Enhancement of Enzymatic Hydrolysis and Klason Lignin Removal of Corn Stover Using Photocatalyst-Assisted Ammonia Pretreatment

Photocatalyst-assisted ammonia pretreatment was explored to improve lignin removal of the lignocellulosic biomass for effective sugar conversion. Corn stover was treated with 5.0–12.5 wt.% ammonium hydroxide, two different photocatalysts (TiO₂ and ZnO) in the presence of molecular oxygen in a batch reactor at 60 °C. Various solid-to-liquid ratios (1:20–1:50) were also tested. Ammonia pretreatment assisted by TiO₂-catalyzed photo-degradation removed 70 % of Klason lignin under the optimum condition (12.5 % ammonium hydroxide, 60 °C, 24 h, solid/liquid?=?1:20, photocatalyst/biomass?=?1:10 with oxygen atmosphere). The enzymatic digestibilities of pretreated corn stover were 85 % for glucan and 75 % for xylan with NH₃-TiO₂-treated solid and 82 % for glucan and 77 % for xylan with NH₃-ZnO-treated solid with 15 filter paper units/g-glucan of cellulase and 30 cellobiase units/g-glucan of β-glucosidase, a 2–13 % improvement over ammonia pretreatment alone.     

Biomass Composition, Lipid Characterization, and Metabolic Profile Analysis of the Fed-Batch Fermentation Process of Two Different Docosahexanoic Acid Producing Schizochytrium sp. Strains

Growth and fermentation characteristics, biomass composition, lipid characterization and metabolic profiling analysis of two different Schizochytrium sp. strains, the original strain and the industrial adaptive strain, were investigated in the fed-batch fermentation process. The final cell biomass, total lipids content, docosahexanoic acid (DHA) content and DHA productivity of the adaptive strain were much higher than those of the original strain. The metabolic distinctions which extensively existed between these two strains were revealed by the score plot of principal component analysis. In addition, potential biomarkers responsible for discriminating different strains were identified as myo-inositol, histidine, alanine, asparagine, cysteine, and oxalic acid. These findings provided new insights into the industrial strain screening and further improvement of DHA production by Schizochytrium sp.     

Improvement of Regio-Specific Production of Myricetin-3-O-α-l-Rhamnoside in Engineered Escherichia coli

Myricetin is an important flavonol whose medically important properties include activities as an antioxidant, anticarcinogen, and antimutagen. The solubility, stability, and other biological properties of the compounds can be enhanced by conjugating aglycon with sugar moieties. The type of sugar moiety also plays a significant role in the biological and physical properties of the natural product glycosides. Reconstructed Escherichia coli containing thymidine diphosphate-α-l-rhamnose sugar gene cassette and Arabidopsis-derived glycosyltransferase were used for rhamnosylation of myricetin. Myricetin (100 μM) was exogenously supplemented to induced cultures of engineered E. coli. The formation of target product—myricetin-3-O-α-l-rhamnoside—was confirmed by chromatographic and NMR analyses. The yield of product was improved by using various mutants and methylated cyclodextrin as a molecular carrier for myricetin in combination with E. coli M3G3. The maximal yield of product is 55.6 μM (3.31-fold higher than the control E. coli MG3) and shows 55.6 % bioconversion of substrate under optimized conditions.     

Recombinant Expression and Characterization of an Organic-Solvent-Tolerant α-Amylase from Exiguobacterium sp. DAU5

The enzyme from halophilic microorganisms often has unique properties such as organic-solvent-tolerance. In this study, a novel organic-solvent-tolerant α-amylase gene was cloned from the mild halophile Exiguobacterium sp. DAU5. The open reading frame (ORF) of the enzyme consisted of 1,545 bp and encoded 514 amino acids, the primary sequence revealed that it belongs to the glycoside hydrolase (GH) family 13. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed an AmyH monomer of 57 kDa. The enzyme exhibited maximal activity at 40 °C in pH?8.5 glycine–NaOH buffer, and the activity was strongly inhibited by Zn²⁺, Cu²⁺, and Fe²⁺. The α-amylase AmyH exhibited high hydrolysis activity toward soluble starch, and the major hydrolysis products were maltose, maltotriose, and maltopentaose; the AmyH could not efficiently hydrolyze oligosaccharides smaller than maltoheptaose, nor could it act on the β-1,4 or α-1,6 glucosidic bonds in xylan or pullulan, respectively. In addition, the α-amylase exhibited better tolerance to organic solvents, as it was stable in the presence of dimethylsulfoxide (DMSO), methanol, ethanol, and acetone. Base on all of these results, the enzyme could be useful for practical application in the bakery industry and in biotechnological processes that occur in the presence of organic solvents.     

Extracellular Enzymes of the White-Rot Fungus Fomes fomentarius and Purification of 1,4-β-Glucosidase

Production of the lignocellulose-degrading enzymes endo-1,4-β-glucanase, 1,4-β-glucosidase, cellobiohydrolase, endo-1,4-β-xylanase, 1,4-β-xylosidase, Mn peroxidase, and laccase was characterized in a common wood-rotting fungus Fomes fomentarius, a species able to efficiently decompose dead wood, and compared to the production in eight other fungal species. The main aim of this study was to characterize the 1,4-β-glucosidase produced by F. fomentarius that was produced in high quantities in liquid stationary culture (25.9 U?ml^?1), at least threefold compared to other saprotrophic basidiomycetes, such as Rhodocollybia butyracea, Hypholoma fasciculare, Irpex lacteus, Fomitopsis pinicola, Pleurotus ostreatus, Piptoporus betulinus, and Gymnopus sp. (between 0.7 and 7.9 U?ml^?1). The 1,4-β-glucosidase enzyme was purified to electrophoretic homogeneity by both anion-exchange and size-exclusion chromatography. A single 1,4-β-glucosidase was found to have an apparent molecular mass of 58 kDa and a pI of 6.7. The enzyme exhibited high thermotolerance with an optimum temperature of 60 °C. Maximal activity was found in the pH range of 4.5–5.0, and K _M and V _max values were 62 μM and 15.8 μmol?min^?1?l^?1, respectively, when p-nitrophenylglucoside was used as a substrate. The enzyme was competitively inhibited by glucose with a K _i of 3.37 mM. The enzyme also acted on p-nitrophenylxyloside, p-nitrophenylcellobioside, p-nitrophenylgalactoside, and p-nitrophenylmannoside with optimal pH values of 6.0, 3.5, 5.0, and 4.0–6.0, respectively. The combination of relatively low molecular mass and low K _M value make the 1,4-β-glucosidase a promising enzyme for biotechnological applications.     

Biological Removal of Phosphate at Low Concentrations Using Recombinant Escherichia coli Expressing Phosphate-Binding Protein in Periplasmic Space

During wastewater treatment, phosphate removal is an important and challenging process; thus, diverse technologies, including those derived from biological means, have been devised for efficient phosphate removal. Although conventional biological methods are effective in decreasing wastewater phosphate levels to ~1 mg/L, long periods of microbial adaptation are required for effective phosphate removal, and the removal efficiency of these methods is relatively poor at lower phosphate concentrations. In the present work, we constructed a recombinant Escherichia coli with periplasmic-expressed phosphate-binding protein (PBP) and investigated its biological removal ability for low phosphate levels. We found that the PBP-expressing recombinant E. coli cells showed efficient (> 94 %) removal of phosphate at low concentrations (0.2–1.0 mg/L) in a treated cell mass-dependent manner. Collectively, we propose that our PBP-expressing recombinant whole-cell system could be successfully used during wastewater treatment for the biological removal of low concentrations of phosphate.     

Glucose Oxidase/Cellulose–Carbon Nanotube Composite Paper as a Biocompatible Bioelectrode for Biofuel Cells

Biofuel cells are devices for generating electrical energy directly from chemical energy of renewable biomass using biocatalysts such as enzymes. Efficient electrical communication between redox enzymes and electrodes is essential for enzymatic biofuel cells. Carbon nanotubes (CNTs) have been recognized as ideal electrode materials because of their high electrical conductivity, large surface area, and inertness. Electrodes consisting entirely of CNTs, which are known as CNT paper, have high surface areas but are typically weak in mechanical strength. In this study, cellulose (CL)–CNT composite paper was fabricated as electrodes for enzymatic biofuel cells. This composite electrode was prepared by vacuum filtration of CNTs followed by reconstitution of cellulose dissolved in ionic liquid, 1-ethyl-3-methylimidazolium acetate. Glucose oxidase (GOx), which is a redox enzyme capable of oxidizing glucose as a renewable fuel using oxygen, was immobilized on the CL–CNT composite paper. Cyclic voltammograms revealed that the GOx/CL–CNT paper electrode showed a pair of well-defined peaks, which agreed well with that of FAD/FADH_2, the redox center of GOx. This result clearly shows that the direct electron transfer (DET) between the GOx and the composite electrode was achieved. However, this DET was dependent on the type of CNTs. It was also found that the GOx immobilized on the composite electrode retained catalytic activity for the oxidation of glucose.     

The Extraction of Polycyclic Aromatic Hydrocarbons from Atmospheric Particulate Matter Samples by Accelerated Solvent Extraction (ASE)

Abstract

The Accelerated solvent extraction (ASE) of PAHs (23 2- to 6-ring species) spiked onto glass fibre filters (GFFs) was studied as a function of variable extraction solvents, pressure, temperature and extraction times. Acceptable recoveries (85% ± 15%) were obtained for certain combinations of conditions and a tentative method (1500 psi, 150°C, 70:30 hexane:acetone mixture, 7 min heat-up time, 5 min static extraction time, 60% flush volume, 2 static cycles was selected for further testing. However, this method did not prove as effective as the traditional Soxhlet method of extraction when these parameters were used to extract native PAHs from ambient atmospheric particulate matter collected on a GFF by Integrated Atmospheric Deposition Network (IADN) sampling protocols. The extraction recovery study for spiked GFFs was repeated using slightly different extraction conditions: 2000 psi, 100°C, 70:30 hexane:acetone, 5 min heat-up time, 5 min static extraction time, 150% flush volume, 3 static cycles. When this method was applied to the extraction of native PAHs from ambient atmospheric particulate matter collected on GFFs, the results showed equivalent or better recoveries to that of the Soxhlet method. The total time of extraction was 25 min requiring only 30 mL of solvent. This ASE method is presently used to quantitatively determine PAHs in IADN particle-phase samples.     

Pressurised solvent extraction of nitrated derivatives of polycyclic aromatic hydrocarbons from roadside dust

An analytical method based upon the pressurised solvent extraction and gas chromatography and mass spectrometry (GC-MS) for analysis of nitrated derivatives of polycyclic aromatic hydrocarbons (nitro-PAHs) in roadside dust has been developed. Five nitro-PAHs (1-nitronaphthalene, 1-nitropyrene, 9-nitroanthracene, 2-nitrofluorene, 6-nitrochrysene) were chosen for this study because of their variable physical properties and effect on human health. Experiments with spiked inert matrix were used for the best extraction conditions finding. Different extraction conditions (temperature, pressure, solvents and duration of extraction cycle) were examined. The highest recoveries were observed at the extraction pressure of 14?MPa, temperature of 100°C and two cycles of 5?min extraction time, when dichloromethane was used as an extraction solvent. These conditions were applied to the extraction of nitro-PAHs from roadside dust. After extraction the nitro-PAHs fraction was isolated by the silica gel column chromatography. The GC-MS detector was used for analyses of the extracts. Recoveries of nitro-PAHs after extraction from roadside dust ranged from 60% for 9-nitroanthracene to 108% for 6-nitrochrysene.     

Calorimetric determination of the effect of additives on cement hydration process

Possibilities of a multicell isoperibolic-semiadiabatic calorimeter application for the measurement of hydration heat and maximum temperature reached in mixtures of various compositions during their setting and early stages of hardening are presented. Measurements were aimed to determine the impact of selected components?? content on the course of ordinary Portland cement (OPC) hydration. The following components were selected for the determination of the hydration behaviour in mixtures: very finely ground granulated blast furnace slag (GBFS), silica fume (microsilica, SF), finely ground quartz sand (FGQ), and calcined bauxite (CB). A commercial polycarboxylate type superplasticizer was also added to the selected mixtures. All maximum temperatures measured for selected mineral components were lower than that reached for cement. The maximum temperature increased with the decreasing amount of components in the mixture for all components except for silica fume. For all components, except for CB, the values of total released heat were higher than those for pure Portland cement samples.