## 2 Materials and Methods
### Small Scale Sand Solidification Test  
Centimeter-scale one-dimensional column specimen tests under different experimental conditions were performed as a preliminary step towards elucidating the potential for strengthening and stabilizing loose beach sand by MICP. Two types of natural beach sands with varying particle size gradations (see Supplementary Materials) collected from Sri Lanka were used in the current study; a well graded sand and a uniformly graded sand (herein after referred to as sand 1 and sand 2, respectively). The particle size distribution curves were obtained by sieve analysis using BS410 standard sieves. The characteristics and chemical composition of the sands (as determined from powdered samples using Energy Dispersive X-ray Fluorescence spectrometer (JSX-31000R II JEOL, Ltd., Tokyo, Japan) at Material Analysis and Structural Analysis Open Unit of Hokkaido University, Japan) are tabulated in Table 1.  
The optical density (OD\({}_{600}\)) of the harvested culture varied between 2.0 and 2.5 and all the solidification tests were conducted at an optimum temperature of 25 \({}^{\circ}\)C for a treatment period of 14 days. 0.5 M and 1.0 M Ca\({}^{2+}\) cementation solutions prepared with distilled water were used to examine the effect of Ca\({}^{2+}\) ion concentration on solidification of sand. The 0.5 M cementation solution composed of 30.0 g/L of urea (CO(NH\({}_{2}\))\({}_{2}\)), 55.0 g/L of CaCl\({}_{2}\), and 3.0 g/L of nutrient broth. A standard size 35 mL syringe (2.5 cm diameter) was used for the sand solidification tests. In each case, sand was oven dried at 105 \({}^{\circ}\)C for 48 h and a predetermined weight of sand was compacted in 3 layers by applying 20 hammer blows on each layer. The bottom face of each column was blocked by a filter paper. After considering the studies pertaining to determine the most favorable and proper treatment method for MICP applications, a method involving cycles of batch treatment of bacterial culture and cementation solution was adopted for this study. Firstly, a 12 mL of bacterial culture was injected from the top of the syringe, excess solutions were drained out at a controlled rate and allowed approximately 2 h for bacteria fixation. Secondly, this was followed by 12 mL of cementation solution, supplying the necessary nutrient and precipitation components. Moreover, in terms of saturation level, the entire length of the sand column was retained under fully saturated condition as an initial test condition. Four test cases were carried out, varying the \begin{table}
\begin{tabular}{l c c c c c c c c c c c c c} \hline \hline \multirow{2}{*}{
\begin{tabular}{c} **Sand** \\ **Type** \\ \end{tabular} } & \multirow{2}{*}{\(\rho\)} & \multirow{2}{*}{\(\text{D}_{50}\)} & \multirow{2}{*}{\(\text{C}_{\text{u}}\)} & \multirow{2}{*}{\(\text{C}_{\text{c}}\)} & \multicolumn{8}{c}{**Elemental Composition (\%)**} \\ \cline{4-14}  & & & & & & **Al\({}_{2}\)O\({}_{3}\)** & **SiO\({}_{2}\)** & **CaO** & **TiO\({}_{2}\)** & **Cr\({}_{2}\)O\({}_{3}\)** & **Fe\({}_{2}\)O\({}_{3}\)** & **NiO** & **CuO** & **SrO** & **PbO** \\ \hline Sand & 2.81 & 0.37 & 3.58 & 1.12 & 3.64 & 75.39 & 4.25 & 8.39 & 0.04 & 8.13 & 0.08 & 0.07 & - & - \\ \hline Sand & 2.68 & 0.61 & 1.51 & 0.93 & - & 24.14 & 71.79 & 0.27 & - & 2.98 & 0.04 & - & 0.61 & 0.06 \\ \hline \hline \end{tabular} \(\varrho\)—specific gravity; D\({}_{50}\)—diameter at which 50% of the sample’s mass is comprised of smaller particles than that value; C\({}_{\text{u}}\)—coefficient of uniformity; C—coefficient of curvature.  
\end{table} 
Table 1: Characteristics of natural beach sand used in the current study and their elemental composition. reactant concentration and frequency of bacteria injection. For cases 1 and 3, the bacterial culture solution was injected only in the beginning (day 0) whereas for cases 2 and 4, the solution was injected twice, on day 0 and day 7. Cementation solution was injected every 24 h for the total treatment period.