Effect of cellulose reducing ends and primary hydroxyl groups modifications on cellulose-cellulase interactions and cellulose hydrolysis.

Cellulose reducing ends are believed to play a vital role in the cellulose recalcitrance to enzymatic conversion. However, their role in insoluble cellulose accessibility and hydrolysis is not clear. Thus, in this study, reducing ends of insoluble cellulose derived from various sources were modified by applying reducing and/or oxidizing agents. The effects of cellulose reducing ends modification on cellulose reducing ends, cellulose structure, and cellulose accessibility to cellulase were evaluated along with the impact on cellulose hydrolysis with complete as well purified cellulase components. Sodium borohydride (NaBH4) reduction and sodium chlorite-acetic acid (SC/AA) oxidation were able to modify more than 90% and 60% of the reducing ends, respectively, while the bicinchoninic acid (BCA) reagent applied for various cycles oxidized cellulose reducing ends to various extents. X-ray diffractograms of the treated solids showed that these treatments did not change the cellulose crystalline structure and the change in crystallinity index was insignificant. Surprisingly, it was found that the cellulose reducing ends modification, either through selective NaBH4 reduction or BCA oxidation, had a negligible impact on cellulose accessibility as well on cellulose hydrolysis rates or final conversions with complete cellulase at loadings as low as 0.5 mg protein/g cellulose. In fact, in contrast to what is traditionally believed, modifications of cellulose reducing ends by these two methods had no apparent impact on cellulose conversion with purified cellulase components and their synergy. However, SC/AA oxidation resulted in significant drop in cellulose conversion (10%-50%) with complete as well purified cellulase components. Nonetheless, further research revealed that the cause for drop in cellulose conversion for the SC/AA oxidation case was due to primary hydroxyl groups (PHGs) oxidation and not the oxidation of reducing ends. Furthermore, it was found that the PHGs modification affects cellulose accessibility and slows the cellulase uptake as well resulting in significant drop in cellulose conversions.

Techno-economic analysis of biomass value-added processing informed by pilot scale de-ashing of paper sludge feedstock.

Paper sludge biomass represents an underutilized feedstock rich in pulped and processed cellulose which is currently a waste stream with significant disposal cost to industry for landfilling services. Effective fractionation of the cellulose from paper sludge presents an opportunity to yield cellulose as feedstock for value-added processes. A novel approach to cellulose fractionation is the sidehill screening system, herein studied at the pilot-plant scale. Composition analysis determined ash removal and carbohydrate retention of both sidehill and high-performance benchtop screening systems. Sidehill screening resulted in greater carbohydrates retention relative to benchtop screening (90% vs 66%) and similar ash removal (95% vs 98%). Techno-economic analysis for production of sugar syrup yielded a minimum selling price of $331/metric ton of sugar syrup including disposal savings, significantly less than a commercial sugar syrup without fractionation. Sensitivity analysis showed that screening conditions played a significant role in economic feasibility for cellulosic yield and downstream processes.

Enzymatic Synthesis of Xylan Microparticles with Tunable Morphologies.

Xylans are a diverse family of hemicellulosic polysaccharides found in abundance within the cell walls of nearly all flowering plants. Unfortunately, naturally occurring xylans are highly heterogeneous, limiting studies of their synthesis and structure-function relationships. Here, we demonstrate that xylan synthase 1 from the charophyte alga Klebsormidium flaccidum is a powerful biocatalytic tool for the bottom-up synthesis of pure ß-1,4 xylan polymers that self-assemble into microparticles in vitro. Using uridine diphosphate-xylose (UDP-xylose) and defined saccharide primers as substrates, we demonstrate that the shape, composition, and properties of the self-assembling xylan microparticles could be readily controlled via the fine structure of the xylan oligosaccharide primer used to initiate polymer elongation. Furthermore, we highlight two approaches for bottom-up and surface functionalization of xylan microparticles with chemical probes and explore the susceptibility of xylan microparticles to enzymatic hydrolysis. Together, these results provide a useful platform for structural and functional studies of xylans to investigate cell wall biosynthesis and polymer-polymer interactions and suggest possible routes to new biobased materials with favorable properties for biomedical and renewable applications.

Single-Laboratory Validation to Extend the Scope of AOAC Official MethodSM 2015.06 to Indian Infant, Child, and Adult Nutritional Matrixes.

BACKGROUND: AOAC Method 2015.06 is a Final Action Official MethodSM for the determination of 12 elements (Na, Mg, P, K, Ca, Cr, Mn, Fe, Cu, Zn, Se, and Mo) in infant formula and adult nutritional products, based on inductively coupled plasma (ICP)-MS. Currently, its scope does not include certain kinds of formulations used in India. The method would likely be used more in Indian laboratories if its performance were characterized on Indian matrixes. OBJECTIVE: In this study we describe a typical single-laboratory validation (SLV) exercise designed to characterize the precision and accuracy of AOAC Method 2015.06 for common Indian nutritional matrixes so that the scope of the method can be extended to include them. METHOD: Six matrixes specific to the Indian market were carried through an SLV and the Standard Method Performance Requirements (SMPRs®) previously published for this method were used to evaluate the results. RESULTS: The method demonstrated typical repeatability (<5% RSD), and intermediate precision (5-8% RSD) on the Indian matrixes, with very few exceptions. Accuracy was demonstrated by overspike recoveries in the range of 90-110% over 3 days for the Indian matrixes, as well as excellent agreement with previously published results for three additional matrixes tested. Some of the new Indian matrixes required alternate sample preparation procedures versus the usual reconstitution prescribed by the SMPRs. CONCLUSIONS: The SLV results showed that AOAC Method 2015.06 can be extended to include these Indian matrices. HIGHLIGHTS: The two special sample preparation procedures can now be considered validated.

Single-Laboratory Validation of AOAC Official Method 2011.10 for Vitamin B12 in 'Indian' Infant and Pediatric Formulas and Adult Nutritionals.

BACKGROUND: Ensuring the quality of infant and pediatric formulas and adult nutritionals is of utmost importance for the health and safety of rapidly urbanizing Indian population. B12 is an important water-soluble vitamin, which is fortified externally in such nutritional formulations. The Bureau of Indian Standards (BIS) has a recommended microbiological assay-based method for determination of vitamin B12 that is not precise and accurate enough to meet the label claim requirements of infant, adult, and/or pediatric nutritionals. The AOAC Official Method 2011.10 was originally developed under the AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN) for the determination of vitamin B12 in infant and pediatric formulas and adult nutritionals. However, those SPIFAN matrixes did not contain malt and other indigenous cereal and legume flour (with or without cocoa powder), which are commonly found in Indian formulations. Thus, there is a need to replace this method with a more precise and accurate method. OBJECTIVE: This study was undertaken to validate the AOAC Official Method 2011.10 on vitamin B12 in 'Indian' infant and pediatric formulas and adult nutritionals. METHODS: The single-laboratory validation (SLV) of AOAC Method 2011.10 was carried out as per the AOAC Guidelines in six Indian pediatric and adult nutritional formulas to verify its fitness for purpose. Cobalamin in the sample was converted to cyanocobalamin on treatment with potassium cyanide. The sample was then subjected to clean up through a C18 cartridge. Vitamin B12 in the eluted extract was separated from other components using size-exclusion column chromatography followed by a C18 column. The HPLC analysis was carried out at 550 nm. RESULTS: Diastase treatment and C18 solid-phase extraction cleanup satisfactorily removed the matrix interference. The relative standard deviation of the determined values in 30 samples each from 6 selected Indian products and NIST SRM 1849a was <20%. The average recoveries for the spiked recovery samples ranged from 91.75 to 101.14%. CONCLUSIONS: Method 2011.10 met the standard method performance requirements set forth by the AOAC SPIFAN. Therefore, we recommend the Method 2011.10 for adoption as the BIS official method for the analysis of vitamin B12 in 'Indian' infant and pediatric formulas and adult nutritionals. HIGHLIGHTS: This was the first SLV project that the AOAC India section undertook to extend the scope of the AOAC Method 2011.10 for vitamin B12 analysis by validating it in 'Indian' infant and pediatric formulas and adult nutritionals.

Simultaneous upgrading of biomass-derived sugars to HMF/furfural via enzymatically isomerized ketose intermediates.

BACKGROUND: Recently, exploring fermentative or chemical pathways that convert biomass-derived sugars to fuels/chemicals has attracted a lot of interest from many researchers. We are investigating a hydrocarbon pathway from mixed sugars via 5-hydroxymethyl furfural (HMF) and furfural intermediates. To achieve this goal, we must first convert glucose and xylose to HMF and furfural in favorable yields. Current processes to produce HMF/furfural generally involve the use of acid catalysts in biphasic systems or solvents such as ionic liquids. However, the yield from transforming glucose to HMF is lower than the yield of furfural from xylose. RESULTS: In this study, we present an efficient chemical pathway simultaneously transforming glucose and xylose to HMF and furfural via ketose intermediates, i.e., fructose and xylulose, which were generated from glucose and xylose via enzymatic isomerization. In the enzymatic isomerization, by adding sodium borate to complex with the ketoses, xylose conversion reached equilibrium after 2 h with a conversion of 91% and glucose conversion reached 84% after 4 h. By enzymatically isomerizing the aldoses to ketoses, the following dehydration reactions to HMF and furfural could be performed at low process temperatures (i.e., 110-120 °C) minimizing the side reactions of the sugars and limiting the degradation of furfurals to humins and carboxylic acids. At 120 °C, pH 0.5, and 15 min reaction time, mixed ketose sugars were converted to HMF and furfural in yields of 77% and 96%, respectively (based on starting aldose concentrations). CONCLUSION: Taken together, our results demonstrate that this combined biological and chemical process could be an effective pathway to simultaneously convert biomass-derived glucose and xylose to HMF and furfural, for use as intermediates in the production of hydrocarbons.

Nanomechanics of cellulose deformation reveal molecular defects that facilitate natural deconstruction.

Technologies surrounding utilization of cellulosic materials have been integral to human society for millennia. In many materials, controlled introduction of defects provides a means to tailor properties, introduce reactivity, and modulate functionality for various applications. The importance of defects in defining the behavior of cellulose is becoming increasingly recognized. However, fully exploiting defects in cellulose to benefit biobased materials and conversion applications will require an improved understanding of the mechanisms of defect induction and corresponding molecular-level consequences. We have identified a fundamental relationship between the macromolecular structure and mechanical behavior of cellulose nanofibrils whereby molecular defects may be induced when the fibrils are subjected to bending stress exceeding a certain threshold. By nanomanipulation, imaging, and molecular modeling, we demonstrate that cellulose nanofibrils tend to form kink defects in response to bending stress, and that these macromolecular features are often accompanied by breakages in the glucan chains. Direct observation of deformed cellulose fibrils following partial enzymatic digestion reveals that processive cellulases exploit these defects as initiation sites for hydrolysis. Collectively, our findings provide a refined understanding of the interplay between the structure, mechanics, and reactivity of cellulose assemblies.

An iterative computational design approach to increase the thermal endurance of a mesophilic enzyme.

BACKGROUND: Strategies for maximizing the microbial production of bio-based chemicals and fuels include eliminating branched points to streamline metabolic pathways. While this is often achieved by removing key enzymes, the introduction of nonnative enzymes can provide metabolic shortcuts, bypassing branched points to decrease the production of undesired side-products. Pyruvate decarboxylase (PDC) can provide such a shortcut in industrially promising thermophilic organisms; yet to date, this enzyme has not been found in any thermophilic organism. Incorporating nonnative enzymes into host organisms can be challenging in cases such as this, where the enzyme has evolved in a very different environment from that of the host. RESULTS: In this study, we use computational protein design to engineer the Zymomonas mobilis PDC to resist thermal denaturation at the growth temperature of a thermophilic host. We generate thirteen PDC variants using the Rosetta protein design software. We measure thermal stability of the wild-type PDC and PDC variants using circular dichroism. We then measure and compare enzyme endurance for wild-type PDC with the PDC variants at an elevated temperature of 60 °C (thermal endurance) using differential interference contrast imaging. CONCLUSIONS: We find that increases in melting temperature (Tm) do not directly correlate with increases in thermal endurance at 60 °C. We also do not find evidence that any individual mutation or design approach is the major contributor to the most thermostable PDC variant. Rather, remarkable cooperativity among sixteen thermostabilizing mutations is key to rationally designing a PDC with significantly enhanced thermal endurance. These results suggest a generalizable iterative computational protein design approach to improve thermal stability and endurance of target enzymes.

Gaze-Aware Streaming Solutions for the Next Generation of Mobile VR Experiences.

This paper presents a novel approach to content delivery for video streaming services. It exploits information from connected eye-trackers embedded in the next generation of VR Head Mounted Displays (HMDs). The proposed solution aims to deliver high visual quality, in real time, around the users' fixations points while lowering the quality everywhere else. The goal of the proposed approach is to substantially reduce the overall bandwidth requirements for supporting VR video experiences while delivering high levels of user perceived quality. The prerequisites to achieve these results are: (1) mechanisms that can cope with different degrees of latency in the system and (2) solutions that support fast adaptation of video quality in different parts of a frame, without requiring a large increase in bitrate. A novel codec configuration, capable of supporting near-instantaneous video quality adaptation in specific portions of a video frame, is presented. The proposed method exploits in-built properties of HEVC encoders and while it introduces a moderate amount of error, these errors are indetectable by users. Fast adaptation is the key to enable gaze-aware streaming and its reduction in bandwidth. A testbed implementing gaze-aware streaming, together with a prototype HMD with in-built eye tracker, is presented and was used for testing with real users. The studies quantified the bandwidth savings achievable by the proposed approach and characterize the relationships between Quality of Experience (QoE) and network latency. The results showed that up to 83% less bandwidth is required to deliver high QoE levels to the users, as compared to conventional solutions.

High activity CAZyme cassette for improving biomass degradation in thermophiles.

BACKGROUND: Thermophilic microorganisms and their enzymes offer several advantages for industrial application over their mesophilic counterparts. For example, a hyperthermophilic anaerobe, Caldicellulosiruptor bescii, was recently isolated from hot springs in Kamchatka, Siberia, and shown to have very high cellulolytic activity. Additionally, it is one of a few microorganisms being considered as viable candidates for consolidated bioprocessing applications. Moreover, C. bescii is capable of deconstructing plant biomass without enzymatic or chemical pretreatment. This ability is accomplished by the production and secretion of free, multi-modular and multi-functional enzymes, one of which, CbCel9A/Cel48A also known as CelA, is able to outperform enzymes found in commercial enzyme preparations. Furthermore, the complete C. bescii exoproteome is extremely thermostable and highly active at elevated temperatures, unlike commercial fungal cellulases. Therefore, understanding the functional diversity of enzymes in the C. bescii exoproteome and how inter-molecular synergy between them confers C. bescii with its high cellulolytic activity is an important endeavor to enable the production of more efficient biomass degrading enzyme formulations and in turn, better cellulolytic industrial microorganisms. RESULTS: To advance the understanding of the C. bescii exoproteome we have expressed, purified, and tested four of the primary enzymes found in the exoproteome and we have found that the combination of three or four of the most highly expressed enzymes exhibit synergistic activity. We also demonstrated that discrete combinations of these enzymes mimic and even  improve upon the activity of the whole C. bescii exoproteome, even though some of the enzymes lack significant activity on their own. CONCLUSIONS: We have demonstrated that it is possible to replicate the cellulolytic activity of the native C. bescii exoproteome utilizing a minimal gene set, and that these minimal gene sets are more active than the whole exoproteome. In the future, this may lead to more simplified and efficient cellulolytic enzyme preparations or yield improvements when these enzymes are expressed in microorganisms engineered for consolidated bioprocessing.

The Multi Domain Caldicellulosiruptor bescii CelA Cellulase Excels at the Hydrolysis of Crystalline Cellulose.

The crystalline nature of cellulose microfibrils is one of the key factors influencing biomass recalcitrance which is a key technical and economic barrier to overcome to make cellulosic biofuels a commercial reality. To date, all known fungal enzymes tested have great difficulty degrading highly crystalline cellulosic substrates. We have demonstrated that the CelA cellulase from Caldicellulosiruptor bescii degrades highly crystalline cellulose as well as low crystallinity substrates making it the only known cellulase to function well on highly crystalline cellulose. Unlike the secretomes of cellulolytic fungi, which typically comprise multiple, single catalytic domain enzymes for biomass degradation, some bacterial systems employ an alternative strategy that utilizes multi-catalytic domain cellulases. Additionally, CelA is extremely thermostable and highly active at elevated temperatures, unlike commercial fungal cellulases. Furthermore we have determined that the factors negatively affecting digestion of lignocellulosic materials by C. bescii enzyme cocktails containing CelA appear to be significantly different from the performance barriers affecting fungal cellulases. Here, we explore the activity and degradation mechanism of CelA on a variety of pretreated substrates to better understand how the different bulk components of biomass, such as xylan and lignin, impact its performance.

Towards an Understanding of Enhanced Biomass Digestibility by In Planta Expression of a Family 5 Glycoside Hydrolase.

In planta expression of a thermophilic endoglucanase (AcCel5A) reduces recalcitrance by creating voids and other irregularities in cell walls of Arabidopsis thaliana that increase enzyme accessibility without negative impacts on plant growth or cell wall composition. Our results suggest that cellulose ß-1-4 linkages can be cut sparingly in the assembling wall and that these minimal changes, made at the proper time, have an impact on plant cell wall recalcitrance without negative effects on overall plant development.

Multifunctional Cellulolytic Enzymes Outperform Processive Fungal Cellulases for Coproduction of Nanocellulose and Biofuels.

Producing fuels, chemicals, and materials from renewable resources to meet societal demands remains an important step in the transition to a sustainable, clean energy economy. The use of cellulolytic enzymes for the production of nanocellulose enables the coproduction of sugars for biofuels production in a format that is largely compatible with the process design employed by modern lignocellulosic (second generation) biorefineries. However, yields of enzymatically produced nanocellulose are typically much lower than those achieved by mineral acid production methods. In this study, we compare the capacity for coproduction of nanocellulose and fermentable sugars using two vastly different cellulase systems: the classical "free enzyme" system of the saprophytic fungus, Trichoderma reesei (T. reesei) and the complexed, multifunctional enzymes produced by the hot springs resident, Caldicellulosiruptor bescii (C. bescii). We demonstrate by comparative digestions that the C. bescii system outperforms the fungal enzyme system in terms of total cellulose conversion, sugar production, and nanocellulose production. In addition, we show by multimodal imaging and dynamic light scattering that the nanocellulose produced by the C. bescii cellulase system is substantially more uniform than that produced by the T. reesei system. These disparities in the yields and characteristics of the nanocellulose produced by these disparate systems can be attributed to the dramatic differences in the mechanisms of action of the dominant enzymes in each system.

Enzymes in Commercial Cellulase Preparations Bind Differently to Dioxane Extracted Lignins.

Commercial fungal cellulases used in biomass-to-biofuels processes can be grouped into three general classes: native, augmented, and engineered. Colorimetric assays for general glycoside hydrolase activities showed distinct differences in enzyme binding to lignin for each enzyme activity. Native cellulase preparations demonstrated low binding of endo- and exocellulases, high binding of xylanase, and moderate binding for ß-D-glucosidases. Engineered cellulase formulations exhibited low binding of exocellulases, very strong binding of endocellulases and ß-D-glucosidase, and mixed binding of xylanase activity. The augmented cellulase had low binding of exocellulase, high binding of endocellulase and xylanase, and moderate binding of ß-D-glucosidase activities. Bound and unbound activities were correlated to general molecular weight ranges of proteins as measured by loss of proteins bands in bound fractions on SDS-PAGE gels. Lignin-bound high molecular weight bands correlated to binding of ß-D-glucosidase activity. Whereas ß-D-glucosidases demonstrated high binding in many cases, they have been shown to remain active. Bound low molecular weight bands correlated to xylanase activity binding. Contrary to other literature, exocellulase activity did not show strong lignin binding. The variation in enzyme activity binding between these three classes of cellulases preparations indicates that it is possible to alter the binding of specific glycoside hydrolase activities during the enzyme formulation process. It remains unclear whether or not loss of endocellulase activity to lignin binding is problematic for biomass conversion.

In situ label-free imaging of hemicellulose in plant cell walls using stimulated Raman scattering microscopy.

BACKGROUND: Plant hemicellulose (largely xylan) is an excellent feedstock for renewable energy production and second only to cellulose in abundance. Beyond a source of fermentable sugars, xylan constitutes a critical polymer in the plant cell wall, where its precise role in wall assembly, maturation, and deconstruction remains primarily hypothetical. Effective detection of xylan, particularly by in situ imaging of xylan in the presence of other biopolymers, would provide critical information for tackling the challenges of understanding the assembly and enhancing the liberation of xylan from plant materials. RESULTS: Raman-based imaging techniques, especially the highly sensitive stimulated Raman scattering (SRS) microscopy, have proven to be valuable tools for label-free imaging. However, due to the complex nature of plant materials, especially those same chemical groups shared between xylan and cellulose, the utility of specific Raman vibrational modes that are unique to xylan have been debated. Here, we report a novel approach based on combining spectroscopic analysis and chemical/enzymatic xylan removal from corn stover cell walls, to make progress in meeting this analytical challenge. We have identified several Raman peaks associated with xylan content in cell walls for label-free in situ imaging xylan in plant cell wall. CONCLUSION: We demonstrated that xylan can be resolved from cellulose and lignin in situ using enzymatic digestion and label-free SRS microscopy in both 2D and 3D. We believe that this novel approach can be used to map xylan in plant cell walls and that this ability will enhance our understanding of the role played by xylan in cell wall biosynthesis and deconstruction.

Direct Production of Propene from the Thermolysis of Poly(ß-hydroxybutyrate) (PHB). An Experimental and DFT Investigation.

We demonstrate a synthetic route toward the production of propene directly from poly(ß-hydroxybutyrate) (PHB), the most common of a wide range of high-molecular-mass microbial polyhydroxyalkanoates. Propene, a major commercial hydrocarbon, was obtained from the depolymerization of PHB and subsequent decarboxylation of the crotonic acid monomer in good yields (up to 75 mol %). The energetics of PHB depolymerization and the gas-phase decarboxylation of crotonic acid were also studied using density functional theory (DFT). The average activation energy for the cleavage of the R'C(O)O-R linkage is calculated to be 163.9 ± 7.0 kJ mol(-1). Intramolecular, autoacceleration effects regarding the depolymerization of PHB, as suggested in some literature accounts, arising from the formation of crotonyl and carboxyl functional groups in the products could not be confirmed by the results of DFT and microkinetic modeling. DFT results, however, suggest that intermolecular catalysis involving terminal carboxyl groups may accelerate PHB depolymerization. Activation energies for this process were estimated to be about 20 kJ mol(-1) lower than that for the noncatalyzed ester cleavage, 144.3 ± 6.4 kJ mol(-1). DFT calculations predict the decarboxylation of crotonic acid to follow second-order kinetics with an activation energy of 147.5 ± 6.3 kJ mol(-1), consistent with that measured experimentally, 146.9 kJ mol(-1). Microkinetic modeling of the PHB to propene overall reaction predicts decarboxylation of crotonic acid to be the rate-limiting step, consistent with experimental observations. The results also indicate that improvements made to enhance the isomerization of crotonic acid to vinylacetic acid will improve the direct conversion of PHB to propene.

New perspective on glycoside hydrolase binding to lignin from pretreated corn stover.

BACKGROUND: Non-specific binding of cellulases to lignin has been implicated as a major factor in the loss of cellulase activity during biomass conversion to sugars. It is believed that this binding may strongly impact process economics through loss of enzyme activities during hydrolysis and enzyme recycling scenarios. The current model suggests glycoside hydrolase activities are lost though non-specific/non-productive binding of carbohydrate-binding domains to lignin, limiting catalytic site access to the carbohydrate components of the cell wall. RESULTS: In this study, we have compared component enzyme affinities of a commercial Trichoderma reesei cellulase formulation, Cellic CTec2, towards extracted corn stover lignin using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and p-nitrophenyl substrate activities to monitor component binding, activity loss, and total protein binding. Protein binding was strongly affected by pH and ionic strength. ß-d-glucosidases and xylanases, which do not have carbohydrate-binding modules (CBMs) and are basic proteins, demonstrated the strongest binding at low ionic strength, suggesting that CBMs are not the dominant factor in enzyme adsorption to lignin. Despite strong adsorption to insoluble lignin, ß-d-glucosidase and xylanase activities remained high, with process yields decreasing only 4-15 % depending on lignin concentration. CONCLUSION: We propose that specific enzyme adsorption to lignin from a mixture of biomass-hydrolyzing enzymes is a competitive affinity where ß-d-glucosidases and xylanases can displace CBM interactions with lignin. Process parameters, such as temperature, pH, and salt concentration influence the individual enzymes' affinity for lignin, and both hydrophobic and electrostatic interactions are responsible for this binding phenomenon. Moreover, our results suggest that concern regarding loss of critical cell wall degrading enzymes to lignin adsorption may be unwarranted when complex enzyme mixtures are used to digest biomass.

Parameter determination and validation for a mechanistic model of the enzymatic saccharification of cellulose-Iß.

Cost-effective production of fuels and chemicals from lignocellulosic biomass often involves enzymatic saccharification, which has been the subject of intense research and development. Recently, a mechanistic model for the enzymatic saccharification of cellulose has been developed that accounts for distribution of cellulose chain lengths, the accessibility of insoluble cellulose to enzymes, and the distinct modes of action of the component cellulases [Griggs et al. (2012) Biotechnol. Bioeng., 109(3):665-675; Griggs et al. (2012) Biotechnol. Bioeng., 109(3):676-685]. However, determining appropriate values for the adsorption, inhibition, and rate parameters required further experimental investigation. In this work, we performed several sets of experiments to aid in parameter estimation and to quantitatively validate the model. Cellulosic materials differing in degrees of polymerization and crystallinity (α-cellulose-Iß and highly crystalline cellulose-Iß ) were digested by component enzymes (EGI/CBHI/ßG) and by mixtures of these enzymes. Based on information from the literature and the results from these experiments, a single set of model parameters was determined, and the model simulation results using this set of parameters were compared with the experimental data of total glucan conversion, chain-length distribution, and crystallinity. Model simulations show significant agreement with the experimentally derived glucan conversion and chain-length distribution curves and provide interesting insights into multiple complex and interacting physico-chemical phenomena involved in enzymatic hydrolysis, including enzyme synergism, substrate accessibility, cellulose chain length distribution and crystallinity, and inhibition of cellulases by soluble sugars.

Agave proves to be a low recalcitrant lignocellulosic feedstock for biofuels production on semi-arid lands.

BACKGROUND: Agave, which is well known for tequila and other liquor production in Mexico, has recently gained attention because of its attractive potential to launch sustainable bioenergy feedstock solutions for semi-arid and arid lands. It was previously found that agave cell walls contain low lignin and relatively diverse non-cellulosic polysaccharides, suggesting unique recalcitrant features when compared to conventional C4 and C3 plants. RESULTS: Here, we report sugar release data from fungal enzymatic hydrolysis of non-pretreated and hydrothermally pretreated biomass that shows agave to be much less recalcitrant to deconstruction than poplar or switchgrass. In fact, non-pretreated agave has a sugar release five to eight times greater than that of poplar wood and switchgrass . Meanwhile, state of the art techniques including glycome profiling, nuclear magnetic resonance (NMR), Simon's Stain, confocal laser scanning microscopy and so forth, were applied to measure interactions of non-cellulosic wall components, cell wall hydrophilicity, and enzyme accessibility to identify key structural features that make agave cell walls less resistant to biological deconstruction when compared to poplar and switchgrass. CONCLUSIONS: This study systematically evaluated the recalcitrant features of agave plants towards biofuels applications. The results show that not only does agave present great promise for feeding biorefineries on semi-arid and arid lands, but also show the value of studying agave's low recalcitrance for developments in improving cellulosic energy crops.

Hydration and saccharification of cellulose Iß, II and III(I) at increasing dry solids loadings.

Crystalline cellulose Iß (Avicel) was chemically transformed into cellulose II and III(I) producing allomorphs with similar crystallinity indices (ATR-IR and XRD derived). Saccharifications by commercial cellulases at arrayed solids loadings showed cellulose III(I) was more readily hydrolysable and less susceptible to increased dry solids levels than cellulose Iß and II. Analysis by dynamic vapor sorption revealed cellulose II has a distinctively higher absorptive capacity than cellulose I and III(I). When equally hydrated (g water/g cellulose), low-field nuclear magnetic resonance (LF-NMR) relaxometry showed that cellulose II, on average, most constrained water while cellulase III(I) left the most free water. LF-NMR spin-spin relaxation time distribution profiles representing distinct water pools suggest cellulose III(I) had the most restricted pool and changes in water distribution during enzymatic saccharification were most dramatic with respect to cellulose III(I) compared to celluloses Iß and II.