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INTERNATIONAL JOURNAL OF ENVIRONMENTAL SCIENCES Volume 2, No 2, 2011 Copyright 2010 All rights reserved Integrated Publishing Association Research

h article ISSN 0976 4402

Microbial diversity in solid waste molasses of Sugar Industry, Aranthangi, Tamilnadu


Jai Shanker Pillai1, Naveen Danesh2, Puttaiah.E.T3, Girish. K4 1-Department of Botany, Gulbarga University, Gulbarga 2-Department of Environmental Science, Gulbarga University, Gulbarga 3-Vice-Chancellor, Gulbarga University, Gulbarga 4-Postgraduate Department of Microbiology, Maharani's Science College for Women, JLB Road, Mysore naveeanu@gmail.com

ABSTRACT Across the world 125 to 130 million tons of sugar is produced every year. About 2/3rd of this is produced from sugarcane and 1/3rd from sugar beet. Sugar industry play an important role in economic growth of country but the effluent releases from the industry posses abrupt changes to water quality and cause water pollution which eventually causes health hazards. The variety of microbes present in the molasses capable to degrade the organic matter in the effluent. The present study reveals the diversity of microorganisms in industrial effluent of molasses. Totally 15 microbial species were isolated from the molasses of Aranthangi sugar industry of Tamilnadu among which six species of fungi, five bacterial species and four species of yeast. Keywords: Bacteria, fungi, yeast sugar industries, effluent 1. Introduction The most important crop from which sugar can be produced in commercial quantity are sugarcane. India is a largest sugar producing country. Today sugarcane is grown in over 110 countries. In 2008 an estimated 1,743 million metric tons were produced worldwide, with about 50 percent of production occurring in Brazil and India (Solomon, 2008). Molasses is one of the major components of growth media used in industrial process. Due to its unique physical and chemical properties molasses has traditionally been used as a major component in compound feeds, livestock feeds and silage additives and most widely used in various industrial processes. Molasses-based distilleries are one of the most polluting industries generating large volumes of high strength wastewater (Y. Satyawali and M. Balakrishnan, 2007). Sugar factory effluent produces obnoxious odour and unpleasant color when released into the environment without proper treatment. Farmers have been using these effluents for irrigation, found that the growth, yield and soil health were reduced. Along with the effects of various industrial effluents on seed germination, growth and yield of crop plants have captivated the attention of many workers (Rahman et al., 2002, Street et al., 2007). Nakajima-Kambe et al., (1999) studied that the various microorganisms were screened for their ability to decolorize molasses wastewater under thermophilic and anaerobic conditions. Isolation and identification of Pseudomonas, Enterobacter, Stenotrophomonas, Aeromonas, Acinetobacter and Klebsiella has more efficiency in reducing the chemical oxygen demand of spent wash. (Ghosh et al., 2004). Bacterial cellulose (BC) production by Acetobacter xylinum subsp,

Received on September 2011 Published on November 2011

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Microbial diversity in solid waste molasses of Sugar Industry, Aranthangi, Tamilnadu

sucrofermentans BPR 2001 using molasses medium was carried out in a jar fermentor (Bae and Shoda 2004). The life in effluent is highly diverse and consists of interacting population of microorganisms and effluent fauna, and their activities affect physical, chemical and biological characteristics of effluent. Some potential fungal strains such as Penicillium pinophilum, Alternaria gaisen, Aspergillus flavus, Fusarium monolifome, A.niger were isolated from sugarcane industrial effluent (Pant and Adholeya 2007b). The aim of this study is to find out pattern of distribution of different microorganisms in sugar industry molasses of Aranthangi sugar industry of Tamilnadu were studied. 2. Materials and Method 2.1 Isolation of bacteria 10ml of the molasses sample was taken in a 250ml conical flask containing 90ml sterile distilled water. The flask was shaken on an electric shaker to get a homogenous suspension and transferring serially 10ml of the molasses suspension to 90ml of sterile distilled water to make different dilutions viz., 10-1, 10-2, 10-3, 10-4 and 10-5. One ml of 10-5 dilution was plated in petridishes containing potato dextrose agar and Nutrient agar medium. The inoculated plates were incubated at 31oC / 24hrs or two days and bacteria appearing over the medium were picked up and mounted on a clean slide, stained with crystal violet, Grams iodine and safranine and observed under the microscope. The bacteria were identified based on colony characteristics. Gram staining methods and by various biochemical tests as given by Bergeys (1984) Manual of Determinative Bacteriology. 2.2 Physiological and Biochemical tests The physiological and biochemical tests were conducted following the methods of Somasegaran and Hoben (1985) and Josey et al., (1979) respectively, as described by Cappuccino and Sherman (1999) to identify the bacteria. 2.3 Isolation of fungi 10ml of the molasses sample was taken in a 250ml conical flask containing 90ml sterile distilled water. The flask was shaken on an electric shaker to get a homogenous suspension and transferring serially 10ml of the molasses suspension to 90ml of sterile distilled water made different dilutions viz., 10-1, 10-2+ and 10-3. One ml of 10-3 dilution was plated in petridishes containing Potato Dextrose Agar medium (PDA). The pH of the medium was adjusted to 5.6. Streptomycin sulphate (100 mgl-1) was added to the media to prevent the bacterial growth. The plates were incubated at 25 + 20C for five days and fungi appearing on the medium were mounted over a clean slide, stained with lacto phenol cotton blue and observed under the microscope photomicrographs were also made. 2.4 Isolation of Yeast 10ml of the molasses sample was taken in a 250-ml conical flask containing 90ml sterile distilled water. The flask was shaken on an electric shaker to get a homogenous suspension and transferring serially 10ml of the molasses suspension to 90ml of sterile distilled water
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made different dilutions viz., 10-1, 10-2 and 10-3. One ml of 10-3 dilution was plated in petridishes containing Sabourauds dextrose agar medium. The pH of the medium was adjusted to 5.6. The fungi and yeast were identified by using standard manuals, such as Manual of soil fungi (Gillman, 1957), More Dematiaceous Hyphomycetes (Ellis, 1976) and Hyphomycetes (Subramanian, 1971). 3. Findings For the present study, the microbial diversity of molasses was carried out. The abundant variation in the types of all living organisms taken together in any geophysical area is called biodiversity. With reference to large organisms (plant, animals etc.,) the distinctive morphological and anatomical features visible to the naked eye enable us to identify the different genera and species and, therefore; we can easily assess the extent of their diversity. Hence, the present study was undertaken to know the bacterial, fungal and yeast speices in industrial molasses. For the present investigation molasses sample was collected from sugar factory. The bacterial, fungal and yeast species were isolated and identified from molasses sample were recorded. 3.1 Bacterial flora in the molasses Bacteria were isolated from the molasses by serial dilution techniques. Then the isolated bacteria were identified through number of various biochemical test (Table. 1). Totally 5 species of bacteria such as Pseudomonas putida, Escherichia Coli, Staphylococcus aureus, Bacillus subtilis and Micrococcus sp were identified from the effluent sample. Ramlake and Bhattacharjee (1992) suggested the polluted habitats found mostly Pseudomonas because it is having ability to degrade various pollutants from water samples. Most studies on the metabolism of organic contaminants have been performed with bacteria especially in the context of bioremediation (Glazer, 1997). Bacteria generally are easier to culture and they grow more quickly than fungi. They are more amenable to molecular genetic manipulations. They are able to metabolize chlorinated and other organic contaminants such as oil and mineralize chemicals using them as carbon or energy source (Glazer, 1997). Dahiya et al. (2001a) isolated Pseudomonas fluorescens from reactor liquid and found that these bacterial strains are capable of decolourizing melanoidin wastewater up to 76% under nonsterile condition and upto 90% in sterile condition. Jain et al. (2002) isolated three bacterial strains from the activated sludge of a distillery effluent identified as a B. megaterium, B. cereus and B. fragairae which were found to remove color and COD from the distillery effluent in the range of 38-58 and 55-68%, respectively. Decolorization of industrial effluents has been achieved by degradation using bacterial and fungal isolates (Suhuttaya Jiranuntipon, 2009). 3.2 Fungal flora in the molasses Totally 6 species of fungi belongs to 5 genus from the molasses were recorded (Table. 1 and fig. 1). Among the genus Aspergillus was recorded as dominant genus with 3 species such as A.niger, A.flavus and A.terrus. The remaining genus such as Penicillium, Fusarium, and Rhizopus were recorded single species each (Table. 3). A total number of 15 species belonging to 9 genera of fungi were isolated during our investigation in various sugarcane industries of Madhya Pradesh by Awasthi et al, (2011). Diverse fungal cultures have been investigated recently for bioremediation process (Aust, 1990 and Bumpus and Aust 1993). By virtue of their aggressive growth, greater biomass production and extensive hyphal reach
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Microbial diversity in solid waste molasses of Sugar Industry, Aranthangi, Tamilnadu

in the environment, fungi have been seen to perform better than bacteria. The high surface to cell ratio of filamentous fungi makes them better degraders under certain niches (Ashoka et al., 2002). In contrast to the bacterial system, the degradative enzymes of fungi are induced by nutrient limitation. Thus, cultivation of the while not fungi on a nutrient limited substrate, will initiate the process (Tuomela, 2002). Biological treatments employing fungi and bacteria have been investigated essentially to decolorize the distillery spent wash. In all cases, it was found necessary to supplement with additional nutrients as well as diluting the effluent for obtaining optimal microbial activity and eventually optimal results (Ohmomo et al., 1988; Sirianuntapiboon et al., 2004a). In recent years, several basidiomycetes and ascomycetes type fungi have been used in the decolourization of natural and synthetic melanoidin in connection with color reduction of wastewaters from distilleries. The fungus have capability to purify the effluent by consumption of organic substances, thus, reducing its COD and BOD, and at the same time to obtain some valuable product, such as fungal biomass for protein-rich animal feed or some specific fungal metabolite. One of the most studied fungus having ability to degrade and decolourize distillery effluent is Aspergillus sps. such as Aspergillus fumigatus G-2-6, A. niger, A. niveus, A. fumigates Ub60 brought about an average of 69-75% decolourization along with 70-90% COD reduction (Ohmomo et al., 1987; Miranda et al.,1996; Jimnez et al., 2003, Radhika Agarwal et al., 2010). 3.3 Yeast flora in the molasses Totally 4 species of yeast such as Saccharomyces cerevisae, Torulopsis glabrata, Candida glabra and Candida albicans were isolated from the molasses (Table. 1 and fig. 2). Yeast Citeromyces was used for treating Municipal Waste Water (melanoidin waste water) and high and stable removal efficiencies in both color intensity and organic matter were obtained. However, the semi-pilot and pilot-scale experiments are to be tested for checking the stability of Citeromyces sp. (Sirianuntapiboon et al., 2003, Radhika Agarwal et al., 2010). Table 1: Biochemical characteristics of isolated bacteria
S.No 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. Biochemical Test Mac Conkey agar test Indole test Methyl red test Voges Proskauer test Citrate utilization test Starch hydrolysis test Urea hydrolysis test Nitrate reduction test H2S production test Cytochrome oxidase test Catalase test P.putida + + + + + + E.coli + + + + + S.aureus + + + + + B.subtilis + + + + + Micrococcus sp + + + -

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It is evident from the available data that microbial composition of effluent sample of industry varied significantly. Maximum numbers of fungal species were recorded compare to bacteria and yeast inhabitants. Determination of microbial diversity is very important because of their economic importance as well as their pathogenicity. So on the basis of reported microbes and their ability of degradation and enzymatic activity of microbial strains they may use in commercial sector. A large number of microbial diversity associated with sugarcane industrial molasses waste and this database created a useful record. Preparation of database provide a base in solving the problems associated with pollution of sugarcane industry and may become a basis for the management of sugarcane industrial effluent.

Figure 1: Fungal flora in the molasses

Figure 2: Yeast flora in the molasses

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Microbial diversity in solid waste molasses of Sugar Industry, Aranthangi, Tamilnadu

4. Conclusion Mechanism of microorganisms in control of environmental pollution is still being explored. However, it is argued that organisms during bioremediation either eat-up/gobble the contaminants especially organic compounds or assimilate heavy metals themselves, thus effectively degrading specific contaminants / harmful compounds and converting them to non-toxic useable by products. 5. References 1. Ashoka, C., Geetha, M.S., Sullia, S.B. (2002), Bioleaching of composite textile dye effluent using bacterial consortia. Asian, J. Microbial Biotechnology and Environmental Science, 4, pp 65 68. Aust, S.D. (1990), Degradation of environmental pollutants, Microb. Ecol., 20, pp 197 209. Awasthi A.K., Pandey.A.K., Rashmi Dubey (2011), Diversity of fungi in effluents of sugar industries of Madhya Pradesh,INTERNATIONAL JOURNAL OF ENVIRONMENTAL SCIENCES.,1(5), pp 834-839. Bae, S.O., Shoda, M. (2004), Production of bacterial cellulose by Acetobacte Xylinum BPR 2001 using molasses medium in a jar fermentor, Chemical Resources Laboratory, Tokyo Institute of Technology.R1-29-4259 Nagatsuta, Midori-ku, Yokohama, Japan. pp 226-8503. Bergeys Manual. (1984), Bergeys Manual of Systematic Bacteriology, Williams & Wilkins, Baltimore: USA. Bumpus, J.A., Aust, S.D. (1993), Biodegradation of DDT (1, 1, 1-trichloro 2, 2 bis0) (4-chlorophenyl) ethane) by the white rot fungus phanerochaete chrysosporium, Appl. Environ. Microb, 53, pp 2001 2008. Cappuccino, J.G., Sherman, N. (1999), Microbiology; A Laboratory Manual, (34d edn.). Rockland Community College, Suffern: New York. Dahiya, J., D. Singh, P. Nigam (2001a), Decolourisation of molasses wastewater by cells of Pseudomonas fluorescens immobilized on porous cellulose carrier, Biores. Technol., 78, pp 111-114. Ellis, M.B (1976), More Dematiaceous Hypomycetes, Commonwealth Mycological Institute Pub., Kew Surrey, England.

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12. Glazer, A.N.(1997), Microbial Biotechnology, WH Freeman and Company. New York. pp. 54-58. 13. Jain, N., Minocha, A.K., Verma C.L. (2002), Degradation of predigested distillery effluent by isolated bacterial strains, Ind. J. Exp. Bot., 40, pp 101-105. 14. Jimenez, A.M., Borja, R., Martin, A. (2003), Aerobic-anaerobic biodegradation of beet molasses alcoholic fermentation wastewater, Process Biochemistry, 38, pp 1275-1284. 15. Josey,D.P.,Beyhon,J.L., Johnson, A.W.B., Beringer, J.E. (1979), Strain identification in Rhizobium using intrinsic antibiotic resistance, J. Appl. Bacteriol., 46, pp 343-350. 16. Miranda, P.M., Benito, G.G., Cristobal, N.S., Nieto, C.H. (1996), Colour elimination from molasses wastewater by Aspergillus niger, Bioresource Technolology, 57, pp 229-235. 17. Nakajima Kambe, T., Shimomura, M., Nomura, N., Chanpornpong, T., Nakahara, T. (1999), Decolorization of molasses wastewater by Bacillus sp. Under themophilic and anaerobic conditions. J Biosci Bioeng, 87(1), pp 119-121. 18. Ohmomo, S. (1988), Screening of anaerobic bacteria with the ability to decolourize molasses melanoidin, Agricultural and biological chemistry, 57, pp 2429-2435. 19. Ohmomo, S. (1987), Decolourization of molasses wastewater by a thermophilic strain Aspergillus fumigatus G-2-6, Agricultural and biological chemistry, 51, pp 3339-3346. 20. Pant, D., Adholeya, A.(2007b), Biological approaches for treatment of distillerywastewater: a review, Bioresource Technology, 98, pp 2321-2334. 21. Radhika Agarwal, Sneh Lata, Meera Gupta,Pratibha Singh.(2010),"Removal of melanoidin present in distillery effluent as a major colorant: A Review, Journal of Environmental Biology, 31, pp 521-528. 22. Rahman, K.S.M., Banat, I.M., Rahman, T.J., Thayumanavan,T., Lakshmanaperumalsamy, P. (2002), Bioremediation of gasoline contaminated soil by a bacterial consortium amended with poultry litter, coir pith and rhamnolipid biosurfactant, Biores. Technol., 81, pp 2532. 23. Ramlake, P.W., Bhattacharjee, J.W. (1992), Bacterial pollution of drinking water sources in north Tripura district, Proc Acad Environ Bio, 1(1), pp 19-26. 24. Satyawali, Y., Balakrishnan, M. (2007), Removal of color from biomethanated distillery spent wash by treatment with activated carbons, Bioresource Technology, 98, pp 2629-2635. 25. Sirianuntapiboon, S., P. Zohsalam, Ohmomo S. (2003), Decolourization of molasses wastewater by Citeromyces sp, WR-43-6. Process Biochemistry, 39, pp 917-924.
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26. Solomon, S.K. (2008), Enviromental pollution and its Management in Sugar Industry in India : An appraisal, Sugar Tech, 7(1), pp 7781. 27. Somasegaran and Hoben. (1985), In : Methods in Legume Rhizobium Technology, University of Hawaii, NIFTAL, Project and Micren, Dept. of Agronomy and Soil., pp 1-300. 28. Street, R.A., Kulkarni, M.G., Strik, W.A., Southway, C., Van Staden, J. (2007), Toxicity of metal elements on germination and seedling growth of widely used medicinal plants belonging to Hyacinthaceae, Bulletin of Environmental Contamination and Toxicolology, 79, pp 371-376. 29. Subramaniam, A., Brown, C.W., Hood, R.R., Carpentor, E.J., Capone, D.G. (2002), Detecting Trichodesmium blooms in Sea WiFS imagery, Deep Sea Res., 49, pp 107-121. 30. Suhuttaya Jiranuntipon. (2009), Decolorization of molasses wastewater from distilleries using bacterial consortium,Dissertation, DAMRONGLERD, Dr.Ing., pp 183 -209. 31. Tuomela, M. (2002), Degradation of lignin and other 14C labeled compounds in compost and soil with an emphasis on white rot fungi, Ph.D Dissertations, Department of Applied Chemistry and Microbiology. University of Helsinki.

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