Bacterial cellulose nanocrystals obtained through enzymatic and acidic routes: A comparative study of their main properties and in vitro biological responses.

Cellulose nanocrystals (CNCs) are crystalline domains isolated from cellulosic fibers. They have been utilized in a wide range of applications, such as reinforcing fillers, antibacterial agents and manufacturing of biosensors. Whitin this context, the aim of this work was to obtain and analyze CNCs extracted from bacterial nanocellulose (BNC) using two distinct methods combined with milling pre-treatment: an acidic hydrolysis using 64 % sulfuric acid and an enzymatic hydrolysis using a commercial cellulase enzyme mixture. The CNCs obtained from the enzymatic route (e-CNCs) were observed to be spherical nanoparticles with diameter of 56 ± 11 nm. In contrast, the CNCs from the acid hydrolysis (a-CNCs) appeared as needle-shaped nanoparticles with a high aspect ratio with lengths/widths of 158 ± 64 nm/11 ± 2 nm. The surface zeta potential (ZP) of the a-CNCs was -30,8 mV, whereas the e-CNCs has a potential of +2.70 ± 3.32 mV, indicating that a-CNCs consisted of negatively charged particles with higher stability in solution. Although the acidic route resulted in nanocrystals with a slightly higher crystallinity index compared to the enzymatic route, e-CNCs was found to be more thermally stable than BNC and a-CNCs. Here, we also confirmed the safety of a-CNCs and e-CNCs using L929 cell line. Lastly, this article describes two different CNCs synthesis approaches that leads to the formation of nanoparticles with different dimensions, morphology and unique physicochemical properties. To the best of our knowledge, this is the first study to yield spherical nanoparticles as a result of BNC enzymatic treatment.

From production to performance: Tailoring moisture and oxygen barrier of cellulose nanomaterials for sustainable applications - A review.

Barrier materials are crucial in preserving product quality, safety and longevity across numerous applications, thereby contributing to sustainability, reducing waste and advancing technology. Among these materials, cellulose nanomaterials (CNs) have emerged as promising alternatives for traditional petroleum-based polymers. However, the wide range of sources and the different methods used to isolate and process CN-based materials can result in significant variations in moisture and oxygen barrier performance. In this review, we provide an in-depth discussion on the latest advancements in CN-based green barrier materials. We begin by offering a critical assessment of the barrier performance of CNs, both in their isolated form and when combined as hybrid materials. This includes their applications as standalone films, fillers and coatings in nanocomposites. This review also covers the influence of the isolation process and the stages of film formation on barrier efficacy. We further discuss the implications of the recycling process on barrier properties of CN-based materials, drawing a connection between barrier characteristics and the product's end-of-life. We conclude by highlighting the significant developments over the past five years, the present challenges, and the prospective future of CN-based materials in barrier applications.

Hydrophobic enzymatic cellulose nanocrystals via a novel, one-pot green method.

Cellulose nanocrystals (CNCs) are a rapidly growing bionanomaterial with remarkable properties that have been harnessed in various applications, including mechanical reinforcement, biomedical materials, and coatings. However, for non-water-based applications, hydrophobization of CNCs while preserving their integrity is crucial. In this study, we propose a new eco-friendly, one-pot surface esterification method for hydrophobizing enzymatic CNCs in aqueous suspension without solvent exchange. By establishing an appropriate set of reaction conditions, it was possible to create a miscibility gradient that enabled a low-cost, and renewable fatty acid to be utilized as an acyl donor and solvent, allowing direct hydrophobic modification of the as-produced aqueous suspension of enzymatic CNC. FT-IR and AFM-IR analyses confirmed the formation of ester groups, while 13C NMR confirmed the emergence of carboxyl groups. XPS revealed a high degree of surface substitution (0.39) in the modified CNC, while a substantial increase in contact angle (from 40 to approximately 90°) quantitatively confirmed the high efficiency of the enzymatic CNC's hydrophobic modification. Additionally, important properties such as morphology remained practically unchanged, except for a slight increase in thermal stability and crystallinity of the CNCs. Therefore, hydrophobic enzymatic CNCs were successfully produced via a simple, scalable, and environmentally friendly approach without compromising their properties. These hydrophobic CNCs have the potential to enhance nanocomposite compatibility, improve packaging performance for electronics and foods, optimize adhesion in coatings, and offer advancements in cosmetics and drug delivery. However, comprehensive studies are needed to confirm their applicability across these sectors.

The emergence of hybrid cellulose nanomaterials as promising biomaterials.

Cellulose nanomaterials (CNs) are promising green materials due to their unique properties as well as their environmental benefits. Among these materials, cellulose nanofibrils (CNFs) and nanocrystals (CNCs) are the most extensively researched types of CNs. While they share some fundamental properties like low density, biodegradability, biocompatibility, and low toxicity, they also possess unique differentiating characteristics such as morphology, rheology, aspect ratio, crystallinity, mechanical and optical properties. Therefore, numerous comparative studies have been conducted, and recently, various studies have reported the synergetic advantages resulting from combining CNF and CNC. In this review, we initiate by addressing the terminology used to describe combinations of these and other types of CNs, proposing "hybrid cellulose nanomaterials" (HCNs) as the standardized classifictation for these materials. Subsequently, we briefly cover aspects of properties-driven applications and the performance of CNs, from both an individual and comparative perspective. Next, we comprehensively examine the potential of HCN-based materials, highlighting their performance for various applications. In conclusion, HCNs have demonstraded remarkable success in diverse areas, such as food packaging, electronic devices, 3D printing, biomedical and other fields, resulting in materials with superior performance when compared to neat CNF or CNC. Therefore, HCNs exhibit great potential for the development of environmentally friendly materials with enhanced properties.

Endoglucanase pretreatment aids in isolating tailored-cellulose nanofibrils combining energy saving and high-performance packaging.

Cellulose nanofibrils (CNFs) have emerged as a potential alternative to synthetic polymers in packaging applications owing to their oxygen and grease barrier performance, as well as their strong mechanical properties. However, the performance of CNF films relies on the inherent characteristics of fibers, which undergo changes during the CNF isolation process. Understanding these variations in characteristics during CNF isolation is crucial for tailoring CNF film properties to achieve optimum performance in packaging applications. In this study, CNFs were isolated by endoglucanase-assisted mechanical ultra-refining. The alterations in the intrinsic characteristics of CNFs and their impact on CNF films were systematically investigated by considering the degree of defibrillation, enzyme loading, and reaction time through a design of experiments. Enzyme loading had a significant influence on the crystallinity index, crystallite size, surface area, and viscosity. Meanwhile, the degree of defibrillation greatly affected the aspect ratio, degree of polymerization, and particle size. CNF films prepared from CNFs isolated under two optimized scenarios (casting and coating applications) exhibited remarkable properties, including high thermal stability (approximately 300 °C), high tensile strength (104 - 113 MPa), excellent oil resistance (kit n°12), and low oxygen transmission rate (1.00 - 3.17 cc·m-2.day-1). Therefore, endoglucanase pretreatment can aid in obtaining CNFs with lower energy consumption, resulting in films that possess higher transmittance, superior barrier performance, and reduced surface wettability compared to control samples without enzymatic pretreatment and other unmodified CNF films reported in the literature, all while maintaining mechanical and thermal performance without significant loss.

High-yield production of rod-like and spherical nanocellulose by controlled enzymatic hydrolysis of mechanically pretreated cellulose.

In this study, a simple and scalable mechanical pretreatment was evaluated as means of enhancing the accessibility of cellulose fibers, with the objective of improving the efficiency of enzymatic reactions for the production of cellulose nanoparticles (CNs). In addition, the effects of enzyme type (endoglucanase - EG, endoxylanase - EX, and a cellulase preparation - CB), composition ratio (0-200UEG:0-200UEX or EG, EX, and CB alone), and loading (0 U-200 U) were investigated in relation to CN yield, morphology, and properties. The combination of mechanical pretreatment and specific enzymatic hydrolysis conditions substantially improved CN production yield, reaching up to 83 %. The production of rod-like or spherical nanoparticles and their chemical composition were highly influenced by the enzyme type, composition ratio, and loading. However, these enzymatic conditions had minimal impact on the crystallinity index (approximately 80 %) and thermal stability (Tmax within 330-355 °C). Overall, these findings demonstrate that mechanical pretreatment followed by enzymatic hydrolysis under specific conditions is a suitable method to produce nanocellulose with high yield and adjustable properties such as purity, rod-like or spherical forms, high thermal stability, and high crystallinity. Therefore, this production approach shows promise in producing tailored CNs with the potential for superior performance in various advanced applications, including, but not limited to, wound dressings, drug delivery, thermoplastic composites, 3D (bio)printing, and smart packaging.

Endoglucanase effects on energy consumption in the mechanical fibrillation of cellulose fibers into nanocelluloses.

Enzymatic processing is considered a promising approach for advancing environmentally friendly industrial processes, such as the use of endoglucanase (EG) enzyme in the production of nanocellulose. However, there is ongoing debate regarding the specific properties that make EG pretreatment effective in isolating fibrillated cellulose. To address this issue, we investigated EGs from four glycosyl hydrolase (GH) families (5, 6, 7, and 12) and examined the roles of the three-dimensional structure and catalytic features, with a focus on the presence of a carbohydrate binding module (CBM). Using eucalyptus Kraft wood fibers, we produced cellulose nanofibrils (CNFs) through mild enzymatic pretreatment, followed by disc ultra-refining. Comparing the results with the control (without pretreatment), we observed that GH5 and GH12 enzymes (without CBM) reduced fibrillation energy by approximately 15 %. The most significant energy reduction, 25 and 32 %, was achieved with GH5 and GH6 linked to CBM, respectively. Notably, these CBM-linked EGs improved the rheological properties of CNF suspensions without releasing soluble products. In contrast, GH7-CBM exhibited significant hydrolytic activity, resulting in the release of soluble products, but did not contribute to a reduction in fibrillation energy. This discrepancy can be attributed to the large molecular weight and wide cleft of GH7-CBM, which led to the release of soluble sugars but had little impact on fibrillation. Our findings suggest that the improved fibrillation observed with EG pretreatment is primarily driven by efficient enzyme adsorption on the substrate and modification of the surface viscoelasticity (amorphogenesis), rather than hydrolytic activity or release of products.

Xylanase increases the selectivity of the enzymatic hydrolysis with endoglucanase to produce cellulose nanocrystals with improved properties.

Enzyme-mediated isolation of cellulose nanocrystals (CNCs) is a promising environment friendly method with expected lower capital and operating expenditures compared to traditional processes. However, it is still poorly understood. In this study, an endoxylanase was applied as accessory enzyme to assess its potential to increase the selectivity of an endoglucanase during cellulose hydrolysis to isolate CNCs with improved properties. Only combinations of the enzymes with xylanase activity equal to or higher than the endoglucanase activity resulted in CNCs with improved properties (i.e., crystallinity, thermostability, uniformity, suspension stability and aspect ratio). The beneficial effects of the accessory enzyme are related to its hydrolytic (xylan and cellulose hydrolysis) and non-hydrolytic action (swelling of cellulose fibers and fiber porosity) and on the ratio of the enzymes, which in turn allows to tailor the properties of the CNCs. In conclusion, compared to the traditional sulfuric acid hydrolysis method, accessory enzymes help to isolate cellulose nanomaterials with improved and customized (sizes, aspect ratio and morphology) properties that may allow for new applications.

Paludibacter propionicigenes GH10 xylanase as a tool for enzymatic xylooligosaccharides production from heteroxylans.

Bioconversion of lignocellulosic biomass into value-added products relies on polysaccharides depolymerization by carbohydrate active enzymes. This work reports biochemical characterization of Paludibacter propionicigenes xylanase from GH10 (PpXyn10A) and its application for enzymatic xylooligosaccharides (XOS) production from commercial heteroxylans and liquor of hydrothermally pretreated corn cobs (PCC). PpXyn10A is tolerant to ethanol and NaCl, and releases xylobiose (X2) and xylotriose (X3) as the main hydrolytic products. The conversion rate of complex substrates into short XOS was approximately 30% for glucuronoxylan and 8.8% for rye arabinoxylan, after only 4 h; while for PCC, PpXyn10A greatly increased unbranched XOS yields. B. adolescentis fermentation with XOS from beechwood glucuronoxylan produced mainly acetic and lactic acids. Structural analysis shows that while the glycone region of PpXyn10A active site is well preserved, the aglycone region has aromatic interactions in the +2 subsite that may explain why PpXyn10A does not release xylose.

High yield biorefinery products from sugarcane bagasse: Prebiotic xylooligosaccharides, cellulosic ethanol, cellulose nanofibrils and lignin nanoparticles.

An integrated biorefining strategy was applied to fractionate Sugarcane bagasse (SCB) into its major constituents, enabling high-yield conversion of the fractionated materials into high-value coproducts alongside cellulosic ethanol. Pilot-scale steam explosion produced a hydrolysate rich in low molecular weight xylooligosaccharides that had a high in vitro efficacy as a prebiotic towards different bifidobacteria. Lignin recovered after alkaline treatment of the steam-exploded SCB was converted into uniform spherical lignin nanoparticles (11.3 nm in diameter) by a green mechanical method. The resulting cellulose was hydrolyzed at 17.5% (w/v) consistency and low enzyme loading (17.5 mg/g) to yield a pure glucose hydrolysate at a high concentration (100 g/L) and a cellulosic solid residue that was defibrillated by disc ultra-refining into homogeneous cellulose nanofibrils (20.5 nm in diameter). Statistical optimization of the cellulosic hydrolysate fermentation led to ethanol production of 67.1 g/L, with a conversion yield of 0.48 g/g and productivity of 1.40 g/L.h.

Effect of chemical treatment of pineapple crown fiber in the production, chemical composition, crystalline structure, thermal stability and thermal degradation kinetic properties of cellulosic materials.

Recently, the growing environmental concerns and economic demands have driven the need to develop effective solutions for the treatment of vegetal fibers to be used as renewable source for various industrial applications. The present study aimed to explore pineapple crown fibers (PCs) as an alternative source of cellulose. The three treatments (alcohol-insoluble residue (AIR), alkaline (AT), and organosolv) evaluated promoted chemical and morphological changes to the PCs. Fresh and treated PCs were characterized by Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), Scanning electron microscopy (SEM), thermogravimetric analysis (TG), and chemical composition. The XRD results showed that the Cellulose-I allomorph was not altered during extraction, and that the crystallinity index of the fibers treated with AT, first bleaching step, second bleaching step, and the second bleaching step followed by KOH treatment (2B_KOH) increased to 77.8; 83.2; 83.5 and 86% when compared with fresh PC (62.3%). Results from the thermal analysis revealed that thermal stability increased for the isolated cellulose, and the maximum degradation for (2B_KOH) is 350 °C. Chemical composition results showed a decrease in the content of hemicellulose, lignin and other soluble materials after alkaline treatment, suggesting high-quality 2B_KOH with 74.6% of cellulose. SEM revealed changes in the morphological structure on fibers. Alkaline treatment followed by H2O2 bleaching is an excellent alternative for the removal of non-cellulosic material and facilitates the isolation of cellulose. These results suggested that there is a potential to isolate cellulose from PC via the sequence of treatment of a methodology by chlorite-free.

Co-production of xylo-oligosaccharides, xylose and cellulose nanofibrils from sugarcane bagasse.

This work investigated the integration of a two-stage hydrothermal treatment for production of xylooligosaccharides (XOS), a high-value product, into the isolation process of cellulose nanofibrils (CNF) from sugarcane bagasse. Under optimized conditions, the first stage yielded a XOS-rich, high purity hydrolysate and in the second stage only a xylose-rich hydrolysate could be obtained at high purity. The resulting solid cellulosic fraction was delignified and bleached to obtain a cellulose-rich pulp, which was mechanically defibrillated by disc ultra-refining to CNF. Except for the viscosity, the sugarcane CNF showed properties (i.e., thermal stability, crystallinity and diameter size) comparable or superior to the CNF prepared from commercial bleached eucalyptus Kraft pulp. In conclusion, the integration of the two-stage hydrothermal treatment is an efficient and promising strategy to obtain hemicellulose-derived high-value co-products in the process of isolating CNF. In addition, lignin was also recovered as a co-product with yield comparable to other biomass fractionation approaches.

A review on commercial-scale high-value products that can be produced alongside cellulosic ethanol.

The demand for fossil derivate fuels and chemicals has increased, augmenting concerns on climate change, global economic stability, and sustainability on fossil resources. Therefore, the production of fuels and chemicals from alternative and renewable resources has attracted considerable and growing attention. Ethanol is a promising biofuel that can reduce the consumption of gasoline in the transportation sector and related greenhouse gas (GHG) emissions. Lignocellulosic biomass is a promising feedstock to produce bioethanol (cellulosic ethanol) because of its abundance and low cost. Since the conversion of lignocellulose to ethanol is complex and expensive, the cellulosic ethanol price cannot compete with those of the fossil derivate fuels. A promising strategy to lower the production cost of cellulosic ethanol is developing a biorefinery which produces ethanol and other high-value chemicals from lignocellulose. The selection of such chemicals is difficult because there are hundreds of products that can be produced from lignocellulose. Multiple reviews and reports have described a small group of lignocellulose derivate compounds that have the potential to be commercialized. Some of these products are in the bench scale and require extensive research and time before they can be industrially produced. This review examines chemicals and materials with a Technology Readiness Level (TRL) of at least 8, which have reached a commercial scale and could be shortly or immediately integrated into a cellulosic ethanol process.

Exploring the action of endoglucanases on bleached eucalyptus kraft pulp as potential catalyst for isolation of cellulose nanocrystals.

Cellulose nanocrystals (CNCs) is a high-value and emerging bionanomaterial with an increasing number of applications. The action of endoglucanases (EGs) from fungal and bacterial sources belonging to three glycosyl hydrolase (GH) families were investigated on bleached eucalyptus kraft pulp as potential catalysts to prepare CNC. Fungal GH7EG was the most efficient in hydrolysis and fiber fragmentation without altering crystallinity and crystallite size. Fiber fragmentation promoted by fungal GH45EG was similar to that observed for GH7EG, although it released a smaller amount of sugar. Bacterial GH5EG resulted in very low hydrolysis yield and practically did not fragment the fibers, resulting in a hydrolysis residue with characteristics very similar to the initial material. GH45EG was the only EG that affected the crystallinity and crystallite size and also the only enzyme capable of isolating nanoparticles. The isolated nanoparticles had very narrow width distribution range of 6-10 nm and length distribution range of 400-600 nm. Supplementation of ß-glucosidase and conventional mechanical refining as a pretreatment did not improve the release of nanoparticles. Despite catalyzing the same biochemical reaction, different EGs displayed very distinct action during hydrolysis. The reported strong binding of GH45EG's CBM to the cellulose and the lack of increased accessibility of the enzyme to new substrate likely allowed continuous hydrolysis of the few fibers available, resulting in the isolation of cellulose nanoparticles.

Kinetic changes in cellulose properties during defibrillation into microfibrillated cellulose and cellulose nanofibrils by ultra-refining.

Defibrillation of cellulose fibers can lead to the isolation of microfibrillated cellulose (MFC) or cellulose nanofibrils (CNF) with intrinsic properties suitable for various applications. However, to what extent these properties are preserved, enhanced, gained or lowered during defibrillation and how they are related remains unclear. In this study, a kinetic study of the ultra-refining of bleached eucalyptus Kraft pulp (BEKP) in a disc ultra-refiner was performed and characterized in terms of physical-structural, morphological and thermal properties and their interactions and compromises. Defibrillation of BEKP to MFC substantially decreased the fiber diameter and increased viscosity, surface area and morphological heterogeneity. It also led to a remarkable increase in transparency and essentially did not alter the thermostability but significantly degraded the crystallinity. A higher degree of defibrillation to isolate CNF led to fibers with smaller diameter and increased diameter uniformity but required a substantial amount of energy to only marginally increase viscosity and transparency. Crystallinity and thermostability were not altered, comparing with CMF. In conclusion, most changes occurred during the defibrillation of BEKP to CMF. Further defibrillation to CNFs with smaller diameters and better uniformity did not significantly reflect on other important structural cellulose physical properties, despite the much higher energy consumption and degree of defibrillation.

Preparation of nanocellulose from Imperata brasiliensis grass using Taguchi method.

Cellulose nanoparticles (CNs) were prepared by acid hydrolysis of the cellulose pulp extracted from the Brazilian satintail (Imperata Brasiliensis) plant using a conventional and a total chlorine free method. Initially, a statistical design of experiment was carried out using Taguchi orthogonal array to study the hydrolysis parameters, and the main properties (crystallinity, thermal stability, morphology, and sizes) of the nanocellulose. X-ray diffraction (XRD), fourier-transform infrared spectroscopy (FTIR), field-emission scanning electron microscopy (FE-SEM), dynamic light scattering (DLS), zeta potential and thermogravimetric analysis (TGA) were carried out to characterize the physical-chemical properties of the CNs obtained. Cellulose nanoparticles with diameter ranging from 10 to 60 nm and length between 150 and 250 nm were successfully obtained at sulfuric acid concentration of 64% (m/m), temperature 35 °C, reaction time 75 min, and a 1:20 (g/mL) pulp-to-solution ratio. Under this condition, the Imperata Brasiliensis CNs showed good stability in suspension, crystallinity index of 65%, and a cellulose degradation temperature of about 117 °C. Considering that these properties are similar to those of nanocelluloses from other lignocellulosics feedstocks, Imperata grass seems also to be a suitable source for nanocellulose production.

The potential of tailoring the conditions of steam explosion to produce xylo-oligosaccharides from sugarcane bagasse.

In this study, the potential of the steam explosion (SE) method to produce high levels XOS from sugarcane bagasse, a xylan-rich hemicellulosic feedstock, was assessed. The effect of different operating conditions on XOS production yield and selectivity were investigated using a mini-pilot scale SE unit. The results show that even under a non-optimized condition (190 °C, 5 min and 0.5% H2SO4 as catalyst), SE led to about 40% xylan recovery as XOS, which was comparable to the well-known, multi-step, enzymatic production of XOS from alkaline-extracted xylan, and other commonly employed chemical methods. In addition, the XOS-rich hydrolysate from SE constituted of greater diversity in the degree of polymerization, which has been shown to be desirable for prebiotic application.

Limitation of cellulose accessibility and unproductive binding of cellulases by pretreated sugarcane bagasse lignin.

BACKGROUND: The effectiveness of the enzymatic hydrolysis of cellulose in plant cell wall is strongly influenced by the access of enzymes to cellulose, which is at least in part limited by the presence of lignin. Although physicochemical treatments preceding the enzymatic catalysis significantly overcome this recalcitrance, the residual lignin can still play a role in the process. Lignin is suggested to act as a barrier, hindering cellulose and limiting the access of the enzymes. It can also unspecifically bind cellulases, reducing the amount of enzymes available to act on cellulose. However, the limiting role of the lignin present in pretreated sugarcane bagasses has not been fully understood yet. RESULTS: A set of sugarcane bagasses pretreated by five leading pretreatment technologies was created and used to assess their accessibility and the unproductive binding capacity of the resulting lignins. Steam explosion and alkaline sulfite pretreatments resulted in more accessible substrates, with approximately 90% of the cellulose hydrolyzed using high enzyme loadings. Enzymatic hydrolysis of alkaline-treated (NaOH) and steam-exploded sugarcane bagasses were strongly affected by unproductive binding at the lowest enzyme loading tested. Analysis of the extracted lignins confirmed the superior binding capacity of these lignins. Sulfite-based pretreatments (alkaline sulfite and acid sulfite) resulted in lignins with lower binding capacities compared to the analogue pretreatments without sulfite (alkaline and acidic). Strong acid groups present in sulfite-based pretreated substrates, attributed to sulfonated lignins, corroborated the lower binding capacities of the lignin present in these substrates. A more advanced enzyme preparation (Cellic CTec3) was shown to be less affected by unproductive binding at low enzyme loading. CONCLUSIONS: Pretreatments that increase the accessibility and modify the lignin are necessary in order to decrease the protein binding capacity. The search for the called weak lignin-binding enzymes is of major importance if hydrolysis with low enzyme loadings is the goal for economically viable processes.

A NaBH4 Coupled Ninhydrin-Based Assay for the Quantification of Protein/Enzymes During the Enzymatic Hydrolysis of Pretreated Lignocellulosic Biomass.

Accurate protein quantification is necessary in many of the steps during the enzymatic hydrolysis of pretreated lignocellulosic biomass, from the fundamental determination of enzyme kinetics to techno-economic assessments, such as the use of enzyme recycling strategies, evaluation of enzyme costs, and the optimization of various process steps. In the work described here, a modified, more rapid ninhydrin-based protein quantification assay was developed to better quantify enzyme levels in the presence of lignocellulosic biomass derived compounds. The addition of sodium borohydride followed by acid hydrolysis at 130 °C greatly reduced interference from monosaccharides and oligosaccharides and decreased the assay time 6-fold. The modified ninhydrin assay was shown to be more accurate as compared to various traditional colorimetric protein assays when commercial cellulase enzyme mixtures were quantified under typical pretreated lignocellulosic biomass enzymatic hydrolysis conditions. The relatively short assay time and microplate-reading capability of the modified assay indicated that the method could likely be used for high-throughput protein determination.

Optimization of chip size and moisture content to obtain high, combined sugar recovery after sulfur dioxide-catalyzed steam pretreatment of softwood and enzymatic hydrolysis of the cellulosic component.

The influence of chip size and moisture content on the combined sugar recovery after steam pretreatment of lodgepole pine and subsequent enzymatic hydrolysis of the cellulosic component were investigated using response surface methodology. Chip size had little influence on sugar recovery after both steam pretreatment and enzymatic hydrolysis. In contrast, the moisture of the chips greatly influenced the relative severity of steam pretreatment and, as a result, the combined sugar recovery from the hemicellulosic and cellulosic fractions. Irrespective of chip size and the pretreatment temperature, time, and SO2 loading that were used, the relative severity of pretreatment was highest at a moisture of 30-40w/w%. However, the predictive model indicated that an elevated moisture content of roughly 50w/w% (about the moisture content of a standard softwood mill chip) would result in the highest, combined sugar recovery (80%) over the widest range of steam pretreatment conditions.