## 4 Materials and Methods
#### Sampling--Isolation--Identification of Microorganisms  
Six samples containing soil were selected from the four study areas in the region of Xi beach, where the surface litter was removed and the soil was dug to a depth of 10 cm with a soil core borer. The samples, which weighed 300 g each, were collected from Lixouri, Kefalonia island. The soils were then placed in plastic bags and refrigerated at 4 \({}^{\circ}\)C until they were transferred to the laboratory for further processing. The soil was air-dried for three days at 30 \({}^{\circ}\)C before being pulverized and sieved (metal, 2 mm 1 mm, Aggelis Equipment, Athens, Greece). The Soil-Plate Method was used to isolate fungi. [41; 42]. In Petri plates with PDA, 0.05 g of dry soil was spread and incubated at 25 \({}^{\circ}\)C. After 3 days, fungal colonies formed, and a tiny sample of mycelium from each colony was moved to a different plate to maintain a pure culture for each fungus for incubation. Total viable count (TVC), total coliforms, _E. coli_, enterococci, S. aureus, _P. aeruginosa_, sulfite-reducing clostitida and fungi were chosen as indicator criteria. For 24 h, the bacterial strains were grown in liquid lysozyme broth (LB) at 37 \({}^{\circ}\)C and 170 rpm. The liquid culture medium consisted of NaCl 1%, Tyrpotene 1% and yeast extract 0.5%. For the solid medium, 1.5% agar was added to the liquid broth. All the ingredients, which were provided by PanReacAppliChem, were diluted in distilled sterile water via stirring. Strains were maintained at \(-\)80 \({}^{\circ}\)C in 20% (\(r/v\)) glycerol in LB broth. Six liquid cultures were prepared and incubated at 37 \({}^{\circ}\)C and 170 rpm. Every 20-30 min, one culture was measured using a spectrophotometer (SHIMADZU, UV-900) through setting the OD at 600 nm. After serial dilutions of the measured culture, 50 \(\mu\)L were plated in LB agar petri dishes and incubated for 24 h at 37 \({}^{\circ}\)C. After incubation, the colony forming units per ml (CFU/mL) were calculated [42]. In this way, inocula suspensions were prepared with approximately 1-2 \(\times\) 10\({}^{8}\) CFU/mL. This bacterial concentration was prescribed based on the Broth Microdilution Method [43]. Fungi were cultured in Sabouraud Dextrose Agar (SDA) petri dishes for fourteen days at 25 \({}^{\circ}\)C. For the preparation of SDA, 64 g of medium (PanReacAppliChem) was diluted in 1 L distilled and sterilized water [44]. \begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Sample** & **Sand (\%)** & **Silt (\%)** & **Clay (\%)** & \
\begin{tabular}{c} **After Folk** \\ **and Ward** \\ \end{tabular} &
\begin{tabular}{c} **Plasticity** \\ **Limit** \\ \end{tabular} \\ \hline S1 & 0.260867 & 69.71377 & 30.16857 & Silt (Z) & 20.52 \\ S3 & 0.324223 & 59.56018 & 35.63327 & Mud (M) & 15.50 \\ S6 & 0.205637 & 73.06384 & 26.6753 & Silt (Z) & 15.93 \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Representative particle size analyses and plasticity limits of studied samples. The Wizard Genomic DNA extraction kit (Promega) was used to extract genomic DNA from the soil samples. The ITS1 (5\({}^{\prime}\)-TCC GTA GGTGAA CCTGCG G-3\({}^{\prime}\)) and ITS4 (5\({}^{\prime}\)-TCC TCC GCT TAT TGA TAT GC-3\({}^{\prime}\)) primers, U341F (5\({}^{\prime}\)-CCT ACG GGR SGC AGC AG-3\({}^{\prime}\)) and 805R (5\({}^{\prime}\)-GAC TAC CAG GGT ATC TAA T-3\({}^{\prime}\)) primers, and NEBNext 2x High-Fidelity Master Mix were used to amplify regions of the 18S rDNA gene for fungi and 16S rRNA gene for bacteria, respectively. The used thermocycling protocol consisted of 30 s at 98 \({}^{\circ}\)C, 25 cycles of 10 s each at 98 \({}^{\circ}\)C, 58 \({}^{\circ}\)C, and 72 \({}^{\circ}\)C, and a final step of 5 min at 72 \({}^{\circ}\)C. Following Illumina's instructions, a second PCR was used to add index Illumina adapters. The enhanced pieces were sanitized using Agencourt's Ampure globules (USA). Mr. DNA Lab performed next-generation sequencing, and online platforms for metagenomic analysis were used to analyze the results.