1 Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran

2 Department of Microbiology, Faculty of Biology, College of Science, University of Tehran, Tehran, Iran

3 Soil Science Department, Faculty of Agricultural Engineering and Technology, University College of Agriculture and Natural Resources, University of Tehran, Tehran, Iran

4 Department of Microbiology, Falavarjan Branch, Islamic Azad University, Isfahan, Iran


The current study was aimed at isolating and identifying the halophilic and halotolerant bacteria which can produce mercuric reductase in Gavkhuni wetland in Iran. Moreover, tracking and sequencing merA gene and kinetic properties of mercuric reductase in the selected strain were performed in this study. Soil samples were taken from Gavkhuni wetland and cultured in nutrient agar medium with 5% NaCl. To examine the tolerance of purified colonies to mercury, agar dilution method was administered. Similarly, the phylogenetic analysis based on 16SrRNA gene sequencing was conducted. To investigate enzyme activity of kinetic parameters, a spectrophotometer was used to measure the NADPH oxidation decrease at 340 n.m. The results showed that among the 21 halophilic and halotolerant strains isolated from Gavkhuni wetland, 4 were resistant to mercuric chloride. A strain designated MN8 was selected for further studies because it showed the highest resistance to mercury. According to phylogenetic sequencing of 16S rRNA gene and phenotypic characteristics, the strain was categorized in the Bacillus genus and nearly related to Bacillus firmus. This strain had merA gene. The mercuric reductase showed Vmax and Km values of 0.106 U/mg and 24.051 µM, respectively. Evaluation of different concentrations of NaCl at 37°C and pH=7.5 in mercuric reductase enzyme activity indicated that the enzyme shows 50% activity in concentration of 1.5 M. Optimum pH and temperature of  enzyme activity were 7.5 and 35 °C, respectively. The results suggested that MN8 strain could be a proper candidate for bioremediation of mercury-contaminated environments such as industrial wastewaters.

Graphical Abstract

Isolation and characterization of mercuric reductase by newly isolated halophilic bacterium, Bacillus firmus MN8


  • The first report of mercury resistant bacteria with molecular and enzymatic characterization of the mercuric reductase from saline soil in the region
  • Exploration Bacillus firmus MN8 as a new mercury transformer bacterium with mercuric reductase activity
  • Temperature and pH were the main factors of controlling mercuric reductase activity.


Main Subjects

Abou-Shanab, R.A.I.; Van Berkum, P.; Angle, J.S., (2007). Heavy metal resistance and genotypic analysis of metal resistance genes in gram-positive and gram-negative bacteria present in Ni-rich serpentine soil and in the rhizosphere of Alyssum murale. Chemosphere, 68(2): 360-367 (8 pages).

Akhavan Sepahy, A.; Sharifian, S.; Zolfaghari, M.R.; Khalily Dermany, M.; Rashedi, H., (2015). Study on Heavy Metal Resistant Fecal Coliforms Isolated From Industrial, Urban Uastewater in Arak, Iran. Int. J. Environ. Res., 9(4):1217-1224 (8 pages).

Amoozegar, M.A.; Ashengroph, M.; Malekzadeh, F.; Razavi, M.R.; Naddaf, S.; Kabiri, M., (2008). Isolation and initial characterization of the tellurite reducing moderately halophilic bacterium, Salinicoccus sp. strain QW6. Microbiol. Res., 163(4): 456-465 (10 pages).

Anjum, R.; Grohmann, E.; Malik, A., (2011). Molecular characterization of conjugative plasmids in pesticide tolerant andmulti-resistant bacterial isolates from contaminated alluvial soil. Chemosphere, 84:175–181(7 pages).

Barkay, T.; Wagner‐Döbler, I., (2005). Microbial transformations of mercury: potentials, challenges, and achievements in controlling mercury toxicity in the environment. Adv. Appl. Microbiol, 57: 1-52 (52 pages).

Barkay, T.; Gillman, M.; Turner, R.R., (1997). Effects of dissolved organic carbon and salinity on bioavailability of mercury. Appl. Environ. Microbiol., 63(11): 4267-4271 (5 pages).

Bisswanger, H., (2014). Enzyme assays. Perspectives in Science, 1(1): 41-55 (15 pages).

Boden, R.; Murrell, J.C., (2011). Response to mercury (II) ions in Methylococcus capsulatus (Bath). FEMS Microbiol. Lett., 324(2): 106-110 (5 pages).

Chatziefthimiou, A.D.; Crespo-Medina, M.; Wang, Y., Vetriani, C.; Barkay, T., (2007). The isolation and initial characterization of mercury resistant chemolithotrophic thermophilic bacteria from mercury rich geothermal springs. Extremophiles, 11(3): 469-479 (11 pages).

Felsenstein, J., (1985). Confidence limits on phylogenies: an approach using the bootstrap. Evolution, 783-791 (9 pages).

Fox, B.; Walsh, C.T.; (1982). Mercuric reductase. Purification and characterization of a transposon-encoded flavoprotein containing an oxidation-reduction-active disulfide. . J. Biol. Chem., 257(5): 2498-2503 (6 pages).

Freedman, Z.; Zhu, C.; Barkay, T., (2012). Mercury resistance and mercuric reductase activities and expression among chemotrophic thermophilic Aquificae. Appl. Environ. Microbiol., 78(18): 6568-6575 (8 pages).

Ghiasian, M.; Akhavan Sepahy; A.; Amoozegar, M.A.; Saadatmand, S.; Shavandi, M. (2017). Bacterial diversity determination using culture-dependent and culture-independent methods. Global J. Environ. Sci. Manage., 3(2): 153-164 (12 pages).

Giovanella, P.; Cabral, L.; Bento, F.M.; Gianello, C.; Camargo, F. A. O., (2016). Mercury (II) removal by resistant bacterial isolates and mercuric (II) reductase activity in a new strain of Pseudomonas sp. B50A. N. Biotechnol, 33(1): 216-223 (8 pages).

Giri, S.; Dash, H.R.; Das, S., (2014). Mercury resistant bacterial population and characterization of Bacillus sp., isolated from sediment of solid waste discharged point of steel industry. Nalt. Acad. Sci. lett. J., 37(3): 237-243 (7 pages).

Gohari, A.; Eslamian, S.; Mirchi, A.; Abedi-Koupaei, J.; Bavani, A. M.; Madani, K., (2013). Water transfer as a solution to water shortage: a fix that can backfire. J. Hydrol, 491: 23-39 (17 pages).

Héry, M.; Herrera, A.; Vogel, T. M.; Normand, P.; Navarro, E., (2005). Effect of carbon and nitrogen input on the bacterial community structure of Neocaledonian nickel mine spoils. FEMS Microbiol. Ecol., 51(3): 333-340 (8 pages).

Järup, L., (2003). Hazards of heavy metal contamination. British Medical Bull., 68(1): 167-182 (16 pages).

Ledwidge, R.; Hong, B.; Dötsch, V.; Miller, S.M., (2010). NmerA of Tn 501 Mercuric Ion Reductase: Structural Modulation of the p K a Values of the Metal Binding Cysteine Thiols. Biochem., 49(41): 8988-8998 (11 pages).

Nakamura, K.; Iwahara, M.; Furukawa, K., (2001). Screening of organomercurial-volatilizing bacteria in the mercury-polluted sediments and seawater of Minamata Bay in Japan. Clean Products Processes, 3(2):104-107 (4 pages).

Olson, G.J.; Porter, F.D.; Rubinstein, J.; Silver, S., (1982). Mercuric reductase enzyme from a mercury-volatilizing strain of Thiobacillus ferrooxidans. J. Bacteriol, 151(3): 1230-1236 (7 pages).

Petrus, A.K.; Rutner, C.; Liu, S.; Wang, Y.; Wiatrowski, H. A., 2015. Mercury reduction and methyl mercury degradation by the soil bacterium Xanthobacter autotrophicus Py2. Appl. Environ. Microbiol., 81(22): 7833-7838 (6 pages).

Raja, C.E.; Selvam, G.S.; Omine, K.I.Y.O.S.H.I.,(2009). Isolation, identification and characterization of heavy metal resistant bacteria from sewage. In Int Joint Symp on Geodisaster Prevention and Geoenvironment in Asia, 205-211(7 pages).

Saitou, N.; Nei, M., (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol, 4(4): 406-425 (20 pages).

Sayed, A.; Ghazy, M.A.; Ferreira, A.J.; Setubal, J.C.; Chambergo, F.S.; Ouf, A.; Siam, R., (2014). A novel mercuric reductase from the unique deep brine environment of Atlantis II in the Red Sea. J. Biol. Chem., 289(3): 1675-1687 (13 pages).

Smibert, R.M.; Krieg, N.R., (1994). Phenotypic characterization. In: Methods for General and Molecular Bacteriology. Gerhardt P, Murray RGE, Wood WA, Krieg NR Eds. American Society for Microbiology, Washington, DC, 1994, pp. 607-654 (48 pages).

Somero, G.N., (1995). Proteins and temperature. Annu. Rev. Physiol., 57 (1): 43-68 (26 pages).

Thompson, J.D.; Gibson, T.J.; Plewniak, F.; Jeanmougin, F.; Higgins, D.G., (1997). The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res., 25(24): 4876-4882 (7 pages).

Vetriani, C.; Chew, Y.S.; Miller, S.M.; Yagi, J.; Coombs, J.; Lutz, R.A.; Barkay, T., (2005). Mercury adaptation among bacteria from a deep-sea hydrothermal vent. Appl. Environ. Microbiol., 71(1): 220-226 (7 pages).

Voica, D.M.; Bartha, L.; Banciu, H.L.; Oren, A., (2016). Heavy metal resistance in halophilic Bacteria and Archaea. FEMS Microbiol. Lett, 363(14), fnw146.

Xiong, J.; He, Z.; Liu, D.; Mahmood, Q.; Yang, X., (2008). The role of bacteria in the heavy metals removal and growth of Sedum alfredii Hance in an aqueous medium. Chemosphere, 70(3): 489-494 (5 pages).

Zeroual, Y.; Moutaouakkil, A.; Dzairi, F. Z.; Talbi, M.; Chung, P.U.; Lee, K.; Blaghen, M., (2003). Purification and characterization of cytosolic mercuric reductase from Klebsiella pneumoniae. Ann. Microbial, 53(2): 149-160 (12 pages).

Letters to Editor

GJESM Journal welcomes letters to the editor for the post-publication discussions and corrections which allows debate post publication on its site, through the Letters to Editor. Letters pertaining to manuscript published in GJESM should be sent to the editorial office of GJESM within three months of either online publication or before printed publication, except for critiques of original research. Following points are to be considering before sending the letters (comments) to the editor.

[1] Letters that include statements of statistics, facts, research, or theories should include appropriate references, although more than three are discouraged.
[2] Letters that are personal attacks on an author rather than thoughtful criticism of the author’s ideas will not be considered for publication.
[3] Letters can be no more than 300 words in length.
[4] Letter writers should include a statement at the beginning of the letter stating that it is being submitted either for publication or not.
[5] Anonymous letters will not be considered.
[6] Letter writers must include their city and state of residence or work.
[7] Letters will be edited for clarity and length.