# Malaysian Virgin Soil Extracts as Natural Growth Enhancer for Targeted Green Microalgae Species
## 2 Materials and Methods
### Microalgae  
The targeted microalgae species utilized in the present study were _Nannochloropsis oceanica_, _Chlorella swokhiniana_, and _Nannochloropsis culdata_ which were kindly supplied by the National Institute for Environmental Studies (NIES), Japan. The preparation of Conway medium from five basic solutions followed the recipe by [PERSON] [18] consisting of:  
1. the mineral solution--100 g of NaNO\({}_{3}\), 45 g of disodium EDTA (C\({}_{6}\)H\({}_{16}\)N\({}_{2}\)O\({}_{8}\)), 33.6 g of H\({}_{3}\)BO\({}_{3}\), 20 g of NaH\({}_{2}\)PO\({}_{4}\)4H\({}_{2}\)O, 1.3 g of FeCl\({}_{3}\)\(\cdot\)6H\({}_{2}\)O, 0.36 g of MnCl\({}_{2}\)4H\({}_{2}\)O, and 1 mL trace metal solution in 1 L of ultrapure water;
2. the trace metal solution--0.21 g of ZnCl\({}_{2}\), 0.2 g of CoCl\({}_{3}\)6H\({}_{2}\)O, 0.09 g of (NH\({}_{4}\))6 MO\({}_{7}\)O\({}_{2}\)4H2O, and 0.2 g of CuSO\({}_{4}\)5H\({}_{2}\)O in 0.1 L of ultrapure water;
3. the vitamin solution--0.2 g of thiamine (B1) and cyanocobalamin (B12) in 0.1 L of ultrapure water;
4. the silicate solution--2 g of Na\({}_{2}\)SiO\({}_{3}\) in 0.1 L of ultrapure water;
5. the nitrate solution--2 g of KNO in 0.1 L of ultrapure water.  
The medium was prepared by adding 1 mL of main mineral, silicate, and nitrate solution to the Schott bottle to prepare a 1 L volume medium. An amount of 1 mL of NH\({}_{4}\)Cl and vitamin solution was added to the cooled autoclaved medium to give a final medium concentration of 5.0 \(\times\) 10\({}^{-4}\) M. The cultures were cultivated at 25 \(\pm\) 0.5 \({}^{\circ}\)C under a light intensity of 33.75 \(\upmu\)mol photons m\({}^{-2}\) s\({}^{-1}\) on a 12 h light:12 h dark cycle.  
The 96-well microplates culturing method was implemented to access the growth of _Nannochloropsis oceanica_, _Chlorella swokhiniana_, and _Nannochloropsis oculata_ supplemented with soil extracts obtained from five different extraction methods, respectively following the methods outlined by [PERSON] [19]. A maximum of 200 \(\upmu\)L of solution can be accommodated by each well in the microplate. As an attempt to prevent evaporation during the course of experimentation, 200 \(\upmu\)L ultrapure water was loaded into the border wells of the microplates. The remaining wells were loaded with 195 \(\upmu\)L of an appropriate medium added with 5 \(\upmu\)L of soil extracts obtained from the autoclave treatment method at 105 \({}^{\circ}\)C once in the second column (as the blank), and the third column was filled with 175 \(\upmu\)L of an appropriate medium added with 5 \(\upmu\)L of soil extracts obtained from autoclave treatment method at 105 \({}^{\circ}\)C once and 20 \(\upmu\)L of microalgae (as the experimental groups) to document the exponential phase of microalgae.  
The same experimental protocols were duplicated in the 4 th to 11 th columns of the microplate with autoclave treatment methods at 105 \({}^{\circ}\)C twice (Column 5), 121 \({}^{\circ}\)C once (Column 7), 121 \({}^{\circ}\)C twice (Column 9), and 24 h-natural extractions (Column 11), respectively. The control experiment containing the Conway medium alone was established to assess the growth of microalgae in other microplates. The air humidity in the microplate wells was preserved and the external contamination before incubation was prevented by sealing the microplates with parafilm after pipetting all the wells in the microplates.  
The microplates were incubated for 9 d and the biomass or growth of microalgae was determined by optical density (OD) at 680 nm for every 24 h using the microplate reader Infinite M200 PRO (Tecan, Austria). The optical density was measured every 24 h for 9 days in each of the wells holding the controls and samples, using an 8-channel Eppendorf pipettor. The homogenization was carried out to thoroughly mix the microalgae suspended in the bottom well with the solution.