BIOACCUMULATION OF LEAD METAL IONS FROM PAINT INDUSTRY EFFLUENTS BY INDIGENOUS BACTERIA

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1 BIOACCUMULATION OF LEAD METAL IONS FROM PAINT INDUSTRY EFFLUENTS BY INDIGENOUS BACTERIA PROJECT REFERENCE NO. : 37S0091 COLLEGE : SAPTHAGIRI COLLEGE OF ENGINEERING,BANGALORE BRANCH : BIOTECHNOLOGY GUIDE : BLESSY B. MATHEW STUDENTS : MONISHA J NARESH A TENZIN TSETEN Keywords: Bioaccumulation, metal toxicity, Biochemical tests, Complexometric titration, Transmission Electron Microscopy Introduction Biosorption can be defined as the selective sequestering of metal soluble species, resulting in the immobilization of the metals for retaining heavy metals from dilute solutions [1], whereas bioaccumulation refers to the high efficiency accumulation of any substances or chemicals such as metals, pesticides or other organic chemicals within a living organism. Bioaccumulation occurs when an organism absorbs a toxic material at a rate greater than that at which the substance is lost. It has a lot of advantages over conventional methods of heavy metal removal. Micro-organisms such as bacteria are very much adaptive in nature and hence they are being opted for bioaccumulation process [2]. Heavy metals are an important class of environmental pollutants. There have been many instances where heavy metal toxicity has led to mass deaths. Heavy metals are also used as additives and pigments in paint industries. Lead is number 2 on the Agency for Toxic Substances and Disease Registry's list (ATSDR) [3]. Lead accounts for most of the cases of pediatric heavy metal poisoning [4]. It is a very soft metal and was used in pipes, drains, smelters and soldering materials for many years [5]. Removal of heavy metal ions from effluents can be achieved by various methods such as reverse osmosis, electro dialysis, ion exchange, ultra filtration, chemical precipitation. The existing technologies for waste water treatment have major 1

2 problems. Costs involved in the construction of waste water treatment plants are uneconomical, it consumes lot of space, and commercially they are unattractive and have disposal problems [1]. Compared to conventional methods of waste water treatment, biological methods such as bioaccumulation have found to be more efficient. Objectives Elemental analysis of paint industry waste water for determination of toxic heavy metals. Determination of indigenous bacterial species and study of toxic metal ion nanoforms and their characterization. Determination of bio-accumulant efficiency in response to various parameters. Methodology Materials required: Distilled water, Lead acetate (Pb(CH 3 COO) 2 ), Disodium EDTA (ethylene diammine tetraacetic acid), Eriochrome Black T Indicator, Tartaric acid, ph 10 buffer, Nutrient agar media, Nutrient broth, Peptone, Beef extract, Meat extract, Phenol red, Hydrogen peroxide, Simmon s citrate agar (hi-media), Methyl red- Voges Proskauer broth, Barrit s reagent A and B, Methyl red indicator, Crystal violet stain, Gram s iodine, Alcohol (95%), Safranin, Compound microscope, ph meter, weighing balance, autoclave, hot air oven, laminar air flow, TEM, EDS, Methodology: Waste water samples were collected from a paint industry located near Sapthagiri College of Engineering, Bangalore. The samples were stored in the refrigerator for future use. Preliminary water analysis was done to measure ph, TDS, TSS, BOD, total hardness, alkalinity, iron, sulphate, calcium, chloride and lead. Followed by EDS analysis which was done to trace out the metals present in the waste water samples. Serial dilution of water samples was done and the samples were inoculated on agar plates for the growth of indigenous bacterial species. Identification of selected colonies was done by Gram s staining and the colonies were confirmed through biochemical tests such as Catalase test, Starch Hydrolysis, VP test, Simmon s Citrate test, Oxidase test, Glucose fermentation test etc. Only two bacterial colonies were selected for carrying out Bioaccumulation studies. Two selected colonies were cultured individually and preserved for carrying out further experiments. Lead metal ions were released into the bacterial pellets and the amount of lead accumulated was determined through complexometric titration of lead with EDTA using Eriochrome T 2

3 indicator. Further, bioaccumulation of lead in both the bacterial species were carried out by varying parameters such as contact time, concentration of lead metal ions, ph, temperature and agitation speed individually. Finally the lead ions were characterized through Transmission Electron Microscope. Results and Conclusions Results: Analysis of paint industry effluents: ph, TDS, TSS, BOD, total hardness, alkalinity, iron, sulphate, calcium, chloride and lead content of paint industry effluent was found to be 6.8, 338, 202, 205.2, 732, 890, 0.054, , 67.2, , 1.44 mg/l respectively. The spectra obtained from Energy Dispersive X-Ray Analysis (EDX), referred to as EDS for paint industry effluents shows the presence of two peaks for lead ions and calcium ions which indicate their presence in abundance. Out of the obtained bacterial colonies only two were selected and their gram s staining showed that one bacterial colony was gram positive and the other was gram negative. By performing various Biochemical tests, the indigenous bacterial colonies were identified as Bacillus subtilis and Pseudomonas aeruginosa. Bioaccumulation of lead was carried out for both the species individually and the amount of lead accumulated was found out through Complexometric titration with EDTA using Erio-T indicator. Bioaccumulation studies for both the bacterial species gave different results by varying many parameters such as concentration of lead solution, contact time, ph, temperature and agitation. Conclusion: Bioaccumulation of lead metal ions was conducted by individual cultures of Bacillus subtilis and Pseudomonas aeruginosa which were isolated from paint industry effluents. The lead bioaccumulation studies using individual cultures of 0.2g of Bacillus subtilis resulted in 60% of accumulation at a concentration of 20mg/l of lead solution, at 48hrs, at ph 6.8, at temperature 27 C and at 250rpm. The lead bioaccumulation studies using individual cultures of 0.2g of Pseudomonas aeruginosa resulted in 40% of accumulation at a concentration of 20mg/l of lead solution, at 48hrs, at ph 6.9, at temperature 27 C and at 250rpm. As per the results obtained Bacillus subtilis was found to be more efficient in bioaccumulation of lead than Pseudomonas aeruginosa. Thus according to this study, we may say that gram positive bacteria would accumulate more lead ions than gram negative bacteria. Scope for future work 3

4 Bioaccumulation application is facing a great challenge and some investigators proposed several suggestions. For the future of bioaccumulation, there are two trends of bioaccumulation development for metal removal. One trend is to use hybrid technology for pollutants removal [6] especially using living cells. Another trend is to develop good commercial bioaccumulants just like a kind of ion exchange resin, and to exploit the market with great endeavor [7]. In future similar studies can be carried out using different types of micro-organisms such as fungi, protozoa, algae, marine micro-orgnisms etc. Indigenous micro-organisms can be used for the process of bioaccumulation. Such micro-organisms would have got adapted to the environment containing high concentrations of heavy metals and hence can accumulate more amounts of heavy metal ions. Exploring such microorganisms can result in efficient removal of heavy metals from the environment [8]. But further studies need to done by varying several other factors and employing high imaging techniques to study the structural depiction and particle distribution in a better way. A variety of researches demonstrated that bioaccumulation is a useful alternative to the conventional systems for the removal of heavy metal ions from aqueous solution. The development of bioaccumulation process requires further investigation in the direction of modeling, regeneration and immobilization of bioaccumulants, and of treating the real industrial wastewater [7,8]. References 1. Jaishankar, M., Mathew, B. B., Shah, M. S., & KR, S. G. Biosorption of Few Heavy Metal Ions Using Agricultural Wastes. Journal of Environment Pollution and Human Health, Vol 2(1), 2014, pp: Bryan, G. W., M. Waldichuk, R. J. Pentreath, and Ann Darracott. Bioaccumulation of marine pollutants [and discussion]. Philosophical Transactions of the Royal Society of London. B, Biological Sciences 286, Vol 1015, 1979, pp: Agency for Toxic Substances and Disease Registry (ATSDR), Toxic Substances for Lead. 4. Malakootian, M., J. Nouri, and H. Hossaini. Removal of heavy metals from paint industry s wastewater using Leca as an available adsorbent. International Journal of Environmental Science & Technology 6, Vol 2, 2009, pp: Technical EIA guidance manual for integrated paint industry. Prepared for the ministry of environment and forests, government of India. 4

5 6. Tsezos M. Biosorption of metals. The experience accumulated and the outlook for technology development. Hydrometallurgy, Vol 59, 2001, pp: Wang, Jianlong, and Can Chen, Biosorbents for heavy metals removal and their future, Biotechnology advances 27, Vol 2, 2009, pp: Volesky B. Biosorption and me, Water Res, Vol 41, 2007, pp: