Dynamic Changes in the Composite Microbial System MC1 During and Following its Rapid Degradation of Lignocellulose

To monitor the dynamics of the composite microbial system MC1 during its degradation of lignocellulose and to improve our understanding of the microbial communities involved in this biomass conversion, MC1 was characterized at eight time points over an 18-day, thermophilic, aerobic, static cultivation. We found the microbial communities to be dynamic, rhythmic consortia capable of changing in response to lignocellulose degradation. The growth curve over 18 days was M-shaped. Based on the quantitative changes in five major components of MC1 (Clostridium straminisolvens CSK-1, Clostridium sp. FG4, Pseudoxanthomonas sp. M1-3, Brevibacillus sp. M1-5, and Bordetella sp. M1-6), reduction in rice straw weight, cellulase (CMCase) activity, xylanase activity, and changes in medium pH, we found that the process comprised two identifiable phases. Rapid degradation occurred from day 0 to day 9, while the post-rapid degradation phase included days 10 to 18. Day 3 and day 12 were two key time points in the rapid degradation phase and post-rapid degradation phase, respectively. Two anaerobes, C. straminisolvens CSK-1 and Clostridium sp. FG4, dominated the MC1 system from day 0 to day 18.     

Gelation of cassia gum by freezing and thawing

Aqueous solution of cassia gum (CG), which is categorized as a galactomannan polysaccharide having mannose/galactose ratio = 5/1, forms hydrogels by freezing and thawing. When frozen CG aqueous solution was thawed, transparent sol was separated from a turbid gel, i.e. syneresis occurred. Gel concentration ({(Mass of dry gel) / (Mass of gel)} × 100) increased with increasing CG concentration. Viscoelastic properties of CG hydrogels formed by freezing and thawing were investigated by thermomechanical analysis (TMA) in water using an oscillation mode at 0.05 Hz. Dynamic modulus (E′) increased from 3 kPa to ca. 5 kPa with increasing freezing rate. In contrast, E′ maintained a constant value regardless of repeating number of freezing and thawing. From TMA results, it is concluded that the density of cross-linking network structure depends on the size of ice formed by freezing. At the same time, the low E′ value of CG gels is ascribed to the fact that association of galactosyl side group is disturbed by the stiff chain attributed to the unsubstituted region of CG.     

Effect of Different Variables on the Efficiency of the Baker's Yeast Cell Disruption Process to Obtain Alcohol Dehydrogenase Activity

Cell disruption process of dry baker's yeast was studied in this work to obtain maximum activity of alcohol dehydrogenase (ADH). Disruption by ultrasonication, glass beads, and combination of these two methods was compared. A 1.8-fold increase of ADH activity can be achieved by combining glass beads with ultrasonication in comparison to ultrasonication. To achieve maximum volume activity of ADH, the effect of different variables on the cell disruption process was investigated (time, glass bead diameter, mass of glass beads, and ultrasound amplitude). Using the Design-Expert© software, 2⁴ factorial experimental design was performed. Two ultrasound probes were tested: MS 73 and KE 76. Optimal conditions (process variables) for cell disruption process were obtained. Optimal ADH activities after cell disruption with MS 73 and KE 76 probes were 1,890.9 and 1,531.7 U cm^?3, respectively. Necessary ultrasonication time and ultrasound amplitude should be at the maximum values in the investigated variable range (30 min and 62 %). Bead size should be at maximum (4 mm) when using MS 73 probe and at minimum (0.3 mm) when using KE 76 probe. Partial purification of the enzyme was carried out and it was kinetically characterized using several oxidation and reduction systems.     

Multi‐responsive methylcellulose/Fe‐alginate‐g‐PVA/PVA/Fe3O4 microgels for immobilizing enzyme

To take advantage of the respective character of methylcellulose (MC), poly(vinyl alcohol) (PVA), and alginate, novel physically cross‐linked methylcellulose/Fe‐alginate‐g‐PVA/PVA (MC/Fe‐Alg‐g‐PVA/PVA) microgels were prepared by emulsification/thermal gelation/freezing–thawing/ionic cross‐linking technique. Subsequently, some ferrous ions bound in the microgels were transformed into magnetite via in situ self oxidation. A model enzyme α‐amylase was immobilized into microgels by direct adsorption. The release behavior of α‐amylase indicated that the obtained complex microgels were magnetic‐, temperature‐, and pH‐ triple sensitive. Copyright © 2010 John Wiley & Sons, Ltd.     

Endoglucanase and Total Cellulase from Newly Isolated Rhizopus oryzae and Trichoderma reesei: Production,Characterization, and Thermal Stability

A multienzymatic complex production was evaluated, as well as endoglucanase and total cellulase characterization, during solid-state fermentation of rice industry wastes with Rhizopus oryzae CCT 7560 (newly isolated microorganism) and Trichoderma reesei QM 9414 (control). R. oryzae produced enzymes with higher activity at 15 h of fermentation (5.1 and 2.3 U g^?1 to endoglucanase and total cellulase), while T. reesei produced them at 55 h (15.3 and 2.8 U g^?1 to endoglucanase and total cellulase). The optimum temperature for total cellulase and endoglucanase was 60 °C. For Trichoderma and Rhizopus, the optimum pH was 5.0 and 6.0 for total cellulase and 6.0 and 5.0 for endoglucanase, respectively. The enzymes produced by Rhizopus presented higher stability at the temperature range evaluated (25–100 °C); the endoglucanase K _M value was 20 times lower than the one found for Trichoderma. The characterization of the cellulolytic enzymes from the fungal species native of rice husk revealed that they can be more efficient than the genetically modified enzymes when rice husk and rice bran are used as substrates.     

Improving the slurry fuel preparation performance to recycle municipal sewage sludge by combined alkali and ultrasonication pretreatment

Municipal wastewater sludge (hereafter, sludge) is economically recycled by mixing with coal to prepare slurry fuels, called coal sludge slurry (CSS). However, raw sludge needs to be pretreated before being prepared as CSS because of its high viscosity. Alkali and ultrasonication pretreatment can effectively disrupt the sludge flocs and improve slurrying performance. The alkali pretreatment method takes a long time but has a low cost, whereas the ultrasonication pretreatment method needs a high energy input but takes a short time. This study combined these two methods with complementary advantages to reduce the sludge pretreatment time and ultrasonication energy demand to improve the slurrying performance for sludge. Results showed that, compared with only alkali and only ultrasonication, the combined alkali and ultrasonication pretreatment method had stronger effects on reducing slurry viscosity. The characteristic viscosity η _c of CSS prepared by raw sludge was 1663.60 mPa s. It was reduced to 1033.44 and 1472.10 mPa s, respectively, after the sludge was only pretreated by NaOH and Ca(OH)₂ at a dosage of 10 % dry sludge (DS) for 2 h, while it was reduced to 1205.43 mPa s after the sludge was only pretreated by ultrasonication of 30 kJ/g DS. After combined pretreatment, η _c of CSS was reduced to 774.98 mPa s (NaOH of 10 % DS for 2 h + ultrasonication of 30 kJ/g DS) and 1083.24 mPa s (Ca(OH)₂ of 10 % DS for 2 h + ultrasonication of 30 kJ/g DS), respectively. Apparently, the combined method can effectively decrease the alkali dosage or the ultrasonication energy input.     

Determination of Five Polyphenols by HPLC/DAD and Discrimination of Apple Varieties

The objective of this study was to set up a method to detect five compounds in fresh smashed apples by HPLC/DAD simultaneously. Different methods have been tested to control browning and ascorbic acid with ultrasonication was adopted. Methanol–water–acetic acid (30:69:1, v/v) containing 2.0 g of ascorbic acid L^?1 was chosen as the extract solvent. The method effectively simplified the sample treatment compared with the traditional ways. And primarily, the results were used to identify between different varieties. The chromatographic separation was performed on an Atlantis C18 (250 mm × 4.5 mm, particle size 5 μm) with a gradient elution program using a mixture of acetonitrile and 2% aqueous acetic acid (v/v) as mobile phase within 20 min at 270 nm wavelength. The variation of the content of five compounds was gallic acid (ND ~1.81 μg g^?1), protocatechuic acid (ND ~1.79 μg g^?1), chlorogenic acid (13.81–189.4 μg g^?1), caffeic acid (6.82–45.02 μg g^?1) and rutin (0.96–18.55 μg g^?1). The results could successfully be used to discriminate between different apple varieties (Gala, Fuji, Delicious, 8th Apple US, Golden Apple, Green Apple and Red Rose); chlorogenic acid and rutin being the polyphenols that contribute most to the differentiation.     

Thermophilic Bacillus coagulans Requires Less Cellulases for Simultaneous Saccharification and Fermentation of Cellulose to Products than Mesophilic Microbial Biocatalysts

Ethanol production from lignocellulosic biomass depends on simultaneous saccharification of cellulose to glucose by fungal cellulases and fermentation of glucose to ethanol by microbial biocatalysts (SSF). The cost of cellulase enzymes represents a significant challenge for the commercial conversion of lignocellulosic biomass into renewable chemicals such as ethanol and monomers for plastics. The cellulase concentration for optimum SSF of crystalline cellulose with fungal enzymes and a moderate thermophile, Bacillus coagulans, was determined to be about 7.5 FPU g^?1 cellulose. This is about three times lower than the amount of cellulase required for SSF with Saccharomyces cerevisiae, Zymomonas mobilis, or Lactococcus lactis subsp. lactis whose growth and fermentation temperature optimum is significantly lower than that of the fungal cellulase activity. In addition, B. coagulans also converted about 80% of the theoretical yield of products from 40 g/L of crystalline cellulose in about 48 h of SSF with 10 FPU g^?1 cellulose while yeast, during the same period, only produced about 50% of the highest yield produced at end of 7 days of SSF. These results show that a match in the temperature optima for cellulase activity and fermentation is essential for decreasing the cost of cellulase in cellulosic ethanol production.     

Improvement of Solvent Production from Xylose Mother Liquor by Engineering the Xylose Metabolic Pathway in Clostridium acetobutylicum EA 2018

Xylose mother liquor (XML) is a by-product of xylose production through acid hydrolysis from corncobs, which can be used potentially for alternative fermentation feedstock. Sixteen Clostridia including 13 wild-type, 1 industrial strain, and 2 genetically engineered strains were screened in XML, among which the industrial strain Clostridium acetobutylicum EA 2018 showed the highest titer of solvents (12.7 g/L) among non-genetic populations, whereas only 40 % of the xylose was consumed. An engineered strain (2018glcG-TBA) obtained by combination of glcG disruption and expression of the d-xylose proton-symporter, d-xylose isomerase, and xylulokinase was able to completely utilize glucose and l-arabinose, and 88 % xylose in XML. The 2018glcG-TBA produced total solvents up to 21 g/L with a 50 % enhancement of total solvent yield (0.33 g/g sugar) compared to that of EA 2018 (0.21 g/g sugar) in XML. This XML-based acetone–butanol–ethanol fermentation using recombinant 2018glcG-TBA was estimated to be economically promising for future production of solvents.     

Highly Thermostable and pH-Stable Cellulases from Aspergillus niger NS-2: Properties and Application for Cellulose Hydrolysis

Optimization of cultural conditions for enhanced cellulase production by Aspergillus niger NS-2 were studied under solid-state fermentation. Significant increase in yields (CMCase 463.9?±?20.1 U/g, FPase 101.1?±?3.5 U/g and β-glucosidase 99?±?4.0 U/g) were obtained under optimized conditions. Effect of different nutritional parameters was studied to induce the maximum production of cellulase complex. Scale-up studies for enzyme production process were carried out. Characterization studies showed that enzymes produced by A. niger NS-2 were highly temperature- and pH stable. At 50 °C, the half life for CMCase, FPase, β-glucosidase were approximately 240 h. Cellulases from A. niger NS-2 were stable at 35 °C for 24 h over a broader pH range of 3.0–9.0. We examined the feasibility of using steam pretreatment to increase the saccharification yields from various lignocellulosic residues for sugar release which can potentially be used in bioethanol production. Saccharification of pretreated dry potato peels, carrot peels, composite waste mixture, orange peels, onion peels, banana peels, pineapple peels by crude enzyme extract from A. niger NS-2, resulted in very high cellulose conversion efficiencies of 92–98 %.     

Chitin/graphene oxide composite films with enhanced mechanical properties prepared in NaOH/urea aqueous solution

Chitin/graphene oxide (GO) composite films with excellent mechanical properties were prepared in NaOH/urea solution using a freezing/thawing method. The structure, thermal stability and mechanical properties of the composite films were investigated. Use of an atomic force microscope and transmission electron microscopy indicated that GO was successfully exfoliated to a single layer by ultrasonication. The results revealed that GO nanosheets were homogeneously dispersed and embedded in the chitin matrix. Due to the strong interactions between GO and the chitin matrix, the tensile strength and elongation at break of the composite film possessing 1.64 wt% GO were significantly improved by 98.7 and 114.5 %, respectively, compared with pure chitin film.     

Cell Immobilization with Polyurethane Foam for Retaining Trichoderma reesei Cells During Foam Fractionation for Cellulase Collection

In situ affinity foam fractionation is a potential powerful tool for continuous, selective removal of products from bioprocesses. When evaluating its applicability to cellulase production by Trichoderma reesei fermentation, we encountered the difficulty of significant removal of fungal mycelia along with the cellulase. To solve this problem, cell immobilization using cut pieces of hydrophilic polyurethane (PU) foam was evaluated. Five commercial PU foams with different pore sizes and porosities were tested. Two were found to support good cell growth, cellulase production, and cell loading (about 0.6 g dry cells per g PU). The PU-immobilized mycelia were successfully retained in the foaming process.     

Simultaneous LC�CMS Analysis of Paclitaxel and Retinoic Acid in Plasma and Tissues from Tumor-Bearing Mice

A novel method has been developed for simultaneous analysis of paclitaxel (PTX) and retinoic acid (ATRA) in mice plasma and tissue homogenates. The analyte was isolated by liquid?Cliquid partitioning, to minimize analyte degradation, and fractions were analyzed by liquid chromatography?Cmass spectrometry (LC?CMS). Liquid?Cliquid extraction with tert-butyl methyl ether was used for sample preparation and docetaxel was used as internal standard (IS). Separation of PTX, ATRA, and the IS was performed on a C₁₈ column with gradient elution and detection by use of a single-quadrupole mass spectrometer in selected-ion-monitoring mode. The method had high extraction recovery (>85%) and accuracy (>90%), with intra-day and inter-day precision <15%, and was rugged with good linearity. The limits of quantitation were determined to be 2 and 4 ng mL^?1 for PTX and ATRA, respectively. Freeze?Cthaw stability, short-term stability, long-term stability, and sample stability in the autosampler tray were examined; this indicated freezing and thawing during bio-sample preparation should be avoided, but no other stability-related problems occurred during sample storage, extraction, and chromatography of PTX and ATRA in plasma and tissue samples. The assay was successfully used for simultaneous analysis of PTX and ATRA in mice plasma and different tissues to support pharmacokinetic and in-vivo distribution studies of the two drugs.     

Application of Endo-β-1,4,d-mannanase and Cellulase for the Release of Mannooligosaccharides from Steam-Pretreated Spent Coffee Ground

Spent coffee ground (SCG), a present waste stream from instant coffee production, represents a potential feedstock for mannooligosaccharides (MOS) production. MOS can be used in nutraceutical products for humans/animals or added to instant coffee, increasing process yield and improving product health properties. The SCG was evaluated for MOS production by steam pretreatment and enzymatic hydrolysis with a recombinant mannanase and a commercial cellulase cocktail (Acremonium, Bioshigen Co. Ltd, Japan). The mannanase was produced using a recombinant strain of Yarrowia lipolytica, used to produce and secrete endo-1,4-β,d-mannanase from Aspergillus aculeatus in bioreactor cultures. Endo-1,4-β,d-mannanase was produced with an activity of 183.5 U/mL and 0.23 mg protein/mL. The enzyme had an optimum temperature of 80 °C, and the activity in the supernatant was improved by 150 % by supplementation with 0.2 % sodium benzoate and 35 % sorbitol as a preservative and stabiliser, respectively. The steam pretreatment of SCG improved the enzymatic digestibility of SCG, thus reducing the required enzyme dosage for MOS release. Combined enzymatic hydrolysis of untreated or steam-pretreated (150, 190 and 200 °C for 10 min) SCG with mannanase and cellulase cocktail resulted in 36–57 % (based on mannan content) of MOS production with a degree of polymerization of up to 6. The untreated material required at least 1 % of both mannanase and cellulase loading. The optimum mannanase and cellulase loadings for pretreated SCG hydrolysis were between 0.3 and 1 and 0.4 and 0.8 %, respectively. Statistical analysis suggested additive effect between cellulase cocktail and mannanase on MOS release, with no indication of synergism observed.     

A Validated LC Method for Determination of Trace Impurities in Technical Triadimefon

A simple and sensitive liquid chromatography with ultraviolet detection (LC?CUV) method was developed for the determination of three impurities with a content over 0.1% (w/w) in technical triadimefon. A Gemini C₁₈ column (5 ??m, 250 mm × 4.6 mm i.d.) was used for the chromatographic separations. The samples were separated by gradient elution with water (solvent A) and methanol (solvent B) using the following conditions: 70% A isocratic for 12 min, linear to 0% A within 8 min, and isocratic for 10 min at 0% A with a flow rate of 1.0 mL min^?1. Chromatograms were recorded at an absorption wavelength of 280 nm. The chromatographic resolutions between triadimefon and its potential impurities A, B, and C were greater than 3. The developed LC method was validated with respect to linearity, accuracy, precision, and robustness. This method was successfully applied to analyze the impurities in commercial technical triadimefon. In addition, the structures of the three impurities were identified to be (A) 4-chlorophenol, (B) 1-(2,4-dichlorophenoxy)-3,3-dimethyl-1-(1H-1,2,4-triazol-1-yl)-2-butanone, and (C) 1,1-bis(4-chlorophenoxy)-3,3-dimethyl-2-butanone.     

A Validated Chiral LC Method for the Enantiomeric Separation of Cinacalcet Hydrochloride

A rapid isocratic chiral LC method has been developed for the separation of (S)-cinacalcet from (R)-cinacalcet. Good resolution with R _S  > 3 was obtained using a Chiralpak-IA column (250 × 4.6 mm, particle size 5 μm) and n-hexane, ethanol and trifluoroacetic acid as the mobile phase (95:5:0.1, v/v) at ambient temperature. Flow rate was kept at 1.0 mL min^–1 and elution was monitored by UV detection at 223 nm. This method was further used to determine the presence of (S)-cinacalcet in enantiopure pharmaceutical formulations containing (R)-cinacalcet. This method allowed for the detection and quantitation of (S)-cinacalcet of levels at 0.04 and 0.16 μg mL^–1, respectively. The method was validated following ICH guidelines.     

Radiation synthesis and characteristics of charcoal filled PVA/PVP hydrogels

Hydrogels from a mixture of charcoal and poly(vinyl alcohol)/poly(N-vinyl pyrrolidone) were made by single “freezing and thawing” or two-step “freezing and thawing” and gamma-ray irradiation. Physical properties such as gel content and adsorption of bacteria were examined.     

UPLC for the Determination of Clopidogrel in Dog Plasma by Tandem Quadrupole MS: Application to a Pharmacokinetic Study

A rapid, sensitive and specific ultra performance liquid chromatography-electrospray ionization tandem quadrupole mass spectrometry method was developed and validated for the determination of clopidogrel in dog plasma. Plasma samples were deproteinized with acetonitrile and separated on a Waters BEH C18 column (1.7 μm, 50 mm × 2.1 mm id.) with isocratic elution at a flow-rate of 0.2 mL min^?1 and mobile phase consisting of acetonitrile and water (containing 0.15% formic acid) (75:25, v/v). The single run analysis was as shorter as 2 min. Electrospray ionization in positive ion mode and multiple reaction monitoring were used for the quantification of clopidogrel with monitored transitions m/z 322 → 212 for clopidogrel and m/z 324 → 217 for internal standard (gliclazide). The intra- and inter-day precisions (RSD%) were less than 6.32 and 7.03%, and accuracy (RE%) between ?9.12 and 9.65% (n = 6). The extraction recovery of clopidogrel was 96.7%. The developed method was successfully applied to the pharmacokinetic study of clopidogrel tablets in dogs following oral administration at a single dose of 75 mg.     

Viscoelastic properties of cross-linked polyvinyl alcohol and surface-oxidized cellulose whisker hydrogels

Reinforcement of polyvinyl alcohol (PVA) hydrogels was achieved by direct chemical cross-linking of surface modified microcrystalline cellulose (MCC) whiskers with PVA. In order to produce hydrogels, the MCC whiskers were first obtained by TEMPO-mediated oxidation of the cellulose substrate and ultrasonication followed by direct cross-linking to PVA (Mw 98,000) via forming acetal bonds and freeze–thawing. The viscoelastic properties of the produced hydrogels were clearly improved following the chemical cross-linking, featuring values for viscous and elastic moduli G′ and G″ on the order of 10 kPa, which is particularly interesting for biomedical orthopedic applications.     

Influence of Forced Internal Air Circulation on Airflow Distribution and Heat Transfer in a Gas Double-dynamic Solid-state Fermentation Bioreactor

Internal air circulation affects the temperature field distribution in a gas double-dynamic solid-state fermentation bioreactor (GDSFB). To enhance heat transfer through strengthening internal air circulation in a GDSFB, we put an air distribution plate (ADP) into the bioreactor and studied the effects of forced internal air circulation on airflow, heat transfer, and cellulase activity of Trichoderma viride L3. Results showed that ADP could help form a steady and uniform airflow distribution, and with gas-guide tubes, air reversal was formed inside the bioreactor, thus resulting in a smaller temperature difference between medium and air by enhancing convective heat transfer inside the bioreactor. Using an ADP of 5.35 % aperture ratio caused a 1 °C decrease in the average temperature difference during the solid-state fermentation process of T. viride L3. Meanwhile, the cellulase activity of T. viride L3 increased by 13.5 %. The best heat-transfer effect was attained when using an ADP of 5.35 % aperture ratio and setting the fan power to 125 V (4.81 W) in the gas double-dynamic solid-state fermentation (GDSF) process. An option of suitable aperture ratio and fan power may be conducive to ADPs’ industrial amplification.