An expert is a person who has made all the mistakes that can be made in a very narrow field.

---- Niels Bohr

Review of Literature

Chapter No. 2.

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2.1. Dyes
Color is an important property of human expression. Ever since the beginning of humankind, people have been using colorants for painting and dyeing of their surroundings, their skins and their clothes.

2.1.1. History All colorants were from natural origin up to middle of the 19th century. In 1856, the English chemist W.H. Perkin synthesized the bluish substance viz. Aniline purple and Tyrian purple with excellent dyeing properties. This invention and kekules discovery of benzene structure strongly stimulated the production of various synthetic dyes. Thus, in the beginning of 20th century, the natural dyestuffs were replaced by synthetic dyestuffs (Welham, 2000).

2.1.2. Dye classification The compounds which absorb electromagnetic energy in the visible range (~350-700 nm) are colored. These colored compounds are known as dyes. Dyes contain chromophores (delocalised electron systems with conjugated double bonds), and auxochromes (electron-withdrawing or electron donating substituents). The chromophore imparts the color to the dye molecule and auxochrome intensifies the color of the chromophore by altering the overall energy of the electron system. Usual chromophores are C=C, C=N, C=O, N=N, -NO2 and quinoid rings. The auxochromes are -NH3, -COOH, -SO3H and -OH (Zollinger, 1987). The dyes are classified on the basis of chemical structure or chromophore viz. azo (monoazo, disazo, triazo, polyazo), anthraquinone, phthalocyanine, triarylmethane, diarylmethane, indigoid, azine, oxazine, thiazine, xanthene, nitro, nitroso, methine, thiazole, indamine, indophenol, lactone, aminoketone, hydroxyketone stilbene and sulphur dyes. In 1924, the Society of Dyers and Colorists and the American Association of Textile Chemists and Colorists classified the dyes on the color, structure and

Chapter No. 2.

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application method in the Color Index (C.I.) which is further revised in every three months. Each dye was given a C.I. generic name on the basis of its application, characteristics and its color. The Color Index discriminate 15 different application classes: I. Acid dyes This is the largest class of dyes in the color index and is referred to as Acid dyes. Acid dyes are anionic compounds that are mainly used for the dyeing nitrogen-containing fabrics like wool, polyamide, silk and modified acryl. They bind to the cationic NH4+ ions of those fibres. Most of the acid dyes are azo, anthraquinone or triarylmethane compounds. The adjective acid refers to the requirement of acidic condition for dyeing these dyes to fibers (cotton, wool, silk and nylon). II. Reactive dyes The dyes, which containing the reactive groups are called as reactive dyes. In the Color Index, this is the second largest dye class, introduced in 1956. The reactive dyes form covalent bonds with -OH, -NH, or -SH groups of fibres. The reactive group is often a heterocyclic aromatic ring substituted with chloride or fluoride or vinyl sulphone. The hydrolysis of reactive group during dyeing lowers the degree of fixation of reactive dyes. These create the problem of colored effluent. III. Metal complex dyes Many metal complex dyes are reported among the acid and reactive dyes. Metal complex dyes are the strong complexes of one metal atom (usually chromium, copper, cobalt or nickel) and one or two dye molecules (1:1 and 1:2 metal complex dyes respectively). About 16% of the azo dyes listed in the color index are metal complexes. The phthalocyanine metal complex dyes are also being used.

Chapter No. 2. IV. Direct dyes

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The direct dyes are the second largest dye class in the color index with respect to the amount of the dyes. Direct dyes are relatively large molecules with high affinity for cellulose fibres. Van der Waals forces make them bind to the fibre. They are mostly azo dyes with more than one azo bond or phthalocyanine, stilbene or oxazine compounds. About 1600 direct dyes are listed but only 30% of them are in current production. V. Basic dyes Basic dyes represent 5% of all dyes listed in the color index. They are cationic compounds and used for the dyeing acid-group containing synthetic fibres like modified polyacryl. They bind to the acid groups of the fibres. Most of the basic dyes are diarylmethane, triarylmethane, anthraquinone or azo compounds. VI. Mordant dyes The dyes which required the mordant for dying it to fibers is called as mordant dyes. They are used for dyeing wool, leather, silk, paper and modified cellulose fibres. Most mordant dyes are azo, oxazine or triarylmethane compounds. The mordants are usually dichromates or chromium complexes. VII. Disperse dyes Disperse dyes form the third largest group of dyes in the color index. Disperse dyes are scarcely soluble dyes, thus it required high temperature or chemical softeners for dying to synthetic fibers viz. cellulose acetate, polyester, polyamide, acryl, etc. Dying takes place in dye baths with fine disperse solutions of these dyes. They are usually small azo or nitro compounds, anthraquinones or metal complex azo compounds. VIII. Pigment dyes These are insoluble non-ionic compounds or insoluble salts retain their crystalline or particulate structure throughout their application. Pigment dyes (i.e. organic pigments) represent a small but increasing fraction of the pigments, the most widely applied group of colorants. About 25% of all commercial dye names

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listed in the color index is pigment dyes. Pigment dyeing required the use of dispersing agents. Pigments are usually used together with thickeners in print pastes for printing diverse fabrics. Most pigment dyes are azo compounds or metal complex phthalocyanines or anthraquinone or quinacridone. IX. Vat dyes Vat dyes are water insoluble dyes that are widely used for dyeing cellulose fibres. Vat refers to the vats that are used for the reduction of indigo plants through fermentation. The dyeing method is based on the solubility of vat dyes in their reduced (leuco) form. Alternate reduction and oxidation process was used for dying these dyes to fibers. Sodium dithionite is used as reducing agent for this process. Almost all vat dyes are anthraquinones or indigoids. X. Anionic dyes and ingrain dyes Azoic dyes and ingrain dyes (naphthol dyes) are the insoluble products of a reaction between a coupling component, including naphthols, phenols or acetoacetylamides and a diazotised aromatic amine. This reaction is carried out on the fibre. All naphthol dyes are azo compounds. XI. Sulphur dyes Sulphur dyes are complex polymeric aromatics with heterocyclic Scontaining rings. This dye group represents about 15% of the global dye production. Dyeing with sulphur dyes involves reduction and oxidation comparable to vat dyeing. They are mainly used for dyeing cellulose fibres. XII. Solvent dyes Solvent dyes are non-ionic dyes that are used for dyeing substrates in which they can dissolve, e.g. plastics, varnish, ink, waxes and fats. They are not often used for textile processing but their use is increasing. Most solvent dyes are diazo compounds that underwent some molecular rearrangement. Also triarylmethane, anthraquinone and phthalocyanine solvent dyes are applied.

Chapter No. 2. XIII. Fluorescent brighteners

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Fluorescent brighteners mask the yellowish tint of natural fibres by absorbing ultraviolet light and weakly emitting visible blue. They are not dyes in the usual sense because they lack intense color. Based on chemical structure, several different classes of fluorescent brighteners are distinguished such as stilbene derivatives, coumarin derivatives, pyrazolines, 1,2-ethene derivatives, naphthalimides and aromatic or heterocyclic ring structures. Many fluorescent brighteners contain triazinyl units and water-solubilising groups. XIV. Other dye classes Apart from the dye classes mentioned above, the color index also lists food dyes and natural dyes. Food dyes are not used as textile dyes and the use of natural dyes viz. anthraquinone, indigoid, flavenol, flavone or chroman compounds that can be used as mordant, vat, direct, acid or solvent dyes in textile processing operations is very limited.

2.1.3. Production and discharge statistics of dyes According to a study on dyes and organic pigments, the worldwide demand for organic colorants (dyes and organic pigments) is projected to increase at $10.6 billion in 2008. Production of dyestuff and pigments in India is close to 80,000 tonnes. India is the second largest exporter of dyestuffs and intermediates among developing countries, after China. The textile industry accounts for the largest consumption of dyestuffs, at nearly 80%. Recent statistics on the global production and use of the dyes and on the relative distribution between the different dye classes are not readily available. The most recent readily available data is from the 1993 SRI report, containing data for 1991 (Ollgaard et al., 1998). The principal route by which dyes enter in the environment is via wastewater. To assess the relative percentage of different dyes classes in textile processing wastewater, the dye consumption data should be considered with the degree of fixation of different dye classes (Table 2.1).

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Table 2.1. Estimated degree of fixation and world wide sale of dyes.

Dye class

Fiber

Degree of fixation (%)

Loss in to effluent (%) 5-20 0-5 5-30 0-10 2-10 10-50 10-40 5-20

World wide sale (tonnes) 100 44 64 157 114 101 40

Acid Basic Direct Disperse Metal complex Reactive Sulfur Vat

Polyamide 80-95 Acrylic Cellulose Polyester Wool Cellulose Cellulose Cellulose 95-100 70-95 90-100 90-98 50-90 60-90 80-95

2.1.4. Toxicity of dyestuffs Many dyes are visible in water at very low (1 mg l-1) concentrations. The dye concentrations in the textile processing wastewaters are in the range of 10-200 mg l-1. Therefore, usually the discharge of highly colored wastewater in open waters presents aesthetic problem. As dyes are designed to be chemically and photolytically stable, they are highly persistent in natural environments. The release of the dyes in a natural environment may cause the ecotoxic hazard. Pollution due to dyes may occur on a significant scale because of their huge usage. The International Agency for Research on Cancer (IARC) has classified various dyes like benzidine as being associated with the cancer in humans (Anonym, 1982). Benzidine is known to be carcinogenic to the variety of mammalian species, including humans. In vitro tests on laboratory animals, two benzidine dyes, Direct blue 6 and Direct black 38, have been reported to be potent carcinogen that induces hepatocellular carcinomas and neoplastic liver nodules in rats after only 13 weeks of exposure (Robens, 1980). A number of dyes have been

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tested for mutagenicity using Ames bioassay. Several of them have been found to be carcinogenic and mutagenic (Venturini and Tamaro, 1979; Mathur et al., 2005). Acute and short term toxicity studies of textile dyestuff and wastewaters on the freshwater fish Gambusia affinis revealed significant reduction in its mortality and erythrocyte count (Sharma et al., 2009). Phytotoxicity studies also revealed the toxic nature of textile dyestuff and effluent (Parshetti et al., 2006).

2.1.5. Dye removal techniques Textile dyestuff and wastewater is recalcitrant to the degradation. Several physicochemical and biological techniques can be employed to remove color from the dye containing wastewaters. Several factors, including dye type, wastewater composition, dose or costs of required chemicals, operation costs, environmental fate and handling costs of generated waste products determine the technical and economic feasibility of each single technique. In general, each technique has its own limitations. The use of one individual process may often not sufficient to achieve complete decolorization.

2.1.5.1. Physicochemical techniques Physicochemical techniques include membrane filtration, coagulation/flocculation, precipitation, flotation, adsorption, ion exchange, ion pair extraction, ultrasonic mineralisation, electrolysis, advanced oxidation (chlorination, bleaching, ozonation, Fenton oxidation and photocatalytic oxidation) and chemical reduction (Table 2.2).

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Table 2.2. Different physicochemical techniques used for the dye decolorization.

Process Membrane filtrationa

Methodology Nanofiltration and reverse osmosis using membranes with a molecular weight cut-off below 10,000 Dalton

Comment A quick method with low spatial requirement. Advantage of this method was reuse of dyes. The disadvantages of this method were flux decline and membrane fouling, necessitating frequent cleaning and regular replacement of the modules. The capital costs of membrane filtration are high. This method was used to partly remove COD and color from raw wastewater.

Coagulation/ flocculationb

Use of coagulants viz. lime, organic polymer, magnesium, iron, and aluminum salts

Adsorptionc

Adsorption of dyes using various adsorbent viz. activated carbon, non-modified cellulose (plant) biomass, modified cellulose biomass, bacterial biomass, yeast biomass, fungal biomass, chitin, soil material,

Equilibrium capacity varies with dyes and adsorbents.

Chapter No. 2.

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Photocatalytic TiO2 catalysts (TiO2)d supported on three different absorbents Ozonation and ultrasound enhanced ozonatione Sonicationf Varying ozone, ultrasound and ultrasound enhanced ozone operational conditions Use of varying frequency of ultrasound waves.

Over 95% color removal; TiO2 supported on absorbents is more efficient than that of bare TiO2. It generates the harmful reactive radicals. First-order rate constant increased to 200% using ultrasonic power inputs compared to ozonation alone. The disadvantage of this technique was generation of highly oxidizing reactive radicals. Complete decolorization and increase in rate of decolorization under spent dye bath conditions. decreased concentration. Decolorization with an efficiency dye increased

Electrolysisg

Applying an electric Dyes were decolorized using combined current to the wastewater using electrodes. electrochemical oxidation, electrochemical reduction, electro-coagulation and electroflotation reaction. It requires the high amount of energy.

a e

Yun et al., 2006; bJoo et al., 2007; cCrini, 2006; dBizani et al., 2006; Gltekin and Ince, 2006; fOkitsu et al., 2005; gDaneshvar et al., 2006.

Chapter No. 2. 2.1.5.2. Biological methods

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Biological dye removal techniques are based on the microbial biotransformation of dyes. The microorganisms used for the decolorization are fungi, actinomycetes, algae and bacteria. Many researchers have demonstrated partial or complete biodegradation of the dyes using fungi, algae, actinomycetes and pure or mixed cultures of bacteria or their enzymes. I. Dye decolorization using fungi Many researchers used the lignolytic and nonlignolytic fungi for the decolorization of dye wastewater. The lignolytic white rot fungi are known to most efficient microorganisms for the dye degradation. Lignolytic fungi, including Phanerochaete chrysosporium, Trichophyton rubrum LSK-27, Ganoderma sp. WR-1, Trametes versicolor, Funalia trogii, Irpex lacteus, etc. are widely used for the decolorization of textile dyes (Novotny et al., 2004; Zille et al., 2005; Nilsson et al., 2006; Yesiladal et al., 2006; Revankar and Lele, 2007; Park et al., 2007; Parshetti et al., 2007). The Myrothecium sp. IMER1, a nonlignolytic fungi and yeast viz. S. cerevisiae, C. tropicalis, K. marxianus, C. zeylanoides, and I. occidentalis are also used for the decolorization of textile dyes (Meehan et al., 2000; Donmez, 2002; Ramalho et al., 2002; Maximo et al., 2003; Jadhav et al., 2006; Zhang et al., 2007). The mechanism of the fugal decolorization was biodegradation or bioadsorption of dyes. The biodegradation of dyes using white rot fungi was associated with an involvement of various lignolytic enzymes, such as lignin peroxidase, manganese peroxidase and laccase. Biodegradation of dyes using nonlignolytic fungi was associated with an involvement of billirubin oxidase, laccase and azoreductase (Pajot et al., 2007; Jadhav et al., 2006; Zhang et al., 2007). II. Dye decolorization using algae Very few reports are available on the use of algae, including Cosmarium sp. and Enteromorpha prolifera for the decolorization of azo and triphenyl methane dyes (Jinqi and Houtian, 1992; Ozer et al., 2005; Daneshvar et al., 2007). The dye

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decolorization using algae was associated with adsorption and degradation (Marungrueng and Pavasant, 2006). III. Dye decolorization using actinomycetes Actinomycetes represent a group of relatively abundant and metabolically diverse bacteria in soils (Labeda and Shearer, 1990). Actinomycetes, including Streptomyces rochei A10, Streptomyces chromofuscus 11, Streptomyces diastaticus A12, Streptomyces diastaticus A13 and Streptomyces rochei A14 were capable of degrading various textile dyes through their extracelluar oxidative enzymes such as lignin peroxidase and manganese peroxidase (Paszczynski et al., 1992). IV. Dye decolorization using bacteria Bacteria are widely used for the decolorization of azo and triphenylmethane dyes. Bacteria degrade the dyes by various mechanisms, including enzymatic and nonenzymatic. i. Dye decolorizing bacteria Most of the potential dye degrading bacteria were isolated from textile dye contaminated soil by enrichment techniques and identified on the basis of 16s rRNA sequence. It was also analyzed on the basis of morphological, biochemical and physiochemical characteristics. However, some microorganisms which are not isolated from textile dye contaminated soil, but showed excellent dye decolorization property (Table 2.3.). ii. Decolorization of dyes under anaerobic conditions Anaerobic azo dye decolorization is the reductive cleavage of an azo bond by the transfer of reducing equivalents resulting in the formation of aromatic amines. The anaerobic decolorization of azo dyes was first investigated using intestinal anaerobic bacteria (Chung et al. 1992). The removal of large amount of azo dyes from wastewater became a major concern. Thus, research on bacterial azo dye reduction has been focused on the activity of (facultative) anaerobic

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Table 2.3. Bacteria used for decolorization of dyes Species

a

Source National Center for Industrial Microorganisms, Pune, India. Microbial Type Culture Collection, Chandigarh, India. Isolated from textile dye

Growth medium g l-1; NaCl 5, peptone 5 and beef extract 3. g l-1; NaCl 5, bacteriological peptone 5, yeast extract 2, beef extract 1. g l-1; peptone 10, NaCl 5, yeast extract g l-1; Peptone 5,

Dye decolorization

Pseudomonas

desmolyticum NCIM

Direct blue-6

Brevibacillus

laterosporus MTCC 2298

Methyl orange

Pseudomonas

Reactive red BLI

sp. SUK1

contaminated soil. 2 and beef extract 1. Isolated from textile dye contaminated soil. Isolated from textile dye contaminated soil. textile dye National Center for Industrial Microorganisms,

Bacillus sp.

NaCl 5, yeast extract Direct brown 2 and beef extract 1. g l-1; peptone 5, beef extract 3, NaCl 5. g l-1; peptone 5, NaCl 5, yeast extract g l-1; NaCl 5, bacteriological peptone 10, and beef Direct red 5B 3RL

UVS

Commamonas

sp. VUS
f

Exiguobacterium Isolated from

sp. RD3
g

Reactive navy blue HE2R Direct brown MR

contaminated soil. 1 and beef extract 3,

Acinetobacter

calcoaceticus NCIM 2890

Chapter No. 2. Pune, India.

h

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Congo red, Pseudomonas Isolated from dye waste water LB broth Methyl orange and Methyl red
i

leutola Enterobacter sp.

(E), (P) and Morganella sp. (M)

Isolated from activated carbon process Microbial Type

g l-1; NaCl 0.007, CaCl2.2H2O 0.004, MgSO4.7H2O2, dye 5 and agar 3. g l-1; beef extract 1, yeast extract 2, peptone 5 and NaCl g l-1; K2HPO4 5.22, KH2PO4 4.08, Malachite green ----

Pseudomonas sp. powdered

Kocuria rosea

Culture Collection,

MTCC 1532

Chandigarh, India 5.

Pseudomonas

Isolated from dye waste water

MgSO4.7-H2O 0.2, CaCl2 0.55, NH4Cl 0.4, and Crystal violet 0.00022.

putida

Crystal violet

Citrobacter sp.

Isolated from dye waste water

Crystal violet LB medium and Methyl red

Kalme et al., 2007; bGomare et al., 2008; cKalyani et al., 2009; dDawkar et al.,

2008; eJadhav et al., 2008; fDhanve et al., 2008; gGhodake et al., 2009a; hHsueh and Chen, 2007; i Barragan et al.,2007; jParshetti et al., 2006; kChen et al., 2007

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bacteria. Bacteria from different origins were used to investigate anaerobic azo dye reduction, including pure cultures, mixed cultures, anaerobic sediments, digester sludge, anaerobic granular sludge and activated sludge (Brown et al., 1983; Weber et al., 1987; Haug et al., 1991; Carliell et al., 1994; Razo Flores et al., 1997; Beydilli et al., 1998; Bromly-Challenor et al., 2000). Anaerobic bacteria were used in a bioreactor for the treatment of textile industry effluent (Maas and Chaudhari, 2005; Isik and Sponza, 2008; Mezohegyi et al., 2008) (Table 2.3). iii. Decolorization of dyes under anoxic condition Pure bacterial strains, such as Pseudomonas luteola, Aeromonas hydrophila, Bacillus subtilis, Pseudomonas sp. and Proteus mirabilis decolorized dyes under anoxic conditions (Chen et al., 1999, 2003; Chang et al., 2001; Yu et al., 2001). Although many of these cultures were able to grow aerobically, but decolorization achieved only under anaerobic conditions. Anoxic decolorization of various dyes using mixed aerobic and facultative anaerobic microbial consortia has been reported (Nigam et al., 1996a; Kapdan et al., 2000; Padmavathy et al., 2003; Khehra et al., 2005; Moosvi et al., 2005). Dye decolorization using pure as well as mixed cultures under anoxic condition required complex organic sources, including yeast extract, peptone, or a combination of complex organic source and carbohydrate (Chen et al., 2003; Khehra et al., 2005). The decolorization of the dyes at an anoxic condition was influenced by various substrates used for the decolorization of azo dyes. Although, azo dye decolorization under anoxic condition was also non-specific, limitations of this method seem to be the requirement for the yeast extract or peptone. To make the process economically feasible for industrial scale, it is necessary to find out the alternate cheaper sources (Nigam et al., 1996b; Moosvi et al., 2005; Chen et al., 2003). iv. Decolorization of dyes under aerobic condition a) Decolorization of dyes under static condition The optimum pH for the decolorization of dyes at static condition was 7.08.0. Isolated pure bacterial culture viz. Pseudomonas sp. SUK 1, Kocuria rosea,

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Bacillus sp.VUS, Commamonas sp. UVS, Exiguobacterium sp. RD3, Proteus sp. SUK7 has efficiently decolorized the azo dyes at static condition than shaking condition (Parshetti et al., 2006; Kalme et al., 2007; Kalyani et al., 2008; Dawkar et al., 2008; Jadhav et al., 2008; Dhanve et al., 2008; Patil et al., 2008). The pure bacterial culture was able to tolerate and degrade the higher concentration (more than 1 g l-1) of azo dyes (Kalyani et al., 2008). The combination of yeast extract with agricultural waste including the baggase powder, wheat bran, rice bran and wood shaving was the efficient substrate for the decolorization of azo dyes at static condition (Jadhav et al., 2008). b) Decolorization of dyes under shaking condition Although for a long time, it was thought that azo dyes remained recalcitrant under aerobic conditions. A bacterial strain S5, derived from Hydrogenophaga palleronii S1 mineralized the sulfonated azo dyes and used as carbon and nitrogen source for growth (Blumel et al. 1998). Pure bacterial culture viz. unidentified KMK4, strain S5, E. coli NO3 and Flavobacterium sp. ATCC39723 and mixed bacterial culture viz. unidentified BF1, BF2 and Pseudomonas putida MTCC 1194 were applied to decolorize the higher concentration of textile dyestuffs (Cao et al., 1993; Chang and Kuo, 2000; Senan and Abraham, 2004; Kodam et al., 2005). v. Anaerobic/aerobic treatment for the degradation of dyes Complete mineralization of textile dyestuffs was possible under the anaerobic-aerobic treatment (Haug et al. 1991). Different reactor configurations used for anaerobic-aerobic steps, including the sequential anaerobic-aerobic reactor system (Zitomer and Speece, 1993) and integrated anaerobic-aerobic reactor system (Field and Brady, 2003). Sequential anaerobic/aerobic conditions can readily be imposed to wastewater by first exposing it to anaerobic conditions and next aerobic conditions using an anaerobic reactor followed by an aerobic reactor. The sequential anaerobic and aerobic degradation has been studied for the conversion of azo dyes by numerous researchers (Van der Zee and Villaverdeb, 2005; Isik and Sponza, 2008). In anaerobic conditions, the reductive cleavage of

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azo dyes was resulted into the formation of aromatic amines. After re-aeration of the synthetic dye wastewater, these amines were further degraded by the same isolates. Thus, total degradation of reactive azo dyes was achieved using an anaerobicaerobic treatment (Isik and Sponza, 2006). There appears to be single report on the successful full-scale implementation of anaerobic technology in combination with aerobic membrane technology for the treatment of more than 1000 l3 wastewaters day-1 from textile processing industry (Stolz, 2001; VA Tech WABAG, 2003). vi. Mechanism of dye decolorization The bacterial degradation of the textile dyestuffs under anaerobic and aerobic conditions occurred by cleavage of chromophoric group. This cleavage may be due to different mechanisms, such as enzymes, low molecular weight redox mediators, chemical reduction by biogenic reductants like sulfide or combination of these. The location of these reactions can be either intracellular or extracellular. a) Direct enzymatic dye decolorization The bacterial decolorization was associated with the involvement of various reductive enzymes viz. azoreductase, NADH-DCIP reductase, MG reductase and oxidative enzymes viz. aminopyrine N-demethylase, lignin peroxidase, laccase and polyphenol oxidase (Parshetti et al., 2006; Kalme et al., 2007; Kalyani et al., 2009). Azoreductase The presence of extracellular oxygen sensitive azoreductase in anaerobic bacteria viz. Clostridium and Eubacterium that decolorized sulfonated azo dyes during growth on solid or complex media was first reported by Rafii et al. (1990). Azoreductases are flavoprotiens (NAD(P)H: flavin oxidoreductase). It is localized intracellular or extracellular site of the bacterial cell membrane. These azoreductases required the NADH or NADPH or FADH as an electron donor for the reduction of an azo bond (Russ et al., 2000). The substrate specificity of

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azoreductases depends on the functional group present near azo bond. The oxygen sensitive Orange II azoreductase from Pseudomonas sp. KF46 showed highest specificity towards the carboxy group substituted sulfophenyl azo dyes. Orange II azoreductase showed lowest Km (0.8 M) for 1-(4-carboxyphenylazo)-2-naphthol and highest Km (14.8 M) for 1-(4-sulfoaminophenylazo)-2-naphthol as substrate. Orange II azoreductase was unable to decolorize the azo dyes which contain charged groups in the proximity to the azo group (Zimmermann et al., 1982). The induction of azoreductase during decolorization of azo dyes under static condition was reported earlier (Dawkar et al., 2008; Dhanve et al., 2008). Azoreductase generated the toxic amines after reduction of an azo bond. The reaction catalyzed by azoreductase is shown in Fig. 2.1.
COOH

N(CH3)2

[A] 2 NADH + H or NADPH + H or FADH2 2 NAD+ or NADP+ or

COOH
+ +

FAD

NH2

H2N

N(CH3)2

[B]

[C]

Fig. 2.1. Reaction catalyzed by azoreductases

[A] = Methyl red. [B] = 2-Amino benzoic acid. [C] = p-dimethyl amino anilline.

NADH-DCIP reductase and MG reductase: NADH-DCIP reductase belongs to the bacterial mixed function oxidase system and takes part in the detoxification of xenobiotic compounds (Salokhe et al., 1999). The NADH-DCIP reductase reduces the DCIP using NADH as an electron donor. DCIP is a blue in its oxidized form and becomes colorless after

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Cl

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NaO

[A]

Cl

NADH + H

NADH + H

NAD

NAD

Cl

NaO

NH

OH

Cl

[B]

Fig. 2.2. Reaction catalyzed by NADH-DCIP reductase.

[A] = Oxidized form of DCIP. [B] = Reduced form of DCIP.

N N

2 NA DH + H
N

2 NA D

[A]

[B]

Fig. 2.3. Reaction catalyzed by MG reductase.

[A] = Malachite green [B] = Leucomalachite green

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reduction. The significant induction of nonspecific reductase in the biodegradation of Malachite green was observed and termed as MG reductase (Parshetti et al., 2006). MG reductase reduces the Malachite green into leucomalachite green using NADH as an electron donor. The reactions catalyzed by NADH-DCIP reductase and MG reductase are shown in Fig. 2.2 and 2.3. Aminopyrine N-demethylase This enzyme belongs to the group of mixed function oxidase system. It catalyzes the oxidative demethylation of N-methyl groups. Lignin peroxidase (LiP) This enzyme belongs to the family of oxidoreductases, specifically those acting on peroxide as an acceptor (peroxidases) and can be included in the broad category of ligninases. The systematic name of this enzyme class is 1,2-bis(3,4dimethoxyphenyl) propane-1,3-diol:hydrogen-peroxide oxidoreductase. LiP is Nglycosylated protein with molecular weight between 38 and 47 kDa. It contains heme in the active site and shows a classical peroxidase mechanism (Tien et al., 1986). LiP catalyzes several oxidations in the side chains of lignin and related compounds by one-electron abstraction to form reactive radicals (Tien and Kirk, 1983; Kersten et al., 1985). The cleavage of an aromatic ring structure is also reported (Umezawa and Higuchi, 1987). The purified LiP from Brevibacillus laterosporous MTCC 2298 and Acinetobacter calcoaceticus NCIM 2890 efficiently decolorized the several sulfonated azo dyes (Gomare et al., 2008; Ghodake et al., 2009b). The LiP from A. calcoaceticus showed the pH optimum at 1.0, broad temperature optimum between 50 and 70 C, H2O2 optimum at 40 mM and substrate optimum at 100 mM for n-propanol oxidation. The pH 1.0 and 40 C temperature was found to be the optimum for the purified LiP from B. laterosporous. The Km value of purified B. laterosporous LiP for n-propanol was 1.6 mM. B. laterosporous LiP degraded the Methyl orange into p-(propanaldehyde) phenol as final product (Gomare et al., 2008). The LiP from A. calcoaceticus decolorized various azo dyes viz. Methyl

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red (98%), Methyl orange (96%), Direct blue GLL (87%), Direct red 5B (79%), Direct brown MR (75%), Reactive red 2 (73%) and Ethylene blue (84%). The classical reaction catalyzed by lignin peroxidase is shown in Fig. 2.4.
OCH3 OCH3 OH OCH3 HO

HO

OH

H 2O 2

OCH3

+
H3CO CHO H3CO OCH3 OCH3

[A]

[B]

[C]

Fig. 2.4. Reaction catalyzed by lignin peroxidase.

[A] = 1,2-bis (3,4-dimethoxyphenyl) propane-1,3-diol. [B] = 3,4-dimethoxybenzaldehyde. [C] = 1-(3,4-dimethoxyphenyl) ethane-1,2-diol.

Laccase Laccases (EC1.10.3.2) are the most numerous members of multicopper oxidase protein family. It catalyzes the oxidation of substituted phenolic and nonphenolic compounds in the presence of oxygen as an electron acceptor (Fig. 2.5) (Sharma et al., 2007). Phylogenetically, these enzymes have developed from small sized prokaryotic azurins to eukaryotic plasma proteins ceruloplasmin (Claus, 2003 and 2004). They contain four histidine rich copper binding domains, which coordinate the types 1, type 2, and type 3 copper atoms that differ in their environment and spectroscopic properties. The molecular weight of laccases varies

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from 60390 kDa (Call and Mucke, 1997). They are classified into two category viz. the blue laccases (presence of type 1 copper site) and laccases that lack the type 1 copper site. The blue laccases showed the absorbance maxima at 610 nm. The laccases that lack type 1 copper site did not show the absorbance maxima at 610 nm. The first report of prokaryotic laccase is from the rhizospheric bacterium Azospirillum lipoferum (Givaudan et al. 1993). Another laccase has been reported from a melanogenic marine bacterium Marinomonas mediterranea producing two different polyphenol oxidases (PPO), an unusual multi-potent PPO that is able to oxidize substrates characteristic of both tyrosinase and laccase (Solano et al. 1997). Laccase-like activity has also been found in other bacteria viz. CopA protein from Pseudomonas syringae and Pedomicrobium sp. (Ridge et al. 2007). Laccase decolorize azo dyes through a highly nonspecific free radical mechanism, thereby avoiding the formation of toxic aromatic amines (Chivikula and Renganathan, 1995). The purified laccase from Pseudomonas desmolyticum NCIM 2112 showed 100% decolorization of various dyes, including Direct blue-6, Green HE4B and Red HE7B (Kalme et al., 2009). The reaction catalyzed by laccase is shown in Fig. 2.6.

Cu O2 Cu O H Cu R e d u c e d la c c a s e H 2O 4 A H Cu

Cu H O Cu O H Cu P e r o x id e - l e v e l i n t e r m e d ia t e 2 Cu

H 2O

4 AH 2 Cu H O Cu O H 2 Cu R e s tin g e n z y m e 2 Cu H O Cu O H 2 Cu N a t iv e in t e r m e d i a t e 2 Cu 2 Cu

Fig. 2.5. Catalytic cycle of laccases.

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HO3S

S N N N N S SO3H

[A]

-e

HO3S

S N N N N S SO3H

[B]

-e
HO3S S N N N N S SO3H

[C]

Fig. 2.6. Reaction catalyzed by laccases.

[A] = ABTS. [B] = Intermediate ABTS radical. [C] = Oxidized form of ABTS.

Polyphenol oxidase Polyphenol oxidase (PPO) enzymes catalyse the o-hydroxylation of monophenols (phenol molecules in which the benzene ring contains a single hydroxyl substituent) to o-diphenols (phenol molecules containing two hydroxyl substituents). They can also further catalyse the oxidation of o-diphenols to produce o-quinones. The amino acid tyrosine contains a single phenolic ring that may be oxidised by the action of PPOs to form o-quinone (Fig. 2.7). Hence, PPOs may also be referred as tyrosinases. Polyphenol oxidases are enzymes that catalyze the oxidation of certain phenolic substrates to quinones in the presence of molecular oxygen. Polyphenol oxidases have been reported in the bacteria viz. Streptomyces glaucescens, Streptomyces antibioticus, Bacillus licheniformis,

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Bacillus natto and Bacillus sphaericus (Whitaker, 1994; Echiqo and Ristuko, 2001). The purified polyphenol oxidase was used for the oxidation of the colored and phenolic substances.
O O O O

H3N

CH CH2

H3N

CH CH2

OH OH OH

[A]

[B]

Fig. 2.7. Reaction catalyzed by polyphenol oxidase.

[A] = L-tyrosine. [B] = o-quinone.

b) Mediated biological dye decolorization High molecular weight sulfonated azo dyes are unable to pass through the cell membrane (Levine, 1991). It was suggested that the reduction of these dyes could occur through the mechanism that is not dependent on the transport in to the cell membrane. The earlier reports showed the role of redox mediators in an azo bond reduction using bacteria under anaerobic condition (Keck et al., 1997; Van der Zee et al., 2001; Dos Santos et al., 2007). Riboflavin in catalytic amounts significantly enhances the reduction of Mordant yellow 10 using anaerobic granular sludge (Field and Brady, 2003). 1-amino 2-napthol, one of the constituent amines of an azo dye, AO7, increased its decolorization rate, possibly by mediating the transfer of reducing equivalents (Mendez-Paz et al., 2005). The addition of synthetic electron carriers such as anthraquinone-2,6-disulphonate could also greatly enhance the decolorization of many azo dyes (Van der Zee et

Chapter No. 2.

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al., 2001). Keck et al. (1997) reported the first example of an anaerobic cleavage of azo dyes by redox mediators formed during the aerobic degradation of a xenobiotic compound. Cell suspensions of Sphingomonas sp. strain BN6 grown aerobically in the presence of 2-naphthyl sulfonate (NS) exhibited a 1020 fold increase in the decolorization rate of an azo dye amaranth under anaerobic conditions. Even the addition of culture filtrates of these cells could enhance anaerobic decolorization by cell suspensions grown in the absence of NS. The redox intermediates generated during aerobic degradation of aromatic compounds could also enhance the dye decolorization (Keck et al., 1997). The addition of culture supernatants containing metabolites of a dye-decolorizing E. coli NO3 strain enhanced its azo dye decolorization rate (Chang et al., 2004). c) Dye decolorization using organic and inorganic compounds Dye decolorization can occur from purely chemical reactions with inorganic compounds such as sulfide and ferrous ion that are formed as end products of metabolic reactions under anaerobic conditions. It has been shown that H2S generation by SRB resulted in the extracellular decolorization of the azo dyes (Yoo et al., 2000; Diniz et al., 2002). Sulfate influenced dye reduction correlated with biogenic sulfide formation under methanogenic conditions. In the absence of sulfur compounds, dye decolorization readily occurred in the presence of granular sludge, demonstrating the importance of enzymatic mechanisms. An analysis of decolorization kinetics in the batch reactors and in the laboratory scale anaerobic sludge bed reactors indicated relative importance of chemical dye reduction mechanisms in high rate anaerobic bioreactors (Van der Zee et al., 2003). Various inducers and stabilizers of oxidoreductive enzymes, such as CaCO3, indole, otolidine, veratrole and vanillin enhanced dye decolorization (Dawkar et al., 2008).

2.1.6. The general characteristics of dyes used for study Thirty different textile dyes from different dye classes have been used for the decolorization experiment. Table 2.4 represents the category, C.I. name, C.I.

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number and CAS number of studied dyes. The chemical structures of dyes of different classes are shown in Table. 2.5. Table 2.4. The category, C.I. name, C.I. number and CAS number of studied dyes. Sr. No. Triphenylmethane dyes 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Crystal violet Methyl violet Malachite green Methyl red Methyl orange Congo red Eriochrome Black T Trypan blue Xylidine ponceau Sudan IV Red HE7B Orange M2R Remazol blue 3R Navy blue HER Navy blue RX Red M5B Red HE3B Crystal violet-3 Basic violet-1 Basic green-4 Staining and indicator dyes Acid red-2 Acid orange-52 Direct red-28 Solochrome black T NA Acid red-18 Solvent red-24 Reactive dyes Reactive red-141 Reactive orange-4 Reactive blue-28 Reactive blue-171 Reactive blue-59 Reactive red-2 Reactive red-120 NA 17757 18260 NA NA NA 18200 25810 61931-52-0 12225-88-6 12225-82-0 12225-45-5 77905-32-5 NA 17804-49-8 61951-82-4 Remazol orange 3R Reactive orange-16 13020 13025 22120 NA NA 16255 26105 63451-28-5 547-58-0 573-58-0 1787-61-7 72-57-1 NA 85-83-6 42555 42535 42000 NA NA NA Comman name C.I. Name C.I. No. CAS No.

Chapter No. 2. 19 20 21 22 23 24 25 26 27 28 29 Red HE8B Turquoise blue H5G Navy blue HE2R Green HE4BD Scarlet RR Brown 3RL Navy blue 3G Direct brown MR Direct red 5B Direct blue GLL

Review of literature Reactive red-152 Reactive blue-25 Reactive blue-172 Reactive green 19 Disperse dyes Disperse red-54 Disperse brown-118 Disperse blue-79:1 Direct dyes Direct brown-2 Direct red-81 Direct blue-71 NA NA NA 11131 11344 NA NA NA NA NA

Page No. - 28 71872-80-5 61951-85-7 NA 85782-76-9 NA NA NA NA NA NA

Golden yellow HER Reactive yellow-84A

11152:2 NA

NA- Not available Table 2.5. The chemical structures of dyes of different dye classes were used for study.

Dyes Methyl violet

Chemical Structure Triphenylmethane dyes

N

H 2N

Chapter No. 2. Crystal violet

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N

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Malachite
N

green

Staining and indicator dyes Methyl orange Methyl red

HO3S N
COOH N N N(CH3)2

N(CH3)2

Congo red

NH 2 N N N N

NH 2

SO 3H

SO 3 H
HO OH N N S O 3H

Eriochrome black T

NO2

Trypan blue
HO3S

NH2

OH N N SO3H H3C N N HO3S

OH

NH2

SO3H

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OH N N N N H3C CH 3

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Reactive dyes Red HE7B

SO3H NN HO3S SO3H HO NH N NH NH HO3S Cl N N N N NN SO3H SO3H Cl N NH OH HO3S

Remazol Orange 3R Red M5B

HO3SO H2C H2C

O S O N N

HO

NHCOCH3 HO3S

HO 3 S

SO 3 H

NH N Cl N N

OH

Cl
NH 2 OH NH 2 O

Navy HE2R

blue
H2C

O S O HO 3 S
Cl NH NH

SO 3 H

Golden Yellow HER

SO3H NN HO3S SO3H

NH

N N

N NH

SO3H N N SO3H

SO3H SO3H

Direct Brown MR
HO

O N N

HO 3S
N

N OH

NH2

HO

Disperse dyes

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N N H N Cl

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N O

Brown 3RL
Cl N N

Cl O O CH2 C O C NH NH

Direct dyes Direct red 5B

HO3S N N N N HO3S OH H O N C
HO3S N N N N OH
H O 3S N N N N N N

Direct MR Direct GLL

brown
HO

NH2

HO

blue

S O 3H

S O 3H S O 3H

2.2 Bisphenol A
Endocrine disruptors induce the adverse effects into wildlife and humans owing to their ability of interfering with an endocrine system. Bisphenol A (2,2bis(4-hydroxyphenyl) propane) has been chosen as model of the endocrine disruptors. Bisphenol A is an organic pollutant which affects the reproductive system of wildlife and humans by mimicking or interfere the action of endogenous gonadal steroid hormones. The chemical characteristics of bisphenol A are shown in Table 2.6.

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Table 2.6. Chemical characteristics of bisphenol A.

Name compound

of Chemical structure
CH3 HO CH3 OH

Molecular formula C15H16

CAS number 80-05-7

IUPAC name 4,4'dihydrox y-2,2diphenyl propane

Bisphenol A

2.2.1. History Bisphenol A was first reported by Dianin (Dianin, 1891). It was prepared by the condensation of acetone with two equivalents of phenol. The reaction catalyzed using an acid, such as hydrochloric acid or a sulfonated polystyrene resin.

2.2.2. Uses Global production of bisphenol A in 2003 was estimated to be over 2 million metric tonnes (Lang et al., 2008). Bisphenol A is used primarily to make plastics. The products containing bisphenol A based plastics have been in commerce for more than 50 years. It is used in the synthesis of polyesters, polysulfones, polyether ketones, as an antioxidant in some plasticizers and polymerization inhibitor in PVC. It is a key monomer in the production of polycarbonate plastic and epoxy resins. Polycarbonate plastic is clear and nearly shatter-proof. It is used to make a variety of common products including baby and water bottles, sports equipment, medical and dental devices, dental fillings and sealants, lenses, CDs, DVDs, and household electronics. Epoxy resins containing bisphenol A is used as inside coatings of almost all food and beverage cans

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(Erickson, 2008). Bisphenol A is also a precursor to the flame retardant and tetrabromobisphenol A. Bisphenol A is used as a fungicide.

2.2.3. Health effect Bisphenol A has low acute toxicity with an oral LD50 of 3250 mg kg-1 in rats, but it is an endocrine disruptor (Okada et al., 2008; vom Saal and Myers, 2008). Low doses of bisphenol A can mimic the bodies own hormones, possibly causing negative health effects (OConnor and Chapin, 2003). Thus it is concern that the long term low dose exposure of bisphenol A may induce chronic toxicity in humans. The endocrine disrupting properties of bisphenol A have been extensively investigated and more than 100 studies have been published "raising health concerns" about the chemical (Table 2.7). These studies have demonstrated developmental toxicity, carcinogenic effects, and possible neurotoxicity at low doses in animal models. Recent studies suggest linkage to obesity by triggering fat cell activity. Bisphenol A exposure during development has carcinogenic effects and produce precursors of breast cancer (Grossman, 2007). Table 2.7. Selected studies on low dose bisphenol A exposure in animals.

Dose (g/kg/day) 0.025

Effects (measured in studies of mice or rats, descriptions (in quotes) are from Environmental Working Group)a,b Permanent changes to genital tract. Changes in breast tissue that predispose cells to hormones and carcinogens.

Study Year 2005c

0.025

2005d

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Increased prostate weight 30%. Lower bodyweight, increase of anogenital

distance in both genders, signs of early puberty and longer estrus.

2002f

2.4 2.5

Decline in testicular testosterone. Breast cells predisposed to cancer. Prostate cells more sensitive to hormones and cancer. Decreased maternal behaviors. Reversed the normal sex differences in brain structure and behavior. U.S. human exposures limit (not a result from an animal study, but a guideline set by EPA).

2004g 2007h

10

2006i

10

2002j

30

2003k

50

1998l

EWG, 2007; bMittelstaedt, 2007; cMarkey et al., 2005; dMunoz-de-Toro et al.,

e

2005;
h l

Nagel et al., 1997; fHonma et al., 2002; gAkingbemi et al., 2004;

Murray et al., 2007; iHo et al., 2006; jPalanza et al., 2002; kKubo et al., 2003;

EPA, 1988.

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2.2.4. Remediation of bisphenol A Increased use of products based on BPA has increased the possibility of environmental contamination. High levels of BPA were identified from leachates of waste landfill (Yamamoto et al., 2001; Filho et al., 2003). The levels of BPA in the leachates of hazardous waste landfill ranged from 1.3 to 17,200 ng ml-1 (average 269 ng ml-1). The leaching of BPA from plastic wastes into water was also reported. The highest levels (9.8 and 139 ng g-1) were identified from polyvinyl chloride products that use BPA as a stabilizer in their manufacturing process (Yamamoto and Yasuhara, 1999). BPA has an acute toxicity in the range of about 1 to 10 mg l-1 for number of freshwater and marine species. Thus, the development of remediation methods to remove BPA is needed urgently. The various physiochemical and biological remediation methods are used to treat the endocrine disruptors, including bisphenol A. 2.2.4.1. Physiochemical methods The physiochemical methods used for degradation of bisphenol A are described in Table 2.8. Table 2.8. Methods used for the treatment of bisphenol A.

Process Photo catalytica

Methodology Irradiation experiments were carried out in a cylindrical reactor, with a 250W metal halide lamp (365 nm) at a fixed flow rate throughout the

Comment This method required the high energy UV radiation. Degradation efficiency decreased with increased bispehnol A concentration. It required the titanium oxide as photo catalyst.

Chapter No. 2. experiment. Photo-Fentonb

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Irradiation reaction was carried out in presence of fenton reagents viz, Fe (II) and H2O2.

Degradation rate were enhanced using the fenton reagent.

Thermal decompositionc

The bisphenol A was subjected to higher temperature (300-500 C).

It required the higher temperature.

Horikoshi et al., 2007; bKatsumata et al., 2004; cJang et al., 2005.

2.2.4.2. Biological methods The bisphenol A is metabolized by microorganisms distributed in the environment. Various enzymes such as lignin peroxidase, Mn peroxidase, laccase and polyphenol oxidase from different microorganisms were involved in the degaradation of bisphenol A. I. Bacteria Many bacteria capable of degrading bisphenol A have been identified from the soils (Sasaki et al., 2005), river (Ike et al., 2000; Kang and Kondo, 2002a and b; Kang and Kondo, 2004) and wastewater treatment plants (Lobos et al., 1992; Spivack et al., 1994) (Table 2.9). Several studies reported about >90% of bisphenol A removal between the influent and the effluent of a wastewater treatment process (Staples et al., 1998; Furhacker et al., 2000). The Kang and Kondo (2002a) found that most bacteria (10 out of 11) isolated from three river waters had bisphenol A biodegradability, but there were differences in removal rates of bisphenol A (1891%) and only two strains viz. Pseudomonas sp. and a Pseudomonas putida strain showed high bisphenol A biodegradability (about 90%).

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The MV1 strain utilized BPA as the sole carbon and energy source through major and minor pathways of BPA metabolism (Spivack et al., 1994). The major pathway produced two primary metabolites, 4-hydroxyacetephenone and 4hydroxybenzoic acid, and the minor pathway also produced two primary metabolites, 2,2-bis(4-hydrozyphenyl)-1-propanol and 2, 3- bis(4-hydroxyphenyl)1,2-propanediol. The total carbon analysis of metabolites obtained after biodegradation of bisphenol A suggests 60% mineralization of the carbon to CO2, 20% associated with the bacterial cells and 20% concerted to soluble organic compounds. Sasaki et al. (2005) reported that the bacterial cytochrome P450 system is involved in the bisphenol A metabolism. The microbial peroxidase and laccase degrade bisphenol A by oxidoreductive mechanism (Sakurai et al., 2001). The products formed after degradation of bisphenol a using laccase did not show the estrogenic activity (Ike et al., 2002).

II. Fungi Many fungi were used for the degradation of bisphenol A, but unable to degrade the high concentration of bisphenol A (Yim et al., 2003; Chai et al., 2005) (Table 2.9). Among the fungi, Fusarium sporotrichioides NFRI-1012, Fusarium moniliforme 2-2, Aspergillus terreus MT-13 and Emericella nidulans MT-98 were more effective for bisphenol A biodegradation (Chai et al., 2005). Bisphenol A biodegradation using fungi caused mainly due to lignin degrading enzymes, such as manganese peroxidase (MnP) and laccase (Hirano et al., 2000; Fukuda et al., 2001; Uchida et al., 2001; Suzuki et al., 2003; Lee et al., 2005). MnP is a heme peroxidase and oxidizes phenolic compounds in the presence of Mn(II) and H2O2. The bisphenol A degradation rate of oxidoreductieve enzymes was higher in the presence of suitable redox mediators viz. 1-hydroxybenxotriaxzole (HBT) and 2,2azino-bis (3- ethylbenzthiazoline-6-sulfonate) than with laccase alone.

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Table 2.9. Microorganisms capable of biodegrading or metabolizing bisphenol A.

Microorganisms Bacteria

Strains Psudomonas paucimobilis FJ-4 Pseudomonas sp. Pseudomonas putida Streptomyces sp. Sphingomonas sp. strain AO-1

References Ike et al., 2000 Kang and Kondo, 2002a Kang and Kondo, 2002a Kang et al., 2004 Sasaki et al., 2005 Hirano et al., 2000 Tsutsumi et al., 2001 Tsutsumi et al., 2001 Fukuda et al., 2001; Uchida et al., 2001 Suzuki et al., 2003 Suzuki et al., 2003 Yim et al., 2003 Chai et al., 2005 Chai et al., 2005 Chai et al., 2005 Chai et al., 2005 Lee et al., 2005 Lee et al., 2005

Fungi

Pleurotus ostreatus O-48 Phanerochaete chrysosporium ME-446 Trametes versicolor IFO-7043 Trametes villosa

Phanerochaete chrysosporum ME-446 Trametes versicolor IFO-6482 Aspergillus fumigatus Fusarium sporotrichioides NFRI1012 Fusarium moniliforme 2-2 Aspergillus terreus MT-13 Emericella nidulans MT-98 Stereum hirsutum Heterobasidium insulare

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2.3. Research objective and thesis outline

The objective of this dissertation is to explore the methods for the complete biodegradation of textile dyestuffs, textile industry effluent and model endocrine disruptor bisphenol A using isolated microorganisms and their enzymes. Three different approaches are considered for the biodegradation. i. Biodegradation of textile dyestuffs and bisphenol A using pure culture of an isolated microorganism. ii. Biodegradation of textile dyestuff and bisphenol A using purified enzymes obtained from isolated microorganism. iii. Biodegradation of textile industry effluent using pure culture of isolated microorganism. The brief objectives of this dissertation were as follows: Isolation of efficient microorganisms from textile dye contaminated site. Identification of microorganism on the basis of biochemical characteristics and 16S RNA sequence. Decolorization of textile dyes and bisphenol A using pure bacterium. Evaluation of enzymatic status viz. lignin peroxidase, laccase, aminopyrine N-demethylase, azoreductase, NADH-DCIP reductase and MG reductase during degradation of dyes and bisphenol A. Kinetics of the dye decolorization. Influence of inorganic and organic compounds on the deolorization of dyes. Purification and characterization of significantly induced enzyme and its application to remove textile dyestuff and bisphenol A. Identification of metabolites obtained after biodegradation using analytical techniques viz. UV-Vis spectroscopy, TLC, HPLC, FTIR and GC-MS.

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Elucidation of proposed pathway of the biodegradation of textile dyestuff and bisphenol A. Application of isolated microorganisms for the treatment of textile industry effluent. Toxicity study of textile dyestuff, textile industry effluent and metabolites obtained after its decolorization.