# Malaysian Virgin Soil Extracts as Natural Growth Enhancer for Targeted Green Microalgae Species
## 4 Conclusions  
Undisturbed soil from Bera Lake Forest extracted via five different methods was supplemented as a growth enhancer for the cultivation of _Nannochloropsis oculata_, _Nannochloropsis ocanica_, and _Chlorella sorokiniana_, respectively. The highest nutrient recovery was observed with the autoclave treatment method at 121 \({}^{\circ}\)C twice, recording the carbon to nitrogen to phosphorus ratio of 240.4:9.5:0.1. However, the optimal growth of _Nannochloropsis ocanica_ and _Chlorella sorokiniana_ was observed when soil extracts obtained from the autoclave treatment method at 121 \({}^{\circ}\)C once were utilized. In addition, soil extracts obtained from natural extraction for 24 h supported the maximum growth of _Nannochloropsis oculata_. These findings indicated different phytoplankton required different needs for nutrients to facilitate their growth process. Nonetheless, the addition of soil extracts indeed enhanced the growth of _Nannochloropsis oculata_, _Nannochloropsis ocanica_, and _Chlorella sorokiniana_ by 478%, 158%, and 261%, respectively, in comparison to the control system containing the medium alone. As a result, soil extracts have a huge potential to be used as an alternative to expensive vitamins to enhance microalgae growth. The 96-well plate culturing method is effective as a high-throughput incubation technique to grow microalgae. However, this method provided a limited area of the microplate wells which could restrict the highest growth rate and biomass growth from cell overpopulation. Hence, further studies concentrating on optimizing the growth of microalgae using statistical modelling such as response surface methodology will be carried out to enhance this technique. Conceptualization, M.F.A. and E.F.H. ; methodology, K.K. ; software, H.A. ; validation, M.N.M., N.S.Y. and V.S.K. ; formal analysis, E.F.H. and M.N.M. ; investigation, M.F.A. and K.K. ; resources, F.S. ; data curation, M.F.A. ; writing--original draft preparation, M.N.M. ; writing--review and editing, M.N.M. ; visualization, N.S.Y. ; supervision, V.S.K. ; project administration, N.S.Y. ; funding acquisition, N.S.Y. All authors have read and agreed to the published version of the manuscript. This study was supported by the Ministry of Higher Education Malaysia (MOHE) and Japan Science and Technology Agency (JST) or Japan International Cooperation Agency (JICA), (Grant No. JPMJSA1509). \begin{table}
\begin{tabular}{c c c c} \hline \hline \multirow{2}{*}{**Modified Soil Extract**} & \multicolumn{3}{c}{**The Division Rate (k)**} \\  & _Nammochloropsis_ & _Nammochloropsis_ & _Chlorella_ \\  & _ocenica_ & _oculata_ & _sorokiniana_ \\ \hline Control medium & 0.046 \({}^{\text{d}}\)\(\pm\) 0.001 & 0.029 \({}^{\text{e}}\)\(\pm\) 0.001 & 0.005 \({}^{\text{e}}\)\(\pm\) 0.001 \\ Medium + 105 \({}^{\circ}\)C once & 0.054 \({}^{\text{c}}\)\(\pm\) 0.005 & 0.103 \({}^{\text{d}}\)\(\pm\) 0.001 & 0.096 \({}^{\text{c}}\)\(\pm\) 0.002 \\ Medium + 105 \({}^{\circ}\)C twice & 0.085 \({}^{\text{b}}\)\(\pm\) 0.003 & 0.152 \({}^{\text{b}}\)\(\pm\) 0.001 & 0.105 \({}^{\text{b}}\)\(\pm\) 0.002 \\ Medium + 121 \({}^{\circ}\)C once & 0.099 \({}^{\text{a}}\)\(\pm\) 0.003 & 0.099 \({}^{\text{d}}\)\(\pm\) 0.003 & 0.116 \({}^{\text{a}}\)\(\pm\) 0.001 \\ Medium + 121 \({}^{\circ}\)C twice & 0.046 \({}^{\text{d}}\)\(\pm\) 0.005 & 0.126 \({}^{\text{c}}\)\(\pm\) 0.007 & 0.109 \({}^{\text{b}}\)\(\pm\) 0.004 \\ Medium + 24 h & 0.053 \({}^{\text{c}}\)\(\pm\) 0.003 & 0.165 \({}^{\text{a}}\)\(\pm\) 0.007 & 0.068 \({}^{\text{d}}\)\(\pm\) 0.005 \\ \hline \hline \end{tabular} Note: The values displayed are the average for replicates of three with standard deviation. \({}^{\text{a}}\)\({}^{\text{c}}\) The overall mean of the same column with distinct superscripts changes significantly (_p_ < 0.05) using ANOVA and Tukey post-hoc.  
\end{table} 
Table 5: The division rate, \(k\) of _Nannochloropsis ocanica_, _Nannochloropsis oculata_, and _Chlorella sorokiniana_ in different modified soil extract medium. Institutional Review Board Statement: Not applicable.