M. Arulmani, K. Murugesan, V. Geetha and
P.T. Kalaichelvan
Centre for Advanced Studies in Botany
University of Madras, Guindy Campus
Chennai - 600 025, Tamil Nadu, India
Email: arulmanim@gmail.com

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India is one of the main producer and consumer of synthetic organic chemicals including synthetic dyes. Synthetic dyes are used extensively in textile dyeing, paper printing and colour photography and also as additives in petroleum products. A wide variety of synthetic dyes namely azo, polymeric, anthraquinone, triphenylmethane and heterocyclic dyes is used in textile dyeing processes. Worldwide more than 10,000 dyes and pigments are used in dyeing and printing industries. The total world colourant production is estimated to be 8,00,000 tonnes per year and at least 10% of the used dyestuff enters the environment through wastes (Levin et al., 2004; Palmieri et al., 2005). The textile industry accounts for two-thirds of the total dyestuff market (Riu et al., 1998) and consumes large volumes of water and chemicals for wet processing of textiles. An estimated, 10-15% of dye is discharged or lost into the effluents during different dyeing processes (Zollinger, 2002).

Wastewaters from textile industries are a complex mixture of many polluting substances like acids, salts, organochlorine-based pesticides, heavy metals, pigments, dyes etc., Due to complex nature and hard-to-treat by conventional methods, textile dyeing industries are facing problems to safe discharge of wastewater. There have been several successful methods developed based on physical and chemical processes for colour removal of textile dyeing effluents. They include coagulation/flocculation, membrane filtration and activated carbon adsorption. Unfortunately, these methods of effluent treatment have high operating costs and limited applicability. Further, these treatment methods produce large quantities of sludge, which again create a problem in waste disposal (Moreira et al., 2000). In recent years, biological decolourization using potential microorganisms capable of decolourizing and detoxifying the synthetic dyes has been considered as a promising and eco-friendly method (Couto et al., 2005; Camarero et al., 2005).

Over the past few decades, numerous microorganisms have been isolated and characterized for decolourization of various groups of synthetic dyes. In general, azo dyes are resistant to bacterial degradation. However, certain bacteria can degrade dyestuff by azoreductase activity (Chung and Stevens, 1993). White rot fungi (WRF), a group of basidiomycetous are the potential organisms capable of mineralizing the complex wood polymer and a wide variety of recalcitrant compounds like xenobiotics, lignin and dyestuff by their extracellular lignolytic enzyme system. WRF offer significant advantages over bacterial system since their extracellular lignolytic enzyme system consisting of lignin peroxidases, manganese dependent peroxidases, manganese independent versatile peroxidases, and laccases and they degrade a wide variety of complex aromatic dyestuffs (Boer et al., 2004; Kamistsuji et al., 2005). White-rot fungi do not require preconditioning to particular pollutants, because enzyme secretion depends on nutrient limitation, nitrogen or carbon, rather than presence of pollutant. The extracellular enzyme system also enables white-rot fungus (WRF) to tolerate high concentration of pollutants (Knapp et al., 1997).

However, the fungi in waste treatment and bioremediation do not always enable the culture conditions for lignolytic to be fulfilled. Other white rot fungi namely Bjerkandera adusta, Irpex lacteus, Plebioa radiata, Pleurotus ostreatus, P.sajor-caju, Ganodema lucidum, Pycnoporus cinnabarinus and Trametes versicolor have been demonstrated for the decomposition of several recalcitrant dyes (Novotny et al., 2001; Murugesan et al., 2006; 2007). The enzymatic treatment of industrial waste has
exhibited several advantages over other physical methods because it can be applied even to compounds, which are biorefractory and it can be operated at varied temperatures, pH and salinities.

Moreover, the enzymatic treatment of wastes does not leave much sludge at the treatment site. Much attention has been focused on the development of processes to treat the wastewaters, solid wastes, hazardous wastes and ameliorate contaminated soils realizing the potential application of enzyme treatments.

Immobilization of enzymes

Biodegradation appears to be a promising technology, particularly the use of oxidative enzymes as biocatalyst included with a microorganism or free enzyme. Laccase has received particular attention because of its ability to catalyze the oxidation of a wide spectrum of molecules containing an aromatic ring substituted with electron withdrawing groups (D’Annibale et al., 1999). Enzyme immobilization usually allows a good preservation of enzyme activity over a long period (D’Annibale et al., 1999). The efficiency of enzyme extract is enhanced by selective adsorption when immobilized, as reported by Tatsumi et al. (1996), in the removal of chlorophenols from wastewaters by peroxidase immobilized on magnetite. In most cases, laccases are immobilized on porous beads. Xenobiotics are degraded in bed-packed column reactors. However, immobilization of enzymes on a membrane and the use of filtration offer several advantages. First it allows the simultaneous downstream separation of the transformation products, when they are insoluble and secondly flow rates can be higher than with packed beads, because all the substrate flows through the support instead of diffusing in the bead pores. Some of the intended applications e.g. kraft pulp bleaching, dye effluent using laccase involve high pH. Among the 40-50 known fungal laccases, a few are active at alkaline pH (Schneider et al., 1999). Being added to alkaline detergents, the laccases are able to oxidize various textile dyes to bleach the undesirable colour in washing solution.

Effluent treatment by immobilized mycelium

The efficiency of immobilized Pleurotus sp. MAK-II for the decolourizing of the textile dye effluent was assessed. Figure 1 shows the steps involved for the textile dyeing effluent treatment with immobilized mycelium of Pleurotus sp. MAK-II. The SEM micrograph of immobilized fungus alginate beads was completely different from that of the beads without fungus. Table 1 shows the physicochemical properties of the untreated and immobilized fungal treated effluents. The initial and final pH of untreated effluent was 9.5-9.8, whereas, the treated effluent pH after 15 days decreased to 7.0-7.2. The values of BOD and COD found high in the untreated effluent, whereas the immobilized mycelium of the test fungus removed up to 75% and 80% of BOD and COD, respectively.

Agar Plate culture	After 1 day		Liquid plate culture	After 5 days		Immobilization	After 10 days
Growth of Immobilized culture at 120 rpm 2days.	After 15 days Note the decolourization of effluent at different days of treatment in Bioreactor.		Bioreactor	Without cell Immobilization With cell Immobilization

Fig. 1 Effluent treatment with immobilized
mycelium of Pleurotus sp. MAK-II

Fig. 2 Declolourization of the dye effluent and
laccase activity by immobilized mucelium
of Pleurotus sp. MAK - II

The fungus removed 55% of the colour on 15^th day and the maximum laccase activity of 27.81 U/mL observed on 12^th day(Fig. 2). Reduction of peak height in the UV spectrum clearly indicate decolourization of effluent (Fig. 3).

Fig. 3 UV-visible spectram of decolourization of effluent
by immobilized myceliam. Spectra after (1)1 day
(2) 5 days (3) 10 days (4) 15 days treatment.

Table 1. Physico-chemical properties of untreated and treated effluents.

Parameter	Effluent and Medium (1:1)
	Untreated	Treated
Colour	Dark blue	Light blue
Odour	Offensive	No Odour
pH	9.5-9.8	7.0-7.2
BOD (mg/L)	4500	1120
COD (mg/L)	14000	2800

 

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