Primary bioreceptivity of limestones from the mediterranean basin to phototrophic microorganisms

Dissertação apresentada para a obtenção do grau de Doutor em Conservação e Restauro pela Universidade Nova de Lisboa, Faculdade de Ciências e TecnologiaThe conservation of historic buildings and monuments from cultural heritage is a major issue in modern societies, both from an economical and cultural point of view. The wide distribution of stone monuments and lithic works of art, and their cultural, artistic and religious importance emphasise the general need to safeguard this praiseworthy cultural heritage. This PhD thesis arises from the growing national and international interest on the biodeterioration of stone cultural heritage as one of the most complex areas of stone conservation and restoration. This study aimed to evaluate the primary bioreceptivity of limestones widely used as building materials in European countries from the Mediterranean Basin. In the first instance, a review of the literature was achieved in order to compare and be acquainted with the most abundant cyanobacteria and green algae detected on stone monuments. Gloeocapsa, Phormidium and Chroococcus, among cyanobacteria, and Chlorella, Stichococcus and Chlorococcum, among chlorophyta, were the most widespread genera identified on outdoor stone monuments. Limestone and marble were the lithotypes presenting the greatest diversity of phototrophic microorganisms. In the second step, five green biofilms were collected from Orologio Tower in Martano (Italy), Santa Clara-a-Velha Monastery (Coimbra) and Ajuda National Palace(Lisbon), both in Portugal, and Seville and Granada Cathedrals from Spain. The biofilm samples were subsequently characterised by molecular biology techniques and cultivated under laboratory conditions. DNA-based molecular analysis of 16S rRNA gene fragments revealed that the biofilms from Orologio Tower and Santa Clara-a-Velha Monastery were dominated by the microalga Chlorella, whereas the cyanobacterium Chroococcidiopsis was the dominating genus from Ajuda National Palace. The biofilms from Seville and Granada Cathedrals (Spain) were both dominated by the cyanobacterium Pleurocapsa. DGGE analysis of the cultivated biofilms revealed a remarkable stability of the microbial components from the Coimbra biofilm. This multiple-species phototrophic culture was further used as inoculum for stone bioreceptivity experiments. Laboratory-based stone colonisations relied on the inoculation of five limestone types with the selected biofilm culture, incubation within a growth chamber and monitoring of photosynthetic biomass through different analytical approaches. Subsequently, the primary bioreceptivity of Ançã (CA) and Lioz (CL) limestones, San Cristobal (SC) and Escúzar (PF) stones and Lecce stone (PL) was determined, evaluating the relationship between stone intrinsic properties and photosynthetic growth. The results were statistically analysed by means of principal component analysis (PCA) and analysis of variance (ANOVA)in an attempt to determine their bioreceptivity to phototrophic microorganisms and to evaluate the direct relationships between stone bioreceptivity and petrophysical properties