Environmental Science and Pollution Research

, Volume 24, Issue 5, pp 4871–4881 | Cite as

Green mitigation strategy for cultural heritage: bacterial potential for biocide production

  • Mara Silva
  • Tânia Rosado
  • Dora Teixeira
  • António Candeias
  • Ana Teresa CaldeiraEmail author
Research Article


Several biosurfactants with antagonistic activity are produced by a variety of microorganisms. Lipopeptides (LPPs) produced by some Bacillus strains, including surfactin, fengycin and iturin are synthesized nonribosomally by mega-peptide synthetase (NRPS) units and they are particularly relevant as antifungal agents. Characterisation, identification and evaluation of the potentials of several bacterial isolates were undertaken in order to establish the production of active lipopeptides against biodeteriogenic fungi from heritage assets. Analysis of the iturin operon revealed four open reading frames (ORFs) with the structural organisation of the peptide synthetases. Therefore, this work adopted a molecular procedure to access antifungal potential of LPP production by Bacillus strains in order to exploit the bioactive compounds synthesis as a green natural approach to be applied in biodegraded cultural heritage context. The results reveal that the bacterial strains with higher antifungal potential exhibit the same morphological and biochemical characteristics, belonging to the genera Bacillus. On the other hand, the higher iturinic genetic expression, for Bacillus sp. 3 and Bacillus sp. 4, is in accordance with the culture antifungal spectra. Accordingly, the adopted methodology combining antifungal screening and molecular data is represent a valuable tool for quick identification of iturin-producing strains, constituting an effective approach for confirming the selection of lipopeptides producer strains.


Green biocides Cultural heritage Bacillus sp. Biosurfactants Lipopeptides Antifungal activity Peptide synthetase 



The authors gratefully acknowledge the following funding sources: “HIT3CH—HERCULES Interface for Technology Transfer and Teaming in Cultural Heritage”, ref. ALT20-03-0246-FEDER-000004, and “MEDUSA—Microorganisms Monitoring and Mitigation—Developing and Unlocking Novel Sustainable Approaches”, ref. ALT20-03-0145-FEDER-000015, co-financed by the European Union through the European Regional Development Fund ALENTEJO 2020 (Regional Operational Programme of the Alentejo)


  1. Arrebola E, Jacobs R, Korsten L (2010) Iturin A is the principal inhibitor in the biocontrol activity of Bacillus amyloliquefaciens PPCB004 against postharvest fungal pathogens. J Appl Microbiol 108(2):386–395. doi: 10.1111/j.1365-2672.2009.04438.x CrossRefGoogle Scholar
  2. Caldeira AT, Arteiro JM, Roseiro JC, Neves J, Vicente H (2011a) An artificial intelligence approach to Bacillus amyloliquefaciens CCMI 1051 cultures: application to the production of anti-fungal compounds. Bioresour Technol 102(2):1496–1502. doi: 10.1016/j.procbio.2011.05.016 CrossRefGoogle Scholar
  3. Caldeira AT, Feio SS, Arteiro JM, Coelho AV, Roseiro JC (2008) Environmental dynamics of Bacillus amyloliquefaciens CCMI 1051 antifungal activity under different nitrogen patterns. J Appl Microbiol 104(3):808–816. doi: 10.1111/j.1365-2672.2007.03601.x CrossRefGoogle Scholar
  4. Caldeira AT, Santos Arteiro JM, Coelho AV, Roseiro JC (2011b) Combined use of LC–ESI-MS and antifungal tests for rapid identification of bioactive lipopeptides produced by Bacillus amyloliquefaciens CCMI 1051. Process Biochem 46(9):1738–1746. doi: 10.1016/j.procbio.2011.05.016 CrossRefGoogle Scholar
  5. Cao X-H, Liao Z-Y, Wang C-L, Yang W-Y, Lu M-F (2009) Evaluation of a lipopeptide biosurfactant from Bacillus natto TK-1 as a potential source of anti-adhesive, antimicrobial and antitumor activities. Braz J Microbiol 40(2):373–379CrossRefGoogle Scholar
  6. Cao Y, Xu Z, Ling N, Yuan Y, Yang X, Chen L, Shen B, Shen Q (2012) Isolation and identification of lipopeptides produced by B. subtilis SQR 9 for suppressing Fusarium wilt of cucumber. Sci Hortic 135:32–39. doi: 10.1016/j.scienta.2011.12.002 CrossRefGoogle Scholar
  7. Das P, Mukherjee S, Sen R (2008) Antimicrobial potential of a lipopeptide biosurfactant derived from a marine Bacillus circulans. J Appl Microbiol 104(6):1675–1684CrossRefGoogle Scholar
  8. Dehghan-Noude G, Housaindokht M, Bazzaz BS (2005) Isolation, characterization, and investigation of surface and hemolytic activities of a lipopeptide biosurfactant produced by Bacillus subtilis ATCC 6633. J Microbiol 43(3):272–276Google Scholar
  9. Drancourt M, Bollet C, Carlioz A, Martelin R, Gayral JP, Raoult D (2000) 16S ribosomal DNA sequence analysis of a large collection of environmental and clinical unidentifiable bacterial isolates. J Clin Microbiol 38(10):3623–3630Google Scholar
  10. Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 1999:95–98Google Scholar
  11. Hsieh FC, Lin TC, Meng M, Kao SS (2008) Comparing methods for identifying Bacillus strains capable of producing the antifungal lipopeptide iturin A. Curr Microbiol 56(1):1–5. doi: 10.1007/s00284-007-9003-x CrossRefGoogle Scholar
  12. Hu LB, Shi ZQ, Zhang T, Yang ZM (2007) Fengycin antibiotics isolated from B-FS01 culture inhibit the growth of Fusarium moniliforme Sheldon ATCC 38932. FEMS Microbiol Lett 272(1):91–98. doi: 10.1111/j.1574-6968.2007.00743.x CrossRefGoogle Scholar
  13. Joshi R, Gardener BB (2006) Identification and characterization of novel genetic markers associated with biological control activities in Bacillus subtilis. Phytopathology 96(2):145–154. doi: 10.1094/phyto-96-0145 CrossRefGoogle Scholar
  14. Kim PI, Ryu J, Kim YH, Chi YT (2010) Production of biosurfactant lipopeptides iturin A, fengycin and surfactin A from Bacillus subtilis CMB32 for control of Colletotrichum gloeosporioides. J Microbiol Biotechnol 20(1):138–145Google Scholar
  15. Li G, Dong Q, Ma L, Huang Y, Zhu M, Ji Y, Wang Q, Mo M, Zhang K (2014) Management of Meloidogyne incognita on tomato with endophytic bacteria and fresh residue of Wasabia japonica. J Appl Microbiol 117(4):1159–1167. doi: 10.1111/jam.12590 CrossRefGoogle Scholar
  16. Mandal S, Sharma S, Pinnaka A, Kumari A, Korpole S (2013) Isolation and characterization of diverse antimicrobial lipopeptides produced by Citrobacter and Enterobacter. BMC Microbiol 13(1):152. doi: 10.1186/1471-2180-13-152 CrossRefGoogle Scholar
  17. Mikkola R, Andersson MA, Grigoriev P, Teplova VV, Saris N-E L, Rainey FA, Salkinoja-Salonen MS (2004) Bacillus amyloliquefaciens strains isolated from moisture-damaged buildings produced surfactin and a substance toxic to mammalian cells. Arch Microbiol 181(4):314–323. doi: 10.1007/s00203-004-0660-x CrossRefGoogle Scholar
  18. Mohkam M, Nezafat N, Berenjian A, Mobasher MA, Ghasemi Y (2016) Identification of bacillus probiotics isolated from soil rhizosphere using 16S rRNA, recA, rpoB gene sequencing and RAPD-PCR. Probiotics Antimicrob Proteins 8(1):8–18. doi: 10.1007/s12602-016-9208-z CrossRefGoogle Scholar
  19. Moyne AL, Cleveland TE, Tuzun S (2004) Molecular characterization and analysis of the operon encoding the antifungal lipopeptide bacillomycin D. FEMS Microbiol Lett 234(1):43–49. doi: 10.16/j.femsle.2004.03.011 CrossRefGoogle Scholar
  20. Moyne AL, Shelby R, Cleveland TE, Tuzun S (2001) Bacillomycin D: an iturin with antifungal activity against Aspergillus flavus. J Appl Microbiol 90(4):622–629. doi: 10.1046/j.1365-2672.2001.01290.x CrossRefGoogle Scholar
  21. Raaijmakers JM, De Bruijn I, Nybroe O, Ongena M (2010) Natural functions of lipopeptides from Bacillus and Pseudomonas: more than surfactants and antibiotics. FEMS Microbiol Rev 34(6):1037–1062. doi: 10.1111/j.1574-6976.2010.00221.x CrossRefGoogle Scholar
  22. Rinta-Kanto J, Ouellette A, Boyer G, Twiss M, Bridgeman T, Wilhelm S (2005) Quantification of toxic Microcystis spp. during the 2003 and 2004 blooms in western Lake Erie using quantitative real-time PCR. Environmental Science & Technology 39(11):4198–4205CrossRefGoogle Scholar
  23. Roongsawang N, Washio K, Morikawa M (2010) Diversity of nonribosomal peptide synthetases involved in the biosynthesis of lipopeptide biosurfactants. Int J Mol Sci 12(1):141–172. doi: 10.3390/ijms12010141 CrossRefGoogle Scholar
  24. Rückert C, Blom J, Chen X, Reva O, Borriss R (2011) Genome sequence of B. amyloliquefaciens type strain DSM7 T reveals differences to plant-associated B. amyloliquefaciens FZB42. J Biotechnol 155(1):78–85. doi: 10.1016/j.jbiotec.2011.01.006 CrossRefGoogle Scholar
  25. Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4(4):406–425Google Scholar
  26. Silva M, Pereira A, Teixeira D, Candeias A, Caldeira AT (2016) Combined use of NMR, LC-ESI-MS and antifungal tests for rapid detection of bioactive lipopeptides produced by Bacillus. Advances in Microbiology 06(10):788–796. doi: 10.4236/aim.2016.610077 CrossRefGoogle Scholar
  27. Silva M, Rosado T, Teixeira D, Candeias A, Caldeira AT (2015) Production of green biocides for cultural heritage. Novel biotechnological solutions. Int J Conserv Sci 6 SI:519–530Google Scholar
  28. Souto G, Correa O, Montecchia M, Kerber N, Pucheu N, Bachur M, Garcia A (2004) Genetic and functional characterization of a Bacillus sp. strain excreting surfactin and antifungal metabolites partially identified as iturin-like compounds. J Appl Microbiol 97(6):1247–1256. doi: 10.1111/j.1365-2672.2004.02408.x CrossRefGoogle Scholar
  29. Stockel S, Meisel S, Elschner M, Rosch P, Popp J (2012) Identification of Bacillus anthracis via Raman spectroscopy and chemometric approaches. Anal Chem 84(22):9873–9880. doi: 10.1021/ac302250t CrossRefGoogle Scholar
  30. Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24(8):1596–1599CrossRefGoogle Scholar
  31. Tsuge K, Akiyama T, Shoda M (2001) Cloning, sequencing, and characterization of the iturin A operon. J Bacteriol 183(21):6265–6273. doi: 10.1128/JB.183.21.6265-6273.2001 CrossRefGoogle Scholar
  32. van Veen SQ, Claas EC, Kuijper EJ (2010) High-throughput identification of bacteria and yeast by matrix-assisted laser desorption ionization-time of flight mass spectrometry in conventional medical microbiology laboratories. J Clin Microbiol 48(3):900–907. doi: 10.1128/JCM.02071-09 CrossRefGoogle Scholar
  33. Varadavenkatesan T, Murty VR (2013) Production and properties of a lipopeptide biosurfactant by B. subtilis subsp. inaquosorum. J Microbiol Biotechnol Res 3(4):63–73Google Scholar
  34. Vardhan S, Kaushik R, Saxena AK, Arora DK (2011) Restriction analysis and partial sequencing of the 16S rRNA gene as index for rapid identification of Bacillus species. Antonie Van Leeuwenhoek 99(2):283–296. doi: 10.1007/s10482-010-9487-4 CrossRefGoogle Scholar
  35. Vos P, Garrity G, Jones D, Krieg N R, Ludwig W, Rainey F A, Schleifer K-H, Whitman W (2011) Bergey’s manual of systematic bacteriology: volume 3: the Firmicutes. Springer Science & Business Media, Vol. 3.Google Scholar
  36. Xu D, Cote JC (2003) Phylogenetic relationships between Bacillus species and related genera inferred from comparison of 3′ end 16S rDNA and 5′ end 16S-23S ITS nucleotide sequences. Int J Syst Evol Microbiol 53(Pt 3):695–704. doi: 10.1099/ijs.0.02346-0 CrossRefGoogle Scholar
  37. Yang D, Wang B, Wang J, Chen Y, Zhou M (2009) Activity and efficacy of Bacillus subtilis strain NJ-18 against rice sheath blight and Sclerotinia stem rot of rape. Biol Control 51(1):61–65. doi: 10.1016/j.biocontrol.2009.05.021 CrossRefGoogle Scholar
  38. Yao S, Gao X, Fuchsbauer N, Hillen W, Vater J, Wang J (2003) Cloning, sequencing, and characterization of the genetic region relevant to biosynthesis of the lipopeptides iturin A and surfactin in Bacillus subtilis. Curr Microbiol 47(4):272–277CrossRefGoogle Scholar
  39. Zhang J, Liu J, Meng L, Ma Z, Tang X, Cao Y, Sun L (2012) Isolation and characterization of plant growth-promoting rhizobacteria from wheat roots by wheat germ agglutinin labeled with fluorescein isothiocyanate. J Microbiol 50(2):191–198. doi: 10.1007/s12275-012-1472-3 CrossRefGoogle Scholar
  40. Zhao X, Zhou Z-j, Han Y, Wang Z-z, Fan J, Xiao H-z (2013) Isolation and identification of antifungal peptides from Bacillus BH072, a novel bacterium isolated from honey. Microbiol Res 168(9):598–606. doi: 10.1016/j.micres.2013.03.001 CrossRefGoogle Scholar
  41. Zwietering M, Jongenburger I, Rombouts F, Van’t Riet K (1990) Modeling of the bacterial growth curve. Appl Environ Microbiol 56(6):1875–1881Google Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Mara Silva
    • 1
    • 2
  • Tânia Rosado
    • 2
  • Dora Teixeira
    • 1
    • 2
  • António Candeias
    • 1
    • 2
  • Ana Teresa Caldeira
    • 1
    • 2
    Email author
  1. 1.Chemistry Department, School of Sciences and TechnologyÉvora UniversityÉvoraPortugal
  2. 2.HERCULES LaboratoryÉvora UniversityÉvoraPortugal

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