# Effects of Microbiota on the Soiling Process of PV Modules in Arid Zones of the Atacama Desert
## 2 Materials and Methods
### Microbiological Characterization of soiling  
For the microbiological characterization of the soiling, three culture media were prepared: Luria-Bertani (LB), Reasoner's 2A agar (R2A), and a low-phosphate Tris-buffered mineral salt (LPTMS) medium, following the recommendations of [PERSON] et al. [10] and [PERSON] et al. [17]. For the isolation of the bacterial strains, a sample of 1 g of soiling collected from the UA and PSDA modules was introduced into sterile 50 mL plastic tubes. Following this, 10 mL of sterile solution of 0.85% NaCl in MilliQ water (18 M\(\Omega\)-cm) was added to each tube and they were agitated in a Biobase shaker model BJI-X-103B for two hours at 230 rpm and at a temperature of 37 \({}^{\circ}\)C. Subsequently, 100 \(\upmu\)L of the supernatant from each tube was extracted with a micropipette and serially diluted from \(10^{-1}\) to \(10^{-6}\) in 0.85% NaCl, and then stored in Eppendorf tubes. A total of 100 \(\upmu\)L of each serial dilution was used to inoculate the respective solid media supplemented with cycloheximide (0.05 g/L) as a fungal growth inhibitor. Finally, the plates were incubated at 30 \({}^{\circ}\)C for 96 h, after which the colony-forming units (CFU) were quantified to determine the microbial density. The colonies grown on different media were selected based on their pigmentation, texture, size, and shape. These isolates were grown on LB-Agar medium, as it yielded the greatest diversity of colonies. They were then incubated in an oven at a constant temperature of 30 \({}^{\circ}\)C for a period of 48 to 96 h. The isolates were individually grown in a liquid medium, preserved in 25% glycerol in 1.5 mL cryotubes, and stored in a Haier freezer at \(-80\)\({}^{\circ}\)C. In total, 34 bacterial strains were isolated: 24 from the UA and 9 from the PSDA. The DNA extraction from the cultivable isolates was performed from the colonies of bacteria grown on LB solid medium using the DNeasy(r) UltraClean(r) Microbial Kit (Qiagen, Inc., Germantown, MD, USA), following the manufacturer's instructions. The metagenomic DNA from the soiling collected from the UA and PSDA was extracted using the DNeasy(r) PowerSoil(r) Kit (Qiagen, Inc.). For this, 0.7 g and 2.3 g of soiling samples from the UA and PSDA were used, respectively. DNA quantification was performed using the Qubit 4 fluorometer (Invitrogen, Carlsbad, CA, USA). The quality of the extracted DNA was assessed by 0.8% agarose gel electrophoresis and visualized with GelRed staining (Sigma-Aldrich, St. Louis, MO, USA) using a UV transilluminator (TS-40, UVP, Jena, Germany). Biofilms were prepared under sterile conditions by mixing 1 g of a soiling sample in 10 mL of 10% LB broth in Falcon tubes and shaking at 230 rpm at 37 \({}^{\circ}\)C for 1 h. Subsequently, 500 \(\upmu\)L of the supernatant was inoculated into three sterile flasks, with each one containing 3 standard 2 cm \(\times\) 2 cm PV glass slides and 50 mL of 10% LB broth. The flasks were kept in agitation at 150 rpm and 37 \({}^{\circ}\)C for 5 days. The metagenomic DNA of the biofilms was extracted from 6-8 PV glass slides using the DNeasy(r) PowerSoil(r) kit (Qiagen, Inc.), following the manufacturer's instructions. For the preparation of the genomic library, 10-15 ng of genomic DNA and the 16S Barcoding kit (1-24) SQK-16S024 (ONT, Oxford Nanopore Technologies, Oxford, UK) were used. PCR amplification was carried out with primers 27F and 1492R using the following protocol: 95 \({}^{\circ}\)C for 1 min; 25 cycles at 95 \({}^{\circ}\)C for 20 s, 55 \({}^{\circ}\)C for 30 s, 65 \({}^{\circ}\)C for 2 min; and finally, an extension at 65 \({}^{\circ}\)C for 5 min and storage at 4 \({}^{\circ}\)C. After the amplification and ligation, the samples were quantified using the Qubit 4 fluorometer (Invitrogen, Carlsbad, CA, USA) and mixed in equimolar amounts according to the manufacturer's instructions.