# Malaysian Virgin Soil Extracts as Natural Growth Enhancer for Targeted Green Microalgae Species
## 3 Results and Discussion
### Effects of Soil Extracts on the Growth of Microalgae
the control system increased from 0.024 on day 0 to 0.236 on day 3, unveiling an increase of 883% in the cell density. However, the growth of _Nannochloropsis oculata_ declined gradually after day 3, reaching an optical density of 0.187 on day 9 in the control system which suggested the inadequacy of using artificial medium alone in the cultivation of microalgae. The experimental groups using the soil extracts obtained from natural extraction (24 h), autoclave 105 \({}^{\circ}\)C once, and autoclave 105 \({}^{\circ}\)C twice treatment methods established the highest growth of _Nannochloropsis oculata_ with an average optical density of 0.484 with no significant difference (\(p>0.05\)). However, these methods differ significantly (\(p<0.05\)) when compared to the autoclave treatment methods at 121 \({}^{\circ}\)C once and 121 \({}^{\circ}\)C twice, which yielded an average optical density of 0.428. The utilization of soil extracts obtained from natural extraction (24 h), autoclave 105 \({}^{\circ}\)C once, and autoclave 105 \({}^{\circ}\)C twice treatment methods supported the growth of _Nannochloropsis oculata_ 1.13-fold higher in contrast to soil extracts obtained from the autoclave treatment methods at 121 \({}^{\circ}\)C once and 121 \({}^{\circ}\)C twice. The growth of _Chorella sorokiniana_ in the control system containing the medium alone showed the lowest value, recording an optical density of 0.104 after 9 d of cultivation (Figure 4). However, there was a spike in the optical density on day 1 (0.125) indicating the occurrence of the exponential phase and from that time on, the optical density decreased to 0.063 on day 3 and became almost consistent throughout the cultivation period of 9 d, although the control system was initiated with the highest inoculation of inoculum (0.066) on day 0. This observation evidently indicated that the use of medium alone did not sustain the growth of the microalgae. Therefore, the virgin soil obtained from Bera Lake Forest was extracted using five different treatment methods and the soil extracts were used as a growth enhancer for _Chlorella sorokiniana_. The growth curves of experimental groups elucidated a typical sigmoid pattern with a lag phase of 1 d, witnessing a gradual increase in the average optical density from 0.012 to 0.04, yielding an increased growth of 70%. The exponential phase was observed from day 1 to day 4 and the mid-log phase appeared around day 2.5. However, the soil extracts obtained from 24 h natural extraction exhibited a more gradual and lower growth surge compared to the rest of the experimental groups. As shown in Figure 4, it was  
Figure 3: Optical density at 680 nm of _Nannochloropsis oculata_. Error bars represent the standard deviation between three replicates (\(n=3\)). rather apparent that by day 4 the early stationary phase was attained, resulting in an optical density of 0.159 \(\pm\) 0.010 when soil extracts from natural extraction (24 h) were used. This method produced the lowest cell density by 98% (\(p\) < 0.05) when compared to that of the autoclave treatment method at 121 \({}^{\circ}\)C once and the autoclave treatment method at 121 \({}^{\circ}\)C twice, respectively. The highest cell density was observed with these autoclave treatment methods, respectively yielding an optical density of 0.316 \(\pm\) 0.003 and 0.315 \(\pm\) 0.009, exhibiting no discernible pattern (\(p\) > 0.05). After the incubation period of 9 d, a substantial difference in terms of growth measured in optical density was observed between the control system containing the medium alone and the experimental groups (\(p\) < 0.05). The autoclave treatment methods at 121 \({}^{\circ}\)C once and at 121 \({}^{\circ}\)C twice emerged as the best methods for effective soil extraction which demonstrated a similar growth curve pattern with an average optical density of 0.316 achieved on day 9, documenting an insignificant difference (\(p\) > 0.05) when compared between the two cell densities.