Biotransformation of Reactive Orange 16 by alkaliphilic bacterium Bacillus flexus VITSP6 and toxicity assessment of biotransformed metabolites

  • P. Saha
  • K. V. B. RaoEmail author
Original Paper


Azo dyes are the largest group of chemically synthesized colorants used in various industries. The hazardous nature of these dyes has led to the development of stringent environmental laws. Thus, mounting pressure exists on the industries for development of cost-effective dye removal systems. The current work focuses on the bioremoval of azo dye Reactive Orange 16 (RO-16) using indigenous bacteria isolated from contaminated sample of a textile industry. The most potent bacterium was capable of decolorizing 89.82% of RO-16 within 24 h under static conditions and was identified as Bacillus flexus VITSP6. When physicochemical parameters were optimized, B. flexus VITSP6 showed efficient decolorization at a temperature of 37 °C, pH 11, glucose as carbon source and peptone as nitrogen source. Interestingly, although B. flexus VITSP6 showed highest decolorization at 37 °C, it was able to maintain its decolorization potency even at elevated temperatures as high as 52 °C. Further the mechanism of dye decolorization was deduced with the help of high-performance liquid chromatography, Fourier transform infrared spectroscopy and liquid chromatography mass spectrometry analyses. These analyses revealed that RO-16 underwent biotransformation to different lower molecular weight aromatic compounds. The toxicity of these biotransformation products were evaluated by conducting phytotoxicity, cytotoxicity and biotoxicity assays. All the toxicity assays indicated that the biotransformed products were non-toxic in nature. The efficient dye decolorization property of B. flexus VITSP6 and its ability to withstand high temperature and alkaliphilic nature make it a highly potential organism to be applied in the dye removal treatments.


Azo dye Biotransformation Decolorization Reactive Orange 16 Toxicity assay 



The authors of this paper wish to thank VIT management for providing a suitable platform to carry out this research work and also CSIR-CLRI for providing with LC–MS service.

Compliance with ethical standards

Conflict of interest

The authors of this paper declare that they have no conflict of interest in the context of this research paper submitted to the journal.

Ethical approval

This article does not contain any studies with human participants. However, invertebrate (Artemia salina) eggs used for lethality assay in this study do not require any ethical committee clearance certificate for usage.


  1. APHA (2005) Standard methods for the examination of water and wastewater, 21st edn. American Public Health Association/American Water Works Association/Water Environment Federation, WashingtonGoogle Scholar
  2. Aravindhan R, Rao JR, Nair BU (2007) Removal of basic yellow dye from aqueous solution by sorption of green alga Caulerpa sealpelliformis. J Hazard Mater 142:68–76. CrossRefGoogle Scholar
  3. Asad A, Amoozegar MA, Pourbabaee AA, Sarbolouki MN, Dastgheib SMM (2007) Decolorization of textile azo dyes by newly isolated halophilic and halotolerant bacteria. Bioresour Technol 98:2082–2088. CrossRefGoogle Scholar
  4. Attschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI BLAST: a new generation of protein database search programs. Nucleic Acid Resour 25(17):3389–3402. CrossRefGoogle Scholar
  5. Ayed L, Mahdhi A, Cheref A, Bakhrouf A (2011) Decolorization and degradation of azo dye methyl red by an isolated Sphingomonas paucimobilis: biotoxicity and metabolites characterization. Desalination 274:272–277. CrossRefGoogle Scholar
  6. Bhatt N, Patel KC, Keharia H, Madamwar D (2005) Decolorization of diazo-dye reactive blue 172 by Pseudomonas aeruginosa NBAR12. J Basic Microbiol 45:407–418. CrossRefGoogle Scholar
  7. Bhavsar S, Dudhagara P, Tank S (2018) R software package based statistical optimization of process components to simultaneously enhance the bacterial growth, laccase production and textile dye decolorization with cytotoxicity study. PLoS ONE 13:e0195795. CrossRefGoogle Scholar
  8. Carballo JL, Hernández-Inda ZL, Pérez P, García-Grávalos MD (2002) A comparison between two brine shrimp assays to detect in vitro cytotoxicity in marine natural products. BMC Biotechnol 2:17. CrossRefGoogle Scholar
  9. Central Pollution Control Board (2010) Pollution control acts, rules and notifications issued thereunder. CPCB, New DelhiGoogle Scholar
  10. Chavan RB (2011) Environmentally friendly dyes. In: Clark M (ed) Wood head publishing series in textiles, handbook of textile and industrial dyeing. Woodhead Publishing, Sawston, pp 515–561CrossRefGoogle Scholar
  11. Chen KC, Wu JY, Liou DJ, Huang SCJ (2003) Decolorization of the textile dyes by newly isolated bacterial strains. J Biotechnol 101:57–68. CrossRefGoogle Scholar
  12. Chen Y, Feng L, Li H, Wang Y, Chen G, Zhang Q (2018) Biodegradation and detoxification of direct black G textile dye by a newly isolated thermophilic microflora. Bioresour Technol 250:650–657. CrossRefGoogle Scholar
  13. Chung KT, Fluk GE, Andrews AE (1981) Mutagenicity testing of some commonly used dyes. Appl Environ Microbiol 42(4):641–648Google Scholar
  14. Dave SR, Patel TL, Tipre DR (2015) Bacterial degradation of azo dye containing wastes. In: Singh S (ed) Microbial degradation of synthetic dyes in wastewaters. Environmental science and engineering. Springer, Cham, pp 57–83Google Scholar
  15. Dawkar VV, Jadhav UU, Jadhav SU, Govindwar SP (2008) Biodegradation of disperse textile dye Brown 3REL by newly isolated Bacillus sp. VUS. J Appl Microbiol 105(1):14–24. CrossRefGoogle Scholar
  16. EPA (2001) Parameters of water quality: interpretation and standards. Environmental Protection Agency, WexfordGoogle Scholar
  17. Evangelista-Barreto NS, Albuquerque CD, Vieira RHSF, Campos-Takaki GM (2009) Cometabolic decolorization of the reactive azo dye orange II by Geobacillus stearothermophilus UCP 986. Text Res J 79:1266–1273. CrossRefGoogle Scholar
  18. Gahlout M, Gupte S, Gupte A (2013) Optimization of culture condition for enhanced decolorization and degradation of azo dye reactive violet 1 with concomitant production of ligninolytic enzymes by Ganoderma cupreum AG-1. 3 Biotech 3:143–152. CrossRefGoogle Scholar
  19. Guo J, Zhou J, Wang D, Tian C, Wang P, Salah Uddin M (2008) A novel moderately halophilic bacterium for decolorizing azo dye under high salt condition. Biodegradation 19:15. CrossRefGoogle Scholar
  20. Haq I, Kumari V, Kumar S, Raj A, Lohani M, Bhargava R (2016) Evaluation of the phytotoxic and genotoxic potential of pulp and paper mill effluent using Vigna radiata and Allium cepa. Adv Biol 2016:10. CrossRefGoogle Scholar
  21. Jain K, Shah V, Chapla D, Madamwar D (2012) Decolorization and degradation of azo dye—reactive violet 5R by an acclimatized indigenous bacterial mixed cultures-SB4 isolated from anthropogenic dye contaminated soil. J Hazard Mater 213–214:378–386. CrossRefGoogle Scholar
  22. Kapdan IK, Oztekin R (2003) Decolorization of textile dyestuff Reactive Orange 16 in fed-batch reactor under anaerobic condition. Enzyme Microb Technol 33:231–235. CrossRefGoogle Scholar
  23. Khan MZ, Singh S, Sreekrishnan TR, Ahammad SZ (2014) Feasibility study on anerobic biodegradation of azo dye Reactive Orange 16. RSC Adv 4(87):46851–46859. CrossRefGoogle Scholar
  24. Lin YH, Leu JY (2008) Kinetics of reactive azo-dye decolorization by Pseudomonas luteola in a biological activated carbon process. Biochem Eng J 39:457–467. CrossRefGoogle Scholar
  25. Mielgo I, Moreira MT, Feijoo G, Lema JM (2001) A packed-bed fungal bioreactor for continuous decolourisation of azo-dyes (Orange II). J Biotechnol 89:99–106. CrossRefGoogle Scholar
  26. Misal SA, Lingojwar DP, Shinde RM, Gawai KR (2011) Purification and characterization of azoreductase from alkaliphilic strain Bacillus badius. Process Biochem 46:1264–1269. CrossRefGoogle Scholar
  27. OECD (2006) Limits on aromatic amines in textiles coloured with azo dyes. In: Environmental requirements and market access. OECD Publishing, ParisCrossRefGoogle Scholar
  28. Pandey AK, Sarada DVL, Kumar A (2016) Microbial decolorization and degradation of reactive red 198 azo dye by a newly isolated alkaligenes species. Proc Natl Acad Sci India Sect B Biol Sci 86:805–815. CrossRefGoogle Scholar
  29. Prasad ASA, Rao KVB (2013) Aerobic biodegradation of Azo dye by Bacillus cohnii MTCC 3616; an obligately alkaliphilic bacterium and toxicity evaluation of metabolites by different bioassay systems. Appl Microbiol Biotechnol 97:7469–7481. CrossRefGoogle Scholar
  30. Prasad ASA, Satyanaryana VSV, Rao KVB (2013) Biotransformation of direct blue 1 by a moderately halophilic bacterium Marinobacter sp. strain HBRA and toxicity assessment of degraded metabolites. J Hazard Mater 262:674–684. CrossRefGoogle Scholar
  31. Pricelius S, Held C, Murkovic M, Bozic M, Kokol V, Cavaco-Paulo A, Guebitz GM (2007) Enzymatic reduction of azo and indigoid compounds. Appl Microbiol Biotechnol 77(2):321–327. CrossRefGoogle Scholar
  32. Rao KVB, Prasad ASA (2014) Biodecolourisation of azo dye reactive red 22 by Bacillus infantis strain AAA isolated from seawater and toxicity assessment of degraded metabolites. Nat Environ Pollut Technol 13:369–374Google Scholar
  33. Roat C, Kadam A, Patel T, Dave S (2016) Biodegradation of diazo dye, reactive blue 160 by isolate Microbacterium sp. B12 mutant: identification of intermediates by LC-MS. Int J Curr Microbiol Appl Sci 5:534–547. CrossRefGoogle Scholar
  34. Rosu CM, Avadanei M, Gherghel D, Mihasan M, Mihai C, Trifan A, Miron A, Vochita G (2018) Biodegradation and detoxification efficiency of azo-dye Reactive Orange 16 by Pichia kudriavzevii CR-Y103. Water Air Soil Pollut 229:15. CrossRefGoogle Scholar
  35. Sahasrabudhe MM, Pathade GR (2011) Biodegradation of sulphonated azo dye C.I. Reactive Orange 16 by Enterococcus faecalis strain YZ 66. Eur J Exp Biol 1:163–173Google Scholar
  36. Sahasrabudhe M, Pathade G (2013) Biodegradation of azo dye C.I. Reactive Orange 16 by an actinobacterium Georgenia sp. CC-NMPT-T3. Int J Adv Res 1:91–99Google Scholar
  37. Saranraj P (2013) Bacterial biodegradation and decolourization of toxic textile azo dyes. Afr J Microbiol Res 7:3885–3890. Google Scholar
  38. Shah MP (2014) Biodegradation of azo dyes by three isolated bacterial strains: an environmental bioremedial approach. J Microbial Biochem Technol S 3:007. Google Scholar
  39. Shah MP, Patel KA, Nair SS, Darji AM (2013) Optimization of environmental parameters on microbial degradation of reactive black dye. J Bioremed Biodeg 4:183–188. Google Scholar
  40. Shobana S, Thangam EB (2012) Biodegradation and decolorization of Reactive Orange 16 by Nocardiopsis alba soil isolate. J Bioremed Biodeg 3:155–162. CrossRefGoogle Scholar
  41. Singh S, Chatterji S, Nandini PT, Prasad ASA, Rao KVB (2015) Biodegradation of azo dye Direct Orange 16 by Micrococcus luteus strain SSN2. Int J Environ Sci Technol 12:2161–2168. CrossRefGoogle Scholar
  42. Solís M, Solís A, Pérez HI, Manjarrez N, Flores M (2012) Microbial decolouration of azo dyes: a review. Process Biochem 47:1723–1748. CrossRefGoogle Scholar
  43. Soni RK, Bhatt NS, Modi HA, Acharya PB (2016) Decolorization, degradation and subsequent toxicity assessment of reactive red 35 by Enterococcus gallinarum. Curr Biotechnol 5:325–336. CrossRefGoogle Scholar
  44. Spagni A, Grilli S, Casu S, Mattioli D (2010) Treatment of a simulated textile wastewater containing the azo-dye Reactive Orange 16 in an anaerobic-biofilm anoxic-aerobic membrane bioreactor. Int Biodeterior Biodegrad 64(7):676–681. CrossRefGoogle Scholar
  45. Teli MD (2016) Environmental textiles: testing and certification. In: Wang L (ed) Woodhead publishing series in textiles, performance testing of textiles: methods, technology and applications. Woodhead Publishing, Sawston, pp 177–192Google Scholar
  46. Telke A, Kalyani D, Jadhav J, Govindwar S (2008) Kinetics and mechanism of reactive red 141 degradation by a bacterial isolate Rhizobium radiobacter MTCC 8161. Acta Chim Slov 55:320–329Google Scholar
  47. Telke AA, Kalyani DC, Dawkar VV, Govindwar SP (2009) Influence of organic and inorganic compounds on oxidoreductive decolorization of sulfonated azo dye CI Reactive Orange 16. J Hazard Mater 172(1):298–309. CrossRefGoogle Scholar
  48. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position- specific gap penalties and weight matrix choice. Nucleic Acids Res 22(22):4673–4680CrossRefGoogle Scholar
  49. Tony BD, Goyal D, Khanna S (2009) Decolorization of textile azo dyes by aerobic bacterial consortium. Int Biodeterior Biodegrad 63:462–469. CrossRefGoogle Scholar
  50. Ventura-Camargo BC, Maltempi PPP, Marin-Morales MA (2011) The use of the cytogenetic to identify mechanisms of action of an azo dye in Allium cepa meristematic cells. J Environ Anal Toxicol 1:109–120. CrossRefGoogle Scholar

Copyright information

© Islamic Azad University (IAU) 2019

Authors and Affiliations

  1. 1.Department of Bio-Medical Sciences, School of Biosciences and TechnologyVIT UniversityVelloreIndia

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