Annals of RSCB

Vol. XVII, Issue 2/2012

PRELIMINARY ANTIFUNGAL INVESTIGATION OF TEN BIOCIDES AGAINST MOULDS FROM TWO DIFFERENT CHURCH FRESCOES Daniela Maxim1, Livia Buca2, Maria Iasmina Moza3, Oana Chachula4
DEPARTMENT OF BIOLOGY, FACULTY OF BIOLOGY,ALEXANDRU IOAN CUZA UNIVERSITY OF IASI, ROMANIA 2 DEPARTMENT OF HISTORY AND PATRIMONY, FACULTY OF HUMAN SCIENCE,LUCIAN BLAGAUNIVERSITY OF SIBIU, ROMANIA 3 DEPARTMENT OF ECOLOGY AND ENVIRONMENTAL PROTECTION, FACULTY OF SCIENCES,LUCIAN BLAGAUNIVERSITY OF SIBIU, ROMANIA 4 NATIONAL MUSEUM OF ROMANIAN HISTORY - NATIONAL CENTRE FOR SCIENTIFIC INVESTIGATION, BUCHAREST, ROMANIA
1

Summary
In this paper, ten biocides: Biotin T, Algophase, C3, P6, Cinnamaldehyde, C2, P2, Preventol, New Des and Capsaicin were investigated for their antifungal activity against some moulds isolated from frescoes of two churches. The tests were conducted in vitro and the inhibitory activity (IA) of biocides was established using the diffusion method. Results indicated that the antifungal activity of biocides is different, depending on moulds type and concentration of each substance used. Biotin T, Algophase and C3 appeared to be the most efficient. Capsaicin seems to have no action on moulds in this case. Keywords: biocides, frescoes, moulds, antifungal activity, biodeterioration

iasmina_moza@yahoo.com

Introduction
The moulds degradable action is very important for the biological cycles of elements (Zarnea, 1970; Tnase and Mititiuc, 2001), but the same action often have a negative role in the deterioration of cultural property and objects (Eggins and Oxley, 2001; Caneva et al., 1991; Tiano, 1999; Ripka et al., 2006). In the context of sustainable development of society, limiting the phenomenon of biodegradation of old buildings and monuments is a continuous challenge (Velea et al., 2011). Problems associated with fungal growth on structures refer to the aesthetic deterioration and potential degradation of the material (Allsopp and Seal, 1986). It was demonstrated that the preference of microorganism for some category of substrate is related to their susceptibility (Guillitte and Dreesen, 1995; Guilliette, 1995; Prieto and Silva, 2005; Miller et al., 2006; Cmara et al., 2008). Frescoes, in special condition, may become a substrate with bioreceptivity for micromicetes (Giacobini et al., 1988). In the past few years, studies reveal that in Romania, fungal attacks appear when exist improper conditions of maintenance, humidity variation and unfit windows for the churches (Buca, 2010; Moza et al, 2012a). Currently scientist use several methods of chemical treatment, physical, mechanical or biological, in such cases, but most of these treatments attack the mineral substrate, pollutes the environment, being effective only in the short term (Velea et al., 2011). The literature recommends also the use of gamma radiation or laser beams to destroy the fungi even though, in some

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cases, they synthesize melanin pigments with aprotective role against radiations (Radvan et al., 2002). In another studies, UV radiation was used for the inactivation of Penicillium spores (Da Silva, 2006; Iseki et al., 2010; Moza et al., 2012b). A modern solution to combat the biodeterioration is to use essential oils as biocides as an environmental alternative (Mironescu et al., 2008, 2009, 2010a, b, c). The use of biocides to control potential microbial contamination is widespread. In some cases, treatment to prevent decay is the only option. However, biocides, by definition, are toxic, and must be used with great care in occupied environments (Burge et al., 1994). Many products are now on the market and the range for biocide is extended in our days; their inhibitory activity is against one or more microorganism, depends of biocide type (Popescu, 2010). Two main categories of biocides are known: those applied before the treatments to eliminate microorganisms

already present, and those that should have a preventive effect that slow down the recolonization of restored surfaces (Urzi et De Leo, 2007). Also, in the field of biocides treatments, even if the results are positive, a constant monitoring is required and preventive methods must be applied to stop the biological attacks (Boldura, 2007). In this paper, the antifungal effect of ten biocides: Biotin T (B), P6, Algophase (AL), C3, Cinnamaldehyde (CI) , C2, P2, Preventol (P) , New Des (ND) and Capsaicin (CS) were investigated against some moulds isolated from mural paintings of two churches from Buzu County: Bbeni and Drgheti. Biocide BiotinT was chosen to be blank for the most powerfull chemical biocide used in restoration and C2, C3 and P2 are biocides that contain plant natural extracts whit antibacterial activity. The study was conducted in vitro and the inhibitory activity (IA) of biocides was determined using the diffusion method.

Material and methods

Several types of fungi were isolated from the two frescoes using microbiological techniques. All the isolated fungi were harvested on Sabouraud medium and incubated at 20 degrees. After 5 days, the colonial appearance was analysed and the microscopic investigation was done (data not shown) in order to indentify the genus that grown on the frescoes from each church apart (table 1). Fungal spores were obtained after incubation for 10 days, followed by collecting the spore in sterile distilled water. From the spore suspension, in each Petri dish, was measured 0.5 ml of sample and MMA was poured over. After the medium solidification, small round paper discs were impregnated with 5l of each biocide and distributed directly on the Petri dishes cultivated with spores. Inhibitory activity of 140 biocide was determinate using the diffusion method, similar with KirbyBauer method, usually used to determine the antibacterial response of different antibiotics (Boyle et al., 1973). The same method was used previously by Mironescu and his collaborators in 2008, 2009 and 2010a.
Table 1.The fungi category find on two types of frescoes investigated. Genus Rhizopus Aspergillus Penicillium Cladosporium Alternaria Fusarium Bbeni Drgheti frescoes frescoes + + + + + + + + + + +

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Solutions of the following biocides: Biotin T, Algophase, C3, P6, Cinnamaldehyde, C2, P2, Preventol, New Des and Capsaicin were diluted in sterile distilled water and for the experiment concentration of 3% and 5 % were used. Only in the case of Capsaicin the concentration was 0.03% and 0.07 %. All the biocides was described in previous research (De Marino et al., 2008;

Balzarotti-Kmmlein, 1999; Mironescu and Georgescu 2010c, Cuzman et al., 2011). Growth was monitored for a period of four months. Minimal inhibitory concentration (MIC) values were measured first, after two weeks and second after four months. After measurement the MIC values were compared to determinate the remanence of each biocide. colonies were developed to their fullest potential. If we refer to remanence efficiency of biocide this values were cosidered to be 100%. Figure 2 reveal the MIC values in case of moulds isolated from Bbeni frescoes; the maximum inhibitory activity is reached by Biotin (B) at the concentration of 3% because, according to the prospectus, the biocide acting at concentrations of 2-5 percent. In this case we have a maximum inhibitory activity of 70% for 3% concentration of substance and 47.5% for the concentration of 5%. AL and C3 have almost the same action here, the inhibition capacity being 35% and respectively 32.5 % for 3% concentration, 40% at the concentration of 5% in the case of AL biocide and 45% at the same concentration for the C3 biocide. Alike, for P6 and CI biocides the IA is also similar: 27.5% and 25% for the minimal concentration and 35% for both biocides at the concentration of 5%. The next inhibitory activity is represent by C2 and P2; at the concentration of 3% their values is 20% and 17.5 % and for the maximum concentration of 5% is 22.5% in case of C2 and 20% in case of P2. For this type of moulds the P is more intense then ND: at the concentration of 3% for P we have 12.5% activity and for ND only 10%, and at maximum concentration for P we have 17.5% and only 15% for ND. The last biocide is the Cs with no inhibition activity in this concentration (0.03% and 0.07%).

Results and discussions

Petri dishes from figure1 present images of the action of ten biocides use in this research. The inhibitory activity is different for each type of fresco and also the substance used is important for treatment against mould attack on painted surface. Also is very well expose the Petri dishes contains the treated with biocides and the dishes contains simple spore represent the blanks samples (figure 1, BLB and BLD) from two type of frescoes. The inhibition areas are clear observed on colonies formed by spores (figure 1, B, P6, ND, AL, P, CS, CI, C2, C3, P2); some type of mould development a protective structures against biocide action (figure 1, B, P6, AL, CI, C2, P2). In the case of Bbeni moulds, a yellow pigmentation appear in all sample; this phenomenon is not present in Drgheti moulds, but here a strong pink pigmentation appear due to species of the Fusarium genus. From these images (figure 1), can be seen easily that the biocides action is different, depending on the moulds type used in this experiment. Similar results for C2 and C3 were obtained with other fungi tested in a recent research (Mironescu et al., 2010a). The antifungal efficiency of biocides tested is expressed as the inhibition zone around each paper disc. In figure 2 and 3 are presented the values of MIC for each type of frescoes apart. After two weeks incubation period, IA is considered the most higher at this time because fungal 141

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Vol. XVII, Issue 2/2012

BLB

BLD

CS

P6

CI

ND

C3

AL

C2

P2

Figure 1.Influence of the treatment with biocide on moulds from each type of frescoes. Legend: BLB- blank from Bbeni frescoes, BLD- blank from Drgheti frescoes, BBiotin, D- New Des, AL- Algophase, P- Preventol, CS-capsaicin, CI- cinnamaldehyde

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120 110 100 90 80 70 60 50 40 30 20 10 0 BL B P6 ND Al P CS CI C3 C2 P2 conc. 5% conc. 3%

Figure 2.IA values in case of Bbeni Church after two weeks

120 110 100 90 80 70 60 50 40 30 20 10 0 BL B P6 ND Al P CS CI C3 C2 P2

conc. 5% conc. 3%

Figure 3.IA values in case of Drgheti Church after two weeks

*For Capsaicin the concentration is 0.03% and 0.05 %

In the Drgheti church case (figure 3), the MIC values are very different. Here the maximum inhibitory activity is reach by C3 biocide with 57.5% for the maximal concentration and 50% when the substance is use in 3% concentration. Next, B and CI have almost the same inhibitory activity: for the conc. of 3% the value is 42.5% in the case of B and 32.5% for CI, and at the 5% concentration IA is 37.5% for B and 47.5% for CI. AL inhibitory activity is the same in this case too, the values for minimal concentration used are 35% and for 5% concentration are 42.5%, more powerful that in previously case studied. For P and C2 biocide, the IA is almost similar: for the concentration of 3% the values are 27.5% 27

and 22.5% and for the maximum concentration used, values are the same for both biocides: 30%. For ND the values are higher than in previous case; their values for IA are17.5 for 3% concentration and 27.5% for 5% concentration. For the other tree biocides, P6, CS and P2 all values are 0%, so in this case they dont present an IA at all. The IA was monitored a period of four months for determination of the after-

effect of biocides used in vitro. The comparative values of MIC for moulds isolated from each type of frescoes are present in figure 4.

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After this period of time, only some of the used biocides have kept their IA. These results are valid for both types of frescoes from where mould was isolated; B biocide maintained his action power with 15% lower after four mounts in the case of Bbeni church and the IA remain almost the same with the first church in the case of Drgheti (with 12.5% lower). For ND biocide, the IA values are with 5% lower for Bbeni church and with 12.5 percent lower in the case of Drgheti church. P is

the only biocide whose remanence decreased whit only few percent even after four mounts of action; the IA values decreased with 5% for the both churches. For C2 percent difference is 2.5 for the Bbeni church and 10% for the Drgheti church. Finaly for P2 the percent difference is 7.5 for the Bbeni church and zero in the case of Drgheti church because here was not any action from the start. For others types of biocides the remanence after four mounts does not exist.

Bbeni

Drghe ti

55

30 10 0 0 0 0 15 12.5 0 0

25 20 20 12.5 0 0 0 0 0 0 0

BL

P6

ND

Al

CS

CI

C3

C2

P2

Figure 4.The comparative values for inhibitory activity (IA) against the mould isolated from the frescoes studied (%).

Based on these results we can deduce that the IA remanence in the case of Drgheti church decreases more drastically than in case of Bbeni church. Seems that moulds from here, are different from Bbeni church, even if they belong to the same genus, or their combination can affect the activity of any type of biocide

against them. This microorganism can develop different defense strategy and their resistance to fungal agents is highly subjective depending on the type of biocide used, its concentration and time of action of biocides and last but not least, on the type of mould or combinations of moulds thereof. tests were conducted in vitro and the inhibitory activity (IA) of biocides was established using the diffusion method. Results indicated that the antifungal activity of biocides is different, depending on moulds type and concentration of each substance used. In the case of Bbeni moulds, a yellow pigmentation appear in 28

Conclusions
In this research, ten biocides: Biotin T, Algophase, C3, P6, Cinnamaldehyde, C2, P2, Preventol, New Des and Capsaicin were investigated for their antifungal activity against some moulds isolated from mural paintings of two churches from Buzu County. The

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Vol. XVII, Issue 2/2012

all sample; this phenomenon is not present in Drgheti moulds, but here a strong pink pigmentation appear due to species of the Fusarium genus. After two weeks of action the power of all biocides is: B>C3>AL>CI>>C2>P>ND>P6>P2>CS for the both frescoes studied. In the case of Drgheti church, was much stronger then Biotin T biocide. The IA was monitored a period of four months for determination the after-effect of biocides used in vitro. After the four months period the order of Acknowledgements Authors transmit their special gratitude and thanks to Dr. Eng. Monica Mironescu which allowed testing the biocides in the Microbiology Laboratory Faculty of Agricultural Sciences, Food Industry and Environmental Protection, Lucian Blaga University of Sibiu,

the power action of biocides who keep their remanence is: P>C2>ND>B>P2. Preventol is the only biocide whose remanence is constant modified regardless of the type of mould tested; also C2 have a strong remanence in both cases studied. In conclusion is essential to identify all the genus of fungi species isolates from the frescoes before restoration and, if is possible to apply the treatment for every species apart to find the better solution to stop this type of attack. Romania from Food Biotechnology Department and to Dr. Physician Gheorghe Niculescu, director of National Centre for Scientific Investigation from Bucharest for all the support.

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