Enzymatic Degradation Of Cellulose Research Articles

ALACEN: A Holistic Herbaceous Biomass Fractionation Process Attaining a Xylose-Rich Stream for Direct Microbial Conversion to Bioplastics.

Lignocellulose biorefining is a promising technology for the sustainable production of chemicals and biopolymers. Usually, when one component is focused on, the chemical nature and yield of the others are compromised. Thus, one of the bottlenecks in biomass biorefining is harnessing the maximum value from all of the lignocellulosic components. Here, we describe a mild stepwise process in a flow-through setup leading to separate flow-out streams containing cinnamic acid derivatives, glucose, xylose, and lignin as the main components from different herbaceous sources. The proposed process shows that minimal degradation of the individual components and conservation of their natural structure are possible. Under optimized conditions, the following fractions are produced from wheat straw based on their respective contents in the feed by the ALkaline ACid ENzyme process: (i) 78% ferulic acid from a mild ALkali step, (ii) 51% monomeric xylose free of fermentation inhibitors by mild ACidic treatment, (iii) 82% glucose from ENzymatic degradation of cellulose, and (iv) 55% native-like lignin. The benefits of using the flow-through setup are demonstrated. The retention of the lignin aryl ether structure was confirmed by HSQC NMR, and this allowed monomers to form from hydrogenolysis. More importantly, the crude xylose-rich fraction was shown to be suitable for producing polyhydroxybutyrate bioplastics. The direct use of the xylose-rich fraction by means of the thermophilic bacteria Schlegelella thermodepolymerans matched 91% of the PHA produced with commercial pure xylose, achieving 138.6 mgPHA/gxylose. Overall, the ALACEN fractionation method allows for a holistic valorization of the principal components of herbaceous biomasses.

Enzymatic degradation of cellulose in soil: A review

Cellulose degradation is a critical process in soil ecosystems, playing a vital role in nutrient cycling and organic matter decomposition. Enzymatic degradation of cellulosic biomass is the most sustainable and green method of producing liquid biofuel. It has gained intensive research interest with future perspective as the majority of terrestrial lignocellulose biomass has a great potential to be used as a source of bioenergy. However, the recalcitrant nature of lignocellulose limits its use as a source of energy. Noteworthy enough, enzymatic conversion of cellulose biomass could be a leading future technology. Fungal enzymes play a central role in cellulose degradation. Our understanding of fungal cellulases has substantially redirected in the past few years with the discovery of a new class of enzymes and Cellulosome. Efforts have been made from time to time to develop an economically viable method of cellulose degradation. This review provides insights into the current state of knowledge regarding cellulose degradation in soil and identifies areas where further research is needed.

Optimized Lytic Polysaccharide Monooxygenase Action Increases Fiber Accessibility and Fibrillation by Releasing Tension Stress in Cellulose Cotton Fibers.

Lytic polysaccharide monooxygenase (LPMO) enzymes have recently shaken up our knowledge of the enzymatic degradation of biopolymers and cellulose in particular. This unique class of metalloenzymes cleaves cellulose and other recalcitrant polysaccharides using an oxidative mechanism. Despite their potential in biomass saccharification and cellulose fibrillation, the detailed mode of action of LPMOs at the surface of cellulose fibers still remains poorly understood and highly challenging to investigate. In this study, we first determined the optimal parameters (temperature, pH, enzyme concentration, and pulp consistency) of LPMO action on the cellulose fibers by analyzing the changes in molar mass distribution of solubilized fibers using high performance size exclusion chromatography (HPSEC). Using an experimental design approach with a fungal LPMO from the AA9 family (PaLPMO9H) and cotton fibers, we revealed a maximum decrease in molar mass at 26.6 °C and pH 5.5, with 1.6% w/w enzyme loading in dilute cellulose dispersions (100 mg of cellulose at 0.5% w/v). These optimal conditions were used to further investigate the effect of PaLPMO9H on the cellulosic fiber structure. Direct visualization of the fiber surface by scanning electron microscopy (SEM) revealed that PaLPMO9H created cracks on the cellulose surface while it attacked tension regions that triggered the rearrangement of cellulose chains. Solid-state NMR indicated that PaLPMO9H increased the lateral fibril dimension and created novel accessible surfaces. This study confirms the LPMO-driven disruption of cellulose fibers and extends our knowledge of the mechanism underlying such modifications. We hypothesize that the oxidative cleavage at the surface of the fibers releases the tension stress with loosening of the fiber structure and peeling of the surface, thereby increasing the accessibility and facilitating fibrillation.

Enzymatic degradation of bacterial cellulose derived carbon nanofibers (BC-CNF) by myeloperoxidase (MPO): Performance evaluation for biosensing

Myeloperoxidase (MPO) is a promising biomarker for early warning of incidence of cardiovascular diseases (CVDs). Among various sensing mechanisms for detection and quantification of MPO, enzymatic degradation of carbon nanoparticles (CNPs) in presence of MPO has unique advantages such as visual change in optical contrast of sample without requiring additional probe chemicals or reagents. While the degradation of various forms of CNPs (including carbon nanotubes and graphene oxide) due to MPO has been reported in literature, the degradation is typically very slow, resulting in response times on the order of multiple hours, and thus presents a significant limitation for CVD diagnosis. Besides, the extent of degradation is highly sensitive to substrate uniformity, necessitating complicated synthesis processes to obtain pristine CNPs. In this work, we have characterized the enzymatic degradation of bacterial cellulose derived carbon nanofibers (BC-CNF) due to MPO in vitro and explored the feasibility of realizing a BC-CNF colorimetric biosensor for measuring MPO activity. The degradation of BC-CNFs is thoroughly characterized using SEM, TEM, Raman and UV–Vis spectroscopy. We report that carboxylic group functionalized BC-CNFs demonstrate noticeable change in optical contrast due to degradation with clinically relevant concentrations of MPO in approximately 1h, and hold great promise for realizing low-cost biosensors for MPO.

Boosting enzymatic degradation of cellulose using a fungal expansin: Structural insight into the pretreatment mechanism

The recalcitrance of cellulosic biomass greatly hinders its enzymatic degradation. Expansins induce cell wall loosening and promote efficient cellulose utilization; however, the molecular mechanism underlying their action is not well understood. In this study, TlEXLX1, a fungal expansin from Talaromyces leycettanus JCM12802, was characterized in terms of phylogeny, synergy, structure, and mechanism of action. TlEXLX1 displayed varying degrees of synergism with commercial cellulase in the pretreatment of corn straw and filter paper. TlEXLX1 binds to cellulose via domain 2, mediated by CH–π interactions with residues Tyr291, Trp292, and Tyr327. Residues Asp237, Glu238, and Asp248 in domain 1 form hydrogen bonds with glucose units and break the inherent hydrogen bonding within the cellulose matrix. This study identified the expansin amino acid residues crucial for cellulose binding, and elucidated the structure and function of expansins in cell wall networks; this has potential applications in biomass utilization.

H2O2 in Liquid Fractions of Hydrothermally Pretreated Biomasses: Implications of Lytic Polysaccharide Monooxygenases

Lytic polysaccharide monooxygenases (LPMOs) are important players in enzyme-aided valorization of lignocellulose. However, the recently discovered dependency of LPMO catalysis on H2O2 along with H2O2-caused inactivation of the enzyme calls for an in-depth understanding of the levels and the dynamics of H2O2 in various streams of the processing of lignocellulosic biomass. Using an LPMO-based sensitive detection, we assessed the dynamics of H2O2 in the liquid fractions (LFs) of hydrothermally pretreated wheat straw (agricultural residue), birch (hardwood), and pine (softwood). Upon contact with air, H2O2 was formed in the LFs of all biomasses. The initially high rate of H2O2 formation decayed with the half-life around 1–2 h to a low but stable plateau value. The rates of H2O2 formation were much higher than the rates of its accumulation, suggesting that H2O2 is an intermediate in aerobic oxidation of the compounds in LFs. Although the general traits were similar, LFs of different biomasses had different rates of H2O2 formation and accumulation. LFs of different biomasses also differed by their effect on the enzymatic degradation of cellulose. Compositional analyses revealed a number of different compounds that were formed and disappeared upon aerobic oxidation of LFs.

Microwave-assisted hydrotropic pretreatment as a new and highly efficient way to cellulosic ethanol production from maize distillery stillage

Aim of the study was to assess the suitability of the combined use of microwave radiation and sodium cumene sulfonate under optimized process conditions for the preparation of maize stillage biomass as a raw material for the production of cellulosic ethanol. The key parameter guaranteeing a high level of lignin removal from biomass (ca. 44%) was concentration of hydrotrope. Even at high biomass concentration (16% w/v) and a cellulase enzyme dose of about 4 filter-paper units/g, maize stillage biomass subjected to microwave-assisted hydrotropic pretreatment was highly susceptible to enzymatic degradation, which resulted in 80% hydrolysis yield. It is possible to obtain a fermentation medium with a very high glucose concentration (up to 80 g/L), without fermentation inhibitors and, as a consequence, to reach a very high level of sugar conversion to ethanol (concentration above 40 g/L), even as much as 95% of theoretical yield. Microwave hydrotropic treatment with sodium cumene sulfonate is a very effective way to prepare waste maize stillage biomass for the production of cellulosic ethanol. The degradation of the lignocellulose structure by the simultaneous use of microwaves and hydrotropes ensured a high degree of conversion of structural polysaccharides to bioethanol. The method provides a high level of enzymatic degradation of cellulose, leading to a medium with high content of released sugars suitable for bioconversion, which is in line with assumptions of the second-generation ethanol production technology.Key points• Microwave-assisted hydrotropic pretreatment is a new way to cellulosic ethanol production.• Microwave-assisted hydrotropic delignification removes 44% of lignin from biomass.• No fermentation inhibitors are obtained after microwave-assisted hydrotropic pretreatment.• High ethanol concentration (above 40 g/L) and fermentation yield (95% of theoretical yield) from biomass after microwave-assisted hydrotropic pretreatment.

Characterization of biomass-degrading enzymes using neutron diffraction and scattering

Efficient enzymatic degradation of cellulose is essential to the low-cost production of cellulosic ethanol and high-value-added chemicals. Fungal lytic polysaccharide monooxygenases (LPMOs), which ...

Sonolytic, sonocatalytic and sonophotocatalytic degradation of carboxymethyl cellulose (CMC): kinetic and mechanisms

In this research, enzymatic degradation of carboxymethyl cellulose (CMC) with different concentration of substrate was studied using stable available parameters of Michaelis-Menton and calculated maximum reaction rate Vmax. We also used effective approaches such as ultrasonic, sonocatalytic and sonophotocatalytic irradiation in the presence of TiO2 nanoparticles as pretreatment. Degradation of the cellulose by means of ultrasound irradiation and its combination with heterogeneous (TiO2) was investigated. The emphasis was on evaluating theeffect of additives on degradation rate constants. Ultrasound irradiation (24 kHz) was provided by a sonicator, while an ultraviolet source of 16 W was used for UV irradiation. The extent of sonolytic degradation increased when ultrasound power (in the range 1560 W) increased, and the presence of TiO2 did not have significant effect on degradation. We should also note that TiO2 sonophotocatalysis led to complete cellulose degradation in 120 min with increasing the catalyst loading. TiO2 sonophotocatalysis was always faster than the respective individual processes due to the enhanced formation of reactive radicals as well as the possible ultrasound-induced increase of the active surface area of the catalyst. Their efficacy on enhancement of reactivity was discussed based on the kinetic parameters including, Michaelis constant Km, maximum reaction rate Vmax and initial reaction rate, correlating with ultrasonic conditions. Also, values of Km and Vmax were calculated in the absence and presence of ultrasonic and sonophoto waves.

Mesoporous silica nanoparticles for β-glucosidase immobilization by templating with a green material: Tannic acid

Bioconversion of lignocellulosic biomass to biofuels through enzymatic hydrolysis has become a promising strategy to reduce our dependence on fossil fuels. β-Glucosidase (BG) is a key enzyme in the enzymatic degradation of cellulose. However, free β-glucosidase is relatively unstable, costly and difficult to recover. Immobilization of β-glucosidase may help to improve its reusability and stability, allowing developing a sustainable low-carbon and resource-efficient circular economy. BG was immobilized by adsorption on synthesised tannic acid templated mesoporous silica nanoparticles (TA-MSNPs). The synthesis procedure adopts a green templating agent cheap and biocompatible. Textural and morphological characterization was performed. We studied the effect induced by the morphology, pore size and surface features of supports, by using different amounts of templating agent, on the catalytic performance of the biocatalyst. The immobilization procedure used preserved most of the secondary structure of the enzyme and consequently its catalytic activity. The biocatalyst exhibiting the best activity and operational stability is obtained with the sorbent prepared with the largest quantity of tannic acid. In this case, the mesopores are too small for the enzyme to enter them, and the large amount of adsorbed enzyme is due to its peculiar surface features, favouring the formation of a multilayer enzyme corona.

Polysaccharide monooxygenase-catalyzed oxidation of cellulose to glucuronic acid-containing cello-oligosaccharides

BackgroundPolysaccharide monooxygenases (PMOs) play an important role in the enzymatic degradation of cellulose. They have been demonstrated to able to C6-oxidize cellulose to produce C6-hexodialdoses. However, the biological function of C6 oxidation of PMOs remains unknown. In particular, it is unclear whether C6-hexodialdoses can be further oxidized to uronic acid (glucuronic acid-containing oligosaccharides).ResultsA PMO gene, Hipmo1, was isolated from Humicola insolens and expressed in Pichia pastoris. This PMO (HiPMO1), belonging to the auxiliary activity 9 (AA9) family, was shown to able to cleave cellulose to yield non-oxidized and oxidized cello-oligosaccharides. The enzyme oxidizes C6 positions in cellulose to form glucuronic acid-containing cello-oligosaccharides, followed by hydrolysis with beta-glucosidase and beta-glucuronidase to yield glucose, glucuronic acid, and saccharic acid. This indicates that HiPMO1 can catalyze C6 oxidation of hydroxyl groups of cellulose to carboxylic groups.ConclusionsHiPMO1 oxidizes C6 of cellulose to form glucuronic acid-containing cello-oligosaccharides followed by hydrolysis with beta-glucosidase and beta-glucuronidase to yield glucose, glucuronic acid, and saccharic acid, and even possibly by beta-eliminative cleavage to produce unsaturated cello-oligosaccharides. This study provides a new mechanism for cellulose cleavage by C6 oxidation of HiPMO1.

Cellulases adsorb reversibly on biomass lignin.

Adsorption of cellulases onto lignin is considered a major factor in retarding enzymatic cellulose degradation of lignocellulosic biomass. However, the adsorption mechanisms and kinetics are not well understood for individual types of cellulases. This study examines the binding affinity, kinetics of adsorption, and competition of four monocomponent cellulases of Trichoderma reesei during adsorption onto lignin. TrCel7A, TrCel6A, TrCel7B, and TrCel5A were radiolabeled for adsorption experiments on lignin‐rich residues (LRRs) isolated from hydrothermally pretreated spruce (L‐HPS) and wheat straw (L‐HPWS), respectively. On the basis of adsorption isotherms fitted to the Langmuir model, the ranking of binding affinities was TrCel5A > TrCel6A > TrCel7B > TrCel7A on both types of LRRs. The enzymes had a higher affinity to the L‐HPS than to the L‐HPWS. Adsorption experiments with dilution after 1 and 24 hr and kinetic modeling were performed to quantify any irreversible binding over time. Models with reversible binding parameters fitted well and can explain the results obtained. The adsorption constants obtained from the reversible models agreed with the fitted Langmuir isotherms and suggested that reversible adsorption–desorption existed at equilibrium. Competitive binding experiments showed that individual types of cellulases competed for binding sites on the lignin and the adsorption data fitted the Langmuir adsorption model. Overall, the data strongly indicate that the adsorption of cellulases onto lignin is reversible and the findings have implications for the development of more efficient cellulose degrading enzymes.

Lignin from hydrothermally pretreated grass biomass retards enzymatic cellulose degradation by acting as a physical barrier rather than by inducing nonproductive adsorption of enzymes

BackgroundLignin is known to hinder efficient enzymatic conversion of lignocellulose in biorefining processes. In particular, nonproductive adsorption of cellulases onto lignin is considered a key mechanism to explain how lignin retards enzymatic cellulose conversion in extended reactions.ResultsLignin-rich residues (LRRs) were prepared via extensive enzymatic cellulose degradation of corn stover (Zea mays subsp. mays L.), Miscanthus × giganteus stalks (MS) and wheat straw (Triticum aestivum L.) (WS) samples that each had been hydrothermally pretreated at three severity factors (log R0) of 3.65, 3.83 and 3.97. The LRRs had different residual carbohydrate levels—the highest in MS; the lowest in WS. The residual carbohydrate was not traceable at the surface of the LRRs particles by ATR-FTIR analysis. The chemical properties of the lignin in the LRRs varied across the three types of biomass, but monolignols composition was not affected by the severity factor. When pure cellulose was added to a mixture of LRRs and a commercial cellulolytic enzyme preparation, the rate and extent of glucose release were unaffected by the presence of LRRs regardless of biomass type and severity factor, despite adsorption of the enzymes to the LRRs. Since the surface of the LRRs particles were covered by lignin, the data suggest that the retardation of enzymatic cellulose degradation during extended reaction on lignocellulosic substrates is due to physical blockage of the access of enzymes to the cellulose caused by the gradual accumulation of lignin at the surface of the biomass particles rather than by nonproductive enzyme adsorption.ConclusionsThe study suggests that lignin from hydrothermally pretreated grass biomass retards enzymatic cellulose degradation by acting as a physical barrier blocking the access of enzymes to cellulose rather than by inducing retardation through nonproductive adsorption of enzymes.

Visualization of the enzymatic degradation of crystalline cellulose at a molecular level

Visualization of the enzymatic degradation of crystalline cellulose at a molecular level

Nondestructive, real-time determination and visualization of cellulose, hemicellulose and lignin by luminescent oligothiophenes

Enabling technologies for efficient use of the bio-based feedstock are crucial to the replacement of oil-based products. We investigated the feasibility of luminescent conjugated oligothiophenes (LCOs) for non-destructive, rapid detection and quality assessment of lignocellulosic components in complex biomass matrices. A cationic pentameric oligothiophene denoted p-HTEA (pentamer hydrogen thiophene ethyl amine) showed unique binding affinities to cellulose, lignin, hemicelluloses, and cellulose nanofibrils in crystal, liquid and paper form. We exploited this finding using spectrofluorometric methods and fluorescence confocal laser scanning microscopy, for sensitive, simultaneous determination of the structural and compositional complexities of native lignocellulosic biomass. With exceptional photostability, p-HTEA is also demonstrated as a dynamic sensor for real-time monitoring of enzymatic cellulose degradation in cellulolysis. These results demonstrate the use of p-HTEA as a non-destructive tool for the determination of cellulose, hemicellulose and lignin in complex biomass matrices, thereby aiding in the optimization of biomass-converting technologies.

Insight into Enzymatic Degradation of Corn, Wheat, and Soybean Cell Wall Cellulose Using Quantitative Secretome Analysis of Aspergillus fumigatus.

Lignocelluloses contained in animal forage cannot be digested by pigs or poultry with 100% efficiency. On contrary, Aspergillus fumigatus, a saprophytic filamentous fungus, is known to harbor 263 glycoside hydrolase encoding genes, suggesting that A. fumigatus is an efficient lignocellulose degrader. Hence the present study uses corn, wheat, or soybean as a sole carbon source to culture A. fumigatus under animal physiological condition to understand how cellulolytic enzymes work together to achieve an efficient degradation of lignocellulose. Our results showed that A. fumigatus produced different sets of enzymes to degrade lignocelluloses derived from corn, wheat, or soybean cell wall. In addition, the cellulolytic enzymes produced by A. fumigatus were stable under acidic condition or at higher temperatures. Using isobaric tags for a relative and absolute quantification (iTRAQ) approach, a total of ∼600 extracellular proteins were identified and quantified, in which ∼50 proteins were involved in lignocellulolysis, including cellulases, hemicellulases, lignin-degrading enzymes, and some hypothetical proteins. Data are available via ProteomeXchange with identifier PXD004670. On the basis of quantitative iTRAQ results, 14 genes were selected for further confirmation by RT-PCR. Taken together, our results indicated that the expression and regulation of lignocellulolytic proteins in the secretome of A. fumigatus were dependent on both nature and complexity of cellulose, thus suggesting that a different enzyme system is required for degradation of different lignocelluloses derived from plant cells. Although A. fumigatus is a pathogenic fungus and cannot be directly used as an enzyme source, as an efficient lignocellulose degrader its strategy to synergistically degrade various lignocelluloses with different enzymes can be used to design enzyme combination for optimal digestion and absorption of corn, wheat, or soybean that are used as forage of pig and poultry.

Synergistic enhancement of cellulase pairs linked by consensus ankyrin repeats: Determination of the roles of spacing, orientation, and enzyme identity

Biomass deconstruction to small simple sugars is a potential approach to biofuels production; however, the highly recalcitrant nature of biomass limits the economic viability of this approach. Thus, research on efficient biomass degradation is necessary to achieve large-scale production of biofuels. Enhancement of cellulolytic activity by increasing synergism between cellulase enzymes holds promise in achieving high-yield biofuels production. Here we have inserted cellulase pairs from extremophiles into hyperstable α-helical consensus ankyrin repeat domain scaffolds. Such chimeric constructs allowed us to optimize arrays of enzyme pairs against a variety of cellulolytic substrates. We found that endocellulolytic domains CelA (CA) and Cel12A (C12A) act synergistically in the context of ankyrin repeats, with both three and four repeat spacing. The extent of synergy differs for different substrates. Also, having C12A N-terminal to CA provides greater synergy than the reverse construct, especially against filter paper. In contrast, we do not see synergy for these enzymes in tandem with CelK (CK) catalytic domain, a larger exocellulase, demonstrating the importance of enzyme identity in synergistic enhancement. Furthermore, we found endocellulases CelD and CA with three repeat spacing to act synergistically against filter paper. Importantly, connecting CA and C12A with a disordered linker of similar contour length shows no synergistic enhancement, indicating that synergism results from connecting these domains with folded ankyrin repeats. These results show that ankyrin arrays can be used to vary spacing and orientation between enzymes, helping to design and optimize artificial cellulosomes, providing a novel architecture for synergistic enhancement of enzymatic cellulose degradation. Proteins 2016; 84:1043-1054. © 2016 Wiley Periodicals, Inc.

Cellular automata modeling depicts degradation of cellulosic material by a cellulase system with single-molecule resolution.

BackgroundEnzymatic hydrolysis of cellulose involves the spatiotemporally correlated action of distinct polysaccharide chain cleaving activities confined to the surface of an insoluble substrate. Because cellulases differ in preference for attacking crystalline compared to amorphous cellulose, the spatial distribution of structural order across the cellulose surface imposes additional constraints on the dynamic interplay between the enzymes. Reconstruction of total system behavior from single-molecule activity parameters is a longstanding key goal in the field.ResultsWe have developed a stochastic, cellular automata-based modeling approach to describe degradation of cellulosic material by a cellulase system at single-molecule resolution. Substrate morphology was modeled to represent the amorphous and crystalline phases as well as the different spatial orientations of the polysaccharide chains. The enzyme system model consisted of an internally chain-cleaving endoglucanase (EG) as well as two processively acting, reducing and non-reducing chain end-cleaving cellobiohydrolases (CBHs). Substrate preference (amorphous: EG, CBH II; crystalline: CBH I) and characteristic frequencies for chain cleavage, processive movement, and dissociation were assigned from biochemical data. Once adsorbed, enzymes were allowed to reach surface-exposed substrate sites through “random-walk” lateral diffusion or processive motion. Simulations revealed that slow dissociation of processive enzymes at obstacles obstructing further movement resulted in local jamming of the cellulases, with consequent delay in the degradation of the surface area affected. Exploiting validation against evidence from atomic force microscopy imaging as a unique opportunity opened up by the modeling approach, we show that spatiotemporal characteristics of cellulose surface degradation by the system of synergizing cellulases were reproduced quantitatively at the nanometer resolution of the experimental data. This in turn gave useful prediction of the soluble sugar release rate.ConclusionsSalient dynamic features of cellulose surface degradation by different cellulases acting in synergy were reproduced in simulations in good agreement with evidence from high-resolution visualization experiments. Due to the single-molecule resolution of the modeling approach, the utility of the presented model lies not only in predicting system behavior but also in elucidating inherently complex (e.g., stochastic) phenomena involved in enzymatic cellulose degradation. Thus, it creates synergy with experiment to advance the mechanistic understanding for improved application.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0463-8) contains supplementary material, which is available to authorized users.

Tunable Semicrystalline Thin Film Cellulose Substrate for High-Resolution, In-Situ AFM Characterization of Enzymatic Cellulose Degradation.

In the field of enzymatic cellulose degradation, fundamental interactions between different enzymes and polymorphic cellulose materials are of essential importance but still not understood in full detail. One technology with the potential of direct visualization of such bioprocesses is atomic force microscopy (AFM) due to its capability of real-time in situ investigations with spatial resolutions down to the molecular scale. To exploit the full capabilities of this technology and unravel fundamental enzyme-cellulose bioprocesses, appropriate cellulose substrates are decisive. In this study, we introduce a semicrystalline-thin-film-cellulose (SCFTC) substrate which fulfills the strong demands on such ideal cellulose substrates by means of (1) tunable polymorphism via variable contents of homogeneously sized cellulose nanocrystals embedded in an amorphous cellulose matrix; (2) nanoflat surface topology for high-resolution and high-speed AFM; and (3) fast, simple, and reproducible fabrication. The study starts with a detailed description of SCTFC preparation protocols including an in-depth material characterization. In the second part, we demonstrate the suitability of SCTFC substrates for enzymatic degradation studies by combined, individual, and sequential exposure to TrCel6A/TrCel7A cellulases (Trichoderma reesei) to visualize synergistic effects down to the nanoscale.

Evaluation of the correlations between biodegradability of lignocellulosic feedstocks in anaerobic digestion process and their biochemical characteristics

Biochemical composition and reactivity are key factors controlling the biodegradability of lignocellulosic residues. In the present study, 14 lignocellulosic substrates including 6 agricultural and 8 forest residues were analyzed for 9 biochemical characteristics, including BioMethane Potential (BMP), Biological Oxygen Demand (BOD), Enzymatic Cellulose Degradation tests (ECD), Van Soest and NREL fractionation methods. The data obtained were exploited by principal component analysis (PCA) and other statistical methods to investigate the possible correlations between the parameters. The study showed that the contents in particular lignin or in non-extractible residues (RES) were the characteristics which influenced most the anaerobic biodegradability (BMP), while the influence of the soluble fraction was quite low. BMP was well correlated with the ratio of the contents in non-lignin over lignin fractions and the cellulose to lignin ratio. Regarding agricultural residues, BMP was better correlated with lignin content than with RES content. Agricultural and forest residues exhibited distinct characteristics of aerobic and anaerobic biodegradability. Good correlation was observed between ECD and lignin content. Finally, it was also observed that Van Soest's and NREL methods did not provide the same results in terms of biochemical composition.