Cellulose Avicel Research Articles

Effect of spo0A, sigE, sigG, and sigK disruption on butanol production and spore formation in Clostridium saccharoperbutylacetonicum strain N1-4 (ATCC13564)

Clostridium saccharoperbutylacetonicum strain N1-4 (ATCC13564) is a butanol-producing strain suitable for application to butanol production from cellulosic materials by co-culture with cellulolytic and thermophilic species, such as Hungateiclostridium thermocellum (synonym: Clostridium thermocellum). The optimal temperature for butanol production by strain N1-4 is 30°C, and the strain is sensitive to a high culture temperature of 37°C. Given that spore formation is observed at high frequency when strain N1-4 is cultivated at 37°C, we assumed in a previous study that the initiation of sporulation is related to a decrease in butanol production. Therefore, to investigate the relationship between butanol production and spore formation, we generated strain N1-4 isolates in which genes related to spore formation were disrupted. The sporulation-related gene disruptants of spo0A, sigE, sigG, and sigK lost the ability to produce heat-resistant spores, irrespective of the culture temperature. Among the gene disruptants produced, only the spo0A disruptant lost butanol-producing ability when cultivated at 30°C. Interestingly, the sigE disruptant maintained butanol productivity similar to that observed at 30°C, even when cultivated at 37°C. In addition, the sigE disruptant successfully produced butanol from Avicel cellulose by co-culture with H.thermocellum at a fermentation temperature of 37°C.

A laboratory-scale compression feed screw for characterizing continuous feeding of dense granular materials

Compression feed screws are commonly used in biorefineries for the continuous feeding of biomass feedstock into reactors. Developing methods to assess the feasibility of feeding granular materials in a compression feed screw is critical for reducing solids handling issues during operation. A laboratory-scale compression feed screw has been built to measure critical process parameters of the feeding operation, namely the driving torque and the mass flow rate. The process parameters of two granular materials, microcrystalline cellulose Avicel PH-200 and milled corn stover are measured using the laboratory-scale feeder. The results show that a larger torque is required to feed corn stover than Avicel PH-200, even though the former consists of more compressible particles. Further, the device can capture real-time fluctuations in the process parameters during operation. The laboratory-scale feeder can be used to compare the feeding of different granular materials and the measured parameters can be used to validate predictive models for a compression screw feeding process.

High-level production of Aspergillus niger prolyl endopeptidase from agricultural residue and its application in beer brewing

BackgroundProlyl endopeptidase from Aspergillus niger (AN-PEP) is a prominent serine proteinase with various potential applications in the food and pharmaceutical industries. However, the availability of efficient and low-cost AN-PEP remains a challenge owing to its low yield and high fermentation cost.ResultsHere, AN-PEP was recombinantly expressed in Trichoderma reesei (rAN-PEP) under the control of the cbh1 promoter and its secretion signal. After 4 days of shaking flask cultivation with the model cellulose Avicel PH101 as the sole carbon source, the extracellular prolyl endopeptidase activity reached up to 16.148 U/mL, which is the highest titer reported to date and the secretion of the enzyme is faster in T. reesei than in other eukaryotic expression systems including A. niger and Komagataella phaffii. Most importantly, when cultivated on the low-cost agricultural residue corn cob, the recombinant strain was found to secret a remarkable amount of rAN-PEP (37.125 U/mL) that is twice the activity under the pure cellulose condition. Furthermore, treatment with rAN-PEP during beer brewing lowered the content of gluten below the ELISA kit detection limit (< 10 mg/kg) and thereby, reduced turbidity, which would be beneficial for improving the non-biological stability of beer.ConclusionOur research provides a promising approach for industrial production of AN-PEP and other enzymes (proteins) from renewable lignocellulosic biomass, which provides a new idea with relevant researchers for the utilization of agricultural residues.

Cellulose esterification with carboxylic acid in deep eutectic solvent pretreatment inhibits enzymatic hydrolysis

Avicel cellulose was pretreated using two commonly used carboxylic acid-based deep eutectic solvents, i.e., choline chloride-lactic acid and choline chloride-formic acid. The pretreatment process resulted in the formation of cellulose esters with lactic acid and formic acid, which was confirmed by infrared and nuclear magnetic resonance spectra. Surprisingly, the esterified cellulose led to a significant decrease in the 48-h enzymatic glucose yield (≥75%) compared to raw Avicel cellulose. Analysis of changes in cellulose properties caused by pretreatment, including crystallinity, degree of polymerization, particle size and cellulose accessibility, contradicted the observed decline in enzymatic cellulose hydrolysis. However, removing the ester groups through saponification largely recovered the reduction in cellulose conversion. The decreased enzymatic cellulose hydrolysis by esterification may be attributed to changes in the interaction between cellulose-binding domain of cellulase and cellulose. These findings provide valuable insights into improving the saccharification of lignocellulosic biomass pretreated by carboxylic acid-based DESs.

Heterologous Expression, Purification and Characterization of an Alkalic Thermophilic β-Mannanase CcMan5C from Coprinopsis cinerea.

A endo-1,4-β-mannanase (CcMan5C) gene was cloned from Coprinopsis cinerea and heterologously expressed in Pichia pastoris, and the recombinant enzyme was purified by Ni-affinity chromatography and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF-MS). CcMan5C hydrolyzed only locust bean gum galactomannan (LBG) but not α-mannan from S. cerevisiae or Avicel cellulose, oat spelt xylan, or laminarin from Laminaria digitata. CcMan5C exhibited distinctive catalytic features that were different from previously reported β-mannanases. (1) CcMan5C is the first reported fungal β-mannase with an optimal alkalic pH of 8.0-9.0 for hydrolytic activity under assay conditions. (2) CcMan5C is the first reported alkalic fungal β-mannase with an optimal temperature of 70 °C for hydrolytic activity under assay conditions. (3) The organic solvents methanol, ethanol, isopropanol, and acetone at concentrations of 10% or 20% did not inhibit CcMan5C activity, while 10% or 20% isopropanol and acetone even enhanced CcMan5C activity by 9.20-34.98%. Furthermore, CcMan5C tolerated detergents such as Tween 20 and Triton X-100, and its activity was even enhanced to 26.2-45.6% by 1% or 10% Tween 20 and Triton X-100. (4) CcMan5C solution or lyophilized CcMan5C exhibited unchanged activity and even increasing activity after being stored at -20 °C or -80 °C for 12 months and retained above 50% activity after being stored at 4 °C for 12 months. These features make CcMan5C a suitable candidate for the detergent industry and paper and pulp industry.

Identification of a Bidirectional Promoter from Trichoderma reesei and Its Application in Dual Gene Expression.

The cellulolytic filamentous fungus Trichoderma reesei has a strong capability in protein synthesis and secretion and is increasingly used as a fungal chassis for the production of heterologous proteins or secondary metabolites. However, bidirectional promoters that would significantly facilitate multiple genes’ expression have not been characterized in T. reesei. Herein, we show that a 767-bp intergenic region between two polyketide synthase encoding genes that were involved in the biosynthesis of the typical yellow pigment served as a bidirectional promoter in T. reesei. This region was shown to be able to drive the simultaneous expression of two fluorescence reporter genes when fused to each end. Quantitative RT-PCR analysis demonstrated that the driving strength of this bidirectional promoter from each direction reached about half of that of the commonly used promoter PgpdA. Moreover, the co-expression of two cellulase genes driven by this bidirectional promoter enabled T. reesei to produce cellulases on glucose and improved the total cellulase activities with cellulose Avicel as the carbon source. Our work identified the first bidirectional promoter in T. reesei, which would facilitate gene co-expression and find applications in synthetic biology using fungal systems.

Cellulolytic Properties of a Potentially Lignocellulose-Degrading Bacillus sp. 8E1A Strain Isolated from Bulk Soil

Cellulolytic enzymes produced by spore-forming bacteria seem to be a potential solution to the degradation of lignocellulosic waste. In this study, several dozen bacterial spore-forming strains were isolated from soil and one of them was selected for further studies. The studied bacterial strain was identified to genus Bacillus (strain 8E1A) by 16S rRNA gene sequencing. Bacillus sp. 8E1A showed an activity of carboxymethyl cellulase (CMCase) with visualization with Congo Red-25 mm (size of clear zone). To study CMCase, filter paper hydrolase (FPase), and microcrystalline cellulose Avicel hydrolase (Avicelase) production, three different cellulose sources were used for bacterial strain cultivation: carboxymethyl cellulose (CMC), filter paper (FP), and microcrystalline cellulose Avicel. The highest CMCase (0.617 U mL−1), FPase (0.903 U mL−1), and Avicelase (0.645 U mL−1) production of Bacillus sp. 8E1A was noted for using CMC (after 216 h of incubation), Avicel cellulose (after 144 h of incubation), and CMC (after 144 h of incubation), respectively. Subsequently, the cellulases’ activity was measured at various temperatures and pH values. The optimal temperature for CMCase (0.535 U mL−1) and Avicelase (0.666 U mL−1) activity was 70 °C. However, the highest FPase (0.868 U mL−1) activity was recorded at 60 °C. The highest CMCase and Avicelase activity was recorded at pH 7.0 (0.520 and 0.507 U mL−1, respectively), and the optimum activity of FPase was noted at pH 6.0 (0.895 U mL−1). These results indicate that the cellulases produced by the Bacillus sp. 8E1A may conceivably be used for lignocellulosic waste degradation in industrial conditions.

Evaluating biohydrogen production by Clostridium hydrogenum sp. nov. strain CUEA01 isolated from mangrove sediments in Thailand

The hydrogen (H 2 ) fermentative Clostridium hydrogenum sp. nov. strain CUEA01 was isolated from a mangrove sediment in Thailand. Genome sequencing and analysis revealed a genome size of 5,501,482 bp that encoded for 3,292 predicted protein coding genes with annotated functional assignments and many genes associated with carbon utilization and H 2 evolution. The H 2 production performance was evaluated in batch fermentation, and revealed that this strain can grow and produce H 2 at a broad range of temperatures (15–40 °C), pH (4–10), and initial glucose concentrations (5–60 g/L). The maximum H 2 yield (3.11 mol H2 /mol glucose ) was obtained at 37 °C, pH 8, and an initial glucose concentration of 10 g/L. Furthermore, this strain could utilize various carbon sources, including xylose, xylan, starch, mannose, glycerol, and avicel cellulose, amongst others. Additionally, CUEA01 was compatible with agro-industrial wastes and could achieve a maximum CHP of 4639 mL/L and 4024 mL/L from sugarcane molasses and cassava pulp, respectively. This demonstrates that CUEA01 has a potential for H 2 fermentation from complex organic wastes since it can secrete enzyme cocktails that consolidate the fermentation process. • Discovery and reported characteristic of Clostridium hydrogenum sp. nov CUEA01. • Draft genome sequencing of CUEA01 isolated from mangrove sediments in Thailand. • CUEA01 was compatible with various carbon sources e.g., glycerol, starch, xylose. • CUEA01 produced maximum H 2 yield of 3.11 mol H2 /mol glucose . • CUEA01 produced cumulative H 2 production of 4639 mL/L from agro-industrial wastes.

Preparation of nanocellulose by hydrolysis with ionic liquids and two-step hydrolysis with ionic liquids and enzymes

The aim of this study was to compare parameters of nanocellulose obtained by two different procedures: hydrolysis with ionic liquids (1-allyl-3-methylimidazolium chloride and 1-ethyl-3-methylimidazolium acetate) and hydrolysis with ionic liquids in combination with hydrolysis using a cellulolytic enzyme from Trichoderma reesei. Avicel cellulose was treated with two ionic liquids: 1-allyl-3-methylimidazolium chloride (AmimCl) and 1-ethyl 3-methylimidazolium acetate (EmimOAc). In the two-step hydrolysis cellulose after treatment with ionic liquids was additionally hydrolyzed with a solution of enzymes. In order to characterize the obtained material, the following analyses were used: infrared spectroscopy, X-ray diffraction and dynamic light scattering. The results indicated that cellulose obtained by two-step nanocellulose production methods (first hydrolysis with ionic liquids and then with enzymes) showed similar parameters (particle size, XRD patterns and degree of crystallinity) as the material after the one-step process, i.e. hydrolysis with ionic liquids.

Parallel proteomic and phosphoproteomic analyses reveal cellobiose‐dependent regulation of lignocellulase secretion in the filamentous fungus Neurospora crassa

AbstractHigh cost of lignocellulases restricts the commercialization of biofuel and bio‐product production from lignocellulosic biomass. Constitutively expressed lignocellulases are considered to degrade cellulose to release small amount of soluble cellodextrins such as cellobiose for further large‐scale production of lignocellulases; however, the underlying mechanism remains to be elucidated. Here, a triple β‐glucosidase mutant of the model fungus Neurospora crassa, which prevents rapid turnover of cellobiose and thus allows the disaccharide to induce lignocellulases, was applied to perform parallel analyses of proteome and phosphoproteome changes in response to cellobiose and Avicel cellulose. The results revealed shared proteome and phosphoproteome responses to cellobiose and Avicel, corroborating the idea that cellobiose mediates the regulation of lignocellulase expression and secretion. The results further suggest that this regulation is achieved at multiple levels, including epigenetic, transcription, post‐transcription, translation, and post‐translation. Proteome profiling revealed that the proteins upregulated by cellobiose and Avicel were over‐represented in cellulose degradation and degradation product transport pathways. Phosphoproteome profiling revealed that the proteins differentially phosphorylated by cellobiose and Avicel were over‐represented by the pathways such as transcriptional control, protein processing and export, cell wall biogenesis, and cellular signaling. Deletion mutation analysis further suggests that the ER chaperon protein Hsp70‐6, the translocation complex subunit Sec66/Sec71, and the signal peptidase subunit Spc2 are involved in lignocellulase secretion, particularly translocation across the endoplasmic reticulum. Altogether, the results offer a new insight into how cellobiose mediates the regulation of lignocellulase expression and secretion, providing a potential strategy for the strain engineering to improve lignocellulase production.

Impact of cellulose properties on its behavior in torrefaction: commercial microcrystalline cellulose versus cotton linters and celluloses extracted from woody and agricultural biomass

Biomass composition on cellulose, hemicelluloses and lignin determinates its behavior in torrefaction (200–350 °C, default-oxygen atmosphere). Up to now, commercial microcrystalline cellulose was typically used to represent cellulose behavior in biomass torrefaction models based on macromolecular components. The objective of this work is to evaluate the impact of cellulose properties on its behavior in torrefaction, so as to identify the most suitable cellulose sample for modelling. To do this, five extracted celluloses from woody and agricultural biomass, commercial microcrystalline cellulose (Avicel) and four cotton linters tailored to different degrees of polymerization (DP) were considered. Cellulosic samples were deeply characterized in terms of fiber analysis, molar mass distribution (MMD), hydrodynamic behavior (SEC-RALS) and allomorphic structure. Cellulosic samples were torrefied in a thermogravimetric analysis up to two temperatures, leading to partial (300 °C) and total (350 °C) cellulose degradation. Extracted celluloses and Avicel cellulose degradation profiles showed strong differences at 300 °C. Polymer MMD, dispersity and the presence of residual hemicellulose sugars, especially xylose, strongly impacted cellulose degradation through torrefaction. Extracted celluloses presented high purity, narrow MMD and high DP, but a cellulose II structure due to mercerization. This cellulosic allomorph was shown to be more reactive, but it led to an identical final solid mass loss. The observed hydrodynamic behavior and the probable preservation of cellulose amorphous areas from native cellulose may also influence polymer structural behavior in torrefaction. Finally, at least one-order-of-magnitude DP variation was shown to be required to see an impact in cellulose degradation profile through torrefaction.

In situ measurements of oxidation\u2013reduction potential and hydrogen peroxide concentration as tools for revealing LPMO inactivation during enzymatic saccharification of cellulose

BackgroundBiochemical conversion of lignocellulosic biomass to simple sugars at commercial scale is hampered by the high cost of saccharifying enzymes. Lytic polysaccharide monooxygenases (LPMOs) may hold the key to overcome economic barriers. Recent studies have shown that controlled activation of LPMOs by a continuous H2O2 supply can boost saccharification yields, while overdosing H2O2 may lead to enzyme inactivation and reduce overall sugar yields. While following LPMO action by ex situ analysis of LPMO products confirms enzyme inactivation, currently no preventive measures are available to intervene before complete inactivation.ResultsHere, we carried out enzymatic saccharification of the model cellulose Avicel with an LPMO-containing enzyme preparation (Cellic CTec3) and H2O2 feed at 1 L bioreactor scale and followed the oxidation–reduction potential and H2O2 concentration in situ with corresponding electrode probes. The rate of oxidation of the reductant as well as the estimation of the amount of H2O2 consumed by LPMOs indicate that, in addition to oxidative depolymerization of cellulose, LPMOs consume H2O2 in a futile non-catalytic cycle, and that inactivation of LPMOs happens gradually and starts long before the accumulation of LPMO-generated oxidative products comes to a halt.ConclusionOur results indicate that, in this model system, the collapse of the LPMO-catalyzed reaction may be predicted by the rate of oxidation of the reductant, the accumulation of H2O2 in the reactor or, indirectly, by a clear increase in the oxidation–reduction potential. Being able to monitor the state of the LPMO activity in situ may help maximizing the benefit of LPMO action during saccharification. Overcoming enzyme inactivation could allow improving overall saccharification yields beyond the state of the art while lowering LPMO and, potentially, cellulase loads, both of which would have beneficial consequences on process economics.

Determining Cellulolytic Activity of Microorganisms

Abstract Decomposition of cellulose to glucose requires complex cooperation of glycoside hydrolase enzymes. As a result of glycoside β-1,4 bonds hydrolysis, shorter chains of cellulose, oligodextrin, cellobiose and glucose are created. A number of bacteria and fungi demonstrate the capacity to degrade cellulose. Their activity can be assessed with the use of qualitative and quantitative methods. Qualitative methods with the use of e.g. Congo red, are used in screening studies, however, they do not provide information about the quantity of the produced enzyme. Spectrophotometric methods are more accurate and they measure the quantities of reducing sugars with the use of appropriate substrates, e.g. carboxymethylcellulose is used to determine endoglucanases, avicel cellulose to determine exoglucanases and Whatman filter paper to determine total cellulolytic activity. Activity of microorganisms depends not only on their species or type but also, among others, on substratum composition, cultivation conditions and the appropriate selection of parameters of the carried out enzymatic reactions.

Enzyme adsorption-desorption and evaluation of various cellulase recycling strategies for steam pre-treated Eucalyptus enzymatic degradation

One of the biggest challenges hindering the successful commercialisation of biofuels is the high dosages of enzymes still required to hydrolyse lignocellulose into fermentable sugars. Enzyme recycling has been recognised as a promising approach to alleviate this problem by reducing enzyme costs and improving the profitability of the bioconversion process. However, developing an effective recycling system is a challenging process and requires an in-depth understanding of enzyme-substrate interactions. In this context, this study investigated the adsorption, desorption and re-adsorption of a cellulase mixture (CelMix) to various lignocellulosic components to gain insights into enzyme-substrate interactions, in order to develop an effective enzyme recycling strategy. Lignin-rich residues exhibited a higher adsorption capacity for CelMix (~ 38% adsorption) compared to Avicel cellulose (15.6% adsorption). The recovery of CelMix from lignin, steam pre-treated Eucalyptus and cellulose by various chemicals and alkaline washing was examined. Alkaline washing with Tris-HCl buffer (pH 9.0; 0.05 M) was the most effective method for promoting enzyme desorption (90.7% activity) and retained a substantial amount of hydrolytic activity after elution. However, minor activity loss was observed due to irreversible binding, which was further confirmed by SDS-PAGE analysis. Using this information, the feasibility of recovering the enzymes from the solid fraction after enzymatic hydrolysis of steam pre-treated Eucalyptus was evaluated by two different approaches: (i) re-adsorption of the entire hydrolysed insoluble biomass fraction (no desorption) to fresh biomass (recycling approach 1-RA1) and (ii) re-adsorption of alkaline elution desorbed enzymes from hydrolysed biomass to fresh biomass (recycling approach 2—RA2). The recycling performance of RA1 and RA2 reached > 95% of the initial sugar liberation for three continuous rounds, whilst successful reduction in enzyme loading by 50% and 40% for RA1 and RA2, respectively, was achieved. Therefore, this study shows that enzyme recycling presents a simple and effective pathway for increasing enzyme productivity, improving the economic feasibility of fermentable sugar production for biofuel.

BILAYER TABLET TENDERED IMMEDIATE RELEASE OF PARACETAMOL AND SUSTAINED RELEASE OF IBUPROFEN FOR QUICK ONSET OF ACTION AGAINST PAIN AND FEVER

Objective: The objective of this research was to formulate bi-layer tablet which contains immediate-release layer of Paracetamol for quick onset of action and sustained release of Ibuprofen for prolonging long period.&#x0D; Materials and Methods: The following chemicals were used: Paracetamol (Combiotic Global Pvt. Ltd., India), Ibuprofen (Combiotic Global Pvt. Ltd., India), microcrystalline cellulose (MCC) (Avicel), and Polyvinylpyrrolidone (PVP) K-30 (Loba Chem). Wet granulation method was adapted to formulate the bilayer tablets. The immediate-release layer of Paracetamol was prepared using sodium starch glycolate as superdisintegrants, MCC as diluent, starch as binder. The sustained release layer of Ibuprofen was prepared using high-performance liquid chromatography E50LV and ethyl cellulose as binder along with other excipients such as MCC, PVP, and magnesium stearate by wet granulation technique.&#x0D; Result and Discussion: The release rate of Paracetamol from Formulations 1 was more than 80% at 40 min. In case of Ibuprofen, sustained-release polymers such as HPMC E50 LV were used to increase the release time.&#x0D; Conclusion: The bilayer tablets were prepared and show good release rate.

Modification of microcrystalline cellulose structural properties by ball-milling and ionic liquid treatments and their correlation to enzymatic hydrolysis rate and yield

This study investigated the characteristics of cellulose Avicel PH-101 (NPA) and Avicel samples treated by either ball milling (BMA) or ionic liquid 1-ethyl-3-methylimidazolium acetate (ILA), which alter cellulose structural properties specifically and, consequently, affect the enzymatic hydrolysis of these materials differently. All materials were submitted to 48 h hydrolysis using the commercial enzymatic preparations Celluclast 1.5 L and Novozymes N188. For ILA hydrolysis, a high yield of 96.8% was observed, which was related to its high specific surface area, low cellulose crystallinity, and high cellulose II content rather than to the particle size. The hydrolysis yields of BMA and NPA were 74.4% and 41.6%, respectively. BMA crystallinity was higher in comparison to ILA and lower in comparison to NPA, whereas the BMA surface area was lower in comparison to ILA and NPA. X-ray diffraction (XRD) analysis suggested the formation of cellulose II by both pretreatments on Avicel, which was reinforced by the samples’ diffractograms and an additional characterization by XRD/crystallinity index of the partial hydrolyzed BMA insoluble residue. In the presence of amorphous cellulose, cellulose II allomorph presented higher hydrolysis recalcitrance. The set of results showed that crystallinity and surface area provided a straightforward correlation with the hydrolysis rate and yield of Avicel cellulose. In addition, the understanding of the effects that can be caused by pretreatments of cellulosic materials was improved and these findings contribute with the interpretation of the kinetics and yields of enzymatic hydrolysis.

The stranded macroalga Ulva lactuca as a new alternative source of cellulose: Extraction, physicochemical and rheological characterization

Cellulose was successfully extracted from Ulva lactuca, an abundant and unexploited green macroalga that causes several environmental disorders by its excessive eutrophication. The extracted cellulose purity was checked by acid hydrolysis and high performance anion exchange chromatography coupled to pulsed amperometric detection, showing mainly glucose, and by Fourier transform infrared spectroscopy analysis, revealing the same functional groups as the commercial cellulose AVICEL and the absence of uronic esters from hemicellulosic contaminants. The crystallinity index was 59% compared to 82% for the AVICEL cellulose, showing the semi crystalline character of the extracted cellulose. The thermal behavior of both celluloses was the same as they showed a degradation peak at 360 °C as demonstrated by the thermogravimetric analysis. Beside the physicochemical properties, the alga cellulose presented several interesting rheological properties such as a high specific surface area (5.74 m2/g of dry matter) and consequently a higher water retention (43.07 g/100g of dry matter). These results show that alga can be an alternative source of cellulose to the conventional ones such as wood.

A novel thermostable GH10 xylanase with activities on a wide variety of cellulosic substrates from a xylanolytic Bacillus strain exhibiting significant synergy with commercial Celluclast 1.5 L in pretreated corn stover hydrolysis

BackgroundCellulose and hemicellulose are the two largest components in lignocellulosic biomass. Enzymes with activities towards cellulose and xylan have attracted great interest in the bioconversion of lignocellulosic biomass, since they have potential in improving the hydrolytic performance and reducing the enzyme costs. Exploring glycoside hydrolases (GHs) with good thermostability and activities on xylan and cellulose would be beneficial to the industrial production of biofuels and bio-based chemicals.ResultsA novel GH10 enzyme (XynA) identified from a xylanolytic strain Bacillus sp. KW1 was cloned and expressed. Its optimal pH and temperature were determined to be pH 6.0 and 65 °C. Stability analyses revealed that XynA was stable over a broad pH range (pH 6.0–11.0) after being incubated at 25 °C for 24 h. Moreover, XynA retained over 95% activity after heat treatment at 60 °C for 60 h, and its half-lives at 65 °C and 70 °C were about 12 h and 1.5 h, respectively. More importantly, in terms of substrate specificity, XynA exhibits hydrolytic activities towards xylans, microcrystalline cellulose (filter paper and Avicel), carboxymethyl cellulose (CMC), cellobiose, p-nitrophenyl-β-d-cellobioside (pNPC), and p-nitrophenyl-β-d-glucopyranoside (pNPG). Furthermore, the addition of XynA into commercial cellulase in the hydrolysis of pretreated corn stover resulted in remarkable increases (the relative increases may up to 90%) in the release of reducing sugars. Finally, it is worth mentioning that XynA only shows high amino acid sequence identity (88%) with rXynAHJ14, a GH10 xylanase with no activity on CMC. The similarities with other characterized GH10 enzymes, including xylanases and bifunctional xylanase/cellulase enzymes, are no more than 30%.ConclusionsXynA is a novel thermostable GH10 xylanase with a wide substrate spectrum. It displays good stability in a broad range of pH and high temperatures, and exhibits activities towards xylans and a wide variety of cellulosic substrates, which are not found in other GH10 enzymes. The enzyme also has high capacity in saccharification of pretreated corn stover. These characteristics make XynA a good candidate not only for assisting cellulase in lignocellulosic biomass hydrolysis, but also for the research on structure–function relationship of bifunctional xylanase/cellulase.

Composite up-conversion luminescent films containing a nanocellulose and SrF2:Ho particles

The synthesis of up-conversion luminescent composite films based on a nanocellulose matrix containing Sr1−xHoxF2+x particles was proposed. The combination of sulfuric acid hydrolysis and ultrasonication allowed us to synthesize a series of stable nanocellulose dispersions from various raw materials (powdered sulphate bleached wood pulp, Blue Ribbon filter paper, and microcrystalline cellulose Avicel). The size distribution of cellulose nanoparticles in the aqueous dispersions was determined. Cellulose nanocrystals (CNC) and/or cellulose nanofibrils (CNF) dispersions were used to fabricate thin films by solution casting followed by solvent evaporation under ambient conditions. The size and shape of cellulose nanoparticles, surface morphology, crystallinity index of nanocellulose, polymerization degree, and optical properties were studied. By mixing aqueous dispersions of CNC with up-conversion Sr1−xHoxF2+x particles, homogeneous suspensions were obtained. Finally, a solution casting technique was used to prepare CNC/Sr1−xHoxF2+x and CNC/CNF/Sr1−xHoxF2+x nanocomposite films. CNC/CNF dispersions were utilized for the production of flexible, durable, transparent composite films. The synthesized nanocomposites demonstrated intense red luminescence upon Ho3+ excitation by 1912 nm laser radiation. The obtained up-conversion luminescent composite films can be considered as a promising material for photonics, in particular for near-IR laser radiation visualization, luminescent labeling and luminescent sensorics.

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

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