Lignocellulose Deconstruction Research Articles

Gut anatomical properties and microbial functional assembly promote lignocellulose deconstruction and colony subsistence of a wood-feeding beetle.

Beneficial microbial associations enhance the fitness of most living organisms, and wood-feeding insects offer some of the most striking examples of this. Odontotaenius disjunctus is a wood-feeding beetle that possesses a digestive tract with four main compartments, each of which contains well-differentiated microbial populations, suggesting that anatomical properties and separation of these compartments may enhance energy extraction from woody biomass. Here, using integrated chemical analyses, we demonstrate that lignocellulose deconstruction and fermentation occur sequentially across compartments, and that selection for microbial groups and their metabolic pathways is facilitated by gut anatomical features. Metaproteogenomics showed that higher oxygen concentration in the midgut drives lignocellulose depolymerization, while a thicker gut wall in the anterior hindgut reduces oxygen diffusion and favours hydrogen accumulation, facilitating fermentation, homoacetogenesis and nitrogen fixation. We demonstrate that depolymerization continues in the posterior hindgut, and that the beetle excretes an energy- and nutrient-rich product on which its offspring subsist and develop. Our results show that the establishment of beneficial microbial partners within a host requires both the acquisition of the microorganisms and the formation of specific habitats within the host to promote key microbial metabolic functions. Together, gut anatomical properties and microbial functional assembly enable lignocellulose deconstruction and colony subsistence on an extremely nutrient-poor diet.

Unraveling the Fate of Lignin from Eucalyptus and Poplar during Integrated Delignification and Bleaching.

Efficient deconstruction of lignocellulose is vitally important for the biorefinery industry because lignin structures play a crucial role in the high value-added conversion of lignin. In this study, an integrated process based on hydrothermal pretreatment (HTP) and Kraft delignification was proposed to deconstruct lignocellulosic biomass. It was found that the HTP not only facilitated the production of xylo-oligosaccharides but also reduced the chemicals dosage of the following delignification. The structural characteristics of lignin obtained from the integrated process were investigated by NMR spectroscopy and gel-permeation chromatography. Additionally, double enzymatic lignins (DELs) isolated from different feedstocks were used as "model lignin" to delineate the structural transformations of lignin during H2 O2 , ClO2 , and O3 bleaching. Significant changes of the lignin structure were observed during the ClO2 bleaching process, including degradation of aromatic rings, enrichment in p-hydroxyphenyl units, and increase of carboxylic groups. A comparison of the structural characteristics of the bleached lignins indicated that HTP benefited the subsequent bleaching process. Enhanced knowledge of lignin chemistry during deconstruction and delignification could provide valuable insight into the current lignocellulose biorefinery.

Ternary deep eutectic solvents for effective biomass deconstruction at high solids and low enzyme loadings

Ternary deep eutectic solvents (DESs) were developed to enable rapid and high-solid biomass pretreatment as well as concentrated sugar hydrolysate production. Six ternary DESs constituted choline chloride (ChCl) or guanidine hydrochloride (GH) as a hydrogen bond acceptor (HBA), ethylene glycol (EG) or propylene glycol (PG) or glycerin (GLY) as a polyol-based hydrogen bond donor (HBD), and p-toluenesulfonic acid (PTSA) as an acidic HBD. GH-EG-PTSA was the most effective, evidenced by 79% xylan and 82% lignin removal in only 6 min at 120 °C and 10 wt% solid loading. Even at 35 wt% solid loading, both GH-EG-PTSA and ChCl-EG-PTSA still removed more than 60% xylan and lignin in 30 min. Using a 20% solid loading and a low enzyme loading of 5 mg protein/g solid, 128 g/L glucose and 20 g/L xylose was obtained, with a glucose yield of 78.4%. Overall, this study demonstrated novel and high-performance ternary DESs for effective lignocellulose deconstruction.

Enhanced depolymerization and utilization of raw lignocellulosic material by co-cultures of Ruminiclostridium thermocellum with hemicellulose-utilizing partners.

Ruminiclostridium thermocellum is one of the most promising candidates for consolidated bioprocessing (CBP) of low-cost lignocellulosic materials to biofuels but it still shows poor performance in its ability to deconstruct untreated lignocellulosic substrates. One promising approach to increase R. thermocellum's rate of hydrolysis is to co-culture this cellulose-specialist with partners that possess synergistic hydrolysis enzymes and metabolic capabilities. We have created co-cultures of R. thermocellum with two hemicellulose utilizers, Ruminiclostridium stercorarium and Thermoanaerobacter thermohydrosulfuricus, both of which secrete xylanolytic enzymes and utilize the pentose oligo- and monosaccharides that inhibit R. thermocellum's hydrolysis and metabolism. When grown on milled wheat straw, the co-cultures were able to solubilize up to 58% more of the total polysaccharides than the R. thermocellum mono-culture control. Repeated passaging of the co-cultures on wheat straw yielded stable populations with reduced R. thermocellum cell numbers, indicating competition for cellodextrins released from cellulose hydrolysis, although these stabilized co-cultures were still able to outperform the mono-culture controls. Repeated passaging on Avicel cellulose also yielded stable populations. Overall, the observed synergism suggests that co-culturing R. thermocellum with other members is a viable option for increasing the rate and extent of untreated lignocellulose deconstruction by R. thermocellum for CBP purposes.

Exploring oyster mushroom (Pleurotus ostreatus) substrate preparation by varying phase I composting time: changes in bacterial communities and physicochemical composition of biomass impacting mushroom yields.

To investigate characterization of the bacterial community composition and functionality and their impact on substrate biodegradation as well as mushroom yield. Bacterial diversity, composition and functionality were accessed by DNA-derived analysis for a sugarcane straw-based substrate composted for either 5, 10 or 15days. In addition, carbon and nitrogen losses, carbohydrate conversion and mushroom yields were measured for the different treatments. Changes were observed in the bacterial community diversity and composition after the process started, but not during the composting process itself. Following phase I, Acinetobacter sequences were recovered in high numbers, and selected genes associated with nitrogen metabolism and lignocellulose deconstruction were mapped. Substrate physicochemical composition showed elevated carbon and nitrogen losses after 10 and 15 days of phase I with reductions in mushroom yield. Acinetobacter species appear to play an important role in substrate degradation processes, and a 5-day phase I period showed a significant higher mushroom yield compared to composting for either 10 or 15 days. This study confers a better understanding of the bacterial community manipulation during the substrate preparation and their influence in substrate selectivity for the Pleurotus ostreatus cultivation.

Secretome profile of Cellulomonas sp. B6 growing on lignocellulosic substrates.

Lignocellulosic biomass deconstruction is a bottleneck for obtaining biofuels and value-added products. Our main goal was to characterize the secretome of a novel isolate, Cellulomonas sp. B6, when grown on residual biomass for the formulation of cost-efficient enzymatic cocktails. We identified 205 potential CAZymes in the genome of Cellulomonas sp. B6, 91 of which were glycoside hydrolases (GH). By secretome analysis of supernatants from cultures in either extruded wheat straw (EWS), grinded sugar cane straw (SCR) or carboxymethylcellulose (CMC), we identified which proteins played a role in lignocellulose deconstruction. Growth on CMC resulted in the secretion of two exoglucanases (GH6 and GH48) and two GH10 xylanases, while growth on SCR or EWS resulted in the identification of a diversity of CAZymes. From the 32 GHs predicted to be secreted, 22 were identified in supernatants from EWS and/or SCR cultures, including endo- and exoglucanases, xylanases, a xyloglucanase, an arabinofuranosidase/β-xylosidase, a β-glucosidase and an AA10. Surprisingly, among the xylanases, seven were GH10. Growth of Cellulomonas sp. B6 on lignocellulosic biomass induced the secretion of a diverse repertoire of CAZymes. Cellulomonas sp. B6 could serve as a source of lignocellulose-degrading enzymes applicable to bioprocessing and biotechnological industries.

Ethanol: A Promising Green Solvent for the Deconstruction of Lignocellulose.

Growing energy demand, environmental impact, energy security issues, and rural economic development have encouraged the development of sustainable renewable fuels. Nonfood lignocellulosic biomass is a suitable source for sustainable energy because the biomass feedstocks are low cost, abundant, and carbon neutral. Recent thermochemical conversion studies are frequently directed at converting biomass into high-quality liquid fuel precursors or chemicals in a single step. Supercritical ethanol has been selected as a promising solvent medium to deconstruct lignocellulosic biomass because ethanol has extraordinary solubility towards lignocellulosic biomass and can be resourced from cellulosic ethanol facilities. This review provides a critical insight into both catalytic and noncatalytic strategies of lignocellulose deconstruction. In this context, the supercritical ethanol deconstruction pathways are thoroughly reviewed; GC-MS, 1D and 2D NMR spectroscopy, and elemental analysis strategies towards liquid biomass deconstruction products are also critically presented. This review aims to provide readers a broad and accurate roadmap of novel biomass to biofuel conversion techniques.

FRET-SLiM on native autofluorescence: a fast and reliable method to study interactions between fluorescent probes and lignin in plant cell wall

BackgroundLignocellulosic biomass is a complex network of polymers making the cell walls of plants. It represents a feedstock of sustainable resources to be converted into fuels, chemicals and materials. Because of its complex architecture, lignocellulose is a recalcitrant material that necessitates some pretreatments and several types of catalysts to be transformed efficiently. In particular, enzymes degrading lignocellulose can become inactivated due to their binding to lignin through non-specific interactions, leading to a loss in catalytic efficiency of industrial processes. Gaining more knowledge in the strength of interactions would allow optimizing enzymes and selecting appropriate pretreatments.ResultsMeasuring interactions directly in plant cell wall can theoretically be performed using confocal fluorescence techniques by evaluating fluorescence resonance energy transfer (FRET) between compatible fluorophores. In this study, autofluorescence of plant cell wall, mainly originating from lignin, was considered as a donor fluorophore while the acceptor was a common rhodamine-based fluorescent probe. To overcome complex plant cell wall fluorescence, which limits FRET analysis by standard techniques, we have developed an original approach, combining spectral and lifetime measurements. It consists in (1) dissecting autofluorescence signal in each spectral channel, (2) optimizing spectral channel choice for lifetime measurements and (3) achieving an unambiguous FRET signature with an autofluorescent donor fluorophore. Interactions between rhodamine-based probes of various sizes and untreated or pretreated wheat sample were evaluated, showing it was possible to discriminate interactions at the nano-scale, revealing some accessibility differences and the effect of pretreatment.ConclusionsSLiM measurement allows precise estimation of the optimal spectral range for FRET measurement. SLiM response allows for the first time doubtless FRET measurements between lignin as a donor, and an acceptor fluorophore with high accuracy and sensitivity related to lifetime decrease studies. As demonstrated, it thus becomes possible to measure interactions of fluorescent probes directly inside plant cell wall samples. This approach can thus be applied to various fields such as lignocellulose deconstruction to optimize the action of enzymes or plant cell wall development to assay in situ the biosynthesis of lignin.

Biomass augmentation through thermochemical pretreatments greatly enhances digestion of switchgrass by Clostridium thermocellum

BackgroundThe thermophilic anaerobic bacterium Clostridium thermocellum is a multifunctional ethanol producer, capable of both saccharification and fermentation, that is central to the consolidated bioprocessing (CBP) approach of converting lignocellulosic biomass to ethanol without external enzyme supplementation. Although CBP organisms have evolved efficient machinery for biomass deconstruction, achieving complete solubilization requires targeted approaches, such as pretreatment, to prepare recalcitrant biomass feedstocks for further biological digestion. Here, differences between how C. thermocellum and fungal cellulases respond to senescent switchgrass prepared by four different pretreatment techniques revealed relationships between biomass substrate composition and its digestion by the two biological approaches.ResultsAlamo switchgrass was pretreated using hydrothermal, dilute acid, dilute alkali, and co-solvent-enhanced lignocellulosic fractionation (CELF) pretreatments to produce solids with varying glucan, xylan, and lignin compositions. C. thermocellum achieved highest sugar release and metabolite production from de-lignified switchgrass prepared by CELF and dilute alkali pretreatments demonstrating greater resilience to the presence of hemicellulose sugars than fungal enzymes. 100% glucan solubilization and glucan plus xylan release from switchgrass were achieved using the CELF–CBP combination. Lower glucan solubilization and metabolite production by C. thermocellum was observed on solids prepared by dilute acid and hydrothermal pretreatments with higher xylan removal from switchgrass than lignin removal. Further, C. thermocellum (2% by volume inoculum) showed ~ 48% glucan solubilization compared to < 10% through fungal enzymatic hydrolysis (15 and 65 mg protein/g glucan loadings) of unpretreated switchgrass indicating the effectiveness of C. thermocellum’s cellulosome. Overall, C. thermocellum performed equivalent to 65 and better than 15 mg protein/g glucan fungal enzymatic hydrolysis on all substrates except CELF-pretreated substrates. CELF pretreatments of switchgrass produced solids that were highly digestible regardless of whether C. thermocellum or fungal enzymes were chosen.ConclusionsThe unparalleled comprehensive nature of this work with a comparison of four pretreatment and two biological digestion techniques provides a strong platform for future integration of pretreatment with CBP. Lignin removal had a more positive impact on biological digestion of switchgrass than xylan removal from the biomass. However, the impact of switchgrass structural properties, including cellulose, hemicellulose, and lignin characterization, would provide a better understanding of lignocellulose deconstruction.

EcXyl43 β-xylosidase: molecular modeling, activity on natural and artificial substrates, and synergism with endoxylanases for lignocellulose deconstruction.

Biomass hydrolysis constitutes a bottleneck for the biotransformation of lignocellulosic residues into bioethanol and high-value products. The efficient deconstruction of polysaccharides to fermentable sugars requires multiple enzymes acting concertedly. GH43 β-xylosidases are among the most interesting enzymes involved in hemicellulose deconstruction into xylose. In this work, the structural and functional properties of β-xylosidase EcXyl43 from Enterobacter sp. were thoroughly characterized. Molecular modeling suggested a 3D structure formed by a conserved N-terminal catalytic domain linked to an ancillary C-terminal domain. Both domains resulted essential for enzymatic activity, and the role of critical residues, from the catalytic and the ancillary modules, was confirmed by mutagenesis. EcXyl43 presented β-xylosidase activity towards natural and artificial substrates while arabinofuranosidase activity was only detected on nitrophenyl α-L-arabinofuranoside (pNPA). It hydrolyzed xylobiose and purified xylooligosaccharides (XOS), up to degree of polymerization 6, with higher activity towards longer XOS. Low levels of activity on commercial xylan were also observed, mainly on the soluble fraction. The addition of EcXyl43 to GH10 and GH11 endoxylanases increased the release of xylose from xylan and pre-treated wheat straw. Additionally, EcXyl43 exhibited high efficiency and thermal stability under its optimal conditions (40°C, pH6.5), with a half-life of 58h. Therefore, this enzyme could be a suitable additive for hemicellulases in long-term hydrolysis reactions. Because of its moderate inhibition by monomeric sugars but its high inhibition by ethanol, EcXyl43 could be particularly more useful in separate hydrolysis and fermentation (SHF) than in simultaneous saccharification and co-fermentation (SSCF) or consolidated bioprocessing (CBP).

Genus-Wide Assessment of Lignocellulose Utilization in the Extremely Thermophilic Genus Caldicellulosiruptor by Genomic, Pangenomic, and Metagenomic Analyses.

Metagenomic data from Obsidian Pool (Yellowstone National Park, USA) and 13 genome sequences were used to reassess genus-wide biodiversity for the extremely thermophilic Caldicellulosiruptor The updated core genome contains 1,401 ortholog groups (average genome size for 13 species = 2,516 genes). The pangenome, which remains open with a revised total of 3,493 ortholog groups, encodes a variety of multidomain glycoside hydrolases (GHs). These include three cellulases with GH48 domains that are colocated in the glucan degradation locus (GDL) and are specific determinants for microcrystalline cellulose utilization. Three recently sequenced species, Caldicellulosiruptor sp. strain Rt8.B8 (renamed here Caldicellulosiruptor morganii), Thermoanaerobacter cellulolyticus strain NA10 (renamed here Caldicellulosiruptor naganoensis), and Caldicellulosiruptor sp. strain Wai35.B1 (renamed here Caldicellulosiruptor danielii), degraded Avicel and lignocellulose (switchgrass). C. morganii was more efficient than Caldicellulosiruptor bescii in this regard and differed from the other 12 species examined, both based on genome content and organization and in the specific domain features of conserved GHs. Metagenomic analysis of lignocellulose-enriched samples from Obsidian Pool revealed limited new information on genus biodiversity. Enrichments yielded genomic signatures closely related to that of Caldicellulosiruptor obsidiansis, but there was also evidence for other thermophilic fermentative anaerobes (Caldanaerobacter, Fervidobacterium, Caloramator, and Clostridium). One enrichment, containing 89.8% Caldicellulosiruptor and 9.7% Caloramator, had a capacity for switchgrass solubilization comparable to that of C. bescii These results refine the known biodiversity of Caldicellulosiruptor and indicate that microcrystalline cellulose degradation at temperatures above 70°C, based on current information, is limited to certain members of this genus that produce GH48 domain-containing enzymes.IMPORTANCE The genus Caldicellulosiruptor contains the most thermophilic bacteria capable of lignocellulose deconstruction, which are promising candidates for consolidated bioprocessing for the production of biofuels and bio-based chemicals. The focus here is on the extant capability of this genus for plant biomass degradation and the extent to which this can be inferred from the core and pangenomes, based on analysis of 13 species and metagenomic sequence information from environmental samples. Key to microcrystalline hydrolysis is the content of the glucan degradation locus (GDL), a set of genes encoding glycoside hydrolases (GHs), several of which have GH48 and family 3 carbohydrate binding module domains, that function as primary cellulases. Resolving the relationship between the GDL and lignocellulose degradation will inform efforts to identify more prolific members of the genus and to develop metabolic engineering strategies to improve this characteristic.

Can We Reduce the Cellulase Enzyme Loading Required To Achieve Efficient Lignocellulose Deconstruction by Only Using the Initially Absorbed Enzymes?

The cost-effective production of sugars from biomass continues to be challenging, partly due to the relatively high enzyme/protein loading required to achieve effective hydrolysis of the insoluble polysaccharides within the pretreated lignocellulosic substrates. Previous work has suggested that those enzymes that initially, strongly adsorb to the insoluble substrate are crucial for effective cellulose hydrolysis. However, most previous work in this topic area has used either purified enzymes or “older” generations of cellulase preparations acting on “model” cellulosic substrates. The results were, in several cases, contradictory or inconclusive. In the work reported here, the roles and functions of the initially adsorbed enzymes in determining the rate and extent of cellulose hydrolysis were assessed when using several different pretreated biomass substrates and the Novozyme enzyme preparation, Cellic CTec3. It was apparent that the initially adsorbed enzymes (irreversible bound to substrate after centrif...

Processive Degradation of Crystalline Cellulose by a Multimodular Endoglucanase via a Wirewalking Mode.

Processive hydrolysis of crystalline cellulose by cellulases is a critical step for lignocellulose deconstruction. The classic Trichoderma reesei exoglucanase TrCel7A, which has a closed active-site tunnel, starts each processive run by threading the tunnel with a cellulose chain. Loop regions are necessary for tunnel conformation, resulting in weak thermostability of fungal exoglucanases. However, endoglucanase CcCel9A, from the thermophilic bacterium Clostridium cellulosi, comprises a glycoside hydrolase (GH) family 9 module with an open cleft and five carbohydrate-binding modules (CBMs) and hydrolyzes crystalline cellulose processively. How CcCel9A and other similar GH9 enzymes bind to the smooth surface of crystalline cellulose to achieve processivity is still unknown. Our results demonstrate that the C-terminal CBM3b and three CBMX2s enhance productive adsorption to cellulose, while the CBM3c adjacent to the GH9 is tightly bound to 11 glucosyl units, thereby extending the catalytic cleft to 17 subsites, which facilitates decrystallization by forming a supramodular binding surface. In the open cleft, the strong interaction forces between substrate-binding subsites and glucosyl rings enable cleavage of the hydrogen bonds and extraction of a single cellulose chain. In addition, subsite -4 is capable of drawing the chain to its favored location. Cellotetraose is released from the open cleft as the initial product to achieve high processivity, which is further hydrolyzed to cellotriose, cellobiose and glucose by the catalytic cleft of the endoglucanase. On this basis, we propose a wirewalking mode for processive degradation of crystalline cellulose by an endoglucanase, which provides insights for rational design of industrial cellulases.

Deciphering lignocellulose deconstruction by the white rot fungus Irpex lacteus based on genomic and transcriptomic analyses

BackgroundIrpex lacteus is one of the most potent white rot fungi for biological pretreatment of lignocellulose for second biofuel production. To elucidate the underlying molecular mechanism involved in lignocellulose deconstruction, genomic and transcriptomic analyses were carried out for I. lacteus CD2 grown in submerged fermentation using ball-milled corn stover as the carbon source.ResultsIrpex lacteus CD2 efficiently decomposed 74.9% lignin, 86.3% cellulose, and 83.5% hemicellulose in corn stover within 9 days. Manganese peroxidases were rapidly induced, followed by accumulation of cellulase and hemicellulase. Genomic analysis revealed that I. lacteus CD2 possessed a complete set of lignocellulose-degrading enzyme system composed mainly of class II peroxidases, dye-decolorizing peroxidases, auxiliary enzymes, and 182 glycoside hydrolases. Comparative transcriptomic analysis substantiated the notion of a selection mode of degradation. These analyses also suggested that free radicals, derived either from MnP-organic acid interplay or from Fenton reaction involving Fe2+ and H2O2, could play an important role in lignocellulose degradation.ConclusionsThe selective strategy employed by I. lacteus CD2, in combination with low extracellular glycosidases cleaving plant cell wall polysaccharides into fermentable sugars, may account for high pretreatment efficiency of I. lacteus. Our study also hints the importance of free radicals for future designing of novel, robust lignocellulose-degrading enzyme cocktails.

NMR studies on lignocellulose deconstructions in the digestive system of the lower termite Coptotermes formosanus Shiraki

Termites represent one of the most efficient lignocellulose decomposers on earth. The mechanism by which termites overcome the recalcitrant lignin barrier to gain access to embedded polysaccharides for assimilation and energy remains largely unknown. In the present study, softwood, hardwood, and grass lignocellulose diets were fed to Coptotermes formosanus workers, and structural differences between the original lignocellulose diets and the resulting feces were examined by solution-state multidimensional nuclear magnetic resonance (NMR) techniques as well as by complementary wet-chemical methods. Overall, our data support the view that lignin polymers are partially decomposed during their passage through the termite gut digestive system, although polysaccharide decomposition clearly dominates the overall lignocellulose deconstruction process and the majority of lignin polymers remain intact in the digestive residues. High-resolution NMR structural data suggested preferential removal of syringyl aromatic units in hardwood lignins, but non-acylated guaiacyl units as well as tricin end-units in grass lignins. In addition, our data suggest that termites and/or their gut symbionts may favor degradation of C–C-bonded β–5 and resinol-type β–β lignin inter-monomeric units over degradation of ether-bonded β–O–4 units, which is in contrast to what has been observed in typical lignin biodegradation undertaken by wood-decaying fungi.

A new screened microbial consortium OEM2 for lignocellulosic biomass deconstruction and chlorophenols detoxification

Recalcitrance limits biomass application in biorefinery. It is even more so when toxic chlorophenols are present. In this study, we screened a microbial consortium, OEM2, for lignocellulose deconstruction and chlorophenols detoxification through a short-term and efficient screening process. Microbial consortium OEM2 had a good buffer capability in the cultivation process and exhibited a high xylanase activity, with over 85% hemicellulose degradation within 12 days. Throughout the treatment process, 41.5% rice straw decomposition on day 12 and around 75% chlorophenols (MCP, 2,4-DCP, 2,4,6-TCP) removal on day 9, were recorded. Moreover, Fourier translation infrared spectroscopy (FTIR) analysis indicated that chemical bonds and groups (eg. hydrogen-bond, β-1,4 glycosidic bond, lignin-carbohydrate cross-linking) in the rice straw were broken. Cuticle and silica layer destruction and subsequent exposed cellulose fibers were observed by scanning electron microscopy (SEM). Microbial consortium OEM2 diversity analysis by 16S rRNA gene sequencing indicated that Proteobacteria (41.3%) was the most abundant phylum and the genera Paenibacillus and Pseudomonas played an important role in the lignocellulose decomposition and chlorophenols detoxification. This study developed a faster and more efficient strategy to screen a specific microbial consortium. And the new microbial consortium, OEM2, makes lignocellulose more accessible and complex pollutants unproblematic in the further biorefinery process.

Lignin-First Biorefining of Lignocellulose: the Impact of Process Severity on the Uniformity of Lignin Oil Composition

In lignin-first biorefining via reductive processes, lignocellulosic materials are deconstructed by the solvent extraction of lignin in the presence of a hydrogenation catalyst. This approach provides a route to the successful extraction and reductive passivation of lignin fragments to produce low molar mass lignin oils together with high-quality pulps. Herein, we present an investigation into the impact of process severity (i.e., cooking temperature) on the reductive processes taking place on the lignin fragments and uniformity of the product mixture. In addition to improving overall delignification yields (up to 87%) and producing low molar mass fragments, higher process temperatures led to the preferential cleavage of hydroxyl groups in monolignol sidechains via hydrodeoxygenation, yielding oils with lower oxygen content. By comparing products from both lignin-first biorefining and organosolv processes at various temperatures, we elucidate key performance differences and outline routes to increased chemical uniformity in lignin streams. Overall, this study outlines clearly the importance of process temperature in the deconstruction of lignocellulose by lignin-first biorefining when producing highly depolymerized lignin products. This study points out a trade-off in the effect of temperature upon delignification and increase in product mixture complexity, which needs to be carefully optimized for the scale-up of lignin-first technologies.

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.

Cleave and couple: toward fully sustainable catalytic conversion of lignocellulose to value added building blocks and fuels.

The structural complexity of lignocellulose offers unique opportunities for the development of entirely new, energy efficient and waste-free pathways in order to obtain valuable bio-based building blocks. Such sustainable catalytic methods - specifically tailored to address the efficient conversion of abundant renewable starting materials - are necessary to successfully compete, in the future, with fossil-based multi-step processes. In this contribution we give a summary of recent developments in this field and describe our "cleave and couple" strategy, where "cleave" refers to the catalytic deconstruction of lignocellulose to aromatic and aliphatic alcohol intermediates, and "couple" involves the development of novel, sustainable transformations for the formation of C-C and C-N bonds in order to obtain a range of attractive products from lignocellulose.

Plant cell wall composition and enzymatic deconstruction

Cellulosic ethanol is one the most prominent technologies capable of replacing the use of fossil fuels in an observable horizon of technological development. The complexity of plant biomass, however, continues to challenge our ability to convert it into biofuels efficiently. Highly complex and cross-linked polysaccharides, hydrophobic and protein adsorbent polymers, and crystalline supramolecular structures comprise some of the structures that shield the plant cell contents (and the shield structures themselves) against predators. In response, a sophisticated enzymatic weaponry, with its associated chemical and physical mechanisms, is necessary to overcome this recalcitrance. Here we describe basic information about chemical composition of lignocellulosic biomass and the enzymatic arsenal for lignocellulose deconstruction into fermentable sugars.