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International Journal of Environmental Biology

Universal Research Publications. All rights reserved

ISSN 2277386X Original Article Biodegradation potential of some bacterial strains isolated from sewage water
Sankar Narayan Sinha* and Dipak Paul Environmental Microbiology Research Laboratory, Department of Botany, University of Kalyani, Kalyani 741235, West Bengal, India *Corresponding author email: sinhasn62@yahoo.co.in Received 20 March 2014; accepted 28 March 2014 Abstract Bacteria are well known for their scavenging activity. They are capable of producing a wide variety of enzymes that can degrade complex organic compounds. The present study aims at the isolation of different bacterial strains that are capable of removing organic matters like protein, carbohydrates etc. and alter BOD and COD values of river water. Highest removal of protein and carbohydrate was brought about by Micrococcus luteus SML-1 followed by SML-4 and SML-5. Micrococcus luteus SML-1 and SML-4 removed maximum COD, while maximum BOD was removed by Staphylococcus aureus SSA-1 isolate. 2014 Universal Research Publications. All rights reserved Keywords: Biodegradation, Sewage, Micrococcus luteus, Staphylococcus aureus biological degradation [7,8]. However, these methods 1. Introduction Water plays an important role in the life of human beings. suffer from the drawbacks of either being costly and/or It is most vital and important resource of our planet. Water might generate secondary pollutants, which shows toxicity is being deteriorated by environmental pollution and also than the parent ones [9,10]. by some other factors [1]. In the last few decades, limitless Accordingly, biological methods are generally preferred for urbanization has caused a serious pollution problem due to being more economical and environmentally friendly the disposal of sewage and industrial effluents to water [11,12]. Bioremediation provides an alternative to chemical bodies. Sewage is waste matter resulting from the discharge treatments. Bioremediation uses naturally occurring into the sewers of human excreta and wastewater microorganisms to degrade various types of wastes. originating from a community and its industries [2]. It has a Contaminants are often potential energy sources for high content of both inorganic and organic matter, as well microorganisms. Microorganisms survive in contaminated as high densities of living biota which include pathogenic, habitat because they are metabolically capable of utilizing commensal and environmental bacteria [3]. In India it is its resources and therefore highly suitable for reduction of reported that about 70% of the available water is polluted. pollutant load of an effluent as microorganisms are capable The chief source of pollution is identified as sewage of oxidizing the organic and inorganic constituents [13,14]. constituting 84 to 92 percent of the waste water [4]. River Thus, the present study was aimed to isolate and identify and aquatic ecosystems continually received huge amount bacteria from the sewage effluent that could produce of sewage and industrial effluents, changing the status of extracellular protease enzyme to enhance bioremediation different water quality parameters such as dissolved process. oxygen, BOD, COD etc [5]. 2. Materials and methods In recent decades the threat due to pollution has become a 2.1 Sampling matter of serious concern. As the quantity of untreated Sewage water samples were collected aseptically in a presewage is enormous, it accumulates in the vicinity of sterilized bottle from municipal area of Jagaddal, North 24 human dwellings, leading to uncontrolled decomposition of parganas, West Bengal and transported immediately to the organic content [6]. Several physico-chemical methods of laboratory. treatment of sewage and industrial effluents are there to 2.2 Isolation, characterization and identification of minimize the pollution load of the aquatic water bodies protease producing bacteria such as adsorption, chlorination, coagulation, flocculation, Heterotrophic bacteria were isolated by dilution plate ozonation, solvent extraction, membrane process and technique on nutrient agar. The bacterial colonies were International Journal of Environmental Biology 2014; 4(2): 107-111

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isolated by repeated streaking on fresh nutrient agar medium at room temperature and maintained at 4C on slants of nutrient agar containing 1% gelatine, which act as an inducer for production of proteolytic enzymes. Identification of protease producing bacterial strains was performed by morphological, biochemical and physiological features comparing with standard references [15]. Physiological, morphological and biochemical tests of the bacterial isolates were carried out for their identification as per the procedures outlined in Bergeys Manual of Systemic Bacteriology [16]. 2.3 Study of biodegradation potential The biodegradation potentials of isolated bacterial strains were determined using the method proposed by Kumari et al [17]. The collected water samples were separately inoculated on sterile nutrient agar plates under aseptic condition. The obtained culture is further sub-cultured by streak plate method. The inoculum was taken from the pure culture grown on nutrient agar slants and inoculated into each test tube containing 5 ml of nutrient broth. The tubes

were then incubated at 37C for 24 h. 5 ml of this uniform suspension of each strain was inoculated as initial inoculum into each 1000 ml Erlenmeyer flask containing 500 ml of sterilized sample. Samples were incubated for 2, 4, 6, 8 and 10 days under room temperature. After desired incubation period the samples were analyzed for their physicochemical parameters, total carbohydrate and protein using standard methods [18,19,20]. 3. Results and discussion Physiological, morphological and biochemical characterization of isolated bacterial strains were presented in Table 1. Based on these characters, the bacterial isolates were identified as Micrococcus luteus and Staphylococcus aureus though molecular analysis based on 16S rRNA gene sequencing are necessary for final characterization. Maximum carbohydrate (30%) and protein (36%) was found to be removed by Micrococcus luteus (SML-1) on 2 days, followed by SML-4 (carbohydrate- 28% and protein33%) and SML-5 (carbohydrate- 24% and protein- 28%) (Figure 1 and 2).

Figure 1: Biodegradation potential of Micrococcus luteus (SML-1, SML-4, SML-5) and Staphylococcus aureus (SSA-1) in carbohydrate removal.

Figure 2: Biodegradation potential of Micrococcus luteus (SML-1, SML-4, SML-5) and Staphylococcus aureus (SSA-1) in protein removal. Staphylococcus aureus (SSA-1) showed maximum removal Waste water containing high BOD and COD are of BOD (24%) on 6 days, while this bacterium removed responsible for a heavy depletion of oxygen levels in the maximum carbohydrate (22%) on 6 days, protein (18%) on particular sector of the water bodies [21,22]. Therefore, 2 days and COD (11%) on 6 days (Figure 3 and 4). High these effluents needed elimination of BOD and COD BOD and COD reduction in the treatment of different through proper treatment methods before discharge. effluents by bacterial species was reported [23,24,25]. Maximal removal of COD (14%) by SML-1 on 4 days and Bioremediation is a microorganism mediated transforma13% COD removed by SML-4 on 4 days were noted. -tion or degradation of contaminants into nonhazardousor

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Figure 3: Biodegradation potential of Micrococcus luteus (SML-1, SML-4, SML-5) and Staphylococcus aureus (SSA-1) in BOD removal

Figure 4: Biodegradation potential of Micrococcus luteus (SML-1, SML-4, SML-5) and Staphylococcus aureus (SSA-1) in COD removal Table 1: Physiological, morphological and biochemical characterization of isolated bacterial strains Bacterial isolates Test Micrococcus luteus SML-1 Circular Convex Entire Yellow Coccus + + + + + Micrococcus luteus Micrococcus luteus SML-4 SML-5 Colony characteristics Circular Circular Convex Convex Entire Entire Yellow Yellow Coccus Coccus + + + + + + + + IMViC test + + Staphylococcus aureus SSA-1 Circular Raised Entire White Coccus + + + + + +

Form Elevation Margin Colour Cell shape Gram reaction Coagulase Catalase Oxidase Urease Indole Methyl red VogesProskauer Citrate H2S production NO3- reduction

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Gelatine liquefaction Starch hydrolysis Casein hydrolysis Lipid hydrolysis Hugh-Leiffson (O/F) reaction Glucose Fructose Sucrose Lactose Maltose Mannose Trehalose Raffinose Cellobiose Xylose Galactose Ribose Mannitol Sorbitol

+ + O + + + -

+ + O Utilization of carbon source + + + -

+ + O + + + -

+ + O + + + + + + + + + + +

+ indicates presence or positive reaction; - indicates absence or negative reaction; O = Oxidation; F= Fermentation

less-hazardous substances. Bioremediation converts the organic matter into CO2 and water. Microorganisms are capable of producing a wide variety of enzymes that can degrade complex organic compounds [26, 27]. The nature of the break down products depends upon both the microorganisms present and the environmental circumstances [28]. There are reports of bioremediation of industrial and domestic waste effluents by microorganisms [29]. The mechanism underline biological treatment is the decomposition of finely dispersed matter, colloidal and dissolved substances by metabolism of aerobic microorganisms. In sewage treatment, the additions of selected microorganisms improve the treatment efficiency of BOD, COD, detergent, oil and grease [30, 31]. The present study indicates the application of Micrococcus and Staphylococcus to removed contaminants from sewage water that is continually polluting the river water. 4. Conclusion The results of this study revealed that the Micrococcus luteus (SML-1, SML-4, SML-5) and Staphylococcus aureus (SSA-1), isolated from the sewage effluent are efficient enough to degrade the components in the sewage and therefore could effectively be used for the treatment of sewage water. This biodegradation study certainly will be helpful to some extent for making a pollution free environment. Acknowledgement The authors are thankful to University of Kalyani, West Bengal, India for providing necessary facilities for doing this research. Authors acknowledge the financial support received under the grant from DST PURSE, New Delhi, India for this study. References 1. P.S. Moschella, R.P.W.M. Laane, S. Back, H. Behrendt, G. Bendoricchio, S. Georgiou, P. M. J.

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