Pretreatment of sweet sorghum straw and its enzymatic digestion: insight into the structural changes and visualization of hydrolysis process.
ABSTRACT: Background:The efficient utilization of lignocellulosic biomass for biofuel production has received increasing attention. Previous studies have investigated the pretreatment process of biomass, but the detailed enzymatic hydrolysis process of pretreated biomass remains largely unclear. Thus, this study investigated the pretreatment efficiency of dilute alkali, acid, hydrogen peroxide and its ultimate effects on enzymatic hydrolysis. Furthermore, to better understand the enzymatic digestion process of alkali-pretreated sweet sorghum straw (SSS), multimodal microscopy techniques were used to visualize the enzymatic hydrolysis process. Result:After pretreatment with alkali, an enzymatic hydrolysis efficiency of 86.44% was obtained, which increased by 99.54% compared to the untreated straw (43.23%). The FTIR, XRD and SEM characterization revealed a sequence of microstructural changes occurring in plant cell walls after pretreatment, including the destruction of lignin-polysaccharide interactions, the increase of porosity and crystallinity, and reduction of recalcitrance. During the course of hydrolysis, the cellulase dissolved the cell walls in the same manner and the digestion firstly occurred from the middle of cell walls and then toward the cell wall corners. The CLSM coupled with fluorescent labeling demonstrated that the sclerenchyma cells and vascular bundles in natural SSS were highly lignified, which caused the nonproductive bindings of cellulase on lignin. However, the efficient delignification significantly increased the accessibility and digestibility of cellulase to biomass, thereby improving the saccharification efficiency. Conclusion:This work will be helpful in investigating the biomass pretreatment and its structural characterization. In addition, the visualization results of the enzymatic hydrolysis process of pretreated lignocellulose could be used for guidance to explore the lignocellulosic biomass processing and large-scale biofuel production.
Project description:Background:Due to the intact structure of lignocellulosic biomass, pretreatment was a prerequisite to improve the enzymatic hydrolysis by disrupting the recalcitrant lignocellulose and increasing the accessibility of cellulose to enzyme. In this study, an alkaline hydrogen peroxide (AHP) pretreatment of sugarcane bagasse with various loadings of H2O2 (1.25-6.25 wt%) at temperatures of 60-160 °C was proposed to degrade hemicellulose/lignin and improve the enzymatic digestibility. Results:It was found that increasing H2O2 loadings during pretreatment lead to the enhancement of substrate digestibility, whereas the alkali (only NaOH)-pretreated solid generated higher glucose yield than that pretreated under AHP pretreatment with lower loading of H2O2. This enhancement of enzymatic digestibility was due to the degradation of hemicellulose and lignin. Furthermore, Tween 80 was added to promote enzymatic digestibility, however, the increased yields were different with various substrates and hydrolysis time. The highest glucose yield of 77.6% was obtained after pretreatment at 160 °C for 60 min with 6.25% H2O2 and the addition of Tween 80, representing 89.1% of glucose in pretreated substrate. Conclusions:This study demonstrated that the AHP pretreatment could greatly enhance the enzymatic saccharification. The addition of Tween 80 played remarkable performances in promoting the glucose yield during enzymatic hydrolysis by stabilizing and protecting the enzyme activity. This study provided an economical feasible and gradual process for the generation of glucose, which will be subsequently converted to bioethanol and bio-chemicals.
Project description:Lignocelluloses from agricultural, industrial, and forest residues constitute a majority of the total biomass present in the world. Environmental concerns of disposal, costly pretreatment options prior to disposal, and increased need to save valuable resources have led to the development of value-added alternate technologies such as bioethanol production from lignocellulosic wastes. In the present study, biologically pretreated (with the fungus, Pleurotus ostreatus HP-1) and chemically pretreated (with mild acid or dilute alkali) wheat straw (WS) and banana stem (BS) were subsequently subjected to enzymatic saccharification (with mixture of 6.0 U/g of filter paper cellulase and 17 U/g of ?-glucosidase) and were evaluated for bioethanol production using Saccharomyces cerevisiae NCIM 3570. Biological and chemical pretreatments removed up to 4.0-49.2 % lignin from the WS and BS which was comparatively higher than that for cellulose (0.3-12.4 %) and for hemicellulose (0.7-21.8 %) removal with an average 5.6-49.5 % dry matter loss. Enzymatic hydrolysis yielded 64-306.6 mg/g (1.5-15 g/L) reducing sugars from which 0.15-0.54 g/g ethanol was produced from Saccharomyces cerevisiae NCIM 3570.
Project description:Various types of lignocellulosic wastes extensively used in biofuel production were provided to assess the potential of EXLX1 as a cellulase synergist. Enzymatic hydrolysis of natural wheat straw showed that all the treatments using mixtures of cellulase and an optimized amount of EXLX1, released greater quantities of sugars than those using cellulase alone, regardless of cellulase dosage and incubation time. EXLX1 exhibited different synergism and binding characteristics for different wastes, but this can be related to their lignocellulosic components. The cellulose proportion could be one of the important factors. However, when the cellulose proportion of different biomass samples exhibited no remarkable differences, a higher synergism of EXLX1 is prone to occur on these materials, with a high proportion of hemicellulose and a low proportion of lignin. The information could be favorable to assess whether EXLX1 is effective as a cellulase synergist for the hydrolysis of the used materials. Binding assay experiments further suggested that EXLX1 bound preferentially to alkali pretreated materials, as opposed to acid pretreated materials under the assay condition and the binding preference would be affected by incubation temperature.
Project description:<h4>Background</h4>Pretreatment is effective in reducing the natural recalcitrance of plant biomass so polysaccharides in cell walls can be accessed for conversion to sugars. Furthermore, lignocellulosic biomass must typically be reduced in size to increase the pretreatment effectiveness and realize high sugar yields. However, biomass size reduction is a very energy-intensive operation and contributes significantly to the overall capital cost.<h4>Results</h4>In this study, the effect of particle size reduction and biomass presoaking on the deconstruction of Alamo switchgrass was examined prior to pretreatment by dilute sulfuric acid (DSA) and Co-solvent Enhanced Lignocellulosic Fractionation (CELF) at pretreatment conditions optimized for maximum sugar release by each pretreatment coupled with subsequent enzymatic hydrolysis. Sugar yields by enzymatic hydrolysis were measured over a range of enzyme loadings. In general, DSA successfully solubilized hemicellulose, while CELF removed nearly 80% of Klason lignin from switchgrass in addition to the majority of hemicellulose. Presoaking and particle size reduction did not have a significant impact on biomass compositions after pretreatment for both DSA and CELF. However, presoaking for 4 h slightly increased sugar yields by enzymatic hydrolysis of DSA-pretreated switchgrass compared to unsoaked samples, whereas sugar yields from enzymatic hydrolysis of CELF solids continued to increase substantially for up to 18 h of presoaking time. Of particular importance, DSA required particle size reduction by knife milling to <?2 mm in order to achieve adequate sugar yields by subsequent enzymatic hydrolysis. CELF solids, on the other hand, realized nearly identical sugar yields from unmilled and milled switchgrass even at very low enzyme loadings.<h4>Conclusions</h4>CELF was capable of achieving nearly theoretical sugar yields from enzymatic hydrolysis of pretreated switchgrass solids without size reduction, unlike DSA. These results indicate that CELF may be able to eliminate particle size reduction prior to pretreatment and thereby reduce overall costs of biological processing of biomass to fuels. In addition, presoaking proved much more effective for CELF than for DSA, particularly at low enzyme loadings.
Project description:<h4>Background</h4>Enzymatic hydrolysis is a key step in the conversion of lignocellulosic polysaccharides to fermentable sugars for the production of biofuels and high-value chemicals. However, current enzyme preparations from mesophilic fungi are deficient in their thermostability and biomass-hydrolyzing efficiency at high temperatures. Thermophilic fungi represent promising sources of thermostable and highly active enzymes for improving the biomass-to-sugar conversion process. Here we present a comprehensive study on the lignocellulosic biomass-degrading ability and enzyme system of thermophilic fungus Malbranchea cinnamomea N12 and the application of its enzymes in the synergistic hydrolysis of lignocellulosic biomass.<h4>Results</h4>Malbranchea cinnamomea N12 was capable of utilizing untreated wheat straw to produce high levels of xylanases and efficiently degrading lignocellulose under thermophilic conditions. Temporal analysis of the wheat straw-induced secretome revealed that M. cinnamomea N12 successively degraded the lignocellulosic polysaccharides through sequential secretion of enzymes targeting xylan and cellulose. Xylanase-enriched cocktail from M. cinnamomea N12 was more active on native and alkali‑pretreated wheat straw than the commercial xylanases from Trichoderma reesei over temperatures ranging from 40 to 75 °C. Integration of M. cinnamomea N12 enzymes with the commercial cellulase preparation increased the glucose and xylose yields of alkali‑pretreated wheat straw by 32 and 166%, respectively, with pronounced effects at elevated temperature.<h4>Conclusions</h4>This study demonstrated the remarkable xylanase-producing ability and strategy of sequential lignocellulose breakdown of M. cinnamomea N12. A new process for the hydrolysis of lignocellulosic biomass was proposed, comprising thermophilic enzymolysis by enzymes of M. cinnamomea N12 followed with mesophilic enzymolysis by commercial cellulases. Developing M. cinnamomea N12 as platforms for thermophilic enzyme mixture production will provide new perspectives for improved conversion yields for current biomass saccharification schemes.
Project description:Generation of biofuels from sugars in lignocellulosic biomass is a promising alternative to liquid fossil fuels, but efficient and inexpensive bioprocessing configurations must be developed to make this technology commercially viable. One of the major barriers to commercialization is the recalcitrance of plant cell wall polysaccharides to enzymatic hydrolysis. Biomass pretreatment with ionic liquids (ILs) enables efficient saccharification of biomass, but residual ILs inhibit both saccharification and microbial fuel production, requiring extensive washing after IL pretreatment. Pretreatment itself can also produce biomass-derived inhibitory compounds that reduce microbial fuel production. Therefore, there are multiple points in the process from biomass to biofuel production that must be interrogated and optimized to maximize fuel production. Here, we report the development of an IL-tolerant cellulase cocktail by combining thermophilic bacterial glycoside hydrolases produced by a mixed consortia with recombinant glycoside hydrolases. This enzymatic cocktail saccharifies IL-pretreated biomass at higher temperatures and in the presence of much higher IL concentrations than commercial fungal cocktails. Sugars obtained from saccharification of IL-pretreated switchgrass using this cocktail can be converted into biodiesel (fatty acid ethyl-esters or FAEEs) by a metabolically engineered strain of E. coli. During these studies, we found that this biodiesel-producing E. coli strain was sensitive to ILs and inhibitors released by saccharification. This cocktail will enable the development of novel biomass to biofuel bioprocessing configurations that may overcome some of the barriers to production of inexpensive cellulosic biofuels.
Project description:<h4>Background</h4> Conventional aqueous dilute sulfuric acid (DSA) pretreatment of lignocellulosic biomass facilitates hemicellulose solubilization and can improve subsequent enzymatic digestibility of cellulose to fermentable glucose. However, much of the lignin after DSA pretreatment either remains intact within the cell wall or readily redeposits back onto the biomass surface. This redeposited lignin has been shown to reduce enzyme activity and contribute to rapid enzyme deactivation, thus, necessitating significantly higher enzyme loadings than deemed economical for biofuel production from biomass. <h4>Results</h4> In this study, we demonstrate how detrimental lignin redeposition on biomass surface after pretreatment can be prevented by employing Co-solvent Enhanced Lignocellulosic Fractionation (CELF) pretreatment that uses THF–water co-solvents with dilute sulfuric acid to solubilize lignin and overcome limitations of DSA pretreatment. We first find that enzymatic hydrolysis of CELF-pretreated switchgrass can sustain a high enzyme activity over incubation periods as long as 5 weeks with enzyme doses as low as 2 mg protein/g glucan to achieve 90% yield to glucose. A modified Ninhydrin-based protein assay revealed that the free-enzyme concentration in the hydrolysate liquor, related to enzyme activity, remained unchanged over long hydrolysis times. DSA-pretreated switchgrass, by contrast, had a 40% drop in free enzymes in solution during incubation, providing evidence of enzyme deactivation. Furthermore, measurements of enzyme adsorption per gram of lignin suggested that CELF prevented lignin redeposition onto the biomass surface, and the little lignin left in the solids was mostly integral to the original lignin–carbohydrate complex (LCC). Scanning electron micrographs and NMR characterization of lignin supported this observation. <h4>Conclusions</h4> Enzymatic hydrolysis of solids from CELF pretreatment of switchgrass at low enzyme loadings was sustained for considerably longer times and reached higher conversions than for DSA solids. Analysis of solids following pretreatment and enzymatic hydrolysis showed that prolonged cellulase activity could be attributed to the limited lignin redeposition on the biomass surface making more enzymes available for hydrolysis of more accessible glucan. <h4>Supplementary Information</h4> The online version contains supplementary material available at 10.1186/s13068-021-01904-2.
Project description:BACKGROUND:Pretreatment is an essential step in the enzymatic hydrolysis of biomass for bio-ethanol production. The dominant concern in this step is how to decrease the high cost of pretreatment while achieving a high sugar yield. Fungal pretreatment of biomass was previously reported to be effective, with the advantage of having a low energy requirement and requiring no application of additional chemicals. In this work, Gloeophyllum trabeum KU-41 was chosen for corn stover pretreatment through screening with 40 strains of wood-rot fungi. The objective of the current work is to find out which characteristics of corn stover pretreated with G. trabeum KU-41 determine the pretreatment method to be successful and worthwhile to apply. This will be done by determining the lignin content, structural carbohydrate, cellulose crystallinity, initial adsorption capacity of cellulase and specific surface area of pretreated corn stover. RESULTS:The content of xylan in pretreated corn stover was decreased by 43% in comparison to the untreated corn stover. The initial cellulase adsorption capacity and the specific surface area of corn stover pretreated with G. trabeum were increased by 7.0- and 2.5-fold, respectively. Also there was little increase in the cellulose crystallinity of pretreated corn stover. CONCLUSION:G. trabeum has an efficient degradation system, and the results indicated that the conversion of cellulose to glucose increases as the accessibility of cellulose increases due to the partial removal of xylan and the structure breakage of the cell wall. This pretreatment method can be further explored as an alternative to the thermochemical pretreatment method.
Project description:Removing alkali-soluble lignin using extractive ammonia (EA) pretreatment of corn stover (CS) is known to improve biomass conversion efficiency during enzymatic hydrolysis. In this study, we investigated the effect of alkali-soluble lignin on six purified core glycosyl hydrolases and their enzyme synergies, adopting 31 enzyme combinations derived by a five-component simplex centroid model, during EA-CS hydrolysis. Hydrolysis experiment was carried out using EA-CS(-) (approx. 40% lignin removed during EA pretreatment) and EA-CS(+) (where no lignin was extracted). Enzymatic hydrolysis experiments were done at three different enzyme mass loadings (7.5, 15 and 30?mg protein?g-1 glucan), using a previously developed high-throughput microplate-based protocol, and the sugar yields of glucose and xylose were detected. The optimal enzyme combinations (based on % protein mass loading) of six core glycosyl hydrolases for EA-CS(-) and EA-CS(+) were determined that gave high sugar conversion. The inhibition of lignin on optimal enzyme ratios was studied, by adding fixed amount of alkali-soluble lignin fractions to EA-CS(-), and pure Avicel, beechwood xylan and evaluating their sugar conversion. The optimal enzyme ratios that gave higher sugar conversion for EA-CS(-) were CBH I: 27.2-28.2%, CBH II: 18.2-22.2%, EG I: 29.2-34.3%, EX: 9.0-14.1%, ?X: 7.2-10.2%, ?G: 1.0-5.0% (at 7.5-30?mg?g-1 protein mass loading). Endoglucanase was inhibited to a greater extent than other core cellulases and xylanases by lignin during enzyme hydrolysis. We also found that alkali-soluble lignin inhibits cellulase more strongly than hemicellulase during the course of enzyme hydrolysis.
Project description:Background:Pretreatment of lignocellulosic biomass (LCB) is a key step for its efficient bioconversion into ethanol. Determining the best pretreatment and its parameters requires monitoring its impacts on the biomass material. Here, we used fluorescent protein-tagged carbohydrate-binding modules method (FTCM)-depletion assay to study the relationship between surface-exposed polysaccharides and enzymatic hydrolysis of LCB. Results:Our results indicated that alkali extrusion pretreatment led to the highest hydrolysis rates for alfalfa stover, cattail stems and flax shives, despite its lower lignin removal efficiency compared to alkali pretreatment. Corn crop residues were more sensitive to alkali pretreatments, leading to higher hydrolysis rates. A clear relationship was consistently observed between total surface-exposed cellulose detected by the FTCM-depletion assay and biomass enzymatic hydrolysis. Comparison of bioconversion yield and total composition analysis (by NREL/TP-510-42618) of LCB prior to or after pretreatments did not show any close relationship. Lignin removal efficiency and total cellulose content (by NREL/TP-510-42618) led to an unreliable prediction of enzymatic polysaccharide hydrolysis. Conclusions:Fluorescent protein-tagged carbohydrate-binding modules method (FTCM)-depletion assay provided direct evidence that cellulose exposure is the key determinant of hydrolysis yield. The clear and robust relationships that were observed between the cellulose accessibility by FTCM probes and enzymatic hydrolysis rates change could be evolved into a powerful prediction tool that might help develop optimal biomass pretreatment strategies for biofuel production.