Immobilization of laccase onto spacer-arm attached non-porous poly(GMA/EGDMA) beads: application for textile dye degradation

Non-porous poly(glycidyl methacrylate/ethyleneglycol dimetacrylate) (poly(GMA/EGDMA)) beads were prepared by suspension polymerization. The enzyme (i.e. laccase) was covalently immobilized onto plain and spacer-arm attached poly(GMA/EGDMA) beads. The amount of immobilized enzyme on the plain and spacer-arm attached beads was determined as 5.6 and 4.9 mg/g, respectively. The maximum activity (V(max)) and Michaelis constant (K(m)) of laccase immobilized on the spacer-arm attached beads, were found to be 77.6 U/min and 0.47 mM, respectively. Finally, the immobilized laccase was operated in a batch system, and textile dye Reactive Red 120 was successfully decolorized in the enzyme reactor.     

Preparation and characterization of epoxy-functionalized magnetic chitosan beads: laccase immobilized for degradation of reactive dyes

Cross-linked magnetic chitosan beads were prepared by phase-inversion technique in the presence of epichlorohydrin under alkaline condition, and used for covalent immobilization of laccase. The activity of the immobilized laccase on the magnetic chitosan was about 260 U (g/dry beads) with an enzyme loading of about 16.33 ± 0.39 mg [(g/dry beads) mg/g]. Kinetic parameters, V _max and K _m values were determined as 21.7 U/mg protein and 9.4 μM for free enzyme, and 15.6 U/mg protein and 19.7 μM for the immobilized laccase, respectively. The operational and thermal stabilities of the immobilized laccase were improved compared to free counterpart. The immobilized laccase was operated in a batch reactor for the decolorization of reactive dyes from aqueous solution. The laccase immobilized on magnetic chitosan beads was very effective for removal of textile dyes from aqueous solution which creates an important environmental problem in the discharged textile dying solutions.     

Immobilized laccase for decolourization of Reactive Black 5 dyeing effluent

Reactive Black 5 industrial dyeing effluent was decolourized by free and immobilized laccase. The stability of the enzyme (194 h free and 79 h immobilized) depended on the dyeing liquor composition and the chemical structure of the dye. In the decolourization experiments with immobilized laccase, two phenomenons were observed – decolourization due to adsorption on the support (79%) and dye degradation due to the enzyme action (4%). Dyeing in the enzymatically recycled effluent provided consistency of the colour with both bright and dark dyes.     

Immobilization of laccase from Streptomyces psammoticus and its application in phenol removal using packed bed reactor

Laccase was produced from Streptomyces psammoticus under solid-state fermentation. The enzyme was partially purified by ammonium sulphate precipitation and was immobilized in alginate beads by entrapment method. Calcium alginate beads retained 42.5% laccase activity, while copper alginate beads proved a better support for laccase immobilization by retaining 61% of the activity. Phenol and colour removal from a phenol model solution was carried out using immobilized laccase. Batch experiments were performed using packed bed bioreactor, containing immobilized beads. Reusability of the immobilized matrix was studied for up to 8 successive runs, each run with duration of 6 h. The system removed 72% of the colour and 69.9% of total phenolics from the phenol model solution after the initial run. The immobilized system maintained 50% of its efficiency after eight successive runs. The degradation of phenolic compounds by immobilized laccase was evaluated and confirmed by Thin layer chromatography and nuclear magnetic resonance spectroscopy.     

Dye decolorization by <Emphasis Type="Italic">Trametes hirsuta</Emphasis> immobilized into alginate beads

Summary The present paper studies the production of laccase by Trametes hirsuta immobilized into alginate beads in an airlift bioreactor. In order to enhance laccase production fresh ammonium chloride was added, which led to the production, of high laccase activities (around 1000 U l⁻¹). The bioreactor operated for 40 days without operational problems and the bioparticles maintained their shape throughout fermentation. Dye decolorization was performed at bioreactor scale operating in the batch mode. High decolorization percentages were obtained in a short time (96% for indigo carmine and 69% for phenol red in 24 h), indicating the suitability of this process for application to synthetic dye decolorization. On the other hand, in vitro decolorization of several industrial azo dyes by crude laccase produced in the above bioreactor was also performed. It was found that some of the dyes needed the addition of 1-hydroxybenzotriazole for their decolorization.     

Affinity separation of immunoglobulin G subclasses on dye attached poly(hydroxypropyl methacrylate) beads

Poly(hydroxypropyl methacrylate) [poly(HPMA)] gel beads with an average size of 150–200 μm were prepared by suspension polymerization of hydroxypropyl methacrylate (HPMA). The poly(HPMA) gel beads were characterized by swelling studies, surface area measurements, scanning electron microscopy (SEM) and elemental analysis. Poly(HPMA) gel beads had a specific surface area of 88.6 m²/g. The dye Reactive Green HE 4BD was chemically attached to yield dye-poly(HPMA) gel beads at an average concentration of 44.3 μmol dye/g bead with a swelling ratio of 75%. These dye attached gel beads were used in the separation of immunoglobulin-G (IgG) through adsorption–elution studies. The non-specific adsorption of IgG on the poly(HPMA) gel beads was 0.5 mg/g. The attachment of Reactive Green HE 4BD significantly increased the adsorption of IgG up to 71 mg/g. The Langmuir adsorption model was found to be applicable in interpretation of data pertaining to the adsorption studies of IgG with Reactive Green HE 4BD attached to the poly(HPMA) gel beads. The adsorption of IgG was found to be optimal at pH 7.0. The adsorption of IgG was observed to decrease by about 76% as the NaCl concentration was increased from 0.001 to 0.1 M. The IgG adsorption capacity of the dye attached poly(HPMA) gel beads was determined for a commercially available IgG solution to be 4.2 mg/g for IgG₁, 64.5 mg/g for IgG₂, 7.1 mg/g for IgG₃ and 10.8 mg/g for IgG₄. The Reactive Green HE 4BD attached poly(HPMA) gel beads have a significant adsorption capacity for IgG₂. The quantity of adsorbed IgG₂ is three times higher than the quantity of the other subclasses, IgG₁, IgG₃ and IgG₄. A similar adsorption behaviour was observed when the albumin free human plasma was used. The quantity of adsorbed IgG₂ is higher than the quantity of the other subclasses, IgG₁, IgG₃ and IgG₄. Adsorption capacities for albumin free human plasma were obtained as 6.4 mg/g for IgG₁, 67.8 mg/g for IgG₂, 5.2 mg/g for IgG₃ and 8.6 mg/g for IgG₄. Significant amount of the adsorbed IgG (up to 95%) was eluted in 1 h in the elution medium containing 2.0 M NaCl. Repeated adsorption/elution processes showed that these dye attached gel beads are suitable for IgG adsorption.     

Immobilized laccase of Cerrena unicolor for elimination of endocrine disruptor micropollutants

The white-rot fungus Cerrena unicolor C-139 produced 450?000 U l⁻¹ of laccase when cultivated in submerged (50 ml) fermentation of wheat bran. Laccase (benzenediol: oxygen oxidoreductase, EC 1.10.3.2.), from C. unicolor C-139 was immobilized covalently on control porosity carrier silica beads. The activity of the immobilized laccase was approximately 15.8 units per gram of silica beads. The pH optimum was between 2.5 and 3.0 for free and immobilized laccase. The immobilization of enzyme appeared to be the main factor for retention of laccase activity at high temperature of 80 °C. The apparent K_m value (100 μmol) of immobilized laccase from C. unicolor C-139 was 6.7 times higher than free laccase (15 μmol) using 2,2-azino-bis-[3-ethylthiazoline-6-sulfonate] (ABTS) as the substrate. Immobilized laccase was able to eliminate 80 % of Bisphenol A, 40 % of Nonylphenol, and 60 % of Triclosan from solutions containing 50 μmol of each micropollutant separately. The experiments were run three times consecutively with the same immobilized laccase without loss of enzyme activity.     

Dye decolorisation by laccase entrapped in copper alginate

A novel immobilisation system was developed for dye decolorisation using laccase produced by Ganoderma sp. KU-Alk4. The enzyme showed high efficiency in dye decolorisation when entrapped in Cu–Al and Cu-alginate beads. The former gave the highest activity but the enzyme activity survived longer in the latter. An experimental design of two 3 × 3 Latin Square experiments was applied to evaluate the effects of three different alginate compositions (low, intermediate and high mannuronate), concentration of alginate, (1.5, 3.0 and 4.5% w/v) and concentration of cross-linking agent, CuSO₄ (0.075, 0.15 and 0.225 M) on the decolorisation of indigo carmine dye and residual laccase activity in beads. The most significant factor for residual activity was the concentration of the cross-linking agent (P < 0.05) followed by alginate composition (P < 0.1). Increasing the alginate concentration resulted in only small increase in the dye decolorisation. However, higher laccase activity remained in 3.0% w/v alginate beads. Maximal dye decolorisation was achieved when 3.6% w/v low mannuronate alginate and 0.15 M CuSO₄ was used. Optimal conditions were confirmed in an extended experimental run. Results are presented from 9 successive batch runs over 12 days, reaching 96% removal of the dye (216 mg/l).     

Bio-remediation of colored industrial wastewaters by the white-rot fungi Phanerochaete chrysosporium and Pleurotus ostreatus and their enzymes

The effect of Phanerochaete chrysosporium and Pleurotus ostreatus whole cells and their ligninolytic enzymes on models of colored industrial wastewaters was evaluated. Models of acid, direct and reactive dye wastewaters from textile industry have been defined on the basis of discharged amounts, economic relevance and representativeness of chemical structures of the contained dyes. Phanerochaete chrysosporium provided an effective decolourization of direct dye wastewater model, reaching about 45% decolourization in only 1 day of treatment, and about 90% decolourization within 7 days, whilst P. ostreatus was able to decolorize and detoxify acid dye wastewater model providing 40% decolourization in only 1 day, and 60% in 7 days. P. ostreatus growth conditions that induce laccase production (up to 130,000 U/l) were identified, and extra-cellular enzyme mixtures, with known laccase isoenzyme composition, were produced and used in wastewater models decolourization. The mixtures decolorized and detoxified the acid dye wastewater model, suggesting laccases as the main agents of wastewater decolourization by P. ostreatus. A laccase mixture was immobilized by entrapment in Cu-alginate beads, and the immobilized enzymes were shown to be effective in batch decolourization, even after 15 stepwise additions of dye for a total exposure of about 1 month.     

High ethanol productivities using small Ca-alginate beads of immobilized cells of Zymomonas mobilis

Summary Zymomonas mobilis cells were immobilized into small 1 mm diameter beads of Ca-alginate in order to minimize mass transfer limitations and maximize immobilized cell activity. A combination of small bead size with a high cell concentration of 58 g dry wt. cell per lit. bead volume resulted in high ethanol productivities using a newly designed packed bed bioreactor system. Steady-state dilution rates ranging from 0.4 h^-1 to 3.9 h^-1 were run resulting in a maximum productivity of 102 g ethanol/l/h for an inlet substrate concentration of 100 g glu/l and 87% conversion. The bioreactor was run continuously at a fixed dilution rate for 384 h and short intermittent treatment of the beads with CaCl₂ temporarily increased ethanol productivity to a maximum of 116 g ethanol/l/h.     

Biosorption of reactive dyes by the mycelium pellets of a new isolate of Penicillium oxalicum

Three reactive dyes were rapidly adsorbed by the mycelium pellets of Penicillium oxalicum. Dye removal of Reactive Blue 19 was up to 60% in 10 min and 91% in 80 min. Dye adsorption isotherms fitted Langmuir model well and the maximum adsorption capacities at 20 °C were calculated to be 160 mg g^–1 for Reactive Blue 19, 122 mg g^–1 for Reactive Red 241 and 137 mg g^–1 for Reactive Yellow 145, respectively. The pellets exhibited a high dye adsorption capacity (80–180 mg g^–1) for all of the 3 dyes over a wide pH range (pH 2–10), and the maximum adsorption was obtained at pH 2. The adsorption capacity was mildly increased by increasing salinity.     

Decolorization of the anthraquinone dye Cibacron Blue 3G-A with immobilized <Emphasis Type="Italic">Coprinus cinereus</Emphasis> in fluidized bed bioreactor

Coprinus cinereus, which was able to decolorize the anthraquinone dye Cibacron Blue 3G-A (CB) enzymatically, was used as a biocatalyst for the decolorization of synthetic solutions containing this reactive dye. Coprinus cinereus was immobilized in both calcium alginate and polyacrylamide gels, and was used for the decolorization of CB from synthetic water by using a fluidized bed bioreactor. The highest specific decolorization rate was obtained when Coprinus cinereus was entrapped in calcium alginate beads, and was of about 3.84 mg g⁻¹ h⁻¹ with a 50% conversion time (t _1/2) of about 2.60 h. Moreover, immobilized fungal biomass in calcium alginate continuously decolorized CB even after 7 repeated experiments without significant loss of activity, while polyacrylamide-immobilized fungal biomass retained only 67% of its original activity. The effects of some physicochemical parameters such as temperature, pH and dye concentration on decolorization performance of isolated fungal strain were also investigated.     

Enhanced production of laccase activity by <Emphasis Type="Italic">Trametes versicolor </Emphasis>immobilized into alginate beads by the addition of different inducers

In the present paper the effect of adding veratryl alcohol and copper sulphate on laccase activity production by Trametes versicolor immobilized into alginate beads has been investigated. Employing copper sulphate as laccase inducer or supplementing the culture medium with veratryl alcohol, led to maximum values of laccase activity. However, the highest laccase activity (around 4,000 U l⁻¹) was obtained in cultures simultaneously supplemented with copper sulphate (3 mM) and veratryl alcohol (20 mM). These values implied a considerable enhancement in relation to␣control cultures without any inducer (around 200 U l⁻¹). The production of laccase by immobilized T. versicolor in a 2-l airlift bioreactor with the optimized inducer has been evaluated. Laccase activities around 1,500 U l⁻¹ were attained. The bioreactor operated for 44 days without operational problems and the bioparticles (fungus grows in alginate beads) maintained their shape throughout the fermentation. Moreover, the extracellular liquid obtained was studied in terms of pH and temperature activity and stability. On the other hand, anthracene was added in two-repeated batches in order to determine the efficiency of this process to degrade pollutants. Near complete degradation was reached in both batches. Moreover, in vitro degradation of several polycyclic aromatic hydrocarbons by crude laccase was also performed.     

De-colourization of textile effluent using immobilized Geotrichum candidum: an insight into mycoremediation

Textile effluent is generally complicated to manage because of its extremely noxious and recalcitrant coloured compositions. Mycoremediation is an extensively used strategy for the competent degradation of hazardous pollutants present in textile effluent. Fungus could be immobilized in synthetic or natural matrices. The current study shows the decolourization of the textile effluent by 85·5 and 98·5% within 6 h using suspended and immobilized fungus, Geotrichum candidum with optimized parameters like inoculum size (5%), pH (4·5), and temperature (30°C). To maintain a high biomass of fungal population and enhance the retention of fungal strain in the contaminated sites, the fungi need to be immobilized. Hence, the fungus was immobilized naturally onto the selected inert support that is, coconut fibres by the means of adsorption, where they grew as active films on the fibres after being grown in the culture broth. The optimized process parameters of inoculum size, fibre quantity and agitation speed for immobilized G. candidum were 5%, 2·2 g l⁻¹ of effluent and 100 rev min⁻¹ respectively. High level of laccase (22 and 25 U l⁻¹ in suspended and immobilized fungal cells treatment respectively) was observed during the process of decolourization and it was found that decolourization was directly proportional to the laccase activity. The UV–vis, FTIR, ¹H NMR and GC-MS analyses of treated textile industrial wastewater revealed the degradation of toxic pollutants in the textile effluent and formation of lower molecular weight intermediates. The study revealed a higher efficacy of immobilized G. candidum in comparison to suspended fungal culture, employing ligninolytic enzyme laccase, which catalyzes the degradation/transformation of aromatic dyes in the textile effluent thus decolourizing it.     

Transformation of arachidonic acid to 19-hydroxy- and 20-hydroxy-eicosatetraenoic acids using Candida bombicola

Candida bombicola (ATCC 22214) and C. apicola (ATCC 96134), grown on glucose (100 g l^–1) and arachidonic acid (5Z, 8Z, 11Z, 14Z-eicosatetraenoic acid; AA), 1.25 g l^–1, synthesized sophorolipid up to 0.93 g l^–1. Acid hydrolysis of sophorolipid yielded 19-hydroxy-5Z, 8Z, 11Z, 14Z-eicosatetraenoic acid (19-HETE) and 20-hydroxy-5Z, 8Z, 11Z, 14Z-eicosatetraenoic acid (20-HETE) which were identified by TLC and GC-MS; the ratio of synthesis was 73:27, respectively. Conversion of AA by immobilized Candida bombicola, suspended in beads of 2% (w/v) calcium alginate for 96 h, gave an 83% conversion of 1 g AA l^–1 to 19- and 20-HETE. There was no significant loss in the efficiency of the immobilized cells after ten uses.     

红平菇产漆酶条件优化及对孔雀绿染料脱色的研究

采用LNAS(低氮天冬酰胺-琥珀酸)培养基添加方式,对红平菇Pleurotus djamor HP1进行培养,检测不同时间培养液对不同底物的氧化作用,进而得到光密度值的变化情况,作为漆酶的产生及活性测定的主要依据。结果表明:在含Cu2+的培养液中漆酶最大酶活为235.4 U/L。含Cu2+的培养液添加底物木屑后漆酶最大酶活为458.8 U/L。提取经优化筛选后的培养基培养出的漆酶粗酶液,对4种具有不同化学结构的染料进行了脱色试验。结果表明:三苯基甲烷类的孔雀绿在6 h时脱色率为87.5%,蒽醌类的SN4R在24 h时脱色率为49.4%,偶氮类的甲基橙在24 h时脱色率为45%,杂环类的中性红在24 h时脱色率为23.6%。因此,显示出红平菇漆酶对孔雀绿染料脱色具有较大的应用潜力,进而对废水处理具有更好的应用前景。     

Decolorization of textile dye RB19 using volcanic rock matrix immobilized <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> cells with surface displayed laccase

A triplicate volcanic rock matrix–Bacillus thuringiensis–laccase WlacD (VRMs–Bt–WlacD) dye decolorization system was developed. WlacD was displayed on the B. thuringiensis MB174 cell surface to prepare a whole-cell laccase biocatalyst by using two repeat N-terminal domains of autolysin Mbg (Mbgn)₂ as the anchoring motif. Immunofluorescence microscopic assays confirmed that the fusion protein (Mbgn)₂–WlacD was anchored on the surface of the recombinant B. thuringiensis MB174. After optimization by a single factor test, L ₉(3⁴)-orthogonal test, Plackett–Burman test, steepest ascent method, and Box–Behnken response surface methodology, the whole-cell specific laccase activity of B. thuringiensis MB174 was improved to 555.2 U L^?1, which was 2.25 times than that of the primary culture condition. Optimized B. thuringiensis MB174 cells were further adsorbed by VRMs to prepare VRMs–Bt–WlacD, an immobilized whole-cell laccase biocatalyst. Decolorization capacity of as-prepared VRMs–Bt–WlacD toward an initial concentration of 500 mg L^?1 of an textile dye reactive blue 19 (RB19) aqueous solution reached 72.36% at a solid-to-liquid ratio of 10 g–100 mL. Repeated decolorization-activation operations showed the high decolorization capacity of VRMs–Bt–WlacD and have the potential for large-scale or continuous operations.     

Fixed-bed biosorption of Cu2+ by polyacrylonitrile-immobilized dead cells of Saccharomyces cerevisiae

Dead cells of Saccharomyces cerevisiae 54 were immobilized by entrappment in polyacrylonitrile. The beads obtained were used to adsorb copper in an up-flow fixed-bed column. The effect of polymer content and cell loading were studied to optimize the porosity and the efficiency in copper removal of the biosorbent beads in a batch system. The optimal concentration of the polyacrylonitrile was assumed to be 12%(w/v) and a concentration of 0.5 g cell dry weight in 1 g polymer was most effective in adsorption of Cu²⁺. The adsorption capacity of this biosorbent was 27 mg Cu²⁺/g dry biomass at 200 mg/l initial concentration of copper ions. Adsorption of Cu²⁺ in a batch system was studied using different initial concentrations of the solute. The optimal conditions in the up-flow column of the following parameters were determined: flow rate, bed height, and initial concentration of Cu²⁺ of the solutions. Results of fixed-bed biosorption showed that breakthrough and saturation time appeared to increase with the bed height, but decrease with the flow rate and the initial concentration. The linearized form of the Thomas equation was used to describe dynamic adsorption of metal ions. As a result, the adsorption capacity of the batch system and the column system was compared. Desorption of copper ions was achieved by washing the column biomass with 0.1 M HCl at an eluent flow rate of 1 ml/min. The reusability of the immobilized biomass was tested in five consecutive adsorption-desorption cycles. The regenerated beads retained over 45% of their original adsorption capacity after five A/D cycles. This revised version was published online in August 2006 with corrections to the Cover Date.     

Adsorptive Immobilization of a Pseudomonas Strain on Solid Carriers for Augmented Decolourization in a Chemostat Bioreactor

Pseudomonas GM3, a highly efficient strain in cleavage of azo bonds of synthetic dyes under anoxic conditions, was immobilized via adsorption on two types of carriers, porous glass beads and solid PVA particles. The cells were cultivated in a nutrient medium, adsorbed on sterile carriers, stabilized as biofilms in repeated batch cultures, and introduced into a chemostat activated sludge reactor for augmented decolourization. The microbial cells were quickly adsorbed and fixed on the PVA surface, compared to a slow and linear immobilization on the glass surface. The porous structure of glass beads provided shelter for the embedded cells, giving a high biomass loading or thick biofilm (13.3 mg VS ml^?1 carrier) in comparison with PVA particles (4.8 mg VS ml^?1 carrier), but the mass transfer of substrate in the biofilm became a significant limiting factor in the thicker biofilms (effectiveness factor η = 0.31). The microbial decolourization rate per volume of carriers was 0.15 and 0.17 mg dye ml^?1 of glass beads and PVA particles, respectively. In augmented decomposition of a recalcitrant azo dye (60 mg l^?1), the immobilized Pseudomonas cells in porous glass beads gave a stable decolourization efficiency (80 - 81%), but cells fixed on solid PVA particles showed an initial high colour removal of 90% which then declined to a stable removal efficiency of 81%. In both cases, the colour removal efficiency of the chemostat bioreactor was increased from < 10% by an activated sludge to ～80% by the augmented system.     

Decolorization of 1:2 metal complex dye Acid blue 193 by a newly isolated fungus, Cladosporium cladosporioides

Summary A fungus Cladosporium cladosporioides isolated from coal sample as a decolorizing microorganism. It decolorized five different azo and triphenylmethane dyes like acid blue 193, acid black 210, crystal violet, reactive black B(S) and reactive black BL/LPR both on solid and in liquid broth medium. Culture broth of this fungus decolorized completely 100 mg of acid blue 193 l⁻¹ in 8 days. The extracellular enzyme of Cladosporium cladosporioides decolorized acid blue 193 on repeated addition to a total (out of 700 mg l⁻¹) concentration of 564 mg l⁻¹ within 168 h without significant decline in the activity, showing the resistant property of Cladosporium cladosporioides to a high concentration of the dye. The optimal temperature 40 °C, pH 5.6 and sugar concentration of 4% required for decolorization of acid blue 193. Cladosporium cladosporioides showed manganese peroxidase activity with 41 U l⁻¹, laccase activity with 1413 U l⁻¹ and lignin peroxidase activity was negligible after day 8 of incubation.