Textile industries have been using synthetic dyes intensively because of their ease and cost effectiveness in synthesis. The textile dyes are highly reactive and there fore during processing it is difficult to treat. The commonly used dyes by the textile industry are azodyes (Orange3R YellowGR and BlackRL). Antroquinone (Blue3R) and copper phthalocyanine (T blue). The textile industries utilize large volume of water in its wet processing operation and there by generates substantial quantities of waste water is the principle route by which dyestuffs enter the soil environment (Elliott, 1999). During the past decade, the use of microbiological degradation methods have been under active development in textile and dyestuff industry (Knapp et al., 1995). Among the microorganisms bacteria are the most commonly used for various bioremediation process. As par as fungi are concerned their reports on bioremediation of textile dyes from the environment by fungi are scanty. There are few reports of using fungi especially white rot fungi is the most commonly used fungi for bioremediation. In the environment there are many microorganisms specially fungi which are abundant but their potential is not utilized completely. The ability of fungi to transport a wide variety of hazardous chemicals has aroused interest in using them in bioremediation (Brar et al., 2006). As can be seen from the literature Phanerochaete chrysosporium, basidiomycetes fungus is the only choice organism reported to be potent decolourizer of the effluent (Krik et al., 1992). In the present study an attempt has been made to utilize the common soil inhabiting fungi isolated indigenously from the soil polluted with textile industry for decolarization of textile dyes.
MATERIALS AND METHODS
The soil samples were collected near the places where the effluents are discharged from the factories such as Sayilakshmi Textiles, Jemini Distillery and South India Paper Mills situated around Nanjanagud Town Mysore, Karnataka, India.
Soil samples were subjected to serial dilution to get dilutions and inoculated onto the sterile petri dishes which consist of sterile, cool, molten, Czapex -Dox agar medium and then we followed the spread plate method and kept the plates for incubation at 30±1°C for 4 days. Fungal colonies were identified using steriobinocular microscope and with the help of standard manuals.
Decolourization study: The dye samples were collected by the textile
industry named by Saayilakshmi Textile Industries situated in Nanjanagud area.
The fungal species which are identified as Aspergillus flavous, Fusarium
moniliforme, Fusarium oxysporum and Trichoderma harzianum. The
isolated fungi were tested for its ability to decolourize textile dyes. Textile
dyes, orange3 R (λm = 493 nm), Blue 3R (λm = 572 nm), yellow GR λm
= 413 nm), Black RL (λm - 574 nm) and T. blue (λm = 664 nm) were used
at 200 mg L1 concentration (Devi and Kaushik, 2005).
Fifty milliliter of C- limited Czapex-Dox sterile medium was amended separately
with each of the textile dyes and subsequently inoculated with 2% (v/v) fungal
spore suspension containing 2.5H106 cfu mL1 (colony forming
unit) spores. The flasks were incubated at 30"1°C for 8 days samples drawn
at 2 days intervals for observation. Samples were centrifuged at 10000Hs for
10 min decolourization was assessed by measuring absorbance of the supernatant
with the help of spectrophotometer at wave length maxima (λm) of respective
dye. Two control flasks (dye + medium without inoculums and medium with inoculums
without dye) were maintained.
The percentage decolourization was calculated using following formula.
% Decolourization = Initial OD-Final OD x 100/ Initial OD.
RESULTS AND DISCUSSION
The soil samples collected from several industries of Nanjanagud where the textile industries are located were screened for fungi subjecting to serial dilution and plating methods. The species identified by its different characteristics and the structural arrangements using stereo microscope with the help of standard manual. (Table 1). The different fungal colony shows different structures with different colors. The Aspergillus Trichoderma and Fusarium species were commonly present in the polluted soil samples of all the industries where the soil is collected. The incidence of fungi in the polluted soils depends on the availability of nutrient, oxygen and biological, chemical and physical features of the pollutant.
The decolourization efficiency of A. flavus and F. oxysporum,
F. moniliforme and T. harzianum was monitored at periodic intervals
by measuring the Optical Density after 2, 4, 6, 8 days of incubation. It is
noticed that there was decrease in the OD in all the four species in all the
four colours as the incubation period increased (Table 2).
||Fungal colonies identified in different industrially polluted
|+: Present; - : Absent
||Percent decolourization of textile dyes by species of Aspergillus,
Fusarium and Trichoderma
Among the four species A. flavous was more effective followed by
F. oxysporum, F. moniliforme and T. harzianum. The percentage
of decolourization of colours by fungi was also calculated. It was found that
all the fungi were found to be efficient decolourizer of Blue the decolourization
of dye amounted to 99.0, 98.8, 98.59 and 98.03%, respectively within 8 day.
Orange was recalcitrant to decolurization. The OD from an initial value of 1.729
was reduced only to 0.831, from 1.729 and 1.839 to 1.0, 1.851 to 1.001 and 1.886
to 1.113 by A. flavous, F. moniliforme, F. oxysporum and
T. harzianum respectively. %decolourization was 54.51, 45.16, 45.92 and
40.98 respectively (Fig. 1). The % decolourization of Yellow
colour is slightly higher than orange colour and the % decolourization of Black
colour is similar to Blue colour (Fig. 1).
As par as F. monilifrome, F. oxysporum and T. harzianum
is concerned there is hardly any literature is available about their use for
decolourization of dyes is concerned. In our study it is proved that these fungi
are also showed the potential to degrade the dyes which is equal to the A.
flavous. There are several advantages in using these species for bioremediation.
T. harzianum has the potential to grow fast and it is non pathogenic.
In addition to decolorize the dyes the fungi especially Trichoderma species
is known to process the complete set of enzymes required to breakdown cellulose
to glucose. It is believed that in the decolourization of reactive dyes is due
to the action of azo-reductase enzymes (Sani and Banerjee, 1999) isolated three
microorganisms from soil samples amended with color effluent and found that
they have great potential to bio-transform the tri-phenyl-methane dyes. Decolonization
of textile dyes by different enzymes has been reported earlier. Copper containing
phthalocyanine dye (200 mg L-1) was found to be completely decolorized
within 7 days by Phanerochaete chrysosporium.
||Optical density of dyes treated with fungal species at different
intervals of incubation
In one study, Aspergillus flavus and A. niger were found to be
efficient decolourizer of T. Blue (Cu-phthalucyanine dye) to the extent of 99%
within 8 days of incubation suggesting that like P. chrysosporium (Conneely
et al., 1999) these species can also be employed. We observed maximum
decolonization of BlackRL followed by Blue3R (anthraquinone dye) and relatively
to less extent of Yellow GR and Orange 3R (Azo dyes) by A. flavus. The
possible mechanism of decolourization may be biosorption, which is dependent
on functional groups in the dye molecule and in fungal biomass, which may also
be playing role in the biosorption of dye (Fu and Viraraghavan, 2002).
Under anaerobic conditions, azo-reductases usually cleave azo dyes into the corresponding amines, many of which are mutagenic and/or carcinogenic (O Neill et al., 2000; Chivukula and Renganathan, 1995; Chung and Stevens, 1993) Furthermore, azo reductases have been shown to be very specific enzymes, thus cleaving only azo bonds of selected dyes (Zimmermann et al., 1984).
Excess production of protein in response of dyes and growth in C-limited medium indicated that the fungus utilized the dyes as the sole source of carbon and produced enzymes to degrade the dyes.
Decolorization of dyes by fungi is mainly ascribed to extracellular activity,
which is in agreement with results reported previously for Trametes hispida
(Rodriguez et al., 1999). 1-Amino-subtituted antraquinoid dyes were good
substrates for the T. hirsuta laccase and they were degraded to a similar
extent. Out of the two azo dyes, Direct Blue 71 was the preferred substrate
for the T. hirsuta laccase, which might be due to limited accessibility
of the -OH and -NH2 groups in Reactive Black 5.
However, for smaller substrates, the electronic contribution of substituents on the aromatic ring seemed to be more important than steric effects (Xu, 1996) Electron-donating methyl and methoxy substituents seemed to enhance laccase activity, while electron-withdrawing chloro, fluoro and nitro substituents inhibited oxidation of azophenols and other substituted phenols and phenol analogs by fungal laccases (Chivukula and Renganatham, 1995).
Similar to our present study on decolourization of dye, Kousar and Charya (2002) also reported for fungi such as Aspergillus niger, Fusarium, Oxysporum, Mucor mucedo isolated from textile and dye contaminated soils were able to decolorize the dye effluent (Nicola et al., 1998). In the biotechnological approach to colour removal suggested that the microbiological treatment is ideal for colour removal as less sludge is produced in comparison to lower daily running cost on anchored in comparison to chemical treatment.