Saccharification Of Corn Stover Research Articles

Sequential pretreatment with hydroxyl radical and manganese peroxidase for the efficient enzymatic saccharification of corn stover

Sequential pretreatment with hydroxyl radical and manganese peroxidase for the efficient enzymatic saccharification of corn stover

Integration of agricultural residues as biomass source to saccharification bioprocess and for the production of cellulases from filamentous fungi.

The production of second-generation bioethanol has several challenges, among them finding cheap and efficient enzymes for a sustainable process. In this work, we analyzed two native fungi, Cladosporium cladosporioides and Penicillium funiculosum, as a source of cellulolytic enzyme production, and corn stover, wheat bran, chickpeas, and bean straw as a carbon source in two fermentation systems: submerged and solid fermentation. Corn stover was selected for cellulase production in both fermentation systems, because we found the highest enzymatic activities when carboxymethyl cellulase activity (CMCase) was assessed using CMC as substrate. C. cladosporioides showed the highest CMCase activity (1.6U/mL), while P. funiculosum had the highest filter paper activity (Fpase) (0.39U/mL). The ß-glucosidase activities produced by both fungi were similar in submerged fermentation using corn stover as substrate. Through in-gel zymography, three polypeptides with cellulolytic activities were identified in each fungus: with molecular weights of ~ 38, 45 and 70kDa in C. cladosporioides and ~ 21, 63 and 100kDa in P. funiculosum. The best results for saccharification (10.11g/L of reducing sugars) of diluted acid pretreated corn stover were obtained after 36h of the hydrolytic process at pH 5 and 50°C using the enzyme extract of P. funiculosum. This is the first report of cellulase identification in C. cladosporioides and the saccharification of corn stover using enzymes of this fungus. Enzymatic extracts of C. cladosporioides and P. funiculosum obtained from low-cost lignocellulosic biomass have great potential for use in the production of second-generation bioethanol.

Combinations of mild chemical and bacterial pretreatment for improving enzymatic saccharification of corn stover

Biological pretreatment of lignocellulosic biomass plays an important role for the enzymatic hydrolysis. In this study, a nitrogen-fixing and lignin-degrading strain of bacteria was isolated from an abandoned termite colony. Then, it was identified and named as R. ornithinolytica RS-1. To improve the degradation and enzymatic saccharification in corn stover, we used pretreatment with strain RS-1 to deplete the lignin, combined with a 2.5% mild NaOH pretreatment to further deplete the hemicellulose. After only seven days, lignin was degraded by the bacteria RS-1 with a 19% decrease, while the relative content of cellulose increased with 21%. Furthermore, the corn stover cellulose conversion was up to 48.58% by a two-stage process with 2.5% NaOH pretreatment. Meanwhile, significant lignin and hemicellulose removal were observed. Manganese peroxidase activity was highest on Day 3 with the value 181.0256 U/L and lignin peroxidase activity was highest on Day 5 with the value 37.473 U/L, respectively. Scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FT-IR) and X-ray diffraction (XRD) analyses showed significant chemical structural changes after the combined pretreatment. Therefore, strain RS-1 provided an efficient strategy to enhance the enzymatic hydrolysis efficiency by pretreatment of corn stover.

Improving enzymatic saccharification of corn stover via thioglycolic acid-mediated Fenton pretreatment

Fenton pretreatment is known as a green and mild approach, but it is usually time-consuming and inefficient in promoting lignocellulose enzymatic hydrolysis. Here we demonstrate that thioglycolic acid (TA) significantly accelerates Fenton pretreatment (FP) of corn stover (CS) and improves enzymatic hydrolysis of cellulose and xylan. The results showed that the reducing sugar yield of CS reached 77.0% with TA-assisted FP in only 16 h, while it was only 34.1% and 38.0% with FP alone in 16 h and 72 h, respectively. Via examination of the changes of composition, physical and chemical characteristics of CS before and after pretreatment, it was found that TA-assisted FP greatly improved the removal of hemicellulose and lignin, resulting in increment of the pore volume, pore size and cellulose exposure of CS substrate, which together promoted the enzymatic saccharification of CS.

Statistical optimization of aqueous ammonia pretreatment and enzymatic saccharification of corn stover for enhancing sugars production

Response surface methodology (RSM) was adopted to optimize aqueous ammonia pretreatment and enzymatic saccharification of corn stover for enhancing sugars production, respectively. Pretreatment variables including pretreatment temperature, aqueous ammonia concentration and pretreatment time and saccharification variables including reaction time, enzyme loading and biomass loading were successfully found to have significant effects on sugars yields. Results indicated that corn stover powder (CSP, particle size < 0.3 mm) pretreated using 13.2 wt % aqueous ammonia solution at 70.0 °C for 7.9 h along with 20.0% ( w/v ) of solid loading was suitable to be applied to saccharification. The optimized pretreatment conditions could result in 98.3% of cellulose recovery, 86.5% of hemicellulose recovery and 60.7% of lignin removal, respectively. After optimization of enzymatic saccharification, maximum yields of reducing sugar (728.33 mg/gds) and xylose (290.43 mg/gds) along with 93.8% of holocellulose conversion and 80.6% of hemicellulose conversion could be obtained while the pretreated CSP was saccharified at 50.0 °C, pH 4.8 for 37.6 h by adopting 12.3 FPU/gds of enzyme loading, 7.6% ( w/v ) of biomass loading and 0.5% ( w/v ) of Tween-80, respectively. This is the first report about systematic optimization of aqueous ammonia pretreatment and enzymatic saccharification of CSP using self production cellulases for enhancing sugars yields in one study. • Aqueous ammonia pretreatment of corn stover was statistically optimized. • Enzymatic saccharification of aqueous ammonia pretreated corn stover was optimized. • Cellulose recovery (98.3%) and hemicellulose recovery (86.5%) were obtained. • Conversions of holocellulose (93.8%) and hemicellulose (80.6%) were obtained.

Enhancement of saccharification of corn stover by cellulolytic enzyme produced from biomass-degrading bacteria

Enzymatic saccharification of corn stover can be enhanced by partially replacing commercial enzymes with bacterial crude enzyme extracts. Thus, in this study, three bacteria (Bacillus sp. A0, Bacillus sp. CH20S1, and Exiguobacterium sp. AS2B) were cultured in a media with corn stover as the substrate to produce crude enzyme extract and saccharify corn stover. The cultural conditions were monitored and optimized to maximize CMCase and xylanase activity in the crude enzyme extracts. After 72 h of hydrolysis of corn stover with diluted crude enzymes (DCE) from the three strains, reducing sugars ranging from 48.2 to 71.7 mg g-1 were released from non-pretreated and pretreated corn stover. Furthermore, the maximum reducing sugars of 316 and 321 mg g-1 were observed when 12 and 4 FPU g-1 of commercial cellulase were added to the DCE of the CH20S1 strain, respectively. It was shown that an effective combination of bacterial DCE with commercial enzymes could achieve higher saccharification of lignocellulosic biomass, which might be cost-efficient compared to their single-use. Overall, this study aims to show the enhanced enzymatic saccharification of corn stover.

Pretreatment Affects Profits From Xylanase During Enzymatic Saccharification of Corn Stover Through Changing the Interaction Between Lignin and Xylanase Protein.

Effective pretreatment is vital to improve the biomass conversion efficiency, which often requires the addition of xylanase as an accessory enzyme to enhance enzymatic saccharification of corn stover. In this study, we investigated the effect of two sophisticated pretreatment methods including ammonium sulfite (AS) and steam explosion (SE) on the xylanase profits involved in enzymatic hydrolysis of corn stover. We further explored the interactions between lignin and xylanase Xyn10A protein. Our results showed that the conversion rates of glucan and xylan in corn stover by AS pretreatment were higher by Xyn10A supplementation than that by SE pretreatment. Compared with the lignin from SE pretreated corn stover, the lignin from AS pretreated corn stover had a lower Xyn10A initial adsorption velocity (13.56 vs. 10.89 mg g−1 min−1) and adsorption capacity (49.46 vs. 27.42 mg g−1 of lignin) and weakened binding strength (310.6 vs. 215.9 L g−1). Our study demonstrated the low absolute zeta potential and strong hydrophilicity of the lignin may partly account for relative weak interaction between xylanase protein and lignin from AS pretreated corn stover. In conclusion, our results suggested that AS pretreatment weakened the inhibition of lignin to enzyme, promoted the enzymatic hydrolysis of corn stover, and decreased the cost of enzyme in bioconversion.

Efficient preparation of soluble inducer for cellulase production and saccharification of corn stover using in-house generated crude enzymes

Although high-level cellulase production by Trichoderma reesei from lignocellulosic biomass in biorefinery has been widely studied, more efficient and economic hydrolysis is required to reduce the production cost. In this study, the recombinant T. reesei PB-3 strain overexpressing the β-glucosidase gene was used to achieve a high cellobiase activity of 78.09 CBU/mL using glucose as the sole substrate using fed‑batch fermentation. The obtained crude cellobiase was then used for transglycosylation, and kinetic studies showed that high concentration of glucose as the substrate is required for the reaction. We found that remarkably shorter transglycosylation time (2 h) than that was commonly reported (72 h), was sufficient when using the optimised conditions. Subsequently, the crude cellobiase from T. reesei PB-3 strain was also used to blend with the crude cellulase produced by T. reesei Rut C30 for hydrolysing corn stover, and a high glucose yield (97.08%) was achieved. The strategy developed in this work benefits reduction of cellulase production cost through in-house cellulase production for both inducer preparation and hydrolysis of the biomass.

Pretreatment of Corn Stover Using an Extremely Low-Liquid Ammonia (ELLA) Method for the Effective Utilization of Sugars in Simultaneous Saccharification and Fermentation (SSF) of Ethanol

Extremely low-liquid ammonia (ELLA) pretreatment using aqueous ammonia was investigated in order to enhance the enzymatic saccharification of corn stover and subsequent ethanol production. In this study, corn stover was treated with an aqueous ammonia solution at different ammonia loading rates (0.1, 0.2, and 0.3 g NH3/g biomass) and various liquid-to-solid (L/S) ratios (0.55, 1.12, and 2.5). The ELLA pretreatment was conducted at elevated temperatures (90–150 °C) for an extended period (24–120 h). Thereafter, the pretreated material was saccharified by enzyme digestion and subjected to simultaneous saccharification and fermentation (SSF) tests. The effects of key parameters on both glucan digestibility and xylan digestibility were analyzed using analysis of variance (ANOVA). Under optimal pretreatment conditions (L/S = 2.5, 0.1 g-NH3/g-biomass, 150 °C), 81.2% glucan digestibility and 61.1% xylan digestibility were achieved. The highest ethanol yield achieved on the SSF tests was 85.4%. The ethanol concentration was 14.5 g/L at 96 h (pretreatment conditions: liquid-to-solid ratio (L/S) = 2.5, 0.1 g-NH3/g-biomass, 150 °C, 24 h. SSF conditions: microorganism Saccharomyces cerevisiae (D5A), 15 FPU/g-glucan, CTech2, 3% w/v glucan, 37 °C, 150 rpm).

Thread Rolling: An Efficient Mechanical Pretreatment for Corn Stover Saccharification

Sugar cane bagasse and corn stalks are rich in lignocellulose, which can be degraded into monosaccharides through enzymatic hydrolysis. Appropriate pretreatment methods can effectively improve the efficiency of lignocellulose enzymatic hydrolysis. To enhance the efficiency of enzymatic hydrolysis, thread rolling pretreatment as a physical pretreatment was applied in this study. The influence of raw material meshes size after pretreatment was also taken as the research target. Specific surface area analysis, Scanning electron microscope (SEM), X-rays diffraction (XRD), and Fourier transform infrared (FT-IR) were used for characterizations. The results showed that, the total monosaccharide recovery rates of the raw materials, 20–40 mesh, 40–60 mesh, and 60–80 mesh enzymolysis substrates were 17.6%, 34.58%, 37.94%, and 50.69%, respectively. The sample after pretreatment showed a better recovery of monosaccharide than that of the raw material. Moreover, the enzymolysis substrates with a larger mesh exhibited a higher recovery of monosaccharide than that of the enzymolysis substrates with smaller meshes. This indicated that thread rolling pretreatment can effectively improve the efficiency of enzymatic hydrolysis.

Oxidative submerged lime pretreatment and high-solids saccharification of corn stover

Economic commercial-scale production of biofuels from lignocellulose depends, in part, on the development of a suitable pretreatment. In pursuit of that goal, this study assesses the repeatability and effectiveness of submerged lime pretreatment (SLP) of two batches of corn stover designated “Field” and “Module.” SLP was performed with 0.15 g Ca(OH)2 g−1 biomass and 10 g H2O g−1 biomass at 50 °C for 4 weeks while purging with air. SLP was repeated 14 times and the results of the mass balances, closures, and compositions of raw and SLP corn stover are presented. The glucan content of corn stover prior to pretreatment was determined to be 36.1 ± 0.7% (Module washed), 37.5 ± 0.4% (Field washed), and 32.6 ± 1.4% (Field unwashed). For washed corn stover (Module and Field), the standard deviation and coefficient of variation were <1%, demonstrating that the measurements were repeatable. For unwashed Field corn stover, because of its higher content of water-soluble components, the variability was slightly higher. The mass closures were above 96% with less than 2% standard deviation. The glucan content of SLP corn stover was 44.3 ± 0.8% (Module washed), 41.5 ± 0.5% (Field washed), and 42.9 ± 2.2% (Field unwashed). This study also presents a reliable analytical method for assessing the enzymatic digestibility of high-solids (15%) lignocellulose. Inositol is used as an internal standard to determine the final reactant volume, which is difficult in high-solids saccharification. After SLP, the overall glucan digestibility was ~80% (standard deviation < 4%, coefficient of variation < 5%), supporting the potential of SLP as a reproducible, cost-effective, and scalable pretreatment.

Protective effects of five surfactants on cellulase in the saccharification of corn stover based on the impeded Michaelis-Menten model

Protective effects of five surfactants were investigated relative to the saccharification of lignocellulose using the impeded Michaelis-Menten model (IMM). The yield of total reducing sugar (Ytrs) and cellulase activity were indexed as the effect of surfactant. The IMM was used to fit the correlation between Ytrs and reaction time to obtain the index (Kobs,0) reflecting the accessibility between cellulose and lignocellulose and the comprehensive index (Ki) reflecting cellulase inactivation and non-specific site adsorption. Results showed that the strongest protective effect was found from polyoxyethylene (80) sorbitan monooleate, followed by rhamnolipid. The surfactants protected cellulase from inactivation and nonspecific site adsorption of lignocellulose in the saccharification, leading to enhanced cellulase activity, especially with respect to carboxymethyl cellulase (CMCase) and filter paper enzyme (FPase) activities. The maximum Ytrs was obtained when the CMCase activity was 136.2 U/mL, while the FPase and β-glucosidase activities should be as high and low as possible, respectively, under the optimized condition. These findings lay the foundation for improving the saccharification efficiency of cellulase and reducing the cost of saccharification of biomass cellulose.

Statistical optimization of simultaneous saccharification fermentative hydrogen production from corn stover

ABSTRACT Corn stovers are rich in carbohydrates and can be used by anaerobic bacteria to produce hydrogen by fermentation. In the present study, using hydrogen production as the main experimental index, the effect of different influential factors on hydrogen production from corn stover saccharification and fermentation was studied, using the response surface method BBD model. The significance of interactions between different influential factors on hydrogen production by simultaneous saccharification and fermentation of corn stover material were investigated and optimized. Results showed that there were several factors affecting simultaneous saccharification fermentative hydrogen production from corn stover, including substrate concentration, inoculation amount, pH value and enzyme concentration. In linear terms, substrate concentration had the greatest influence on hydrogen production by anaerobic simultaneous saccharification and fermentation. In terms of multi-factor interactions, the interaction between pH and enzyme concentration was the most significant. The optimal hydrogen production conditions established from the BBD model were as follows: substrate concentration of 25 mg/mL, inoculation amount proportion of 32.62%, initial pH value of 6.50 and enzyme concentration of 172.08 mg/g, resulting in the maximum hydrogen production of 55.29 mL/g TS. The actual maximum hydrogen production reached 56.66 mL/g TS, with these experimental results consistent with the predicted value established from equation fitting. This study provides a reference for hydrogen production by anaerobic synchronous saccharification fermentation using corn stover as substrate and lays a foundation and provides technical support for the industrialization of biological hydrogen production using corn stover as substrate.

Improving cellulase recycling efficiency by decreasing the inhibitory effect of unhydrolyzed solid on recycled corn stover saccharification

Recycling enzymes associated with unhydrolyzed solid residue offers a potential strategy to reduce the amount of enzyme used during production of biofuels from lignocellulosic biomass. In this work, the hydrolytic efficiency of the enzymes associated with unhydrolyzed corn stover residue is evaluated and strategies to enhance their recycling efficiency are developed. We found whether direct recycling of corn stover residue could boost the recycling hydrolysis was dependent on the dosage of fresh cellulases. Under the investigated conditions, the addition of 5 FPU/g DM cellulases and BSA (2.5 mg/mL) could take the most advantage of the bound enzymes and suppress the negative effects caused by recalcitrant solid residues. The enzyme loading could be reduced by approximately 50% to achieve the same glucose yields (approximately 85%). This provided a highly promising technology for the optimization of solid residue recycling process and achieved the ultimate goal of cheaper and more efficient hydrolysis of lignocellulosic biomass.

Effect of crystallinity on pretreatment and enzymatic hydrolysis of lignocellulosic biomass based on multivariate analysis

In this work, multivariate data analysis was employed to correlate variables of pretreatment process of lignocellulosic biomass. Principal component analysis and partial least square methods were performed to get the inner-relationship and data interpretation between the crystallinity and other parameters of mechanical refining-assisted sodium hydroxide pretreatment followed by enzymatic saccharification of corn stover. The PCA and PLS models showed that Sodium hydroxide dosage, mechanical refining treatment, lignin removal rate and crystallinity had close inner-related relationship with the efficiency of pretreatment and enzymolysis. Alkaline reaction and mechanical refining treatment had strong influence on the crystallinity. Multivariate data analysis revealed that pretreated corn stover samples with lower crystallinity were more easily hydrolyzed by enzyme and could get more final reducing sugar. This work could offer a new methodology to get further understanding of effect of crystallinity on the crop residue lignocellulosic biomass conversion process.

Neural Network Prediction of Corn Stover Saccharification Based on Its Structural Features.

The classic assay for a large population biomass is time-consuming, labor intensive, and chemically expensive. This paper would find out a rapid assay for predicting biomass digestibility from biomass structural features without hydrolysis. We examined the 62 representative corn stover accessions that displayed a diverse cell-wall composition and varied biomass digestibility. Correlation analysis was firstly to detect effects of cell-wall compositions and wall polymer features on corn stover digestibility. Based on the dependable relationship of structural features and digestibility, a neural networks model has been developed and successfully predicted the corn stover saccharification based on the features without enzymatic hydrolysis. The actual measured and net-simulated predicted corn stover saccharification had good results as mean square error of 1.80E-05, coefficient of determination of 0.942 and average relative deviation of 3.95. The trained networks satisfactorily predicted the saccharification results based on the features of corn stover. Predicting the corn stover saccharification without hydrolysis will reduce capital and operational costs for corn stover purchasing and storage.

One-pot NaOH/urea pretreatment and saccharification of corn stover for fermentable sugar production

Conversion of lignocellulose into fermentable sugars and other chemicals usually requires multi-step unit operations, such as pretreatment, filtration/washing, and enzymatic saccharification and fermentation, which are the core steps responsible for increased operating expenses. A low-temperature NaOH/urea solution was shown to be an efficient cellulose solvent for overcoming the recalcitrance of lignocellulose by partially or fully converting rigid cellulose I crystallite into the more easily digestible cellulose II structure and by extracting a majority portion of the lignin and xylan from the lignocellulose. Higher yields of fermentable sugars were produced directly from corn stover in one vessel. This one-pot production of fermentable sugars was achieved via a combination process, including pretreatment with low-temperature NaOH/urea solution, pH adjustment, and enzymatic saccharification in a single reactor. This one-pot process liberated 86.3% of glucose and 71.3% of xylose in 24 h at an enzyme loading of 10 FPU/g and solid loading of 5%. Surfactant addition further enhanced enzymatic saccharification. The combination of low-temperature NaOH/urea pretreatment and enzymatic saccharification into a one-pot process is an efficient method for the conversion of lignocellulose into fermentable sugars suitable for conversion into fuels and other chemicals. Further studies related to lignin recovery and economical evaluation will be conducted.

Structure and saccharification of corn stover pretreated with sulfur trioxide micro-thermal explosion and dilute alkali (STEX-DA)

Due to its different organizational structures, dense hard husk, and loose soft core, corn stover (CS) is more resistant to transformation into monosaccharides for biofuel production in comparison to rice straw or wheat straw. In this paper, before pretreatment with sulfur trioxide micro-thermal explosion and dilute alkali (STEX-DA), CS was cut into 2 cm to 3 cm lengths in the transverse direction and cross opening in the vertical direction. During the process of STEX-DA, the structures of cores of corn stover (CS-C) and husks of corn stover (CS-H) were separately studied via scanning electron microscopy (SEM), X-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FT-IR) analysis. Furthermore, the components content and reducing sugars yield were calculated. The results showed that the CS-C achieved 66.7% of lignin removal rate, and the reducing sugars yield (calculated by original dry weight of straw) was 70.8%, while the CS-H displayed values of 64.7% and 55.3%, respectively. This result indicated that STEX-DA pretreatment could facilitate corn stover usage as a renewable energy source.

Pretreatment of Corn Stover Using Organosolv with Hydrogen Peroxide for Effective Enzymatic Saccharification

In this study, the chemical pretreatment method using ethanol organosolv with hydrogen peroxide was investigated to improve enzymatic saccharification of corn stover. The pretreatment method using ethanol with hydrogen peroxide in a flow-through reaction was proposed to lower the reaction severity such as the pretreatment temperature. With the same reaction time, the pretreatment process using organosolv (30 wt.% ethanol) containing 1 wt.% hydrogen peroxide at 150 °C resulted in a similar conversion yield as the result of the alkali pretreatment method using 15 wt.% aqueous ammonia at 170 °C. When corn stover was pretreated with 30 wt.% ethanol solution containing 5 wt.% hydrogen peroxide, a glucose conversion yield of 69.7 wt.% and glucose production of 23.8 g were achieved.

Effective Saccharification of Corn Stover Using Low-Liquid Aqueous Ammonia Pretreatment and Enzymatic Hydrolysis.

Low-liquid aqueous ammonia (LLAA) pretreatment using aqueous ammonia was investigated to enhance enzymatic saccharification of corn stover. In this method, ground corn stover was simply contacted with aqueous ammonia mist (ammoniation step), followed by pretreatment at elevated temperature (90–150 °C) for an extended period (24–120 h) at different solid/liquid (S/L) ratios (0.29, 0.47 or 0.67), termed a pretreatment step. After that, excess (unreacted) ammonia was removed by evaporation, and the pretreated material was immediately saccharified by an enzyme without a washing step. The effects of key reaction parameters on both glucan digestibility and XMG digestibility were evaluated by analysis of variance (ANOVA). Under the best pretreatment conditions [S/L = 0.47, 0.16 (g NH3)/(g biomass), 90 °C, 24 h], LLAA pretreatment enhanced enzymatic digestibility from 23.1% for glucan and 11.3% for XMG (xylan + galactan + mannan) of untreated corn stover to 91.8% for glucan and 72.6% for XMG in pretreated solid.