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) [9] . Bacillus cells were maintained on Nutrient Agar (HIMEDIA) slants and stored at 4 °C. The fungal cultures were maintained on malt extract agar (HIMEDIA) slants and used as test microorganisms. The strains Mucor sp., Alternaria sp., Penicillium sp. and Cladosporium sp., belonging to the laboratory collection (HERCULES-Biotec laboratory, Évora University), were isolated from biodegraded mural paintings, being also associated with known pathologies [4] [13] .

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) [14] [15] .

Several studies have described that bacteria of genera Bacillus are worthy to be used as antifungal substances producer including extracellular lipopeptides for versatile applications [16] - [19] . To detect these biosurfactants several qualitative tests are available [20] .

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. (Table 1). These fungal strains represent different fungi genera commonly found in biodegraded heritage materials and their inhibition is an important issue in this context.

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 (Figure 1 and Table 1). Results obtained in this work

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 [10] [17] [21] [22] related to the presence of lipopeptides.

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 (Figure 2). Additionally we can observe multiple sets of A₂B₂ coupling pattern protons of benzene rings at δ 6.6 - 7.1 ppm (each d, J ~ 8.0 Hz), methylene protons of long aliphatic chains at δ 1.0 - 1.3 ppm and terminal methyl protons at δ 0.7 - 1.0 ppm. These patterns of signals are consistent with the presence, almost exclusive, of lipopeptides, compounds made of amino acids and a fatty acid chain [14] [23] - [25] .

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 (Figure 3). These peaks differ by 14 Da suggesting a series of homologous molecules with different lengths of fatty acid chain. In the literature bacterial lipopeptides such as iturin A, surfactin and fengycin exhibited the same peak profile that the obtained with Bacillus sp. CCMI1053. Kim et al. (2010) described that the strains of Bacillus subtillis CMB32 produced lipopeptides with molecular masses estimated by 1080, 1486 and 1044 Da, corresponding to the antifungal lipopeptides iturin A, fengycin and surfactin A, respectively [26] . Caldeira et al. (2011) reported that Bacillus amyloliquefaciens CCMI1051 exhibit high levels of antagonistic properties against filamentous fungi duo to the production of compounds of masses between 1000 and 1100 Da, comparable to that of iturin and surfactin compounds between 1436 and 1478 Da, compatibles with fengycin [17] .

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 [9] (m/z 1031, 1045 and 1463, 1477) (Figures 3(a)-(d)). Several isoforms exist for each li-

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 [27] .