Skip to main content

Insight into the bacterial diversity of fermentation woad dye vats as revealed by PCR-DGGE and pyrosequencing

Abstract

The bacterial diversity in fermenting dye vats with woad (Isatis tinctoria L.) prepared and maintained in a functional state for approximately 12 months was examined using a combination of culture-dependent and -independent PCR-DGGE analyses and next-generation sequencing of 16S rRNA amplicons. An extremely complex ecosystem including taxa potentially contributing to both indigo reduction and formation, as well as indigo degradation was found. PCR-DGGE analyses revealed the presence of Paenibacillus lactis, Sporosarcina koreensis, Bacillus licheniformis, and Bacillus thermoamylovorans, while Bacillus thermolactis, Bacillus pumilus and Bacillus megaterium were also identified but with sequence identities lower than 97%. Dominant operational taxonomic units (OTUs) identified by pyrosequencing included Clostridium ultunense, Tissierella spp., Alcaligenes faecalis, Erysipelothrix spp., Enterococcus spp., Virgibacillus spp. and Virgibacillus panthothenicus, while sub-dominant OTUs included clostridia, alkaliphiles, halophiles, bacilli, moderately thermophilic bacteria, lactic acid bacteria, Enterobacteriaceae, aerobes, and even photosynthetic bacteria. Based on the current knowledge of indigo-reducing bacteria, it is considered that indigo-reducing bacteria constituted only a small fraction in the unique microcosm detected in the natural indigo dye vats.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2

References

  1. 1.

    Aino K, Narihiro T, Minamida K, Kamagata Y, Yoshimune K, Yumoto I (2010) Bacterial community characterization and dynamics of indigo fermentation. FEMS Microbiol Ecol 74:174–183

    CAS  Article  PubMed  Google Scholar 

  2. 2.

    Aquilanti L, Santarelli S, Babini V, Osimani A, Clementi F (2013) Quality evaluation and discrimination of semi-hard and hard cheeses from the Marche region (Central Italy) using chemometric tools. Int Dairy J 29:42–52

    CAS  Article  Google Scholar 

  3. 3.

    Blackburn RS, Bechtold T, John P (2009) The development of indigo reduction methods and pre-reduced indigo products. Coloration Technol 125:193–207

    CAS  Article  Google Scholar 

  4. 4.

    Cardinali F, Taccari M, Milanović V, Osimani A, Polverigiani S, Garofalo C, Foligni R, Mozzon M, Zitti S, Raffaelli N, Clementi F, Aquilanti L (2016) Yeast and mould dynamics in Caciofiore della Sibilla cheese coagulated with an aqueous extract of Carlina acanthifolia All. Yeast 33:403–414

    CAS  Article  PubMed  Google Scholar 

  5. 5.

    Ercolini D (2004) PCR-DGGE fingerprinting: novel strategies for detection of microbes in food. J Microbiol Methods 56:297–314

    CAS  Article  PubMed  Google Scholar 

  6. 6.

    Ercolini D (2013) High-throughput sequencing and metagenomics: moving forward in the culture-independent analysis of food microbial ecology. Appl Environ Microbiol 79(10):3148–3155

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  7. 7.

    Garofalo C, Bancalari E, Milanović V, Cardinali F, Osimani A, Savo Sardaro ML, Bottari B, Bernini V, Aquilanti L, Clementi F, Neviani E, Gatti M (2017) Study of the bacterial diversity of foods: PCR-DGGE versus LH-PCR. Int J Food Microbiol 242:24–36

    CAS  Article  PubMed  Google Scholar 

  8. 8.

    Garofalo C, Osimani A, Milanović V, Aquilanti L, De Filippis F, Stellato G, Di Mauro S, Turchetti B, Buzzini P, Ercolini D, Clementi F (2015) Bacteria and yeast microbiota in milk kefir grains from different Italian regions. Food Microbiol 49:123–133

    CAS  Article  PubMed  Google Scholar 

  9. 9.

    Garofalo C, Silvestri G, Aquilanti L, Clementi F (2008) PCR-DGGE analysis of lactic acid bacteria and yeast dynamics during the production processes of three varieties of Panettone. J Appl Microbiol 105(1):243–254

    CAS  Article  PubMed  Google Scholar 

  10. 10.

    Han X, Wei Wang W, Xiao X (2008) Microbial biosynthesis and biotransformation of indigo and indigo-like pigments. Chin J Biotechnol 24:921–926

    CAS  Article  Google Scholar 

  11. 11.

    Hartl A, Gaibor ANP, van Bommel MR, Hofmann-de Keijzer R (2015) Searching for blue: experiments with woad fermentation vats and an explanation of the colours through dye analysis. J Archaeol Sci 2:9–39

    Google Scholar 

  12. 12.

    Hirota K, Aino K, Nodasaka Y, Yumoto I (2013) Oceanobacillus indicireducens sp. nov., a facultative alkaliphile that reduces an indigo dye. Int J Syst Evol Microbiol 63:1437–1442

    Article  PubMed  Google Scholar 

  13. 13.

    Hirota K, Aino K, Yumoto I (2013) Amphibacillus iburiensis sp. nov., an alkaliphile that reduces an indigo dye. Int J Syst Evol Microbiol 63:4303–4308

    CAS  Article  PubMed  Google Scholar 

  14. 14.

    Hirota K, Aino K, Yumoto I (2016) Fermentibacillus polygoni gen. nov., sp. nov., an alkaliphile that reduces indigo dye. Int J Syst Evol Microbiol 66:2247–2253

    Article  PubMed  Google Scholar 

  15. 15.

    Hirota K, Okamoto T, Matsuyama H, Yumoto I (2016) Polygonibacillus indicireducens gen nov., sp. nov., an indigo-reducing and obligate alkaliphile isolated from indigo fermentation liquor for dyeing. Int J Syst Evol Microbiol. doi:10.1099/ijsem.0.001405

    Google Scholar 

  16. 16.

    Hynes WL, Ferretti JJ, Gilmore MS, Segarra RA (1992) PCR amplification of streptococcal DNA using crude cell lysates. FEMS Microbiol Lett 94:139–142

    CAS  Article  Google Scholar 

  17. 17.

    John P, Arghyros S, Nicholson S (2008) Indigo reducing bacteria from the medieval woad (Isatis tinctoria L.) vat: some aspects of their interaction with indigo. In: Dyes in History and Archaeology, pp. 45–50. ISBN 9781904982074

  18. 18.

    Khelifi E, Touhami Y, Thabet OBD, Ayed L, Bouallagui H, Fardeau ML, Hamdi M (2012) Exploring bioaugmentation strategies for the decolourization of textile wastewater using a two species consortium (Bacillus cereus and Bacillus pumilus) and characterization of produced metabolites. Desalt Water Treat 45:48–54

    CAS  Article  Google Scholar 

  19. 19.

    Lu L, Zhao M, Wang TN, Zhao LY, Du MH, Li TL, Li DB (2012) Characterization and dye decolorization ability of an alkaline resistant and organic solvents tolerant laccase from Bacillus licheniformis LS04. Bioresour Technol 115:35–40

    CAS  Article  PubMed  Google Scholar 

  20. 20.

    Miller D (1984) Indigo from seed to dye. Indigo Press APTOS, Santa Cruz

    Google Scholar 

  21. 21.

    Muyzer G, DeWall EC, Uitterlinden AG (1993) Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Appl Environ Microbiol 59:695–700

    CAS  PubMed  PubMed Central  Google Scholar 

  22. 22.

    Nakajima K, Hirota K, Nodasaka Y, Yumoto I (2005) Alkalibacterium iburiense sp. nov., an obligate alkaliphile that reduces an indigo dye. Int J Syst Evol Microbiol 55:1525–1530

    CAS  Article  PubMed  Google Scholar 

  23. 23.

    Nicholson SK, John P (2005) The mechanism of bacterial indigo reduction. Appl Microbiol Biotechnol 68:117–123

    CAS  Article  PubMed  Google Scholar 

  24. 24.

    Oberthür C, Schneider B, Graf H, Hamburger M (2004) The elusive indigo precursors in woad (Isatis tinctoria L.) Identification of the major indigo precursor, isatan a, and a structure revision of isatan B. Chem Biodivers 1:174–182

    Article  PubMed  Google Scholar 

  25. 25.

    Orsini R, Aquilanti L, Osimani A, Santilocchi R (2012) Isatis tinctoria L.: biomass production and indigo dye yield as influenced by mineral and organic nitrogen fertilization. Agrochimica 6:292–308

    Google Scholar 

  26. 26.

    Osimani A, Aquilanti L, Baldini G, Silvestri G, Butta A, Clementi F (2012) Implementation of a biotechnological process for vat dyeing with woad. J Ind Microbiol Biotechnol 39:1309–1319

    CAS  Article  PubMed  Google Scholar 

  27. 27.

    Osimani A, Garofalo C, Aquilanti L, Milanović V, Clementi F (2015) Unpasteurised commercial boza as a source of microbial diversity. Int J Food Microbiol 194:62–70

    CAS  Article  PubMed  Google Scholar 

  28. 28.

    Padden AN, Dillon VM, Edmonds J, Collins MD, Alvarez N, John P (1999) An indigo-reducing moderate thermophile from a woad vat, Clostridium isatidis sp. nov. Int Syst Bacteriol 49:1025–1031

    CAS  Article  Google Scholar 

  29. 29.

    Padden AN, Dillon VM, John P, Edmonds J, Collins MD, Alvarez N (1998) Clostridium used in medieval dyeing. Nature 396:225

    CAS  Article  Google Scholar 

  30. 30.

    Padden AN, John P, Collins MD, Hutson R, Hall AR (2000) Indigo-reducing Clostridium isatidis isolated from a variety of sources, including a 10th-century viking dye vat. J Archaeol Sci 27:953–956

    Article  Google Scholar 

  31. 31.

    Randazzo CL, Torriani S, Akkermans ADL, De Vos WM, Vaughan EE (2002) Diversity, dynamics, and activity of bacterial communities during production of an artisanal Sicilian cheese as evaluated by 16S rRNA analysis. Appl Environ Microbiol 68:1882–1892

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  32. 32.

    Regar RK, Gaur VK, Mishra G, Jadhao S, Kamthan M, Manickam N (2016) Draft genome sequence of Alcaligenes faecalis strain IITR89, an indole-oxidizing bacterium. Genome Announc 4(2):e00067-16

    Article  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Roshan P (2015) Denim. Manufacture, finishing and applications. Woodhead publishing series in textiles -number 164. Elsevier, Cambridge

    Google Scholar 

  34. 34.

    Shipkowski S, Brenchley JE (2005) Characterization of an unusual cold-active beta-glucosidase belonging to family 3 of the glycoside hydrolases from the psychrophilic isolate Paenibacillus sp. strain C7. Appl Environ Microbiol 71:4225–4232

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Takahara Y, Tanabe O (1960) Studies on the reduction of indigo in industrial fermentation vat (XXI). J Ferment Technol 38:293–297

    CAS  Google Scholar 

  36. 36.

    Yang G, Zhou S (2014) Sinibacillus soli gen. nov., sp. nov., a moderately thermotolerant member of the family Bacillaceae. Int J Syst Evol Microbiol 64:1647–1653

    CAS  Article  PubMed  Google Scholar 

  37. 37.

    Yumoto I, Hirota K, Nodasaka Y, Yokota Y, Hoshino T, Nakajima K (2004) Alkalibacterium psychrotolerans sp. nov., a psychrotolerant obligate alkaliphile that reduces an indigo dye. Int J Syst Evol Microbiol 54:2379–2383

    CAS  Article  PubMed  Google Scholar 

  38. 38.

    Yumoto I, Hirota K, Nodasaka Y, Tokiwa Y, Nakajima K (2008) Alkalibacterium indicireducens sp. nov., an obligate alkaliphile that reduces indigo dye. Int J Syst Evol Microbiol 58:901–905

    CAS  Article  PubMed  Google Scholar 

Download references

Acknowledgements

The authors wish to thank Dr. Francesca De Filippis of the Dipartimento di Agraria, Universita degli Studi di Napoli Federico II, Italy, for her valuable assistance in pyrosequencing analyses.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Lucia Aquilanti.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Milanović, V., Osimani, A., Taccari, M. et al. Insight into the bacterial diversity of fermentation woad dye vats as revealed by PCR-DGGE and pyrosequencing. J Ind Microbiol Biotechnol 44, 997–1004 (2017). https://doi.org/10.1007/s10295-017-1921-4

Download citation

Keywords

  • Microbial diversity
  • Indigo reduction
  • Spore-forming bacteria
  • Fermentation liquor
  • Isatis tinctoria L