# Cleaning of Phototrophic Biofilms in a Show Cave: The Case of Tesoro Cave, Spain
## 2 Materials and Methods  
Phototrophic biofilm samples were scraped off with a sterile scalpel, placed into 1.5 mL sterile Eppendorf tubes and stored at 4 \({}^{\circ}\)C until arrival at the laboratory. Taxonomic identifications were based on the morphological characters of the whole biofilm and on colonies grown on 15% agar plates with medium BG11 (Sigma-Aldrich, Steinheim, Germany) or Bold's Basal Medium (BBM) (Sigma-Aldrich, Steinheim, Germany) and incubated at room temperature and a low light intensity (semi-closed window oriented to the north) [17; 18]. The identification of the different microalgae and cyanobacteria was based on the available taxonomic keys and confirmed in AlgaeBase [19; 20; 21; 22; 23; 24]. Molecular biology techniques were applied  
Figure 1: Map of Tesoro Cave. T1–T3 represent the three sectors into which the cave was divided for study purposes. for the identification of _Cyanidium_ sp. DNA was extracted using the FastDNASPIN for Soil Kit (MP Biomedicals, Illkirch, France). The amplification of 16S gene sequences was performed using the cyanobacteria-specific primer pair, Cya106F (5\({}^{\prime}\)-CGGACGGTGGAGAACGGTG-3\({}^{\prime}\)) and Cya 781R (5\({}^{\prime}\)-GACTACTGGGGTATCTAATCCWTT-3\({}^{\prime}\)) [25]. The PCR amplification protocol consisted of the following thermal conditions: 95 \({}^{\circ}\)C for 2 min; 35 cycles of 95 \({}^{\circ}\)C for 15 s, 60 \({}^{\circ}\)C for 15 s, 72 \({}^{\circ}\)C for 2 min; and a final step of 72 \({}^{\circ}\)C for 10 min. DNA libraries of PCR-amplified products were constructed using the TOPO-TA cloning kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's recommendations. Plasmids were extracted with the JetQuick Plasmid Miniprep Spin kit (Genomed, Lohne, Germany), following the manufacturer's protocol, and sequenced by Secugen Sequencing Services (CSIC, Madrid, Spain). The identification of phylogenetic neighbors was determined using the BLASTN algorithm [26]. The 16S rRNA gene sequences of clones and its closest related sequences were multiplied and aligned using CLUSTAL W [27]. The phylogenetic tree was constructed using the maximum-likelihood method [28] and [PERSON]'s two-parameter model with a discrete Gamma distribution in MEGA version 11 [29]. A bootstrap analysis of 1000 re-samplings was used to evaluate the robustness of the tree. All sequences were submitted to GenBank with consecutive accession numbers ON619559-ON619566. The direct observation of phototrophic biofilms and isolate colonies from enrichment cultures was carried out using an Axioplan microscope (Carl Zeiss, Oberkochen, Germany) and the images captured with an AxioCam MRc5 digital camera and processed with Axiolan LE software and a scanning electron microscope (Quanta 200 FEI \(\Phi\) EDAX) [13; 14]. The samples were subjected to pre-fixation with acrolein vapor and post-fixation with osmium tetroxide vapor. The samples were double coated with carbon and sputtered gold. Several treatments were tested for biofilm removal and cleaning. The treatments applied for cleaning the whole cave included mechanical removal by brush and cleaning with liquid nitrogen, with hydrogen peroxide and/or sodium hypochlorite. Physicochemical cleaning was a two-phase treatment: the first physical and the second chemical. The first consisted of the mechanical removal of the thickest layers of biofilms using a spatula and a brush. This treatment was conditioned by the type of biofilm and the rock surface. In fact, this was applied to dense biofilms showing abundance of hyrophytes and/or algae that could easily be removed from the rock surfaces. The application was never done dry, but rather by means of previous hydration of the surfaces. Once finished, a chemical treatment was applied, which was conditioned by the typology of the surface (solid rock surfaces), the type of biofilms (residual algae and/or hyrophyte protonema not removed by brushing) and the environmental conditions (dry or wet surfaces). The products used were as follows.