Conversion Of Cellulosic Biomass Research Articles

Carboxymethyl Cellulase Production by Extremotolerant Bacteria in Low-Cost Media and Application in Enzymatic Saccharification of Stevia Biomass

The aim of this study was to characterize novel cellulase-producing bacteria isolated from desertic environments in Tunisia, and then applied for cellulosic ethanol production. In the first instance, 27 alkali-thermo-halotolerant bacterial strains, belonging to Bacillaceae family, were screened on agar plates containing carboxymethyl cellulose as the sole carbon source. The selected high cellulase producing bacteria were identified as Bacillus mojavensis AG21, Bacillus vallismortis J77, Bacillus sonorensis 7.26 and Bacillus safiensis AJ49. Production of cellulolytic enzymes by selected bacteria was further tested in submerged cultures with Posidonia oceanica leaves, Peganum Harmala biomass, Stevia rebaudiana leaves (SRL), Olive cake, Spent coffee ground and peanut hulls as low-cost cellulosic substrates. Response surface methodology was applied to evaluate the effect of SRL concentration, NaCl concentration, cultivation pH and temperature on carboxymethyl cellulase production and to optimize enzyme yields by selected bacteria. Maximum cellulase production yields of about 1890, 1642, 1516 and 1455 IU/L were recorded for B. mojavensis AG21, B.vallismortis J77, B. sonorensis 7.26 and B. safiensis AJ49, respectively. Production of reducing sugars by hydrolysis of Stevia rebaudiana leaves with bacterial cellulases and commercially fungal cellulases was investigated and compared. The overall reducing sugar yields of B. mojavensis, B.vallismortis, B. sonorensis, B. safiensis, Aspergillus niger and Trichoderma reesei treated- SSR biomasses reached 3620 ± 181, 3040 ± 164, 3656 ± 164, 2654 ± 106, 2646 ± 145 and 2253 ± 123 mg/L respectively. The unique property of cellulase-producing extremotolerant bacteria, proves the potential candidature for current mainstream agricultural cellulosic biomass conversion into fuel ethanol.

Stoichiometric Conversion of Cellulosic Biomass by in Vitro Synthetic Enzymatic Biosystems for Biomanufacturing

Cellulosic biomass is the earth’s most abundant renewable resource, which is considered to be a promising feedstock for manufacturing biofuels and biochemicals. In this study, stoichiometric enzymatic phosphorolysis of cellulosic biomass for manufacturing biochemicals or biofuels by in vitro synthetic enzymatic biosystems was designed. Three cascade phosphorolytic enzymes, cellodextrin phosphorylase, cellobiose phosphorylase, and polyphosphate-dependent glucokinase, were used for the biotransformation of cellodextrins to high-energy phosphorylated sugars (that is, glucose 1-phosphate and glucose 6-phosphate). A series of downstream exergonic reactions then converted these high-energy phosphorylated sugars into myo-inositol, resulting in a near-stoichiometric conversion of cellodextrins with a high product yield of 98% (w/w). Moreover, this enzymatic biosystem can even work for the acid-treated biomass hydrolysate containing microorganism-toxic compounds. The construction of this in vitro synthetic enzymat...

Catalytic dehydration of hexose sugars to 5‐hydroxymethylfural

AbstractThe catalytic conversion of cellulosic biomass to downstream products provides a viable solution to alleviate the energy crisis and global warming. This article provides a relatively comprehensive review of the dehydration of glucose (the monomer of cellulose) and fructose considering various catalyst and solvent systems. Biphasic solvents and heterogeneous systems are recommended from the point of view of industrial applications. A two‐step catalytic conversion of glucose via fructose has been envisaged: first, isomerization of glucose to fructose, which has been extensively discussed in this review, and then transformation of fructose to downstream products. Two‐step tandem catalytic reactions from glucose to downstream products (i.e., one‐pot synthesis via fructose) are also attractive for industrial applications and should be given more attention. © 2018 Society of Chemical Industry and John Wiley & Sons, Ltd

Heterobimetallic catalysis for lignocellulose to ethylene glycol on nickel-tungsten catalysts: Influenced by hydroxy groups

The superficial chemical environment of the catalyst supports played an important role in heterometallic catalysts preparation. The functions of hydroxy groups on the surface of SiO2 nanosphere was investigated in catalyst preparing and cellulosic biomass conversion to ethylene glycol (EG). The results demonstrated that 15%Ni-20%W/SiO2-OH catalyst was favorable for EG production, with the highest yield of 63.1 and 42.1% from microcrystalline cellulose and hardwood pulp, respectively. The characterization and activity tests suggested that the existence of hydroxy groups could promote the formation of more Ni-W alloys (e.g. NiW, Ni4W) under the same preparing condition, which supplemented and enhanced the incorporation of intrinsic nickel and tungsten catalytic activity, especially for enhancement of retro-aldol condensation reaction. Moreover, the metallic Ni0 was generated in higher amount when supported on hydroxy-rich SiO2, boosting hydrogenation of the unsaturated intermediates. Finally, the reusability investigation of the catalyst implied that embedded Ni and W in alloys could inhibit leaching of metals in pressurized hot water.

Streamlined hydrogen production from biomass

Hydrogen is playing an increasingly larger role in clean energy technologies and the emerging hydrogen economy. However, efficient and selective H2 production from renewable resources is rare so far. Herein, we describe a dehydrogenation route that is applicable to various kinds of non-food-related biomass and daily waste, such as wheat straw, corn straw, rice straw, reed, bagasse, bamboo sawdust, cardboard and newspaper. H2 yields up to 95% were achieved by a one-pot, two-step reaction with a 69 ppm molecularly defined iridium catalyst bearing an imidazoline moiety from formic acid, which was in turn obtained via a 1 v% dimethyl sulfoxide-promoted hydrolysis–oxidation of biomass. Formation of the unwanted side products CO and CH4 was no more than 22 and 2 ppm, respectively, while CO2 was captured as carbonate. The resulting hydrogen gas can be directly applied in proton exchange membrane fuel cells. Access to renewable hydrogen represents an important target for the success of the hydrogen economy. Now, a one-pot method is presented for the conversion of cellulosic biomass into hydrogen via formic acid as the intermediate, followed by its application to a fuel cell.

Biochemical characterization of rice xylan O-acetyltransferases.

Rice xylan is predominantly monoacetylated at O-2 and O-3, and 14 rice DUF231 proteins were demonstrated to be xylan acetyltransferases. Acetylated xylans are the principal hemicellulose in the cell walls of grass species. Because xylan acetylation impedes the conversion of cellulosic biomass into biofuels, knowledge on acetyltransferases catalyzing xylan acetylation in grass species will be instrumental for a better utilization of grass biomass for biofuel production. Xylan in rice (Oryza sativa) is predominantly monoacetylated at O-2 and O-3 with a total degree of acetylation of 0.19. In this report, we have characterized 14 rice DUF231 proteins (OsXOAT1 to OsXOAT14) that are phylogenetically grouped together with Arabidopsis xylan acetyltransferases ESK1 and its close homologs. Complementation analysis demonstrated that the expression of OsXOAT1 to OsXOAT7 in the Arabidopsis esk1 mutant was able to rescue its defects in 2-O- and 3-O-monoacetylation and 2,3-di-O-acetylation. Activity assay of recombinant proteins revealed that all 14 OsXOATs exhibited acetyltransferase activities capable of transferring acetyl groups from acetyl-CoA to the xylohexaose acceptor with 10 of them having high activities. Structural analysis of the OsXOAT-catalyzed products showed that the acetylated structural units consisted mainly of 2-O- and 3-O-monoacetylated xylosyl residues with a minor amount of 2,3-di-O-acetylated xylosyl units, which is consistent with the acetyl substitution pattern of rice xylan. Further kinetic studies revealed that OsXOAT1, OsXOAT2, OsXOAT5, OsXOAT6 and OsXOAT7 had high affinity toward the xylohexaose acceptor. Our results provide biochemical evidence indicating that OsXOATs are acetyltransferases involved in xylan acetylation in rice.

Integration of Enzymatic and Heterogeneous Catalysis for One‐Pot Production of Fructose from Glucose

The search for efficient routes for the production of fructose from biomass-derived glucose is of great interest and importance, as fructose is a highly attractive substrate in the conversion of cellulosic biomass into biofuels and chemicals. In this study, a one-pot, multistep procedure involving enzyme-catalyzed oxidation of glucose at C2 and Ni/C-catalyzed hydrogenation of d-glucosone at C1 selectively gives fructose in 77 % yield. Starting from upstream substrates such as α-cellulose and starch, fructose was also generated with similar efficiency and selectivity by the combination of enzymatic and heterogeneous catalysis. This method constitutes a new means of preparing fructose from biomass-derived substrates in an efficient fashion.

Low-temperature, Low-Energy, and High-Efficiency Pretreatment Technology for Large Wood Chips with a Redox Couple Catalyst.

The pretreatment of lignocellulosic biomass plays a vital role in the conversion of cellulosic biomass to bioethanol, especially for softwoods and hardwoods. Although many pretreatment technologies have been reported so far, only a few pretreatment methods can handle large woodchips directly. To improve the efficiency of pretreatment, existing technologies require the grinding of the wood into small particles, which is an energy-consuming process. Herein, for the first time, we report a simple, effective, and low-temperature (≈100 °C) process for the pretreatment of hardwood (HW) and softwood (SW) chips directly by using a catalytic system of FeCl3 /NaNO3 (FCSNRC). The pretreatment experiments were conducted systematically, and a conversion of 71.53 and 70.66 % of cellulose to sugar could be obtained for the direct use of large HW and SW chips. The new method reported here overcomes one of the critical barriers in biomass-to-biofuel conversion, and both grinding and thermal energies can be reduced significantly.

Formulation and validation of a computational model for a dilute biomass slurry undergoing rotational mixing

In this paper we develop a computational model for the mixing and transport of a dilute biomass slurry. The objective was to create a sufficiently simple and efficient model for biomass transport that can be coupled with reaction models for the study of conversion of cellulosic biomass into fermentable sugars. Our target system is 5%-by-mass α-cellulose, which is our proxy for more complex lignocellulosic biomass. In the authors’ previous work, an experimental investigation with α-cellulose under two vane-mixer configurations showed a bifurcation between a settling regime, for which settling effects dominate, and a suspended regime, for which solids are mostly suspended. Here, a mixed-fluid model was chosen, for which the model for the mixture-velocity field is the incompressible Navier-Stokes equations under the Boussinesq approximation for buoyancy. Solids transport includes solids motion due to diffusion, settling, advection, and shear. Comparison of simulated and experimental results show good agreement in the suspended regime, and in capturing the bifurcation rate. While the model captured well the distribution of solids in the settling regime, the model was incapable of capturing the high torque values seen in experiments with vanishing mixer rotation rate.

Physico-chemical oxidative cleavage strategy facilitates the degradation of recalcitrant crystalline cellulose by cellulases hydrolysis

BackgroundEfficient enzymatic conversion of recalcitrant crystalline cellulose is critical for enabling cost-effective industrial conversion of cellulosic biomass to biofuels and chemicals. Fully understanding enzyme digestion mechanism is paving a new way to design efficient process for biomass conversion. Accordingly, a continuing drive is inspiring to discover new routes to promote crystalline cellulose disruption.ResultsHerein, a physico-chemical oxidative cleavage strategy of irradiation oxidation/post-reduction (IOPR) was employed to treat crystalline cellulose I to cleave glycosidic bonds association with some new oxidized and reduced chain ends, thus boosting downstream degradation by cellulases from Trichoderma reesei. The hydrolysis performance of treated crystalline cellulose was conducted with either T. reesei Cel7A (TrCel7A) alone, or a cellulase enzyme mixture (90% Celluclast 1.5 L, 10% β-glucosidase). 81.6 and/or 97% of conversion efficiency have been reached for 24-h and 48-h cellulase hydrolysis, respectively. The high efficient conversion of crystalline cellulose after IOPR is mainly attributed to generating some new chain ends, which are identified by MAIDI-TOF–MS and HPLC. Furthermore, the nanoscale architectures of crystalline cellulose before and after IOPR are systematically investigated by XRD, EPR, ATR- FTIR, GPC, and XPS techniques. Together with TEM images, the results reveal a fascinating digestion mechanism of “peel-off” and “cavity-formation” paradigms toward degrading crystalline cellulose by cellulase mixtures after IOPR treatment.ConclusionsThis encouraging results show that the proposed IOPR approach will become a potential competitive alternative to current biomass pretreatment. It opens a new avenue toward the implementation of pretreatment and the design of enzyme cocktails in lignocellulosic biorefinery.

Steered molecular dynamics simulations reveal the role of Ca2+ in regulating mechanostability of cellulose-binding proteins.

The conversion of cellulosic biomass into biofuels requires degradation of the biomass into fermentable sugars. The most efficient natural cellulase system for carrying out this conversion is an extracellular multi-enzymatic complex named the cellulosome. In addition to temperature and pH stability, mechanical stability is important for functioning of cellulosome domains, and experimental techniques such as Single Molecule Force Spectroscopy (SMFS) have been used to measure the mechanical strength of several cellulosomal proteins. Molecular dynamics computer simulations provide complementary atomic-resolution quantitative maps of domain mechanical stability for identification of experimental leads for protein stabilization. In this study, we used multi-scale steered molecular dynamics computer simulations, benchmarked against new SMFS measurements, to measure the intermolecular contacts that confer high mechanical stability to a family 3 Carbohydrate Binding Module protein (CBM3) derived from the archetypal Clostridium thermocellum cellulosome. Our data predicts that electrostatic interactions in the calcium binding pocket modulate the mechanostability of the cellulose-binding module, which provides an additional design rule for the rational re-engineering of designer cellulosomes for biotechnology. Our data offers new molecular insights into the origins of mechanostability in cellulose binding domains and gives leads for synthesis of more robust cellulose-binding protein modules. On the other hand, simulations predict that insertion of a flexible strand can promote alternative unfolding pathways and dramatically reduce the mechanostability of the carbohydrate binding module, which gives routes to rational design of tailormade fingerprint complexes for force spectroscopy experiments.

Metagenomic insights into lignocellulose-degrading genes through Illumina-based de novo sequencing of the microbiome in Vietnamese native goats' rumen.

The scarcity of enzymes having an optimal activity in lignocellulose deconstruction is an obstacle for industrial-scale conversion of cellulosic biomass into biofuels. With the aim of mining novel lignocellulolytic enzymes, a ~9 Gb metagenome of bacteria in Vietnamese native goats' rumen was sequenced by Illumina platform. From the data, 821 ORFs encoding carbohydrate esterases (CEs) and polysaccharide lyases (PLs) serving for lignocellulose pre-treatment, 816 ORFs encoding 11 glycoside hydrolase families (GHs) of cellulases, and 2252 ORFs encoding 22 GHs of hemicellulases, were mined. The carbohydrate binding module (CBM) was also abundant with 763 ORFs, of which 480 ORFs are located with lignocellulolytic enzymes. The enzyme modularity analysis showed that CBMs are usually present in endoglucanase, endo 1,3-beta-D-glucosidase, and endoxylanase, whereas fibronectin 3-like module (FN3) mainly represents in GH3 and immunoglobulin-like domain (Ig) was located in GH9 only. Every domain located in each ORF was analyzed in detail to contribute enzymes' modularity which is valuable for modelling, to study the structure, and for recombinant production. With the aim of confirming the annotated results, a mined ORF encoding CBM63 was highly expressed in E. coli in soluble form. The purified recombinant CBM63 exhibited no cellulase activity, but enhanced a commercial cellulase activity in the destruction of a paper filter.

Advanced Cloning Tools for Construction of Designer Cellulosomes.

Cellulose deconstruction is achieved in nature through two main enzymatic paradigms, i.e., free enzymes and enzymatic complexes (called cellulosomes). Gaining insights into the mechanism of action and synergy among the different cellulases is of high interest, notably in the field of renewable energy, and specifically, for the conversion of cellulosic biomass to soluble sugars, en route to biofuels. In this context, designer cellulosomes are artificially assembled, chimaeric protein complexes that are used as a tool to comparatively study cellulose degradation by different enzymatic paradigms, and could also serve to improve cellulose deconstruction. Various molecular biology techniques are employed in order to design and engineer the various components of designer cellulosomes. In this chapter, we describe the cloning processes through which the appropriate modules are selected and assembled at the molecular level.

Multi-Scale Structural Studies of Sequential Ionic Liquids and Alkali Pretreated Corn Stover and Sugarcane Bagasse

The complexity of plant cell walls impedes the conversion of cellulosic biomass to sugars. Pretreatment becomes a necessity to increase the digestibility of biomass. An in-depth understanding of the structure and underlying mechanisms governing deconstruction process is important. In the present study, the comprehensive investigation of morphological and structural changes in corn stover and sugarcane bagasse following ionic liquids dissolution and alkaline extraction was done using Fourier transform infrared spectroscopy, Thermogravimetric analysis, Confocal scanning laser microscopy, Atomic force microscopy and Dynamic light scattering studies. Both the substrates were pretreated with ILs 1-ethyl-3-methylimidazolium acetate and 1-butyl-3-methylimidazolium acetate followed by alkaline extraction. The pronounced changes such as lignin, hemicelluloses removal and decreased cellulose crystallinity after the pretreatments lead to the structural transformation of matrix polymers. The enzymatic hydrolysis showed 90% theoretical sugar yield in case of sugarcane bagasse and 80% in corn stover following synergistically combined pretreatments.

Supercritical extraction of waxes and lipids from biomass: A valuable first step towards an integrated biorefinery

The decline in petrochemical wax supply coupled with the ever-growing demand for bio-products means that the development of a sustainable process to renewably sourced waxes is paramount. This review focuses on recent advances in supercritical extraction as a clean efficient process for extracting waxes from waste biomass as a part of a holistic biorefinery. The use of supercritical carbon dioxide leads to reductions in solvent waste and leaves no solvent residues meaning that the biomass can further processed without the need for energy-intensive solvent removal steps. This technology crucially improves the downstream conversion of residual cellulosic biomass for the sustainable production of sugars, consumer products and biofuels (up to a 40% increase in ethanol production) leading to higher energy efficiencies and higher economic returns. Supercritical carbon dioxide extraction is not only an important technology for the cleaner production of waxes, it is a sustainable pre-treatment of biomass as part of an integrated holistic biorefinery and significantly, it can improve the safety of products, e.g. less off gassing of wood pellets.

Metabolome analysis reveals a role for glyceraldehyde 3-phosphate dehydrogenase in the inhibition of C. thermocellum by ethanol

BackgroundClostridium thermocellum is a promising microorganism for conversion of cellulosic biomass to biofuel, without added enzymes; however, the low ethanol titer produced by strains developed thus far is an obstacle to industrial application.ResultsHere, we analyzed changes in the relative concentration of intracellular metabolites in response to gradual addition of ethanol to growing cultures. For C. thermocellum, we observed that ethanol tolerance, in experiments with gradual ethanol addition, was twofold higher than previously observed in response to a stepwise increase in the ethanol concentration, and appears to be due to a mechanism other than mutation. As ethanol concentrations increased, we found accumulation of metabolites upstream of the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) reaction and depletion of metabolites downstream of that reaction. This pattern was not observed in the more ethanol-tolerant organism Thermoanaerobacterium saccharolyticum. We hypothesize that the Gapdh enzyme may have different properties in the two organisms. Our hypothesis is supported by enzyme assays showing greater sensitivity of the C. thermocellum enzyme to high levels of NADH, and by the increase in ethanol tolerance and production when the T. saccharolyticum gapdh was expressed in C. thermocellum.ConclusionsWe have demonstrated that a metabolic bottleneck occurs at the GAPDH reaction when the growth of C. thermocellum is inhibited by high levels of ethanol. We then showed that this bottleneck could be relieved by expression of the gapdh gene from T. saccharolyticum. This enzyme is a promising target for future metabolic engineering work.

Modular Organization of the Thermobifida fusca Exoglucanase Cel6B Impacts Cellulose Hydrolysis and Designer Cellulosome Efficiency.

The protein engineering of the modular arrangement of a key exoglucanase from a highly cellulolytic bacterium, Thermobifida fusca, served to explore and compare three major enzymatic paradigms for cellulose degradation. This approach revealed highly active chimaeric forms of the exoglucanase that act in synergy together with a potent endoglucanase in bifunctional enzymes or divalent pseudo-cellulosome-like complexes. Such engineered enzymes could be further integrated into larger enzymatic complexes, thereby providing a significant step forward towards conversion of the entire T. fusca free cellulolytic system into the cellulosomal modex and the enhanced conversion of cellulosic biomass into soluble sugars.

Unique organization and unprecedented diversity of the Bacteroides (Pseudobacteroides) cellulosolvens cellulosome system

Background(Pseudo) Bacteroides cellulosolvens is an anaerobic, mesophilic, cellulolytic, cellulosome-producing clostridial bacterium capable of utilizing cellulose and cellobiose as carbon sources. Recently, we sequenced the B. cellulosolvens genome, and subsequent comprehensive bioinformatic analysis, herein reported, revealed an unprecedented number of cellulosome-related components, including 78 cohesin modules scattered among 31 scaffoldins and more than 200 dockerin-bearing ORFs. In terms of numbers, the B. cellulosolvens cellulosome system represents the most intricate, compositionally diverse cellulosome system yet known in nature.ResultsThe organization of the B. cellulosolvens cellulosome is unique compared to previously described cellulosome systems. In contrast to all other known cellulosomes, the cohesin types are reversed for all scaffoldins i.e., the type II cohesins are located on the enzyme-integrating primary scaffoldin, whereas the type I cohesins are located on the anchoring scaffoldins. Many of the type II dockerin-bearing ORFs include X60 modules, which are known to stabilize type II cohesin–dockerin interactions. In the present work, we focused on revealing the architectural arrangement of cellulosome structure in this bacterium by examining numerous interactions between the various cohesin and dockerin modules. In total, we cloned and expressed 43 representative cohesins and 27 dockerins. The results revealed various possible architectures of cell-anchored and cell-free cellulosomes, which serve to assemble distinctive cellulosome types via three distinct cohesin–dockerin specificities: type I, type II, and a novel-type designated R (distinct from type III interactions, predominant in ruminococcal cellulosomes).ConclusionsThe results of this study provide novel insight into the architecture and function of the most intricate and extensive cellulosomal system known today, thereby extending significantly our overall knowledge base of cellulosome systems and their components. The robust cellulosome system of B. cellulosolvens, with its unique binding specificities and reversal of cohesin–dockerin types, has served to amend our view of the cellulosome paradigm. Revealing new cellulosomal interactions and arrangements is critical for designing high-efficiency artificial cellulosomes for conversion of plant-derived cellulosic biomass towards improved production of biofuels.

Microbial β-Glucosidases: Screening, Characterization, Cloning and Applications

Cellulose is the most abundant biomaterial in the biosphere and the major component of plant biomass. Cellulase is an enzymatic system required for conversion of renewable cellulose biomass into free sugar for subsequent use in different applications. Cellulase system mainly consists of three individual enzymes namely: endoglucanase, exoglucanase and β-glucosidases. β-Glucosidases are ubiquitous enzymes found in all living organisms with great biological significance. β-Glucosidases have also tremendous biotechnological applications such as biofuel production, beverage industry, food industry, cassava detoxification and oligosaccharides synthesis. Microbial β-glucosidases are preferred for industrial uses because of robust activity and novel properties exhibited by them. This review aims at describing the various biochemical methods used for screening and evaluating β-glucosidases activity from microbial sources. Subsequently, it generally highlights techniques used for purification of β-glucosidases. It then elaborates various biochemical and molecular properties of this valuable enzyme such as pH and temperature optima, glucose tolerance, substrate specificity, molecular weight, and multiplicity. Furthermore, it describes molecular cloning and expression of bacterial, fungal and metagenomic β-glucosidases. Finally, it highlights the potential biotechnological applications of β-glucosidases.

Developing elite Neurospora crassa strains for cellulosic ethanol production using fungal breeding

The demand for renewable and sustainable energy has generated considerable interest in the conversion of cellulosic biomass into liquid fuels such as ethanol using a filamentous fungus. While attempts have been made to study cellulose metabolism through the use of knock-out mutants, there have been no systematic effort to characterize natural variation for cellulose metabolism in ecotypes adapted to different habitats. Here, we characterized natural variation in saccharification of cellulose and fermentation in 73 ecotypes and 89 laboratory strains of the model fungus Neurospora crassa. We observed significant variation in both traits among natural and laboratory generated populations, with some elite strains performing better than the reference strain. In the F1 population N345, 15% of the population outperformed both parents with the top performing strain having 10% improvement in ethanol production. These results suggest that natural alleles can be exploited through fungal breeding for developing elite industrial strains for bioethanol production.