 and ATP conversion related gene ADK2 contributed to enhanced cellobiose conversion. Thereafter, the effectiveness of MXR1 and ADK2 deletions was demonstrated in artificial hydrolysate and rice straw hydrolysate, respectively.

Re-examination of dilute acid hydrolysis of lignocellulose for production of cellulosic ethanol after de-bottlenecking the inhibitor barrier.

Dilute acid hydrolysis of lignocellulose biomass had been used for production of cellulosic ethanol since 1940 s. The major technical barrier is the acid catalyzed dehydration of monosaccharides to furan aldehydes (furfural and 5-hydroxymethylfurfural), resulting in the high loss of fermentable sugars and significant inhibition on the fermentability of ethanologenic strains. This study re-examined the dilute acid hydrolysis of corn stover and cellulosic ethanol fermentation after a novel biodetoxification approach was introduced to de-bottleneck the inhibitor barrier. The cocktail of sulfuric acid, phosphoric acid and oxalic acid hydrolyzed corn stover to the 51.1 g/L of glucose (0.50 g/g cellulose) and 18.1 g/L of xylose (0.22 g/g xylan). The furfural, 5-hydroxymethylfurfural and acetic acid in the corn stover hydrolysate were completely removed by Paecilomyces variotii FN89, leading to the successful ethanol fermentation of 24.2 g/L, corresponding to 72.6 kg per metric ton of dry corn stover. No wastewater streams, solid wastes and toxic compounds were generated in hydrolysis, biodetoxification and fermentation. The techno-economic evaluations suggest that the cost reduction of replacing cellulase enzyme with cheap acid catalysts compensated the partial ethanol loss of sugar conversion to inhibitors (21.5-89.1%). The re-examination of acid hydrolysis process reveals that a substantial breakthrough in highly active and selective acid catalyst is required for acid hydrolysis to compete with enzymic hydrolysis for cellulosic ethanol fermentation.

Manipulating cell flocculation-associated protein kinases in Saccharomyces cerevisiae enables improved stress tolerance and efficient cellulosic ethanol production.

Cell self-flocculation endows yeast strains with improved environmental stress tolerance that benefits bioproduction. Exploration of the metabolic and regulatory network differences between the flocculating and non-flocculating cells is conducive to developing strains with satisfactory fermentation efficiency. In this work, integrated analyses of transcriptome, proteome, and phosphoproteome were performed using flocculating yeast Saccharomyces cerevisiae SPSC01 and its non-flocculating mutant grown under acetic acid stress, and the results revealed prominent changes in protein kinases. Overexpressing the mitogen-activated protein kinase Hog1 upregulated by flocculation led to reduced ROS accumulation and increased glutathione peroxidase activity, leading to improved ethanol production under stress. Among the seven genes encoding protein kinases that were tested, AKL1 showed the best performance when overexpressed, achieving higher ethanol productivity in both corncob hydrolysate and simulated corn stover hydrolysate. These results provide alternative strategies for improving cellulosic ethanol production by engineering key protein kinases in S. cerevisiae.

Effects of Engineered Saccharomyces cerevisiae Fermenting Cellobiose through Low-Energy-Consuming Phosphorolytic Pathway in Simultaneous Saccharification and Fermentation.

Until recently, four types of cellobiose-fermenting Saccharomyces cerevisiae strains have been developed by introduction of a cellobiose metabolic pathway based on either intracellular ß-glucosidase (GH1-1) or cellobiose phosphorylase (CBP), along with either an energy-consuming active cellodextrin transporter (CDT-1) or a non-energy-consuming passive cellodextrin facilitator (CDT-2). In this study, the ethanol production performance of two cellobiose-fermenting S. cerevisiae strains expressing mutant CDT-2 (N306I) with GH1-1 or CBP were compared with two cellobiose-fermenting S. cerevisiae strains expressing mutant CDT-1 (F213L) with GH1-1 or CBP in the simultaneous saccharification and fermentation (SSF) of cellulose under various conditions. It was found that, regardless of the SSF conditions, the phosphorolytic cellobiose-fermenting S. cerevisiae expressing mutant CDT-2 with CBP showed the best ethanol production among the four strains. In addition, during SSF contaminated by lactic acid bacteria, the phosphorolytic cellobiose-fermenting S. cerevisiae expressing mutant CDT-2 with CBP showed the highest ethanol production and the lowest lactate formation compared with those of other strains, such as the hydrolytic cellobiose-fermenting S. cerevisiae expressing mutant CDT-1 with GH1-1, and the glucose-fermenting S. cerevisiae with extracellular ß-glucosidase. These results suggest that the cellobiose-fermenting yeast strain exhibiting low energy consumption can enhance the efficiency of the SSF of cellulosic biomass.

Spent mushroom substrates for ethanol production - Effect of chemical and structural factors on enzymatic saccharification and ethanolic fermentation of Lentinula edodes-pretreated hardwood.

Spent mushroom substrates (SMS) from cultivation of shiitake (Lentinula edodes) on three hardwood species were investigated regarding their potential for cellulose saccharification and for ethanolic fermentation of the produced hydrolysates. High glucan digestibility was achieved during enzymatic saccharification of the SMSs, which was related to the low mass fractions of lignin and xylan, and it was neither affected by the relative content of lignin guaiacyl units nor the substrate crystallinity. The high nitrogen content in SMS hydrolysates, which was a consequence of the fungal pretreatment, was positive for the fermentation, and it ensured ethanol yields corresponding to 84-87% of the theoretical value in fermentations without nutrient supplementation. Phenolic compounds and acetic acid were detected in the SMS hydrolysates, but due to their low concentrations, the inhibitory effect was limited. The solid leftovers resulting from SMS hydrolysis and the fermentation residues were quantified and characterized for further valorisation.