November 2013 January 2014, Vol. 4, No. 1 ; 812-817.

E- ISSN: 2249 1929

Journal of Chemical, Biological and Physical Sciences

An International Peer Review E-3 Journal of Sciences

Available online atwww.jcbsc.org

Section D: Environmental Sciences
CODEN (USA): JCBPAT Research Article

Heavy Metal Tolerance and Accumulation by Bacterial Strains Isolated from Waste Water
Sankar Narayan Sinha* and Dipak Paul
Environment Microbiology Research Laboratory, Department of Botany, University of Kalyani, Kalyani-741235, West Bengal, India
Received: 16 December 2013; Revised: 27 December 2013; Accepted: 3 January 2014

Abstract: Heavy metal pollution for the last few decades is spreading throughout the world along with rapid industrialization. A large number of bacteria and their products can be highly efficient bio-accumulators of different forms of metals. The present study deals with isolation, enumeration and characterization of heavy metal tolerant bacteria isolated from the metal factory effluent. The three potential metal tolerating isolates were morphologically, physiologically and biochemically characterized. All isolates were found to be Gram positive cocci demonstrating physiological characteristics primarily indicative of the genus Aerococcus, though it needs further characterization. The study indicated the potentiality of the isolate GM1 to tolerate and accumulate significance amount of lead, which is indicative of use of this strain for bioremediation of lead pollution in the river Ganga in those metal contaminated area. Keywords: Heavy metal, Bio-accumulation, Waste water, Aerococcus, Bioremediation

INTRODUCTION Presence of heavy metals in the industrial waste water is a potential hazard to aquatic system, animal and human. High concentrations of heavy metals often pose a serious threat to biota and the environment of any ecosystem 1. Heavy metal pollution can be a much more serious problem because they cannot be degraded by natural processes and persist in soil and sediment from where they are released gradually into water bodies as sink. Distributions of such heavy metals through food chain affect ecological community. Microorganisms, owing to their large surface volume ratio and high metabolic activity, are important vectors in introducing heavy metals into food chain. Immediate removal or detoxifications of toxic heavy metals from the ecosystem are of utmost importance. Several reports are there about the 812 J. Chem. Bio. Phy. Sci. Sec. D, Nov. 2013-Jan .2014; Vol.4, No.1; 812-817.

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interaction between microbes and metal pollutants 2-5. Microorganisms have acquired a variety of mechanisms for adaptation to the presence of toxic heavy metals. Among the various adaptation mechanisms, metal sorption, uptake and accumulation, mineralization, extracellular precipitation and enzymatic oxidation or reduction to a less toxic form, and efflux of heavy metals from the cell has been reported 6-8. Conventional methods for removal of heavy metals are electrochemical treatment, chemical precipitation, reverse osmosis, ion exchange, evaporation, and sorption, but each method has been observed to have technical and economical constrains. The purpose of the present study was to screen the heavy metal resistant (Pb, Cr and Cd) bacterial population and to determine the heavy metals resistance of bacteria and biochemical characteristics were used to exploit these isolates for clean-up of industrial wastes for environmental cleanup. MATERIALS AND METHODS Sampling: Effluent samples were collected from a metal factory effluent and also from the lower stretch of the river Ganga near the effluent discharge point of metal and steel factory, Ichhapore, West Bengal, India. Samples were collected in sterilized glass sampling bottles aseptically and transported to the laboratory in an ice-bucket within six hours of collection for heavy metal analysis and bacteriological study. Determination of heavy metals in the effluent and river water: For the analysis of heavy metals like lead (Pb), chromium (Cr) and cadmium (Cd) water samples (200 mL) were digested with 5 mL of di-acid mixture (HNO3:HClO4=9:4 ratio) on a hot plate and filtered by Whatman no. 1 filter paper and made up the volume to 50 mL by double distilled water. The analysis for the heavy metals was done using atomic absorption spectrophotometer (Varian AA240). Heavy metal resistant bacteria isolation: Heavy metal resistant bacteria were isolated from the discharged effluent in a culture medium which contained the following ingredients: casein 20 g, dextrose 10 g, sodium chloride 5 g, sodium formaldehyde sulfoxylate 1g, methylyne blue 2 mg, agar agar 20g, distilled water 1000ml, pH 7.20.2. The Petri plates containing the above media were amended with heavy metals such as lead, chromium and cadmium (200 g ml-1 and 400 g ml-1) by standard spread plate technique 9,10. The inoculated plates were incubated at 242oC for 5 days. After incubation bacterial colonies were purified on agar plates. All the cultures were stored and maintained at 20oC. Determination of metal tolerating activities: The bacterial colonies developed on agar plate were examined for their resistance to chromate both by agar dilution method 11 and broth dilution method 12. In agar dilution method, freshly prepared anaerobic agar plates amended with lead, chromium and cadmium as lead chloride, potassium dichromate and cadmium chloride at various concentrations ranging from 50 to 800 g ml-1 were inoculated with overnight (24 h) grown cultures. All the plates were then incubated at 242oC for 48 h. The minimal concentration of metal in plate inhibiting complete bacterial growth was taken as minimum inhibitory concentration (MIC). Determination of accumulated metal in the cells of bacterial isolates: One ml of 16 h grown cell culture of the isolated bacterial strains was inoculated into 100 ml of nutrient broth in 250 ml Erlenmeyer flask and incubated at 37oC for 72 h. Sterilized metal salt solution were added separately. The metal salts were added in an amount to get the final concentration of 15 ppm respective metal separately. For each treatment three replicas were maintained. The control one was added with same volume of sterilized distilled water. After 72 h, the cell mass was harvested and washed twice with tris-HCl buffer (0.1 M, pH 7.2) by centrifugation (8000 rpm for 10 min at 4oC). The washed cell pellet digested with 10 ml 60% 813 J. Chem. Bio. Phy. Sci. Sec. D; Nov. 2013-Jan .2014; Vol.4, No.1; 812-817.

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HNO3, 0.5 ml of concentrated H2SO4 and 4-5 drops of HClO4 at 140oC until the digest become clear. The cell pellets of the control were similarly digested. The blank consisted of digested samples were made a volume up to 10 ml with di-ionised water. The amount of metal accumulated in the cell is reflected from the amount of metal present in the digest. The metal content of the digest was determined by atomic absorption spectrophotometer. The data were presented as g of metal per mg of dry weight of cell mass. Characterization of bacterial isolates: Bacterial isolates were characterized according to Bergeys manual of systematic bacteriology 13. RESULTS AND DISCUSSION Table 1 indicates the heavy metal concentration in effluent and river water samples. High amount of lead (Pb) was observed in the effluent as well as river water followed by chromium (Cr) and cadmium (Cd). High counts of lead, chromium and cadmium tolerating bacteria were screened from the metalcontaminated effluents discharged into the river Ganga. Among these, lead tolerant bacteria were found (Table 2). A majority (75%) of the isolates were observed tolerant to concentration of lead >400 g ml-1. Around 35% isolates were observed tolerant to lead level of >800 g ml-1 (Table 3). Out of several isolates screened, three potential strains (GM1, GM2 and GM3) showing a high degree of metal resistance accumulated heavy metals in considerable amount (Figure 1). Table-1: Heavy metal concentrations in the metal and steel factory effluents and effluent mixing zone of the river Ganga. Heavy metals Lead (Pb) Chromium (Cr) Cadmium (Cd) Concentration of heavy metals (g ml-1) Effluent 14.061.07 12.521.18 1.170.01 River water 3.200.36 2.920.32 0.580.01

indicates standard error

Table-2: Isolation of heavy metals tolerant bacteria from metal contaminated effluent. Heavy metals Lead (Pb) Chromium (Cr) Cadmium (Cd) Concentration of heavy metals (g ml-1) cfu/ml 200 400 200 400 200 400 4.52.82 x 106 5.23.10 x 105 4.23.23 x 106 5.03.32 x 105 3.02.22 x 106 3.22.18 x 105

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Heavy Metal... Table-3: MIC of heavy metals of twenty heavy metal resistant isolates Heavy metals Lead (Pb) Concentration of heavy metals (g ml-) <400 >400 >800 Chromium (Cr) <400 >400 >800 Cadmium (Cd) <400 >400 >800

Sinha and Paul.

No. of isolates 5 8 7 8 7 5 11 6 3

45 40 35 30 25 20 15 10 5 0 GM1 GM2 GM3

Cellularmetalaccumulation (gml1)

Pb Cr Cd

Figure 1: Accumulation of heavy metals by bacterial isolates Cells of isolate GM1 accumulated significance amount of lead and less amount of chromium and cadmium. On the other hand, isolates GM2 and GM3 accumulated significance amount of chromium and cadmium respectively. The three potential metal tolerating isolates were morphologically, physiologically and biochemically characterized (Table 4). All isolates were found to be Gram positive cocci demonstrating physiological characteristics primarily indicative of the genus Aerococcus, though it needs further characterization. Present study indicated the potentiality of the isolate GM1 to tolerate and accumulate significance amount of lead, which is indicative of use of this strain for bioremediation of lead pollution in the river Ganga in those metal contaminated area. Natural habitats are generally characterized by the co-existence of a large number of toxic and non-toxic cations and therefore it is necessary to study multiple metal effects on the physiology and biochemistry of microorganisms 14. The potential three isolates in this 815 J. Chem. Bio. Phy. Sci. Sec. D; Nov. 2013-Jan .2014; Vol.4, No.1; 812-817.

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study were found tolerant to lead, chromium and cadmium. These observations assume great significance because effluent from any metal related industry contains several metal ions or contaminants and application of these metal tolerate bacteria are found to be important for the recovery and reuse of heavy metals from industrial wastes. Table-4: Morphological, physiological and biochemical characteristics of the isolates Characteristics GM1 Gram character Cell shape Endospore formation Motility Aerobic growth Anaerobic growth Indole production Nitrate reduction Oxidase test Catalase test O/F test H2S production Gelatin liquefaction Carbohydrate utilization Glucose Sucrose Lactose + + + + + + + + + + Coccus + + F + Bacterial isolates GM2 + Coccus + + F + GM3 + Coccus + + F +

+ indicates presence or positive reaction, - indicates absence or negative reaction

CONCLUSION The present study revealed the bio-accumulation of heavy metals viz. lead, chromium and cadmium by heavy metal resistant bacteria isolated from the metal contaminated site of the river. Isolate GM1 was found to tolerate and accumulate significant amount of lead which is indicative of the strain for bioremediation of lead pollution in heavy metal contaminated site of the river. ACKNOWLEDGEMENTS The authors are thankful to Department of Botany, University of Kalyani, and West Bengal, India for providing infrastructure support to carry out the research. Thanks are also due to DST-PURSE for providing financial support.

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Heavy Metal... REFERENCES

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Corresponding author: Sankar Narayan Sinha; Environment Microbiology Research Laboratory, Department of Botany, University of Kalyani, Kalyani-741235, West Bengal, India

817 J. Chem. Bio. Phy. Sci. Sec. D; Nov. 2013-Jan .2014; Vol.4, No.1; 812-817.