# Biological Control of Phototrophic Biofilms in a Show Cave: The Case of Nerja Cave
## 2 Materials and Methods  
Nerja Cave was discovered in 1959 and open to public visits in 1960. The cave attracts about 450,000 visitors per year due to its location in Costa del Sol, a major tourist area, at 50 km from Malaga, Spain. The cave has an extension of almost 5 km, but the tourist trail is restricted to one-third of the cave. The cave is protected as a site of cultural interest due to its rock art paintings [3]. In addition to the cave entrance, this cave has two sinkholes that connect with the outside. The natural lighting that penetrates through the two sinkholes and the electric lighting installed in the tourist trail have allowed the development of photosynthetic-based biofilms on cave speleothems and walls. Currently, the electric lighting system consists of a trail, which runs along a circular route of about 600 m, a scenic lighting, and an emergency system. Some biofilms coating the speleothems close to light sources show extensive detachments and/or rounded areas without phototrophic microorganisms. The biofilm samples were collected in November 2018 from two speleothems located in the so-called Tourist Galleries (Figure 1A). Unfortunately, sampling was restricted to a very limited number of samples due to the cataloging of the cave as a site of cultural interest. However, we believe that the data provided were representative of the cave microbiome and shed light on biofilms composition and trophic interactions. Microclimatic data registered in the Tourist Galleries showed that cave air temperature ranges from 19.8 to 18.1 \({}^{\circ}\)C with a mean value of 19.0 \({}^{\circ}\)C, the relative humidity ranges from 98.6 to 67.5% with a mean value of 89.4% and the air CO\({}_{2}\) ranges from 1313 to 360 ppm with a mean value of 679 ppm [3]. Both biofilms studied in this work were exposed to white low pressure commercial fluorescent lamps (Roblan, Casarrubios del Monte, Spain, model ES27, 18W with a color temperature of 4845 K and 1030 lm), located 1 m away from speleothems. All the samples were collected at the same time by carefully scraping the biofilms with sterile scalpels; samples were kept at -80 \({}^{\circ}\)C until processing. A total of six samples were collected from two speleothems displaying detachment patterns. In the sampling site 1 (Figure 1B), three areas were collected: with biofilms (1A), with and without biofilms (1B), and a zone of rounded detachments (1C). Sampling site 2 (Figure 1C) comprised: a biofilm sample (2A), an extensive detachment (2B), and the interface between the biofilm and the detached zone (2C). Genomic DNA was extracted from 250-500 mg of biofilms, using the FastDNA SPIN Kit for soil (MP Biomedicals, Illkirch, France) according to the manufacturer's instructions. The DNA concentrations, ranging from 6.0-104.0 ng/\(\mu\)L depending on each sample, were quantified by using a Qubit 2.0 fluorometer (Invitrogen, Carlsbad, CA, USA). The extracted DNA of all samples was analyzed by Next Generation Sequencing (NGS). We investigated the bacterial V3 and V4 regions of the 165 rRNA gene and V4 region of the 185 rRNA gene using Illumina MiSeq and 2 \(\times\) 250 paired-end sequencing, according to the Illumina metagenomic library preparation protocol used by Macrogen (Seoul, Korea). The primer sequences used in this study were 341F (CCTACGGGNGGCWGCAG) and 805R (GACTACHVGGGTATCTAATCC) for bacteria, Reuk454 FWD1 (CCAGCASCYGCGGTAATTCC) and V4r (ACTTTCGTTTGTAT) for eukaryotes [14]. Raw data were processed in QIIME 1.9.1. Quality control and trimming were performed using FASTQC ([[http://www.bioinformatics.babraham.ac.uk/projects/fastqc/](http://www.bioinformatics.babraham.ac.uk/projects/fastqc/)]([http://www.bioinformatics.babraham.ac.uk/projects/fastqc/](http://www.bioinformatics.babraham.ac.uk/projects/fastqc/))) and Trimmomatic (0.36 version), respectively. Paired-end reads were assembled using PEAR. Operational Taxonomic Units (OTUs) were clustered at 97% cutoff using UCLUST.