Decolorization of a kraft mill effluent with fungal mycelium immobilized in calcium alginate gel

A white-rot fungus Coriolus versicolor was immobilized by entrapment in calcium alginate beads. Treatment of a kraft mill effluent with the immobilized fungus in the presence of sucrose resulted in 80% loss of color of the effluent within 3 days. The minimal concentration of sucrose required for the decolorization was 10 mM. Other carbon sources (xylose, glucose, glycerol, and ethanol) could also be used.     

Bioremediation of pulp and paper mill effluent by a novel fungal consortium isolated from polluted soil

Two basidiomycetous fungi (Merulius aureus syn. Phlebia sp. and an unidentified genus) and a deuteromycetous fungus (Fusarium sambucinum Fuckel MTCC 3788) were isolated from soils affected with effluents of a pulp and paper mill over several years. These isolates were immobilized on nylon mesh and the consortium was used for bioremediation of pulp and paper mill effluent in a continuously aerated bench-top bioreactor. The treatment resulted in the reduction of color, lignin and COD of the effluent in the order of 78.6%, 79.0% and 89.4% in 4 days. A major part of reductions in these parameters occurred within first 24h of the treatment, which was also characterized by a steep decline in the pH of the effluent. During this period, total dissolved solids, electrical conductivity and salinity of the effluent also registered marked decline. It is pertinent to note that this is the first report of bioremediation of pulp and paper mill effluent by an immobilized fungal consortium.     

Decolorization and detoxification of extraction-stage effluent from chlorine bleaching of kraft pulp by Rhizopus oryzae

Rhizopus oryzae, a zygomycete, was found to decolorize, dechlorinate, and detoxify bleach plant effluent at lower cosubstrate concentrations than the basidiomycetes previously investigated. With glucose at 1 g/liter, this fungus removed 92 to 95% of the color, 50% of the chemical oxygen demand, 72% of the adsorbable organic halide, and 37% of the extractable organic halide in 24 h at temperatures of 25 to 45 degrees C and a pH of 3 to 5. Even without added cosubstrate the fungus removed up to 78% of the color. Monomeric chlorinated aromatic compounds were removed almost completely, and toxicity to zebra fish was eliminated. The fungal mycelium could be immobilized in polyurethane foam and used repeatedly to treat batches of effluent. The residue after treatment was not further improved by exposure to fresh R. oryzae mycelium.     

疏水吸附固定化天冬氨酸酶及其性质的研究

本文论述了一种N-烷基琼脂珠衍生物的合成方法,研究了pH,离子强度、载体上疏水基团含量等因素对载体吸附天冬氨酸酶的影响以及固定化天冬酸酶的性质。结果表明邻甲苯胺基琼脂珠在pH5.5,0.1mol/L磷酸缓冲液(含有0.25mol/LKCL)中,每克湿载体可吸附15—25mg酶蛋白,酶活力回收达90％以上。固定化酶的性质有所改变,其热稳定性和操作稳定性明显增强。 固定化天冬氨酸酸柱可以用于连续化生产L-天冬氮酸,在pH8.0,1.0mol/L及丁烯二酸铵(含0.02mol/L MgCl_2),30℃条件下,以空间流速SV=3.5操作2个月,固定化酶活力仍保持79.5％。     

Sucrose hydrolysis by thermostable immobilized inulinases from aspergillus ficuum

The possibility of using thermostable inulinases from Aspergillus ficuum in place of invertase for sucrose hydrolysis was explored. The commercial inulinases preparation was immobilized onto porous glass beads by covalent coupling using activation by a silane reagent and glutaraldehyde before adding the enzyme. The immobilization steps were optimized resulting in a support with 5,440 IU/g of support (sucrose hydrolysis) that is 77% of the activity of the free enzyme. Enzymatic properties of the immobilized inulinases were similar to those of the free enzymes with optimum pH near pH 5.0. However, temperature where the activity was maximal was shifted of 10 degrees C due to better thermal stability after immobilization with similar activation energies. The curve of the effect of sucrose concentration on activity was bi-phasic. The first part, for sucrose concentrations lower than 0.3 M, followed Michaelis-Menten kinetics with apparent K(M) and Vm only slightly affected by immobilization. Substrate inhibition was observed at values from 0.3 to 2 M sucrose. Complete sucrose hydrolysis was obtained for batch reactors with 0.3 and 1 M sucrose solutions. In continuous packed-bed reactor 100% (for 0.3 M sucrose), 90% (1 M sucrose) or 80% sucrose conversion were observed at space velocities of 0.06-0.25 h(-1). The operational half-life of the immobilized inulinases at 50 degrees C with 2 M sucrose was 350 days.     

Studies on decolourization, degradation and detoxification of chlorinated lignin compounds in kraft bleaching effluents by Ceriporiopsis subvermispora

Ceriporiopsis subvermispora CZ-3, a wood degrading white rot fungus, was able to decolourize and degrade the first extraction stage effluent from kraft pulp bleaching at lower co-substrate concentration than the basidiomycetes previously investigated. With glucose at 1 g l⁻¹, this fungus removed up to 90% colour, 45% COD, 62% lignin, 32% AOX, and 36% EOX in 48 h at temperatures of 30–35°C and pH 4.0–4.5. In the absence of glucose, the fungus removed up to 62% of the colour. Significant reduction in chlorinated aromatic compounds was observed and toxicity to zebra fish was completely eliminated. The fungal mycelium could be immobilized in polyurethane foam and used repeatedly to treat batches of effluent. The molecular weight of chlorolignins was substantially reduced.     

Immobilization of β-Galactosidase by Polyacrylamide in the Presence of Protective Agents

β-Galactosidase from Aspergillus oryzae was immobilized in crosslinked polyacrylamide gel beads. The presence of the enzyme inhibitor, such as glucono-δ-lactone or galactono-γ-lactone, during polymerization procedure enhanced the residual enzymatic activity in the polymer beads, and activity yield attained up to 45%. Such enhancement effect was also observed when bovine serum albumin, dithiothreitol or glutathione was added during polymerization. Temperature and pH optima were not affected by the immobilization. The Michaelis constants for free and immobilized β-galactosidase were comparable. Lyophilized beads exhibited good stability without loss of enzymatic activity when stored at 4°C for 47 days.     

Immobilization of pycnoporus coccineus laccase on Eupergit C: Stabilization and treatment of olive oil mill wastewaters

The use of olive oil mill wastewaters (OMW) as an organic fertilizer is limited by their phytotoxic effect, due to the high concentration of phenolic compounds. As an alternative to physico-chemical methods for OMW detoxification, the laccase from Pycnoporus coccineus, a white-rot fungus with the ability to decrease the chemical oxygen demand (COD) and color of the industrial effluent, is being studied. In this work, the P. coccineus laccase was immobilized on two acrylic epoxy-activated resins, Eupergit C and Eupergit C 250L. The highest activity was obtained with the macroporous Eupergit C 250L, reaching 110 U g^?1 biocatalyst. A substantial stabilization effect against pH and temperature was obtained upon immobilization. The soluble enzyme maintained ≥80% of its initial activity after 24 h at pH 7.0–10.0, whereas the immobilized laccase kept the activity in the pH range 3.0–10.0. The free enzyme was quickly inactivated at temperatures >50°C, whereas the immobilized enzyme was very stable up to 70°C. Gel filtration profiles of the OMW treated with the immobilized enzyme (for 8 h at room temperature) showed both degradation and polymerization of the phenolic compounds.     

黄曲霉毒素解毒酶的固定化及其性质的研究

黄曲霉毒素是农作物常见的受污染的霉菌毒素,毒性大,稳定性高,是潜在的肝癌致癌物,对人的危害较大。该毒素的解毒与去毒一直是受到关注的问题。黄曲霉毒素解毒酶对黄曲霉毒素有特殊的去毒和降解作用,但是该酶的稳定性离解决实际问题尚有一段距离。报道了对黄曲霉毒素解毒酶的固定化,并对固定化处理后酶的稳定性、性质、催化活性、解毒活性进行了测定。结果表明,通过固定化操作酶的解毒活性被保留下来,酶的酸碱稳定性、热稳定性、放置稳定性等均得到显著的提高。     

Efficient biodegradation of cyanide and ferrocyanide by Na-alginate beads immobilized with fungal cells of Trichoderma koningii

Cyanide or metal cyanide contaminations have become serious environmental and food-health problems. A fungal mutant of Trichoderma koningii, TkA8, constructed by restriction enzyme-mediated integration, has been verified to have a high cyanide degradation ability in our previous study. In this study, the mutant cells were entrapped in sodium-alginate (Na-alginate) immobilization beads to degrade cyanide and ferrocyanide in a liquid mineral medium. The results showed that the fungus in immobilization beads consisting of 3% Na-alginate and 3% CaCl2 could degrade cyanide more efficiently than a nonimmobilized fungal culture. For maximum degradation efficiency, the optimal ratio of Na-alginate and wet fungal biomass was 20:1 (m/m) and the initial pH was 6.5. In comparison, cell immobilization took at least 3 and 8 days earlier, respectively, to completely degrade cyanide and ferrocyanide. In addition, we showed that the immobilized beads could be easily recovered from the medium and reused for up to 5 batches without significant losses of fungal remediation abilities. The results of this study provide a promising alternative method for the large-scale remediation of soil or water systems from cyanide contamination.     

Immobilization of a proteinase from the extremely thermophilic organism Thermus Rt41A

An extracellular proteinase from Thermus strain Rt41A was immobilized to controlled pore glass (CPG) beads. The properties of the free and CPG-immobilized enzymes were compared using both a large (azocasein) and a small (peptidase) substrate. The specific activity of the immobilized proteinase was 5284 azoU/mg with azocasein and 144 sucU/mg for SucAAPFpNA. The percentage recovery of enzyme activity was unaffected by pore size when it was immobilized at a fixed level of activity/g of beads, whereas it increased with increasing pore size when added at a fixed level/m(2) of support. Saturation of the CPG beads was observed at 540 azoU/m(2) of 105-nm beads. Lower levels (50 azoU/m(2) of 50-nm beads) were used in characterization experiments. The pH optimum of the immobilized Rt41A proteinase was 8.0 for azocasein and 9.5 for SucAAPFpNA, compared with the free proteinase which was 10.5 for both substrates. The immobilized enzyme retained 65% of its maximum activity against azocasein at pH 12, whereas the free proteinase retained less than 10% under the same conditions. Stability at 80 degrees C increased on immobilization at all pH values between 5 and 11, the greatest increase in half-life being approximately 12-fold at pH 7.0. Temperature-activity profiles for both the free and immobilized enzymes were similar for both substrates. The stability of the immobilized proteinase, however, was higher than that of the free enzyme in the absence and presence of CaCl(2). Overall, the results show that low levels of calcium (10 muM) protect against thermal denaturation, but that high calcium or immobilization are required to protect against autolysis. (c) 1994 John Wiley & Sons, Inc.     

Preparation and activity of carbonic anhydrase immobilized on porous silica beads and graphite rods

Techniques for the immobilization of bovine carbonic anhydrase (BCA) on porous silica beads and graphite are presented. Surface coverage on porous silica beads was found to be 1.5 x 10(-5) mmol BCA/m(2), and on graphite it was 1.7 x 10(-3) mmol BCA/m(2) nominal surface area. Greater than 97% (silica support) and 85% (graphite support) enzyme activity was maintained upon storage of the immobilized enzyme for 50 days in pH 8 buffer at 4 degrees C. After 500 days storage, the porous silica bead immobilized enzyme exhibited over 70% activity. Operational stability of the enzyme on silica at 23 degrees C and pH 8 was found to be 50% after 30 days. Catalytic activity expressed as an apparent second-order rate constant K'(Enz) for the hydrolysis of p-nitrophenyl acetate (p-NPA) catalyzed by BCA immobilized on silica beads and graphite at pH 8 and 25 degrees C is 2.6 x 10(2) and 5.6 x 10(2) M(-1)s(-1) respectively. The corresponding K(ENZ) value for the free enzyme is 9.1 x 10(2) M(-1)s(-1). Activity of the immobilized enzyme was found to vary with pH in such a manner that the active site pK, on the porous silica bead support is 6.75, and on graphite it is 7.41. Possible reasons for a microenvironmental influence on carbonic anhydrase pK(a), are discussed. Comparison with literature data shows that the enzyme surface coverage on silica beads reported here is superior to previously reported data on silica beads and polyacrylamide gels and is comparable to an organic matrix support. Shifts in BCA-active site pK(a) values with support material, a lack of pH dependent activity studies in the literature, and differing criteria for reporting enzyme activity complicate literature comparisons of activity; however, immobilized BCA reported here generally exhibits comparable or greater activity than previous reports for immobilized BCA.     

Solubilization of fish proteins using immobilized microbial cells

Cells of Bacillus megaterium, Aeromonas hydrophila, and Pseudomonas marinoglutinosa were immobilized in calcium alginate. The immobilized cells secreted protease when held in fish meat suspension in water. The enzyme synthesis by the entrapped cells was supported by small amounts of soluble nutrients present in the meat. The secreted protease solubilized the fish meat, solubilization being optimum at pH range of 7.5 to 9.5 and at 50 degrees C. Under these conditions immobilized B. megaterium was most efficient giving 30% solubilization of the meat, followed by A. hydrophila (18%), while immobilized P. marinoglutinosa was less effective. The optimum ratio of fish meat to beads was about 4:3 for B. megaterium and A. hydrophila. The beads had a storage life of 30 days at 4 degrees C. The results suggested potential for use of immobilized microbial cells having extracellular protease activity to enhance solubility of waste proteins. A prototype reactor with beads holding assembly was fabricated which could recover the beads from the meat slurry after the treatment.     

Transformation of 2,4,6-trichlorophenol by free and immobilized fungal laccase

Laccase from the white rot fungus Coriolus versicolor was immobilized on Celite R-637 by covalent binding with glutaraldehyde. After a sharp primary decline in activity (up to 50%), the retained enzyme activity was stable over a storage period of 33 days at 4 degrees C. A comparative study of soluble and immobilized laccases revealed the increased resistance of immobilized enzyme to the unfavourable effects of alkaline pH, high temperature and the action of inhibitors. A combination of these properties of immobilized laccase resulted in the ability to oxidize 2,4,6-trichlorophenol (2,4,6-TCP) at 50 degrees C at pH 7.0. The reactions of soluble and immobilized laccase with 2,4,6-TCP were examined in the presence and absence of redox mediators. 3,5-Dichlorocatechol, 2,6-dichloro-1,4-benzoquinone and 2,6-dichloro-1,4-hydroquinone were found to be the primary products of 2,4,6-TCP oxidation by laccase; oligo- and polymeric compounds were also found.     

Peroxidase-catalyzed color removal from bleach plant effluent

Effluent from the caustic extraction stage of a bleach plant is highly colored due to the presence of dissolved products from lignin chlorination and oxidation. Color removal from the effluent by hydrogen peroxide at neutral pH was catalyzed by addition of horseradish peroxidase. The catalysis with peroxidase (20 mg/L) was observed over a wide range of peroxide concentrations (0.1mM-500mM), but the largest effect was between 1mM and 100mM. The pH optimum for catalysis was around 5.0, while the basal rate of noncatalyzed peroxide color removal simply increased with pH within the range tested (3-10). Peroxidase catalysis at pH 7.6 reached a maximum at 40 degrees C in 4 h assays with 10mM peroxide, and disappeared above 60 degrees C. Compared with mycelial color removal by Coriolus versicolor, the rate of color removal by peroxide plus peroxidase was initially faster (first 4 h), but the extent of color removal after 48 h was higher with the fungal treatment. Further addition of peroxidase to the enzyme-treated effluent did not produce additional catalysis. Thus, the peroxide/peroxidase system did not fully represent the metabolic route used by the fungus.     

Improved stability and catalytic activity of graphene oxide/chitosan hybrid beads loaded with porcine liver esterase

Graphene oxide/chitosan and reduced graphene oxide/chitosan (GO/CS and RGO/CS) beads were prepared by precipitation with NaOH. Porcine liver esterase was immobilized on these beads to give GO/CS/E and RGO/CS/E beads. The optimum conditions for the maximum activity of RGO/CS/E beads were pH 8 and 50°C. The stability of the enzyme immobilized on GO/CS/E and RGO/CS/E was high in the pH range of 5–8. The GO/CS/E beads showed superior stability compared to that of the free enzyme and CS/E beads between 20 and 50°C. Kinetic analysis showed that GO/CS/E was a better catalyst than the RGO/CS/E beads with a lower K_m value of 0.9?mM. The hybrid beads also retained more than 95% activity after 10 consecutive cycles. The GO/CS/E and RGO/CS/E beads retained 84% and 87% activity after 40 days at 4°C. The GO/CS/E beads were used for the successful hydrolysis of methyl 4-hydroxy benzoate.     

固定化重组β-葡萄糖苷酶转化大豆异黄酮的研究

以壳聚糖微球为载体,采用交联-吸附法固定重组β -葡萄糖苷酶.研究考察了该酶的固定化条件及固定化酶转化大豆异黄酮类底物黄豆黄苷的最适反应体系和系统稳定性.结果显示该固定化酶能够有效转化大豆异黄酮的三种糖苷,黄豆黄苷最适转化条件为pH 6.4,45℃.在pH6.4的缓冲液中4℃贮存25 d后,酶活力仍保持85％以上.固定化重组酶在重复使用10批次的情况下,底物转化率仍能保持在85％左右.     

Enhanced β-lactam antibiotic production by coimmobilization of fungus and alga

Summary It was determined thatCephalosporium acremonium, an oxygenconsuming fungus, had a symbiotic relationship withChlorella pyrenoidosa, an oxygen-generating alga. The fungus and the alga were coimmobilized by entrapment in calcium alginate beads. The production rate of -lactam antibiotics was evaluated at various ratios of the fungus and the alga cells in the free and immobilized states. When the ratio of fungus to alga was one to eight in free mixed culture, the production rate of -lactam antibiotics was 240% of the rate in the presence of fungus alone in the free state. In coimmobilized cell system, the increased amount was 370% in comparison with immobilized fungus alone.     

A new alternative process for Kraft E1 effluent treatment

Lentinus edodes (UEC-2019 strain) was selected after screening 51 ligninolytic strains of fungi for their ability to decolorize phenolic industrial effluent with high content of lignin peroxidases, Mn-peroxidases and beta-glucosidases. This strain removed 73 % of color in theEucalyptus Kraft E1 effluent in 5 days without any additional carbon sources. A 13% mycelial adsorption was found. Correlation between mass loss, COD, TOC and decolorization was observed. When an effluent pre-irradiated (10 min) in the presence of ZnO was treated withL. edodes, a marked enhancement of the decolorization at 48 h was obtained.L. edodes is an active fungus in this pre-treatment and biobleaching process. The combined photo-biological decolorization procedure appears to be an efficient decontamination method with great potential in industrial effluent treatment.Abbreviation COD Chemical oxygen demand - TOC Total organic carbon     

Decoloration of a Carpet Dye Effluent Using Trametes Versicolor

Although a non-sterile, undiluted carpet dye effluent (containing two anthraquinone dyes) did not support growth of Trametes versicolor, the pre-grown fungus removed 95% of its color in shake-flasks after 10 h of incubation. After decoloration, the COD of the cell-free supernatant increased and the toxicity was unchanged as determined by the Microtox assay using Vibrio fischeri. Decoloration rates decreased when either glucose alone or Mn2+ and glucose were added. T. versicolor, immobilized on jute twine in a rotating biological contacting reactor, also decolorized four successive batches of the effluent. There was no decoloration in any of the uninoculated, non-sterile controls.