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

Environ Sci Pollut Res (2017) 24:4871–4881 DOI 10.1007/s11356-016-8175-y RESEARCH ARTICLE Green mitigation strategy for cultural heritage: bacterial potential for biocide production Mara Silva 1,2 & Tânia Rosado 2 & Dora Teixeira 1,2 & António Candeias 1,2 & Ana Teresa Caldeira 1,2 Received: 15 August 2016 / Accepted: 28 November 2016 / Published online: 17 December 2016 # Springer-Verlag Berlin Heidelberg 2016 Abstract 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 Responsible editor: Philippe Garrigues * Ana Teresa Caldeira 1 Chemistry Department, School of Sciences and Technology, Évora University, Rua Romão Ramalho 59, 7000-671 Évora, Portugal 2 HERCULES Laboratory, Évora University, Largo Marquês de Marialva 8, 7000-809 Évora, Portugal effective approach for confirming the selection of lipopeptides producer strains. Keyword Green biocides . Cultural heritage . Bacillus sp. . Biosurfactants . Lipopeptides . Antifungal activity . Peptide synthetase Introduction Biosurfactants are biological surface-active compounds largely produced by a wide variety of microorganisms (DehghanNoude et al. 2005; Das et al. 2008; Cao et al. 2009). An extensive range of structurally different biosurfactants have been identified, including glycolipids, lipoproteins, polysaccharides, proteins and lipopeptides (Souto et al. 2004; Roongsawang et al. 2010). Lipopeptides molecules are commonly made up of a hydrophobic portion composed by fatty acids (saturated, unsaturated or hydroxylated), which are linked to a short linear or cyclic oligopeptide, that mark the hydrophilic portion of the molecule (Raaijmakers et al. 2010). On the basis of the structural relationships, the lipopeptides that have been identified in some Bacillus strains are generally classified into three groups: the surfactin group (Yao et al. 2003; Mikkola et al. 2004), the fengycin group (Hu et al. 2007; Arrebola et al. 2010) and the iturin group (Moyne et al. 2001; Kim et al. 2010; Mandal et al. 2013). The members of the surfactin and fengycin groups are composed of one β-hydroxy fatty acid and 7 or 10 α-amino acids, respectively, while the members of the iturin group consist of one β-amino fatty acid and 7 α-amino acids. The presence of the β-amino fatty acid is the most striking characteristic of the iturin A family and distinguishes this from the other two families (Tsuge et al. 2001). 4872 These compounds have been received significant attention for their antimicrobial and surfactant properties, especially the nonribosomally synthesized lipopeptides surfactin, iturin and fengycin (Caldeira et al. 2011a). In contrast to the structural diversity of these lipopeptides, their biosynthetic mechanism is basically conserved (Zhao et al. 2013). The lipopeptides are synthesized nonribosomally by a mega-peptide synthetase (NRPS) unit which is composed by several cooperating multifunctional modules, with capacity to perform 1 cycle of peptide elongation (Roongsawang et al. 2010). NRPSs have a modular structure, and each module possesses approximately 1000 amino acids, which catalysed the incorporation of one amino acid in the peptide product. The order of the modules corresponds to the order in which the amino acids are incorporated in the peptide product (Roongsawang et al. 2010). The iturin A synthetase operon shares the same organisation as the mycosubtilin operon and spans a region more than 38 kb long, which is composed by four open reading frames: ituA, ituB, ituC and ituD (Tsuge et al. 2001). The ituD gene encodes a putative malonyl co-enzyme A transacylase, whose disruption results in a specific deficiency in iturin A production (Hsieh et al. 2008). In this study a characterisation, identification and evaluation of the antibiotic potential of a set of bacterial isolates were undertaken with the assistance of molecular approaches. Thus, this work intend (i) the microorganism selection with antifungal potential and (ii) the identification of the bacterial strains with higher activity against biodeteriogenic fungi isolated from biodegraded cultural heritage artefacts. Materials and methods Bacteria strains and culture media Bacillus sp. used in this study was selected in a range of 21 bacterial isolates from biodegraded artworks and healthy Quercus suber (south of Portugal) that are currently stored in the culture collection of HERCULES Laboratory, Biotech laboratory, Évora University. The strains were characterized by the morphological, physiological and biochemical characteristics based on the Bergey’s Manual of Systematic Bacteriology (Vos et al. 2011) and by 16S rDNA sequencing (outsourcing service). The bacterial strains were maintained on nutrient agar slants and stored at 4 °C. Antifungal activity assessment For the production of potential bioactive compounds, the Bacillus sp. cells were inoculated in 100 mL of NB (Nutrient Broth) medium. The culture was incubated for 48 h at 30 °C in an orbital shaker at 150 rpm (IKA KS 4000 i control, Germany). The bacterial cells were removed from Environ Sci Pollut Res (2017) 24:4871–4881 the culture broth by centrifugation (1000×g for 10 min at 4 °C). The supernatant was maintained at −20 °C for further analysis of antifungal activity. Fungal spore suspension of Fusarium oxysporum, Aspergillus niger, Cladosporium sp., Penicillium sp. 1, Mucor sp. Rhodotorula sp., Penicillium sp. 2, Penicillium sp. 3 and Alternaria sp. was prepared by adding a loopful of hyphae and spores from a malt extract agar (MEA) slant, incubated at 25 °C for 7 days, in 5 mL of NaCl 0.85% solution. The suspension was (...truncated)