Cellulose solvent-based biomass pretreatment breaks highly ordered hydrogen bonds in cellulose fibers of switchgrass

The switchgrass (SG) samples pretreated by cellulose solvent‐ and organic solvent‐based lignocellulose fractionation were characterized by enzymatic hydrolysis, substrate accessibility assay, scanning electron microscopy, X‐ray diffraction (XRD), cross polarization/magic angle spinning (CP/MAS) ¹³C nuclear magnetic resonance (NMR), and Fourier transform infrared spectroscopy (FTIR). Glucan digestibility of the pretreated SG was 89% at hour 36 at one filter paper unit of cellulase per gram of glucan. Crystallinity index (CrI) of pure cellulosic materials and SG before and after cellulose solvent‐based pretreatment were determined by XRD and NMR. CrI values varied greatly depending on measurement techniques, calculation approaches, and sample drying conditions, suggesting that the effects of CrI data obtained from dried samples on enzymatic hydrolysis of hydrated cellulosic materials should be interpreted with caution. Fast hydrolysis rates and high glucan digestibilities for pretreated SG were mainly attributed to a 16.3‐fold increase in cellulose accessibility to cellulase from 0.49 to 8.0 m²/g biomass, because the highly ordered hydrogen‐bonding networks in cellulose fibers of biomass were broken through cellulose dissolution in a cellulose solvent, as evidenced by CP/MAS ¹³C‐NMR and FTIR. Biotechnol. Bioeng. 2011; 108:521–529. © 2010 Wiley Periodicals, Inc.     

Improved effects of okara atomized by a water jet system on α-amylase inhibition and butyrate production by Roseburia intestinalis

ABSTRACT

Improving the physicochemical properties of okara for various applications in foods is of great importance. Here, okara and microcrystalline cellulose (MCC) were atomized using a water jet (WJ) system. The WJ-treated okara and MCC dispersed homogeneously in water, and their median sizes in particle size distribution were 6.6 μm and 9.5 μm, respectively. The dispersions of WJ-treated okara and MCC showed high apparent viscosity and shear thinning behavior. Moreover, the inhibition of α-amylase activities by WJ-treated okara was more effective than that by untreated MCC and cellulose. Furthermore, the production of short-chain fatty acids by 32 dominant species of human gut microbes was determined. An increase in butyrate production by Roseburia intestinalis was observed in the presence of WJ-treated okara, but not in untreated okara or WJ-treated MCC. These results demonstrate that WJ system can be used on okara to increase inhibited α-amylase activities and butyrate production by gut microbiota.     

A new approach to obtain cellulose nanocrystals and ethanol from eucalyptus cellulose pulp via the biochemical pathway

The feasibility of integration of cellulosic ethanol production with the manufacture of cellulose nanofibers (CNF) and cellulose nanocrystals (CNC) was evaluated using eucalyptus cellulose pulp as feedstock and employing the biochemical route alone. For the enzymatic hydrolysis step, experimental central composite design (CCD) methodology was used as a tool to evaluate the effects of solids loading (SL) and enzymatic loading (EL) on glucose release and cellulose conversion. Glucose concentrations from 45 to 125 g/L were obtained after 24 h, with cellulose conversions from 35 to 96%. Validation of the statistical model was performed at SL of 20% and EL of 10 mg protein/g, which was defined by the desirability function as the optimum condition. The sugars released were used for the production of ethanol by Saccharomyces cerevisiae, resulting in 62.1 g/L ethanol after 8 h (yield of 95.5%). For all the CCD experimental conditions, the residual solids presented CNF characteristics. Moreover, the use of a new strategy with temperature reduction from 50 to 35°C after 24 h of enzymatic hydrolysis enabled CNC to be obtained after 144 h. The CNC showed a crystallinity index of 83%, length of 260 nm, diameter of 15 nm, and aspect ratio (L/D) of 15. These characteristics are suitable for many applications, such as reinforcement in polymeric materials and other lower volume higher value bio‐based products. The findings indicate the viability of obtaining ethanol and CNC using the biochemical route exclusively, potentially contributing to the future implementation of forest biorefineries. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1085–1095, 2017     

Biomass‐water interactions correlate to recalcitrance and are intensified by pretreatment: An investigation of water constraint and retention in pretreated spruce using low field NMR and water retention value techniques

The underlying mechanisms of the recalcitrance of biomass to enzymatic deconstruction are still not fully understood, and this hampers the development of biomass based fuels and chemicals. With water being necessary for most biological processes, it is suggested that interactions between water and biomass may be key to understanding and controlling biomass recalcitrance. This study investigates the correlation between biomass recalcitrance and the constraint and retention of water by the biomass, using SO₂ pretreated spruce, a common feedstock for lignocellulosic biofuel production, as a substrate to evaluate this relationship. The water retention value (WRV) of the pretreated materials was measured, and water constraint was assessed using time domain Low Field Nuclear Magnetic Resonance (LFNMR) relaxometry. WRV increased with pretreatment severity, correlating to reduced recalcitrance, as measured by hydrolysis of cellulose using commercial enzyme preparations. Water constraint increased with pretreatment severity, suggesting that a higher level of biomass‐water interaction is indicative of reduced recalcitrance in pretreated materials. Both WRV and water constraint increased significantly with reductions in particle size when pretreated materials were further milled, suggesting that particle size plays an important role in biomass water interactions. It is suggested that WRV may be a simple and effective method for measuring and comparing biomass recalcitrance. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:146–153, 2017     

Natural cellulose fibers from switchgrass with tensile properties similar to cotton and linen

We report the production and characteristics of natural cellulose fibers obtained from the leaves and stems of switchgrass. In this paper, the composition, structure and properties of fibers obtained from the leaves and stem of switchgrass have been studied in comparison to the common natural cellulose fibers, such as cotton, linen and kenaf. The leaves and stems of switchgrass have tensile properties intriguingly similar to that of linen and cotton, respectively. Fibers were obtained from the leaves and stems of switchgrass using a simple alkaline extraction and the structure and properties of the fibers were studied. Fibers obtained from switchgrass leaves have crystallinity of 51%, breaking tenacity of 5.5 g per denier (715 MPa) and breaking elongation of 2.2% whereas the corresponding values for fibers obtained from switchgrass stems are 46%, 2.7 g per denier and 6.8%, respectively. Switchgrass is a relatively easy to grow and high yield biomass crop that can be source to partially substitute the natural and synthetic fibers currently in use. We hope that this research will stimulate interests in using switchgrass as a novel fiber crop in addition to being promoted as a potential source for biofuels.     

Liquid hot water and alkaline pretreatment of soybean straw for improving cellulose digestibility

Soybean straw was pretreated with either liquid hot water (LHW) (170-210 °C for 3-10 min) or alkaline soaking (4-40 g NaOH/100 g dry straw) at room temperature to evaluate the effects on cellulose digestibility. Nearly 100% cellulose was recovered in pretreated solids for both pretreatment methods. For LHW pretreatment, xylan dissolution from the raw material increased with pretreatment temperature and time. Cellulose digestibility was correlated with xylan dissolution. A maximal glucose yield of 70.76%, corresponding to 80% xylan removal, was obtained with soybean straw pretreated at 210 °C for 10 min. NaOH soaking at ambient conditions removed xylan up to 46.37% and the subsequent glucose yield of pretreated solids reached up to 64.55%. Our results indicated LHW pretreatment was more effective than NaOH soaking for improving cellulose digestibility of soybean straw.     

Enhancement of cellulose saccharification kinetics using an ionic liquid pretreatment step

Hydrolysis of cellulose to glucose in aqueous media catalyzed by the cellulase enzyme system suffers from slow reaction rates due in large part to the highly crystalline structure of cellulose and inaccessibility of enzyme adsorption sites. In this study, an attempt was made to disrupt the cellulose structure using the ionic liquid (IL), 1-n-butyl-3-methylimidazolium chloride, in a cellulose regeneration strategy which accelerated the subsequent hydrolysis reaction. ILs are a new class of non-volatile solvents that exhibit unique solvating properties. They can be tuned to dissolve a wide variety of compounds including cellulose. Because of their extremely low volatility, ILs are expected to have minimal environmental impact on air quality compared to most other volatile solvent systems. The initial enzymatic hydrolysis rates were approximately 50-fold higher for regenerated cellulose as compared to untreated cellulose (Avicel PH-101) as measured by a soluble reducing sugar assay.     

Increased water content in bacterial cellulose synthesized under rotating magnetic fields

The current study describes properties of bacterial cellulose (BC) obtained from Komagataeibacter xylinus cultures exposed to the rotating magnetic field (RMF) of 50 Hz frequency and magnetic induction of 34 mT for controlled time during 6 days of cultivation. The experiments were carried out in the customized RMF exposure system adapted for biological studies. The obtained BC displayed an altered micro-structure, degree of porosity, and water-related parameters in comparison to the non-treated, control BC samples. The observed effects were correlated to the duration and the time of magnetic exposure during K. xylinus cultivation. The most preferred properties in terms of water-related properties were found for BC obtained in the setting, where RMF generator was switched off for the first 72 h of cultivation and switched on for the next 72 h. The described method of BC synthesis may be of special interest for the production of absorbent, antimicrobial-soaked dressings and carrier supports for the immobilization of microorganisms and proteins.     

Application of high throughput pretreatment and co‐hydrolysis system to thermochemical pretreatment. Part 1: Dilute acid

Because conventional approaches for evaluating sugar release from the coupled operations of pretreatment and enzymatic hydrolysis are extremely time and material intensive, high throughput (HT) pretreatment and enzymatic hydrolysis systems have become vital for screening large numbers of lignocellulosic biomass samples to identify feedstocks and/or processing conditions that significantly improve performance and lower costs. Because dilute acid pretreatment offers many important advantages in rendering biomass highly susceptible to subsequent enzymatic hydrolysis, a high throughput pretreatment and co‐hydrolysis (HTPH) approach was extended to employ dilute acid as a tool to screen for enhanced performance. First, a single‐step neutralization and buffering method was developed to allow effective enzymatic hydrolysis of the whole pretreated slurry. Switchgrass and poplar were then pretreated with 0.5% and 1% acid loadings at a 5% solids concentration, the resulting slurry conditioned with the buffering approach, and the entire mixture enzymatically hydrolyzed. The resulting sugar yields demonstrated that single‐step neutralizing and buffering was capable of adjusting the pH as needed for enzymatic saccharification, as well as overcoming enzyme inhibition by compounds released in pretreatment. In addition, the effects of pretreatment conditions and biomass types on susceptibility of pretreated substrates to enzymatic conversion were clearly discernible, demonstrating the method to be a useful extension of HTPH systems. Biotechnol. Bioeng. 2013; 110: 754–762. © 2012 Wiley Periodicals, Inc.     

Visualization of biomass solubilization and cellulose regeneration during ionic liquid pretreatment of switchgrass

Auto‐fluorescent mapping of plant cell walls was used to visualize cellulose and lignin in pristine switchgrass (Panicum virgatum) stems to determine the mechanisms of biomass dissolution during ionic liquid pretreatment. The addition of ground switchgrass to the ionic liquid 1‐n‐ethyl‐3‐methylimidazolium acetate resulted in the disruption and solubilization of the plant cell wall at mild temperatures. Swelling of the plant cell wall, attributed to disruption of inter‐ and intramolecular hydrogen bonding between cellulose fibrils and lignin, followed by complete dissolution of biomass, was observed without using imaging techniques that require staining, embedding, and processing of biomass. Subsequent cellulose regeneration via the addition of an anti‐solvent, such as water, was observed in situ and provided direct evidence of significant rejection of lignin from the recovered polysaccharides. This observation was confirmed by chemical analysis of the regenerated cellulose. In comparison to untreated biomass, ionic liquid pretreated biomass produces cellulose that is efficiently hydrolyzed with commercial cellulase cocktail with high sugar yields over a relatively short time interval. Biotechnol. Bioeng. 2009; 104: 68–75 Published 2009 Wiley Periodicals, Inc.     

Wetland environmental bioreactor system contributes to the decomposition of cellulose

Recently, numerous species of aquatic invertebrates inhabiting wetlands have been shown to possess endogenous cellulase, following the discovery that termites have cellulase genes encoded in their own genome rather than relying on symbiotic bacteria for decomposing cellulose. Wetlands have been empirically shown to play an important role in the decomposition of land‐originating hard‐to‐degrade polysaccharides such as cellulose. However, the mechanism that connects the cellulase producer and the wetlands remains unknown, which makes it very difficult to evaluate the ecological function of wetlands. Here we found that a macrobenthic bivalve, Corbicula japonica, secretes its cellulase to the wetland sediment. Secreted cellulases are immobilized in the components of the sediment. Moreover, adding cellulose or glucose to C. japonica could trigger its cellulase secretion level. These findings suggest a novel wetland cellulose decomposition mechanism. The decomposition ability of wetlands was previously ascribed only to microbes and/or invertebrates that contain cellulases. Our findings suggest that benthic animals supply wetlands with their enzymes as decomposition agents, while wetland sediments serve as immobilization scaffolds for the enzymes. This system, which was named by us an “environmental bioreactor system,” could provide a key function in wetlands.     

Preparation of an injectable doxorubicin surface modified cellulose nanofiber gel and evaluation of its anti‐tumor and anti‐metastasis activity in melanoma

Cellulose nanofibers (Cel‐NFs) gel can be considered as a useful drug carrier because of its biocompatibility, high specific surface area, and high loading capacity of drugs. Injectable Cel‐NFs gel could deliver doxorubicin (DOX) for localized chemotherapy of melanoma and suppress melanoma cells migration because of the physical barrier property of Cel‐NFs. We prepared DOX surface modified Cel‐NFs (DOX‐Cel‐NFs) gel by the electrostatic attachment of DOX molecules on the surface of Cel‐NFs. The increase in the zeta potential of nanofibers and the changes in the FTIR spectra of DOX‐Cel‐NFs compared to Cel‐NFs proved this attachment. DOX‐Cel‐NFs showed nano‐fibrous structure with an average diameter of 22.32 ± 10.66 nm after analyzing using field emission scanning electron microscopy. The suitable injectability of DOX‐Cel‐NFs gel verified its promising application for the localized chemotherapy. DOX‐Cel‐NFs gel exhibited a sustained drug release manner. The cytotoxicity results showed that DOX‐Cel‐NFs were more cytotoxic against melanoma cancer cells than the free DOX during 48 h incubation period. Moreover, DOX‐Cel‐NFs gel can suppress the melanoma cancer cells migration efficiently. Thus our results emphasize the potential of DOX‐Cel‐NFs gel as a chemotherapeutic agent for local delivery of DOX in order to treat melanoma and prevent its metastasis. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:537–545, 2018     

Cleavage of cellulose by a CBM33 protein

Bacterial proteins categorized as family 33 carbohydrate-binding modules (CBM33) were recently shown to cleave crystalline chitin, using a mechanism that involves hydrolysis and oxidation. We show here that some members of the CBM33 family cleave crystalline cellulose as demonstrated by chromatographic and mass spectrometric analyses of soluble products released from Avicel or filter paper on incubation with CelS2, a CBM33-containing protein from Streptomyces coelicolor A3(2). These enzymes act synergistically with cellulases and may thus become important tools for efficient conversion of lignocellulosic biomass. Fungal proteins classified as glycoside hydrolase family 61 that are known to act synergistically with cellulases are likely to use a similar mechanism.     

Reconstitution of cellulose and lignin after [C2mim][OAc] pretreatment and its relation to enzymatic hydrolysis

Although the effects of cellulose crystallinity and lignin content as two major structural features on enzymatic hydrolysis have been extensively studied, debates regarding their effects still exist. In this study, reconstitution of cellulose and lignin after 1‐ethyl‐3‐methylimidazolium acetate ([C₂mim][OAc]) pretreatment was proposed as a new method to study their effects on enzymatic digestibility. Different mechanisms of lignin content for reduction of cellulose hydrolysis were found between the proposed method and the traditional method (mixing of cellulose and lignin). The results indicated that a slight change of the crystallinity of the reconstituted materials may play a minor role in the change of enzyme efficiency. In addition, the present study suggested that the lignin content does not significantly affect the digestibility of cellulose, whereas the conversion of cellulose fibers from the cellulose I to the cellulose II crystal phase plays an important role when an ionic liquid pretreatment of biomass was conducted. Biotechnol. Bioeng. 2013; 110: 729–736. © 2012 Wiley Periodicals, Inc.     

Room temperature ionic liquids as emerging solvents for the pretreatment of lignocellulosic biomass

Room temperature ionic liquids (RTILs) are emerging as attractive and green solvents for lignocellulosic biomass pretreatment. The unique solvating properties of RTILs foster the disruption of the 3D network structure of lignin, cellulose, and hemicellulose, which allows high yields of fermentable sugars to be produced in subsequent enzymatic hydrolysis. In the current review, we summarize the physicochemical properties of RTILs that make them effective solvents for lignocellulose pretreatment including mechanisms of interaction between lignocellulosic biomass subcomponents and RTILs. We also highlight several recent strategies that exploit RTILs and generate high yields of fermentable sugars suitable for downstream biofuel production, and address new opportunities for use of lignocellulosic components, including lignin. Finally, we address some of the challenges that remain before large-scale use of RTILs may be achieved.     

预处理对木质纤维素生物质细胞壁超微结构的影响

预处理是提高木质纤维素生物质向生物燃料转化的有效途径,但生物质的天然抗降解屏障严重阻碍了这一转化的进行,因此全面了解预处理过程中植物细胞壁的微观结构及区域化学变化是实现农林生物质高效转化的基础。本文总结了多种预处理方法对植物细胞壁超微结构影响的研究进展,对生物质科学研究可能有一定的促进和指导作用。