Optimization of Solid-State Fermentation Parameters for the Production of Xylanase by \u3cem\u3eTrichoderma longibrachiatum\u3c/em\u3e on Wheat Bran

Solid-state fermentation has the potential to produce inexpensive enzymes for use in high-volume industrial applications. Process parameters such as substrate moisture content and length of fermentation can have a significant effect on the amount and timing of enzyme production. This study was conducted in two stages, a screening stage and an optimization stage, to determine the effects of moisture content of the substrate, surfactant addition upon inoculation, depth of the substrate, and duration of fermentation on xylanase activity produced by Trichoderma longibrachiatum. Screening fermentations were conducted at 25°C, 50 and 75% wet basis moisture content (w.b.), 0.0 and 0.2% v/v surfactant addition, 0.5 and 1.5 cm depth of wheat bran, and 5 and 10 days of fermentation. Optimization fermentations were conducted at 25°C, 45, 55, and 65% moisture content (w.b.), 1.0, 1.5, and 2.0 cm depth of wheat bran, and three and five days of fermentation. Experiments were conducted as full factorial experiments with three replications of each treatment. The optimal values of the process variables were selected based on the units of xylanase activity produced per gram of wheat bran (U/g). Moisture content, depth of substrate, and duration of fermentation had significant main effects on the production of enzyme activity. Surfactant addition upon inoculation had interaction effects with moisture content, and the duration of fermentation by moisture content interaction also was significant. The treatment of 55% moisture content, 1.5 cm depth of substrate, and five days of fermentation resulted in the highest average xylanase activity (716 U/g wheat bran)

Optimization of Solid-State Fermentation Parameters for the Production of Xylanase by \u3cem\u3eTrichoderma longibrachiatum\u3c/em\u3e on Wheat Bran in a Forced Aeration System

The effect of aeration on the production of xylanase by Trichoderma longibrachiatum on wheat bran in a solid-state fermentation (SSF) system has not been investigated. This study was conducted to investigate the interactive effects of aeration, initial moisture content of the substrate, and depth of the substrate on xylanase activity produced in a tray fermenter. The experiment was conducted as a split plot experiment with factorial treatments and three replications of each treatment combination. The whole plot treatment was aeration rate (0, 2.9, 5.7 L/min/kg bran). Initial moisture content (45, 55, 65% w.b.) and depth of substrate (1.0 and 2.5 cm) were investigated factorially. Trays of wheat bran were assayed after fermentation by Trichoderma longibrachiatum to determine the production of xylanase activity. Aeration rate had a significant nonlinear effect on enzyme activity with highest yields obtained at an aeration rate of 2.9 L/min/kg bran (738 U/g, averaged over all initial moisture contents and depth of substrates). Initial moisture content of the substrate also had a significant nonlinear effect on enzyme activity with the highest yields at 55% (556 U/g, averaged over all airflow rates and depths of substrate). Depth of substrate had no significant statistical effect on enzyme activity. The treatment combination of 2.9 L/min/kg airflow rate and 55% moisture content resulted in the highest yields (948 U/g, averaged over depth of substrate)

Effects of Growth Media pH and Reaction Water Activity on the Conversion of Acetophenone to (S)-1-Phenylethanol by \u3cem\u3eSaccharomyces cerevisiae\u3c/em\u3e Immobilized on Celite 635 and in Calcium Alginate

Biologically catalyzed reactions often produce enantiomers of the product; however, only one configuration is desired. Reaction conditions are known to affect enantiomer ratios and reaction kinetics, but little is known regarding the effect of processing conditions on whole-cell biocatalysis. Saccharomyces cerevisiae cells were grown in batch on glucose at pH = 4, 5, and 7, and then immobilized on Celite beads or in calcium alginate beads and used as the biocatalyst for the conversion of acetophenone in hexane to (S)-1-phenylethanol at water activities of 0.37, 0.61, and 0.80. S. cerevisiae was used as a model microorganism for the whole-cell catalyzed reaction. The initial reaction rate (IRR) and the final (S)-1-phenylethanol concentration were quantified for each treatment. The highest IRR value (94.9 µmol/h) and the highest final concentration of (S)-1-phenylethanol (17.8 mM) were observed on Celite-immobilized cells grown at pH 5 or 7, with the main effect of growth medium pH highly statistically significant. The main effect of water activity and the interactions of the two were not statistically significant (a = 0.05). The cells immobilized in calcium alginate beads favored a water activity of 0.61, resulting in an IRR of 916.2 µmol/h/g dcw, averaged over pH. The highest final concentration of (S)-1-phenylethanol (4.8 mM) was achieved with cells grown at pH 5 or 7. Calcium alginate beads gave the highest initial reaction rate with a growth pH of 7 and a water activity of 0.61. However, pH of 5 and water activity of 0.61 resulted in the highest final concentration of (S)-1-phenylethanol

Preparation and Characterization of Multimodal Hybrid Organic and Inorganic Nanocrystals of Camptothecin and Gold

We demonstrate a novel inorganic-organic crystalline nanoconstruct, where gold atoms were imbedded in the crystal lattices as defects of camptothecin nanocrystals, suggesting its potential use as simultaneous agents for cancer therapy and bioimaging. The incorporation of gold, a potential computed tomography (CT) contrast agent, in the nanocrystals of camptothecin was detected by transmission electron microscope (TEM) and further quantified by energy dispersive X-ray spectrometry (EDS) and inductively coupled plasma-optical emission spectrometers (ICP-OES). Due to gold\u27s high attenuation coefficient, only a relatively small amount needs to be present in order to create a good noise-to-contrast ratio in CT imaging. The imbedded gold atoms and clusters are expected to share the same biological fate as the camptothecin nanocrystals, reaching and accumulating in tumor site due to the enhanced permeation and retention (EPR) effect

Toward Biochemical Conversion of Lignocellulose On-Farm: Pretreatment and Hydrolysis of Corn Stover \u3cem\u3eIn Situ\u3c/em\u3e

High-solids lignocellulosic pretreatment using NaOH followed by high-solids enzymatic hydrolysis was evaluated for an on-farm biochemical conversion process. Increasing the solids loadings for these processes has the potential for increasing glucose concentrations and downstream ethanol production; however, sequential processing at high-solids loading similar to an on-farm cellulose conversion system has not been studied. This research quantified the effects of high-solids pretreatment with NaOH and subsequent high-solids enzymatic hydrolysis on cellulose conversion. As expected, conversion efficiency was reduced; however, the highest glucose concentration (40.2 g L-1), and therefore the highest potential ethanol concentration, resulted from the high-solids combined pretreatment and hydrolysis. Increasing the enzyme dosage improved cellulose conversion from 9.6% to 36.8% when high-solids loadings were used in both unit operations; however, increasing NaOH loading and pretreatment time did not increase the conversion efficiency. The enzyme-to-substrate ratio had a larger impact on cellulose conversion than the NaOH pretreatment conditions studied, resulting in recommendations for an on-farm bioconversion system

On-Farm Integrated High-Solids Processing System for Biomass

A method for on-farm processing a biomass feedstock into a useful industrial chemicals includes the steps of (a) delignifying the biomass feedstock to produce a delignified biomass, (b) subjecting the deliguified biomass to cellulase production, (c) subjecting the deliguified biomass with attached cellulase to simultaneous cellulolytic and solventogenic reactions to produce useful industrial chemicals (d) collecting and separating the useful industrial chemical from the fermentation broth and (e) collecting the fermentation residues

The Confounding Effects of Particle Size and Substrate Bulk Density on \u3cem\u3ePhanerochaete chrysosporium\u3c/em\u3e Pretreatment on \u3cem\u3ePanicum virgatum\u3c/em\u3e

Phanerochaete chrysosporium treatment is less effective as a biological pretreatment on feedstock with larger particle sizes. We hypothesized that the improved effectiveness of the pretreatment when smaller particle sizes are used may be due to the inherently higher bulk density with smaller particle sizes. The effects of substrate bulk density and particle size on the efficacy of P. chrysosporium pretreatment of switchgrass (Panicum virgatum) was tested experimentally. Phanerochaete chrysosporium was grown on senesced switchgrass (2 different particle sizes) with various bulk densities. In all treatments, the fungal-pretreated samples released more glucose during enzymatic saccharification than the control sample. Substrate bulk density was a statistically significant factor in explaining the variation in the amount of glucose released per gram of substrate used. However, the particle size was not found to be a significant factor. On-farm switchgrass pretreatment may not require particle size reduction if the switchgrass is supplied in high-density bales

Cytotoxic Activity of Triazole-Containing Alkyl ß-D-Glucopyranosides on a Human T-Cell Leukemia Cell Line

BACKGROUND: Simple glycoside surfactants represent a class of chemicals that are produced from renewable raw materials. They are considered to be environmentally safe and, therefore, are increasingly used as pharmaceuticals, detergents, and personal care products. Although they display low to moderate toxicity in cells in culture, the underlying mechanisms of surfactant-mediated cytotoxicity are poorly investigated. RESULTS: We synthesized a series of triazole-linked (fluoro)alkyl β-glucopyranosides using the copper-catalyzed azide-alkyne reaction, one of many popular click reactions that enable efficient preparation of structurally diverse compounds, and investigate the toxicity of this novel class of surfactant in the Jurkat cell line. Similar to other carbohydrate surfactants, the cytotoxicity of the triazole-linked alkyl β-glucopyranosides was low, with IC50 values decreasing from 1198 to 24 μM as the hydrophobic tail length increased from 8 to 16 carbons. The two alkyl β-glucopyranosides with the longest hydrophobic tails caused apoptosis by mechanisms involving mitochondrial depolarization and caspase-3 activation. CONCLUSIONS: Triazole-linked, glucose-based surfactants 4a-g and other carbohydrate surfactants may cause apoptosis, and not necrosis, at low micromolar concentrations via induction of the intrinsic apoptotic cascade; however, additional studies are needed to fully explore the molecular mechanisms of their toxicity. Graphical AbstractTriazole-linked, glucose-based surfactants cause apoptosis, and not necrosis, at low micromolar concentrations via induction of the intrinsic apoptotic cascade

Cytotoxic Activity of Triazole-Containing Alkyl β-D-Glucopyranosides on a Human T-Cell Leukemia Cell Line

Simple glycoside surfactants represent a class of chemicals that are produced from renewable raw materials. They are considered to be environmentally safe and, therefore, are increasingly used as pharmaceuticals, detergents, and personal care products. Although they display low to moderate toxicity in cells in culture, the underlying mechanisms of surfactant-mediated cytotoxicity are poorly investigated

Strategy for Conjugating Oligopeptides to Mesoporous Silica Nanoparticles Using Diazirine-Based Heterobifunctional Linkers

Successful strategies for the attachment of oligopeptides to mesoporous silica with pores large enough to load biomolecules should utilize the high surface area of pores to provide an accessible, protective environment. A two-step oligopeptide functionalization strategy is examined here using diazirine-based heterobifunctional linkers. Mesoporous silica nanoparticles (MSNPs) with average pore diameter of ~8 nm and surface area of ~730 m2/g were synthesized and amine-functionalized. Tetrapeptides Gly-Gly-Gly-Gly (GGGG) and Arg-Ser-Ser-Val (RSSV), and a peptide comprised of four copies of RSSV (4RSSV), were covalently attached via their N-terminus to the amine groups on the particle surface by a heterobifunctional linker, sulfo-succinimidyl 6-(4,4′-azipentanamido)hexanoate (sulfo-NHS-LC-diazirine, or SNLD). SNLD consists of an amine-reactive NHS ester group and UV-activable diazirine group, providing precise control over the sequence of attachment steps. Attachment efficiency of RSSV was measured using fluorescein isothiocyanate (FITC)-tagged RSSV (RSSV-FITC). TGA analysis shows similar efficiency (0.29, 0.31 and 0.26 mol peptide/mol amine, respectively) for 4G, RSSV and 4RSSV, suggesting a generalizable method of peptide conjugation. The technique developed here for the conjugation of peptides to MSNPs provides for their attachment in pores and can be translated to selective peptide-based separation and concentration of therapeutics from aqueous process and waste streams