Introduction

Several electroplating industries including agricultural sector industries, sewage treatment and mining operations all combine to generate hazardous waste water containing enormous amounts of toxic heavy metal^1,2,3,4. The accumulation of these toxic heavy metals in human beings, animal through food chain. Many methods for treatment of heavy metal contaminated wastewater have been used in the world. Among them, chemical precipitation⁷, ion exchange⁸, reduction⁹, adsorption¹⁰, electrochemical precipitation¹¹, solvent extraction¹², membrane separation¹³, Cementation, evaporation, reverse osmosis, foam separation and freeze separation^14,15. Despite effective treatment, these methods are expensive with the high cost of equipment, chemical and manpower. So, the use of microbial method that minimizes metal contaminants has been considerably interested now. In this study bioremediation investigator solved heavy metal problem in wastewater through microorganism. Efficiency for removal of heavy metal from wastewater using microorganisms has been varied from one to another microorganism.  The present work describes the isolation and screening of the heavy metal resistant microorganisms from wastewater, sewage, sludge, industrial effluents and testing of efficient bacterial isolates for removal of heavy metals (Pb , Cd, Cr and Ni).

Materials and Methods

Collection of samples: Samples of sewage, sludge and industrial effluents were collected in sterilized containers from sewage treatment plants at Karnal, Panipat and electroplating industry at Sonepat. These samples were brought to laboratory and kept in refrigerator at 4 ^oC.

Isolation of Bacteria: Bacterial isolates were isolated from samples of sewage, sludge and industrial effluents by serial dilution method using Nutrient agar medium containing 25 ppm of Pb, Ni, Cr, and Cd individually. The 1000 ppm stock solutions of Pb, Ni, Cr and Cd were made in double distilled water using Pb(NO₃)₂, NiCl₂ .6 H₂O, CdCl_2, and K₂Cr₂O₇. The 25 ppm solution of these heavy metals was prepared from 1000 ppm stock solution by dilution with double distilled water. The stock solution of heavy metals were sterilized separately through bacteriological filters and added to sterilize Nutrient medium (Hi Media Pvt. Ltd, Mumbai) to make its concentration 25 ppm. A serial dilution of each sample was made up to 10^-6 and one ml of dilution 10^-4 and 10^-6 was added in sterilized Petri plates. 20 ml Nutrient agar medium containing 25 ppm of one of these heavy metals individually was poured and incubated at 37 ^oC for 48 hours. The colonies of predominant genera of bacteria were picked and purified by serial dilution method⁴.

Screening of bacterial isolates for tolerance to heavy metals: The isolated heavy metal tolerant (25 ppm) bacterial isolates were further screened for tolerance to Pb, Ni, Cr and Cd at 50, 100 and 400 ppm of heavy metals individually on Nutrient agar medium. All the bacterial isolates were streaked on Nutrient agar medium containing 50, 100 and 400 ppm of each of the four heavy metals separately. Streaking of bacterial isolates on normal Nutrient medium served as control (normal growth) for comparison of growth of bacterial isolates on Nutrient medium containing different concentration of heavy metals. Observations on growth of bacterial isolate were made after 48 hours of incubation of inoculated Petri plates.

Uptake of heavy metals by bacterial isolates from liquid media: The highly tolerant bacterial isolates to different heavy metals were evaluated for uptake in Nutrient broth medium containing 50 ppm concentration of different heavy metals Pb, Ni, Cr and Cd individually. Nutrient broth containing 50 ppm of one of the heavy metals was dispensed in100 ml lots to 250 ml conical flask and sterilized at 15lbs/psi for 15 min. These flasks were inoculated with 1 ml of bacterial cell suspension (10⁶ to10⁷ cells/ml) of each bacterial isolate and put on shaker at 150 rpm at 37 ^oC for 48 hours. Uninoculated flasks containing Nutrient broth of 50 ppm concentration of different heavy metals served as control. Bacterial growth was centrifuged  at 5000 rpm for 10 minutes after 48 hours and washed with phosphate buffer saline (PBS).The harvested bacterial biomass was rinsed with double distilled water and dried in hot air oven at 80 ⁰C for 18 hours. The dried bacterial biomass was weighed and heavy metal concentration in it was estimated by digestion with nitric acid and perchloric acid (3:1 ratio)¹⁶. The digestion mixture was filtered through Whatman filter No. 42. and made the  volume of filtrate to 50ml in volumetric flask. The heavy metals concentration in filtrate was estimated by Atomic Absorption Spectrophotometer (AAS)¹⁶.

Results and Discussion

Bacterial growth and uptake of heavy metals by bacterial isolates:

Total 60, 16, 24, 41 isolates selected for tolerance towards the Pb, Cd, Cr and Ni respectively (data not shown). Out of these isolates only 14, 4, 4 and 10 isolates tolerant 400 ppm Pb , Cd , Cr and Ni respectively (Table1).

Table 1: Screening of different bacterial isolates on nutrient agar media containing different concentration of heavy metals.

The maximum dry weight (0.361 g) was observed by bacterial isolate BPb23 followed by BPb24 (0.335 g) in Nutrient broth containing 50 ppm of Pb¹⁷. The minimum dry weight (0.024g) was observed by bacterial isolate BPb10, 15. The maximum uptake (55.88 mg/g) of Pb was observed in bacterial isolate BPb11. Minimum uptake Pb (0.52 mg/g) found in bacterial isolate BPb19 (Table 2)¹⁸.

Table 2: Uptake of Pb by different bacterial strains from liquid media containing 50 ppm Pb.

The maximum dry weight (0.155g) was observed by bacterial isolate BCd9 in Nutrient broth containing 50 ppm of Cd. The minimum dry weight (0.040g) was observed in BCd3. The maximum uptake (2.42 mg/g) of Cd was observed in BCd4. Minimum uptake of Cd (0.34mg/g) was observed in BCd9 (Table 3)^19,22,23,24.

Table 3: Uptake of Cd by different bacterial strains from liquid media containing 50 ppm Cd.

The maximum dry weight (0.098g) was observed in BCr12. The minimum dry weight (0.023g) was found in bacterial isolate BCr10. The maximum uptake (4.29 mg/g) and minimum uptake of Cr (0.02) was observed in bacterial isolate BCr24 and BCr11,16,18 in Nutrient broth containing 50 ppm of Cr respectively (Table 4)^24,25,27,28.

Table 4: Uptake of Cr by different bacterial strains from liquid media containing 50 ppm Cr.

The maximum dry weight (0.95g) was observed in BNi15 in Nutrient broth containing 50 ppm of Ni. The minimum dry weight (0.025g) was found in bacterial isolate BNi3. The maximum uptake (5.39 mg/g) and minimum uptake of Ni (0.16 mg/g) from Nutrient broth containing 50 ppm of Ni respectively (Table 5)^4,26,28.

Table 5: Uptake of Ni by different bacterial strains from liquid media containing 50 ppm Ni.

The above data of dry weight of bacterial isolate and uptake of heavy metal by bacterial isolate indicate, in general, that where there is high dry weight of bacterial isolate, there is less uptake of heavy metal and vice versa. Similar results have been reported by earlier workers⁴. The bacterial isolates were tested in liquid media containing 50 ppm of Pb, Cd, Cr and Ni. Those bacterial isolates which are showing maximum uptake capacity for heavy metals (Pb, Cd, Cr, Ni) are useful for removal of heavy metals from wastewater and industrial effluents.

References

1. Standard of water quality: Indian standard Drinking water quality specification  Bureau of Indian Standard: SI 50010-1991
2. Gadd G.M., Microbiology of extreme environments Metal tolerance, In C. Edwards (edu.). McGraw-Hill, New York,178 (1990)
3. Gadd G.M. and White C., Trends Biochem. Technol., 11, 353 (1993).
4. Congeevaram S., Dhanarani S., Park J., Dexilin M., and Thamaraiselvi K., J. Hazard. Mater., 146, 270 (2007).
5. Alvarez E. A., Mochon M.C., Sanchez J.C., and Rodriguez M.T., Chemosphere, 47, 765 (2002).
6. Singh V., Singh A., and Chandel C.P.,  J Environ. Sci. Eng., 48,103 (2006).
7. Zhou X., Korenaga T., Takahashi T., Moriwake  T. and Shinoda S., Water Res., 27,1049 (1993).
8. Tiravanti G., Petruzzelli D. and  Passino R., Water Sci. Technol., 36,197 (1997).
9. Seaman J.C., Bertsch P.M., and Schwallie L., Environ. Sci. Technol., 33, 938 (1999).
10. Dahbi S., Azzi M.D., and Guardia M., J. Anal. Chem. 363, 404 (1999).
11. Kongsricharoern N. and  Polprasert C., Water Sci. Technol., 34, 109 (1996).
12.  Pagilla K. and Canter L.W., J Environ. Eng., 125, 243 (1999).
13. Chakravarti A.K., Chowdhury S.B., Chakrabarty S., Chakrabarty T. and Mukherjee D.C., Physicochem. Eng. Aspects, 103, 59 (1995).
14. Aksu Z. and Kutsal T., Environ. Technol., 11, 979 (1990).
15. Aksu  Z., Ozer D.,  Ekiz H., Kutsal T. and Calar A., Environ. Technol., 17, 215 (1996).
16. Greenberg A.E., Trussell R.R., Clesceri L.S. and Franson M.A., Standard Methods for the Examination of Water and Wastewater, Amer. Public Health Assoc., Washington DC, USA, 149  (1985).
17. Scott J.A. and Palmer S.J., Biotechnol. Lett., 10, 21 (1988).
18. Kefala M.I., Zouboulis A.I. and Matis K.A., Environ. Pollut., 104, 283 (1999).
19.  Veglio F. and Beolcini F.,Hydrometallurgy,  44, 301 (1997).
20.  Volesky B., Biosorption of heavy metals. Boca Raton, FL: CRC Press, (1990).
21. Ledin M., Earth Sci. Rev., 51, 1 (2000).
22. Puranik P.R. and Paknikar K.M., Biotechnol. Prog., 5, 228 (1998).
23. Gourdon R., Bhende S., Rus E. and Sofers S., Biotechnol. Lett., 12, 839 (1990)
24. Katircioğlu H., Aslim B., Rehber Türker A., Atici T. and Beyatli Y., Bioresour. Technol., 99, 4185 (2008).
25. Nicholas R.A., Stenberg S.P.K. and Kathryn C., Biores. Technol., 89, 41 (2003).
26. Srinath T., Garg S.K. and Ramteke P.W., Indian J. Microbiol, 42, 141 (2002).
27. Yi-Tin W. and Changsong X., Water Res., 11, 2467 (1995).
28. Rani G. and Harapriya M., Indian J. Exp. Biol., 41, 945 (2003).