International Journal of Environmental Bioremediation & Biodegradation
ISSN (Print): 2333-8628 ISSN (Online): 2333-8636 Website: Editor-in-chief: Apply for this position
Open Access
Journal Browser
International Journal of Environmental Bioremediation & Biodegradation. 2013, 1(2), 37-42
DOI: 10.12691/ijebb-1-2-1
Open AccessArticle

Microbial Decolorization of Reactive Black by Pseudomonas stutzeri ETL-79

Maulin P Shah1, , Kavita A Patel1 and A M Darji1

1Industrial Waste Water Research Laboratory, Applied & Environmental Microbiology Lab, Enviro Technology Limited (CETP), Gujarat, India

Pub. Date: November 01, 2013

Cite this paper:
Maulin P Shah, Kavita A Patel and A M Darji. Microbial Decolorization of Reactive Black by Pseudomonas stutzeri ETL-79. International Journal of Environmental Bioremediation & Biodegradation. 2013; 1(2):37-42. doi: 10.12691/ijebb-1-2-1


Azo reductase are often associated with decolorization of non-degradation of non-degradable azo dyes via cleavage azo bonds. In this study, Pseudomonas stutzeri ETL-79 bacterium was used for the decolorization of Reactive Black dye. The highest activity of azoreductase was obtained during the end of log phase. Azoreductase produced intracellularly had the highest specific activity of 0.0334 U/mg compared to the culture supernatant (Extracellular), resting cell and cell debris with low enzyme activity of 0.034 U/mg, 0.010 U/mg and 0.200 U/mg respectively. The optimum assay condition for the maximum azoreductase activity were at 37°C, pH 7, Reactive Black dye concentration of 100 mg/L and NADH concentration of 0.2 Mm by using phosphate buffer as a medium for the enzyme reaction. Alternatively the azoreductase assay was also carried out using ionic liquid that may function to enhance the activity and stability of azoreductase. Results using phosphate buffer (pH-7) showed higher enzyme activity twice that of the ionic liquid besides enhancing the stability of enzyme. Under the optimum assay condition upto 93% of decolorization was achieved after 8h of incubation. In addition, growth of bacteria was also concurrently observed during decolorization of Reactive Black.

azoreductase Pseudomonas stutzeri ETL-79 Reactive Black 5 decolourisation azoreductase assay

Creative CommonsThis work is licensed under a Creative Commons Attribution 4.0 International License. To view a copy of this license, visit


Figure of 6


[1]  Jin, X. C., Liu, G. Q., Xu, Z. H., and Tao, W. Y. 2007. Decolourization of a Dye Industry Effluent by Aspergillus fumigates XC6. Applied Microbiology and Biotechnology. 74: 239-243.
[2]  Jadhav, J. P., Kalyani, D. C., Telke, A. A., Phugare, S. S., and Govindwar, S. P. 2010. Evaluation of the Efficacy of a Bacterial Consortium for the Removal of Colour, Reduction of Heavy Metals and Toxicity from Textile Dye Effluent. Bioresources Technology. 101: 165-173.
[3]  Forgacs, E., Cserhati, T., and Oros, G. 2004. Removal of Synthetic Dyes from Wastewaters. A Review. Environmental International. 30: 953-971.
[4]  Ghosh, D. K., Mandal, A., and Chaudhuri, J. 1992. Purification and Partial Characterization of Two Azoreductases from Shigella Dysenteriae Type1, FEMS Microbiology Letters. 77: 229-233.
[5]  Moutaouakkil, A., Zeroual, Y., Dzayri, F. Z., Talbi, M., Lee, K., and Blaghen, M. 2003. Purification and Partial Characterization of Azoreductase From Enterobacter agglomerans. Archieves of Biochemistry and Biophysics. 413: 139-146.
[6]  Chen, H., Hopper, S. L., and Cerniglia, C. E. 2005. Biochemical And Molecular Characterization of an Azoreductase From Staphylococcus aureus, a tetrameric NADPH-Dependent Flavoprotein. Microbiology. 151: 1433-1441.
[7]  Olukanni, O. D., Osuntoki, A. A., and Gbenle, G. O. 2009. Decolourization of Azo Dye by a Strain of Micrococcus Isolated from a Refuse Dump Soil. Biotechnology. 8: 442-448.
[8]  Nakanishi, M., Yatome, C., Ishida, N., and Kitade, Y. 2001. Putative ACP Phosphodiesterase Gene (acpD) Encodes an Azoreductase. Journal of Biological Chemistry. 276: 46394-46399.
[9]  Kirby, N., Marchant, R. and McMullan, G. 2000. Decolourization of Synthetic Textile Dyes by Phlebia tremellosa. FEMS Microbiology Letters. 188: 93-96.
[10]  Ramalho, P. A., Cardoso, M. H., Paulo, A. C., and Ramalho, M. T. 2004. Characterization of Azo Reduction Activity in a Novel Ascomycetes Yeast Strain. Applied and Environmental Microbiology. 70: 2279-288.
[11]  Maier, J., Kandelbauer, A., Erlacher, A., Cavaco-Paulo, A., and Gubitz, G. M. 2004. A New Alkali-Thermostable Azoreductase from Bacillus sp. Strain SF. Applied and Environmental Microbiology. 70: 837-844.
[12]  Cull, S. G., Holbrey, J. D., Vargas-Mora, V., Seddon, K. R., and Lye, G. J. 2000. Room-temperature Ionic Liquids as Replacements For Organic Solvents in Multiphase Bioprocess Operations. Biotechnology and Bioengineering. 69: 227-233.
[13]  Santafé, A. A., Doumèche, B., Blum, L. J., Girard-Egrot, A. P., Marquette, C. A. 2010. 1-Ethyl-3-methylimidazolium ethylsulfate/copper Catalyst for the Enhancement of Glucose Chemiluminescent Detection: Effects on Light Emission and Enzyme Activity. Analytical Chemistry. 82: 2401-4.
[14]  Tavares, A. P. M., Rodriguez, O., and Macedo, E. A. 2008. Ionic Liquids as Alternative Co-solvents for Laccase: Study of Enzyme Activity and Stability. Biotechnology and Bioengineering. 101: 201-207.
[15]  Yang, Z., Yue, Y. J., Huang, W. C., Zhuang, Z. M., Chen, Z. T., and Xing, M. 2009. Importance of the Ionic Nature of Ionic Liquids in Affecting Enzyme Performance. Journal of Biochemistry. 145: 355-364.
[16]  Lowry, O. H., Rosenbrough, N. J., Farr, A. I., and Randall, R. J. 1951. Protein Measurement with the Folin Phenol Reagent. Journal of Biology and Chemistry. 193: 265-275.
[17]  Gottlieb, A., Shaw, C., Smith, A., Wheatley, A., and Forsythe, S. 2001. The Toxicity of Textile Reactive Azo Dyes after Hydrolysis and Decolourisation. Journal of Biotechnology. 101: 49-56.
[18]  Wang, H., Zheng, X. W., Su, J. Q., Tian, Y., Xiong, X. J., and Zheng, T. L. 2009. Biological Decolourisation of The Reactive Dyes Reactive Black 5 by A Novel Isolated Bacterial Strain Enterobacter sp. EC3. Journal of Hazardous Materials. 171: 654-659.
[19]  Choong, L. Y. 2004. Enzymatic Studies on Azoreductase from Enterococcus Strain C1 that Decolourizes Azo Dyes. B. Sc. Thesis, Universiti Teknologi Malaysia, Johor Bahru.
[20]  Ooi, T., Shibata, T., Sato, R., Ohno, H., Kinoshita, S., and Thuoc, T.L. et al. 2007. An Azoreductase, Aerobic NADH-Dependent Flavoprotein Discovered From Bacillus sp. Functional Expression and Enzymatic Characterization. Applied Microbiology and Biotechnology. 75: 377-386.
[21]  Panswad, T., and Luangdilok, W. 2000. Decolourisation of Reactive Dyes with Different Molecular Structures Under Different Environmental Conditions. Water Research. 34: 4177-4184.
[22]  Basso, A., Cantone, S., Linda, P., and Ebert, C. 2005. Stability and Activity of Immobilsed Penicilin G Amidase In Ionic Liquids At Controlled aw. Green Chemistry. 7: 671-676.
[23]  Yang, Z., Yue, Y., and Xing, M. 2008. Tyrosinase Activity in Ionic Liquids. Biotechnology Letters. 30: 153-158.
[24]  Duplissa, L., Andreescu, S., Baltus, R., and Njagi, J. 2010. Interactions of Room Temperature Ionic Liquids with Enzymes: Characterization and Activity Studies. Chemical Engineering REU. 53: 432-436.