Biosorption of 2,4-dichlorophenol by immobilized white-rot fungus Phanerochaete chrysosporium from aqueous solutions

The fungus Phanerochaete chrysosporium was immobilized in several polymer matrices: Ca-alginate, Ca-alginate-polyvinyl alcohol (PVA) and pectin, and was then used as a biosorbent for removing 2,4-dichlorophenol (2,4-DCP) in wastewater. Immobilization of P. chrysosporium onto pectin was less efficient than that onto other matrices because of its poor mechanical strength and low adsorption efficiency. Ca-alginate immobilized fungal beads with biocompatibility exhibited good mechanical strength and adsorption efficiency over 60%. Among the different biomass dosages in Ca-alginate immobilized fungal beads, 1.25% (w/v) was the optimum. The adsorption data of 2,4-DCP on the blank Ca-alginate beads, free, and immobilized fungal biomass could be described by the Langmuir and Freundlich isotherms very well. Desorption operation was efficiently completed by using distilled water as eluant, and the desorption efficiency reached 82.16% at an optimum solid/liquid ratio of 14.3. The consecutive adsorption/desorption cycles studies employing the Ca-alginate immobilized fungal beads demonstrated that the immobilized fungal biomass could be reused in five cycles without significant loss of adsorption efficiency and adsorbent weight.     

Comparative studies of physiological and environmental effects on the production of cyclosporin A in suspended and immobilized cells of Tolypocladium inflatum

Process improvement of the production of cyclosporin A (Cy A), a powerful immunosuppressive fungal metabolite, has been undertaken by analyzing suspended and immobilized cell cultures in parallel. Conidiospores of the producer microorganism, Tolypocladium inflatum, were entrapped into porous celite particles. Easier germination of the entrapped spores and more active growth of the immobilized cells were manifested when compared with free cell cultures initiated with spores or with mycelial inocula. Significant differences in precursor flow between the immobilized and free cell systems were evident when the effects of L-valine (a constituent amino acid of the Cy A molecule) on Cy A biosynthesis were compared in the two systems. For the freely suspended cells, L-valine supplemented early in the fermentation served as a possible precursor or stimulator of Cy A biosynthesis. A significant increase in specific production and Cy A yield on carbon source was observed in this system relative to suspended cultures supplemented with L-valine during or after exponential growth. In contrast to the free cell cultures, the addition of L-valine during the initial stage of immobilized cell growth had a negative effect on Cy A production but resulted in somewhat increased cell growth. This suggests an incompatibility between primary and secondary metabolic networks involved in Cy A biosynthesis in the immobilized state upon external addition of the amino acid.     

Removal of glucose from egg prior to spray drying by fermentation with immobilized yeast cells

Summary Baker's yeast cells (40% w/v) were immobilized in 2% Ca-alginate and were used in a batch process for the removal of glucose from egg melange by fermentation. Immobilized cells (10 g) could completely remove the glucose from 135 ml melange in 60 min. The immobilized preparation was repeatedly used in 30 batches without decrease in the activity.     

Preparation and some properties of immobilized Penicillium funiculosum 258 dextranase

The production of dextranase was investigated in static cultures of Penicillium funiculosum 258. Maximal enzyme productivity was attained at pH 8.0, with 3.5% (w/v) dextran (MW, 260 000) as carbon source, NaNO₃ (1%, w/v) and yeast extract (0.2%, w/v) as nitrogen source, 0.4% (w/v) K₂HPO₄ and 0.06% (w/v) MgSO₄. It was possible to increase the productivity of dextranase to 41.8 units ml⁻¹ in the modified medium. The enzyme was immobilized on different carriers by different techniques of immobilization. The enzyme prepared by covalent binding on chitosan using glutaraldehyde had the highest activity, the immobilized enzyme retaining 63% of its original specific activity. Compared with the free dextranase, the immobilized enzyme exhibited: a higher pH optimum, a higher optimal reaction temperature and energy of activation, a higher Michaelis constant, improved thermal stability and higher values of deactivation rate constant. The immobilized enzyme retained about 80% of the initial catalytic activity even after being used for 12 cycles.     

Determination Method for l-Ascorbic Acid in Foods with Immobilized Ascorbate Oxidase

Pumpkin (Cucurbita moschata) ascorbate oxidase was entrapped within 6% (w/v) Ca-alginate gel beads, and then the beads were treated with 1% (w/v) glutaraldehyde for 20 hr at 4°C. The immobilized ascorbate oxidase was much more stable than the free form. Under the optimum conditions, the immobilized enzyme remained fully active for 3 months and after 50 assays. A linear relationship was found between immobilized ascorbate oxidase activity and l-ascorbic acid concentration in the range of 2 ~ 20 μg/ml. The immobilized preparation could be employed for the simple and rapid determination of l-ascorbic acid in foods.     

Preparation of lactose free milk by fermentation using immobilizedSaccharomyces fragilis

Summary Saccharomyces fragilis cells (40% w/v) were immobilized in 2% Ca-alginate and were used in a batch process for the removal of lactose from milk by fermentation. Immobilized cells (10 g) could completely desugarate 100 mL of milk in 3.5 h. The immobilized preparation was used repeatedly in 15 batches without decrease in the activity.     

A novel immobilised design for the production of the heterologous protein lysozyme by a genetically engineered Aspergillus niger strain

Abstract A novel immobilisation design for increasing the final concentration of the heterologous protein lysozyme by a genetically engineered fungus, Aspergillus niger B1, was developed. A central composition design was used to investigate different immobilised polymer types (alginate and pectate), polymer concentration [24% and 4% (w/v)], inoculum support ratios (1:2 and 1:4) and gel-inducing agent concentration [CaCl₂, 2% and 3.5% (w/v)]. Studies of the kinetics of production showed that optimum lysozyme productivity occurred after 10 days. Lysozyme production was significantly affected by polymer type, polymer concentration, and inoculum support ratio. Overall, immobilisation in Ca-pectate resulted in higher lysozyme production compared to that in Ca-alginate. Similar effects were observed when the polymer concentration was reduced. Regardless of polymer type and concentration, increasing the fungal inoculum level increased lysozyme production. A significantly higher lysozyme yield was achieved with Ca-pectate in comparison to Ca-alginate (approximately 20–23 mg l^–1 and 0.5–2 mg l^–1, respectively). The maximum lysozyme yield achieved was about 23 mg l^–1 by immobilisation in Ca-pectate 2% (w/v) with 33% (v/v) mycelium and 3.5% (w/v) gel-inducing agent (CaCl₂). Response surface methodology was used to investigate the effect of pH and water activity (a_w). The best medium pH was 4.5–5.0, and bead a_w for optimum lysozyme yield was 0.94, regardless of polymer type.     

Production of alcohol from sugar beet molasses without heat or filter sterilization

Among three esters of p-hydroxybenzoate, n-butyl p-hydroxybenzoate was selected as the best antimicrobial substance. Molasses medium sterilized by this ester was used as a substrate for ethanol production. n-Butyl p-hydroxybenzoate (0.15% w/v) completely inhibited the growth of free yeast cell inoculum, Ca-alginate immobilized yeast inoculum and bacterial contaminants. Immobilization of the yeast cell inoculum in Ca-alginate with castor oil (6% v/v) offered a yeast cell protection against the inhibitory effect of n-butyl p-hydroxybenzoate. The presence of castor oil in this immobilization system did not affect the metabolic activity of the yeast in beads compared to the cells immobilized without castor oil. The yeast cell beads in this system completely utilized up to 25% molasses sugar with an ethanol yield of 10.58%, equal to 83% of its theoretical value. The beads were stable and could be used successfully for seven cycles of batch fermentation. The optimum fermentation temperature using this system was 35°C. Received 21 January 1997/ Accepted in revised form 05 May 1997     

Citric acid production using immobilized conidia of Aspergillus niger TMB 2022

Conidia of Aspergillus niger TMB 2022 were immobilized in calcium alginate for the production of citric acid. A 1-mL conidia suspension containing ca. 2.32 x 10(8) conidia were entrapped into sodium alginate solution in order to prepare 3% Ca-alginate (w/v) gel bead. Immobilized conidia were inoculated into productive medium containing 14% sucrose, 0.25% (NH(4))(2)CO(3), 0.25% KH(2)PO(4), and 0.025% MgSO(4).7H(2)O with addition of 0.06 mg/L CuSO(4).5H(2)O, 0.25 mg/L ZnCl(2), 1.3 mg/L FeCl(3).6H(2)O, pH 3.8, and incubated at 35 degrees C for 13 days by surface culture to produce 61.53 g/L anhydrous citric acid. Under the same conditions with a batchwise culture, it was found that immobilized conidia could maintain a longer period for citric acid production (31 days): over 70 g/L anhydrous citric acid from runs No. 2-4, with the maximum yield for anhydrous citric acid reaching 77.02 g/L for run No. 2. In contrast, free conidia maintained a shorter acid-producing phase, ca. 17 days; the maximum yield for anhydrous citric acid was 71.07 g/L for run No. 2 but dropped quickly as the run number increased.     

Preparation and properties of an immobilized pectinlyase for the treatment of fruit juices

Pectinlyase, present in different commercial pectinases used in juice technology, was immobilized on alginate beads. The optimal conditions were: 0.17 g alginate ml(-1), 1.2% (w/v or v/v) enzyme concentration and acetic-HCl/glycine-HCl buffer at pH 3.6 or tris-HCl/imidazole buffer at pH 6.4. Maximum percentage of immobilization (10.6%) was obtained with Rapidase C80. Kinetic parameters of free and immobilized pectinlyase were also determined. The pH and temperature at which activity of soluble and immobilized enzyme was maximum were 7.2 and 55 degrees C. Thermal stability was not significantly altered by immobilization, especially at 40 degrees C, showing two periods of different stability. Free and immobilized preparation reduced the viscosity of highly esterified pectin from 1.09 to 0.70 and 0.72 mm(2) s(-1), respectively, after 30 min at 40 degrees C. Furthermore, the immobilized enzyme could be re-used through 4 cycles and the efficiency loss in viscosity reduction was found to be only 9.2%.     

Construction a hybrid biosorbent using Scenedesmus quadricauda and Ca-alginate for biosorption of Cu(II), Zn(II) and Ni(II): kinetics and equilibrium studies

The potential use of the immobilized fresh water algae (in Ca-alginate) of Scenedesmus quadricauda to remove Cu(II), Zn(II) and Ni(II) ions from aqueous solutions was evaluated using Ca-alginate beads as a control system. Ca-alginate beads containing immobilized algae were incubated for the uniform growth at 22 degrees C for 5d ays. Adsorption of Cu(II), Zn(II) and Ni(II) ions on the immobilized algae showed highest values at around pH 5.0. Adsorption of Cu(II), Zn(II) and Ni(II) ions on the immobilized algae increased as the initial concentration of metal ions increased in the medium. The maximum adsorption capacities of the immobilized algal biosorbents for Cu(II), Zn(II) and Ni(II) were 75.6, 55.2 and 30.4 mg/g (or 1.155, 0.933 and 0.465 mmol/g) biosorbent, respectively. When the heavy metal ions were in competition, the amounts of adsorbed metal ions were found to be 0.84 mol/g for Cu(II), 0.59 mol/g for Ni(II) and 0.08 mol/g for Zn(II), the immobilised algal biomass was significantly selective for Cu(II) ions. The adsorption-equilibrium was also represented with Langmuir, Freundlich and Dubinin-Radushkevich adsorption isotherms. The adsorption of Cu(II), Zn(II) and Ni(II) ions on the immobilized algae followed second-order kinetic.     

Production and characterization of a thermostable chitinase from a new alkalophilic Bacillus sp. BG-11

An alkalophilic, environmental micro-organism, Bacillus sp. BG-11, has been isolated and characterized. It produced 76 U ml-1 of chitinase in liquid batch fermentation after 72 h of incubation at 50 degrees C using chitin-enriched medium. The molecular weight of purified chitinase was estimated to be 41 kDa by SDS-PAGE. The pH and temperature optima of chitinase immobilized on chitosan and calcium alginate were 8.5 and 50 degrees C, respectively, which were same as that of free enzyme. The pH and thermostability of immobilized chitinase were enhanced significantly. The chitinase immobilized on chitosan was stable between pH 5.0 and 10.0, and the half-life of chitosan-immobilized enzyme at 70, 80 and 90 degrees C was 90, 70 and 60 min, respectively. The end-products formed during the enzyme-substrate reaction were identified by 13C-NMR, and N-acetyl-D-glucosamine was found to be the major end-product. GlcNAc (GlcNAc)2 and (GlcNAc)3 inhibited the chitinase activity by 32, 25 and 18%, respectively, at a concentration of 10 mmol l-1. The shelf-life of chitinase (retained 100% activity) at 4 degrees C was 8 weeks in the presence of either sodium azide (100 microgram ml-1), sodium metabisulphite (0.1% w/v) or KCl (15% w/v). The enzyme was resistant to the action of proteases and allosamidin.     

Conversion of quinate to 3-dehydroshikimate by Ca-alginate-immobilized membrane of Gluconobacter oxydans IFO 3244 and subsequent asymmetric reduction of 3-dehydroshikimate to shikimate by immobilized cytoplasmic NADP-shikimate dehydrogenase

The membrane fraction of Gluconobacter oxydans IFO 3244, involving membrane-bound quinoprotein quinate dehydrogenase and 3-dehydroquinate dehydratase, was immobilized into Ca-alginate beads. The Ca-alginate-immobilized bacterial membrane catalyzed a sequential reaction of quinate oxidation to 3-dehydroquinate and its spontaneous conversion to 3-dehydroshikimate under neutral pH. An almost 100% conversion rate from quinate to 3-dehydroshikimate was observed. NADP-Dependent cytoplasmic enzymes from the same organism, shikimate dehydrogenase and D-glucose dehydrogenase, were immobilized together with different carriers as an asymmetric reduction system forming shikimate from 3-dehydroshikimate. Blue Dextran 2000, Blue Dextran-Sepharose-4B, DEAE-Sephadex A-50, DEAE-cellulose, and hydroxyapatite were effective carriers of the two cytoplasmic enzymes, and the 3-dehydroshikimate initially added was converted to shikimate at 100% yield. The two cytoplasmic enzymes showed strong affinity to Blue Dextran 2000 and formed a soluble form of immobilized catalyst having the same catalytic efficiency as that of the free enzymes. This paper may be the first one on successful immobilization of NAD(P)-dependent dehydrogenases.     

Ethanol and acetic acid tolerance in free and immobilized cells of Saccharomyces cerevisiae and Acetobacter aceti

Saccharomyces cerevisiae and Acetobacter aceti cells were immobilized by entrapment in Ca-alginate or by adsorption on to preformed cellulose beads and were treated with 0-20% (v/v) ethanol and 0-10% (v/v) acetic acid. At 20% (v/v) ethanol, lethal for free yeast cells, 62-72% of the immobilized cells survived. In 10% (v/v) acetic acid, free and adsorbed Acetobacter aceti cells ceased to grow but 69% of entrapped cells survived. Cells released from the carrier showed an intermediate survival (20-60%).     

A new immobilized cell system with protection against toxic solvents

A new immobilized cell system providing protection against toxic solvents was investigated so that normal fermentations could be carried out in a medium containing toxic solvents. The system consists of immobilized growing cells in Ca-alginate gel beads to which vegetable oils, which are inexpensive absorbents of solvents, had been added. The ethanol fermentation of Saccharomyces cerevisiae ATCC 26603 was used as a model fermentation to study the protection afforded by the system against solvent toxicities. The fermentation was inhibited by solvents such as 2-octanol, benzene, toluene, and phenol. Ethanol production of one batch was not finished even after 35 h using immobilized growing yeast cells in conventional Ca-alginate gel beads in an ethanol production medium (5% glucose) containing 0.1% 2-octanol, which is used as a solvent for liquid-liquid extraction and is one of the most toxic solvents in our experiments. With the new immobilized growing cell system using vegetable oils, however, four repeated batch fermentations were completed in 35 h. Castor oil provided even more protection than soy bean, olive, and tung oils, and it was possible to complete six repeated batches in 35 h. The immobilized cell system with vegetable oils also provided protection against other toxic solvents such as benzene and toluene. A possible mechanism for the protective function of the new immobilized cell system is discussed.     

Acrylamide synthesis using agar entrapped cells of <Emphasis Type="Italic">Rhodococcus rhodochrous</Emphasis> PA-34 in a partitioned fed batch reactor

The nitrile hydratase (Nhase) induced cells of Rhodococcus rhodochrous PA-34 catalyzed the conversion of acrylonitrile to acrylamide. The cells of R. rhodochrous PA-34 immobilized in 2% (w/v) agar (1.76 mg dcw/ml agar matrix) exhibited maximum Nhase activity (8.25 U/mg dcw) for conversion of acrylonitrile to acrylamide at 10°C in the reaction mixture containing 0.1 M potassium phosphate buffer (pH 7.5), 8% (w/v) acrylonitrile and immobilized cells equivalent to 1.12 mg dcw (dry cell weight) per ml. In a partitioned fed batch reaction at 10°C, using 1.12 g dcw immobilized cells in a final volume of 1 l, a total of 372 g of acrylonitrile was completely hydrated to acrylamide (498 g) in 24 h. From the above reaction mixture 87% acrylamide (432 g) was recovered through crystallization at 4°C. By recycling the immobilized biocatalyst (six times), a total of 2,115 g acrylamide was produced.     

Immobilization of fructosyltransferase by chitosan and alginate for efficient production of fructooligosaccharides

The effective system of reusing mycelial fructosyltransferase (FTase) immobilized with two polymers, chitosan and alginate were evaluated for continuous production of fructooligosaccharides (FOS). The alginate beads were successfully developed by maintaining spherical conformation of using 0.3% (w/v) sodium alginate with 0.1% (w/v) of CaCl₂ solution for highest transfructosylating activity. The characteristics of free and immobilized FTase were investigated and results showed that optimum pH and temperature of FTase activity were altered by immobilized materials. A successive production of FOS by FTase entrapped alginate beads was observed at an average of 62.96% (w/w) up to 7 days without much losing its activity. The data revealed by HPLC analysis culminate 67.75% (w/w) of FOS formation by FTase entrapped alginate beads and 42.79% (w/w) by chitosan beads in 36 h of enzyme substrate reaction.     

Bacterial cellulose–alginate composite sponge as a yeast cell carrier for ethanol production

A bacterial cellulose–alginate (BCA) sponge, fabricated by a freeze-drying process, was successfully used as a yeast cell carrier for ethanol fermentation. The BCA sponge exhibited several advantageous properties, such as high porosity, appropriate pore size, strong hydrophilicity and high mechanical, chemical and thermal stabilities. BCA has an asymmetric structure, with a thin, dense outer layer covering an interior of interconnected macropores that are distributed throughout the sponge, which is effective for yeast immobilization. At 48 h of the fermentation, the maximum ethanol concentration produced by the immobilized culture (IC) in the BCA carrier was about 100 g/L, which was approximately 13% and 45% higher than that from the suspended culture (SC) and from IC in Ca-alginate matrix, respectively. Repeated-batch ethanol productions using IC in BCA carriers were also more stable than those using SC or IC in Ca-alginate matrix. The results of a 15 cycle repeated batch operation demonstrated that the system with IC in BCA exhibited superior long-term stability for ethanol fermentation with the average ethanol productivity at 1.9 g/L h and the immobilized yield at 86%. The improved ethanol fermentation performance was mainly due to the water uptake ability and properly interconnected pore structure, which help to overcome limiting mass transfer.     

Use of immobilized Candida cells on xylitol production from sugarcane bagasse

In this study we used the yeast Candida guilliermondii FTI 20037 immobilized by entrapment in Ca-alginate beads (2.5-3 mm diameter) for xylitol production from concentrated sugarcane bagasse hemicellulosic hydrolysate in a repeated batch system. The fermentation runs were carried out in 125- and 250-ml Erlenmeyer flasks placed in an orbital shaker at 30 degrees C and 200 rpm during 72 h, keeping constant the proportion between work volume and flask total volume. According to the results, cell viability was substantially high (98%) in all fermentative cycles. The values of parameters xylitol yield and volumetric productivity increased significantly with the reutilization of the immobilized biocatalysts. The highest values of xylitol final concentration (11.05 g/l), yield factor (0.47 g/g) and volumetric productivity (0.22 g/lh) were obtained in 250-ml Erlenmeyer flasks containing 80 ml of medium plus 20 ml of immobilized biocatalysts. The support used in this study (Ca-alginate) presented stability in the experimental conditions used. The results show that the use of immobilized cells is a promising approach for increasing the xylitol production rates.     

Sanitization of immobilized biocatalysts for the production of 6‐aminopenicillanic acid

Five different chemical reagents and γ‐rays were tested for the sanitization of immobilized biocatalysts with high penicillin G acylase (PGA) activity. The most effective chemical reagents were N‐cetyl‐N,N,N‐trimethylammonium bromide (CTAB) and 2‐isopropyl‐5‐methylphenol (thymol). The optimum concentration of CTAB for the treatment of the immobilized enzyme was 0.25% [w/v] and 1 h, for immobilized cells 0. [w/v] and 3 h. The optimum concentration of thymol for the immobilized enzyme was found to be 0.1% [w/v] and 1 h, for immobilized cells 0.27% [w/v] and 2 h. The optimum dose of γ‐rays for the sanitization of the immobilized enzyme was established as 3.2 kGy, for immobilized cells as 4.5 kGy.