Killing them gently; control of phototrophic biofilms growing on stone monuments using plant products.

Although lithic archaeological sites and confined environments are relatively harsh environments for microorganisms to inhabit, they are still subjected to biodeterioration by microorganisms able to attack rocks and give rise to a microbial succession (cyanobacteria, bacteria, microalgae, mosses and fungi). These organisms can survive and even grow on minimal amounts of light, water and nutrients, mainly supplied by the substrate and unwitting visitors. Both structural and aesthetic damage to these valuable surfaces occur as a result of physical and chemical processes governed by the biofilm, which result in discoloration and material impairment. Standard physical and chemical treatments to remove biofilms can themselves cause surface damage as well as being health hazard. Moreover, the employment of chemical biocides seems to be selective for resistant microorganisms. Therefore, more sustainable and safer practices are being sought. Rome, 64 AD, Emperor Nero ordered the reconstruction of his residence, with the Domus Aurea forming part of the project to transform Rome into a new city. The main part of the building was located on the Palatine and Oppian Hills and was famous for its sumptuous decoration in which gold and precious stone coverings were added to the stuccos, paintings and coloured marbles, while mosaics, normally restricted to floors were also placed in some of the vaulted ceilings. Nero’s successors were embarrassed of the Domus’ opulence and so stripped it of its marble, its jewels and its ivory within a decade. Within 40 years, the Domus Aurea was completely obliterated and filled in with soil. Surprisingly, this in-filling ensured the survival of the wall paintings by protecting them from damp. After 20 years of excavation and restoration it was opened to the public in 1999, only to close six years later due to detachments and security problems. The doors opened again to the tourist in February of 2007 with restricted visitation. However, due to artificial lighting and high humidity levels many surfaces have been heavily infested by both heterotrophic and phototrophic biofilms. So now, among the main restoration efforts, the disinfection of biodeteriogens growing on the walls and ceilings has become fundamental. Data on the biocidal efficacy of some plant-based products is showing great promise, so it was decided to carry out tests on biofilms sampled at the Domus Aurea using the essential oil from Lavandula angustifolia, alcohol extracts from Glycyrrhizza glabra leaves (Trifolio-M GmbH) and Capsicum sp, singularly or mixed. Biofilm samples were collected in ‘room 93’ from an undecorated wall and were then homogenised and inoculated on agarized BG11 growth medium. Observations showed that the cyanobacterium Scytonema julianum was the dominant species, and has often been described from other hypogean environments, such as Roman Catacombs, and known to deteriorate substrate integrity by dissolution of minerals from the substrate and the precipitation of calcium carbonate on its sheaths. Identification of bacteria by r-DNA16S sequencing revealed the presence of Proteobacteria (6 spp.), Actinobacteria (2 spp.) and Bacteroidetes (1 sp.). Three fungal strains were also isolated and are to be identified. The biofilms were treated twice with the extracts on day 1 and 5, and the photosynthetic response of the biofilm was followed for five days with a mini-PAM portable fluorometer. Photosynthesis is highly susceptible to this kind of treatment, so measurements of rates were used as a proxy for cell health. Changes in photosynthetic activity of the samples treated with the extracts were compared to control biofilms receiving no treatment. Results showed that the essential oil of L. angustifolia and G. glabra leaves extract at 30% had the highest photosynthesis inhibition potential, followed by G. glabra extract 10%. Capsicum extract was the least efficient. These first results are encouraging enough to continue beyond the laboratory trials in the hope to realise an eco-friendly, non-toxic and sustainable strategy for the conservation of lithic cultural heritage