# Black on White: Microbial Growth Darkens the External
## Materials and Methods
### Cultivation, Morphological Analysis and Identification of Microorganisms and Lichens  
To cultivate green algae and cyanobacteria, aliquots (10 mg) of the collected marble powder were immersed in the liquid nutrient medium BG-11 prepared according to [PERSON] et al. (2014) and adjusted only with 5 mL/L NaNO\({}_{3}\) instead of 15 mL in order to allow the growing of nitrogen fixing cyanobacteria, as well. After 2 months, the main phototrophic biodiversity was observed under a Zeiss Axio Scope A1 microscope (Zeiss, Oberkochen, Germany) equipped with a Zeiss Axio Cam ICc3 (Zeiss, Oberkochen, Germany), and the morphological characterization was executed according to [PERSON] et al. (2015) and [PERSON] (2016).  
In a previous work [3], we performed various lab tests to find suitable conditions for a high recovery of fungal colonies from SMFC marble. Such conditions will be applied in future evaluations of biocide treatments. Among the nutrient media used, Malt Extract Agar (MEA) gave the best results, with a viable titer up to \(1.5\times 10^{4}\) CFU/g at the NW site and an apparently high biodiversity. MEA was also modified by adding 0.1% and 1% marble powder, and 1% aqueous marble extract and no significant differences were observed in viable titer [3]. Consequently, we used MEA at the subsequent sampling. To cultivate fungi, 10 mg of marble particulate, obtained by mixing three samplings from each area, were suspended in 1 mL of Phosphate Buffered Saline (PBS; 8 g/L NaCl, 0.2 g/L KCl, 1.44 g/L Na\({}_{2}\)HPO\({}_{4}\), g/L 0.24 KH\({}_{2}\)PO\({}_{4}\), pH 7.4) added with 0.001% Tween 80 and vortexed. Then, 0.1 mL of suspension were plated in quadruplicate on MEA (Oxoid) supplemented with 10 \(\upmu\)g/mL of chloramphenicol to prevent bacterial growth and then incubated at 30 \({}^{\circ}\)C for at least seven days. Viable titer was calculated as mean value of the number of Colony Formant Units (CFUs) per gram of marble particulate. Morphological analysis of colonies was conducted by observing them under the stereomicroscope Olympus SZX9 (Olympus, Tokyo, Japan). Colonies with the same morphological characteristics were grouped in the same morphotype. For each morphotype, at least one strain was re-isolated on MEA and then stored on slant. Identification of fungal morphotypes was based on rDNA analysis. Genomic DNA of isolated strains was extracted using Wizard(r) Genomic DNA Purification kit (Promega, Madison, WI, USA) following the yeast protocol and modifying the cell lysis step as follows: about 1 cm\({}^{2}\) mycelium from an isolated colony was cut, suspended in 500 \(\upmu\)L physiological solution, and centrifuged. The pellet was then suspended in 47.5 mM EDTA + 0.5 mg/mL zymolyase (Sunrise Science Products, Knoxville, TN, USA) and incubated at 37 \({}^{\circ}\)C for 45 min. Amplification of the internal transcribed spacer (ITS) region of rRNAgenes was performed using primers ITS1D (5'-GTTTTCCGTAGGTGAACCTGC-3') and ITS4 (5'-TCCTCCGCTTATTGTATGT-3'), and PCRBIO Taq Polymerase (PCRBioSystem, London, UK). PCR conditions consisted of an initial denaturation step at 95 \({}^{\circ}\)C for 2 min followed by 30 cycles at 95 \({}^{\circ}\)C for 30 s, 60 \({}^{\circ}\)C for 1 min, 72 \({}^{\circ}\)C for 1 min, and a final extension at 72 \({}^{\circ}\)C for 5 min. The Sanger sequencing was performed by Bio-Fab Research s.r.l. (Rome, Italy). To identify the isolates, the nucleotide sequences were analyzed by BLAST (Basic Local Alignment Search Tool) using the National Center of Biotechnology Information (NCBI) database [47]. Newly generated ITS rDNA sequences were deposited at the NCBI database under the accession numbers from MW361278 to MW361325.  
Lichens were identified directly in situ using the portable stereomicroscope. The identification key of [PERSON] et al. [48] was used as a reference. Nomenclature follows Nimis [49].