INTRODUCTION:

Our biosphere is under constant threat from constituting environmental pollution. Water pollution is a state of deviation from pure conditions partially, wholly or largely as a byproduct of human activity through direct or indirect effects of changes in energy patterns, chemical and physical composition in nature and abundance of organisms. The function and properties affecting the quality of water is of vital concern for humanity, since it is directly linked with human welfare. Water supply sources like ground water and surface water are related and interconnected by the hydrological cycles. The quality of life in earth is linked inextricably to the overall quality of the environment. Whether water is used as a habitat or to meet drinking and irrigation demands, the maintenance of quality of water is crucial for the survival of life [1].

The textile industry is one of the industries that generate a high volume of waste water. Strong colour of the textile waste water is the most serious problem of the textile waste effluent. The disposal of these wastes into receiving water causes damage to the environment. Dyes may significantly affect photosynthetic activity in aquatic habitat because of reduced light penetration and may also be toxic to some aquatic life due to the presence of aromatics, metals, chlorides and other toxic compounds [2].

Azo dyes represent the largest and most versatile class of synthetic dyes. More than 3000 different varieties of azo dyes are extensively used in the textile, paper, food, cosmetics and pharmaceutical industries [3]. Approximately 10 - 15% of the dyes are release into the environment during manufacturing and usage. Since some of the dyes are harmful, dye-containing wastes poor important environmental problems. These dyes are poorly biodegrabale because of their structures and treatment of wastewater containg dyes usually involves physical and/or chemical methods such as adsorption, coagulation, flocculation, oxidation, filtration and electrochemical methods. Over the past decades, Biological decolonization has been investigated as a method to transform, degrade or mineralize azo dyes. Moreover, such decolonization and degradation is an environmentally friendly and cost competitive alternative to chemical decomposition possess. Unfortunately, most azo dyes are recalcitrant to aerobic degradation by bacterial cells. However, there are few known microorganisms that have the ability to reductively cleave azo bonds under aerobic conditions.

Dye:-AZO DYE DIRECT ORANGE 102/ORANGE 102

DIRECT ORANGE 102

Chemical Name: Trisodium 4-((1-hydroxy-6-((((5-hydroxy-6-(phenylazo)-7-sulphonato-2-naphthyl) amino) carbonyl)amino)-3-sulphonato-2-naphthyl)azo)benzoate;

Molecular Formula: C₃₄H₂₁N₆Na₃O₁₁S₂

Formula Weight: 822.66345

λ_max: 480 nm

Uses of Direct Orange 102 Dyes

Ink Dyestuffs, Leather Dyestuffs, Paint. Direct Orange 102 dyes are usually cheap and easily applied for use on cotton and other cellulose fibres such as cotton, rayon, and linen.

MATERIALS AND METHODS:

Direct Orange 102 was purchased from Nagpur (MH). All microbiological media and medium ingredients were purchased from HiMedia Laboratories (Mumbai, MH, India). Soil sample was collected in autoclaved bottle from dumping area near the Nagpur (MH) India and kept in cold condition till the sample was brought to the laboratory. For lignin source bagasse particles were used.

Isolation and Identification Fungi

Soil sample were subjected to serial dilution and performed pour plate technique by adding sterile cool molten Potato Dextrose medium containing Direct Orange 102 Dye on plate and kept the plate for incubation in dark for 4 days. Appearance of zone of clearance around growth on potato dextrose agar with stress amount of dye confirmed the presence of dye degrading fungal colonies.

Microscopic study

The fungal colonies obtained on potato dextrose agar were taken on clean grees free slide and slam with lactophenol cotton blue the slide was identified under microscope [4].

Organism and culture condition

Isolated and identified fungi were maintained at 4⁰C and frequently cultured on Potato Dextrose agar slant. The decolourization experiments were carried out at room temperature for fungi.

Enzyme Assay

Variety of biotransformation enzymes were studied viz ligninperoxidase, NADH-dichlorophenol indophenols reductase (NADH-DCIP reductase), and tyrosinase. Activities of ligninperoxidases, were assayed spectrophotometrically in the cell free extract at room temperature where blank contained all the components except enzyme [4].

Enzyme assayed method

The fungus was tested for extracellular secretion of ligninperoxidase in liquid culture growth medium. The growth medium consist of 10 g glucose, 1.32 g ammonium tartarate, 0.2 g KH₂PO₄, 50 mg MgSO₄, 10 mg CaCI₂, 10 µg thiamin per liter and 1 ml of a solution containing per liter 3g MgSO₄.7H₂O, 0.5 g MnSO₄.H₂O, 1 g NaCI, 100 mg FeSO₄.7H₂O, 185 mg CoCI₂.6H₂O, 80 mg CaCI₂, 180 mg ZnSO₄.7H₂O, 10 mg CuSO₄.5H₂O, 10 mg AIK(SO₄), 10 mg H₃BO₃, 12 mg Na₂MoO₄, 1.5 mg nitrilotriacetate. The pH of basal medium was adjusted to 0.4 with 20 mM dimethyl succinate. Growth media containing natural lignin source like bagasse particles was prepared by adding 1 g of bagasse particle to 40 ml of growth medium in 100 ml culture flasks which were sterilized. The sterilized growth medium was inoculated with small pieces of mycelium of isolated fungus under aseptic condition and the fungal culture was incubated at room temperature for 5 days. After the incubation of 5 days aliquots of growth medium were withdrawn followed by filtrations by filter paper. The filtrate was used as a source of enzyme.

Assay of ligninperoxidase activity[5]

The ligninperoxidase activity was assayed using n-propanol as the substrate, H₂O₂ (0.4 mM) and 50 mM of tartaric acid/disodium tartarate (pH 2.5) at 25ºC and monitoring the formation of propionaldehyde at wavelength 300 nm with UV/Vis spectrophotometer. The value of steady state velocities were average of triplicate measurements. The protocols were as follows,

Table-1 Protocol for assay of ligninperoxidase All additions are in ‘ml’

Reagents	Blank	1	2	3
Enzyme solution	0	0.4	0.4	0.4
Substrate(n-propanol)	0.8	0.8	0.8	0.8
H₂O₂	0.4	0.4	0.4	0.4
Tartaric acid/disodium tartarate Buffer	3.8	3.4	3.4	3.4

Stand for few minutes and read absorbance at 300 nm

Enzymatic Characteristics of ligninperoxidase

Determination of pH and temperature optima using n-propanol as substrate and monitoring the formation of propionaldehyde at wavelength 300 nm spectrophotometrically performed by following protocol.

Table-2 Protocol for pH

All additions are in ‘ml’

Reagents	pH
Reagents	Blank	2	4	7	9	10	11
Enzyme solution	0	0.4	0.4	0.4	0.4	0.4	0.4
Substrate (n-propanol)	0.8	0.8	0.8	0.8	0.8	0.8	0.8
H₂O₂	0.4	0.4	0.4	0.4	0.4	0.4	0.4
Tartaric acid/disodium tartarate Buffer	3.8	3.4	3.4	3.4	3.4	3.4	3.4

Maintaining the pH of the reaction mixture at pH-2, pH-4, pH-7, pH-9, pH-10 and pH-11 respectively by using tartaric acid/disodium tartarate buffer (50 mM) and read absorbance at 300nm.

The optimum pH value of an enzyme catalyzed reaction was determined at different pH values and a plot of absorbance vs. pH was drawn.

Table-3 Protocol for temperature

All additions are in ‘ml’

Reagents	Temperature (⁰C)
Reagents	Blank	5	20	30	40	50
Enzyme solution	0	0.4	0.4	0.4	0.4	0.4
Substrate(n-propanol)	0.8	0.8	0.8	0.8	0.8	0.8
H₂O₂	0.4	0.4	0.4	0.4	0.4	0.4
Tartaric acid/disodium tartarate Buffer	3.8	3.4	3.4	3.4	3.4	3.4

Maintaining the temperature of the reaction mixture at 5⁰C, 20⁰C, 30⁰C, 40⁰C and 50⁰C respectively by using tartaric acid/disodium tartarate buffer (50 mM) and read absorbance at 300nm.

The optimum temperature value of an enzyme catalyzed reaction was determined at different temperature values and a plot of absorbance vs. temperature was drawn.

Decolorization study in fungus

The isolated fungus were tested for its ability to decolorize dye, textile dye Direct Orange 102. 100 ml of Sabouraud Maltose broth sterile medium with dye was inoculated with fungi spore. The flasks were incubated at room temperature for 4 days. Samples were centrifuge at 9000 rpm for 5 min. Decolorization was assayed by measuring absorbance of the supernatant. Initial and final absorbances were recorded.

Immobilization of fungus

Dissolve 30 gm of sodium alginate in 1 lit to make 3% solution. Mix isolated fungus with 50 ml of 3% sodium alginate solution. The beads were formed by dripping the polymer solution into 50 ml of 0.2 M CaCl₂ solution with dropper. Kept beads at 0⁰C for overnight [7].

Decolorization with the help of immobilized fungus

100 ml of sterile Sabouraud Maltose broth contain 30 mg of dye Direct Orange 102 was inoculated with 100 beads contain immobilized fungi and incubated at room temperature for 4 days with shaking. Sample was centrifuge at 9000 rpm for 5 min. Decolorization was assayed by measuring absorbance of the supernatant. Initial and final absorbances were recorded.

RESULTS AND DISCUSSION:

Present study deals with the screening of the microorganism resulted in isolation of fungus capable of degrading azo dye. The sample collections for isolation of microorganism from soil waste water indicate the natural adaptation of these microorganisms to survive in the presence of toxic dye. The isolated fungus was able to decolourize dye. The isolated fungus were co-immobilized in Ca-alginate beads and its decolourization ability were determined. Enzyme assay showed production of extracellular enzyme ligninperoxidase responsible for decolourization.

Isolation of fungus

The soil sample collected were screened for fungus subjected to serial dilution and pour plate method was performed. Zone of decolourization with trace amount of dye was observed. The species identified by its different characteristic and structural arrangement.

Identification of fungus

Macroscopic features of isolated green fungus

Growth rate is rapid and texture of colonies varies from downy to powdery. Surface colony color is gray to green.

Macroscopic features of isolated white fungus

Colonies varied in color from white to cream and were velvety with cottony tufts, with irregular margin with pale reverse color.

Microscopic features of isolated green fungus

The conidiophore is septate and ends in a whorl of short branches, the metulae, each of which bears a whorl of little branches. The spore heads are seen to be looser. They are more brush-like in appearance.

Microscopic features of isolated white fungus

Hyaline septate hyphae; simple, well-differentiated conidiophores; catenate, hyaline, smooth-walled, single-cell arthroconidia produced at the end of conidiophores either in clusters or in irregular groups.

Enzyme Assay

The enzyme assay was used to study the secretion of ligninperoxidase from isolated fungus by using bagasse particles as a natural lignin source using n-propanol as a substrate. Assay of ligninperoxidase in presence of bagasse particles at a wavelength 300 nm spectrophotometrically. The ligninperoxidase activity secreted by fungus in presence of bagasse particles in liquid culture growth medium was assayed at absorbant value 0.646 and 0.783 of green and white fungus (Table-4). Ligninperoxidase activity was assayed by modifying the procedure in which veratryl alcohol (2 mM) was used as substrate [8], enzyme activities were calculated using the extinction coefficients of veratraldehyde (9,300 M-1 cm-1) at 310 nm [9].

TABLE-4: Assay of ligninperoxidase activity secreted by isolated fungus

Sr.No.

Fungus

Absorbance at 300 nm

Average

Green fungi

0.660

0.639

0.638

0.646

White fungi

0.788

0.803

0.759

0.783

Enzymatic characteristic of ligninperoxidase

Enzymatic characteristics (pH and temperature) of ligninperoxidase were determined using n-propanol as a substrate and monitoring the formation of propionaldehyde at wavelength 300 nm spectrophotometrically.

Effect of pH on Ligninperoxidase activity secreted by green and white fungus

The optimum pH value for ligninperoxidase secreted by green and white fungus of the present work was found to be 7 (Table-5, 6 and Fig no.1, 2) which is different from the pH value such as 4.0 and 2.3 respectively.

TABLE-5: Effect of pH on ligninperoxidase secreted by Green fungi

Sr. No.	Solution	pH	Absorbance at 300 nm
1.	B		0.000
2.	S1	2	0.543
3.	S2	4	0.683
4.	S3	7	0.826
5.	S4	9	0.649
6.	S5	10	0.450
7.	S6	11	0.412

Fig no.1: Effect of pH on lignin peroxide secreted by Green fungi

TABLE-6: Effect of pH on ligninperoxidase secreted by White fungi

Sr. No.	Solution	pH	Absorbance at 300 nm
1.	B		0.000
2.	S1	2	0.665
3.	S2	4	0.761
4.	S3	7	0.840
5.	S4	9	0.740
6.	S5	10	0.235
7.	S6	11	0.114

Fig no.2: Effect of pH on lignin peroxidase secreted by white fungi

Effect of temperature on ligninperoxidase activity secreted by green and white fungus

The optimum temperature value for ligninperoxidase secreted by green and white fungus of the present work was found to be 30⁰C and 20⁰C (Table-7, 8 and Fig no.3, 4) which is similar to the temperature value such as 30⁰C, 25⁰C and 22⁰C respectively.

TABLE-7: Effect of temperature on ligninperoxidase secreted by Green fungi

Sr. No.	Solution	Temperature	Absorbance at 300 nm
1.	B		0.000
2.	S1	5⁰C	0.531
3.	S2	20⁰C	0.663
4.	S3	30⁰C	0.683
5.	S4	40⁰C	0.645
6.	S5	50⁰C	0.562

Fig no.3: Effect of temperature on lignin peroxidase secreted by Green fungi

TABLE-8: Effect of temperature on ligninperoxidase secreted by White fungi

Sr. No.	Solution	Temperature	Absorbance at 300 nm
1.	B		0.000
2.	S1	5⁰C	0.741
3.	S2	20⁰C	0.833
4.	S3	30⁰C	0.898
5.	S4	40⁰C	0.841
6.	S5	50⁰C	0.762

Fig no.4: Effect of temperature on lignin peroxidase secreted by white fungi

Dye Decolorization by Immobilized Isolated fungus

The dye degradation (%) by isolated fungus co-immobilized in Ca-alginate beads was found to be 18.87% and 19.61% respectively for white and green fungus.

Photo plate.1 Isolated fungus

Photo plate.2 Isolated White fungus showing decolourization zone

Photo plate.3 Isolated Green fungus showing decolourization zone

CONCLUSION:

The dye degradation property was exhibited by fungus. The fungus possesses enzyme ligninperoxidase for dye degradation. The isolated fungus were potent for the secretion of ligninperoxidase in liquid culture growth medium using bagasse particles as a lignin source. The assay and enzymatic characteristics (pH and temperature) of ligninperoxidase was carried out using n-propanol as substrate. Ligninperoxidase having pH 7 and temperature 30⁰C is more suited for its activity. The isolated bacteria and fungus were immobilized and their dye decolourizing activity was determined

REFERENCE:

1. Manivannan M, Reetha D and Ganesh P. Decolourization of Textile Azo Dyes by using Bacteria Isolated from Textile Dye Effluent. Journal of Ecobiotechnology. 3(8): 2011:29-32

2. Husseiny M. Biodegradation of the Reactive and Direct dyes using Egyptian isolates. Journal of Applied Science Research 4(6): 2008; 599-606.

3. Maximo, C., Amorim, M.T.P., and Costa Ferreira, M. Biotransformation of industrial reactive azo dye by Geotrichum sp. Enzyme and Microbial Technology 32: 2003; 145-151.

4. Chaube P., Moghe S., Indurkar H. Biodegradation and decolourisation of Direct violet 51 and Tetrazine dye from isolated fungus (Type I) Asiatic J. Biotech. Res. 01(03): 2010;220-226.

5. Dhanve RS, Shedbalkar UU, Jadhav JP. Biodegradation of diazo reactive dye Navy Blue HE2R (Reactive Blue 172) by an isolated Exiguobacterium sp. RD3. Biotechnol Bioproc E. 13: 2008;53–60.

6. Shanmugam V, Kumari M, Yadav KD. n-Propanol as a substrate for assaying the lignin peroxidase activity of Phanerochaete chrysoporium. Indian J. Biochem. and Biophys. 36: 1999. 39-43.

7. Meera Yadav, Sunil Kumar Singh, Kamlesh Singh Yadav and Kapil Deo Singh Yadav. “Purification of lignin peroxidase from Hexagona tenuis MTCC-1119 and it’s kinetic properties in aqueous medium containing miscible organic solvents”. Indian Journal of Chemistry. Vol 49B: 2010;489-494.

8. Meera Yadav and K D S Yadav. “Enzymatic characteristics of ligninperoxidase from Penicillum citrinum, Fusarium oxysporum and Aspergillus terreum using n-propanol as substrate”. Indian J. Biochem. and Biophys. Vol 43: 2006;48-51.

9. Rajeshwari Sivaraj,C. Agnes Mariya Dorthy and R Venckatesh - Isolation, Characterization and growth kinetics of bacteria metabolizig textile effluent. J Biosci Tech. 2(4) : 2011; 324-330.

Received on 21.02.2014 Accepted on 12.03.2014

Asian J. Pharm. Tech. 2014; Vol. 4: Issue 1, Pg 22-27