# Black on White: Microbial Growth Darkens the External
## 5 Discussion  
The studied marbles consist of almost pure calcium carbonate; however, trace amounts of silicates and gypsum were revealed, in almost all samples, by the FT-IR analyses. The presence of gypsum is generally imputed to the sulphation process. This consists of the reaction of sulfuric acid, coming from the air pollutant SO\({}_{2}\), with the insoluble calcium carbonate of marble, which is consequently transformed into the sulfate dihydrate or gypsum (CaSO\({}_{4}\) * 2H\({}_{2}\)O). The process is considered one of the main causes of carbonic stone deterioration in urban environments and is known to be associated to the blackening of outdoor marble artworks (e.g., [52]). Since gypsum is soluble in water, it is usually washed away in rain-exposed areas, whereas in sheltered areas, its crystals form networks that entrap particles of dirt and airborne pollutants, such as carbonaceous particles, to form black crusts [24]. The Cathedral of Santa Maria del Fiore is exposed to the urban polluted air of Florence and, among pollutants, to SO\({}_{2}\). The annual average values of SO\({}_{2}\) emissions detected in Florence from 2007 to 2012 were around 1-2 \(\upmu\)g/m\({}^{3}\). Since 2014, the annual average values showed a reduction from 3 \(\upmu\)g/m\({}^{3}\) to 1 \(\upmu\)g/m\({}^{3}\) detected in 2019 [53] possibly due to the pedestrianization of this area since October 2009. Due to the long-term SO\({}_{2}\) exposure, the external marbles of the Cathedral display black crusts in several areas [3]; however, they are not present in the studied areas, as demonstrated by microscopic, mineralogical, and chemical investigations. The NW and SE study sites are rain-exposed, thus, soluble salts including gypsum are washed away; this is in agreement with the low amounts of gypsum found by FT-IR. The blackening of marble can thus be imputed to microbial colonization. The dark spots and patches on marble contain in fact dark colonies often present as multispecies aggregates mainly consisting of dematiaceous fungi and dark cyanobacteria, as shown by in situ (Figure 2) and ex situ microscopic observation of samples (Supplementary Figure S3). The presence of black fungi in these \begin{table}
\begin{tabular}{c c} \hline \hline
**Morphotype** & **Organisms with the Most Similar ITS Sequences** & **Similarity (\%)** \\ \hline SN & _Elaphocordycps_ sp. 4  & 98.85 \\ _SO_ & _Aureobasidium pullulans_ & 99.64 \\ _Sud P_ & _Aureobasidium pullulans_ & 99.82 \\ NT-S-1 & _Phaeosphareiopsis pseudoadagracoearum_ & 99.81 \\ _NT-S-5_ & _Dudlidomycps_ sp. & 99.45 \\
**NT-S-6** & _Cidasporium rantomcellum/C. pugue_ & 98.82 \\
**NT-S-7** & _Alternaria citri_ & 99.81 \\
**NT-S-8** & _Cidasporium sinuousum/C. tencllum/C. herbarum_ & 99.80 \\ NT-S-9 & _Paracontiolbrium havaitiense_ & 100 \\ NT-S-10 & _Coprimellus miscaeus_ & 99.69 \\ \hline \hline \end{tabular}  
* \(L\)_chartarum_ is the teleomorphic form, _P. chartarum_ is the anamorphic form. They were considered as a single species. 2 Some _Phoma_ species are the anamorphs and their telecomorphic forms are described in genera _Didymella_ and _Contibrrimen_. They were considered as a single species. 3 _Cellikolus_ is the teleomorphic form, _Bipolaris_ is the anamorphic form. They were considered as a single species. 4 _Dipredadium_ genus includes some asexual morphs of _Elaphocordycps_ species. They were considered as a single species.  
\end{table} 
Table 1: Cont. aggregates was further confirmed by the growth of a meristematic colony, strain M1, from a black spot. On the other hand, cultivation confirmed the presence of dematiaceous fungi and dark cyanobacteria. Even if dark patinas show different levels of development at the SE and NW sampling sites, they can be attributed to the same biological cause. Indeed, a complex multi-kingdom microbial community inhabits the SMFC marble. Cyanobacteria, green algae, and lichens have been detected and characterized by morphological criteria. The fungal community was deeper investigated through isolation and identification by molecular methods.