A polyphasic approach to the study of EPS-producing heterocystous cyanobacteria from biofilms in Roman hypogean monuments

Roman hypogea (e.g. the Roman catacombs) are man-made low-light environments of great archaeological and cultural interest. Few species of cyanobacteria are able to colonise the lithic surfaces inside the catacombs, where they live in close vicinity to the artificial lamps associated with green algae, diatoms and bacteria in the life-form of biofilms. Biofilm formation is due to the production of an exopolymeric matrix mostly composed of exopolysaccharides and it is well known that these polymers are involved in the aesthetic and structural alteration of the properties of artistic surfaces (biodeterioration), particularly when rich in negatively charged groups. Based on this information, the aim of the present work was firstly the identification of the cyanobacterial species involved in biodeterioration process and dominating the biofilms collected in Roman hypogea, and secondly the characterisation of the exopolysaccharides that they secrete to form the mucous matrix of the biofilm. For the isolation of new cyanobacterial strains, selected sites of the Roman catacombs of St. Callistus and Domitilla were chosen for biofilm collection and recording of physico-chemical parameters. Taxonomical identification of five heterocystous cyanobacterial strains previously isolated (Scytonema julianum CP6, Scytonema julianum CSC7-21, Scytonema ocellatum CP8-2, Fischerella sp. CSC9d* and Fischerella maior NAV10bis) was done using a “polyphasic approach”, combining cytomorphological observations at light and transmission electron microscopy and molecular phylogeny based on the 16S rRNA gene analysis. Exopolysaccharides (EPS) from different heterocystous cyanobacterial strains were characterised using different methods: i) cytochemical techniques to evidence the different distribution of EPS; ii) testing different exopolysaccharide extraction protocols; iii) CD (circular dichroism) and RP-HPLC on EPS to characterised their physico-chemical and biochemical properties; iv) quantitative analysis to determine the influence of different light conditions and incubation period on EPS production. Twenty cyanobacterial taxa were isolated and are maintained in controlled conditions of light, temperatures and humidity and will be added to the “Tor Vergata” culture collection being available for further cytomorphological, ecophysiological and molecular studies. Cytomorphological and cytochemical studies were useful to determine the diacritical features of the genera. Moreover, the two species Scytonema julianum and S. ocellatum could be distinguished on the ground of cytomorphological features and molecular tools based on sequence similarities of the 16S rRNA genes confirmed this assignment. The same investigations done on the five isolates showed that all the strains but CP6 and CSC7-21 belong to different species. The two phylogenetic trees (maximum parsimony and neighbour joining) highlighted that: i) the two strains CP6 and CSC7-21 clustered with other terrestrial Scytonema species; ii) Fischerella maior NAV10bis and Scytonema ocellatum CP8-2 clustered together and shared a percent sequence similarity over 95%, suggesting that they possibly belong to the same genus, not yet defined; iii) the two strains of Fischerella belong to different species and possibly to different genera, sharing a low percent sequence similarity (90%). Thus, the assignment of these strains to the genus Fischerella needs a revision. This conclusion could not be drawn only on the ground of cytomorphological studies. The ultrastructural and cytochemical studies showed in the strains CP8-2 and NAV10bis the presence of a bi-stratified sheath, in both species rich in negative charges (sulphated and carboxylic groups). The protocol for EPS extraction using sulphuric acid 0.5 M was quantitatively the most effective, and caused the complete removal of the sheath. CD spectra on exopolysaccharides from the studied strains did not show any conformational transition by varying pH and temperatures, concluding that the polymers follow a “random coil” model, as shown for similar macromolecules. The highest production of EPS under different cultural conditions was obtained from the 8 month old cultures under high irradiances of the strains S. ocellatum CP8 and Fischerella sp. CSC9d*. Moreover, both uronic acids (galacturonic and glucuronic) were produced in each 8 month old culture grown under high irradiance, confirming that these groups in EPS increase with incubation period and metabolic stress. Since the presence of uronic acids is detrimental for the lithic surfaces because of their ability to scavenge cations from the substratum, the most damaging species among those analysed are the Fischerella strains, being the best producer of these negatively charged groups. In conclusion, this study improves the knowledge on poorly studied cyanobacterial strains from archaeological underground sites by increasing the “Tor Vergata” culture collection with twenty new isolates and using the polyphasic approach to describe five heterocystous cyanobacterial strains. This approach is into line with current trends on the taxonomy of cyanobacteria. Moreover, useful information for the development of new strategies for the control and prevention of the biodeterioration were obtained. In fact, the quantification of EPS secretion (in particular with regard to the uronic acid) and the characterisation of the climatic conditions that sustain the over-production of these macromolecules can be useful to identify the most damaging cyanobacterial taxa. To this purpose, the new, effective extraction protocol tested will facilitate the complete removal of exopolysaccharides from the little biomass available, being the cyanobacterial biofilms from catacombs usually very thin.