The science and technology interact with the art in several ways. Biotechnological coupled with analytical approaches can play an important role in protecting and preserving cultural heritage for future generations. Many microorganisms influenced by environmental conditions are the main responsible for biological contamination in built heritage. Biocides based on chemical compounds have been used to mitigate this problem. Thus, it is vitally important to develop proper remediation actions based on environmentally innocuous alternative. Bacillus specie is emerging as an optimistic alternative for built heritage treatment due to their capacity to produce secondary metabolites with antagonistic activities against many fungal pathogens. Therefore, the intent of this work was to access a rapid evaluation of antifungal potential of bioactive metabolites produced by Bacillus strains and simultaneously their characterization using spectroscopic (NMR) and chromatographic techniques (LC - ESI-MS). The high antifungal activity obtained for Bacillus sp. active compounds produced in this study confirm s the great potential to suppress biodeteriogenic fungi growth on historical artworks. Additionally, the proposed methodology allowed to access bioactive metabolites produced without need of the laborious total previous isolation and c ould be used as a viable alternative to be employed for screening and production of new green biocides.
The problems caused by lack of proper preservation of historical built heritage often only come to people’s attention when a tragedy occurs such as a fire or collapse. Far from the eyes of most, silence threats lurking permanently our cultural heritage. Microorganisms, including bacteria, fungi, algae and lichens, influenced by environmental conditions are the main biodeteriogens agents responsible for esthetical damage and structural damage [
The procedure of controlling biodegradation/biodeterioration of artworks with minimal environmental impact it is a major challenge for the professionals responsible for the conservation and preservation of built heritage [
Bacillus species produce a great diversity of secondary metabolites with biological activity [
Some strains of Bacillus subtilis and Bacillus amyloliquefaciens have been referred to produce antifungal peptides [
The aim of this study was to access a rapid evaluation of antifungal potential of bioactive metabolites produced by Bacillus strains and simultaneously their characterization using NMR and LC-ESI-MS analysis without need of previous isolation.
The strains of Bacillus sp. CCMI 1051, CCMI 1052 and CCMI 1053 were isolated from healthy Quercussuber in the south of Portugal and identified according with morphological, physiological and biochemical characteristics and by 16S rDNA sequence analysis (accession number AY785773, AY785775 and AY785774, respectively) [
For the production of bioactive compounds Bacillus sp. CCMI 1051, CCMI 1052 and CCMI 1053 cells were cultivated in 2 L of Nutrient Broth (HIMEDIA) medium and incubated for 48 hours at 30˚C in an orbital shaker at 150 rpm (IKA KS 4000 I control). After 48 hours (stationary-phase) of culture growth, the bacterial cells were removed from the culture broth by centrifugation (10,000× g for 10 min at 4˚C). The supernatant were stored at −80˚C and lyophilized for further analysis.
Antimicrobial compounds in lyophilized cell-free supernatant cultures were solubilized in CHCl3/CH3OH/H2O (65:35:5) and purified by flash column chromatography on silica gel (Merck, Kieselgel 60 with 0.040 - 0.063 mm) using the system CHCl3/CH3OH/ H2O (65:35:5) as eluent. The bioactive compounds purification was monitored by thin layer chromatography (Merck, Kieselgel GF 254, 0.2 mm) using the same eluent.
Fungal spore suspension of Mucor sp., Alternaria sp., Penicillium sp. and Cladosporium sp. were prepared by adding loopful of hyphae and spores from a Malt Extract Agar (MEA) slant incubated at 25˚C for 7 days, in 15 mL of NaCl 0.85% solution. The suspension was filtered by sterilized cotton or triple gauze. A 106 CFU/mL spore suspension was obtained through dilutions and fungal suspensions were incorporated in MEA at 45˚C in Petri dishes. Filter paper discs (Macherey-Nagel 827 ATD) with 13 millimeters of diameter impregnated with 20 μL of Bacillus culture broth after cells removed were placed on the agar. The Petri dishes were incubated at 25˚C for 48 - 72 h. Antifungal activity was indicated by the formation of an inhibition halos around the discs.
Before and after compounds separation, the different fractions obtained was filtered with a 0.45 μm nylon filter (VWR International, West Chester, PA, USA), and a 10 μL sample was analysed by liquid chromatography coupled to mass spectrometry (LC-MS).
LC-ESI-MS analyses were carried out in a LCQ Advantage Thermo Finnigan mass spectrometer equipped with an electrospray ionization (ESI) source and using an ion trap mass analyzer. The conditions of lipopetides analysis were: capillary temperature 300 ºC, source voltage 5.0 kV, source current 100.0 A, and capillary voltage 22 V, in positive mode. The mass spectrometer equipment was coupled to an HPLC system with autosampler (Surveyor ThermoFinnigan). The analytical column was a reversed phase Zorbax Eclipse (C18, particle size 5.0 μm, 150 mm × 2.4 mm). The chromatographic separation was performed with a gradient program using acetonitrile as eluent A and water acidified with 0.1% (v/v) formic acid as eluent B, at a flow rate of 0.3 mL∙min−1. The elution program was as following: linear gradient from 20% to 50% of A (0 - 10 min) and from 50% to 100% of A (10 - 40 min).
1H NMR spectra were recorded on a Bruker Avance III HD 400 spectrometer at 400 MHz in DMSO-d6 (Euriso-top). 1H shifts are reported relative to the 1H signal of DMSO-d6 (δ = 2.50 ppm) [
Several studies have described that bacteria of genera Bacillus are worthy to be used as antifungal substances producer including extracellular lipopeptides for versatile applications [
In this work a new methodological scheme using analytical and biological approaches was established in order to access bioactive metabolites produced by Bacillus strains and simultaneously their characterization without need of the laborious total previous isolation.
The final propose is the production of a new alternative for commercial toxic biocides used in heritage context thought the development of green biocides produced by harmful Bacillus species.
After 48 h of Bacillus sp. CCMI 1053 culture, the supernatants of liquid cultures were tested against the selected biodeteriogenic fungal strains isolated from biodegraded mural paintings: Mucor sp., Alternaria sp., Penicillium sp. and Cladosporium sp. (
Antifungal assays show a high inhibition zone for all free cells culture broths produced against biodeteriogenic fungi. Nevertheless the Bacillus sp. CCMI1053 display a major antifungal potential against Penicillium sp., confirmed by the formation of an inhibition halo of 37.44 mm (
Bacillus sp. | Inhibition halos (mm) | |||
---|---|---|---|---|
Mucorsp. | Alternaria sp. | Penicilliumsp. | Cladosporium sp. | |
CCMI 1051 | 25.67 ± 1.00 | 23.11± 0.93 | 23.67 ± 0.50 | 22.11 ± 0.19 |
CCMI 1052 | 18.22 ± 0.97 | 17.89 ± 0.33 | 18.89 ± 1.05 | 17.33 ± 0.03 |
CCMI 1053 | 27.33 ± 0.87 | 22.33 ± 0.71 | 37.44 ± 1.83 | 26.67 ± 1.41 |
accomplish a ground-breaking research for the utilization of the compounds produced for application in heritage materials contaminated by fungal communities and confirm previous studies reporting that Bacillus strains have a height antifungal activity [
The 1H-NMR spectrum of the bioactive compounds showed signals for N-binding protons at δ 6.8 - 8.6 ppm, α-protons at δ 4.0 - 4.5 ppm and β/γ-protons at δ 1.4 - 4.0 ppm of peptide bonds (
A LC-ESI-MS spectral analysis of the cell free supernatant showed a cluster containing molecules that were observed at m/z 1031, 1045 1463 and 1477 (
Thus, the peaks with m/z 1031, 1045, 1463 and 1477 could be assigned to lipopeptides. The mass spectra obtained in ESI mode shows a cluster containing molecules with a difference of 14 Da, corresponding to the loss of CH2 in the lipidic chain [
popeptide due to diverse producer strains and different nutritional conditions. These environments affect the substitution of amino acids in the peptidic ring and the length of lipidic chain [
In this work, a combined use of NMR and LC-ESI-MS allows a rapid identification of lipopeptides produced by Bacillus sp. CCMI1053. The proposed methodology allowed to access bioactive metabolites produced without need of the laborious total previous isolation and could be used as a viable alternative to be employed for screening and production of new green biocides.
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-Microrganisms 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).
Silva, M., Pereira, A., Teixeira, D., Candeias, A. and Caldeira, A.T. (2016) Combined Use of NMR, LC-ESI- MS and Antifungal Tests for Rapid Detection of Bioactive Lipopeptides Produced by Bacillus. Advances in Microbiology, 6, 788-796. http://dx.doi.org/10.4236/aim.2016.610077