Journal of Microbiology and Biotechnology Research

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J. Microbiol. Biotech. Res., 2012, 2 (1):57-62
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ISSN : 2231 –3168

CODEN (USA) : JMBRB4

Decolorisation of textile dye effluent using fungal microflora isolated

from spent mushroom substrate (SMS)
N. Manikandan, S. Surumbar Kuzhali and *R. Kumuthakalavalli

Department of Biology, Gandhigram Rural Institute, Deemed University, Gandhigram, India

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ABSTRACT

Decolorisation study was carried out by treating the textile dye effluent with the fungal strains
namely Aspergillus niger, Pencillium spp., Rhizopus spp isolated from spent mushroom substrate
(SMS). The fungal strains were inoculated directly into the effluent, nourished with two
nutritional sources namely with czpexdox medium with normal carbon source and czpexdox
medium with limited carbon source. The fungi treated in carbon limited media showed higher
rate of dye decolorisation. Of the three fungal strains tested Aspergillus niger recorded higher
efficiency of decolorisation. This study indicates that dye decolorisation using Aspergillus niger,
Penicillium spp., Rhizopus spp., can be implement for its viability, user friendly, non
conventional and low cost method.

Key words: Aspergillus niger, Penicillium spp, Rhizopus spp, czapex-dox media, decolorisation.
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INTRODUCTION

A major environmental problem faced due to the textile dyeing and finishing industry is that the
industry produces large volumes of high strength of aqueous wastes. The discharge of waste
water containing recalcitrant residue into rivers and lakes lead to higher Biological Oxygen
Demond (BOD) causing serious threat to native aquatic life [1]. Azo dyes constitute the largest
and the versatile class of synthetic dyes used for textile dyeing and other industrial applications.
They are the aromatic components that are very recalcitrant to biodegradation process [2] [3].
The presence of even very low concentrations of dyes in effluents is highly visible and
degradation products of these textile dyes are often carcinogenic [4]. Currently textile effluents
are treated by physicochemical methods that are often quite expensive; in addition, these
methods don’t generally degrade the pollutants thereby causing accumulation of dyes as sludge
that creates a disposal problem. Over the past decade, biological decolorisation has been
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investigated as method to transform, degrade or mineralized azo dyes. Moreover such
decolorisation and degradation is an eco friendly method and cost comparative alternative to
chemical degradation process [5].Numerous bacteria and fungi are capable of dye
decolorisation, either in pure culture and in consortia, have been reported [6].

MATERIALS AND METHODS

Effluent
Reactive dyes are the dyes, which are mostly used in the textile industries. The effluent samples
were collected from the textile industries of Chinnalapatti, Dindigul district of Tamil Nadu and
immediately subjected to physico-chemical analysis following standard methods [7].

Culture media
To grow the fungal isolates Czapex-dox broth and Czapex-dox medium were used. Czapex-dox
broth ingredients (g/l): Sucrose-5.0g, NaNo3-2.0g, K2HPO4 1.0g, MgSO4.7H2O-0.5g, KCl-
0.5g, FeSO4.7H2O 0.01 with pH 7.0. Czapex-dox agar medium ingredients (g/l): Sucrose-5.0g,
NaNo3-2.0g, K2HPO4 1.0g, MgSO4.7H2O-0.5g, KCl-0.5g, FeSO4.7H2O 0.01 with pH 7.0. In
normal medium 5.0g/l sucrose was added; in carbon limited medium 1.2g/l sucrose was added.
[8].

Enrichment and isolation of dye decolorizing fungi from SMS

Isolation of dye decolorizing fungi from SMS was carried out by enriching the SMS suspension.
One gram of the spent mushroom substrate was suspended in 100 ml of saline (0.85%) solution
and kept in shaker for 30 min. 10 ml of this suspension was added to 250ml conical flasks
containing 40 ml of Czapex-dox broth and 50ml of dye effluent. This mixture was incubated at
28+10 0C on a rotary shaker at 120 rpm for four days and observed for its decolorisation. If there
is no decolorisation, the content of the flask was transferred to a fresh Czapex-dox broth and the
screening procedure in the liquid culture was conducted repeatedly until decolorized culture
appeared. Subsequently an aliquot of 0.2 ml of decolorized broth was spread on Czapex-dox agar
media containing plates supplemented with dye effluent and incubated at 35oC for 4 days.
Colonies showing maximum decolorizing zones were selected and identification of fungal
isolates has been carried out. The identified fungal isolates of SMS were developed in Czapex-
dox broth, incubated for three days at 29+1 oC and used for further study.

Decolorisation experiments and biomass production using fungal mycelia

The textile effluent was treated with the reported isolates in two different treatments. In the first
treatment (T1) 10 ml of czapex-dox broth was mixed with 100ml textile effluent and 5ml of
fungal mycelium. In the second treatment (T2) 10 ml of carbon limited Czapex-dox broth was
mixed with 100ml textile effluent and 5ml of fungal inoculum. Both the treated mixtures were
incubated at 29+1oC for 16 days in shaker at 120 rpm. Raw effluent was treated as control.
Decolorisation efficiency was measured at 4,8,12, & 16th day of incubation.

Determination of decolorisation[9]
The 5ml treated textile dye effluent was centrifuged at 10,000 rpm for 10 min and decolorisation
was assessed by measuring the absorbance of the supernatant at 570nm using spectrophotometer.

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The percentage of decolorisation was calculated using the following formula:

% of Decolorisation = Initial OD-Final OD x 100/Intial OD

RESULTS AND DISCUSSION

The physicochemical characteristics of effluent from textile dye effluent and the recommended
level of NEQS are given in Table 1a, 1b. The textile effluent was highly alkaline in nature with
pH was 8.2 which is below (6-9). Temperature was 260 C which is below the NEQS
recommended level (400 C). Effluent was highly colored, showing presence of high
concentrations of unused dye. Total suspended solids were 5310 mg/l, and BOD was310.18mg/l.

Table I a: Physical properties of dye effluent

S. NO Parameters Value NEQS

1 pH 8.2 6-9
2 Temperature(0C) 260C 400C
3 Colour Dark brown Colourless
4 Odour Unpleasant Odourless
5 Electrical Conductivity(mhos) 2400 80-450

Table I b: Chemical properties of dye effluent

S. NO Parameters Value NEQS

1 Hardness (mg/l) 640 -
2 Calcium(mg/l) 360 -
3 Chlorides(mg/l) 34 -
4 Sulphates (mg/l) 68 -
5 Total Solids(mg/l) 52.43 -
6 Total Dissolved Solids(mg/l) 5310 3500
7 Total Suspended Solids(mg/l) 1278 -
8 Dissolved Oxygen(mg/l) 5252 -
9 Alkalinity(mg/l) 18 -
10 Chemical Oxygen Demand(mg/l) 870 156-400
11 Biological Oxygen Demand(mg/l) 310.18 80-250

Similar findings in pH ,BOD and COD were reported in dye effluent[10].

Enrichment and isolation of dye decolorizing fungi

Enrichment technique had led to the isolation of three fungal isolates viz; Rhizopus spp,
Penicillium spp and Aspergillus niger from SMS.

SMS is the left over substrate after harvesting mushrooms; it is harboring both bacteria and fungi
[11]. The microorganisms from SMS were characterised as Enterobacter sp, Bacillus polymxa,
Micrococcus roseus, Citrobacter fruedi, Bacillus subtilis, Clostridium perfringens, Pseudomonas
aeruginosa, Bacillus cereus, Bacillus licheniformis and Escherichia coli and the fungi from
SMS were characterized as Trichoderma hazianum, Trichoderma polysporum, Trichoderma
viride, Pencillium janthinellum, Mycogene perniciosa and Aspergillus bisporus [12].

More recently, it has been reported that not only the fungus Phanerochaete chrysosporium but
also several other fungi like Geotrichum candidum, Trametes versicolar, Bjerkandera adusta,
Penicillium spp., Pleurotus ostereatus, Pycnoprus cinnabarinus and Pyricularia oryzae are able
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to decolorize rather complex azo dyes [13]. Nicola et al. 1998[14] also reported that fungi such
as Aspergillus niger , Fusarium, oxysporium, Mucor muce isolated from textile and dye
contaminated soils were also able to decolorize the dye effluent. Fu and Tiraragahavan 2001[15]
reported that there are various fungi other than white rot fungi such as Aspergillus niger, which
can also decolorize and /or biosorb diverse dyes. .

Percentage of dye decolorisation of fungal isolates

The percentage of decolourisation of Rhizopus spp inoculated in Czapex-dox medium with
normal carbon 1; 4; 8; 12 and 16th days were recorded as 1.40+ 0.04; 9.30+ 0.30%; 16.30 +
0.68%; 35.80 + 0.39%; 51.70 + 0.78%, respectively. The percentage of decolourisation of
Rhizopus spp in carbon limited Czapex-dox media on 1;4;8;12; 16th days were recorded as
3.90+0.18; 13.53 +1.48; 21.1 + 0.46; 42.0 +0.56; 55 .36 + 0.46 respectively.

The percentage of decolourisation of Pencillium spp inoculated in Czapex-dox medium with

normal carbon on 1; 4; 8; 12 and 16th days were recorded 2.20+0.13; 13.92 +0.92%;
27.30+0.15;45.80 +0.43% and 55.25 +0.26% respectively. The percentage of decolourization of
Penicillium spp in carbon limited Czapex-dox media 1; 4; 8;12 and 16th days were recorded as
3.20+0.22%; 23.1+0.55%; 30.0+0.61%; 53.25 +0.16% and 66.57+0.30% respectively.

The percentage of decolourisation of Aspergillus niger inoculated in Czapex-dox media with

normal carbon 1; 4; 8; 12 and 16th days were recorded 3.00 + 0.03%, 39.9 +0.45%,52.3 +0.52%,
74.5+0.50%, and 77.9 +0.5%, respectively. The percentage of decolourization of Aspergillus
niger inoculated in Czapex-dox media with limited carbon 1; 4; 8; 12 and 16th days were
recorded as 4.56+0.14%; 40.40+0.72%; 55.67+0.47%; 77.68+0.33% and 89.2+0.45 respectively
(Table 3).

Among these three fungal strains Aspergillus niger showed high percentage of decolourization. It
showed the decolourization up to 77.9 +0.50% in Czapex-dox media with normal carbon and
89.2+0.45 in Czapex-dox media with limited carbon..

Table 3: Percentage of dye decolorisation using fungal strains

Percentage of dye decolorisation

1st day 4th day 8th day 12th day 16th day
Name of Media Media Media Media Media Media Media Media Media Media
fungi with with with with with with with with with with
normal limited normal limted normal limited normal limited normal limited
Carbon Carbon Carbon Carbon Carbon Carbon Carbon Carbon Carbon Carbon
Rhizopus 1.40+ 3.90+ 9.30 13.50 16.30 21.10 35.80 42.50 51.70 55.36+
spp 0.04 0.18 +0.30 +1.48 +0.68 +0.46 +0.39 +0.36 +0.78 0.46
Pencillium 2.20 3.20 13.92+ 23.10 27.30 30.00 45.80 53.25 55.25+ 66.57+
spp +0.13 +0.22 0.92 +0.55 +0.15 +0.61 +0.43 +0.16 0.26 0.30
Aspergillus 3.00+ 4.56+ 39.90+ 40.40+ 52.30+ 55.67 74.50+ 77.68+ 77.90+ 89.20+
.niger 0.23 0.14 0.45 0.72 0.52 +0.47 0.50 0.33 0.50 0.45

Aspergillus niger and Aspergillus flavus isolated from the site of dye effluent were reported as
efficient strains in decolourization of textile dyes [16]. In invitro conditions Aspergillus niger
was found to be efficient in decolorizing textile dyes and dry biomass of Aspergillus niger has
been found to be an effective biosorbent of dyes namely methyl violet and basic fuchsin.

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Rhizopus Spp and Aspergillus Spp were two predominant genera found in highly alkaline
effluent. Aspergillus terreus and Aspergillus niger demonstrated nickel uptake capability from
aqueous solution [17]. The decolorization efficiency of fungi may be due to their cell wall rich
in chitin with hydroxyl and amino groups which make them an efficient adsorbent of dye
effluent [18]. The catalytic stability is often improved by immobilization, microorganisms may
degrade higher concentration of toxic components than their free cell counterparts [19].

CONCLUSION

All the three SMS fungal isolates namely Penicillum spp, Rhizopus spp and Aspergillus niger
have potential for dye decolorization. Among these Aspergillus niger showed greater
decolorisation production during 16 days incubation. Decolorisation may be due biosorption,
which is dependent on functional groups in the dye molecule in fungal biomass. The SMS
isolates are able to decolorize and detoxify highly concentrated effluent and therefore the
proposed method has high applicability at industrial scale.

Acknowledgement
The authors acknowledge the UGC MRP, New Delhi for financial support and Gandhigram
Rural Institute for laboratory facilities.

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