Molecular identification and optimized production of violacein by Chromobacterium vaccinii Strain NFML 5214 isolated from Arunachal Pradesh, India
This study aimed to screen and isolate a purple-violet pigment-producing bacterium from soil samples collected from Talley Valley Wildlife Sanctuary, India, and develop an optimized procedure for enhanced pigment production for potential industrial, agricultural, and medicinal applications. A preliminary study was also conducted to evaluate textile-dyeing properties of the pigment produced by the bacterium. The purple–violet-colored bacterium was isolated on a nutrient agar plate, and a pure culture was prepared and maintained for further studies. The bacterium’s identity was established using both morphological and molecular (16S rRNA gene sequence analysis) approaches. An optimum culture medium was developed for enhanced pigment production by adjusting various growth factors, such as pH, temperature, incubation period, and concentration of media components, for maximum pigment production. The isolated bacterium strain NFML 5214 (PV613353.1) was identified as Chromobacterium vaccinii based on 16S rRNA gene sequence analysis, which showed 99.07% similarity to C. vaccinii strain 21-1 (CP017707.1). The bacterium produced the maximum amount of pigment on the 6th day of incubation at pH 6 and 30°C with optimized concentrations of peptone (7 g/L), beef extract (4 g/L), and NaCl (2 g/L). The total yield of pigments in the optimized medium (192 mg/L) showed a 69.42% increase compared with the yield of the basal medium (113 mg/L). A hot water extract dyeing process of white muslin cloth revealed dark purple fastness. Since C. vaccinii is reported to produce violacein and deoxy-violacein, further studies are needed to characterize the pigments and evaluate their potential medicinal properties, such as antimicrobial, antidiabetic, and antioxidant properties, and industrial value, such as pharmaceutical and textile dyes as an alternative to synthetic dyes.
Yama T, Shrivastava K, Singh SS. Molecular identification and optimized production of violacein by Chromobacterium vaccinii Strain NFML 5214 isolated from Arunachal Pradesh, India. J App Biol Biotech 2026. Article in Press. http://doi.org/10.7324/JABB.2026.272361
1. Anshi, Kapil S, Goswami L, Sharma V. Unveiling the intricacies of microbial pigments as sustainable alternatives to synthetic colorants: Recent trends and advancements. Micro. 2024;4:621-40. https://doi.org/10.3390/micro4040038
2. Kanade Y, Mohan W, Patwardhan R. Violacein: A promising bacterial secondary metabolite. Res J Chem Environ. 2022;26:165-77. https://doi.org/10.25303/2606rjce165177
3. Banc R, Filip L, Cozma-Petru? A, Ciobârc? D, Miere D. Yellow and red synthetic food dyes and potential health hazards: A mini review. Bull Univ Agric Sci Vet Med. 2024;81(1):1-17. https://doi.org/10.15835/buasvmcn-fst:2024.0005
4. Das S, Maulik SR. Recent Approaches and Advancements in the Use of Natural Dyes. Berlin: Springer International Publishing; 2023. p. 63-78. https://doi.org/10.1007/978-3-031-47471-24
5. Jain Savinay K, Prakash D, Akash S, Hema JN. Production, characterization and optimization of red pigment echinenone produced by Micrococcus sp., isolated from soil. Nat Life Sci Commun. 2023;22(2):e2023025. https://doi.org/10.12982/NLSC.2023.025
6. Barreto JVO, Casanova LM, Neves Junior A, Mansur MCPP, Vermelho AB. Biotechnological applications of microbial pigments. Biol Biotechnol. 2023;11(12):2920. https://doi.org/10.20944/preprints202310.0121.v1
7. Banerjee D, Chatterjee S, Banerjee UC, Guha AK, Ray L. Green pigments from Bacillus cereus M(1)(16) (MTCC 5521): Production parameters and antibacterial activity. Appl Biochem Biotechnol. 2011;164:767-79. https://doi.org/10.1007/s12010-011-9172-8
8. Anahas AM, Kumaran S, Kandeel M, Panagal M, Pugazhvendan SR, Suresh G, et al. Application of natural violet pigments from halophilic Chromobacterium violaceum PDF23 for textile dyeing with antimicrobial and antioxidant potentials. J Nanomater. 2022;2022:3885396. https://doi.org/10.1155/2022/3885396
9. Aranda S, Montes-Borrego M, Landa BB. Purple-pigmented violacein-producing Duganella spp. inhabit the rhizosphere of wild and cultivated olives in Southern Spain. Microb Ecol. 2011;62:446-59. https://doi.org/10.1007/s00248-011-9840-9
10. Fatima M, Anuradha K. Isolation, characterization, and optimization studies of bacterial pigments. J Pure Appl Microbiol. 2022;16:1039- 48. https://doi.org/10.22207/JPAM.16.2.28
11. Bhagwat A, Padalia U. Optimization of prodigiosin biosynthesis by Serratia marcescens using unconventional bioresources. J Genet Eng Biotechnol. 2020;18:26. https://doi.org/10.1186/s43141-020-00045-7
12. Hamada MA, Mohamed ET. Characterization of Serratia marcescens (OK482790)’ prodigiosin along with in vitro and in silico validation for its medicinal bioactivities. BMC Microbiol. 2024;24:495. https://doi.org/10.1186/s12866-024-03634-5
13. Nemer G, Louka N, Blandin PR, Maroun RG, Vorobiev E, Rossignol T, et al. Purification of natural pigments Violacein and Deoxyviolacein produced by fermentation using Yarrowia lipolytica. Molecules. 2023;28:4292. https://doi.org/10.3390/molecules28114292
14. Berti IR, Gantner ME, Rodriguez S, Islan GA, Fávaro WJ, Talevi A, et al. Potential biocide roles of violacein. Front Nanotechnol. 2023;5:1186386. https://doi.org/10.3389/fnano.2023.1186386
15. Bilsland E, Tavella TA, Krogh R, Stokes JE, Roberts A, Ajioka J, et al. Antiplasmodial and trypanocidal activity of Violacein and deoxyviolacein produced from synthetic operons. BMC Biotechnol. 2018;18:22. https://doi.org/10.1186/s12896-018-0428-z
16. Durán N, Castro GR, Portela RW, Fávaro WJ, Durán M, Tasic L, et al. Violacein and its antifungal activity: Comments and potentialities. Lett Appl Microbiol. 2022;75(4):796-803. https://doi.org/10.1111/lam.13760
17. Antonisamy P, Ignacimuthu S. Immunomodulatory, analgesic and antipyretic effects of violacein isolated from Chromobacterium violaceum. Phytomedicine. 2010;17(3-4):300-8. https://doi.org/10.1016/j.phymed.2009.05.018
18. Masuelli L, Pantanella F, Regina G, Benvenuto M, Fantini M, Mattera R, et al. Violacein, an indole-derived, purple-colored natural pigment produced by Janthinobacterium lividum, inhibits the growth of head and neck carcinoma cell lines both in vitro and in vivo. Tumor Biol. 2015;37:3705-17. https://doi.org/10.1007/s13277-015-4207-3
19. Abedin SMM, Tarafdar MR, Saha A, Atiqua, Rahim S, Karim MM, et al. Isolation and characterization of Chromobacterium violaceum and its metabolite violacein antibacterial activities of its metabolite violacein. Dhaka Univ J Biol Sci. 2024;33(1):109-19. https://doi.org/10.3329/dujbs.v33i1.72487
20. Andrighetti-Frohner CR, Antonio RV, Creczynski-Pasa TB, Barardi CR, Simões CM. Cytotoxicity and potential antiviral activity of violacein produced by Chromobacterium violaceum. Mem Inst Oswaldo Cruz. 2003;98(6):843-8. https://doi.org/10.1590/S0074- 02762003000600020
21. Antonisamy P, Kannan P, Ignacimuthu S. Anti-diarrheal and ulcer-protective effects of violacein isolated from Chromobacterium violaceum in Wistar rats. Fundam Clin Pharmacol. 2009;23(4):483- 90. https://doi.org/10.1111/j.1472-8206.2009.00701.x
22. Martin PA, Soby MS. Insecticidal Strains of Chromobacterium vaccinii sp. nov. for Insect Control (US Patent No. 9,339,039 B1). U.S. Patent and Trademark Office; 2016.
23. Cheng KC, Hsiao HC, Hou YC, Hsieh CW, Hsu SH, Chen HY, et al. Improvement in violacein production by formic acid to induce quorum sensing in Chromobacterium violaceum. Antioxidants. 2022;11:849. https://doi.org/10.3390/antiox11050849
24. Muhammad G, Zhao A, Mofijur M, Xu J, Alam MA. Sustainable production of microalgae-derived lutein, an underexplored commercially relevant pigment. Biomass Convers Biorefin. 2024;14:7255-76. https://doi.org/10.1007/s13399-022-03349-5
25. Soby SD, Gadagkar SR, Contreras C, Caruso FL. Chromobacterium vaccinii sp. nov., isolated from native and cultivated cranberry (Vaccinium macrocarpon Ait.). Int J Syst Evol Microbiol. 2013;63:1840-6. https://doi.org/10.1099/ijs.0.045161-0
26. Egorova DA, Voronina OL, Solovyev AI, Kunda MS, Aksenova EI, Ryzhova NN, et al. Integrated into the environmental biofilm Chromobacterium vaccinii survives winter with the support of the bacterial community. Microorganisms. 2020;8:1696. https://doi.org/10.3390/microorganisms8111696
27. Verma N, Choksket S, Singla R, Pinnaka AK, Korpole S. Chromobacterium indicum sp. nov., a pigment-producing bacterium isolated from soil. Curr Microbiol. 2024;81:385. https://doi.org/10.1007/s00284-024-03910-7
28. Park H, Park S, Yang YH, Choi KY. Microbial synthesis of violacein pigment and its potential applications. Crit Rev Biotechnol. 2021;41(6):879-901.
29. World Health Organization. Laboratory Biosafety Manual. 4th ed. Geneva: World Health Organization; 2020. Available from: https://www.who.int/publications/i/item/9789240011311 [Last accessed on 2025 Jul 15].
30. Sondhi S, Karmakar T, Sondhi Y, Kunte K. Moths of tale wildlife sanctuary, Arunachal Pradesh, India with 17 additions to the moth fauna of India (Lepidoptera: Heterocera). Trop Lepid Res. 2021;31(2):1-53. https://10.5281/zenodo.5062572
31. Saikia B, Sinha B. On the Liurana (Anura: Ceratobatrachidae) of India with the description of three new species from Talley Valley Wildlife Sanctuary in Arunachal Pradesh, Eastern Himalayas. Rec Zool Surv India. 2019;119(4):303-15. https://doi.org/10.26515/rzsi/v119/i4/2019/141629
32. Cappuccino JG, Sherman North Microbiology: A Laboratory Manual. Singapore: Pearson Education (Singapore Pvt. Ltd.); 2004.
33. Loeffler F, Gaffky G. On the Method of Pure Cultures of Bacteria. In: Reports from the Imperial Health Office. Vol. 1; 1881. p. 1-15.
34. De Souza Rabello VB, Corrêa-Moreira D, Santos C, Abreu Pinto TC, Procopio-Azevedo AC, Boechat J, et al. Preservation methods in Sporothrix isolates characterized by the polyphasic approach. J Fungi. 2023;9(1):34. https://doi.org/10.3390/jof9010034
35. Bhumbla U. Gram Staining. 1st ed. New Delhi: Jaypee Brothers Medical Publishers (P) Ltd.; 2018. p. 30. https://doi.org/10.5005/JP/BOOKS/14206_7
36. Sambrook J, Russell DW. Purification of nucleic acids by extraction with phenol: Chloroform. CSH Protoc. 2006;2006(1):pdb.prot4455. https://doi.org/10.1101/pdb.prot4455
37. Afshari M, Shahidi F, Mortazavi SA, Tabatabai F, Eshagi Z. Investigating the influence of pH, temperature and agitation speed on yellow pigment production by Penicillium aculeatum ATCC 10409. Nat Prod Res. 2015;29(14):1300-6. https://doi.org/10.1080/1478641 9.2014.999059
38. Bhat SV, Khan SS, Amin T. Isolation and characterization of pigment producing bacteria from various foods for their possible use as biocolours. Int J Recent Sci Res. 2013;4(10):1605-9. https://doi.org/10.1016/s0020-013-009
39. Shaba AM, Oyeleke SB, Ijah UJ, Oyewole OA, Adamu BB, Okeke KS, et al. Optimization of growth conditions of Serratiamarcescens for prodigiosin production. UMYU J Microbiol Res. 2017;2(2):27-37. https://doi.org/10.47430/ujmr.1722.005
40. Aftab A, Muhammad ST, Akbar N, Khaliq S, Sajjad A, Kakar MA. Pigment production in Penicillium: Different optimization methods for submerged fermentation. Pak Euro J Med Life Sci. 2021;4(Special Issue 1):S77-95. https://doi.org/10.31580/pjmls.v4iSpecialIs.2105
41. Kumar S, Stecher G, Sanderford M, Sharma S, Tamura K. Mega12: Molecular evolutionary genetic analysis version 12 for adaptive and green computing. Mol Biol Evol. 2024;41(12):msae263. https://doi.org/10.1093/molbev/msae263
42. Nei M, Kumar S. Molecular Evolution and Phylogenetics. United Kingdom: Oxford University Press; 2000. https://doi.org/10.1093/oso/9780195135848.001.0001
43. Zwe YH, Yadav M, Ten MM, Srinivasan M, Jobichen C, Sivaraman J, et al. Bacterial antagonism of Chromobacterium haemolyticum and characterization of its putative type VI secretion system. Res Microbiol. 2022;173:103918. https://doi.org/10.1016/j.resmic.2021.103918
44. Abedin SM, Tarafdar MR, Saha A, Atiqua, Rahim MM, Karim SN, et al. Isolation and characterization of Chromobacterium violaceum and antibacterial activities of its metabolite violacein. Dhaka Univ J Biol Sci. 2024;33(1):109-19. https://doi.org/10.3329/dujbs. v33i1.72487
45. Vishnu TS, Palaniswamy M. Isolation and identification of Chromobacterium sp. from different ecosystems Asian J Pharm Clin Res. 2016;9(Suppl 3):253-7. https://doi.org/10.22159/ajpcr.2016. v9s3.14847
46. Sandrasaigaran P, Rajandrai P, Loon MW, Hasan H. Isolation and characterization of Chromobacterium sp. from Lake water at Manipal International University. Malays J Med Health Sci. 2021;17(Suppl 4):53-7. https://doi.org/10.47836/mjmhs17.s4.11
47. Sharma SK, Dhyani R, Ahmad E, Maurya PK, Yadav M, Yadav VK, et al. Characterization and low-cost preservation of Chromobacterium violaceum strain TRFM-24 isolated from Tripura state, India. J Genet Eng Biotechnol. 2021;19:146. https://doi.org/10.1186/s43141-021- 00241-z
48. Kumar S, Kumar V, Ambika AA, Nag D, Kumar V, Darnal S, et al. Microbial pigments: Learning from Himalayan perspective to industrial applications. J Ind Microbiol Biotechnol. 2022;49(5):kuac017. https://doi.org/10.1093/jimb/kuac017
49. Cassarini M, Crônier D, Besaury L, Besaury L Remond C. Protein-rich agro-industrial co-products are key substrates for growth of Chromobacterium vaccinii and its violacein bioproduction. Waste Biomass Valorization. 2022;13:4459-68. https://doi.org/10.1007/s12649-022-01798-7
50. Gohil N, Bhattacharjee G, Gayke M, Narode H, Alzahrani KJ, Singh V. Enhanced production of violacein by Chromobacterium violaceum using agro-industrial waste soybean meal. J Appl Microbiol. 2022;132:1121-33. https://doi.org/10.1111/jam.15277
51. Nathan VK, Rajam KS, Rani ME, Rathinasamy G, Dhriviamkannan N. Surface culturing of Chromobacterium violaceum MTCC 2656 for violacein production and prospecting its bio-activities. In: Sivasankari B, Tomazzetto G, Verma M, editors. Current Research in Microbiology. Vol. 3., Ch. 3. 2018. Available from: https://openaccessebooks.com/current-research-in-microbiology-volume-3.html [Last accessed on 2025 Nov 06].
52. Musa NN, Yusof NZ. Chemical and physical parameters affecting bacterial pigment production. Mater Today Proc. 2019;19(4):1608- 17. https://doi.org/10.1016/j.matpr.2019.11.189
53. Bhat MR, Marar T. Optimization, extraction, and partial characterization of an orange pigment from Salinicoccus sp. MKJ 997975. Int J Life Sci Biotechnol Pharma Res. 2015;4(2):85-9.
54. El Sayed GH, Fadel M, Fouad R, Ahmed HM, Hamed AA. Improving natural red pigment production by Streptomyces phaeolivaceus strain GH27 for functionalization of textiles with in silico ADME prediction. BMC Microbiol. 2024;24:137. https://doi.org/10.1186/s12866-024-03697-4
55. Parmar RS, Singh C, Kumar A. Optimization of cultural parameters for pigment production from Streptomyces flavofuscus ARITM02, isolated from rhizosphere soil. Int J Curr Microbiol Appl Sci. 2017;6(2):961-6. https://doi.org/10.20546/ijcmas.2017.602.108
56. Shirata A, Tsukamoto T, KuyYasui H, Hata T, Hayasaka S, Kojima A, et al. Isolation of bacteria producing Bluish-Purple pigment and use dyeing. Jpn Agric Res Q. 2000;34:131-40. https://www.jircas.go.jp/sites/default/files/publication/jarq/34-2-131-140_0.pdf
57. Momen AZ, Hoshino T. Biosynthesis of violacein: Intact incorporation of the tryptophan molecule on the oxindole side, with intramolecular rearrangement of the indole ring on the 5-hydroxyindole side. Biosci Biotechnol Biochem. 2000;64(3):539-49. https://doi.org/10.1271/bbb.64.539
58. Loginove LI, Manuilova VP, Tolstikov VP. Content of free amino acids in peptone and the dynamics of their consumption in the microbiological synthesis of dextran. Pharm Chem J 1974;4:249-51. https://doi.org/10.1007/BF00777001
59. Goswami B, Bhowal J. Identification and characterization of extracellular red pigment producing bacteria isolated from soil. Int J Curr Microbiol Appl Sci. 2014;3:169-76. https://www.ijcmas.com/vol-3-9/Bhaswati%20Goswami%20and%20Jayati%20Bhowal.pdf
60. Said GH, Fadel M, Fouad R, Ahmed HM, Hamed AA. Improving natural red pigment production by Streptomyces phaeolivaceus strain GH27 for functionalization of textiles with in silico ADME prediction. BMC Microbiol. 2025;25:19. https://doi.org/10.1186/s12866-024-03697-4
61. Kanade YB, Abhyankar PS, Patwardhan RB. Studies on Violacein Extracted from Chromobacterium violaceum with its application in textile dyeing. Bull Environ Pharmacol Life Sci. 2014;13:88-93. https://bepls.com/beplsjune2024/12.pdf
62. Ahmed A, Ahmad A, Li R, Ansi WA, Fatima M, Mushtaq BS, et al. Recent advances in synthetic, industrial and biological applications of violacein and its heterologous production. J Microbiol Biotechnol. 2021;31(11):1465-80. https://doi.org/10.4014/jmb.2107.07045
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