Title of Invention	A PROCESS FOR REMOVAL OF DYES IN DYE-CONTAINING WASTE-WATERS AND SOIL
Abstract	Title: A process for removal of dyes such as azo dyes, hetrocyclic & polymeric dyes from dye-containing waste-waters and soil A process for removal of dyes such as azo dyes, hetrocyclic & polymeric dyes from dye-containing waste-waters and soil which comprises, growing the white-rot fungus strain Flavodon flavus such as here in described, any conventional nutrient medium containing assimilable C and N source, with optinally having salinity up to 15 parts per thousand(ppm) for at least 4 days, then contacting the said biomass with waste-waters or soil containing dyes for a minimum period of 5 days followed by removing the fungal biomass by conventional methods in case of waste-waters to get water devoid of dyes

Title of Invention

A PROCESS FOR REMOVAL OF DYES IN DYE-CONTAINING WASTE-WATERS AND SOIL

Abstract

Title: A process for removal of dyes such as azo dyes, hetrocyclic & polymeric dyes from dye-containing waste-waters and soil A process for removal of dyes such as azo dyes, hetrocyclic & polymeric dyes from dye-containing waste-waters and soil which comprises, growing the white-rot fungus strain Flavodon flavus such as here in described, any conventional nutrient medium containing assimilable C and N source, with optinally having salinity up to 15 parts per thousand(ppm) for at least 4 days, then contacting the said biomass with waste-waters or soil containing dyes for a minimum period of 5 days followed by removing the fungal biomass by conventional methods in case of waste-waters to get water devoid of dyes

Full Text	This invention relates to a process for removal of dyes in dye containing waste-waters and soil. The present invention particularly relates to a process for removal of synthetic dyes in dye containing waste-waters and soil using the lignin-modifying white-rot fungus, Flavodon flavus (Kl) Ryv., NIOCC isolate 312. Further this invention relates to 80-100% removal within four days after addition of synthetic dyes such as azo dyes, heterocyclic and polymeric dyes to the 4 day old culture of Flavodon flavus. Textile and dyestuff industries are the major contributors to industrial pollutants containing dyes. These dyes are highly stable in light and are also resistant to microbial attack. Most of these dyes are azo dyes and under anaerobic conditions, the azo linkages are reduced to form aromatic amines and these are toxic and carcinogenic (Meyer, U. 1981. Biodegradation of synthetic organic colorants. Federation of European Microbiological Societies Symposium. 12: 371-385). Due to the. importance attached to prevention of environmental pollution, environmental agencies all over the world are imposing strict regulations for mitigation of pollution from industries. The effluents from the textile industries containing fast colored dyes are a major source of concern for environmentalist since such dyes beside causing aesthetic damage to sites, are toxic and carcinogenic. Industrial effluents from textiles, paper and pulp industries and leather industries contain chromogenic substances as well as high concentrations of salts, especially chlorides and sulfates (Public Health Engineering-Design in Metric waste-water treatment by R. E. Bartlett, 1971, Applied Science Publishers Ltd., London). Remediation of such dye containing waste-waters using biological methods is termed bioremediation Thus, this invention particularly relates to degradation of synthetic dyes in the presence of salts by the fungus F. flavus deposited at the National Institute of Oceanography, Dona Paula, Goa 400004, India and having the accession number NIOCC 312, in textile waste-water treatment, and bioremediation of soil. The said fungus can be grown in synthetic media prepared with either distilled water or 50% artificial sea water. The said fungus can also be grown in conventional media or in powdered sugarcane bagasse suspended in distilled water or 50% artificial sea water to raise large biomass of the fungus for application in field trials for bioremediation in the presence of sea water or fresh water. The said fungus thus grown can be immobilized by conventional methods and used for removal of various synthetic dyes from contaminated soil as well as aquatic habitats. The said fungus, F. flavus produces lignin-modifying enzymes such as manganese-dependent peroxidase E. C. 1.11.1.7 (MNP), lignin peroxidase, E. C. 1.11.1.7 (LIP) and laccase, E. C. 1.10.3.2 when grown on sugarcane bagasse suspended in plain distilled water or in 50% artificial sea water or in conventional media prepared with distilled water or 50% artificial sea water. By virtue of these lignin-modifying enzymes which break down a broad range of polymeric substrates, this fungus is useful in degradation of pollutants such as synthetic dyes. Thus degradation of synthetic dyes in waste-waters can be achieved by using the fungus F. flavus in fresh water as well as estuarine conditions. Normally, the textile dye waste-waters disposal includes physical-chemical treatment, waste minimization and biological treatment. Biological treatment includes biological pretreatment with activated sludge of textile waste-waters, and treatment in stabilization ponds (Groff, K.A. 1992, Textile waste. Water-Environment Research, 64: 725-729.). Unfortunately, waste-water treatment facilities are often unable to completely remove commercial dyestuff from contaminated waters and thus contribute to pollution of aquatic habitats. Some of these synthetic dyes are carcinogenic and are suggested to be responsible for tumor growth in some species of fish (see Bumpus J. A., B. J. Brock. 1988. Biodegradation of crystal violet by the white-rot fungus Phanerochaete chrysosporium. Applied and Environmental Microbiology, 54:1143-1150). Various organisms have been tried for degradation of dyes in textile waste-waters and bioremediation. (i) Reference may be made to the Japanese patent wherein a green alga Chlorella vulgaris is used for degradation of methylene blue by irradiating a microalga fermentor to generate OH radicals which in turn help in degradation of the dye (Japanese patent JP06047394, Title: Organic dye degradation in waste-water, Date of issue: 22.02.94). The method has disadvantage as it involves irradiating the fermentor containing microalga and thus becomes expensive. (ii) Reference may be made to a U.S. patent wherein, living, dead, free, immobilized white-rot fungi Myrothecium or Ganoderma spp. have been employed for adsorption, dye degradation and color removal (US patent US5091089, Title: Decolorization of dye-containing waste-water, Date of issue: 25.02.92). These fungi have not been tested for their efficiency in color removal in the presence of sea salts. (iii) Reference may be made to US patent wherein, white-rot fungus Phanerochaete chrysosporium and actinomycetes Streptomyces spp are used in degradation of low nitrogen medium. Fig. 6c. Shows production of laccase by the isolate # 312 when grown in the low nitrogen medium. Fig. 7a. shows production of manganese-dependent peroxidase by the isolate # 312 when grown in the low nitrogen medium prepared with half-strength artificial sea water. Fig. 7b. shows production of leaccase by the isolate # 312 when grown in the low nitrogen medium prepared with half-strength artificial sea water. Fi. 8a. shows production of manganese-dependent peroxidase by the isolate # 312 when grown in sugarcane bagasse suspended in distilled water. Fig. 8b. shows production of lignin peroxidase by the isolate # 312 when grown in sugarcane bagasse suspended in distilled water. Fig. 8c. shows production of laccase by the isolate # 312 when grown in sugarcane bagassse suspended in distilled water. Fig. 9a. shows production of manganese-dependent peroxidase by the isolate # 312 when grown in sugarcane bagasse suspended in half-strength artificial sea water. Fig. 9b. shows production of laccase by the isolate # 312 when grown in sugarcane bagasse suspended in half-strength artificial sea water. Although, the fungus strain used here has the same characteristics as already described in the literature, the novelty lies in of novel strain in the process of decolorization of synthetic dyes as described here. Accordingly, the present invention provides a process for removal of dyes such as azo dyes, hetrocyclic & polymeric dyes from dye-containing waste-waters and soil which comprises, growing the white-rot fungus strain Flavodon flavus (as such or immobilized) such as here in described, on any conventional nutrient medium containing assimilable Carbon and Nitrogen source, with optinally having salinity up to 15 parts per thousand(ppm) for at least 4 days, then contacting the said biomass with waste-waters or soil containing dyes for a minimum period of 5 days followed by removing the fungal biomass by conventional methods in case of waste-waters to get water devoid of dyes. The main objective of the present invention is to provide a process for removal of dyes using the lignin-modifying white-rot fungus, Flavodon flavus, NIOCC 312 for possible use in dye-containing waste-water and in saline soils, which obviates the drawbacks as detailed above. The said fungus can be efficiently utilized for the above-mentioned usage in fresh water as well as under estuarine conditions because of its tolerance to sea salts. Another objective of the present invention is to provide a process for growing the fungus on a large scale using an inexpensive raw material such as sugarcane bagasse suspended in distilled water or 50% artificial sea water or simple medium like malt extract broth prepared with fresh water as well as 50% artificial sea water. The biomass of the fungus prepared in this manner can be used for seeding soil contaminated with synthetic dyes for treating dye-containing wastwater under normal conditions or under estuarine conditions. The biomass of the said fungus thus raised can also be immobilized using conventional methods used for immobilizing fungi and used for bioremediation of dye-containing aquatic systems. In the drawings accompanying this specification, Fig. 1a. represents decolorization of Azure-B by the fungus Flavodon flavus, NIOCC isolate # 312, grown in the malt extract broth. The results are given as the difference in percent decolorization between heat-killed control and the experimental live cultures. Fig. 1b. represents decolorization of Brilliant green by the fungus Flavodon flavus, NIOCC isolate # 312, grown in the malt extract broth. The results are given as the difference in percent decoicrization between heat-killed control and the experimenta' live cultures. Fig. 1c. represents decolorization of Congo red by the fungus Flavodon flavus, NIOCC isolate # 312, grown in the malt extract broth. The results are given as the difference in percent decolorization between heat-killed control and the experimental live cultures. Fig. 1d. represents decolorization of Crystal violet by the fungus Flavodon flavus, NIOCC isolate # 312, grown in the malt extract broth. The results are given as the difference in percent decolorization between heat-killed control and the experimental live cultures. Fig. 1e. represents decolorization of Poly-B by the fungus Flavodon flavus, NIOCC isolate # 312, grown in the malt extract broth. The results are given as the difference in percent decolorization between heat-killed control and the experimental live cultures. Fig. 1f. represents decolorization of Poly-R by the fungus Flavodon flavus, NIOCC isolate # 312, grown in the malt extract broth. The results are given as the difference in percent decolorization between heat-killed control and the experimental live cultures. Fig. 1g. represents decolorization of Remazol Brilliant Blue R by the fungus Flavodon flavus, NIOCC isolate # 312, grown in the malt extract broth. The results are given as the difference in percent decolorization between heat-killed control and the experimental live cultures. Fig. 2a. represents decolorization of Azure-B by the fungus Flavodon flavus, NIOCC isolate # 312, grown in low nitrogen medium. The results are given as the difference in percent decolorization between heat-killed control and the experimental live cultures. Fig. 2b. represents decolorization of Brilliant green by the fungus Flavodon flavus, NIOCC isolate # 312, grown in low nitrogen medium. The results are given as the difference in percent decolorization between heat-killed control and the experimental live cultures. Fig. 2c. represents decolorization of Congo red by the fungus Flavodon flavus, NIOCC isolate # 312, grown in low nitrogen medium. The results are given as the difference in percent decolorization between heat-killed control and the experimental live cultures. Fig. 2d. represents decolorization of Crystal violet by the fungus Flavodon flavus, NIOCC isolate # 312, grown in low nitrogen medium. The results are given as the difference in percent decolorization between heat-killed control and the experimental live cultures. Fig. 2e. represents decolorization of Poly-R by the fungus Flavodon flavus, NIOCC isolate # 312, grown in low nitrogen medium. The results are given as the difference in percent decolorization between heat-killed control and the experimental live cultures. Fig. 2f. represents decolorization of Poly-B by the fungus Flavodon flavus, NIOCC isolate # 312, grown in low nitrogen medium. The results are given as the difference in percent decolorization between heat-killed control and the experimental live cultures. Fig. 2g. represents decolorization of Remazol Brilliant Blue R by the fungus Flavodon flavus, NIOCC isolate # 312, grown in low nitrogen medium. The results are given as the difference in percent decolorization between heat-killed control and the experimental live cultures. Fig. 3a. represents- decolorization of Azure-B by the fungus Flavodon flevus NIOCC isolate # 312, grown in low nitrogen medium prepared with half-strength artificial sea water. The results are given as the difference in percent decolorization between heat-killed control and the experimental live cultures. Fig. 3b. represents decolorization of Brilliant green by the fungus Flavodon flavus, NIOCC isolate # 312, grown in low nitrogen medium prepared with half-strength artificial sea water. The results are given as the difference in percent decolorization between heat-killed control and the experimental live cultures. Fig. 3c. represents decolorization of Congo red by the fungus Flavodon flavus, NIOCC isolate # 312, grown in low nitrogen medium prepared with half-strength artificial sea water. The results are given as the difference in percent decolorization between heat-killed control and the experimental live cultures Fig. 3d. represents decolorization of Crystal violet by the fungus Flavodon flavus, NIOCC isolate # 312, grown in low nitrogen medium prepared with half-strength artificial sea water. The results are given as the difference in percent decolorization between heat-killed control and the experimental live cultures, Fig. 3e. represents decolorization of Poly-R by the fungus Flavodon flavus, NIOCC isolate # 312, grown in low nitrogen medium prepared with half-strength artificial sea water. The results are given as the difference in percent decolorization between heat killed control and the experimental live cultures. Fig. 3f. represents decolorization of Poly-B by the fungus Flavodon flavus, NtOCC isolate # 312, grown in low nitrogen medium. The results are given as the difference in percent decolorization between heat-killed control and the experimental live cultures. Fig. 3g. represents decolorization of Remazol Brilliant Blue R by the fungus Flavodon flavus, NIOCC isolate # 312, grown in low nitrogen medium prepared with half-strength artificial sea water. The results are given as the difference in percent decolorization between heat-killed control and the experimental live cultures. Fig. 4 a. shows spectrum of undegraded Azure-B in control heat-killed culture and the same after degradation in live culture of the isolate # 312. Fig. 4 b. shows spectrum of undegraded Brilliant green in control heat-killed culture and the same after degradation in live culture of the isolate # 312. Fig. 4 c. shows spectrum of undegraded Crystal violet in control heat-killed culture and the same after degradation in live culture.of the isolate #312. Fig. 4 d. shows spectrum of undegraded Congo red in control heat-killed culture and the same after degradation in live culture of the isolate # 312. Fig.'4 e. shows spectrum of undegraded Remazol Brilliant Blue R in control heat-killed culture and the same after degradation in live culture of the isolate # 312. Fig. 5a. shows production of manganese-dependent peroxidase by the isolate # 312 when grown in the malt extract broth medium. Fig. 5b. shows production of lignin peroxidase by the. isolate # 312 when grown in the malt extract broth medium. Fig. 5c. shows production of laccase by the isolate # 312 when grown in the malt extract broth medium. Fig. 6a. shows production of manganese-dependent peroxidase by the isolate # 312 when grown in the low nitrogen medium. Fig. 6b. shows production of lignin peroxidase by the isolate # 312 when grown in the low nitrogen medium. Fig. 6c. Shows production of laccase by the isolate # 312 when grown in the low nitrogen medium. Fig. 7a. shows production of manganese-dependent peroxidase by the isolate # 312 when grown in the low nitrogen medium prepared with half-strength artificial sea water. Fig. 7b. shows production of leaccase by the isolate # 312 when grown in the low nitrogen medium prepared with half-strength artificial sea water. Fi. 8a. shows production of manganese-dependent peroxidase by the isolate # 312 when grown in sugarcane bagasse suspended in distilled water. Fig. 8b. shows production of lignin peroxidase by the isolate # 312 when grown in sugarcane bagasse suspended in distilled water. Fig. 8c. shows production of laccase by the isolate # 312 when grown in sugarcane bagassse suspended in distilled water. Fig. 9a. shows production of manganese-dependent peroxidase by the isolate # 312 when grown in sugarcane bagasse suspended in half-strength artificial sea water. Fig. 9b. shows production of laccase by the isolate # 312 when grown in sugarcane bagasse suspended in half-strength artificial sea water. Although, the fungus strain used here has the same characteristics as already described in the literature, the novelty lies in of novel strain in the process of decolorization of synthetic dyes as described here. Accordingly, the present invention provides a process for removal of dyes such as azo dyes, hetrocyclic & polymeric dyes from dye-containing waste-waters and soil which (OSSUiJ^fft ii^^obiytir comprises, growing the white-rot fungus strain Flavodon flavusj^such as here in described, on any conventional nutrient medium containing assimilable Carbon and Nitrogen source, with optinally having salinity up to 15 parts per thousand(ppm) for at least 4 days, then contacting the said biomass with waste-waters or soil containing dyes for a minimum period of 5 days followed by removing the fungal biomass by conventional methods in case of waste-waters to get water devoid of dyes. In an embodiment of the present -invention, the carbon source lor growing the fungus used is selected from glucvose or sugarcane bagasse at a concentration of at least 1%.In another embodiment of the present invention, the proportion of nitrogen used soluble low and preferably be ammonium tartrate. In yet another embodiment of the present invention, the salinity of the medium or growing the fungus ranges between 0 to 15 parts per thousand. In yet another embodiment of the present invention, the age of the said fungus is at least 4 days to get maximum decolorization of the said dyes. In yet another embodiment of the present invention, the fungal biomas is immobilized on a conventional support such as nylon mesh and like in any conventional manner such as immobilization or adsorption. In yet another embodiment of the present invention, the dyes such as azo, heterocyclic and polymeric dyes are removed by contacting with the said fugal biomass. In yet another embodiment of the present invention, the removal of fungal biomass is carried out by manual removal or filtration. The organism given in the present invention is a white-rot basidiomycete fungus isolated from a decaying marine plant from a coastal marine environment and identified as Flavodon flavus. The said fungus F. flavus can be grown in malt extract broth containing 2% malt extract and 0.3% peptone in distilled water. The fungal mat grown this way can be macerated using a homogeniser and used as starter inoculum for the experimental cultures of synthetic media prepared in distilled water or in 50% artificial seawater. The synthetic media can be prepared in distilled water or 50% artificial sea water containing 1% glucose as carbon source, 2.4 mM ammonium tartrate as the nitrogen source, thiamine, trace metal solution, macro element solution containing' potassium and manganese salts Tween 80, veratryl alcohol and 20 mM sodium acetate buffer at pH 4.5. This medium is referred to as low nitrogen medium. An example of the process for dye degradation involves addition of various synthetic dyes such as Azure B, Brilliant Green, Congo Red, Crystal Violet, Remazol Brilliant Blue R and polymeric dyes such as Poly B-411 and Poly R-478 at a final concentration of 0.02% to 3 day old cultures of F.flavus growing in malt extract broth, synthetic medium prepared with distilled water or 50% artificial sea water. The degradation of dyes, can be monitored spectrophotometrically by removing an aliquot of sample from these cultures and measuring changes in absorbance at respective wavelengths of various dyes every alternate day up to 10 days. Heat-killed cultures serve as controls, where no decolorization or degradation of dyes is observed Moreover, the said fungus can be grown on a large scale using an inexpensive raw material such as sugarcane bagasse suspended in distilled water or in 50% artificial sea water. The said fungus produces lignin-modifying enzymes such as manganese-dependent peroxidase (MNP), lignin peroxidase (LIP) and laccase, in conventional media prepared with distilled water as well as in media prepared with 50% artificial sea water. The said fungus Flavodon flavus, NIOCC isolate 312, is capable of growing in the presence of salts whose concentration is similar to that found in half-strength sea water. Most of the industrial effluents from textiles, dyestuff, paper and pulp and leather industries contain chromogenic substances as well as high concentrations of salts, especially chlorides and sulfates (Public Health Engineering - Design in Metric waste-water treatment by R. E. Bartlett, 1971, Applied Science Publishers Ltd., London). In light of this, salt tolerant organisms are better suited for such waste-water treatments. Most of the fungi used for bioremediation of such colored waste-waters have not been tested for their salt tolerance. In view of this, the present process has an advantage over the conventional processes referred to in various patents discussed above. White-rot fungi are unique among eukaryotic microbes in possessing powerful lignin-degrading oxidative enzymes such as MNP, LIP and lacasses which have a broad substrate specificity and are thus able to oxidize several environmental pollutants. Results from several laboratories have shown that the ability of white-rot fungi such as Phanerochaete chrysosporium and Trametes versicolor to degrade an array of pollutants including synthetic dyes such as axo, heterocyclic and polycyclic dyes is due to the lignin-modifying/degrading system (C A. Reddy. 1995. The potential for white-rot fungi in the treatment of pollutants. Current Opinion in Biotechnology, 6: 320-328). Our fungal isolate F. flavus, NIOCC isolate 312, obtained from marine environment also produces lignin-modifying enzymes such as MNP LIP and laccase. It produces these enzymes in natural media such as ME broth, conventional synthetic medium prepared with distilled water or 50% artificial sea water and also in powdered sugarcane bagasse suspended in distilled water or in 50% artificial sea water, and degrades synthetic dyes such as Azure B, Brilliant Green, Crystal Violet, Congo Red, Remazol Brilliant Blue R and polymeric dyes such as Poly B and Poly R. The following examples are given by way of illustration of the present invention and therefore should not be construed to limit the scope of the present invention. EXAMPLE-1 A non-spbrulating form of the fungus was isolated from decaying brown leaves of sea grass from the: intertidal of a lagoon in Kavaratti island of the Lakshadweep archipelago (10°30'-11.0° N Lat, 72.0°-73.0°E, Long.) off the west coast of India in the Arabian sea. Discs this synthetic medium. However, it produced fertile basidiomata on medium containing alpha cellulose or after a long incubation in malt extract agar medium. Based on fertile basidiomata using the key of Ryvarden and Johansen, it was identified as Flavodon flavus (Kl) Ryv. Since the strain #312 was isolated from a marine habitat, the effect of salinity on its growth can be studied by growing in medium prepared with 100% sea water, 50% sea water and in distilled water. Growth in natural medium such as malt extract broth and synthetic media were compared for this purpose. Malt extract broth contains 2% malt extract and 0.3% peptone in distilled water or seawater of various salinities. Instead of natural-seawater which may not be available to every one, synthetic .sea salts 'of reputed companies can be used in preparing artificial sea water. We used Instant Ocean-salts obtained from Aquarium Systems, Mentor Ohio, USA. By dissolving 15 g and 30 g of Instant Ocean salts in 1 litre of distiHed water, salinity equivalents of 1.5 and 3.0% were obtained. The former is referred to as 50% artificial sea water or half-strength sea water. The synthetic media can be prepared in distilled water or-50% artificial sea water containing 1% glucose as carbon source,., 2.4mM ammonium tartrate as the nitrogen source, thiamine, trace metal solution, macro element solution containing sodium, potassium and manganese salts, Tween 80, veratryl alcohol and 20mM sodium acetate buffer at pH 4,5. This synthetic medium is referred to as low nitrogen medium. Growth of the fungus is measured as mycelial dry weight produced at the end of 10 days incubation in various media. For determination of dry weight, the contents of three flasks are vacuum filtered through tared Whatmen GF/C filter papers, rinsed with 100ml of distilled water to remove salts and dried to a constant weight and the net mycelial dry weight is calculated. Table 1.* (Table Removed) Figures represent mg dry weight of fungus per 10 ml of medium in 10 days. The example given here shows that the fungus F. flavus grows much better in the presence of 50% artificial sea water and thus can be used in presence of synthetic salts or in estuarine conditions. EXAMPLE-2 The ability of F. flavus, NIOCC isolate 312, to degrade and decolorize synthetic dyes was tested in nutrient rich medium such as malt extract broth. Preparation of medium was done as described in Example 1. Sterile media are dispensed in. 9 ml amounts into 125 ml sterile rubber-stoppered Erlenmeyer flasks. The flasks are inoculated with 1ml of fungal inoculum. For inoculum preparation, mycelial mats grown in malt extract broth (25 ml in 250 ml foam-plugged Erlenmeyer flasks) for 10 days, at room temperature were washed twice with 150 ml of sterile water and blended in 20 ml of low nitrogen medium using a Sorvall blender (with four 30-seconds spurts). One ml of this blended mycelium (equivalent to 3 mg initial dry weight of mycelium) was used for inoculating media in 125 ml Erlenmeyer flasks. The flasks are flushed with 100% 02 at the time of inoculation and every alternate day thereafter. Culture flasks are incubated at room temperature. Individual dye solutions were added to 4-day-old cultures of F. flavus, to give final dye concentrations of 0.02%. The dyes used were Azure B, Brilliant Green, Crystal Violet, Congo Red, Remazol Brilliant Blue R, Poly B- 411 and Poly R-478. Stock solutions of dyes were prepared in distilled water and filter sterilized. Autoclaved cultures supplemented with dyes served as heat-killed controls. Aliquots (0.5ml) of culture supematants from experimental cultures and from heat-killed controls were removed aseptically at 2-day intervals, diluted appropriately with distilled water and the changes in absorbance maxima for the different dyes were measured using a Varian Cary 1 Bio UVA/isible spectrophotometer (Varian, Australia). Azure B was measured at 647 nm, Brilliant Green at 625 nm, Congo Red at 495 nm, Crystal Violet at 590 nm, and Remazol Brilliant Blue R at 595 nm. (Heinfling, A., M. Bergbauer, and U. Szewzyk. 1997. Biodegradation of azo and phthalocyanine dyes by Trametes versicolor and- Bjerkandera adusta. Applied Microbiology and Biotechnology. 48:261- 266). Decolorization of Poiy B and Poly R was monitored by determining the absorption ratio at 593/483 nm and 513/362 nm respectively (Gold, M.H., J.K. Glenn, and M. Alic. 1988. Use of polymeric dyes in lignin biodegradation assays. Methods in Enzymology. 161: 74-78). The results are calculated as the difference in percent decolorization between heat-killed and experimental cultures. Degradation of various dyes was also monitored by comparing the changes in the visible spectra of native dyes in heat-killed culture fluids with that of decolorized dyes in experimental'culture fluids at 400-800 nm. This was carried out at the end of the experiment. Accordingly, Figure 1a-1g shows the percentage of decolorization of various dyes by F. flevus, NIOCC isolate 312 when grown in simple natural medium such as malt extract broth. Azure B and Remazol Brilliant Blue R were decolorized up to 60% within 7 days. Decolorization of Poly B is delayed initially but the final percentage of decolorization is 60%. EXAMPLE-3 Decolorization of various dyes by F. flavus, NIOCC isolate 312, was tested in synthetic medium such as low nitrogen medium prepared with distilled water. Preparation of synthetic medium was done as described in Example-1. Inoculation, oxygenation and incubation were done as described in Example-2. Experimental procedures of addition of dyes and monitoring the decolorization were same as described in Example-2. Accordingly, Figure 2a-2g show that about 70% decolorization of all the dyes took place within 7 days in low nitrogen medium prepared with distilled water. EXAMPLE-4 Decolorization of various dyes by F. flavus, NIOCC isolate 312, was tested in synthetic medium such as low nitrogen medium prepared with half-strength artificial sea water. Artificial sea water was prepared by dissolving 15g of Instant Ocean salts as per the manufacturer's instructions (Instant Ocean of Aquarium Systems, Mentor, OH) in 1 litre of distilled water to get salinity equivalent of 15 parts per thousand and filtered through a GF/C filter paper. The normal salinity of sea water is 30 parts per thousand. Preparation of low nitrogen medium was done as described in Example-1. Inoculation, oxygenation and incubation were done as described in Example-2. Experimental procedures for addition of dyes and monitoring the decolorization were same as described in Example-2. Accordingly, Figure 3a-3g show decolorization of various dyes by F. flavus, NIOCC isolate 312, when grown in low nitrogen medium prepared with 50% artificial sea water. More than 80% decolorization of all dyes was achieved within 7 days in this medium. The fungus F. flavus also degraded the various dyes as seen by changes in the visible spectra of native dyes in experimental cultures in comparison with heat-killed control culture fluids. Figure 4a-4e shows complete degradation of some of the dyes by the fungus F. flavus when grown in low nitrogen medium with 50% artificial sea water. EXAMPLE-5 In order to show that degradation of dyes is brought about by production of. lignin-modifying enzymes in malt extract broth, the fungus was grown in this medium as described in Example-2.; Inoculation, oxygenation and incubation were carried out as described in Example-2. . Gdntents df three flasks were filtered on different days of incubation using Whatman GF/C filters and the extracellular fluids were analysed for lignin-modifying enzymes such as manganese-dependent peroxidase (MNP), lignin peroxidase (LIP) and laccase. A convenient assay for MNP involves monitoring the enzyme's oxidation of Mn(ll) to Mn(lll). The reaction mixture contains enzyme, 0.1 M sodium tartrate buffer (pH 5.0), 0.1 M H2O2 and 0.1 mM MnS04. Reactions are initiated by the addition of H2O2 and increase in absorbance at 238 nm is monitored during the first 5-30 sec of reaction. One unit of MNP oxidizes 1 µmol of Mn(ll)/min (Paszczyniski, A, R. L. Crawford, and V.-B. Huynh. 1988. Manganese peroxidase of Phanerochaete chrysosporium: purification. Methods in Enzymoiogy. 161: 264-270). Lignin peroxidase catalyzes oxidation of veratryl alcohol to veratraldehyde by H202, The reaction mixture for assay contains veratryi alcohol, tartaric acid at pH 3.0, 8 mM H2O2 and enzyme solution. Increase in absorbance at 310 nm is monitored for 1 min. One unit of enzyme is defined as 1 µmol of veratryl alcohol oxidized per min (Tien, M., and T. K. Kirk. 1988. Lignin peroxidase of Phanerochaete chrysosporium. Methods in Enzymoiogy. 161; 239-249). Lacasse activity was determined by using the substrate 2,21azino-bis{3- ethylbenzthiazoline-6-sulfonic acid), in 0.1 M glycine buffer at pH 3.0. The reaction was monitored by measuring change in absorbance at 405 nm for 5 min. The enzyme units are expressed as nkatals/L (Niku-Paavola, M-L, E. Karhunen, P. Salola and V. Raunio. 1988. Ligninolytic enzymes of the white-rot fungus Phlebia radiata. Biochemical Journal. 254: 877-884). One katal is defined as one molar extinction coefficient of product formed per second. Accordingly, Figure 5a represents production of MNP in malt extract broth prepared with distilled water by the fungus F. fiavus, NIOCC isolate 312. Accordingly Figure 5b represents production of LIP in malt extract broth prepared with distilled water by the fungus F. fiavus, NIOCC isolate 312. Accordingly, Figure 5c represents production of laccase in malt extract broth prepared with distilled water by the fungus f. fiavus, NIOCC isolate 312. EXAMPLE-6 In order to show that degradation of dyes is brought about by production of lignin-modifying enzymes in synthetic medium such as low nitrogen medium, the fungus was grown in the low nitrogen medium prepared with distilled water as described in Example-1. Inoculation, incubation, oxygenation and sampling of culture filtrates were done as described in Example-5. Enzyme assays of the culture fluids were carried out as described in Example-5. Accordingly Figure 6a represents production of MNP in low nitrogen medium prepared with distilled water by the fungus F. flavus, NIOCC isolate 312. Accordingly, Figure 6b represents production of LIP in this medium by the fungus F. flavus, NIOCC isolate 312. Accordingly, Figure 6c represents production of laccase in this medium-prepared with distilled water by the fungus F. flavus, NIOCC isolate 312. EXAMPLE-7 In order to show that degradation of dyes is brought about by production of lignin-modifying enzymes in the presence of salts, the fungus was grown in the low nitrogen medium prepared with half-strength artificial sea water as described in Example-1. Inoculation, incubation, oxygenation and sampling of culture filtrates were done as described in Example-5. Enzyme assays of the culture fluids were carried out as described in Example-5. Accordingly, Figure 7a represents production of. MNP in low nitrogen medium prepared with 50% artificial sea water by the fungus F. flavus, NIOCC isolate 312. No LIP production was detected in this medium. Accordingly, Figure 7b represents production of laccase in low nitrogen medium prepared with 50% artificial sea water by the fungus F. flavus, NIOCC isolate 312. EXAMPLE-8 Wood and other lignocellulosics are natural substrates for ligninolytic fungi. Therefore, lignin-modifying enzyme production was compared in cultures grown in sugarcane bagasse, which is an inexpensive raw material for large-scale application of this fungus for its potential use in field trials. As the above examples 5, 6, and 7 showed, the degradation of dyes is brought about in various media by production of lignin-modifying enzymes in these media, we wanted to show production of these enzymes in bagasse medium also if it were to be used for application in field trials. For preparing media with lignocellulosics as the sole carbon/nitrogen/energy source, sugarcane bagasse powder (16 screen mesh/sq. in) was added at 1% final concentration in distilled water. To minimize contamination by residual sugars, the sugarcane bagasse was washed under running tap water for 12 h, followed by a wash with three volumes of distilled water and dried at room temperatures. The sugarcane bagasse medium was. inoculated, oxygenated, incubated and sampled as described in Example-5. The extracellular culture fluids were assayed for MNP, LIP and laccase as described in Example-5. Accordingly, Figure 8a represents production of MNP in sugarcane bagasse medium prepared with distilled water by the fungus F. flavus, NIOCC isolate 312. Accordingly, Figure 8b represents production of LIP in sugarcane bagasse medium prepared with distilled water by the fungus F. flavus, NIOCC isolate 312. Accordingly, Figure 8c represents production of laccase in sugarcane bagasse medium prepared with distilled water by the fungus F. flavus, NIOCC isolate 312. It is of interest that MNP, which has been shown to be important in delignification of Kraft pulp (Paice, M.G., l.d. Reid, R. Bourbonnais, F. S. Archibald and L. Jurasek. 1993. Manganese peroxidase produced by Trametes versicolor during pulp bleaching, demethylates and delignifies kraft pulp. Applied Environmental Microbiology. 59:260-265), and in decolorizing Kraft bleach plant effluents (Michel, F. C. Jr., S. B. Dass, E.A. Grulke, and C.A. Reddy. 1991. Role of manganese peroxidases and lignin peroxrdases of Phanerochaete chrysosporium in decolorization of kraft bleach plant effluent. Applied Environmental Microbiology. 57: 2368-2375) was produced in highest titres in cultures of F. flavus grown with sugarcane bagasse. These results further suggest that sugarcane bagasse can potentially be used for bulk production of biomass of F. flavus, NIOCC isolate 312, for possible application in field trials for removal of dyes in dye-containing waste-waters and soil. EXAMPLE-9 As the previous example showed production of lignin-modifying enzymes in sugarcane bagasse suspended in distilled .water, for its application in presence of salts we can similarly grow the fungus in sugarcane bagasse suspended in half-strength artificial sea water. The medium was inoculated, oxygenated, incubated and sampled as described in the Example-5. The extracellular culture fluids were assayed for MNP, LIP and lacasse as described in Example-5. Accordingly, Figure 9a represents production of MNP in sugarcane bagasse medium prepared with half-strength artificial sea water by the fungus F. flavus, NIOCC isolate 312. No LIP production was detected in this medium. Accordingly, Figure 9b represents production of laccase in sugarcane bagasse medium prepared with half-strength artificial sea water by the fungus F. flavus, NIOCC isolate 312. Examples 1-9 illustrate that the said fungus Flavodon flavus, NIOCC isolate 312, can be grown in conventional media prepared with distilled water or half-strength artificial sea water and in sugarcane bagasse suspended in distilled water or half-strength artificial sea water and the biomass of the fungus thus obtained can be used for removal of synthetic dyes in freshwater and estuarine conditions and in soil. The fungus can be immobilized using conventional methods and can be used for removal of synthetic dyes in aquatic habitats and soil. The above mentioned examples further illustrate that the degradation of synthetic dyes by the said fungus is brought about by production of lignin-modifying enzymes such as MNP, LIP and taccase in conventional media or in sugarcane bagasse medium prepared either with distilled water or half-strength artificial sea water. The main advantages of the present invention are : 1. The fungus can be grown on large scale using inexpensive raw materials such as sugarcane bagasse suspended in distilled water or half-strength artificial sea water for any biotechnological application. 2. The said fungus decolorizes about 80% of various dyes within 7 days in media prepared with artificial sea water and about 60-70% in media prepared with distilled water, and thus can be used for treating dye containing waste-waters in the presence of salts as well. 3. The biomass of the fungus obtained on sugarcane bagasse can be used for seeding soil contaminated with synthetic dyes in fresh water, estuarine conditions or soil. , 4. The said fungus produces lignin-modifying enzymes in malt extract broth, low nitrogen medium prepared either with distilled water or half-strength artificial sea water and in sugarcane bagasse suspended in distilled water or half-strength artificial sea water, 5. The said fungus-grows better in the presence of half-strength artificial sea water and thus can tolerate salinity up to 15 parts per thousand and can be applied for removal of synthetic dyes in sea water of appropriate salinity, in estuarine conditions. 1. A process for removal of dyes such as azo dyes, hetrocyclic & polymeric dyes from dye-containing waste-waters and soil which comprises, growing the white-rot fungus strain Flavodon flavus such as here in described, any conventional nutrient medium containing assimilable C and N source, with optinally having salinity up to 15 parts per thousand(ppm) for at least 4 days, then contacting the said biomass with waste-waters or soil containing dyes for a minimum period of 5 days followed by removing the fungal biomass by conventional methods in case of waste-waters to get water devoid of dyes. 2. A process as claimed in claims 1 wherein, the carbon source for growing the fungus used is selected from glucose or sugarcane bagasse at a concentration of at least 1%. 3. A process is claimed in claim 1 & 2 wherein, the proportion of nitrogen used should be low and preferably be ammonium tartrate 2.4 mM. 4. A process is claimed in claims 1 to 3 wherein, the salinity of the medium for growing the fungus ranges between 0 to 15 parts per thousand. 5. A process is claimed in claims 1 o 4 wherein, the age of the said fungus is 4 days to get maximum decolorization of the said dyes. 6. A process is claimed in claims 1 to 5 wherein, the fungal biomass is immobilized on a conventional support selected from nylon mesh or immobilization or adsorption. 7. A process for removal of dyes in dye containing waste-waters and soil substantially as herein described with reference to examples 1-9 and figures 1-9.

Full Text

This invention relates to a process for removal of dyes in dye containing waste-waters and soil. The present invention particularly relates to a process for removal of synthetic dyes in dye containing waste-waters and soil using the lignin-modifying white-rot fungus, Flavodon flavus (Kl) Ryv., NIOCC isolate 312. Further this invention relates to 80-100% removal within four days after addition of synthetic dyes such as azo dyes, heterocyclic and polymeric dyes to the 4 day old culture of Flavodon flavus.
Textile and dyestuff industries are the major contributors to industrial pollutants containing dyes. These dyes are highly stable in light and are also resistant to
microbial attack. Most of these dyes are azo dyes and under anaerobic conditions, the
azo linkages are reduced to form aromatic amines and these are toxic and carcinogenic
(Meyer, U. 1981. Biodegradation of synthetic organic colorants. Federation of European Microbiological Societies Symposium. 12: 371-385). Due to the. importance attached to prevention of environmental pollution, environmental agencies all over the world are imposing strict regulations for mitigation of pollution from industries. The effluents from the textile industries containing fast colored dyes are a major source of concern for environmentalist since such dyes beside causing aesthetic damage to sites, are toxic and carcinogenic. Industrial effluents from textiles, paper and pulp industries and leather industries contain chromogenic substances as well as high concentrations of salts, especially chlorides and sulfates (Public Health Engineering-Design in Metric waste-water treatment by R. E. Bartlett, 1971, Applied Science Publishers Ltd., London). Remediation of such dye containing waste-waters using biological methods is termed bioremediation Thus, this invention particularly relates to
degradation of synthetic dyes in the presence of salts by the fungus F. flavus deposited
at the National Institute of Oceanography, Dona Paula, Goa 400004, India and having
the accession number NIOCC 312, in textile waste-water treatment, and bioremediation
of soil. The said fungus can be grown in synthetic media prepared with either distilled
water or 50% artificial sea water. The said fungus can also be grown in conventional
media or in powdered sugarcane bagasse suspended in distilled water or 50% artificial
sea water to raise large biomass of the fungus for application in field trials for
bioremediation in the presence of sea water or fresh water. The said fungus thus grown
can be immobilized by conventional methods and used for removal of various synthetic
dyes from contaminated soil as well as aquatic habitats. The said fungus, F. flavus
produces lignin-modifying enzymes such as manganese-dependent peroxidase E. C.
1.11.1.7 (MNP), lignin peroxidase, E. C. 1.11.1.7 (LIP) and laccase, E. C. 1.10.3.2
when grown on sugarcane bagasse suspended in plain distilled water or in 50%
artificial sea water or in conventional media prepared with distilled water or 50%
artificial sea water. By virtue of these lignin-modifying enzymes which break down a
broad range of polymeric substrates, this fungus is useful in degradation of pollutants
such as synthetic dyes. Thus degradation of synthetic dyes in waste-waters can be
achieved by using the fungus F. flavus in fresh water as well as estuarine conditions.
Normally, the textile dye waste-waters disposal includes physical-chemical treatment, waste minimization and biological treatment. Biological treatment includes biological pretreatment with activated sludge of textile waste-waters, and treatment in stabilization ponds (Groff, K.A. 1992, Textile waste. Water-Environment Research, 64:
725-729.). Unfortunately, waste-water treatment facilities are often unable to completely remove commercial dyestuff from contaminated waters and thus contribute to pollution of aquatic habitats. Some of these synthetic dyes are carcinogenic and are suggested to be responsible for tumor growth in some species of fish (see Bumpus J. A., B. J. Brock. 1988. Biodegradation of crystal violet by the white-rot fungus Phanerochaete chrysosporium. Applied and Environmental Microbiology, 54:1143-1150).
Various organisms have been tried for degradation of dyes in textile waste-waters and bioremediation.
(i) Reference may be made to the Japanese patent wherein a green alga Chlorella vulgaris is used for degradation of methylene blue by irradiating a microalga fermentor to generate OH radicals which in turn help in degradation of the dye (Japanese patent JP06047394, Title: Organic dye degradation in waste-water, Date of issue: 22.02.94). The method has disadvantage as it involves irradiating the fermentor containing microalga and thus becomes expensive.
(ii) Reference may be made to a U.S. patent wherein, living, dead, free, immobilized white-rot fungi Myrothecium or Ganoderma spp. have been employed for adsorption, dye degradation and color removal (US patent US5091089, Title: Decolorization of dye-containing waste-water, Date of issue: 25.02.92). These fungi have not been tested for their efficiency in color removal in the presence of sea salts.
(iii) Reference may be made to US patent wherein, white-rot fungus Phanerochaete chrysosporium and actinomycetes Streptomyces spp are used in degradation of
low nitrogen medium.
Fig. 6c. Shows production of laccase by the isolate # 312 when grown in the low nitrogen medium.
Fig. 7a. shows production of manganese-dependent peroxidase by the isolate # 312 when grown in the low nitrogen medium prepared with half-strength artificial sea
water.
Fig. 7b. shows production of leaccase by the isolate # 312 when grown in the low
nitrogen medium prepared with half-strength artificial sea water.
Fi. 8a. shows production of manganese-dependent peroxidase by the isolate # 312
when grown in sugarcane bagasse suspended in distilled water.
Fig. 8b. shows production of lignin peroxidase by the isolate # 312 when grown in
sugarcane bagasse suspended in distilled water.
Fig. 8c. shows production of laccase by the isolate # 312 when grown in sugarcane
bagassse suspended in distilled water.
Fig. 9a. shows production of manganese-dependent peroxidase by the isolate # 312
when grown in sugarcane bagasse suspended in half-strength artificial sea water.
Fig. 9b. shows production of laccase by the isolate # 312 when grown in sugarcane
bagasse suspended in half-strength artificial sea water.
Although, the fungus strain used here has the same characteristics as already described in the literature, the novelty lies in of novel strain in the process of decolorization of synthetic dyes as described here.
Accordingly, the present invention provides a process for removal of dyes such as azo dyes, hetrocyclic & polymeric dyes from dye-containing waste-waters and soil which comprises, growing the white-rot fungus strain Flavodon flavus (as such or immobilized) such as here in described, on any conventional nutrient medium containing assimilable Carbon and Nitrogen source, with optinally having salinity up to 15 parts per thousand(ppm) for at least 4 days, then contacting the said biomass with waste-waters or soil containing dyes for a minimum period of 5 days followed by removing the fungal biomass by conventional methods in case of waste-waters to get water devoid of dyes.
The main objective of the present invention is to provide a process for removal of dyes using the lignin-modifying white-rot fungus, Flavodon flavus, NIOCC 312 for possible use in dye-containing waste-water and in saline soils, which obviates the drawbacks as detailed above. The said fungus can be efficiently utilized for the above-mentioned usage in fresh water as well as under estuarine conditions because of its tolerance to sea salts.
Another objective of the present invention is to provide a process for growing the fungus on a large scale using an inexpensive raw material such as sugarcane bagasse suspended in distilled water or 50% artificial sea water or simple medium like malt extract broth prepared with fresh water as well as 50% artificial sea water. The biomass of the fungus prepared in this manner can be used for seeding soil contaminated with synthetic dyes for treating dye-containing wastwater under normal conditions or under estuarine conditions. The biomass of the said fungus thus raised can also be immobilized using conventional methods used for immobilizing fungi and used for bioremediation of dye-containing aquatic systems.
In the drawings accompanying this specification, Fig. 1a. represents decolorization of Azure-B by the fungus Flavodon flavus, NIOCC isolate # 312, grown in the malt extract broth. The results are given as the difference in percent decolorization between heat-killed control and the experimental live cultures. Fig. 1b. represents decolorization of Brilliant green by the fungus Flavodon flavus, NIOCC isolate # 312, grown in the malt extract broth. The results are given as the difference in percent decoicrization between heat-killed control and the experimenta'
live cultures.
Fig. 1c. represents decolorization of Congo red by the fungus Flavodon flavus, NIOCC isolate # 312, grown in the malt extract broth. The results are given as the difference in percent decolorization between heat-killed control and the experimental live cultures. Fig. 1d. represents decolorization of Crystal violet by the fungus Flavodon flavus, NIOCC isolate # 312, grown in the malt extract broth. The results are given as the difference in percent decolorization between heat-killed control and the experimental live cultures.
Fig. 1e. represents decolorization of Poly-B by the fungus Flavodon flavus, NIOCC isolate # 312, grown in the malt extract broth. The results are given as the difference in percent decolorization between heat-killed control and the experimental live cultures. Fig. 1f. represents decolorization of Poly-R by the fungus Flavodon flavus, NIOCC isolate # 312, grown in the malt extract broth. The results are given as the difference in percent decolorization between heat-killed control and the experimental live cultures. Fig. 1g. represents decolorization of Remazol Brilliant Blue R by the fungus Flavodon flavus, NIOCC isolate # 312, grown in the malt extract broth. The results are given as the difference in percent decolorization between heat-killed control and the experimental live cultures.
Fig. 2a. represents decolorization of Azure-B by the fungus Flavodon flavus, NIOCC isolate # 312, grown in low nitrogen medium. The results are given as the difference in percent decolorization between heat-killed control and the experimental live cultures. Fig. 2b. represents decolorization of Brilliant green by the fungus Flavodon flavus,
NIOCC isolate # 312, grown in low nitrogen medium. The results are given as the difference in percent decolorization between heat-killed control and the experimental live cultures.
Fig. 2c. represents decolorization of Congo red by the fungus Flavodon flavus, NIOCC isolate # 312, grown in low nitrogen medium. The results are given as the difference in percent decolorization between heat-killed control and the experimental live cultures. Fig. 2d. represents decolorization of Crystal violet by the fungus Flavodon flavus, NIOCC isolate # 312, grown in low nitrogen medium. The results are given as the difference in percent decolorization between heat-killed control and the experimental live cultures.
Fig. 2e. represents decolorization of Poly-R by the fungus Flavodon flavus, NIOCC isolate # 312, grown in low nitrogen medium. The results are given as the difference in percent decolorization between heat-killed control and the experimental live cultures. Fig. 2f. represents decolorization of Poly-B by the fungus Flavodon flavus, NIOCC isolate # 312, grown in low nitrogen medium. The results are given as the difference in percent decolorization between heat-killed control and the experimental live cultures. Fig. 2g. represents decolorization of Remazol Brilliant Blue R by the fungus Flavodon flavus, NIOCC isolate # 312, grown in low nitrogen medium. The results are given as the difference in percent decolorization between heat-killed control and the experimental live cultures.
Fig. 3a. represents- decolorization of Azure-B by the fungus Flavodon flevus NIOCC isolate # 312, grown in low nitrogen medium prepared with half-strength artificial sea
water. The results are given as the difference in percent decolorization between heat-killed control and the experimental live cultures.
Fig. 3b. represents decolorization of Brilliant green by the fungus Flavodon flavus, NIOCC isolate # 312, grown in low nitrogen medium prepared with half-strength artificial sea water. The results are given as the difference in percent decolorization between heat-killed control and the experimental live cultures. Fig. 3c. represents decolorization of Congo red by the fungus Flavodon flavus, NIOCC isolate # 312, grown in low nitrogen medium prepared with half-strength artificial sea water. The results are given as the difference in percent decolorization between heat-killed control and the experimental live cultures
Fig. 3d. represents decolorization of Crystal violet by the fungus Flavodon flavus, NIOCC isolate # 312, grown in low nitrogen medium prepared with half-strength artificial sea water. The results are given as the difference in percent decolorization between heat-killed control and the experimental live cultures, Fig. 3e. represents decolorization of Poly-R by the fungus Flavodon flavus, NIOCC isolate # 312, grown in low nitrogen medium prepared with half-strength artificial sea water. The results are given as the difference in percent decolorization between heat killed control and the experimental live cultures.
Fig. 3f. represents decolorization of Poly-B by the fungus Flavodon flavus, NtOCC isolate # 312, grown in low nitrogen medium. The results are given as the difference in percent decolorization between heat-killed control and the experimental live cultures. Fig. 3g. represents decolorization of Remazol Brilliant Blue R by the fungus Flavodon
flavus, NIOCC isolate # 312, grown in low nitrogen medium prepared with half-strength
artificial sea water. The results are given as the difference in percent decolorization
between heat-killed control and the experimental live cultures.
Fig. 4 a. shows spectrum of undegraded Azure-B in control heat-killed culture and the
same after degradation in live culture of the isolate # 312.
Fig. 4 b. shows spectrum of undegraded Brilliant green in control heat-killed culture
and the same after degradation in live culture of the isolate # 312.
Fig. 4 c. shows spectrum of undegraded Crystal violet in control heat-killed culture and
the same after degradation in live culture.of the isolate #312.
Fig. 4 d. shows spectrum of undegraded Congo red in control heat-killed culture and
the same after degradation in live culture of the isolate # 312.
Fig.'4 e. shows spectrum of undegraded Remazol Brilliant Blue R in control heat-killed
culture and the same after degradation in live culture of the isolate # 312.
Fig. 5a. shows production of manganese-dependent peroxidase by the isolate # 312
when grown in the malt extract broth medium.
Fig. 5b. shows production of lignin peroxidase by the. isolate # 312 when grown in the
malt extract broth medium.
Fig. 5c. shows production of laccase by the isolate # 312 when grown in the malt
extract broth medium.
Fig. 6a. shows production of manganese-dependent peroxidase by the isolate # 312
when grown in the low nitrogen medium.
Fig. 6b. shows production of lignin peroxidase by the isolate # 312 when grown in the low nitrogen medium.
Fig. 6c. Shows production of laccase by the isolate # 312 when grown in the low nitrogen medium.
Fig. 7a. shows production of manganese-dependent peroxidase by the isolate # 312
when grown in the low nitrogen medium prepared with half-strength artificial sea
water.
Fig. 7b. shows production of leaccase by the isolate # 312 when grown in the low
nitrogen medium prepared with half-strength artificial sea water.
Fi. 8a. shows production of manganese-dependent peroxidase by the isolate # 312
when grown in sugarcane bagasse suspended in distilled water.
Fig. 8b. shows production of lignin peroxidase by the isolate # 312 when grown in
sugarcane bagasse suspended in distilled water.
Fig. 8c. shows production of laccase by the isolate # 312 when grown in sugarcane
bagassse suspended in distilled water.
Fig. 9a. shows production of manganese-dependent peroxidase by the isolate # 312
when grown in sugarcane bagasse suspended in half-strength artificial sea water.
Fig. 9b. shows production of laccase by the isolate # 312 when grown in sugarcane
bagasse suspended in half-strength artificial sea water.
Although, the fungus strain used here has the same characteristics as already described in the literature, the novelty lies in of novel strain in the process of decolorization of synthetic dyes as described here.
Accordingly, the present invention provides a process for removal of dyes such as azo dyes, hetrocyclic & polymeric dyes from dye-containing waste-waters and soil which
(OSSUiJ^fft ii^^obiytir
comprises, growing the white-rot fungus strain Flavodon flavusj^such as here in described, on any conventional nutrient medium containing assimilable Carbon and Nitrogen source, with optinally having salinity up to 15 parts per thousand(ppm) for at least 4 days, then contacting the said biomass with waste-waters or soil containing dyes for a minimum period of 5 days followed by removing the fungal biomass by conventional methods in case of waste-waters to get water devoid of dyes.
In an embodiment of the present -invention, the carbon source lor growing the fungus used is selected from glucvose or sugarcane bagasse at a concentration of at least 1%.In another embodiment of the present invention, the proportion of nitrogen used soluble low and preferably be ammonium tartrate.
In yet another embodiment of the present invention, the salinity of the medium or growing the fungus ranges between 0 to 15 parts per thousand.
In yet another embodiment of the present invention, the age of the said fungus is at least 4 days to get maximum decolorization of the said dyes.
In yet another embodiment of the present invention, the fungal biomas is immobilized on a conventional support such as nylon mesh and like in any conventional manner such as immobilization or adsorption.
In yet another embodiment of the present invention, the dyes such as azo, heterocyclic and polymeric dyes are removed by contacting with the said fugal biomass.
In yet another embodiment of the present invention, the removal of fungal biomass is carried out by manual removal or filtration.
The organism given in the present invention is a white-rot basidiomycete fungus
isolated from a decaying marine plant from a coastal marine environment and identified
as Flavodon flavus. The said fungus F. flavus can be grown in malt extract broth
containing 2% malt extract and 0.3% peptone in distilled water. The fungal mat grown
this way can be macerated using a homogeniser and used as starter inoculum for the
experimental cultures of synthetic media prepared in distilled water or in 50% artificial
seawater. The synthetic media can be prepared in distilled water or 50% artificial sea
water containing 1% glucose as carbon source, 2.4 mM ammonium tartrate as the
nitrogen source, thiamine, trace metal solution, macro element solution containing'
potassium and manganese salts Tween 80, veratryl alcohol and 20 mM sodium
acetate buffer at pH 4.5. This medium is referred to as low nitrogen medium. An
example of the process for dye degradation involves addition of various synthetic dyes
such as Azure B, Brilliant Green, Congo Red, Crystal Violet, Remazol Brilliant Blue R
and polymeric dyes such as Poly B-411 and Poly R-478 at a final concentration of
0.02% to 3 day old cultures of F.flavus growing in malt extract broth, synthetic medium
prepared with distilled water or 50% artificial sea water. The degradation of dyes, can
be monitored spectrophotometrically by removing an aliquot of sample from these
cultures and measuring changes in absorbance at respective wavelengths of various
dyes every alternate day up to 10 days. Heat-killed cultures serve as controls, where no
decolorization or degradation of dyes is observed Moreover, the said fungus can be grown on a large scale using an inexpensive raw material such as sugarcane bagasse suspended in distilled water or in 50% artificial sea water. The said fungus produces lignin-modifying enzymes such as manganese-dependent peroxidase (MNP), lignin peroxidase (LIP) and laccase, in conventional media prepared with distilled water as well as in media prepared with 50% artificial sea water.
The said fungus Flavodon flavus, NIOCC isolate 312, is capable of growing in the presence of salts whose concentration is similar to that found in half-strength sea water. Most of the industrial effluents from textiles, dyestuff, paper and pulp and leather industries contain chromogenic substances as well as high concentrations of salts, especially chlorides and sulfates (Public Health Engineering - Design in Metric waste-water treatment by R. E. Bartlett, 1971, Applied Science Publishers Ltd., London). In light of this, salt tolerant organisms are better suited for such waste-water treatments. Most of the fungi used for bioremediation of such colored waste-waters have not been tested for their salt tolerance. In view of this, the present process has an advantage over the conventional processes referred to in various patents discussed above. White-rot fungi are unique among eukaryotic microbes in possessing powerful lignin-degrading oxidative enzymes such as MNP, LIP and lacasses which have a broad substrate specificity and are thus able to oxidize several environmental pollutants. Results from several laboratories have shown that the ability of white-rot fungi such as Phanerochaete chrysosporium and Trametes versicolor to degrade an array of pollutants including synthetic dyes such as axo, heterocyclic and polycyclic dyes is due to the lignin-modifying/degrading system (C A. Reddy. 1995. The potential for white-rot
fungi in the treatment of pollutants. Current Opinion in Biotechnology, 6: 320-328). Our fungal isolate F. flavus, NIOCC isolate 312, obtained from marine environment also produces lignin-modifying enzymes such as MNP LIP and laccase. It produces these enzymes in natural media such as ME broth, conventional synthetic medium prepared with distilled water or 50% artificial sea water and also in powdered sugarcane bagasse suspended in distilled water or in 50% artificial sea water, and degrades synthetic dyes such as Azure B, Brilliant Green, Crystal Violet, Congo Red, Remazol Brilliant Blue R and polymeric dyes such as Poly B and Poly R.
The following examples are given by way of illustration of the present invention and therefore should not be construed to limit the scope of the present invention.
EXAMPLE-1
A non-spbrulating form of the fungus was isolated from decaying brown leaves of sea grass from the: intertidal of a lagoon in Kavaratti island of the Lakshadweep archipelago (10°30'-11.0° N Lat, 72.0°-73.0°E, Long.) off the west coast of India in the Arabian sea. Discs this synthetic medium. However, it produced fertile basidiomata on medium containing alpha cellulose or after a long incubation in malt extract agar medium. Based on fertile basidiomata using the key of Ryvarden and Johansen, it was identified as Flavodon flavus (Kl) Ryv.
Since the strain #312 was isolated from a marine habitat, the effect of salinity on
its growth can be studied by growing in medium prepared with 100% sea water, 50%
sea water and in distilled water. Growth in natural medium such as malt extract broth
and synthetic media were compared for this purpose. Malt extract broth contains 2%
malt extract and 0.3% peptone in distilled water or seawater of various salinities.
Instead of natural-seawater which may not be available to every one, synthetic .sea
salts 'of reputed companies can be used in preparing artificial sea water. We used
Instant Ocean-salts obtained from Aquarium Systems, Mentor Ohio, USA. By
dissolving 15 g and 30 g of Instant Ocean salts in 1 litre of distiHed water, salinity
equivalents of 1.5 and 3.0% were obtained. The former is referred to as 50% artificial
sea water or half-strength sea water. The synthetic media can be prepared in distilled
water or-50% artificial sea water containing 1% glucose as carbon source,., 2.4mM
ammonium tartrate as the nitrogen source, thiamine, trace metal solution, macro
element solution containing sodium, potassium and manganese salts, Tween 80,
veratryl alcohol and 20mM sodium acetate buffer at pH 4,5. This synthetic medium is
referred to as low nitrogen medium. Growth of the fungus is measured as mycelial dry
weight produced at the end of 10 days incubation in various media. For determination
of dry weight, the contents of three flasks are vacuum filtered through tared Whatmen
GF/C filter papers, rinsed with 100ml of distilled water to remove salts and dried to a constant weight and the net mycelial dry weight is calculated. Table 1.*

(Table Removed)
Figures represent mg dry weight of fungus per 10 ml of medium in 10 days.
The example given here shows that the fungus F. flavus grows much better in
the presence of 50% artificial sea water and thus can be used in presence of synthetic salts or in estuarine conditions.
EXAMPLE-2
The ability of F. flavus, NIOCC isolate 312, to degrade and decolorize synthetic dyes was tested in nutrient rich medium such as malt extract broth. Preparation of medium was done as described in Example 1. Sterile media are dispensed in. 9 ml amounts into 125 ml sterile rubber-stoppered Erlenmeyer flasks. The flasks are inoculated with 1ml of fungal inoculum. For inoculum preparation, mycelial mats grown in malt extract broth (25 ml in 250 ml foam-plugged Erlenmeyer flasks) for 10 days, at room temperature were washed twice with 150 ml of sterile water and blended in 20 ml of low nitrogen medium using a Sorvall blender (with four 30-seconds spurts). One ml of this blended mycelium (equivalent to 3 mg initial dry weight of mycelium) was used for inoculating media in 125 ml Erlenmeyer flasks. The flasks are flushed with 100% 02 at the time of inoculation and every alternate day thereafter. Culture flasks are
incubated at room temperature. Individual dye solutions were added to 4-day-old
cultures of F. flavus, to give final dye concentrations of 0.02%. The dyes used were
Azure B, Brilliant Green, Crystal Violet, Congo Red, Remazol Brilliant Blue R, Poly B-
411 and Poly R-478. Stock solutions of dyes were prepared in distilled water and filter
sterilized. Autoclaved cultures supplemented with dyes served as heat-killed controls.
Aliquots (0.5ml) of culture supematants from experimental cultures and from heat-killed
controls were removed aseptically at 2-day intervals, diluted appropriately with distilled
water and the changes in absorbance maxima for the different dyes were measured
using a Varian Cary 1 Bio UVA/isible spectrophotometer (Varian, Australia). Azure B
was measured at 647 nm, Brilliant Green at 625 nm, Congo Red at 495 nm, Crystal
Violet at 590 nm, and Remazol Brilliant Blue R at 595 nm. (Heinfling, A., M. Bergbauer,
and U. Szewzyk. 1997. Biodegradation of azo and phthalocyanine dyes by Trametes
versicolor and- Bjerkandera adusta. Applied Microbiology and Biotechnology. 48:261-
266). Decolorization of Poiy B and Poly R was monitored by determining the absorption
ratio at 593/483 nm and 513/362 nm respectively (Gold, M.H., J.K. Glenn, and M. Alic.
1988. Use of polymeric dyes in lignin biodegradation assays. Methods in Enzymology.
161: 74-78). The results are calculated as the difference in percent decolorization
between heat-killed and experimental cultures. Degradation of various dyes was also
monitored by comparing the changes in the visible spectra of native dyes in heat-killed
culture fluids with that of decolorized dyes in experimental'culture fluids at 400-800 nm.
This was carried out at the end of the experiment.
Accordingly, Figure 1a-1g shows the percentage of decolorization of various
dyes by F. flevus, NIOCC isolate 312 when grown in simple natural medium such as malt extract broth. Azure B and Remazol Brilliant Blue R were decolorized up to 60% within 7 days. Decolorization of Poly B is delayed initially but the final percentage of decolorization is 60%.
EXAMPLE-3
Decolorization of various dyes by F. flavus, NIOCC isolate 312, was tested in synthetic medium such as low nitrogen medium prepared with distilled water. Preparation of synthetic medium was done as described in Example-1. Inoculation, oxygenation and incubation were done as described in Example-2. Experimental procedures of addition of dyes and monitoring the decolorization were same as described in Example-2.
Accordingly, Figure 2a-2g show that about 70% decolorization of all the dyes took place within 7 days in low nitrogen medium prepared with distilled water.
EXAMPLE-4
Decolorization of various dyes by F. flavus, NIOCC isolate 312, was tested in synthetic medium such as low nitrogen medium prepared with half-strength artificial sea water. Artificial sea water was prepared by dissolving 15g of Instant Ocean salts as per the manufacturer's instructions (Instant Ocean of Aquarium Systems, Mentor, OH) in 1 litre of distilled water to get salinity equivalent of 15 parts per thousand and filtered through a GF/C filter paper. The normal salinity of sea water is 30 parts per thousand. Preparation of low nitrogen medium was done as described in Example-1. Inoculation, oxygenation and incubation were done as described in Example-2. Experimental
procedures for addition of dyes and monitoring the decolorization were same as described in Example-2.
Accordingly, Figure 3a-3g show decolorization of various dyes by F. flavus, NIOCC isolate 312, when grown in low nitrogen medium prepared with 50% artificial sea water. More than 80% decolorization of all dyes was achieved within 7 days in this medium.
The fungus F. flavus also degraded the various dyes as seen by changes in the visible spectra of native dyes in experimental cultures in comparison with heat-killed control culture fluids. Figure 4a-4e shows complete degradation of some of the dyes by the fungus F. flavus when grown in low nitrogen medium with 50% artificial sea water.
EXAMPLE-5
In order to show that degradation of dyes is brought about by production of.
lignin-modifying enzymes in malt extract broth, the fungus was grown in this medium as
described in Example-2.; Inoculation, oxygenation and incubation were carried out as
described in Example-2. .
Gdntents df three flasks were filtered on different days of incubation using Whatman GF/C filters and the extracellular fluids were analysed for lignin-modifying enzymes such as manganese-dependent peroxidase (MNP), lignin peroxidase (LIP) and laccase. A convenient assay for MNP involves monitoring the enzyme's oxidation of Mn(ll) to Mn(lll). The reaction mixture contains enzyme, 0.1 M sodium tartrate buffer (pH 5.0), 0.1 M H2O2 and 0.1 mM MnS04. Reactions are initiated by the addition of
H2O2 and increase in absorbance at 238 nm is monitored during the first 5-30 sec of reaction. One unit of MNP oxidizes 1 µmol of Mn(ll)/min (Paszczyniski, A, R. L. Crawford, and V.-B. Huynh. 1988. Manganese peroxidase of Phanerochaete chrysosporium: purification. Methods in Enzymoiogy. 161: 264-270). Lignin peroxidase catalyzes oxidation of veratryl alcohol to veratraldehyde by H202, The reaction mixture for assay contains veratryi alcohol, tartaric acid at pH 3.0, 8 mM H2O2 and enzyme solution. Increase in absorbance at 310 nm is monitored for 1 min. One unit of enzyme is defined as 1 µmol of veratryl alcohol oxidized per min (Tien, M., and T. K. Kirk. 1988. Lignin peroxidase of Phanerochaete chrysosporium. Methods in Enzymoiogy. 161; 239-249). Lacasse activity was determined by using the substrate 2,21azino-bis{3-
ethylbenzthiazoline-6-sulfonic acid), in 0.1 M glycine buffer at pH 3.0. The reaction
was monitored by measuring change in absorbance at 405 nm for 5 min. The enzyme
units are expressed as nkatals/L (Niku-Paavola, M-L, E. Karhunen, P. Salola and V.
Raunio. 1988. Ligninolytic enzymes of the white-rot fungus Phlebia radiata.
Biochemical Journal. 254: 877-884). One katal is defined as one molar extinction
coefficient of product formed per second.
Accordingly, Figure 5a represents production of MNP in malt extract broth
prepared with distilled water by the fungus F. fiavus, NIOCC isolate 312.
Accordingly Figure 5b represents production of LIP in malt extract broth
prepared with distilled water by the fungus F. fiavus, NIOCC isolate 312.
Accordingly, Figure 5c represents production of laccase in malt extract broth
prepared with distilled water by the fungus f. fiavus, NIOCC isolate 312.
EXAMPLE-6
In order to show that degradation of dyes is brought about by production of lignin-modifying enzymes in synthetic medium such as low nitrogen medium, the fungus was grown in the low nitrogen medium prepared with distilled water as described in Example-1. Inoculation, incubation, oxygenation and sampling of culture filtrates were done as described in Example-5. Enzyme assays of the culture fluids were carried out as described in Example-5.
Accordingly Figure 6a represents production of MNP in low nitrogen medium prepared with distilled water by the fungus F. flavus, NIOCC isolate 312.
Accordingly, Figure 6b represents production of LIP in this medium by the fungus F. flavus, NIOCC isolate 312.
Accordingly, Figure 6c represents production of laccase in this medium-prepared with distilled water by the fungus F. flavus, NIOCC isolate 312.
EXAMPLE-7
In order to show that degradation of dyes is brought about by production of lignin-modifying enzymes in the presence of salts, the fungus was grown in the low nitrogen medium prepared with half-strength artificial sea water as described in Example-1. Inoculation, incubation, oxygenation and sampling of culture filtrates were done as described in Example-5. Enzyme assays of the culture fluids were carried out as described in Example-5.
Accordingly, Figure 7a represents production of. MNP in low nitrogen medium prepared with 50% artificial sea water by the fungus F. flavus, NIOCC isolate 312. No
LIP production was detected in this medium.
Accordingly, Figure 7b represents production of laccase in low nitrogen medium prepared with 50% artificial sea water by the fungus F. flavus, NIOCC isolate 312.
EXAMPLE-8
Wood and other lignocellulosics are natural substrates for ligninolytic fungi. Therefore, lignin-modifying enzyme production was compared in cultures grown in sugarcane bagasse, which is an inexpensive raw material for large-scale application of this fungus for its potential use in field trials. As the above examples 5, 6, and 7 showed, the degradation of dyes is brought about in various media by production of lignin-modifying enzymes in these media, we wanted to show production of these enzymes in bagasse medium also if it were to be used for application in field trials.
For preparing media with lignocellulosics as the sole carbon/nitrogen/energy source, sugarcane bagasse powder (16 screen mesh/sq. in) was added at 1% final concentration in distilled water. To minimize contamination by residual sugars, the sugarcane bagasse was washed under running tap water for 12 h, followed by a wash with three volumes of distilled water and dried at room temperatures.
The sugarcane bagasse medium was. inoculated, oxygenated, incubated and sampled as described in Example-5. The extracellular culture fluids were assayed for MNP, LIP and laccase as described in Example-5.
Accordingly, Figure 8a represents production of MNP in sugarcane bagasse medium prepared with distilled water by the fungus F. flavus, NIOCC isolate 312.
Accordingly, Figure 8b represents production of LIP in sugarcane bagasse
medium prepared with distilled water by the fungus F. flavus, NIOCC isolate 312.
Accordingly, Figure 8c represents production of laccase in sugarcane bagasse medium prepared with distilled water by the fungus F. flavus, NIOCC isolate 312.
It is of interest that MNP, which has been shown to be important in delignification of Kraft pulp (Paice, M.G., l.d. Reid, R. Bourbonnais, F. S. Archibald and L. Jurasek. 1993. Manganese peroxidase produced by Trametes versicolor during pulp bleaching, demethylates and delignifies kraft pulp. Applied Environmental Microbiology. 59:260-265), and in decolorizing Kraft bleach plant effluents (Michel, F. C. Jr., S. B. Dass, E.A. Grulke, and C.A. Reddy. 1991. Role of manganese peroxidases and lignin peroxrdases of Phanerochaete chrysosporium in decolorization of kraft bleach plant effluent. Applied Environmental Microbiology. 57: 2368-2375) was produced in highest titres in cultures of F. flavus grown with sugarcane bagasse. These results further suggest that sugarcane bagasse can potentially be used for bulk production of biomass of F. flavus, NIOCC isolate 312, for possible application in field trials for removal of dyes in dye-containing waste-waters and soil.
EXAMPLE-9
As the previous example showed production of lignin-modifying enzymes in sugarcane bagasse suspended in distilled .water, for its application in presence of salts we can similarly grow the fungus in sugarcane bagasse suspended in half-strength artificial sea water. The medium was inoculated, oxygenated, incubated and sampled as described in the Example-5. The extracellular culture fluids were assayed for MNP, LIP and lacasse as described in Example-5.
Accordingly, Figure 9a represents production of MNP in sugarcane bagasse medium prepared with half-strength artificial sea water by the fungus F. flavus, NIOCC isolate 312. No LIP production was detected in this medium.
Accordingly, Figure 9b represents production of laccase in sugarcane bagasse medium prepared with half-strength artificial sea water by the fungus F. flavus, NIOCC isolate 312.
Examples 1-9 illustrate that the said fungus Flavodon flavus, NIOCC isolate 312, can be grown in conventional media prepared with distilled water or half-strength artificial sea water and in sugarcane bagasse suspended in distilled water or half-strength artificial sea water and the biomass of the fungus thus obtained can be used for removal of synthetic dyes in freshwater and estuarine conditions and in soil. The fungus can be immobilized using conventional methods and can be used for removal of synthetic dyes in aquatic habitats and soil. The above mentioned examples further illustrate that the degradation of synthetic dyes by the said fungus is brought about by production of lignin-modifying enzymes such as MNP, LIP and taccase in conventional media or in sugarcane bagasse medium prepared either with distilled water or half-strength artificial sea water.
The main advantages of the present invention are :
1. The fungus can be grown on large scale using inexpensive raw materials such as
sugarcane bagasse suspended in distilled water or half-strength artificial sea water
for any biotechnological application.
2. The said fungus decolorizes about 80% of various dyes within 7 days in media
prepared with artificial sea water and about 60-70% in media prepared with distilled
water, and thus can be used for treating dye containing waste-waters in the
presence of salts as well.
3. The biomass of the fungus obtained on sugarcane bagasse can be used for seeding
soil contaminated with synthetic dyes in fresh water, estuarine conditions or soil. ,
4. The said fungus produces lignin-modifying enzymes in malt extract broth, low
nitrogen medium prepared either with distilled water or half-strength artificial sea
water and in sugarcane bagasse suspended in distilled water or half-strength
artificial sea water,
5. The said fungus-grows better in the presence of half-strength artificial sea water
and thus can tolerate salinity up to 15 parts per thousand and can be applied for
removal of synthetic dyes in sea water of appropriate salinity, in estuarine
conditions.

1. A process for removal of dyes such as azo dyes, hetrocyclic & polymeric dyes from
dye-containing waste-waters and soil which comprises, growing the white-rot
fungus strain Flavodon flavus such as here in described, any conventional nutrient
medium containing assimilable C and N source, with optinally having salinity up to
15 parts per thousand(ppm) for at least 4 days, then contacting the said biomass
with waste-waters or soil containing dyes for a minimum period of 5 days followed
by removing the fungal biomass by conventional methods in case of waste-waters
to get water devoid of dyes.
2. A process as claimed in claims 1 wherein, the carbon source for growing the fungus
used is selected from glucose or sugarcane bagasse at a concentration of at least
1%.
3. A process is claimed in claim 1 & 2 wherein, the proportion of nitrogen used should
be low and preferably be ammonium tartrate 2.4 mM.
4. A process is claimed in claims 1 to 3 wherein, the salinity of the medium for growing
the fungus ranges between 0 to 15 parts per thousand.
5. A process is claimed in claims 1 o 4 wherein, the age of the said fungus is 4 days
to get maximum decolorization of the said dyes.
6. A process is claimed in claims 1 to 5 wherein, the fungal biomass is immobilized on
a conventional support selected from nylon mesh or immobilization or adsorption.
7. A process for removal of dyes in dye containing waste-waters and soil substantially
as herein described with reference to examples 1-9 and figures 1-9.

Documents:

494-del-1999-abstract.pdf

494-del-1999-claims.pdf

494-del-1999-correspondence-others.pdf

494-del-1999-correspondence-po.pdf

494-del-1999-description (complete).pdf

494-del-1999-drawings.pdf

494-del-1999-form-1.pdf

494-del-1999-form-19.pdf

494-del-1999-form-2.pdf

494-del-1999-form-3.pdf

494-del-1999-petition-137.pdf

494-del-1999-petition-138.pdf

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Patent Number

215594

Indian Patent Application Number

494/DEL/1999

PG Journal Number

11/2008

Publication Date

14-Mar-2008

Grant Date

27-Feb-2008

Date of Filing

31-Mar-1999

Name of Patentee

COUNCIL OF SCIENTIFIC & INDUSTRIAL RESEARCH

Applicant Address

RAFI MARG, NEW DELHI-110 001,INDIA

Inventors:

#	Inventor's Name	Inventor's Address
1	TREVOR M.D'SOUZA	DEPARTMENT OF MICROBIOLOGY, MICHIGAN STATE UNIVERSITY, EAST LANSING, MI 48824, U.S.A
2	R.GREG THORN	DEPARTMENT OF BOTANY, UNIVERSITY OF WYOMING, LARAMIE, WY 82071 U.S.A
3	C. A. REDDY	DEPARTMENT OF MICROBIOLOGY AND NSF CENTER FOR MICROBIAL ECOLOGY, MICHIGAN STATE UNIVERSITY, EAST LANSING, MI 48824, U.S.A
4	CHANDRALATA RAGHUKUMAR	NATIONAL INSTITUTE OF OCEANOGRAPHY, DONA PAULA GOA 403 004,INDIA

PCT International Classification Number

C08J 11/04

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA