Multistep Structural and Chemical Evaluation of Sugarcane Baggase, Pretreated With Alkali for Enhancing the Enzymatic Saccharification by Cellulase and Xylanase of the Pseudomonas sp. CVB-10 (MK443365) and Bacillus paramycoides T4 (MN370035) Mix-Culture System

The enzymatic saccharification of sugarcane bagasse was significantly increased by alkali pretreatment under mild conditions. The effectiveness of different concentrations of alkali and acid pretreatment of sugarcane bagasse for improving the enzymatic saccharification of lignocellulose has been evaluated. The sugarcane bagasse was characterized to contain 39.52% celluloses, 25.63% hemicelluloses, and 30.36% lignin. After that, sugarcane bagasse was pretreated with 5 and 10% of H2SO4 and NaOH at 121°C for 60 min. FTIR, XRD, and SEM analyses also showed significant molecular and surface structure changes of the sugarcane bagasse with 10% NaOH. Maximum saccharification was 489.5 mg/g from 10% NaOH pretreatment followed by 322.75, 301.25, and 276.6 mg/g from 10% H2SO4, 5% NaOH, and 5% H2SO4, respectively, which were 55.1, 32.0, 27.1, and 20.6 times higher than the that of the control. Cellulase and xylanase produced by Pseudomonas sp. CVB-10 (MK443365) and Bacillus paramycoides T4 (MN370035) were used to hydrolyze the pretreated bagasse, and the optimal condition was determined to be 30 h of the enzymatic reaction with the 3:1 ratio of enzymes under the temperature of 55°C, pH 5.0, and substrate concentration of 3%, leading to celluloses and hemicelluloses conversion in the enzymatic hydrolysis/saccharification that is more proficient.

One-Pot Ionic Liquid-Mediated Bioprocess for Pretreatment and Enzymatic Hydrolysis of Rice Straw with Ionic Liquid-Tolerance Bacterial Cellulase

Ionic liquid (IL) pretreatment of lignocellulose is an efficient method for the enhancement of enzymatic saccharification. However, the remaining residues of ILs deactivate cellulase, therefore making intensive biomass washing after pretreatment necessary. This study aimed to develop the one-pot process combining IL pretreatment and enzymatic saccharification by using low-toxic choline acetate ([Ch][OAc]) and IL-tolerant bacterial cellulases. Crude cellulases produced from saline soil inhabited Bacillus sp. CBD2 and Brevibacillus sp. CBD3 were tested under the influence of 0.5–2.0 M [Ch][OAc], which showed that their activities retained at more than 95%. However, [Ch][OAc] had toxicity to CBD2 and CBD3 cultures, in which only 32.85% and 12.88% were alive at 0.5 M [Ch][OAc]. Based on the specific enzyme activities, the sugar amounts produced from one-pot processes using 1 mg of CBD2 and CBD3 were higher than that of Celluclast 1.5 L by 2.0 and 4.5 times, respectively, suggesting their potential for further application in the biorefining process of value-added products.

A Hydrotrope Pretreatment for Stabilized Lignin Extraction and High Titer Ethanol Production

The biomass pretreatment strategies using organic acids facilitate lignin removal and enhance the enzymatic digestion of cellulose. However, lignin always suffers a severe and irreversible condensation. The newly generated C-C bonds dramatically affect its further upgrading. In this study, we used a recyclable hydrotrope (p-Toluenessulfonic acid, p-TsOH) to dissolve lignin under mild condition and stabilized lignin with a quenching agent (formaldehyde, FA) during extraction, achieving both value-added lignin extraction and efficient enzymatic saccharification of cellulose. Approximately 63.7% of lignin was dissolved by 80% (wt. %) p-TsOH with 1.5% FA addition at 80 o C, 30 min. The obtained lignin was characterized by FTIR spectroscopy, TGA, 2D HSQC NMR spectroscopy, and GPC. The results indicated that the extracted lignin exhibited excellent properties, such as light color, a low molecular weight (Mw, 5371 g/mol), and a narrow polydispersity (Mw/Mn, 1.63). The pretreated substrate was converted to ethanol via a quasi-simultaneous saccharification and fermentation process (Q-SSF). After fermentation of 60 h, the ethanol concentration reached 38.7±3.3 g/L which was equivalent to a theoretical ethanol yield of 82.9±2.2% based on the glucan content, while the residual glucose concentration was only 4.69±1.4 g/L. In short, this pretreatment strategy protected lignin to form new C-C linkages and improved the enzymatic saccharification of glucan for high-titer ethanol production.

Microwave Assisted Pretreatment of Szarvasi (Agropyron elongatum) Biomass to Enhance Enzymatic Saccharification and Direct Glucose Production

Biomass from perennial plants can be considered a carbon-neutral renewable resource. The tall wheatgrass hybrid Szarvasi-1 (Agropyron elongatum, hereafter referred to as “Szarvasi”) belongs to the perennial Poaceae representing a species, which can grow on marginal soils and produce large amounts of biomass. Several conventional and advanced pretreatment methods have been developed to enhance the saccharification efficiency of plant biomass. Advanced pretreatment methods, such as microwave-assisted pretreatment methods are faster and use less energy compared to conventional pretreatment methods. In this study, we investigated the potential of Szarvasi biomass as a biorefinery feedstock. For this purpose, the lignocellulosic structure of Szarvasi biomass was investigated in detail. In addition, microwave-assisted pretreatments were applied to Szarvasi biomass using different reagents including weak acids and alkali. The produced pulp, hydrolysates, and extracted lignin were quantitatively characterized. In particular, the alkali pretreatment significantly enhanced the saccharification efficiency of the pulp 16-fold compared to untreated biomass of Szarvasi. The acid pretreatment directly converted 25% of the cellulose into glucose without the need of enzymatic digestion. In addition, based on lignin compositional and lignin linkage analysis a lignin chemical model structure present in Szarvasi biomass could be established.

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.

Improving the Substrate Affinity and Catalytic Efficiency of β-Glucosidase Bgl3A from Talaromyces leycettanus JCM12802 by Rational Design

Improving the substrate affinity and catalytic efficiency of β-glucosidase is necessary for better performance in the enzymatic saccharification of cellulosic biomass because of its ability to prevent cellobiose inhibition on cellulases. Bgl3A from Talaromyces leycettanus JCM12802, identified in our previous work, was considered a suitable candidate enzyme for efficient cellulose saccharification with higher catalytic efficiency on the natural substrate cellobiose compared with other β-glucosidase but showed insufficient substrate affinity. In this work, hydrophobic stacking interaction and hydrogen-bonding networks in the active center of Bgl3A were analyzed and rationally designed to strengthen substrate binding. Three vital residues, Met36, Phe66, and Glu168, which were supposed to influence substrate binding by stabilizing adjacent binding site, were chosen for mutagenesis. The results indicated that strengthening the hydrophobic interaction between stacking aromatic residue and the substrate, and stabilizing the hydrogen-bonding networks in the binding pocket could contribute to the stabilized substrate combination. Four dominant mutants, M36E, M36N, F66Y, and E168Q with significantly lower Km values and 1.4–2.3-fold catalytic efficiencies, were obtained. These findings may provide a valuable reference for the design of other β-glucosidases and even glycoside hydrolases.