## 3 Results and Discussions
### Associated Heterotrophic Microorganisms  
The microbial load composed of heterotrophic bacteria in the collected biofilm was 5.55 \(\pm\) 0.6 (\(\times 10^{7}\) CFU/g of wet biofilm), 5.4 \(\pm\) 0.8 (\(10^{7}\) CFU/g of wet biofilm) and 3.6 \(\pm\) 0.3 (\(10^{5}\) CFU/g of wet biofilm) on TSA, MM and PDA mediums, respectively. Based on colony morphologies, ten bacterial strains were isolated and identified (Table 1), belonging to three main phyla: Proteobacteria (class Alpha and Gamma), Actinobacteria and Bacteroidetes.  
Only a few colonies of fungal strains were grown on PDA plates, but not yet identified.  
\begin{table}
\begin{tabular}{c c c c} \hline \hline
**Strain Name** & **Phylum** & **Species** & **ID \%** \\ \hline DA1 & Bacteroidetes & _Chriseobacterium plytrichastri_ & 98 \\ DA2 & Proteobacteria & _Enseifer adhaerens_ & 98 \\ DA3 & Proteobacteria & _Inquilius ginsengi_ & 96 \\ DA4 & Proteobacteria & _Pseudomonas aspeliiii_ & 97 \\ DA5 & Proteobacteria & _Pseudomonas glareae_ & 97 \\ DA6 & Proteobacteria & _Pseudomonas helmanticensis_ & 99 \\ DA7 & Proteobacteria & _Pseudomonas vancourevenensis_ & 99 \\ DA8 & Actinobacteria & _Rhodococcus jostii_ & 98 \\ DA9 & Actinobacteria & _Rhodocossus korensis_ & 98 \\ DA10 & Actinobacteria & _Streptomyces sporraveus_ & 99 \\ \hline \hline \end{tabular}
\end{table}
Table 1: Phylogenetic affiliation by 16S r-DNA sequencing of the bacterial strains co-occurring in the sampled biofilm. The strains have been named with the abbreviation DA followed by a progressive number.  
Figure 3: Biofilm sample taken from the wall of Room 93 and observed at the Confocal Laser Scanning Microscope (CLSM); the biofilm is dominated by _S. julianum_ (thick filaments) and _Leptolyngbya_ sp. (thin filaments). Scale bar = 40 \(\upmu\)m.  
The presence of both heterotrophic and phototrophic microorganisms within the biofilm suggests that they may be acting in synergy and could actually be causing the enhancement of hypogean biodeterioration processes. Bacteria and fungi can proliferate using the organic matter released by phototrophs and in turn secreting acidic organic compounds that can solubilize the minerals of the substratum, which are in turn utilised by the algal component [35]. The strain _Ensifer aldaerens_ DA2 participate as a nitrogen-fixing agent.  
In Roman Catacombs Actinobacteria (especially _Streptomyces_) and filamentous fungi (e.g., _Sporotrichum, Aspergillus, Cladosporium, Penicillin_, etc.) are commonly detected together with photosynthetic microbes [36]. They are widely distributed on stone monuments because of their filamentous growth and their ability to use a large range of nitrogen and carbon sources [37]. Moreover, in this study we have isolated some slime-forming bacteria, belonging to the genus _Pseudomonas_, already described in surface samples of the Roman Catacombs of St. Calliktus and Domitilla [37; 38]. Slime-forming bacteria play an essential role in biofilm establishment, producing extracellular complex carbohydrates which ensure adhesion of microbial cells to surfaces, provide protection from environmental antimicrobials, serve as reservoirs for nutrients and create distinct architectures to facilitate further microbial adherence [39; 40]. Members of Alpha Proteobacteria and Bacteroidetes were frequently found as heterotrophic components of biofilms affecting stone monuments [41; 42].