## 2 Materials and Methods
### Laboratory Analysis
#### 2.2.4 Isolation and Identification of a Suitable Urease Active Bacterium  
We hypothesized that there was likely to be a close biomineralization relationship between algae and other agents in microbial mats. Moreover, to identify the microbial activity, a 5.0 g sample of beachrock was mixed with 10 mL of autoclaved artificial seawater (Aquamarine, Yashima Drug Company, Osaka, Japan; Table 1), then diluted \(10^{1}\)-\(10^{4}\) times with artificial seawater. Next, 10 \(\mu\)L ofeach dilution was added to ZoBell2216E agar medium (for marine bacteria, pH 7.7) and NH\({}_{4}\)-YE agar medium for inland bacteria (20 g/L yeast extract; 10 g/L di-ammonium sulfate (NH\({}_{4}\))\({}_{2}\)SO\({}_{4}\); 0.13 M Tris buffer for pH 8.0), and 20 g/L agar amended to isolate micro bacteria resistant strains as target study of this research. To avoid contamination, every step of the investigation was conducted inside a clean bench. After incubation at 35 \({}^{\circ}\)C for 3 d, about \(\sim\)53 colonies from each location were isolated from the plates. A urease activity test was then conducted on the colonies to identify ureolytic bacteria. Each selected colony (diameter: 2-3 mm) was mixed with 20 mL of solution (20 mL/L cresol red solution, 0.4 g/L cresol red with distilled water, and 25 g/L CO(NH\({}_{2}\))\({}_{2}\) with distilled water) in a 20 mL bottle. The samples were then sealed, mixed by shaking, and then incubated at 45 \({}^{\circ}\)C for 2 h. To determine whether or not the colonies have urease activity, we observed the color of the solution after 2 h. There was a change in the cresol red, from yellow to purple as pH increased from 7.2 to 8.8. Gene amplification (16S rRNA) and sequencing was carried out for selected ureolytic isolates. The analysis of the DNA sequences was performed using the DB-BA 12.0 and International Nucleotide Sequence Database created by the Techno Suruga Laboratory, Shizuoka, Japan. The ureolytic bacteria isolated were identified using 16S rRNA sequence analysis. DNA extracts were amplified using two sets of primers targeting the region-specific 16S rRNA sequences for almost all bacterial 16S sequences: the F9 (5\({}^{\prime}\)-GAGTTTGATCCTGGCTCAG-3\({}^{\circ}\)) and R1451 (5\({}^{\prime}\)-AAGAGGTGATCCCAGCC-3\({}^{\prime}\)) primers. The PCR amplification cycle consisted of an initial denaturation step lasting 5 min at 94 \({}^{\circ}\)C, followed by 25 cycles lasting 1 min at 94 \({}^{\circ}\)C, 2 min at 60 \({}^{\circ}\)C, 1 min at 72 \({}^{\circ}\)C, and a final extension step of 30 min at 72 \({}^{\circ}\)C. The amplicons were separated through gel electrophoresis, and the resulting DNA bands were extracted and purified using the FastGene(tm) PCR extraction kit according to manufacturer instructions (Nippon Genetics Co., Ltd., Tokyo, Japan). The extracted DNA was sent to the Eurofins Genomics Laboratory (Eurofins Genomics, Tokyo, Japan) for sequencing. Subsequent phylogenetic analysis was conducted by TechnoSuruga Laboratory (TechnoSuruga Laboratory Company Ltd., Tokyo, Japan). The latter used the BLAST algorithm to find related sequences in the GeneBank Database, DNA Data Bank of Japan, and the European Molecular Biology Laboratory. PCR sequencing was conducted to identify the species of ureolytic bacteria isolated from the sites at Krakal-Sadranan. 2.5. Cultivation of Bacteria and Assessing the Potential for Microbial Induced Carbonate Precipitation  
Standard media (Zobell 2216E for marine bacteria and NH\({}_{4}\)-YE medium for land bacteria) were used for culturing under sterile aerobic conditions. Cells were precultured separately in 5 mL of each medium at 37 \({}^{\circ}\)C and 160 rpm for 24 h. One milliliter of the preculture was inoculated with 100 mL of the fresh medium and incubated under the same conditions. The microbial cell growth of the isolates was determined based on optical density at a wavelength of 600 nm (OD\({}_{600}\)) using a UV-vis spectrophotometer (V-730, JASCO Corporation, Tokyo, Japan).