Digestibility of Bacillus firmus K-1 pretreated rice straw by different commercial cellulase cocktails.

Removal of xylan in plant biomass is believed to increase cellulose hydrolysis by uncovering cellulose surfaces for cellulase adsorption and, in turn, catalysis reaction. Herein, we describe an eco-friendly method by culturing a xylanolytic Bacillus firmus K-1 on rice straw to remove xylan. The bacterium was grown on 2.5% (w/v) rice straw with different biomass particle sizes for two days at 37 °C. We found that the particle sizes ranged from <1 to 5 mm gave a similar xylan removal degree (about 21%). Besides, the porosity and disintegration of the rice straw fibers were observed at the molecular level. The digestibility of pretreated rice straw was tested with different commercial cellulase cocktails. We found that the pretreated rice straw was more susceptible to enzymatic hydrolysis, giving 30-70% glucan conversion than the untreated one. The degree of cellulose hydrolysis depended strongly on the kinds of enzyme and their formulations. HighlightCulturing B. firmus K-1 on rice straw yielded about 21% removal of xylan.Particle sizes (of 1-5 mm) had negligible effects on xylan removal efficiency.The degree of glucan conversion in pretreated biomass relied on enzyme formulation.

Chemical Pretreatment-Independent Saccharifications of Xylan and Cellulose of Rice Straw by Bacterial Weak Lignin-Binding Xylanolytic and Cellulolytic Enzymes.

Complete utilization of carbohydrate fractions is one of the prerequisites for obtaining economically favorable lignocellulosic biomass conversion. This study shows that xylan in untreated rice straw was saccharified to xylose in one step without chemical pretreatment, yielding 58.2% of the theoretically maximum value by Paenibacillus curdlanolyticus B-6 PcAxy43A, a weak lignin-binding trifunctional xylanolytic enzyme, endoxylanase/ß-xylosidase/arabinoxylan arabinofuranohydrolase. Moreover, xylose yield from untreated rice straw was enhanced to 78.9% by adding endoxylanases PcXyn10C and PcXyn11A from the same bacterium, resulting in improvement of cellulose accessibility to cellulolytic enzyme. After autoclaving the xylanolytic enzyme-treated rice straw, it was subjected to subsequent saccharification by a combination of the Clostridium thermocellum endoglucanase CtCel9R and Thermoanaerobacter brockii ß-glucosidase TbCglT, yielding 88.5% of the maximum glucose yield, which was higher than the glucose yield obtained from ammonia-treated rice straw saccharification (59.6%). Moreover, this work presents a new environment-friendly xylanolytic enzyme pretreatment for beneficial hydrolysis of xylan in various agricultural residues, such as rice straw and corn hull. It not only could improve cellulose saccharification but also produced xylose, leading to an improvement of the overall fermentable sugar yields without chemical pretreatment.IMPORTANCE Ongoing research is focused on improving "green" pretreatment technologies in order to reduce energy demands and environmental impact and to develop an economically feasible biorefinery. The present study showed that PcAxy43A, a weak lignin-binding trifunctional xylanolytic enzyme, endoxylanase/ß-xylosidase/arabinoxylan arabinofuranohydrolase from P. curdlanolyticus B-6, was capable of conversion of xylan in lignocellulosic biomass such as untreated rice straw to xylose in one step without chemical pretreatment. It demonstrates efficient synergism with endoxylanases PcXyn10C and PcXyn11A to depolymerize xylan in untreated rice straw and enhanced the xylose production and improved cellulose hydrolysis. Therefore, it can be considered an enzymatic pretreatment. Furthermore, the studies here show that glucose yield released from steam- and xylanolytic enzyme-treated rice straw by the combination of CtCel9R and TbCglT was higher than the glucose yield obtained from ammonia-treated rice straw saccharification. This work presents a novel environment-friendly xylanolytic enzyme pretreatment not only as a green pretreatment but also as an economically feasible biorefinery method.

A novel GH6 cellobiohydrolase from Paenibacillus curdlanolyticus B-6 and its synergistic action on cellulose degradation.

We recently discovered a novel glycoside hydrolase family 6 (GH6) cellobiohydrolase from Paenibacillus curdlanolyticus B-6 (PcCel6A), which is rarely found in bacteria. This enzyme is a true exo-type cellobiohydrolase which exhibits high substrate specificity on amorphous cellulose and low substrate specificity on crystalline cellulose, while this showed no activity on substitution substrates, carboxymethyl cellulose and xylan, distinct from all other known GH6 cellobiohydrolases. Product profiles, HPLC analysis of the hydrolysis products and a schematic drawing of the substrate-binding subsites catalysing cellooligosaccharides can explain the new mode of action of this enzyme which prefers to hydrolyse cellopentaose. PcCel6A was not inhibited by glucose or cellobiose at concentrations up to 300 and 100 mM, respectively. A good synergistic effect for glucose production was found when PcCel6A acted together with processive endoglucanase Cel9R from Clostridium thermocellum and ß-glucosidase CglT from Thermoanaerobacter brockii. These properties of PcCel6A make it a suitable candidate for industrial application in the cellulose degradation process.

Bioconversion of Untreated Corn Hull into L-Malic Acid by Trifunctional Xylanolytic Enzyme from Paenibacillus curdlanolyticus B-6 and Acetobacter tropicalis H-1.

L-Malic acid (L-MA) is widely used in food and non-food products. However, few microorganisms have been able to efficiently produce L-MA from xylose derived from lignocellulosic biomass (LB). The objective of this work is to convert LB into L-MA with the concept of a bioeconomy and environmentally friendly process. The unique trifunctional xylanolytic enzyme, PcAxy43A from Paenibacillus curdlanolyticus B-6, effectively hydrolyzed xylan in untreated LB, especially corn hull to xylose, in one step. Furthermore, the newly isolated, Acetobacter tropicalis strain H1 was able to convert high concentrations of xylose derived from corn hull into L-MA as the main product, which can be easily purified. The strain H1 successfully produced a high L-MA titer of 77.09 g/l, with a yield of 0.77 g/g and a productivity of 0.64 g/l/h from the xylose derived from corn hull. The process presented in this research is an efficient, low-cost and environmentally friendly biological process for the green production of L-MA from LB.

Interplays of enzyme, substrate, and surfactant on hydrolysis of native lignocellulosic biomass.

Tracking enzyme, substrate, and surfactant interactions to reach maximum reducing sugar production during enzymatic hydrolysis of plant biomass may provide a better understanding of factors that limit the lignocellulosic material degradation in native rice straw. In this study, enzymes (Cellic Ctec2 cellulase and Cellic Htec2 xylanase) and Triton X-100 (surfactant) were used as biocatalysts for cellulose and xylan degradation and as a lignin blocking agent, respectively. The response surface model (R2 = 0.99 and R2-adj = 0.97) indicated that Cellic Ctec2 cellulase (p < 0.0001) had significant impacts on reducing sugar production, whereas Cellic Htec2 xylanase and Triton X-100 had insignificant impacts on sugar yield. Although FTIR analysis suggested binding of Triton X-100 to lignin surfaces, the morphological observation by SEM revealed similar surface features (i.e., smooth surfaces with some pores) of rice straw irrespective of Triton X-100. The reducing sugar yields from substrate hydrolysis with or without the surfactant were comparable, suggesting similar exposure of polysaccharides accessible to the enzymes. The model analysis and chemical and structural evidence suggest that there would be no positive effects on enzymatic hydrolysis by blocking lignins with Triton X-100 if high lignin coverage exists in the substrate due to the limited availability of hydrolyzable polysaccharides.

A cellulolytic and xylanolytic enzyme complex from an alkalothermoanaerobacterium, Tepidimicrobium xylanilyticum BT14.

A cellulolytic and xylanolytic enzyme complex-producing alkalothermoanaerobacterium strain, Tepidimicrobium xylanilyticum BT14 is described. The cell was Gram-positive, rod-shaped and endospore-forming. Based on 16S rRNA gene analysis and various lines of biochemical and physiological properties, the strain BT14 was a new member of the genus Tepidimicrobium. The strain BT14 cells had ability to bind to Avicel, xylan and corn hull. The pH and temperature optima for growth were 9.0 and 60 degrees C, respectively. The strain BT14 was able to use a variety of carbon sources. When the bacterium was grown on corn hulls under an anaerobic condition, a cellulolytic and xylanolytic enzyme complex was produced. Crude enzyme containing cellulase and xylanase of the strain BT14 was active in broad ranges of pH and temperature. The optimum conditions for cellulase and xylanase activities were pH 8.0 and 9.0 at 60 degrees C, respectively. The crude enzyme had ability to bind to Avicel and xylan. The analysis of native-PAGE and native-zymograms indicated the cellulose-binding protein showing both cellulase and xylanase activities while SDS-PAGE zymograms showed 4 bands of cellulases and 5 bands of xylanases. Evidence of cohesin-like amino acid sequence seemed to indicate that the protein complex shared direct relationship to the cellulosome of Clostridium thermocellum. The crude enzyme from the strain BT14 showed effective degradation of plant biomass. When grown on corn hulls at pH 9.0 and 60 degrees C under anaerobic conditions, the strain BT14 produced ethanol and acetate as the main fermentation products.

Effect of microwave-assisted ionic liquid/acidic ionic liquid pretreatment on the morphology, structure, and enhanced delignification of rice straw.

In the present study, was investigated an environmentally friendly method for pretreating lignocellulosic rice straw (RS) by using 1-butyl-3-methylimidazolium chloride ([Bmim]Cl) as an ionic liquid (IL) and 1-butyl-3-methylimidazolium hydrogen sulfate ([Bmim]HSO4) as an acidic-IL (Acidic-IL) under microwave irradiation (microwave-[Bmim]Cl and microwave-[Bmim]HSO4). The conversion of lignocellulosic biomass into simple sugars requires both efficient pretreatment and hydrolysis enzymes to produce biofuels and specialty chemicals. Therefore, the applied [Bmim]Cl, [Bmim]HSO4, microwave-[Bmim]Cl, and microwave-[Bmim]HSO4 to improve hydrolysis yields. Structural analyses of the pretreated solids were performed to understand the synergistic effects of [Bmim]Cl, and [Bmim]HSO4 pretreatment under microwave irradiation (microwave-[Bmim]Cl and microwave-[Bmim]HSO4) on the efficiencies of enzymatic hydrolyses. The results of a chemical composition analysis of untreated and all pretreated RS samples by using the difference pretreatment methods showed that significant lignin removal was achieved using microwave-[Bmim]Cl (57.02 ±â€¯1.24%), followed by [Bmim]Cl only (41.01 ±â€¯2.67%), microwave-[Bmim]HSO4 (20.77 ±â€¯1.79%), and [Bmim]HSO4-only (16.88 ±â€¯1.14%). The highest glucan yield and xylan conversion achieved through the enzymatic saccharification of microwave-[Bmim]Cl-regenerated cellulose was consistent with the observations obtained from a structural analysis, which indicated a more disrupted, amorphous structure, with lowered crystallinity index (CrI) and lateral order index (LOI) of cellulose polymers. Thus results demonstrated that the pretreatment of lignocellulosic biomass with [Bmim]Cl under microwave irradiation has potential as an alternative method for pretreating lignocellulosic materials.

Structural changes and enzymatic response of Napier grass (Pennisetum purpureum) stem induced by alkaline pretreatment.

Napier grass is a promising energy crop in the tropical region. Feasible alkaline pretreatment technologies, including NaOH, Ca(OH)2, NH3, and alkaline H2O2 (aH2O2), were used to delignify lignocellulose with the aim of improving glucose recovery from Napier grass stem cellulose via enzymatic saccharification. The influences of the pretreatments on structural alterations were examined using SEM, FTIR, XRD, and TGA, and the relationships between these changes and the enzymatic digestibility of cellulose were addressed. The extensive removal of lignin (84%) in NaOH-pretreated fibre agreed well with the high glucan conversion rate (94%) by enzymatic hydrolysis, while the conversion rates for fibre pretreated with Ca(OH)2, NH3, and aH2O2 approached 60%, 51%, and 42%, respectively. The substantial solubilisation of lignin created porosity, allowing increased cellulose accessibility to cellulases in NaOH-pretreated fibre. In contrast, high lignin content, lignin redeposition on the surface, and residual internal lignin and hemicellulose impeded enzymatic performance in Ca(OH)2-, NH3-, and aH2O2-pretreated fibres, respectively.