Applied Biochemistry and Biotechnology

, Volume 180, Issue 7, pp 1328–1344 | Cite as

Heavy Metal Resistances and Chromium Removal of a Novel Cr(VI)-Reducing Pseudomonad Strain Isolated from Circulating Cooling Water of Iron and Steel Plant

  • Jian-Kun ZhangEmail author
  • Zhen-Hua Wang
  • Yun Ye


Three bacterial isolates, GT2, GT3, and GT7, were isolated from the sludge and water of a circulating cooling system of iron and steel plant by screening on Cr(VI)-containing plates. Three isolates were characterized as the members of the genus Pseudomonas on the basis of phenotypic characteristics and 16S rRNA sequence analysis. All isolates were capable of resisting multiple antibiotics and heavy metals. GT7 was most resistant to Cr(VI), with a minimum inhibitory concentration (MIC) of 6.5 mmol L−1. GT7 displayed varied rates of Cr(VI) reduction in M2 broth, which was dependent on pH, initial Cr(VI) concentration, and inoculating dose. Total chromium analysis revealed that GT7 could remove a part of chromium from the media, and the maximum rate of chromium removal was up to 40.8 %. The Cr(VI) reductase activity of GT7 was mainly associated with the soluble fraction of cell-free extracts and reached optimum at pH 6.0∼8.0. The reductase activity was apparently enhanced by external electron donors and Cu(II), whereas it was seriously inhibited by Hg(II), Cd(II), and Zn(II). The reductase showed a K m of 74 μmol L−1 of Cr(VI) and a V max of 0.86 μmol of Cr(VI) min−1 mg−1 of protein. The results suggested that GT7 could be a promising candidate for in situ bioremediation of Cr(VI).


Pseudomonas sp. Heavy metal resistance Chromate reduction Chromium removal Chromate reductase Bioremediation 



This study was supported by the Fundamental Research Funds for the Central Universities, Wuhan University of Technology, Wuhan, Hubei, P. R. China (2011-IA-032) and by the Research and Development Project, General Administration of Quality Supervision, Inspection and Quarantine of the PRC, Beijing, P. R. China (2015IK123).


  1. 1.
    Kotaś, J., & Stasicka, Z. (2000). Chromium occurrence in the environment and methods of its speciation. Environmental Pollution, 107(3), 263–283.CrossRefGoogle Scholar
  2. 2.
    Malik, A. (2004). Metal bioremediation through growing cells. Environment International, 30, 261–278.CrossRefGoogle Scholar
  3. 3.
    Narayani, M., & Shetty, K. V. (2013). Chromium-resistant bacteria and their environmental condition for hexavalent chromium removal: a review. Critical Reviews in Environmental Science and Technology, 43(9), 955–1009.CrossRefGoogle Scholar
  4. 4.
    Thatoi, H., Das, S., Mishra, J., Rath, B. P., & Das, N. (2014). Bacterial chromate reductase, a potential enzyme for bioremediation of hexavalent chromium: a review. Journal of Environmental Management, 146, 383–399.CrossRefGoogle Scholar
  5. 5.
    Bae, W. C., Lee, H. K., Choe, Y. C., Jahng, D. J., Lee, S. H., Kim, S. J., Lee, J. H., & Jeong, B. C. (2005). Purification and characterization of NADPH-dependent Cr(VI) reductase from Escherichia coli ATCC 33456. Journal of Microbiology, 43(1), 21–27.Google Scholar
  6. 6.
    Bopp, L. H., & Ehrlich, H. L. (1988). Chromate resistance and reduction in Pseudomonas fluorescens strain LB300. Archives of Microbiology, 150(5), 426–431.CrossRefGoogle Scholar
  7. 7.
    Camargo, F. A. O., Okeke, B. C., Bento, F. M., & Frankenberger, W. T. (2003). In vitro reduction of hexavalent chromium by a cell-free extract of Bacillus sp. ES 29 stimulated by Cu2+. Applied Microbiology and Biotechnology, 62, 569–573.CrossRefGoogle Scholar
  8. 8.
    Dey, S., & Paul, A. K. (2012). Optimization of cultural conditions for growth associated chromate reduction by Arthrobacter sp. SUK 1201 isolated from chromite mine overburden. Journal of Hazardous Materials, 213, 200–206.CrossRefGoogle Scholar
  9. 9.
    Opperman, D. J., Piater, L. A., & van Heerden, E. (2008). A novel chromate reductase from Thermus scotoductus SA-01 related to old yellow enzyme. Journal of Bacteriology, 190(8), 3076–3082.CrossRefGoogle Scholar
  10. 10.
    Sultan, S., & Hasnain, S. (2006). Characterization of an Ochrobactrum intermedium strain STCr-5 manifesting high level Cr(VI) resistance and reduction potential. Enzyme and Microbial Technology, 39, 883–888.CrossRefGoogle Scholar
  11. 11.
    Terahara, T., Xu, X., Kobayashi, T., & Imada, C. (2015). Isolation and characterization of Cr(VI)-reducing Actinomycetes from estuarine sediments. Applied Biochemistry and Biotechnology, 175(7), 3297–3309.CrossRefGoogle Scholar
  12. 12.
    Ganguli, A., & Tripathi, A. (2002). Bioremediation of toxic chromium from electroplating effluent by chromate-reducing Pseudomonas aeruginosa A2Chr in two bioreactors. Applied Microbiology and Biotechnology, 58(3), 416–420.CrossRefGoogle Scholar
  13. 13.
    Singh, R., Bishnoi, N. R., & Kirrolia, A. (2013). Evaluation of Pseudomonas aeruginosa an innovative bioremediation tool in multi metals ions from simulated system using multi response methodology. Bioresource Technology, 138, 222–234.CrossRefGoogle Scholar
  14. 14.
    Ishibashi, Y., Cervantes, C., & Silver, S. (1990). Chromium reduction in Pseudomonas putida. Applied and Environmental Microbiology, 56(7), 2268–2270.Google Scholar
  15. 15.
    Viti, C., Pace, A., & Giovannetti, L. (2003). Characterization of Cr (VI)-resistant bacteria isolated from chromium-contaminated soil by tannery activity. Current Microbiology, 46(1), 1–5.CrossRefGoogle Scholar
  16. 16.
    Garg, S. K., Tripathi, M., Singh, S. K., & Singh, A. (2013). Pentachlorophenol dechlorination and simultaneous Cr6+ reduction by Pseudomonas putida SKG-1 MTCC (10510): characterization of PCP dechlorination products, bacterial structure, and functional groups. Environmental Science and Pollution Research, 20(4), 2288–2304.CrossRefGoogle Scholar
  17. 17.
    Pal, A., & Paul, A. K. (2004). Aerobic chromate reduction by chromium-resistant bacteria isolated from serpentine soil. Microbiological Research, 159(4), 347–354.CrossRefGoogle Scholar
  18. 18.
    Christl, I., Imseng, M., Tatti, E., Frommer, J., Viti, C., Giovannetti, L., & Kretzschmar, R. (2012). Aerobic reduction of chromium(VI) by Pseudomonas corrugate 28: influence of metabolism and fate of reduced chromium. Geomicrobiology Journal, 29(2), 173–185.CrossRefGoogle Scholar
  19. 19.
    Dogan, N. M., Kantar, C., Gulcan, S., Dodge, C. J., Yilmaz, B. C., & Mazmanci, M. A. (2011). Chromium(VI) bioremoval by Pseudomonas bacteria: role of microbial exudates for natural attenuation and biotreatment of Cr(VI) contamination. Environmental Science & Technology, 45, 2278–2285.CrossRefGoogle Scholar
  20. 20.
    Dong, G., Wang, Y., Gong, L., Wang, M., Wang, H., He, N., & Li, Q. (2013). Formation of soluble Cr(III) end-products and nanoparticles during Cr(VI) reduction by Bacillus cereus strain XMCr-6. Biochemical Engineering Journal, 70, 166–172.CrossRefGoogle Scholar
  21. 21.
    Puzon, G. J., Roberts, A. G., Kramer, D. M., & Xun, L. (2005). Formation of soluble organo−chromium(III) complexes after chromate reduction in the presence of cellular organics. Environmental Science & Technology, 39(8), 2811–2817.CrossRefGoogle Scholar
  22. 22.
    McLean, J., & Beveridge, T. J. (2001). Chromate reduction by a pseudomonad isolated from a site contaminated with chromated copper arsenate. Applied and Environmental Microbiology, 67(3), 1076–1084.CrossRefGoogle Scholar
  23. 23.
    Dong, X., & Cai, M. (2001). Manual for the identification of bacteria. Beijing: Scientific and Technological Press.Google Scholar
  24. 24.
    Holt, J. G., Krieg, N. R., Sneath, P. H. A., Staley, J. T., & Williams, S. T. (1994). Bergey’s manual of determinative bacteriology. Baltimore: Williams and Wilkins.Google Scholar
  25. 25.
    Hongoh, Y., Yuzawa, H., Ohkuma, M., & Kudo, T. (2003). Evaluation of primers and PCR conditions for the analysis of 16S rRNA genes from a natural environment. FEMS Microbiology Letters, 221(2), 299–304.CrossRefGoogle Scholar
  26. 26.
    Philip, L., Iyengar, L., & Venkobachar, C. (1998). Cr(VI) reduction by Bacillus coagulans isolated from contaminated soils. Journal of Environmental Engineering, 124(12), 1165–1170.CrossRefGoogle Scholar
  27. 27.
    Clesceri, L., Greenberg, E., & Trussel, R. (1989). Standard methods for the examination of water and wastewater. 17th ed. Washington: American Public Health Association.Google Scholar
  28. 28.
    Viti, C., Mini, A., Ranalli, G., Lustrato, G., & Giovannetti, L. (2006). Response of microbial communities to different doses of chromate in soil microcosms. Applied Soil Ecology, 34, 125–139.CrossRefGoogle Scholar
  29. 29.
    Paul, C. D., & Ehrlich, L. H. (1994). Reduction of hexavalent chromium by Pseudomonas fluorescens LB300 in batch and continuous cultures. Applied Microbiology and Biotechnology, 40(5), 756–759.CrossRefGoogle Scholar
  30. 30.
    McLean, J. S., Beveridge, T. J., & Phipps, D. (2000). Isolation and characterization of a chromium-reducing bacterium from a chromated copper arsenate-contaminated site. Environmental Microbiology, 2(6), 611–619.CrossRefGoogle Scholar
  31. 31.
    Mishra, R. R., Dhal, B., Dutta, S. K., Dangar, T. K., Das, N. N., & Thatoi, H. N. (2012). Optimization and characterization of chromium(VI) reduction in saline condition by moderately halophilic Vigribacillus sp. isolated from mangrove soil of Bhitarkanika, India. Journal of Hazardous Materials, 227, 219–226.CrossRefGoogle Scholar
  32. 32.
    Das, S., Mishra, J., Das, S. K., Pandey, S., Rao, D. S., Chakraborty, A., & Thatoi, H. (2014). Investigation on mechanism of Cr(VI) reduction and removal by Bacillus amyloliquefaciens, a novel chromate tolerant bacterium isolated from chromite mine soil. Chemosphere, 96, 112–121.CrossRefGoogle Scholar
  33. 33.
    Ziagova, M. G., Koukkou, A. I., & Liakopoulou-Kyriakides, M. (2014). Optimization of cultural conditions of Arthrobacter sp. Sphe3 for growth-associated chromate(VI) reduction in free and immobilized cell systems. Chemosphere, 95(5), 535–540.CrossRefGoogle Scholar
  34. 34.
    Shukla, V. Y., Tipre, D. R., & Dave, S. R. (2014). Optimization of chromium(VI) detoxification by Pseudomonas aeruginosa and its application for treatment of industrial waste and contaminated soil. Bioremediation Journal, 18(2), 128–135.CrossRefGoogle Scholar
  35. 35.
    Park, C. H., Keyhan, M., Wielinga, B., Fendorf, S., & Matin, A. (2000). Purification to homogeneity and characterization of a novel Pseudomonas putida chromate reductase. Applied and Environmental Microbiology, 66(5), 1788–1795.CrossRefGoogle Scholar
  36. 36.
    Cheung, K., Lai, H., & Gu, J. (2006). Membrane-associated hexavalent chromium reductase of Bacillus megaterium TKW3 with induced expression. Journal of Microbiology and Biotechnology, 16(6), 855–862.Google Scholar
  37. 37.
    Desai, C., Jain, K., & Madamwar, D. (2008). Hexavalent chromate reductase activity in cytosolic fractions of Pseudomonas sp. G1DM21 isolated from Cr(VI) contaminated industrial landfill. Process Biochemistry, 43(7), 713–721.CrossRefGoogle Scholar
  38. 38.
    Majumder, A., Bhattacharyya, K., Bhattacharyya, S., & Kole, S. C. (2013). Arsenic-tolerant, arsenite-oxidising bacterial strains in the contaminated soils of West Bengal, India. Science of the Total Environment, 463(64), 1006–1014.CrossRefGoogle Scholar
  39. 39.
    Martinez, J. L., Sánchez, M. B., Martínez-Solano, L., Hernandez, A., Garmendia, L., Fajardo, A., & Alvarez-Ortega, C. (2009). Functional role of bacterial multidrug efflux pumps in microbial natural ecosystems. FEMS Microbiology Reviews, 33(2), 430–449.CrossRefGoogle Scholar
  40. 40.
    Das, S., Dash, H. R., & Chakraborty, J. (2016). Genetic basis and importance of metal resistant genes in bacteria for bioremediation of contaminated environments with toxic metal pollutants. Applied Microbiology and Biotechnology, 100, 2967–2984.CrossRefGoogle Scholar
  41. 41.
    Das, S., Ram, S. S., Sahu, H. K., Rao, D. S., Anindita, C., Sudarshan, M., & Thatoi, H. N. (2013). A study on soil physico-chemical, microbial and metal content in Sukinda chromite mine of Odisha India. Environment and Earth Science, 69(8), 2487–2497.CrossRefGoogle Scholar
  42. 42.
    Tambekar, D. H., & Gayakwad, S. S. (2013). Studies on bioremediation of chromium [VI] by bacteria isolated from alkaline Lonar Lake (MS) India. Science Research Reporter, 3(1), 87–90.Google Scholar
  43. 43.
    Sarangi, A., & Krishnan, C. (2008). Comparison of in vitro Cr(VI) reduction by CFEs of chromate resistant bacteria isolated from chromate contaminated soil. Bioresource Technology, 99(10), 4130–4137.CrossRefGoogle Scholar
  44. 44.
    Xu, L., Luo, M., Jiang, C., Wei, X., Kong, P., Liang, X., Zhao, J., Yang, L., & Liu, H. (2012). In vitro reduction of hexavalent chromium by cytoplasmic fractions of Pannonibacter phragmitetus LSSE-09 under aerobic and anaerobic conditions. Applied Biochemistry and Biotechnology, 66(4), 933–941.CrossRefGoogle Scholar
  45. 45.
    Pal, A., Dutta, S., & Paul, A. K. (2005). Reduction of hexavalent chromium by cell-free extract of Bacillus sphaericus AND 303 isolated from serpentine soil. Current Microbiology, 51(5), 327–330.CrossRefGoogle Scholar
  46. 46.
    Elangovan, R., Chandraraj, P., & Ligy, K. (2010). Hexavalent chromium reduction by free and immobilized cell-free extract of Arthrobacter rhombi-RE. Applied Biochemistry and Biotechnology, 160(1), 81–97.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Department of Biological Science and Biotechnology, School of Chemistry, Chemical Engineering and Life SciencesWuhan University of TechnologyWuhanPeople’s Republic of China
  2. 2.Technological Center of Inspection and Quarantine, Hubei Entry-Exit Inspection and Quarantine BureauWuhanPeople’s Republic of China

Personalised recommendations