A novel knotted cotton-thread carrier:Its potential use in achieving the biomass renewal of Phanerochaete chrysosporium in an immobilized growth system

In order to achieve an innovative strategy to renew the biomass of Phanerochaete chrysosporium in an immobilized growth system which can maintain white-rot fungi biomass, a novel knotted cotton-thread carrier was designed and made. By using a high-speed stirring apparatus under the conditions of 1400 r/min stirring speed for 6 min, mycelia immobilized on the knotted cotton-thread carriers were exfoliated completely and homogenized to a proper size. Furthermore, the homogenized mycelia from the immobilized m...     

High production of β-glucosidase from a marine Aspergillus niger immobilized on towel gourd vegetable sponges

To produce β-glucosidase by consecutive batch fermentation, a marine Aspergillus niger was immobilized on a natural carrier, towel gourd vegetable sponges. The immobilized mycelia were 0.15 g/g carrier with the immobilized biomass percentage of over 95%. The immobilized mycelia possessed the long durability(22.5 days). The maximum production occurred 1.5 day earlier by the immobilized mycelia than by the free mycelia. β-Glucosidase production of five consecutive batches was over 110 U/m L. At high salinity,the activity and stability of β-glucosidase from the marine A. niger increased remarkable. Immobilizing the marine A. niger on the novel natural carrier achieved the efficient production of β-glucosidase.     

Production and characterization ofPhanerochaete chrysosporium lignin peroxidases for pulp bleaching

The production of lignin peroxidase fromPhanerochaete chrysosporium was studied using immobilized mycelia in nylon-web cubes in semicontinuous fermentation using glucose pulses or ammonium tartrate pulses. Consistent enzyme production was achieved when glucose pulses were used, leading to an average activity of 253 U/L. The crude enzyme was added to eucalyptus kraft pulp before conventional and ECF bleaching sequences. Optimization of the enzymatic pretreatment led to the following operational conditions: enzyme load of 2 U/g of pulp, hydrogen peroxide addition rate of 10 ppm/h, and reaction time of 60 min. Pulp final characteristics were dependent on the chemical treatment sequence that followed enzymatic pretreatment. The chief advantage of enzymatic pretreatment was pulp viscosity preservation, which was observed in most of the experiments carried out with seven different chemical treatment sequences     

SiO2 Xerogel: A Suitable Inert Support for Microbial Growth

A silica xerogel was prepared at pH 2 by the hydrolysis-condensation reactions of the sol-gel method. Silica xerogel was used as a support for the growth of two filamentous fungi: Aspergillus niger ATCC 9642 and Phanerochaete chrysosporium A594. In both cases, an apparent abundant mycelia growth (5.5 mg biomass/g dry xerogel and 4.7 mg biomass/g dry xerogel respectively) was observed. A phase of rapid consumption of glucose which lasted until 96 h of culture with sugar consumption rate of 0.075 mg sugar/g support h and 0.042 mg sugar/g support h respectively, was also observed. Scanning electron microscopy (SEM) showed a superficial colonisation of both strains even in the occasional imperfections and crevices of the xerogel. This novel application of sol-gel metallic oxide systems suggests the potential use of an inert and versatile support which could be valuable, for example for solid state fermentation fundamental studies.     

A technique for mycelial development of ectomycorrhizal fungi on agar media

A technique was established to study ectomycorrhizal fungi on agar media. Petri dishes, 60 mm in diameter, containing 10 mL of culture medium covered with a cellophane disk were used for easy collection of the mycelium after growth. For analysis of fungal biomass production, a sterilized cellophane sheet was placed on the medium’s surface. Inoculation was achieved by placing a mycelial block onto the center of the cellophane sheet and then incubating at 25°C in the dark. Colony radial growth was measured and biomass dry wt was determined. Fresh mycelia were homogenized with 10 mL of acetate buffer (pH 5.5) for enzyme analysis. A crude extract was obtained by adding all culture medium to 90 mL of distilled water and homogenizing in a Potter. Reducing sugars, enzyme concentration, and pH were determined. Three fungal strains, Suillus collinitus, Pisosithus arrhizus, and Hebeloma cylindrosporum, were grown in different culture media (potato dextrose agar or Pintro’s medium). Parameters measured over time included glucose concentration, phosphatase activity, biomass, and pH.     

Biosorption of mercury by carboxymethylcellulose and immobilized Phanerochaete chrysosporium

Phanerochaete chrysosporium basidiospores immobilized onto carboxymethylcellulose were used for the removal of mercury ions from aqueous solutions. The biosorption of Hg(II) ions onto carboxymethylcellulose and both immobilized live and heat-inactivated fungal mycelia of Phanerochaete chrysosporium was studied using aqueous solutions in the concentration range 30-700 mg l⁻¹. The biosorption of Hg(II) ions by the carboxymethylcellulose and both live and heat-inactivated immobilized preparations increased as the initial concentration of mercury ions increased in the medium. Maximum biosorption capacity for immobilized live and heat-inactivated fungal mycelia of Phanerochaete chrysosporium was found to be 83.10 and 102.15 mg Hg(II) g⁻¹, respectively, whereas the amount of Hg(II) ions adsorbed onto the plain carboxymethylcellulose beads was 39.42 mg g⁻¹. Biosorption equilibria were established in approximately 1 h and the correlation regression coefficients show that the adsorption process can be well defined by a Langmuir equation. Temperature changes between 15 and 45 °C did not affect the biosorption capacity. The effect of pH was also investigated and the maximum adsorption of Hg(II) ions onto the carboxymethylcellulose and both live and heat-inactivated immobilized fungal mycelia was observed at pH 6.0. The carboxymethylcellulose-fungus beads could be regenerated using 10 mM HCl, with up to 95% recovery. The biosorbents were used in three biosorption-desorption cycles and no significant loss in the biosorption capacity was observed.     

Lipase Production in Solid-State Fermentation Monitoring Biomass Growth of Aspergillus niger Using Digital Image Processing

The aim of this study was to monitor the biomass growth of Aspergillus niger in solid-state fermentation (SSF) for lipase production using digital image processing technique. The strain A. niger 11T53A14 was cultivated in SSF using wheat bran as support, which was enriched with 0.91% (m/v) of ammonium sulfate. The addition of several vegetable oils (castor, soybean, olive, corn, and palm oils) was investigated to enhance lipase production. The maximum lipase activity was obtained using 2% (m/m) castor oil. In these conditions, the growth was evaluated each 24 h for 5 days by the glycosamine content analysis and digital image processing. Lipase activity was also determined. The results indicated that the digital image process technique can be used to monitor biomass growth in a SSF process and to correlate biomass growth and enzyme activity. In addition, the immobilized esterification lipase activity was determined for the butyl oleate synthesis, with and without 50% v/v hexane, resulting in 650 and 120 U/g, respectively. The enzyme was also used for transesterification of soybean oil and ethanol with maximum yield of 2.4%, after 30 min of reaction.     

β-Galactosidase from <Emphasis Type="Italic">Penicillium canescens</Emphasis>. Properties and immobilization

β-galactosidase from Penicillium canescens was immobilized on chitosan, sepharose-4B, foamable polyurethane and some other carriers. The highest yield of immobilization (up to 98%) was obtained by using chitosan as a carrier. The optimum pH and temperature were not significantly altered by immobilization. High stability of immobilized β-galactosidase during storage was demonstrated. Efficient lactose saccharification (over 90%) in whey was achieved by using immobilized β-galactosidase.     

Effects of pH and Temperature on Recombinant Manganese Peroxidase Production and Stability

The enzyme manganese peroxidase (MnP) is produced by numerous white-rot fungi to overcome biomass recalcitrance caused by lignin. MnP acts directly on lignin and increases access of the woody structure to synergistic wood-degrading enzymes such as cellulases and xylanases. Recombinant MnP (rMnP) can be produced in the yeast Pichia pastoris αMnP1-1 in fed-batch fermentations. The effects of pH and temperature on recombinant manganese peroxidase (rMnP) production by P. pastoris αMnP1-1 were investigated in shake flask and fed-batch fermentations. The optimum pH and temperature for a standardized fed-batch fermentation process for rMnP production in P. pastoris αMnP1-1 were determined to be pH 6 and 30 °C, respectively. P. pastoris αMnP1-1 constitutively expresses the manganese peroxidase (mnp1) complementary DNA from Phanerochaete chrysosporium, and the rMnP has similar kinetic characteristics and pH activity and stability ranges as the wild-type MnP (wtMnP). Cultivation of P. chrysosporium mycelia in stationary flasks for production of heme peroxidases is commonly conducted at low pH (pH 4.2). However, shake flask and fed-batch fermentation experiments with P. pastoris αMnP1-1 demonstrated that rMnP production is highest at pH 6, with rMnP concentrations in the medium declining rapidly at pH less than 5.5, although cell growth rates were similar from pH 4–7. Investigations of the cause of low rMnP production at low pH were consistent with the hypothesis that intracellular proteases are released from dead and lysed yeast cells during the fermentation that are active against rMnP at pH less than 5.5.     

Immobilized cells in microbial nitrate reduction

The microorganismPseudomonas denitrificans was immobilized in alginate. These immobilized cells were capable of reducing 0.8 mg NO ₃ ^- /min/g wet weight of cells.     

Screening thermotolerant white-rot fungi for decolorization of wastewaters

To select a thermotolerant fungal strain for decolorization of wastewaters, ligninolytic enzyme production (lignin peroxidase, manganese peroxidase [MnP], and laccase), decolorization, and removal of total phenol and chemical oxygen demand (COD) were detected. Thirty-eight fungal strains were studied for enzyme production at 35 and 43°C on modified Kirk agar medium including 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and MnCl₂. Thirteen strains grew on manganese-containing agar and provided green color on ABTS-containing agar plates under culture at 43°C. Decolorization of wastewater from alcohol distillery (WAD) by these strains was compared under static culture at 43°C, and Pycnoporus coccineus FPF 97091303 showed the highest potential. Thereafter, immobilized mycelia were compared with free mycelia for WAD decolorization under culture conditions of 43°C and 100 rpm. The immobilized mycelia on polyurethane foam enhanced the ligninolytic enzyme production as well as total phenol and color removal. At about the same COD removal, MnP and laccase produced by immobilized mycelia were 2 and 19 times higher than by free mycelia; the simultaneous total phenol and color removal were 3.1 and 1.5 times higher than the latter. Moreover, decolorization of synthesis dye wastewater was carried out at 43°C and 100 rpm. More than 80% of 300 mg/L of reactive blue-5 was decolorized by the immobilized mycelia within 1 to 2 d for four cycles.     

Gibberellic acid production by free and immobilized cells in different culture systems

Gibberellic acid production was studied in different fermentation systems. Free and immobilized cells of Gibberella fujikuroi cultures in shakeflask, stirred and fixed-bed reactors were evaluated for the production of gibberellic acid (GA₃). Gibberellic acid production with free cells cultured in a stirred reactor reached 0.206 g/L and a yield of 0.078 g of GA₃/g biomass.     

Enhanced Production of High-Quality Biomass, δ-Aminolevulinic Acid,Bilipigments, and Antioxidant Capacity of a Food Alga <Emphasis Type="Italic">Nostochopsis lobatus</Emphasis>

The growing interest in natural food has raised the global demand for nutraceuticals. We studied enhanced production of biomass, delta-aminolevulinic acid (delta-ALA), bili pigments and antioxidant capacity of a food alga Nostochopsis lobatus in a full-factorial (three level) design with supplemental Zn, glutamine, and Zn + glutamine in batch culture. Production of biomass, pigments, and antioxidant capacity all were higher under immobilized cell cultures in comparison to free cell cultures. Maximum biomass (2,390 mg dry wt l(-1)), delta-ALA (2.715 microg mg(-1) dry wt h(-1)), phycocyanin (98.50 mg g(-1) dry wt), phycoerythrin (158.0 mg g(-1) dry wt), and antioxidant capacity (140.50 mumoles ascorbic acid equivalent capacity g(-1) fresh wt) were recorded when Zn and glutamine were supplemented together in the growth medium at pH 7.8. These effects were found to be significantly related to the activities of glutamine synthetase (GS(max): 490.2 nmoles mg protein(-1) min(-1)), glutamate synthase (GOGAT(max): 27.0 nmoles mg protein(-1) min(-1)), and glutamate dehydrogenase (GDH(max): 159.9 nmoles mg protein(-1) min(-1)). This study shows that N. lobatus could be a promising bioresource for the production of nutritionally rich biomass, delta-ALA, bili pigments, and antioxidants. Use of immobilized cells in batch culture supplemented with Zn and glutamine could be an effective approach for scaling up production for commercial use.     

Optimization of protease immobilization by covalent binding using glutaraldehyde

Immobilization of a protease, Flavourzyme, by covalent binding on various carriers was investigated. Lewatit R258-K, activated with glutaraldehyde, was selected among the tested carriers, because of the highest immobilized enzyme activity. The optimization of activation and immobilization conditions was performed to obtain high recovery yield. The activity recovery decreased with increasing carrier loading over an optimal value, indicating the inactivation of enzymes by their reaction with uncoupled aldehyde groups of carriers. The buffer concentrations for carrier activation and enzyme immobilization were optimally selected as 500 and 50 mM, respectively. With increasing enzyme loading, the immobilized enzyme activity increased, but activity recovery decreased. Immobilization with a highly concentrated enzyme solution was advantageous for both the immobilized enzyme activity and activity recovery. Consequently, the optimum enzyme and carrier loadings for the immobilization of Flavourzyme were determined as 1.8 mg enzyme/mL and 0.6 g resin/mL, respectively.     

Production of 2-O-α-glucopyranosyl l-ascorbic acid from ascorbic acid and β-cyclodextrin using immobilized cyclodextrin glycosyltransferase

Cyclodextrin glycosyltransferase (CGTase) isolated and purified from Paenibacillus sp. A11 was immobilized on various carriers by covalent linkage using bifunctional agent glutaraldehyde. Among tested carriers, alumina proved to be the best carrier for immobilization. The effects of several parameters on the activation of the support and on the immobilization of enzyme were optimized. The best preparation of immobilized CGTase retained 31.2% of its original activity. After immobilization, the enzymatic properties were investigated and compared with those of the free enzyme. The optimum pH of the immobilized CGTase was shifted from 6.0 to 7.0 whereas optimum temperature remained unaltered (60°C). Free and immobilized CGTase showed similar pH stability profile but the thermal stability of the immobilized CGTase was 20% higher. Kinetic data (K _M and V _max) for the free and immobilized enzymes were determined from the rate of β-CD formation and it was found that the immobilized form had higher K _M and lower V _max. The immobilized CGTase also exhibited higher stability when stored at both 4°C and 25°C for 2 months. The enzyme immobilized on alumina was further used in a batch production of 2-O-α-glucopyranosyl-l-ascorbic acid (AA-2G) from ascorbic acid and β-cyclodextrin. The yield of AA-2G was 2.92% and the immobilized CGTase retained its activity up to 74.4% of the initial catalytic activity after being used for 3 cycles. The immobilized CGTase would have a promising application in the production of various transglycosylated compounds and in the production of cyclodextrin by the hydrolysis of starch.     

Increased synthesis of ajmalicine and catharanthine by cell suspension cultures ofCatharanthus roseus in response to fungal culture-filtrates

The ammonium sulfate-precipitated fraction from mycelia and culture-filtrates and the crude, cell-free culture filtrates from the growth medium of the fungiChrysosporium palmorum, Eurotium rubrum, Micromucor isabellina, andPythium aphanidermatum when aseptically added to cell suspensions ofCantharanthus roseus caused a rapid and dramatic increase in indole alkaloid biosynthesis. Up to 400 μg/L ajmalicine and 600 μg/L catharanthine were detected in C.roseus cell suspension grown in the presence of theM. isabellina fungal culture filtrate for 3 d. Untreated cells produced only trace levels of ajmalicine and catharanthine per liter of cell suspension after 15 d of culture.     

A novel knotted cotton-thread carrier: its potential use in achieving the biomass renewal of Phanerochaete chrysosporium in an immobilized growth system

Science China Chemistry - In order to achieve an innovative strategy to renew the biomass of Phanerochaete chrysosporium in an immobilized growth system which can maintain white-rot fungi biomass,...     

Immobilization of glucose isomerase and its application in continuous production of high fructose syrup

Bilayer glucose isomerase was immobilized in porousp-trimethylamine-polystyrene (TMPS) beads, through a molecular deposition technique. Some of the factors that influence the activity of immobilized glucose isomerase were optimized, with the enzyme concentration of 308 IU/mL, enzyme:matrix ratio of 924 IU/g wet carrier, and hexamethylenebis(trimethylammonium iodine) concentration of 15 mg/mL, giving the maximum catalytic activity (2238 IU/g dry gel) of the immobilized bilayer glucose isomerase, retaining 68.5% of the initially added activity. The half-life of the immobilized bilayer glucose isomerase was approx 45 d at pH 8.5, 60°C, with 50% (w/v) glucose as substrate. The specific productivity of the immobilized bilayer glucose isomerase was 223 g dry D-glucose/g dry immobilized enzyme per day.     

Use of steam explosion liquor from sugar cane bagasse for lignin peroxidase production by Phanerochaete chrysosporium

The possibility of using two by-products of the sugar cane industry, molasses and bagasse steam explosion liquor (SEL), for lignin peroxidase (LiP) production by Phanerochaete chrysosporium was investigated. For comparison, the fungus was initially cultivated in synthetic media containing either glucose, sucrose, xylose, or xylan as sole carbon sources. The effect of veratryl alcohol (VA) was also investigated in relation to the enzyme activity levels. Results showed that sucrose was not metabolized by this fungus, which precluded the use of molasses as a carbon source. Glucose, xylose, and xylan promoted equivalent cell growth. Enzyme levels in the absence of VA were lower than 28 UI/L and in the presence of VA reached 109 IU/L with glucose and 85 IU/L with xylose or xylan. SEL was adequate for P. chrysosporium LiP production as LiP activity reached 90 IU/L. When VA was added to this medium, enzyme concentration increased to 155 IU/L.     

Study of operational variables in the submerged growth ofPleurotus ostreatus mushroom mycelium

Pleurotus ostreatus mushroom mycelium was cultivated in submerged culture in shake-flask experiments with acid extract from peat and yeast extract as nutrient sources. Different concentrations of water-diluted peat extract were tested in an attempt to overcome the effect of growth inhibitors apparently present in nondiluted peat extracts. The best results were obtained with a ratio of one part of peat extract diluted with one part of water. Several operating variables were studied to optimize the growth of mycelial biomass ofP. ostreatus. The best results produced approximately 5 g/L dry biomass with a yield of 60% and an efficiency of 33%. These results were obtained in 8 d at 5% (v/v) inoculum ratio, 28°C, pH of 5.0, and 150 rpm.