# Biodeterioration of Salon de Reinos, Museo Nacional del Prado, Madrid, Spain
## 3 Results and Discussion
### Cleaning and Biocide Treatment  
When treating a biodeterioration, two different aspects must be taken into account: the elimination of the existing microorganisms and the prevention of new colonization. The elimination of the effects of a biological attack is a delicate problem. If the growth is superficial, a mechanical cleaning and a soft biocide treatment should be effective. If the attack affected deep layers of the mural paintings, the fungal hyphae will be difficult to eliminate without affecting the painting, and for these situations an appropriate biocide is recommended. The ideal biocide should have the following characteristics: broad spectrum of action at low concentrations, stability in solution, low vapor pressure to reduce evaporation, stability over time, and physical and chemical neutrality against the components of paints without action on the colors and texture of the paint. One of the biocides most used by restoreers and conservators in Italy [56; 57; 58] and also tested in Spain [59; 60] is Preventol RI 80, based on a quaternary ammonium salt. It is active against a wide variety of bacteria, fungi, algae, and lichens on stones and building materials [57; 59; 61; 62; 63]. Nevertheless, quaternary ammonium salts have shown, in a few cases, to be unsuitable, such as in subterranean environments (caves with rock art paintings) with high and constant relative humidity (above 90%) [64; 65]. However, these conditions are not present in the _Salon de Reinos_. For the prevention of new biological attacks, an environmental control of the hall is needed. The main causes that favored the biodeterioration in the _Salon de Reinos_ were the existence of fissures in the walls and water filtration that provoked the microbial growth. The restoration to be carried out will circumvent these problems. Considering the variations due to the different compositions of the materials, the ideal conditions in which works of art (including wall paintings) should be preserved are a relative humidity of 40-65% and a temperature of 16-18 \({}^{\circ}\)C. These, or close, environmental  
Figure 3: Metabolic activities of isolates. (**A**) Gelatin hydrolysis by _Bacillus_ _panicheniformis_. (**B**) Lipolytic activity by _Pristia endophyta. (**C**) Casein hydrolysis by _Pristia argubhattai_. (**D**) Calcite solubilization by _Penicillium fuscoglacum_. conditions will prevent the growth and re-colonization of airborne microorganisms that can be deposited on the painted surfaces. Bacteria need relative humidity >95% and fungi need 75-95%. In certain cases, some xerophilic microorganisms can grow at lower relative humidity. For this reason, the humidity limit in the _Salon_ must never exceed values of 70% and the temperature must be maintained between 16 and 20 \({}^{\circ}\)C. If these conditions are maintained, the colonization and growth of microorganisms will be prevented. Once these conditions have been fixed, an additional precaution is the periodic cleaning of the painted surfaces (all this, of course, after the consolidation and restoration processes) to avoid the accumulation of dust particles and volatile and suspended organic matter as well as the deposition of airborne microorganisms. As reported by Agora, S.L., the microorganisms were eliminated using a protocol consisting of their mechanical removal with a natural bristle brush and the application of Preventol RI 80 at 0.5 % in demineralized water, which has a broad spectrum in low concentrations on bacteria, fungi, algae, and lichens. This biocide is effective because it interferes with the enzymatic processes of the cell and modifies the permeability of its membrane. However, the sensitivity to quaternary ammonium salts depends on the species tested [55]. The intervention at the _Salon de Reinos_ was focused mainly on the areas of superficial fungal colonies, showing black stains, and in specific areas of the lintels of three windows that were blinded on the south side of the hall. One of the treatments that wall paintings may require is fixing and consolidation.