Dye Degrading Mycoflora from Industrial Effluents
The present study deals with the distribution of fungal species in Cuddalore dye industrial waste (Lat 11°42`N; Long 79°46`E) and their dye degrading activity. Totally 13 species under 17 genera were isolated and screened for their decolorization activity against methylene blue, gentian violet, crystal violet, cotton blue, Sudan black, malachite green, methyl red and corbol fushion in mineral salt medium and Czepex-Dox broth. In agar medium, decolorization began with the formation of zone of clearance around the colonies. Aspergillus ochraceus, A. terreus, A. niger, Penicillium citrinum and Fusarium moniliforme decolorized maximum number of dyes to a great extent. Mucor racemosus, Cladosporium cladosporioides, Penicillium oxalicum and Trichoderma viride did not decolorize any of the dyes tested. In liquid medium, decolorizing activity was measured spectrophotometrically. Aspergillus ochraceus, A. terreus, A. niger, Penicillium citrinum and Fusarium moniliforme registered maximum color reduction, where as Mucor racemosus, Cladosporium cladosporioides, Penicillium oxalicum and Trichoderma viride expressed very low amount of color reduction. Biomass and the extent of dye removal are directly propositional. Among the 13 species of fungal isolates, Aspergillus ochraceus, A. terreus, A. niger, Fusarium moniliforme and Penicillium citrinum seems to be potential candidates for dye degradation. These strains can be used for the bioremediation of environs polluted with dye effluents.
Explosion of population coupled with industrial revolution results in pollution
of water, air and soil. The discharge of pollutants from various industries
poses threat to the biodiversity of the earth. Among various types of environmental
pollution, water pollution is of major concern in which, dye based industries
are first and foremost. Dyes are released into the environment through industrial
effluents from three major sources such as textile, dyestuff manufacturing and
paper industries (Cripps et al., 1990). One of the most pressing environmental
problems related to dye effluents is the improper disposal of waste water from
dyeing industry. With regard to their color removal by conventional treatment
methods lead to severe water pollution, thus developing cost effective clean-up
operations (Willmott et al., 1998). Globally, it is estimated that, 10%
of the total dyestuffs used (Maguire, 1992), or about 7x105 tones
per annum are released into the environment. Not only the color is aesthetically
unacceptable, it also affect aquatic ecosystem by decreasing the light penetration
and solubility of gases (Saranaik and Kanekar, 1995). Further, some synthetic
dyes, such as azo dyes are carcinogenic or mutagenic (Spadaro et al.,
1992). Microbial degradation seems to be promising compared to other organisms
and the method of application are simpler compared to other available methods.
Several combined anaerobic and aerobic microbial treatments had been suggested
to enhance the degradation of textile dyes (Haug et al., 1991). A great
number of white rot fungi had been reported to produce various lignin degrading
enzymes like laccase, lignin peroxidase and manganese peroxidase or at least
any one of these enzymes (Dey et al., 1994). To degrade the complex lignin
molecule, white rot fungi produce various extra cellular enzymes with low specificity
and strong oxidative activity. Numbers of laboratories are investigating the
ability of the white rot fungus like Phanerochaete chrososporium to degrade
xenobiotic compounds. The present work is an earnest attempt to assess the ability
of various fungi in degradation of commonly used dyes and develop potential
biobleaching agents from superior catabolic strains.
MATERIALS AND METHODS
The fungal strains were isolated from industrial effluent water and sediment samples collected from SIPCOT (Small Industries Promotion Corporation of Tamil Nadu, India) effluent discharge points (Lat 11°42N; Long 79°46E) located in Uppanar estuary at December 2005 work was carried out in CAS in Marine Biology. Diluted sample was taken from 10-3 and 10-4 dilutions and plated on potato dextrose agar (g L-1 potato infusion 200, dextrose 20, agar 15, Distilled water 1000 mL) and incubated at 28°C for 3 to 8 days. To eliminate the bacterial contamination 8 mL of 1% streptomycin was added to the medium. Isolated fungal strains were tested for decolorization activity against commercially important dyes such as methylene blue, gentian violet, crystal violet, cotton blue, Sudan black, malachite green, methyl red and corbol fuchsion in broth cultures as well as in agar medium. Dye degradation in broth culture was done by following the method described by Jothimani and Prabakaran (2003). The fungal cultures were inoculated into the potato dextrose broth and incubated at 27°C for 3 days. After 3 days, 1 mL of the culture suspension was transferred to 50 mL of Czepex-Dox broth (g L-1 Sucrose 30, Sodium nitrate 2, K2HPO4 1 g, MgSO4 0.5, KCl 0.5, FeSO4 0.01 and Distilled water 1000 mL) in 250 mL Erlenmeyer flasks. These flasks were incubated in a shaker at 200 rpm at room temperature for 4-8 days. On the fourth day, an aliquot of one of the aforementioned dye (20 mg) was added in to broth culture and incubated for 5 days. Uninoculated flasks served as controls to assess the abiotic decolorization. OD values were measured spectrophotometrically at 594 nm to estimate the decolorization process. The rate of decolorization was calculated using the following formula as described by Sani and Banerjee (1999).
Dye degradation in solid medium was done by adopting the method of Swamy and Ramsay (1999). Solid mineral salt media (g L-1 sucrose 30, sodium nitrate 2, KH2PO4 1, MgSO4 0.5, KCl 0.5, FeSO4 0.01, Thiamine hydrochloride 0.01, distilled water 1000 mL and micronutrient solution 1.0 mL (mg L-1 B4O7Na2 100, MnSO4 10, (NH4)6 Mo7O24 10, Na2SO4 10, CuSO4 50 and distilled water) was prepared and 20 mg of filter sterilized dye was added after sterilization of the medium. Fungal strains were inoculated in to the plates and incubated at 28°C for 8 days along with control plates. The extent of clear zone formation around the colonies was observed and recorded. For biomass estimation mycelial mat recovered from the Whatman No.1 filter paper was washed with distilled water and dried at 70°C for 48 h and weighed (Yesilada and Ozcan, 1998). Biomass was expressed as dry weight.
RESULTS AND DISCUSSION
Total number of fungal species isolated included 2 members of Zygomycotina
represented by Mucor racemosus and Rhizopus stolonifer and 11
species from 5 genera of Deuteromycotina represented by Hyphomycetes such as,
Aspergillus niger, A. fumigatus, A. japanicus, A. ochraceus
A. flavus, A. terreus, Penicillium oxalicum, P. citrinum,
Fusarium moniliforme, Cladosporium cladosporioides and Trichoderma
|| Decolorization of dyes
|| Growth of fungal strains in different dyes
Total number of fungi isolated in the sediment sample was relatively higher
than water sample indicating that, the sediments act as reservoir of nutrients
for fungal growth. Complete decolorization was represented by the total disappearance
of color without any visible sorption into the biomass (Table
1). Mucor racemosus, Cladosporium cladosporioides, Penicillium
oxalicum and Trichoderma viride did not decolorize any of the dyes
tested. During 8 days of incubation period, it was found that, Aspergillus
ochraceus, A. terreus, A. niger, Penicillium citrinum
and Fusarium moniliforme decolorized maximum number of dyes to a greater
extent and the percentage of color removal increased with incubation period.
It was similar to the earlier findings of Jothimani and Prabakaram (2003) and
Ryu and Weon (1992). From the results, it was noted that, at least 48 h of contact
time is required for the initial acclimatization of the fungal species to establish
in the stimulated environment with dyes. Growth study revealed that, biomass
and dye removal are directly proportional, which may be attributed to the fact
that, the increase of biomass gave more surface area for sorption of the dye
molecules available (Table 2). Aspergillus ochraceus,
A. terreus, A. niger, A japonicus, A. flavus, A.
fumigatus, Rhizopus stolonifer, Penicillium citrinum, P.
oxalicum, Fusarium moniliforme, Mucor racemosus, Cladosporium
cladosporioides and Trichoderma viride shows good decolorization
activity against all the dyes tested (i.e.) methylene blue, gentian violet,
crystal violet, cotton blue, Sudan black, malachite green, methyl red and corbol
fuchsion. Results obtained in this study are testimony to the potential biodegrading
capability of these strains. Decolorization of azo, anthroquinone, heterocyclic,
triphenylmethane and polymeric dyes with this fungus had been reported (Paszczynski
and Crawford, 1992), among the fungal strains tested, Aspergillus ochraceus
and A. terreus were potential against the dyes, which are confirmed
by the larger zone of clearance in the dye containing medium.
|| Decolorization of synthetic dyes by Deuteromycotina and Zygomycotina
in agar medium
|+: - Zone of clearance 0.2 to 0.5 mm, ++: - Zone of clearance
0.6 to 0.8 mm and +++, - Zone of clearance 0.9 to 1.2 mm
Aspergillus ochraceus, A. terreus, A. niger, F. moniliforme
and P. citrinum were able to decolorize methylene blue, sudan black,
cotton blue, malachite green and gentian violet (Table 3).
However, with synthetic dyes like methyl red, corbol fuchsion and crystal violet
a complete decolorization could not be achieved. This may be due to the chemical
functionalities of dyes and a clear relationship between dye structures and
fungal dye biodegradability had not been established so far (Fu and Viraraghavan,
2001). Among the fungal strains Aspergillus ochraceus, A. terreus,
A. niger, Penicillium citrinum and Fusarium moniliforme showed
maximum decolorization (80%), where as Mucor racemosus, Cladosporium
cladosporioides, Penicillium oxalicum and Trichoderma viride showed
very low decolorization (25%). Among the dyes used, cotton blue, methylene blue,
gentian violet, sudan black and malachite green were completely decolorized,
where as 40-60% decolorization was observed with crystal violet, methyl red
and corbol fuchsion. Based on the mycelial weight, Aspergillus ochraceus,
A. terreus, A. niger, Penicillium citrinum and Fusarium
moniliforme were well grown in dye containing liquid medium, where as Rhizopus
stolonifer, Mucor racemosus, A. flavus, A. fumigatus and
A. japonicus showed limited growth. Finally, Cladosporium cladosporioides,
Penicillium oxalicum and Trichoderma viride were found with very
low or negligible growth in dye containing liquid medium. In liquid culture,
rapid dye decolorization by the fungal strain was observed within 24 h. It was
mainly due to the high adsorption of the dye in the mycelium. In subsequent
days, dye decolorization may be due to production of extra cellular enzyme (Haglund,
1999). However further works on their enzyme systems are needed to understand
the decolorizing mechanism of there fungal isolates. The study concluded that,
these fungal consortia on their own can offer a cost effective, easily applicable
and an environmentally sound solution to dye effluents.
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