Isolation of Spirosoma foliorum sp. nov. from the fallen leaf of Acer palmatum by a novel cultivation technique.

In the effort of isolating novel microbial species, the strain PL0132T was isolated from a fallen leaf under fresh water at a stream, which glided when grown on a tap water medium (without nutrients). The strain was determined to be Gram-negative, strictly aerobic, and rod-shaped, which grew optimally at 25 °C, pH 6-7, and the strain tolerates 1% (w/v) NaCl concentration. The complete genome of strain PL0132T comprises one contig with a sequencing depth of 76×, consisting of 8,853,064 base pairs and the genomic DNA G + C content was 46.7% (genome). 16S rRNA gene sequence analysis revealed that strain PL0132T represents a member of the phylum Bacteroidetes and is affiliated with the genus Spirosoma. Based on genomic, phenotypic, and chemotaxonomic characteristics, the strain PL0132T represents a novel species of the genus Spirosoma, for which the name Spirosoma foliorum sp. nov. is proposed (= KCTC 72228 T = InaCC B1447T).

Reactive Green 19 dye-ligand immobilized on the aminated nanofiber membranes for efficient adsorption of lysozyme: Process development and optimization in batch and flow systems.

The performance of lysozyme adsorption by the aminated nanofiber membrane immobilized with Reactive Green 19 (RG19) dyes was evaluated in batch and flow systems. The physicochemical properties of the dye-immobilized nanofiber membrane were characterized. The parameters of batch-mode adsorption of lysozyme (e.g., pH, initial dye concentration, and lysozyme concentration) were optimized using the Taguchi method. In a flow process, the factors influencing the dynamic binding performance for lysozyme adsorption in the chicken egg white (CEW) solution include immobilized dye concentration, adsorption pH value, feed flow rate, and feed CEW concentration. The impact of these operating conditions on the lysozyme purification process was investigated. Under optimal conditions, the recovery yield and purification factor of lysozyme achieved from the one-step adsorption process were 98.52% and 143 folds, respectively. The dye-affinity nanofiber membrane also did not exhibit any significant loss in its binding capacity and purification performance after five consecutive uses.

Studies of Protein Wastes Adsorption by Chitosan-Modified Nanofibers Decorated with Dye Wastes in Batch and Continuous Flow Processes: Potential Environmental Applications.

In this study, reactive green 19 dye from wastewater was immobilized on the functionalized chitosan nanofiber membranes to treat soluble microbial proteins in biological wastewater. Polyacrylonitrile nanofiber membrane (PAN) was prepared by the electrospinning technique. After heat treatment, alkaline hydrolysis, and chemically grafted with chitosan to obtain modified chitosan nanofibers (P-COOH-CS), and finally immobilized with RG19 dye, dyed nanofibers were generated (P-COOH-CS-RG19). The synthesis of P-COOH-CS and P-COOH-CS-RG19 are novel materials for protein adsorption that are not deeply investigated currently, with each of the material functions based on their properties in significantly improving the adsorption efficiency. The nanofiber membrane shows good adsorption capacity and great recycling performance, while the application of chitosan and dye acts as the crosslinker in the nanofiber membrane and consists of various functional groups to enhance the adsorption of protein. The dyed nanofibers were applied for the batch adsorption of soluble protein (i.e., lysozyme), and the process parameters including chitosan's molecular weight, coupling pH, chitosan concentration, dye pH, dye concentration, and lysozyme pH were studied. The results showed that the molecular weight of chitosan was 50 kDa, pH 5, concentration 0.5%, initial concentration of dye at 1 mg/mL dye and pH 12, lysozyme solution at 2 mg/mL at pH 8, and the maximum adsorption capacity was 1293.66 mg/g at a temperature of 318 K. Furthermore, thermodynamic, and kinetic studies suggested that the adsorption behavior of lysozyme followed the Langmuir adsorption isotherm model and the pseudo-second-order kinetic model. The optimal adsorption and desorption conditions based on batch experiments were directly applied to remove lysozyme in a continuous operation. This study demonstrated the potential of dyed nanofibers as an efficient adsorbent to remove approximately 100% of lysozyme from the simulated biological wastewater.

Electrospinning of Quaternized Chitosan-Poly(vinyl alcohol) Composite Nanofiber Membrane: Processing Optimization and Antibacterial Efficacy.

N-(2-hydroxy) propyl-3-trimethylammonium chitosan chloride (HTCC) is a type of quaternary ammonium chitosan derivative with an antibacterial activity superior to the pristine chitosan, but its electrospinnability is limited. In this study, polyvinyl alcohol (PVA) was blended with HTCC to improve the electrospinnability of nanofibers. The electrospinning of PVA-HTCC nanofiber membranes was optimized in terms of structural stability and antimicrobial performance. Based on scanning electron microscopic analysis, the morphology and diameter of the produced nanofibers were influenced by the applied voltage, flow rate of the feed solution, and weight ratio of the polymer blend. An increase in the HTCC content decreased the average nanofiber diameter. The maximum water solubility of the PVA-HTCC nanofibers reached the maximum value of 70.92% at 12 h and 25 °C. The antibacterial activity of PVA-HTCC nanofiber membranes against Escherichia coli was ~90%, which is significantly higher than that of PVA-chitosan nanofiber membrane. Moreover, the antibacterial efficiency of PVA-HTCC nanofiber membranes remained unaffected after 5 cycles of antibacterial treatment. The good antibacterial performance and biocompatibility of PVA-HTCC nanofiber membrane makes them attractive for biomedical and biochemical applications that necessitate sterile conditions.

Removal of Ionic Dyes by Nanofiber Membrane Functionalized with Chitosan and Egg White Proteins: Membrane Preparation and Adsorption Efficiency.

Electrospun polyacrylonitrile (PAN) nanofiber membrane was functionalized with chitosan and proteins for use in the treatment of dye-containing wastewater. The PAN nanofiber membrane was subjected to alkaline hydrolysis, before being grafted with chitosan and subsequently the proteins from chicken egg white. The resultant nanofiber membrane (P-COOH-CS-CEW) was comprehensively characterized using thermogravimetric analysis, Fourier-transform infrared spectroscopy, and scanning electron microscopy. The efficiency of P-COOH-CS-CEW in removing cationic dye toluidine blue O (TBO) and anionic dye acid orange 7 (AO7) in aqueous solution was evaluated. Based on the performance of model fitting, Langmuir and pseudo-second-order kinetic model could be used to describe the performance of P-COOH-CS-CEW in the removal of TBO (pH 10) and AO7 (pH 2) from the dye solutions. The adsorbed TBO and AO7 dyes can be completely desorbed by an elution solution made of 50% (v/v) ethanol and 1 M sodium chloride. After five consecutive adsorption-desorption cycles, the efficiency of dye removal by P-COOH-CS-CEW was maintained above 97%.

Kinetic and Thermodynamic Studies of Lysozyme Adsorption on Cibacron Blue F3GA Dye-Ligand Immobilized on Aminated Nanofiber Membrane.

The polyacrylonitrile (PAN) nanofiber membrane was prepared by the electrospinning technique. The nitrile group on the PAN nanofiber surface was oxidized to carboxyl group by alkaline hydrolysis. The carboxylic group on the membrane surface was then converted to dye affinity membrane through reaction with ethylenediamine (EDA) and Cibacron Blue F3GA, sequentially. The adsorption characteristics of lysozyme onto the dye ligand affinity nanofiber membrane (namely P-EDA-Dye) were investigated under various conditions (e.g., adsorption pH, EDA coupling concentration, lysozyme concentration, ionic strength, and temperature). Optimum experimental parameters were determined to be pH 7.5, a coupling concentration of EDA 40 µmol/mL, and an immobilization density of dye 267.19 mg/g membrane. To understand the mechanism of adsorption and possible rate controlling steps, a pseudo first-order, a pseudo second-order, and the Elovich models were first used to describe the experimental kinetic data. Equilibrium isotherms for the adsorption of lysozyme onto P-EDA-Dye nanofiber membrane were determined experimentally in this work. Our kinetic analysis on the adsorption of lysozyme onto P-EDA-Dye nanofiber membranes revealed that the pseudo second-order rate equation was favorable. The experimental data were satisfactorily fitted by the Langmuir isotherm model, and the thermodynamic parameters including the free energy change, enthalpy change, and entropy change of adsorption were also determined accordingly. Our results indicated that the free energy change had a negative value, suggesting that the adsorption process occurred spontaneously. Moreover, after five cycles of reuse, P-EDA-Dye nanofiber membranes still showed promising efficiency of lysozyme adsorption.

Ganoderma lucidum stimulates autophagy-dependent longevity pathways in Caenorhabditis elegans and human cells.

The medicinal fungus Ganoderma lucidum is used as a dietary supplement and health tonic, but whether it affects longevity remains unclear. We show here that a water extract of G. lucidum mycelium extends lifespan of the nematode Caenorhabditis elegans. The G. lucidum extract reduces the level of fibrillarin (FIB-1), a nucleolar protein that correlates inversely with longevity in various organisms. Furthermore, G. lucidum treatment increases expression of the autophagosomal protein marker LGG-1, and lifespan extension is abrogated in mutant C. elegans strains that lack atg-18, daf-16, or sir-2.1, indicating that autophagy and stress resistance pathways are required to extend lifespan. In cultured human cells, G. lucidum increases concentrations of the LGG-1 ortholog LC3 and reduces levels of phosphorylated mTOR, a known inhibitor of autophagy. Notably, low molecular weight compounds (<10 kDa) isolated from the G. lucidum water extract prolong lifespan of C. elegans and the same compounds induce autophagy in human cells. These results suggest that G. lucidum can increase longevity by inducing autophagy and stress resistance.

Antibacterial efficacy of quaternized chitosan/poly (vinyl alcohol) nanofiber membrane crosslinked with blocked diisocyanate.

N-[(2-hydroxyl-3-trimethylammonium) propyl] chitosan chloride (HTCC), which is a type of chitosan derivative with quaternary ammonium groups, possesses a higher antibacterial activity as compared to the pristine chitosan. The nanofiber membranes made of HTCC are attractive for applications demanding for antibacterial function. However, the hydrophilic nature of HTCC makes it unsuitable for electrospinning of nanofibers. Hence, biodegradable polyvinyl alcohol (PVA) was proposed as an additive to improve the electrospinnability of HTCC. In this work, PVA/HTCC nanofiber membrane was crosslinked with the blocked diisocyanate (BI) to enhance the stability of nanofiber membrane in water. Microbiological assessments showed that the PVA/HTCC/BI nanofiber membranes possessed a good antibacterial efficacy (∼100 %) against E. coli. Moreover, the biocompatibility of PVA/HTCC/BI nanofiber membrane was proven by the cytotoxicity test on mouse fibroblasts. These promising results indicated that the PVA/HTCC/BI nanofiber membrane can be a promising material for food packaging and as a potential wound dressing for skin regeneration.

Antibacterial efficacy of poly(hexamethylene biguanide) immobilized on chitosan/dye-modified nanofiber membranes.

This study aimed to develop a novel electrospun polyacrylonitrile (PAN) nanofiber membrane with the enhanced antibacterial property. The PAN nanofiber membrane was first subjected to alkaline hydrolysis treatment, and the treated membrane was subsequently grafted with chitosan (CS) to obtain a CS-modified nanofiber membrane (P-COOH-CS). The modified membrane was then coupled with different dye molecules to form P-COOH-CS-Dye membranes. Lastly, poly(hexamethylene biguanide) hydrochloride (PHMB) was immobilized on the modified membrane to produce P-COOH-CS-Dye-PHMB. Physical characterization studies were conducted on all the synthesized nanofiber membranes. The antibacterial efficacies of nanofiber membranes prepared under different synthesis conditions were evaluated systematically. Under the optimum synthesis conditions, P-COOH-CS-Dye-PHMB was highly effective in disinfecting a high concentration of Escherichia coli, with an antibacterial efficacy of approximately 100%. Additionally, the P-COOH-CS-Dye-PHMB exhibited an outstanding wash durability as its antibacterial efficacy was only reduced in the range of 5%-7% even after 5 repeated cycles of treatment. Overall, the experimental results of this study suggested that the P-COOH-CS-Dye-PHMB is a promising antibacterial nanofiber membrane that can be adopted in the food, pharmaceutical, and textile industries.

Fabrication of bio-based polyamide 56 and antibacterial nanofiber membrane from cadaverine.

A green bioprocess for the fabrication of nanofiber membranes from the biomaterial polyamide 56 (PA56) via electrospinning was proposed. Cadaverine, as the precursor of PA56, was first produced from recombinant Escherichia coli using the whole-cell biotransformation of lysine. PA56 was then fabricated by mixing adipic acid with purified cadaverine obtained from solvent extraction and distillation. The thermal properties of the fabricated PA56 are as follows: a melting point of 250 °C, a crystallization point of 220 °C, and a degradation temperature of 410 °C. A PA56 nanofiber membrane (PAM) was further prepared via electrospinning. Dyed membranes (P-Dye) were obtained by the reaction of Reactive Red 141 dye with the amino group of PAM. Poly-(hexamethylene biguanide) (PHMB) was attached to the P-Dye to create P-Dye-PHMB. On the other hand, PAM with alginate, used to facilitate PHMB attachment (P-Alg-PHMB), was compared with P-Dye-PHMB in terms of antibacterial activity against pathogenic strains of E. coli and Pseudomonas putida. P-Alg-PHMB showed excellent antibacterial efficiency for E. coli (97%) and P. putida (100%). The proposed bioprocess can be used to fabricate novel membranes for biomedical applications and functional textiles.

Removal of cationic dye waste by nanofiber membrane immobilized with waste proteins.

Water pollution caused by dyes has been a serious problem affecting human health and environment. The surface of polyacrylonitrile (PAN) nanofiber membranes was modified by mild hydrolysis and coupled with bovine serum albumin (BSA) obtained from the laboratory wastes, resulting in the synthesis of P-COOH and P-COOH-BSA nanofibers. The nanofibers with specific functional groups may enhance their potential applications toward the removal of ionic dyes in wastewater. Toluidine blue O (TBO) was applied as an example of cationic dye to evaluate the removal efficiency of P-COOH-BSA nanofiber. Results showed that the equilibrium dissociation constant and maximum removal capacity were 0.48 mg/mL and 434.78 mg/g, respectively, at pH 12, where the TBO removal can be explained based on Langmuir isotherm and pseudo-second-order model. Desorption studies have shown that TBO adsorbed on P-COOH-BSA protein membrane can be completely eluted with either 1 M NaCl or 50% glycerol. The results of repeated studies indicated that after five consecutive adsorption/desorption cycles, the removal efficiency of TBO can be maintained at ~97%. P-COOH-BSA has shown to be promising adsorbent in TBO dye removal from dye wastewater.

Antibacterial efficacy of chitosan- and poly(hexamethylene biguanide)-immobilized nanofiber membrane.

In this study, polyacrylonitrile (PAN) nanofiber membrane was prepared by an electrospinning technique. After alkaline hydrolysis, the ion-exchange nanofiber membrane (P-COOH) was grafted with chitosan molecules to form a chitosan-modified nanofiber membrane (P-COOH-CS). Poly(hexamethylene biguanide) (PHMB) was then covalently immobilized on P-COOH and P-COOH-CS to form P-COOH-PHMB and P-COOH-CS-PHMB, respectively. The nanofiber membranes were subjected to various surface analyses as well as to the evaluations of antibacterial activity against Escherichia coli. The optimal modification conditions for P-COOH-CS-PHMB were attained by water-soluble chitosan at 50 kDa of molecular weight, coupling pH at 7, and 0.05% (w/w) of PHMB. Within 10 min of treatment, the antibacterial rate was close to 100%. Under the similar conditions of antibacterial treatment, the P-COOH-CS-PHMB exhibited a better antibacterial efficacy than the P-COOH-PHMB. When the number of bacterial cells was increased by 2000 folds, both types of nanofiber membranes still maintained the antibacterial rate close to 100%. After five cycles of repeated antibacterial treatment, the antibacterial efficacy of P-COOH-PHMB was 96%, which was higher than that of P-COOH-CS-PHMB (83%). The experimental results revealed that the PHMB-modified nanofiber membranes can be suitably applied in water treatment such as water disinfection and biofouling control.

Crosslinked on novel nanofibers with thermophilic carbonic anhydrase for carbon dioxide sequestration.

The recombinant Sulfurihydrogenibium yellowstonense carbonic anhydrase (SyCA) was covalently bonded on novel polyacrylonitrile (PAN) and polyethylene terephthalate (PET) nanofibers (PAN-PET-PAN donated as AEA) that was first fabricated by electrospinning. The resulting composite materials further crosslinked by the glutaraldehyde, which significantly increased thermostability up to 89.8% and 18.0% after heating at 60 °C for 1 h for immobilized crude and pure SyCA, respectively. After four repetitive attempts in the demonstration of CO2 sequestration, immobilized crude and pure SyCA on AEA also effectively improved the total CaCO3 yields to be 5.8 folds and 2.2 folds compared to free enzyme. Furthermore, the endurance of immobilized crude was investigated on flue gasses, which was retained its activity up to 57% on 50 mM NOx and 61% on 50 mM SOx presence. This is the first report of immobilized thermophilic SyCA on a novel nanofiber at the reusability, durability, sequestration of carbon dioxide, tolerant to sulfur oxides (SOx) and nitrogen oxides (NOx) toxic gases and to prevent air pollution.

ARduino-pH Tracker and screening platform for characterization of recombinant carbonic anhydrase in Escherichia coli.

Carbonic anhydrase (CA, EC 4.2.1.1) is an ancient enzyme with zinc ion as its active site, which catalyzes the chemical reaction of carbon dioxide (CO2 ) to react with water and form bicarbonate ions. Due to its high catalytic efficiency on CO2 assimilation, CA is expected to use for carbon sequestration in industry. However, the protein expression level, thermostability and high-throughput screening of an active CA are still with difficulty. In this study, the CA from Sulfurihydrogenibium yellowstonense (denoted as SyCA) was selected for overexpressed in Escherichia coli by different pET vectors. The enzymatic properties including thermo-stability, pH tolerance, effect of metal ion, and kinetic parameters were characterized through a novel ARduino-pH Tracker (ART) for monitoring online effectively. The SyCA is thermophilic and acidophilic as it maintains 100% activity at 50°C, while the residual activity is 34.8% after heating at 80°C for 150 min and the optimal pH is 3-5. The kinetic analysis by ART system showed that the k cat /K m of free enzyme was 4.4-folds that that of whole cell. On the other hand, the screening platforms as Wilbur-Anderson unit, phenol red indicator and size of colony forming unit have been established to explore CA with higher activity. The high-throughput screening platform is support in direct evolution of CA and further used in the industry.

Antibacterial activity of quaternized chitosan modified nanofiber membrane.

The electrospinning PAN nanofiber membrane (P-CN) was hydrolysed to convert carboxylic groups as reaction sites and covalently graft chitosan molecule. The chitosan derivatives with quaternary ammonium groups exerted greater efficiency against bacteria as compared to pure chitosan. Hence, the chitosan modified membrane (P-CS), can be functionalized with quaternary amine (i.e., glycidyl trimethyl ammonium chloride, GTMAC) to form quaternized chitosan nanofiber membrane (designated as P-HTCC) under various conditions (acidic, neutral, and alkaline). N-quaternized derivatives of chitosan modified membrane (N-HTCC) showed 72% and 60% degree of quaternization (DQ) under acidic and neutral conditions, respectively. Under alkaline condition, additional quaternization of N, O-HTCC via its amino and hydroxyl groups, has improved up to 90% DQ of the chitosan. The antibacterial activity of the quaternized chitosan modified membrane prepared from acetic acid medium is stronger than that prepared from water and alkaline media. Also, antibacterial activity of quaternized chitosan is stronger than chitosan modified membrane against E. coli. The microbiological assessments showed that the water-stable P-HTCC nanofiber membrane under modification in acidic medium exerted antibacterial activity up to 99.95% against E. coli. Therefore, the P-HTCC membrane exhibited high potential to be integrated into microfiltration membrane to effectively disinfect E. coli.

Pinicolol B from Antrodia cinnamomea induces apoptosis of nasopharyngeal carcinoma cells.

ETHNOPHARMACOLOGICAL RELEVANCE: The medicinal mushroom Antrodia cinnamomea possesses anticancer properties but the active compounds responsible for these effects are mostly unknown. AIM OF THE STUDY: We aimed to identify novel A. cinnamomea compounds that produce cytotoxic effects on cancer cells. MATERIALS AND METHODS: Using ethanol extraction and chromatography, we isolated the lanostanoid compound lanosta-7,9(11),24-trien-3ß,15α,21-triol (1) from cultured A. cinnamomea mycelium. Cytotoxicity and pro-apoptotic effects of compound 1 were evaluated using the MTS assay and flow cytometry analysis, respectively. RESULTS: Compound 1 produced cytotoxic effects on the nasopharyngeal carcinoma cell lines TW02 and TW04, with IC50 values of 63.3 and 115.0µM, respectively. On the other hand, no cytotoxic effects were observed on non-tumorigenic nasopharyngeal epithelial cells (NP69). In addition, compound 1 induced apoptosis in TW02 and TW04 cells as revealed by flow cytometry analysis. CONCLUSIONS: Our results demonstrate for the first time the presence of pinicolol B in A. cinnamomea mycelium and suggest that this compound may contribute to the anticancer effects of A. cinnamomea.

Isolation, Culture and Characterization of Hirsutella sinensis Mycelium from Caterpillar Fungus Fruiting Body.

The caterpillar fungus Ophiocordyceps sinensis (previously called Cordyceps sinensis) has been used for centuries in Asia as a tonic to improve health and longevity. Recent studies show that O. sinensis produces a wide range of biological effects on cells, laboratory animals and humans, including anti-fatigue, anti-infection, anti-inflammatory, antioxidant, and anti-tumor activities. In view of the rarity of O. sinensis fruiting bodies in nature, cultivation of its anamorph mycelium represents a useful alternative for large-scale production. However, O. sinensis fruiting bodies harvested in nature harbor several fungal contaminants, a phenomenon that led to the isolation and characterization of a large number of incorrect mycelium strains. We report here the isolation of a mycelium from a fruiting body of O. sinensis and we identify the isolate as O. sinensis' anamorph (also called Hirsutella sinensis) based on multi-locus sequence typing of several fungal genes (ITS, nrSSU, nrLSU, RPB1, RPB2, MCM7, ß-tubulin, TEF-1α, and ATP6). The main characteristics of the isolated mycelium, including its optimal growth at low temperature (16°C) and its biochemical composition, are similar to that of O. sinensis fruiting bodies, indicating that the mycelium strain characterized here may be used as a substitute for the rare and expensive O. sinensis fruiting bodies found in nature.