Department of Biology, Gandhigram Rural Institute, Deemed University, ... from the textile industries of Chinnalapatti, Dindigul district of Tamil Nadu and.
Journal of Microbiology and Biotechnology Research
Scholars Research Library J. Microbiol. Biotech. Res., 2012, 2 (1):57-62
(http://scholarsresearchlibrary.com/archive.html) 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 ______________________________________________________________________________ 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. ______________________________________________________________________________ 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 57 Available online at www.scholarsresearchlibrary.com
R. Kumuthakalavalli et al J. Microbiol. Biotech. Res., 2012, 2 (1):57-62 ______________________________________________________________________________ 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, KCl0.5g, 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 Czapexdox 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. 58 Available online at www.scholarsresearchlibrary.com
R. Kumuthakalavalli et al J. Microbiol. Biotech. Res., 2012, 2 (1):57-62 ______________________________________________________________________________ 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 1 2 3 4 5
Parameters pH Temperature(0C) Colour Odour Electrical Conductivity(mhos)
Value 8.2 260C Dark brown Unpleasant 2400
NEQS 6-9 400C Colourless Odourless 80-450
Table I b: Chemical properties of dye effluent S. NO 1 2 3 4 5 6 7 8 9 10 11
Parameters Hardness (mg/l) Calcium(mg/l) Chlorides(mg/l) Sulphates (mg/l) Total Solids(mg/l) Total Dissolved Solids(mg/l) Total Suspended Solids(mg/l) Dissolved Oxygen(mg/l) Alkalinity(mg/l) Chemical Oxygen Demand(mg/l) Biological Oxygen Demand(mg/l)
Value 640 360 34 68 52.43 5310 1278 5252 18 870 310.18
NEQS 3500 156-400 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 59 Available online at www.scholarsresearchlibrary.com
R. Kumuthakalavalli et al J. Microbiol. Biotech. Res., 2012, 2 (1):57-62 ______________________________________________________________________________ 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
Name of fungi
Rhizopus spp Pencillium spp Aspergillus .niger
1st day Media Media with with normal limited Carbon Carbon 1.40+ 3.90+ 0.04 0.18 2.20 3.20 +0.13 +0.22 3.00+ 4.56+ 0.23 0.14
Percentage of dye decolorisation 4th day 8th day 12th day Media Media Media Media Media Media with with with with with with normal limted normal limited normal limited Carbon Carbon Carbon Carbon Carbon Carbon 9.30 13.50 16.30 21.10 35.80 42.50 +0.30 +1.48 +0.68 +0.46 +0.39 +0.36 13.92+ 23.10 27.30 30.00 45.80 53.25 0.92 +0.55 +0.15 +0.61 +0.43 +0.16 39.90+ 40.40+ 52.30+ 55.67 74.50+ 77.68+ 0.45 0.72 0.52 +0.47 0.50 0.33
16th day Media Media with with normal limited Carbon Carbon 51.70 55.36+ +0.78 0.46 55.25+ 66.57+ 0.26 0.30 77.90+ 89.20+ 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. 60 Available online at www.scholarsresearchlibrary.com
R. Kumuthakalavalli et al J. Microbiol. Biotech. Res., 2012, 2 (1):57-62 ______________________________________________________________________________ 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. REFERENCES [1] McMullan, G., Meehan, C., Conneely, A.,Nirby, N.,Robinson Nigam. P., Banat, IM. and Marchant, SWF.(2001). Appl Microbiol Biotechnol 56:81-87. [2] Zollinger, H.(1991). Color chemistry: syntheses, Properties and Applications of organic Dyes and Pigments, second ed. VHC Publishers, New york. [3] Stolz, A.(1991). Appl Microbiol Biotechnol 56(1-2):69-80. [4] Kim, HT., Lee, Y., Yang, J., Lee, B., Park, CH. and Kim, S. 2004. Desalination. 168,287293. [5] Verma, P. and Madamwar, D. (2003). World Journal of Microbiology and Biotechnology 19,615-618. [6] Yatome, C., Ogawa, T., Koga, D.and Idaka ,E.( 1981). J Soc Dyers Colour. 97:166-168. [7] Trivedy, RK. and Goel, PK. 1981. Chemical and Biological methods of water pollution studies, Environmental Publication, Karad.. [8] Cappuccino, JG and Sherman,N.(1999). Microbiology A Laboratory Manual.4th Ed, Addison Wesley Publication, California. [9] Chen, KC., Hung, WT., Wu, JY. and Houng, JY.(1999). J. Industrial Microbiol. Biotechnology 23:686-690. [10] Sekar, P., Hariprasad, S., Deccaraman, M. (2008). Journal of Applied science Research 4(11):1526-1533. [11] Chium, J., Bergsten-Torralba, LR., Nishikawa, MM., Baptista, DF., Magalhães, DP. and DeSilva, M.( 2000). Brazilian Journal of Microbiology 40: 808-817. [12] Viji K, Sumathi S, Manju BS 2002. Enzyme Microb Technol 27:347-355. [13] Assadi, MMK., Rostami Shahvali, M. and Azin, M. 2001. Desalination.141:331-336. [14] Nicola, W., Guthrie, H. and G. Belson 1998. JSBC.114:38-41. [15] Fu,Y.and Tiraragahavan,Y. (2001). Bioresource, technology 79;251-262. 61 Available online at www.scholarsresearchlibrary.com
R. Kumuthakalavalli et al J. Microbiol. Biotech. Res., 2012, 2 (1):57-62 ______________________________________________________________________________ [16] Maya Devi. and Kaushik, BD.( 2005). India J. of Microbiology Vol. 45:41-44. [17] Dias, M., Lacerda, I., Pimentel, P., De-castro. and Rosa, H. (1980). Lett. Appl. Microbiology 34: 46-50. [18] Annadurai ,G., Chellapandian. and Krishnan, MRV.(1999). Environ Monitor Assess 59;111119. [19] D’Souza, SF. and Nadkarni, GB.(1980). Biotechnol Bioeng 22,191.
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