M.K. Sabullah, M.F. Rahman, S.A. Ahmad, M.R. Sulaiman, M.S. Shukor, N.A. Shamaan, M.Y. Shukor


Bioremediation of pollutants including heavy metals and xenobiotics is an economic and environmentally friendly process. A novel molyb-denum-reducing bacterium with the ability to utilize the pesticide glyphosate as a carbon source is reported. The characterization works were carried out utilizing bacterial resting cells in a microplate format. The bacterium reduces molybdate to Mo-blue optimally between pH 6.3 and 6.8 and at 34oC. Glucose was the best elec-tron donor for supporting molybdate reduction followed by lactose, maltose, melibiose, raffinose, d-mannitol, d-xylose, l-rhamnose, l-arabinose, dulcitol, myo-inositol and glycerol in descending order. Other requirements include a phosphate concentration at 5.0 mM and a molybdate concentration between 20 and 30 mM. The molybdenum blue exhibited an absorption spec-trum resembling a reduced phospho-molybdate. Molybdenum reduction was inhibited by mercury, silver, cadmium and copper at 2 ppm by 45.5, 26.0, 18.5 and 16.3%, respectively. Biochemical analysis identified the bacterium as Klebsiella oxytoca strain Saw-5. To conclude, the capacity of this bacterium to reduce molybdenum into a less toxic form and to grow on glyphosate is novel and makes the bacterium an important instrument for bioremediation of these pollutants.


bioreduction; glyphosate; Klebsiella oxytoca; molybdenum blue; molybdenum-reducing bacterium

Full Text:



Abo-Shakeer, L.K.A., S.A. Ahmad, M.Y. Shukor, N.A. Shamaan and M.A. Syed. 2013. Isolation and characterization of a molybdenum-reducing Bacillus pumilus strain lbna. Journal of Environmental Microbiology and Toxicology. 1(1): 9-14.

Achiorno, C.L., Cd. Villalobos and L. Ferrari. 2008. Toxicity of the herbicide glyphosate to Chordodes nobilii (Gordiida, Nemato-morpha). Chemosphere. 71(10): 1816-1822. crossref

Ahmad, S.A., M.Y. Shukor, N.A. Shamaan, W.P. Mac Cormack and M.A. Syed. 2013. Molybdate reduction to molybdenum blue by an Antarctic bacterium. BioMed Research International. pp. 1-10. crossref

Auffret, M.D., E. Yergeau, D. Labbé, F. Fayolle-Guichard and C.W. Greer. 2015. Importance of Rhodococcus strains in a bacterial consortium degrading a mixture of hydrocarbons, gasoline, and diesel oil additives revealed by meta-transcriptomic analysis. Appl. Microbiol. Biotechnol. 99(5): 2419-2430. crossref

Barceló, D. and M-C. Hennion. 1995. On-line sample handling strategies for the trace-level determination of pesticides and their degradation products in environ-mental waters. Anal. Chim. Acta. 318(1), 1–41. crossref

Bates, N. and N. Edwards. 2013. Glyphosate toxicity in animals [letter]. Clinical Toxicology. 51(10): 1243.

Bazot, S. and T. Lebeau. 2008. Simultaneous mineralization of glyphosate and diuron by a consortium of three bacteria as free- and/or immobilized-cells formulations. Applied Microbiology and Biotechnology. 77(6): 1351-1358.

Bernal, M.H., K.R. Solomon and G. Carrasquilla. 2009. Toxicity of formulated glyphosate (glyphos) and cosmo-flux to larval and juvenile Colombian frogs 2. Field and laboratory microcosm acute toxicity. J. Toxicol. Environ. Health. A. 72: 966-973. crossref

Campbell, A.M., A. del Campillo-Campbell and D.B. Villaret. 1985. Molybdate reduction by Escherichia coli K-12 and its chl mutants. Proc. Natl. Acad. Sci. USA 82(1): 227-231.

Carpentier, W., L.D. Smet, J.V. Beeumen and A. Brigé. 2005. Respiration and growth of Shewanella oneidensis MR-1 using vanadate as the sole electron acceptor. J. Bacteriol. 187(10): 3293–3301. crossref

Chaturvedi, V. and A. Kumar. 2011. Diversity of culturable sodium dodecyl sulfate (SDS) degrading bacteria isolated from detergent contaminated ponds situated in Varanasi city, India. International Biodeterioration Biodegradation. 65(7): 961–971. crossref

Chirwa, E.N. and Y-T Wang. 2000. Simultaneous chromium (VI) reduction and phenol degradation in an anaerobic consortium of bacteria. Water Res. 34(8): 2376-2384. crossref

Chung, J., B.E. Rittmann, W.F. Wright and R.H. Bowman. 2007. Simultaneous bio-reduction of nitrate, perchlorate, selenate, chromate, arsenate, and dibromo-chloropropane using a hydrogen-based membrane biofilm reactor. Biodegra-dation 18(2): 199–209.

Costin, S. and S. Ionut. 2015. ABIS online - bacterial identification software, http://, database version: Bacillus 022012-2.10. Accessed on March 2015.

Davis, G.K. 1991. Molybdenum. In Merian, E. (ed) Metals and their compounds in the environment, occurrence, analysis and biological relevance. New York: VCH Weinheim. pp. 1089-1100.

Deepananda, K.H.M.A., D. Gajamange, W.A.J.P. De Silva and H.C.E. Wegiriya. 2011. Acute toxicity of a glyphosate herbicide, Roundup®, to two freshwater crusta-ceans. J. Natl. Sci. Found. Sri. 39(2): 169–173. crossref

Duke, S.O. and S.B. Powles. 2008. Glyphosate: a once-in-a-century herbicide. Pest Management Science. 64(4): 319-325. crossref

Ermis, U.B. and N. Demir. 2009. Toxicity of glyphosate and ethoxysulfuron to the green microalgae (Scenedesmus obliquus). Asian J. Chem. 21(3): 2163–2169.

Filizadeh, Y. and H.R. Islami. 2011. Toxicity determination of three sturgeon species exposed to glyphosate. Iran. J. Fish. Sci. 10(3): 383–392.

Ghani, B., M. Takai, N.Z. Hisham, N. Kishimoto, A.K.M. Ismail, T. Tano and T. Sugio. 1993. Isolation and characterization of a Mo6+-reducing bacterium. Appl. Environ. Microbiol. 59(4): 1176–1180.

Giesy, J.P., S. Dobson and K.R. Solomon. 2000. Ecotoxicological risk assessment for Roundup® herbicide. In Ware, D.G.W. (ed) Reviews of environmental contami-nation and toxicology. New York: Springer. pp. 35-120.

Glenn, J.L. and F.L. Crane. 1956. Studies on metalloflavoproteins: V. The action of silicomolybdate in the reduction of cyto-chrome c by aldehyde oxidase. Biochim. Biophy. Acta. 22(1): 111–115. crossref

Hadi, F., A. Mousavi, K.A. Noghabi, H.G. Tabar and A.H. Salmanian. 2013. New bacterial strain of the genus Ochrobactrum with glyphosate-degrading activity. J. Environ. Sci. Health B. 48(3): 208–213. crossref

Haley, C.L., J.A. Colmer-Hamood and A.N. Hamood. 2012. Characterization of biofilm-like structures formed by Pseudomonas aeruginosa in a synthetic mucus medium. BMC Microbiol. 12: 181. crossref

Halmi, M.I.E., S.W. Zuhainis, M.T. Yusof, N.A. Shaharuddin, W. Helmi, Y. Shukor, M.A. Syed and S.A. Ahmad. 2013. Hexavalent molybdenum reduction to Mo-blue by a sodium-dodecyl-sulfate- degrading Klebsiella oxytoca strain DRY14. BioMed Res. Int. pp. 1-8. crossref

Holt, J.G., N.R. Krieg, P.H.A. Sneath, J.T. Staley and S.T. Williams. 1994. Bergey’s manual of determinative bacteriology (9th edition). Baltimore: Williams and Wilkins.

Hori, T., M. Sugiyama and S. Himeno. 1988. Direct spectrophotometric determination of sulphate ion based on the formation of a blue molybdosulphate complex. Analyst. 113: 1639–1642. crossref

Iyamu, E.W., T. Asakura and G.M. Woods. 2008. A colorimetric microplate assay method for high-throughput analysis of arginase activity in vitro. Anal. Biochem. 383(2): 332–334. crossref

Khan, A., M.I.E. Halmi and M.Y. Shukor. 2014. Isolation of Mo-reducing bacteria in soils from Pakistan. J. Environ. Microbiol. Toxicol. 2(1): 38–41.

Kincaid, R.L. 1980. Toxicity of ammonium molybdate added to drinking water of calves. J. Dairy Sci. 63(4): 608–610. crossref

Kononova, S.V. and M.A. Nesmeyanova. 2002. Phosphonates and their degradation by microorganisms. Biochem. (Mosc.). 67 (2): 220-233.

Kryuchkova, Y.V., G.L. Burygin, N.E. Gogoleva, Y.V. Gogolev, M.P. Chernyshova, O.E. Makarov, E.E. Fedorov and O.V. Turkovskaya. 2014. Isolation and charac-terization of a glyphosate-degrading rhizosphere strain, Enterobacter cloacae K7. Microbiol. Res. 169(1): 99–105. crossref

Lim, H.K., M.A. Syed and M.Y. Shukor. 2012. Reduction of molybdate to molybdenum blue by Klebsiella sp. strain hkeem. J. Basic Microbiol. 52(3): 296–305. doi: 10.1002/jobm.201100121

Lim, K.T., M.Y. Shukor and H. Wasoh. 2014. Physical, chemical, and biological methods for the removal of arsenic compounds. BioMed Res. Int. pp. 1-9. crossref

Llovera, S., R. Bonet, M.D. Simon-Pujol and F. Congregado. 1993. Chromate reduction by resting cells of Agrobacterium radiobacter EPS-916. Appl. Environ. Microbiol. 59 (10): 3516–3518.

Losi, M.E. and W.T.F. Jr. 1997. Reduction of selenium oxyanions by Enterobacter cloacae strain SLD1a-1: Reduction of selenate to selenite. Environ. Toxicol. Chem. 16(9): 1851–1858. crossref

Masdor, N., M.S.A. Shukor, A. Khan, M.I.F bin Halmi, S.R.S. Abdullah, N.A. Shamaan and M.Y. Shukor. 2015. Isolation and characterization of a molybdenum-reducing and SDS- degrading Klebsiella oxytoca strain Aft-7 and its bioreme-diation application in the environment. Biodiversitas 16 (2): 238–246. crossref

McAuliffe, K.S., L.E. Hallas and C.F. Kulpa. 1990. Glyphosate degradation by Agro-bacterium radiobacter isolated from activated sludge. J. Industrial Microbiol. 6(3): 219–221. crossref

Moneke, A.N., G.N. Okpala and C.U. Anyanwu. 2010. Biodegradation of glyphosate herbicide in vitro using bacterial isolates from four rice fields. Afr. J. Biotech. 9 (26): 4067-4074.

Neunhäuserer, C., M. Berreck and H. Insam. 2001. Remediation of soils contaminated with molybdenum using soil amendments and phytoremediation. Water Air Soil Poll. 128: 85–96.

Othman, A.R., N.A. Bakar, M.I.E. Halmi, W.L.W. Johari, S.A. Ahmad, H. Jirangon, M.A. Syed and M.Y. Shukor. 2013. Kinetics of molybdenum reduction to molybdenum blue by Bacillus sp. strain A.rzi. BioMed Res. Int. p. 1-9. crossref

Piola, L., J. Fuchs, M.L. Oneto, S. Basack, E. Kesten and N. Casabé. 2013. Compar-ative toxicity of two glyphosate-based formulations to Eisenia andrei under laboratory conditions. Chemosphere 91 (4): 545–551. crossref

Rahman, M.F.A., M.Y. Shukor, Z. Suhaili, S. Mustafa, N.A Shamaan and M.A. Syed. 2009. Reduction of Mo(VI) by the bacterium Serratia sp. strain DRY5. J. Environ. Biol. 30(1): 65–72.

Raj, J., S. Prasad, N.N. Sharma and T.C. Bhalla. 2010. Bioconversion of acrylonitrile to acrylamide using polyacrylamide en-trapped cells of Rhodococcus rhodo-chrous PA-34. Folia Microbiol. (Praha) 55 (5): 442-446. crossref

Runnells, D.D., D.S Kaback and E.M. Thurman. 1976. Geochemistry and sampling of molybdenum in sediments, soils, and plants in Colorado. In: Chappel, W.R. and K.K. Peterson (Eds.), Molybdenum in the environment. New York: Marcel and Dekker.

Sabullah, M.K., M.R. Sulaiman, M.Y.A. Shukor, M.A. Syed, N.A. Shamaan, A. Khalid, and S.A. Ahmad. 2014. The assessment of cholinesterase from the liver of Puntius javanicus as detection of metal ions. Sci. World J. p. 9. crossref

Sabullah, M.K., S.A. Ahmad, M.Y. Shukor, A.J. Gansau, M.A. Syed, M.R. Sulaiman and N.A. Shamaan. 2015. Heavy metal bio-marker: Fish behavior, cellular alteration, enzymatic reaction and proteomics approaches. Int. Food Res. J. 22 (2): 435-454. Available at http://www.ifrj.

Schowanek, D. and W. Verstraete. 1990. Phos-phonate utilization by bacterial cultures and enrichments from environmental samples. Appl. Environ. Microbiol. 56 (4): 895–903.

Sedighi, M. and F. Vahabzadeh. 2014. Kinetic modeling of cometabolic degradation of ethanethiol and phenol by Ralstonia eutropha. Biotechnol. Bioprocess Eng. 19: 239–249. crossref

Shukor, M.Y., N.A. Shamaan, M.A. Syed, C.H. Lee and M.I.A. Karim. 2000. Charac-terization and quantification of molyb-denum blue production in Enterobacter cloacae strain 48 using 12-molybdo-phosphate as the reference compound. Asia Pacific J. Mol. Biol Biotechnol. 8 (2): 167–172.

Shukor, M.Y., C.H. Lee, I. Omar, M.I.A. Karim, M.A. Syed and N.A. Shamaan. 2003. Isolation and characterization of a molybdenum-reducing enzyme in Enterobacter cloacae strain 48. Pertanika J. Sci. Technol. 11(2): 261–272.

Shukor, Y., H. Adam, K. Ithnin, I. Yunus, N.A. Shamaan and M.A. Syed. 2007. Molybdate reduction to molybdenum blue in microbe proceeds via a phosphomolybdate inter-mediate. J. Biol Sci. 7(8): 1448–1452.

Shukor, M.Y., S.H. Habib, M.F. Rahman, H. Jirangon, M.P. Abdullah, N.A. Shamaan and M.A. Syed. 2008a. Hexavalent molybdenum reduction to molybdenum blue by S. marcescens strain Dr. Y6. Appl. Biochem. Biotechnol. 149(1): 33–43. doi: crossref

Shukor, M.Y., M.F. Rahman, N.A. Shamaan, C.H. Lee, M.I. Karim and M.A. Syed. 2008b. An improved enzyme assay for molybdenum-reducing activity in bacteria. Applied Biochemistry and Biotechnology 144 (3): 293–300.

Shukor, M.Y., M.F. Rahman, N.A. Shamaan and M.A. Syed. 2009a. Reduction of molybdate to molybdenum blue by Enterobacter sp. strain Dr.Y13. J. Basic Microbiol. 49 Suppl. 1, S43–S54. crossref

Shukor, M.Y., M.F. Rahman, Z. Suhaili, N.A. Shamaan and M.A. Syed. 2009b. Bacterial reduction of hexavalent molybdenum to molybdenum blue. World J. Microbiol. Biotechnol. 25, 1225–1234. crossref

Shukor, M.Y. and M.A. Syed. 2010. Microbio-logical reduction of hexavalent molyb-denum to molybdenum blue. In: Mendez-Vilas A. (Eds.), Current research, technology and education topics in applied microbiology and microbial biotechnology. Formatex Research Center. Badajoz, Spain.

Shukor, M.Y., S.A. Ahmad, M.M. Nadzir, M.P. Abdullah, N.A. Shamaan and M.A. Syed. 2010a. Molybdate reduction by Pseudo-monas sp. strain DRY2. J. Appl. Microbiol. 108(6): 2050–2058. crossref

Shukor, M.Y., M.F. Rahman, Z. Suhaili, N.A. Shamaan and M.A. Syed. 2010b. Hexavalent molybdenum reduction to Mo-blue by Acinetobacter calcoaceticus. Folia Microbiol. (Praha). 55(2), 137–143. crossref

Shukor, M.S. and M.Y. Shukor. 2014. A microplate format for characterizing the growth of molybdenum-reducing bacteria. J. Environ. Microbiol. Toxicol. 2(2): 42–44.

Shukor, M.Y., M.I.E. Halmi, M.F.A. Rahman, N.A. Shamaan and M.A. Syed. 2014. Molyb-denum reduction to molybdenum blue in Serratia sp. strain DRY5 is catalyzed by a novel molybdenum-reducing enzyme. BioMed Res. Int. pp. 1-8.crossref

Sims, R.P.A. 1961. Formation of heteropoly blue by some reduction procedures used in the micro-determination of phosphorus. The Analyst. 86(1026): 584–590. crossref

Steiert, J.G., J.J. Pignatello and R.L. Crawford. 1987. Degradation of chlorinated phenols by a pentachlorophenol-degrading bac-terium. Applied and Environmental Mi-crobiology. 53(5): 907–910.

Sugiura, Y. and Y. Hirayama. 1976. Structural and electronic effects on complex formation of copper(II) and nickel(II) with sulfhydryl-containing peptides. Inorganic Chemistry. 15(3): 679–682. crossref

Walter, M., J.M. Guthrie, S. Sivakumaran, E. Parker, A. Slade, D. McNaughton and K.S.H. Boyd-Wilson. 2003. Screening of New Zealand Native White-Rot Isolates for PCP Degradation. Bioremediation Journal. 7: 119-128

Yamaguchi, S., C. Miura, A. Ito, T. Agusa, H. Iwata, S. Tanabe, B.C. Tuyen and T. Miura. 2007. Effects of lead, molyb-denum, rubidium, arsenic and organo-chlorines on spermatogenesis in fish: Monitoring at Mekong Delta area and in vitro experiment. Aquatic Toxicology. 83 (1): 43–51. crossref

Yoshimura, K., M. Ishii and T. Tarutani. 1986. Microdetermination of phosphate in water by gel-phase colorimetry with molyb-denum blue. Anal. Chem. 58(3): 591–594. crossref

Yunus, S.M., H.M. Hamim, O.M. Anas and S.M. Arif. 2009. Mo (VI) reduction to molyb-denum blue by Serratia marcescens strain Dr. Y9. Polish Journal of Microbiology 58(2): 141–147.

Zboinska, E., B. Lejczak and P. Kafarski. 1992. Organophosphonate utilization by the wild-type strain of Pseudomonas fluorescens. Applied and Environmental Microbiology 58(9): 2993–2999.

Zeng, G-M., L. Tang, G-L. Shen, G-H. Huang and C-G. Niu. 2004. Determination of trace chromium (VI) by an inhibition-based enzyme biosensor incorporating an electropolymerized aniline membrane and ferrocene as electron transfer mediator. Int. J. Environ. Anal. Chem. 84 (10): 761–774. crossref

Zhang, Y.L., F.J. Liu, X.L. Chen, Z.Q. Zhang, R.Z. Shu, X.L. Yu, X.W. Zhai, L.J. Jin, X.G. Ma, Q. Qi and Z.J. Liu. 2013. Dual effects of molybdenum on mouse oocyte quality and ovarian oxidative stress. Syst. Biol. Reprod. Med. 59(6): 312-318. crossref


Copyright (c) 2016 AGRIVITA Journal of Agricultural Science

License URL: