Alkali Pretreated Corn Stover Research Articles

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.

Conversion of Corn Stover for Microbial Enzymes Production by Phanerochaete chrysosporium.

Phanerochaete chrysosporium, a white rot fungus, exhibits remarkable capabilities in producing various extracellular enzymes. These microbial enzymes find extensive applications in disrupting the intricate structure of plant cell walls, decolorizing synthetic dyes, and facilitating pulp extraction, among other functions. The process of solid-state fermentation stands out as an economical and sustainable approach, ideal for achieving high yields in enzyme production using lignocellulosic biomass as a substrate. In this research paper, both untreated and alkali pretreated corn stover materials served as substrates for enzyme production, leveraging the fungal strain's capacity to generate enzymes like cellulases and manganese peroxidase. The maximum production of endoglucanase was notably observed, reaching 121.21 ± 0.90 U/gds on the 9th day for untreated biomass and 79.75 ± 0.57 U/gds on the 6th day for treated biomass. Similarly, the peak exoglucanase production was recorded at 2.46 ± 0.008 FPU/ml on the 3rd day for untreated biomass and 0.92 ± 0.002 FPU/ml on the 6th day for treated biomass. Furthermore, the highest production of manganese peroxidase was achieved, with values of 5076.81 U/l on the 6th day for untreated biomass and 1127.58 ± 0.23 U/l on the 3rd day for treated biomass. These results collectively emphasize the potential of corn stover as a renewable and promising substrate for the production of essential enzymes.

Engineering Trichoderma reesei for the hyperproduction of cellulose induced protein 1 (Cip1) on a sophorose-containing inducer to efficiently saccharify alkali-pretreated corn stover

Trichoderma reesei was induced to produce cellulase by a combination of glucose and β-disaccharide; however, lower levels of auxiliary proteins for degrading lignocellulosic biomass were detected by iTRAQ analysis compared with cellulose as an inducer, especially cellulose induced protein 1 (CIP1). In this study, A pdc1 promoter-driven overexpression of the endogenous Trcip1 gene was observed in T. reesei Rut C30, and the Trcip1 transcription levels of the two transformants, T. reesei OE-cip1-1 and OE-cip1-2, demonstrated 31.2- and 164.6-fold increases, respectively, but there was no significant change in cellobiohydrolase, endoglucanase and filter paper activity at 48 h. The crude enzyme was then used to hydrolyze corn stover. For T. reesei OE-cip1-1 and OE-cip1-2, the hydrolysis efficiency increased by 25.0 and 28.6% with a solid loading of 5% at 2 h, respectively. Simultaneously, 85.5 and 85.2 g/L glucose were released using a cellulase cocktail at high solid loading (20%), and these glucose release rates were significantly greater than that of T. reesei Rut C30 cellulase (77.4 g/L) at 120 h. Furthermore, scanning electron microscopy (SEM) and X-ray diffraction (XRD) showed that the enhanced hydrolysis efficiency was primarily triggered by the decrease in the crystallinity of lignocellulose, and the fiber structure had varying degrees of loosening and disintegration.

Rational engineering of xylanase hyper-producing system in Trichoderma reesei for efficient biomass degradation

BackgroundFilamentous fungus Trichoderma reesei has been widely used as a workhorse for cellulase and xylanase productions. Xylanase has been reported as the crucial accessory enzyme in the degradation of lignocellulose for higher accessibility of cellulase. In addition, the efficient hydrolysis of xylan needs the co-work of multiple xylanolytic enzymes, which rise an increasing demand for the high yield of xylanase for efficient biomass degradation.ResultsIn this study, a xylanase hyper-producing system in T. reesei was established by tailoring two transcription factors, XYR1 and ACE1, and homologous overexpression of the major endo-xylanase XYNII. The expressed xylanase cocktail contained 5256 U/mL xylanase activity and 9.25 U/mL β-xylosidase (pNPXase) activity. Meanwhile, the transcription level of the xylanolytic genes in the strain with XYR1 overexpressed was upregulated, which was well correlated with the amount of XYR1-binding sites. In addition, the higher expression of associated xylanolytic enzymes would result in more efficient xylan hydrolysis. Besides, 2310–3085 U/mL of xylanase activities were achieved using soluble carbon source, which was more efficient and economical than the traditional strategy of xylan induction. Unexpectedly, deletion of ace1 in C30OExyr1 did not give any improvement, which might be the result of the disturbed function of the complex formed between ACE1 and XYR1. The enzymatic hydrolysis of alkali pretreated corn stover using the crude xylanase cocktails as accessory enzymes resulted in a 36.64% increase in saccharification efficiency with the ratio of xylanase activity vs FPase activity at 500, compared to that using cellulase alone.ConclusionsAn efficient and economical xylanase hyper-producing platform was developed in T. reesei RUT-C30. The novel platform with outstanding ability for crude xylanase cocktail production would greatly fit in biomass degradation and give a new perspective of further engineering in T. reesei for industrial purposes.

Establishment of a highly efficient and low cost mixed cellulase system for bioconversion of corn stover by Trichoderma reesei and Aspergillus niger

The high cost of cellulase production is one of the main obstacles for the conversion of lignocellulose into high valued products. As a widely existing biomass resource, corn stover can be used to reduce the cost of cellulase production. In this study, Parameters of producing cellulase by Trichoderma reesei were optimized using Response Surface methodology, the optimum conditions were as follows: 3.84% solid acid pretreated corn stover, 1.19% yeast extract, initial pH of 5.01, 0.12% glycerol monostearate, 0.25% Na2HPO4, with 3% inoculum content for 12 days at 30 °C. Filter paper activity (FPA) of Trichoderma reesei was increased by 180% to 13.44 IU/mL. Enzymatic hydrolysis experiments were performed on different pretreated corn stovers with Trichoderma reesei cellulase produced under optimal conditions, and the highest cellulose conversion efficiency reached 62.39%. In addition, a blend of Trichoderma reesei cellulase at a concentration of 30 FPU/g cellulose and Aspergillus niger beta-glucosidase at a concentration of 50 FPU/g cellulose could effectively hydrolyse alkali pretreated corn stover, resulting in an improvement in the enzymatic hydrolysis efficiency by 17.26% compared to Trichoderma reesei cellulase alone. In parallel, the mixed enzymes achieved an equivalent enzymatic effect with that of commercial cellulase (Novozymes, Cellic Ctec2) at the same concentration. These data suggested that solid acid pretreated corn stover was a promising carbon source for fungal cellulase production for effective saccharification of corn stover biomass. Glycerol monostearate was first found to have a strong induction effect on enzyme production. The development of a high efficiency and low-cost mixed enzyme system had a potential application in future industrialization scale.

Enhance photocatalytic hydrogen evolution by using alkaline pretreated corn stover as a sacrificial agent

In this study, alkali pretreated corn stover was added as a sacrificial agent to the suspension of Pt/TiO2 to significantly enhanced photocatalytic hydrogen evolution. The changes in structural characteristics of corn stover before and after alkali treatment and photocatalytic reactions were studied. According to the results, the removal of lignin and hemicellulose, as well as the changes in surface morphology, structural components, and functional groups of alkali pretreated corn stover may affect the photocatalytic hydrogen production process. Next, the effects of NaOH concentration, pretreatment time and temperature on hydrogen production were also investigated. Among them, within the scope of the experimental conditions, the optimal hydrogen production is 25.84 μmol h−1. Moreover, the enhancement of photocatalytic hydrogen production is also achieved by using the waste liquid of alkali pretreated. The output of this study may provide a reference for the reuse of alkali-treated biomass residues and waste liquor in some industries, and make more comprehensive, efficient and green use of native lignocellulosic biomass.

Low-Cost Cellulase-Hemicellulase Mixture Secreted by Trichoderma harzianum EM0925 with Complete Saccharification Efficacy of Lignocellulose.

Fermentable sugars are important intermediate products in the conversion of lignocellulosic biomass to biofuels and other value-added bio-products. The main bottlenecks limiting the production of fermentable sugars from lignocellulosic biomass are the high cost and the low saccharification efficiency of degradation enzymes. Herein, we report the secretome of Trichoderma harzianum EM0925 under induction of lignocellulose. Numerously and quantitatively balanced cellulases and hemicellulases, especially high levels of glycosidases, could be secreted by T. harzianum EM0925. Compared with the commercial enzyme preparations, the T. harzianum EM0925 enzyme cocktail presented significantly higher lignocellulolytic enzyme activities and hydrolysis efficiency against lignocellulosic biomass. Moreover, 100% yields of glucose and xylose were obtained simultaneously from ultrafine grinding and alkali pretreated corn stover. These findings demonstrate a natural cellulases and hemicellulases mixture for complete conversion of biomass polysaccharide, suggesting T. harzianum EM0925 enzymes have great potential for industrial applications.

Microbial lipid production from dilute acid and dilute alkali pretreated corn stover via Trichosporon dermatis

Microbial lipid production from lignocellulosic biomass has attracted much attention recently. In this study, T. dermatis 32903 was selected from eleven promising oleaginous yeast strains. Carbon to nitrogen ratio (C/N) was investigated and optimized to maximize lipid production. Dilute acid (DA) pretreated corn stover (CS) and dilute alkali (AL) pretreated CS were then used for microbial lipid production, resulting in lipid concentrations of 7.46 g/L and 6.81 g/L, with sugar to lipid yields reached 0.104 g/g and 0.101 g/g, respectively. Washing of DA-CS and AL-CS enhanced lipid production to 11.43 g/L and 20.36 g/L with sugar to lipid yields improved to 0.156 g/g and 0.186 g/g, respectively. As degradation products in pretreated biomass showed severe inhibition on lipid fermentation, eight typical degradation products were further investigated for their effects on lipid fermentation. T. dermatis 32903 exhibited high tolerance to furan derivatives and week acids, but lower tolerance to phenolic compounds.

Developing fast enzyme recycling strategy through elucidating enzyme adsorption kinetics on alkali and acid pretreated corn stover

BackgroundAlthough various pre-treatment methods have been developed to disrupt the structure of lignocellulosic biomass, high dosage of cellulases is still required to hydrolyze lignocellulose to fermentable sugars. Enzyme recycling via recycling unhydrolyzed solids after enzymatic hydrolysis is a promising strategy to reduce enzyme loading for production of cellulosic ethanol.ResultsTo develop effective enzyme recycling method via recycling unhydrolyzed solids, this work investigated both enzymatic hydrolysis kinetics and enzyme adsorption kinetics on dilute acid and dilute alkali pre-treated corn stover (CS). It was found that most of the hydrolysable biomass was hydrolyzed in the first 24 h and about 40% and 55% of the enzymes were adsorbed on unhydrolyzed solids for dilute alkali-CS and dilute acid-CS, respectively, at 24 h of enzymatic hydrolysis. Lignin played a significant role in such adsorption and lignin materials derived from dilute acid-CS and dilute alkali-CS possessed different enzyme adsorption properties. Enzyme recycling was performed by recycling unhydrolyzed solids after 24 h enzymatic hydrolysis for five successive rounds, and successfully reduced 40% and 50% of the enzyme loadings for hydrolysis of dilute alkali-CS and for hydrolysis of dilute acid-CS, respectively.ConclusionsThis study presents that the enzymes adsorbed on the unhydrolyzed solids after short-time hydrolysis could be recycled effectively for efficient enzymatic hydrolysis. Lignin derived from dilute acid-CS has higher enzyme adsorption capacity than the lignin derived from dilute alkali-CS, which led to more enzymes recycled. By applying the enzyme recycling strategy developed in this study, the enzyme dosage needed for effective cellulose hydrolysis can be significantly reduced.

Optimization of cellulolytic enzyme components through engineering Trichoderma reesei and on-site fermentation using the soluble inducer for cellulosic ethanol production from corn stover

BackgroundCellulolytic enzymes produced by Trichoderma reesei are widely studied for biomass bioconversion, and enzymatic components vary depending on different inducers. In our previous studies, a mixture of glucose and disaccharide (MGD) was developed and used to induce cellulase production. However, the enzymatic profile induced by MGD is still not defined, and further optimization of the enzyme cocktail is also required for efficient ethanol production from lignocellulosic biomass.ResultsIn this study, cellulolytic enzymes produced by T. reesei Rut C30 using MGD and alkali-pretreated corn stover (APCS) as inducer were compared. Cellular secretome in response to each inducer was analyzed, which revealed a similar enzyme profile. However, significant difference in the content of cellulases and xylanase was detected. Although MGD induction enhanced β-glucosidase production, its activity was still not sufficient for biomass hydrolysis. To overcome such a disadvantage, aabgl1 encoding β-glucosidase in Aspergillus aculeatus was heterologously expressed in T. reesei Rut C30 under the control of the pdc1 promoter. The recombinant T. reesei PB-3 strain showed an improved β-glucosidase activity of 310 CBU/mL in the fed-batch fermentation, 71-folds higher than that produced by the parent strain. Meanwhile, cellulase activity of 50 FPU/mL was detected. Subsequently, the crude enzyme was applied for hydrolyzing corn stover with a solid loading of 20% through separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation, respectively, for ethanol production. Better performance was observed in the SHF process, through which a total of 119.9 g/L glucose was released within 12 h for concomitant ethanol production of 54.2 g/L.ConclusionsThe similar profile of cellulolytic enzymes was detected under the induction of MGD and APCS, but higher amount of cellulases was present in the crude enzyme induced by MGD. However, β-glucosidase activity induced by MGD was not sufficient for hydrolyzing lignocellulosic biomass. High titers of cellulases and β-glucosidase were achieved simultaneously by heterologous expression of aabgl1 in T. reesei and fed-batch fermentation through feeding MGD. We demonstrated that on-site cellulase production by T. reesei PB-3 has a potential for efficient biomass saccharification and ethanol production from lignocellulosic biomass.

Multi-Scale Structural Studies of Sequential Ionic Liquids and Alkali Pretreated Corn Stover and Sugarcane Bagasse

The complexity of plant cell walls impedes the conversion of cellulosic biomass to sugars. Pretreatment becomes a necessity to increase the digestibility of biomass. An in-depth understanding of the structure and underlying mechanisms governing deconstruction process is important. In the present study, the comprehensive investigation of morphological and structural changes in corn stover and sugarcane bagasse following ionic liquids dissolution and alkaline extraction was done using Fourier transform infrared spectroscopy, Thermogravimetric analysis, Confocal scanning laser microscopy, Atomic force microscopy and Dynamic light scattering studies. Both the substrates were pretreated with ILs 1-ethyl-3-methylimidazolium acetate and 1-butyl-3-methylimidazolium acetate followed by alkaline extraction. The pronounced changes such as lignin, hemicelluloses removal and decreased cellulose crystallinity after the pretreatments lead to the structural transformation of matrix polymers. The enzymatic hydrolysis showed 90% theoretical sugar yield in case of sugarcane bagasse and 80% in corn stover following synergistically combined pretreatments.

Paenibacillus panacisoli enhances growth of Lactobacillus spp. by producing xylooligosaccharides in corn stover ensilages

The knowledge about the association of lignocellulosic biomass-degrading microbes with lactic acid bacteria (LAB) in ensilages is still limited. Paenibacillus strains are important microbes in sustainable agriculture. Here, P. panacisoli SDMCC050309 was isolated from ensiled corn stover and used as an example to investigate the effects on LAB. This strain produced at least 7 xylanases, and two of them were purified and characterized. Temperature and pH optima were determined to be 55 °C and 8.0 for Xyn10 and 40 °C and 7.0 for Xyn11, respectively. They could degraded larch wood xylan and alkali-pretreated corn stover into xylooligosaccharides (XOS). Using the produced XOS to culture Lactobacillus brevis SDMCC050297 and L. parafarraginis SDMCC050300, both of them grew well with high level of acetic acid production. The same phenomenon was observed when co-culturing those two Lactobacillus strains with P. panacisoli SDMCC050309. Therefore, P. panacisoli enhances growth of LAB by producing XOS in corn stover ensilages.

A β-xylosidase hyper-production Penicillium oxalicum mutant enhanced ethanol production from alkali-pretreated corn stover

β-Xylosidase activity is deficient in most cellulase enzymes secreted by filamentous fungi, which limits effective enzymatic hydrolysis of hemicellulose in lignocellulose materials and resulted in accumulation of xylo-oligosaccharides that inhibit the cellulase and xylanase activitives. An endogenous β-xylosidase gene, xyl3A, was overexpressed using two types of promoters in cellulolytic P. oxalicum RE-10. The mutants RXyl, RGXyl-1 and RGXyl-2 displayed higher β-xylosidase production than native strain RE-10 besides higher cellulase and xylanase activities, especially RGXyl-1, showing the highest β-xylosidase activity of 15.05±1.79IU/mL, about 29 folds higher than native strain, more than the highest level reported by literature. Enzymatic hydrolysis results indicated the cellulase RGXyl-1 not only increased glucose and xylose yields and thus resulted in high ethanol yield during the simultaneous saccharification and fermentation, but decreased the total enzyme loading compared to starting RE-10, which indicated a good prospect of industrial application in bioconversion of lignocellulose.

On-site cellulase production and efficient saccharification of corn stover employing cbh2 overexpressing Trichoderma reesei with novel induction system

Although on-site cellulase production offers cost-effective saccharification of lignocellulosic biomass, low enzyme titer is still a barrier for achieving robustness. In the present study, a strain of T. reesei was developed for enhanced production of cellulase via overexpression of Cellobiohydrolase II. Furthermore, optimum enzyme production was achieved using a novel inducer mixture containing synthesized glucose-sophorose (MGD) and alkali pre-treated corn stover (APCS). Within 60h, a remarkably higher cellulase productivity and activity were achieved in the fed-batch fermentation using the optimized ratio of MGD and APCS in the inducer mixture, compared to those reported using cellulosic biomass as the sole inducer. After the enzyme production, APCS was added directly into the fermentation broth at 20% solid loading, which produced 122.5g/L glucose and 40.21g/L xylose, leading to the highest yield reported so far. The improved enzyme titers during on-site cellulase production would benefit cost-competitive saccharification of lignocellulosic biomass.

Utilizing Anaerobic Fungi for Two-stage Sugar Extraction and Biofuel Production from Lignocellulosic Biomass.

Lignocellulosic biomass is a vast and underutilized resource for the production of sugars and biofuels. However, the structural complexity of lignocellulosic biomass and the need for multiple pretreatment and enzymatic steps for sugar release renders this process economically challenging. Here, we report a novel approach for direct, single container, exogenous enzyme-free conversion of lignocellulosic biomass to sugars and biofuels using the anaerobic fungal isolate strain C1A. This approach utilizes simple physiological manipulations for timely inhibition and uncoupling of saccharolytic and fermentative capabilities of strain C1A, leading to the accumulation of sugar monomers (glucose and xylose) in the culture medium. The produced sugars, in addition to fungal hyphal lysate, are subsequently converted by Escherichia coli strain K011 to ethanol. Using this approach, we successfully recovered 17.0% (w/w) of alkali-pretreated corn stover (20.0% of its glucan and xylan content) as sugar monomers in the culture media. More importantly, 14.1% of pretreated corn stover (17.1% of glucan and xylan content) was recovered as ethanol at a final concentration of 28.16 mM after the addition of the ethanologenic strain K011. The high ethanol yield obtained is due to its accumulation as a minor fermentation end product by strain C1A during its initial growth phase, the complete conversion of sugars to ethanol by strain K011, and the possible conversion of unspecified substrates in the hyphal lysate of strain C1A to ethanol by strain K011. This study presents a novel, versatile, and exogenous enzyme-free strategy that utilizes a relatively unexplored group of organisms (anaerobic fungi) for direct biofuel production from lignocellulosic biomass.

Constitutive cellulase production from glucose using the recombinant Trichoderma reesei strain overexpressing an artificial transcription activator

The high cost of cellulase production presents biggest challenge in biomass deconstruction. Cellulase production by Trichoderma reesei using low cost carbon source is of great interest. In this study, an artificial transcription activator containing the Cre1 binding domain linked to the Xyr1 effector and binding domains was designed and constitutively overexpressed in T. reesei RUT C30. The recombinant strain T. reesei zxy-2 displayed constitutive cellulase production using glucose as a sole carbon source, and the production titer was 12.75-fold of that observed with T. reesei RUT C30 in shake flask culture. Moreover, FPase and xylanase titers of 2.63 and 108.72IU/mL, respectively, were achieved using glucose as sole carbon source within 48h in a 7-L fermenter by batch fermentation using T. reesei zxy-2. The crude enzyme obtained was used to hydrolyze alkali pretreated corn stover, and a high glucose yield of 99.18% was achieved.

Oleaginous fungal lipid fermentation on combined acid- and alkali-pretreated corn stover hydrolysate for advanced biofuel production.

A combined hydrolysis process, which first mixed dilute acid- and alkali-pretreated corn stover at a 1:1 (w/w) ratio, directly followed by enzymatic saccharification without pH adjustment, has been developed in this study in order to minimize the need of neutralization, detoxification, and washing during the process of lignocellulosic biofuel production. The oleaginous fungus Mortierella isabellina was selected and applied to the combined hydrolysate as well as a synthetic medium to compare fungal lipid accumulation and biodiesel production in both shake flask and 7.5L fermentor. Fungal cultivation on combined hydrolysate exhibited comparable cell mass and lipid yield with those from synthetic medium, indicating that the integration of combined hydrolysis with oleaginous fungal lipid fermentation has great potential to improve performance of advanced lignocellulosic biofuel production.

The Role of Polymeric Micelles on Chemical Changes of Pretreated Corn Stover, Cellulase Structure, and Adsorption

Enzymatic hydrolysis of lignocellulosic biomass is limited by rapid cellulase deactivation, consequently requiring large amounts of enzyme to maintain acceptable biomass conversion. In this study, a new approach to improve lignocellulose hydrolysis was investigated. Performing enzymatic hydrolysis of corn stover (CS) in the presence of polymeric–surfactant micelles (PMs) was demonstrated to improve hydrolysis yield to a greater extent than using only surfactant micelles. Application of 2 % (w/w) of polyethylene glycol (PEG 6000) with casein, Tween-20, and Triton X-100 at levels above the critical micelle concentrations increased the hydrolysis yield of CS containing high-bound lignin (extrusion-pretreated) by up to 87.8, 11.7, and 7.5 %, respectively. These PMs were not effective during enzymatic hydrolysis of biomass lacking lignin (Avicel) or alkali-pretreated CS (7.2 % lignin). The main reasons for the enhanced cellulase activity observed due to PEG-casein, PEG-Tween, and PEG-Triton were enhanced cellulase solubilization; reformation of α-helix substructure; and combination of induced cellulase solubilization, α-helix reformation, and chemical changes in the microstructure of biomass, respectively. Deformation of the cellulase substructure during hydrolysis of biomass and its subsequent reformation in the presence of surfactants were shown in this study for the first time. Chemical changes in the microstructure of biomass (e.g., lignin side changes, C–O bonds, and amorphous cellulose) were found to be another potential reason for the effectiveness of surfactants when they are incubated at above 6 g/L for 72 h with biomass.

Fungal Cellulase/Xylanase Production and Corresponding Hydrolysis Using Pretreated Corn Stover as Substrates

Three pretreated corn stover (ammonia fiber expansion, dilute acid, and dilute alkali) were used as carbon source to culture Trichoderma reesei Rut C-30 for cellulase and xylanase production. The results indicated that the cultures on ammonia fiber expansion and alkali pretreated corn stover had better enzyme production than the acid pretreated ones. The consequent enzymatic hydrolysis was performed applying fungal enzymes on pretreated corn stover samples. Tukey's statistical comparisons exhibited that there were significant differences on enzymatic hydrolysis among different combination of fungal enzymes and pretreated corn stover. The higher sugar yields were achieved by the enzymatic hydrolysis of dilute alkali pretreated corn stover.

Complex Physiology and Compound Stress Responses during Fermentation of Alkali-Pretreated Corn Stover Hydrolysate by an Escherichia coli Ethanologen

The physiology of ethanologenic Escherichia coli grown anaerobically in alkali-pretreated plant hydrolysates is complex and not well studied. To gain insight into how E. coli responds to such hydrolysates, we studied an E. coli K-12 ethanologen fermenting a hydrolysate prepared from corn stover pretreated by ammonia fiber expansion. Despite the high sugar content (∼6% glucose, 3% xylose) and relatively low toxicity of this hydrolysate, E. coli ceased growth long before glucose was depleted. Nevertheless, the cells remained metabolically active and continued conversion of glucose to ethanol until all glucose was consumed. Gene expression profiling revealed complex and changing patterns of metabolic physiology and cellular stress responses during an exponential growth phase, a transition phase, and the glycolytically active stationary phase. During the exponential and transition phases, high cell maintenance and stress response costs were mitigated, in part, by free amino acids available in the hydrolysate. However, after the majority of amino acids were depleted, the cells entered stationary phase, and ATP derived from glucose fermentation was consumed entirely by the demands of cell maintenance in the hydrolysate. Comparative gene expression profiling and metabolic modeling of the ethanologen suggested that the high energetic cost of mitigating osmotic, lignotoxin, and ethanol stress collectively limits growth, sugar utilization rates, and ethanol yields in alkali-pretreated lignocellulosic hydrolysates.