Hydrolysis Of Corn Stover Research Articles

Deep eutectic solvents assisted laccase pretreatment for improving enzymatic hydrolysis of corn stover.

The efficient and eco-friendly removal of lignin is a critical challenge for bioethanol production from lignocellulosic biomass. Herein, we report the integration of laccase with deep eutectic solvents (DESs) for the pretreatment of corn stover to enhance the production of reducing sugars. Three betaine-based DESs were prepared and tested for their effects on the activity and stability of a bacterial laccase from Bacillus amyloliquefaciens LC02. The aqueous solution of DESs showed no adverse influence on laccase activity, and the laccase thermostability was improved in the presence of DESs. More than 95% of the laccase activity was retained in the DESs solution during the first hour of incubation at 70°C. A red shift in the fluorescence spectra was observed for the laccase in the presence of DESs, indicating conformational changes. The laccase was able to degrade a dimeric lignin model compound by cleaving its β-O-4 bond. The transformation products were identified using LC-MS. The maximal lignin removal from corn stover was achieved by pretreatment using laccase in combination with the betaine-glycerol DES, which also resulted in a yield of fermentable sugar that was 130% higher than the control. This combination strategy provides guidance on the application of laccase and DESs in the pretreatment of lignocellulosic biomass.

Acetate production from corn stover hydrolysate using recombinant Escherichia coli BL21 (DE3) with an EP-bifido pathway

BackgroundAcetate is an important chemical feedstock widely applied in the food, chemical and textile industries. It is now mainly produced from petrochemical materials through chemical processes. Conversion of lignocellulose biomass to acetate by biotechnological pathways is both environmentally beneficial and cost-effective. However, acetate production from carbohydrate in lignocellulose hydrolysate via glycolytic pathways involving pyruvate decarboxylation often suffers from the carbon loss and results in low acetate yield.ResultsEscherichia coli BL21 (DE3) was confirmed to have high tolerance to acetate in this work. Thus, it was selected from seven laboratory E. coli strains for acetate production from lignocellulose hydrolysate. The byproduct-producing genes frdA, ldhA, and adhE in E. coli BL21 (DE3) were firstly knocked out to decrease the generation of succinate, lactate, and ethanol. Then, the genes pfkA and edd were also deleted and bifunctional phosphoketolase and fructose-1,6-bisphosphatase were overexpressed to construct an EP-bifido pathway in E. coli BL21 (DE3) to increase the generation of acetate from glucose. The obtained strain E. coli 5K/pFF can produce 22.89 g/L acetate from 37.5 g/L glucose with a yield of 0.61 g/g glucose. Finally, the ptsG gene in E. coli 5K/pFF was also deleted to make the engineered strain E. coli 6K/pFF to simultaneously utilize glucose and xylose in lignocellulosic hydrolysates. E. coli 6K/pFF can produce 20.09 g/L acetate from corn stover hydrolysate with a yield of 0.52 g/g sugar.ConclusionThe results presented here provide a promising alternative for acetate production with low cost substrate. Besides acetate production, other biotechnological processes might also be developed for other acetyl-CoA derivatives production with lignocellulose hydrolysate through further metabolic engineering of E. coli 6K/pFF.

Engineering a xylose fermenting yeast for lignocellulosic ethanol production.

Lignocellulosic ethanol is produced by yeast fermentation of lignocellulosic hydrolysates generated by chemical pretreatment and enzymatic hydrolysis of plant cell walls. The conversion of xylose into ethanol in hydrolysates containing microbial inhibitors is a major bottleneck in biofuel production. We identified sodium salts as the primary yeast inhibitors, and evolved a Saccharomyces cerevisiae strain overexpressing xylose catabolism genes in xylose or glucose-mixed medium containing sodium salts. The fully evolved yeast strain can efficiently convert xylose in the hydrolysates to ethanol on an industrial scale. We elucidated that the amplification of xylA, XKS1 and pentose phosphate pathway-related genes TAL1, RPE1, TKL1, RKI1, along with mutations in NFS1, TRK1, SSK1, PUF2 and IRA1, are responsible and sufficient for the effective xylose utilization in corn stover hydrolysates containing high sodium salts. Our evolved or reverse-engineered yeast strains enable industrial-scale production of lignocellulosic ethanol and the genetic foundation we uncovered can also facilitate transfer of the phenotype to yeast cell factories producing chemicals beyond ethanol.

Systematic optimization of cellulases production and extraction by Sporotrichum thermophile mutant strain TH3-9 and application in enzymatic hydrolysis of corn stover

Enzymatic hydrolysis of cellulose generally occurs at 40-50 ℃, which can result in slow hydrolysis rate, low sugar yield with incomplete hydrolysis and easy microbial contamination. The limitations can be resolved by adopting cellulases from thermophilic microorganisms. In this work, response surface methodology was used for optimizing cellulases production and extraction by Sporotrichum thermophile mutant strain TH3-9 in solid state fermentation. Results indicated that ammonium sulphate, initial pH and inoculum amount had significant effects on cellulases production. Maximal cellulases activities (endoglucanase activity, 718.03 U/g; filter paper activity, 126.51 U/g; β-glucosidase activity, 444.33 U/g) could be obtained by adopting corn stover (10.0g) and wheat bran (5.0g) as substrates, ammonium sulphate 1.63g, initial pH 5.1 and inoculum amount 13.8% (v/w). Compared with cellulases activities in the initial conditions, optimization resulted in 5.86-fold, 4.29-fold and 6.06-fold increase for endoglucanase activity, filter paper activity and β-glucosidase activity, respectively. During optimization of cellulases extraction, filter paper activity (FPA) was used as the response and results indicated that variables including volume of solvent, rotation speed and time had significant effects on FPA of the crude cellulases solution. Maximal FPA (24.53U/mL) could be obtained while extraction was performed at 30 ℃, 135rpm for 38min with the use of distill water as solvent. In addition, the crude cellulases solution was primarily applied in hydrolysis of alkaline hydrogen peroxide pretreated corn stover and the reducing sugar yield (418.47mg/g) could be obtained during hydrolysis at 50 ℃ for 48h.

Separation of 2,3-Butanediol from Fermentation Broth via Cyclic and Simulated Moving Bed Adsorption Over Nano-MFI Zeolites.

The biomass-based platform molecule 2,3-butanediol (2,3-BDO) has a wide range of applications in production of sustainable fuels, chemicals, synthetic rubber, and others. However, the selective separation of 2,3-BDO from multicomponent fermentation broths presents challenges due to its low concentration, high solubility in water, high boiling point, and presence of many other species. Here, we demonstrate remarkably selective enrichment and recovery of 2,3-BDO from a corn stover hydrolysate fermentation broth by a pure-silica nano-MFI-type zeolite adsorbent. By means of cyclic and simulated moving bed adsorption processes, we obtained concentrated aqueous 2,3-BDO streams from the fermentation process stream with ∼93% purity and 3-fold enrichment, and >98% purity and 8-fold enrichment, respectively. These findings provide strong support for large-scale adsorptive separation for biobased 2,3-BDO production.

Enhanced enzymatic hydrolysis of corn stover by γ-valerolactone pretreatment and the characteristics of enzymatic residues

Efficient pretreatment methods are crucial for the generation of fermentable sugars from the renewable lignocellulose. In this study, γ-valerolactone (GVL), a biomass-derived green solvent, was used to fractionate corn stover (CS). The impact of different additives on the enzymatic hydrolysis and pretreatment of CS within the GVL/water system was carefully investigated. The results demonstrate that the use of H2SO4-assisted GVL/H2O (80:20, w/w) pretreatment at mild conditions successfully facilitates the depolymerization of CS, thereby improving the cellulase accessibility. Furthermore, by increasing the GVL pretreatment temperature to 160 °C with 100 mM H2SO4 addition, the pretreated CS led to nearly 100 % of hydrolysis yield at 2 % (w/v) solid load. Finally, the thermogravimetric analysis (TGA) was performed to evaluate the thermal decomposition characteristics of untreated CS and enzymatic residues based on the H2SO4-assisted GVL/H2O pretreatment. The results indicate that the enzymatic residues had two depolymerization stages from 200 °C to 500 °C with a final residual mass of 22.73 %, which was higher than that of untreated CS (20.56 %). Overall, this study provides an efficient lignocellulose pretreatment for fermentable sugars production and valorization of enzymatic residues.

Rewiring the reductive TCA pathway and glyoxylate shunt of Escherichia coli for succinate production from corn stover hydrolysate using a two-phase fermentation strategy

Succinate was found extensive applications in the food additives, pharmaceutical, and biopolymers industries. However, the succinate biosynthesis in E. coli required IPTG, lacked NADH, and produced high yields only under anaerobic conditions, unsuitable for cell growth. To overcome these limitations, the glyoxylate shunt and reductive TCA pathway were simultaneously enhanced to produce succinate in both aerobic and anaerobic conditions and achieve a high cell growth meanwhile. On this basis, NADH availability and sugars uptake were increased. Furthermore, an oxygen-dependent promoter was used to dynamically regulate the expression level of key genes of reductive TCA pathway to avoid the usage of IPTG. The final strain E. coli Mgls7-32 could produce succinate from corn stover hydrolysate without an inducer, achieving a titer of 72.8 g/L in 5 L bioreactor (1.2 mol/mol of total sugars). Those findings will aid in the industrial production of succinate.

Enhanced glucose-1-phosphate production from corn stover using cellulases with reduced β-glucosidase activity via Trbgl1 gene knockout in Trichoderma reesei Rut C30

The scarcity of cellulases with low β-glucosidase activity poses a significant technological challenge in precisely controlling the partial hydrolysis of lignocellulose to cellobiose, crucial for producing high-value chemicals such as starch, inositol, and NMN. Trichoderma reesei is a primary strain in cellulase production. Therefore, this study targeted the critical β-glucosidase gene, Trbgl1, resulting in over an 86 % reduction in β-glucosidase activity. However, cellulase production decreased by 19.2 % and 20.3 % with lactose or cellulose inducers, respectively. Notably, transcript levels of cellulase genes and overall yield remained unaffected with an inducer containing sophorose. This indicates that β-glucosidase BGL1 converts lactose or cellulose to sophorose through transglycosylation activity, inducing cellulase gene transcription. The resulting enzyme cocktail, comprising recombinant cellulase and cellobiose phosphorylase, was applied for corn stover hydrolysis, resulting in a 24.3 % increase in glucose-1-phosphate yield. These findings provide valuable insights into obtaining enzymes suitable for the high-value utilization of lignocellulose.

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.

Fungal combined with ammoniation pretreatment to promote enzymatic hydrolysis of corn stover

Fungal combined with ammoniation pretreatment to promote enzymatic hydrolysis of corn stover

Rapid fractionation of corn stover by microwave-assisted protic ionic liquid [TEA][HSO4] for fermentative acetone–butanol–ethanol production

BackgroundThe use of ionic liquids (ILs) to fractionate lignocelluloses for various bio-based chemicals productions is in the ascendant. On this basis, the protic ILs consisting of triethylammonium hydrogen sulfate ([TEA][HSO4]) possessed great promise due to the low price, low pollution, and high efficiency. In this study, the microwave-assistant [TEA][HSO4] fractionation process was established for corn stover fractionation, so as to facilitate the monomeric sugars production and supported the downstream acetone–butanol–ethanol (ABE) fermentation.ResultsThe assistance of microwave irradiation could obviously shorten the fractionation period of corn stover. Under the optimized condition (190 W for 3 min), high xylan removal (93.17 ± 0.63%) and delignification rate (72.90 ± 0.81%) were realized. The mechanisms for the promotion effect of the microwave to the protic ILs fractionation process were ascribed to the synergistic effect of the IL and microwaves to the depolymerization of lignocellulose through the ionic conduction, which can be clarified by the characterization of the pulps and the isolated lignin specimens. Downstream valorization of the fractionated pulps into ABE productions was also investigated. The [TEA][HSO4] free corn stover hydrolysate was capable of producing 12.58 g L−1 of ABE from overall 38.20 g L−1 of monomeric sugars without detoxification and additional nutrients supplementation.ConclusionsThe assistance of microwave irradiation could significantly promote the corn stover fractionation by [TEA][HSO4]. Mass balance indicated that 8.1 g of ABE and 16.61 g of technical lignin can be generated from 100 g of raw corn stover based on the novel fractionation strategy.

Taurine-mediated gene transcription and cell membrane permeability reinforced co-production of bioethanol and Monascus azaphilone pigments for a newly isolated Monascus purpureus

BackgroundTaurine, a semi-essential micronutrient, could be utilized as a sulfur source for some bacteria; however, little is known about its effect on the accumulation of fermentation products. Here, it investigated the effect of taurine on co-production of bioethanol and Monascus azaphilone pigments (MonAzPs) for a fungus.ResultsA newly isolated fungus of 98.92% identity with Monascus purpureus co-produced 23.43 g/L bioethanol and 66.12, 78.01 and 62.37 U/mL red, yellow and orange MonAzPs for 3 d in synthetic medium (SM). Taurine enhanced bioethanol titer, ethanol productivity and ethanol yield at the maximum by 1.56, 1.58 and 1.60 times than those of the control in corn stover hydrolysates (CSH), and red, yellow and orange MonAzPs were raised by 1.24, 1.26 and 1.29 times, respectively. Taurine was consumed extremely small quantities for M. purpureus and its promotional effect was not universal for the other two biorefinery fermenting strains. Taurine intensified the gene transcription of glycolysis (glucokinase, phosphoglycerate mutase, enolase and alcohol dehydrogenase) and MonAzPs biosynthesis (serine hydrolases, C-11-ketoreductase, FAD-dependent monooxygenase, 4-O-acyltransferase, deacetylase, NAD(P)H-dependent oxidoredutase, FAD-dependent oxidoredutase, enoyl reductase and fatty acid synthase) through de novo RNA-Seq assays. Furthermore, taurine improved cell membrane permeability through changing cell membrane structure by microscopic imaging assays.ConclusionsTaurine reinforced co-production of bioethanol and MonAzPs by increasing gene transcription level and cell membrane permeability for M. purpureus. This work would offer an innovative, efficient and taurine-based co-production system for mass accumulation of the value-added biofuels and biochemicals from lignocellulosic biomass.

Acetic acid- and furfural-based adaptive evolution of Saccharomyces cerevisiae strains for improving stress tolerance and lignocellulosic ethanol production

Acetic acid and furfural are known as prevalent inhibitors deriving from pretreatment during lignocellulosic ethanol production. They negatively impact cell growth, glucose uptake and ethanol biosynthesis of Saccharomyces cerevisiae strains. Development of industrial S. cerevisiae strains with high tolerance towards these inhibitors is thus critical for efficient lignocellulosic ethanol production. In this study, the acetic acid or furfural tolerance of different S. cerevisiae strains could be significantly enhanced after adaptive evolution via serial cultivation for 40 generations under stress conditions. The acetic acid-based adaptive strain SPSC01-TA9 produced 30.5 g·L–1 ethanol with a yield of 0.46 g·g–1 in the presence of 9 g·L–1 acetic acid, while the acetic acid/furfural-based adaptive strain SPSC01-TAF94 produced more ethanol of 36.2 g·L–1 with increased yield up to 0. 49 g·g–1 in the presence of both 9 g·L–1 acetic acid and 4 g·L–1 furfural. Significant improvements were also observed during non-detoxified corn stover hydrolysate culture by SPSC01-TAF94, which achieved ethanol production and yield of 29.1 g·L–1 and 0.49 g·g–1, respectively, the growth and fermentation efficiency of acetic acid/furfural-based adaptive strain in hydrolysate was 95% higher than those of wildtype strains, indicating the acetic acid- and furfural-based adaptive evolution strategy could be an effective approach for improving lignocellulosic ethanol production. The adapted strains developed in this study with enhanced tolerance against acetic acid and furfural could be potentially contribute to economically feasible and sustainable lignocellulosic biorefinery.

Optimization of poly(3-hydroxybutyrate) production with simulating corn stover hydrolysates by Paraburkholderia sacchari

In this study, poly(3-hydroxybutyrate) production by Paraburkholderia sacchari was investigated using glucose-xylose mixtures as simulated corn stover hydrolysates to optimize the culture media. Box-Behnken Design (BBD) was selected to comprehensively understand the effects of four variables including sugar concentration, molar C/N ratio, phosphorus concentration, and sugar ratio on P(3HB) production. The optimized parameters were identified as initial sugar concentration (23 g/L), molar C/N ratio (44), phosphorus concentration (8.7 g/L), and sugar ratio (1.9∶1). Under the optimal conditions, P. sacchari achieved the maximum P(3HB) accumulation of 55.8 % with a P(3HB) titer of 5.16 g/L (0.23 g/g Sugar). The extracted polymer was confirmed as P(3HB) and exhibited enhanced thermostability. Furthermore, P(3HB) was successfully synthesized from ball-milled corn stover hydrolysate under optimized conditions with the P(3HB) titer reached 7.66 g/L (0.33 g/g Sugar), which was higher than the pure mixed sugar system and other reports on lignocellulose hydrolysates with batch fermentation (<5 g/L). Therefore, P. sacchari exhibits the potential for P(3HB) production using corn stover hydrolysates as an inexpensive substrate.

A sustainable synthesis of polyhydroxyalkanoate from stubble waste as a carbon source using Pseudomonas putida MTCC 2475.

Polyhydroxyalkanoates (PHAs) are biodegradable polymers that can be produced from lignocellulosic biomass by microorganisms. Cheap and readily available raw material, such as corn stover waste, has the potential to lessen the cost of PHA synthesis. In this research study, corn stover is pretreated with NaOH under conditions optimized for high cellulose and low lignin with central composite design (CCD) followed by characterization using Fourier-transform infrared spectroscopy (FTIR), thermal gravimetric analysis (TGA), and scanning electron microscopy (SEM). Design expert software performed further optimization of alkali pretreated corn stover for high total reducing sugar (TRS) enhancement using CCD using response surface methodology (RSM). The optimized condition by RSM produced a TRS yield of 707.19mg/g. Fermentation using corn stover hydrolysate by Pseudomonas putida MTCC 2475 gave mcl-PHA detected through gas c hromatography - t andem m ass s pectrometry (GC-MS/MS) and characterization of the PHA film by differential scanning calorimetry (DSC), FTIR, and nuclear magnetic resonance (NMR). Thus, this research paper focuses on using agriculture (stubble) waste as an alternative feedstock for PHA production.

Mutation breeding of high-stress resistant strains for succinic acid production from corn straw

The production of succinic acid from corn stover is a promising and sustainable route; however, during the pretreatment stage, byproducts such as organic acids, furan-based compounds, and phenolic compounds generated from corn stover inhibit the microbial fermentation process. Selecting strains that are resistant to stress and utilizing nondetoxified corn stover hydrolysate as a feedstock for succinic acid production could be effective. In this study, A. succinogenes CICC11014 was selected as the original strain, and the stress-resistant strain A. succinogenes M4 was obtained by atmospheric and room temperature plasma (ARTP) mutagenesis and further screening. Compared to the original strain, A. succinogenes M4 exhibited a twofold increase in stress resistance and a 113% increase in succinic acid production when hydrolysate was used as the substrate. By conducting whole-genome resequencing of A. succinogenes M4 and comparing it with the original strain, four nonsynonymous gene mutations and two upstream regions with base losses were identified.Key points• A high-stress-resistant strain A. succinogenes M4 was obtained by ARTP mutation• The production of succinic acid increased by 113%• The mutated genes of A. succinogenes M4 were detected and analyzedGraphical

Extraction, recovery, and characterization of lignin from industrial corn stover lignin cake

Lignin utilization in value-added co-products is an important component of enabling cellulosic biorefinery economics. However, aqueous dilute acid pretreatments yield lignins with limited applications due to significant modification during pretreatment, low solubility in many solvents, and high content of impurities (ash, insoluble polysaccharides). This work addresses these challenges and investigates the extraction and recovery of lignins from lignin-rich insoluble residue following dilute acid pretreatment and enzymatic hydrolysis of corn stover using three extraction approaches: ethanol organosolv, NaOH, and an ionic liquid. The recovered lignins exhibited recovery yields ranging from 30% for the ionic liquid, 44% for the most severe acid ethanol organosolv condition tested, and up to 86% for the most severe NaOH extraction condition. Finally, the fractional solubilities of different recovered lignins were assessed in a range of solvents and these solubilities were used to estimate distributions of Hildebrand and Hansen solubility parameters using a novel approach.

Monitoring Yeast Cultures Grown on Corn Stover Hydrolysate for Lipid Production

Microbial oils can be used as an alternative sustainable and renewable feedstock to fossil reserves for producing lubricants and polyurethane materials. Two oleaginous yeasts were grown on non-detoxified corn stover hydrolysate supplemented with corn steep liquor and mineral medium in shake flasks. Trichosporon oleaginosus DSM 11815 displayed the highest lipid production. This strain was further cultivated in a bench bioreactor, using the same culture medium, under a batch regime. Flow cytometry was used to monitor the T. oleaginosus culture using the dual staining technique (SYBR Green and PI) for cell membrane integrity detection. Values of 42.28% (w/w) and 0.06 g/Lh lipid content and lipid productivity, respectively, were recorded for T. oleaginosus cultivated in the bench bioreactor operated under a batch regime. During the cultivation, most of the yeast cells maintained their integrity. T. oleaginosus has the potential to be used as an oil microbial source for a wide range of industrial applications. In addition, it is robust in adverse conditions such as lignocellulosic hydrolysate exposure and oxygen-limiting conditions. Flow cytometry is a powerful and useful tool for monitoring yeast cultivations on lignocellulosic hydrolysates for cell count, size, granularity, and membrane integrity detection.

Dynamic regulation and cofactor engineering of escherichia coli to enhance production of glycolate from corn stover hydrolysate

Glycolic acid is widely employed in chemical cleaning, the production of polyglycolic acid-lactic acid, and polyglycolic acid. Currently, the bottleneck of glycolate biosynthesis lies on the imbalance of metabolic flux and the deficiency of NADPH. In this study, a dynamic regulation system was developed and optimized to enhance the metabolic flux from glucose to glycolate. Additionally, the knockout of transhydrogenase (sthA), along with the overexpression of pyridine nucleotide transhydrogenase (pntAB) and the implementation of the Entner-Doudoroff pathway, were performed to further increase the production of the NADPH, thereby increasing the titer of glycolate to 5.6 g/L. To produce glycolate from corn stover hydrolysate, carbon catabolite repression was alleviated and glucose utilization was accelerated. The final strain, E. coli Mgly10-245, is inducer-free, achieving a glycolate titer of 46.1 g/L using corn stover hydrolysate (77.1 % of theoretical yield). These findings will contribute to the advancement of industrial glycolate production.

Pilot-scale acetone-butanol-ethanol fermentation from corn stover

Biobutanol is an advanced biofuel that can be produced from excess lignocellulose via acetone-butanol-ethanol (ABE) fermentation. Although significant technological progress has been made in this field, attempts at large-scale lignocellulosic ABE production remain scarce. In this study, 1 m3 scale ABE fermentation was investigated using high inhibitor tolerance Clostridium acetobutylicum ABE-P1201 and steam-exploded corn stover hydrolysate (SECSH). Before expanding the fermentation scale, the detoxification process for SECSH was simplified by process engineering. Results revealed that appropriate pH management during the fed-batch cultivation could largely decrease the inhibition of the toxic components in undetoxified SECSH to the solventogenesis phase of the ABE-P1201 strains, avoiding “acid crash”. Therefore, after naturalizing the pH by Ca(OH)2, the undetoxified SECSH, without removal of the solid components, reached 17.68 ± 1.30 g/L of ABE production with 0.34 ± 0.01 g/g of yield in 1 L scale bioreactor. Based on this strategy, the fermentation scale gradually expanded from laboratory-scale apparatus to pilot-scale bioreactors. Finally, 17.05 ± 1.20 g/L of ABE titer and 0.32 ± 0.01 g/g of ABE yield were realized in 1 m3 bioreactor, corresponding to approximately 145 kg of ABE production from 1 t of dry corn stover. The pilot-scale ABE fermentation demonstrated excellent stability during repeated operations. This study provided a simplified ABE fermentation strategy and verified the feasibility of the pilot process, providing tremendous significance and a solid foundation for the future industrialization of second-generation ABE plants.