Isolation and identification of heavy metal tolerant bacteria from volta battery effluent , Hattar , Haripur Pakistan

Use of microorganisms to remediate contaminated water is cheap, rapid, easy and eco friendly methodology. In present investigation bacterial isolates from Volta Battery effluent Hattar, Haripur are studied for tolerance to Copper, Cobalt, Lead and Zinc salts. Four isolates i.e. Staphylococcus aureus, Pseudomonas aeruginosa, Citrobacterfreundii and Rahnella selected after initial screening were further assayed by time kill assay. Growth kinetics of bacterial cultures and metal accumulation capability are also examined.Pseudomonas aeruginosawas found resistant to tested metals at varying concentrations however Zinc badly influenced bacterial growth. Highest metals uptake efficiency by strains was noted for Cobalt followed by Zinc; 87 and 81 %, respectively. Staphylococcus aureus accumulated metals at greater extent as compared to other strains tested. The results suggests that species isolated from Volta Battery effluent can further be investigated for mechanisms that make them able to grow in high metal concentrations and can be applied to contaminated sites for remediation.


Introduction
Industrial effluents is one of the major source for addition of heavy metals into the environment [1].Heavy metal pollution to wastewater is of great concern to environmentalists [2].Water bodies are the main target sites for disposal of industrial waste water.Heavy metals present in the waste keep on accumulating in the environment and eventually accumulate in food chain therefore causing long lasting health and ecological problems [3].
Various physical and chemical methods can be used for the removal of heavy metals from contaminated sites and water bodies but bioremediation is becoming popular because it is environment friendly and less expensive.It is a process in which potentials of living organisms are used to extract pollutants from contaminated area [4,5].Microorganisms are naturally present in polluted environment.These microorganisms have adopted themselves to resist, detoxify or biodegrade the heavy metals from the environment [6, 7].Heavy metal uptake and efflux in ionic forms involve redox reactions involving the metals therefore bacteria play an important role in biogeochemical cycles [8].Keeping in view the potential of microbes to uptake and degrade environmental pollutants there is a need to isolate, identify and study their response to heavy metal applications in vitro.The aims of the present study were to isolate bacteria from Volta battery effluent, Hattar Haripur, to check these microorganisms for resistance against various concentrations of heavy metals like Cobalt (Co), Zinc (Zn), Lead (Pb) and Copper (Cu) and to determine uptake efficiency of isolated bacterial strains for these metals.

Isolation of bacteria
Effluent samples were collected from Volta battery effluent in sterile plastic containers and transported to laboratory for physicochemical and bacteriological analysis.Different physico-chemical parameters such as pH, temperature, total dissolved solids, and electrical conductance were analyzed.The pH was measured by using an ion specific pH electrode, electrical conductance of the effluent were measured by using a DDS Microprocessor conductivity meter, model-110202 (Bante).

Isolation and identification of bacterial strains
Different dilutions of effluent samples were inoculated on Nutrient Agar plates.The plates were incubated overnight at 37 ○ C in incubator model-WP25A.Different colonies appeared on the media were selected based on morphological characteristics for purification and further studies.Bacterial colonies were examined for their morphological characteristics like color, size and shape.Gram staining and microscopic observation was done for identification of isolates.Biochemical analysis was done for the activities of oxidase, catalase and coagulase test.Further identification was confirmed by API 20E kit and available literature.

Growth kinetics of bacterial isolates
Exponentially growing cultures of the selected bacterial isolates were inoculated into nutrient broth supplemented with 0(control), 20, 40, 60, 80, 100 and 150 ppm of Zn, Pb, Cu and Co. Salts of ZnSO4, Pb(NO3)2, CuSO4.2H2O, and CoSO4 were used as source of Zn, Pb, Cu and Co respectively.Cultures were incubated at 37 ○ C in Heidolph (Unimax) shaker incubator at 150 rpm.Growth of cultures was determined after 12 and 24 hrs of incubation by measuring turbidity of media using 721spectrophotometer at 600 nm.Heavy metal uptake studies Two isolates were checked for heavy metal uptake by adding four heavy metal salts ZnSO4, Pb(NO3)2, CuSO4.2H2O, and CoSO4 at concentration of 100ppm in sterile nutrient broth.The flasks were inoculated with 1 ml of 24 hr old culture (106CFU/ml) and inoculated at 37˚C for 72 hrs at shaker incubator on 150 rpm.After 72 hours, the broth was centrifuged at 5000 rpm for 15 min and supernatant after filtration by membrane filters was tested for determination of heavy metal concentration by Atomic Absorption Spectrophotometer [9].Isolates were incubated in triplicates for each individual metal.On the basis of results obtained by AAS heavy metal uptake efficiency of both isolated species was calculated for each metal.

Statistical analysis
All experiments were performed in triplicate and the results are expressed as the means with standard deviation.The means are further analyzed by ANOVA and LSD at significance level p<0.05.

Physico-chemical characteristic of industrial effluent
The industrial effluent collected from Volta batteries Industry, Hattar Haripur was light brown in color and unpleasant smell.Mean temperature of effluent was 36 ˚C.The average pH of the effluent was approx.8.05.
The mean electrical conductivity and total dissolved solids were 4.5 dS and 4300 mg/L, respectively (Table 1).Results indicate that electrical conductivity and total dissolved solids of battery effluent was higher than National Environment Quality Standards (NEQS), Pakistan.

Heavy metal uptake efficiency of isolates at 72 hrs of inoculation Conclusion
[18]lts of study suggest that both the isolated strains can uptake Co from the solution efficiently.Both the strains are equally competent in removing Zn from the media while S2 can better uptake Pb from the solution.Because of continuous exposure to heavy metals bacterial species can develop resistance to heavy metals and thus can be utilized to detoxify and remove heavy metals from polluted environments[18].On the basis of results of the study it can be concluded that isolated bacterial strains can be utilized for bioremediation of effluents containing heavy metals.Authors' contributionsConceived and designed the experiments: S Nisa, Performed the experiments: I Khan, Analyzed the data: M Zia, Contributed reagents/ materials/ analysis tools: N