## 3 Results and Discussion
### Effect of Storage Conditions  
According to [12] (2023), the manufacturing cost has been identified as a significant obstacle to the widespread implementation of biofloculants in industrial settings. Consequently, there is a pressing need to explore strategies to mitigate these costs. The current study examined crude biofloculant as a cost-effective alternative in downstream solid-liquid separation processes. These processes often include using chemicals, resulting in additional processes and, thus, expenses that may be avoided. Numerous methods are employed for the downstream processing of biofloculant, i.e., the purification step, which utilizes various chemicals. Chemicals such as chloroform and n-butyl-alcohol in the proportion of 5:2 (\(v/v\)) [29], 2% hexadecyltrimethylammonium bromide [30], chloroform and methanol at a ratio of 2:1 (\(v/v\)) [31] and 32% (\(w/v\)) sulfuric acid [32], have been used for biofloculation purification processes. When manufacturing biofloculants, it is necessary to avoid using an excessive amount of these chemicals, mainly if the production is to  
Figure 1: FTIR spectrum of biofloculant produced by _Bacillus_ sp. D2 (from _Bacillus megaterium_).  
be scaled up for wastewater treatment. Therefore, the storage conditions of the crude biofoculant were evaluated to determine the biofiloculants' viability.  
Figure 2 shows the differences in flocculation activity of the biofoculant when stored under chilled, frozen, and room temperature conditions over twenty days. The results show reduced efficiency as the days progressed in terms of the various conditions. The worst form of preservation was observed at room temperature, with activity above 80% on day 0 and down to below 30% within five days of storage. The flocculants lost their efficiency as the days progressed to days 10 and 15. This may have been caused by the proliferation of microorganisms in the crude biofoculant, as centrifugation may not have eliminated all cells. Overall, ambient temperature is conducive to the growth of various microorganisms. It is close to the optimal temperature for cultivating the microorganisms used in the experiments, thereby increasing the likelihood of survival and growth. In addition, _Bacillus_ spp. are known to be spore-forming [33], so they may have resurrected and produced EPS in response to the conditions they were exposed to, rendering the biofoculant ineffective. The chilled flocculant lost efficiency gradually over time until there was no activity. This could be because the temperature (4 \({}^{\circ}\)C) was unfavorable for the rapid revival of spores and the rapid growth of microorganisms if there were any remaining cells. It was noted that contamination risks exist at ambient temperature and in chilled environments. However, the frozen biofoculants maintained a reasonable flocculation efficacy over time (\(n\) = 20 days). The minor decrease in flocculation efficiency may have been due to water accumulation under frigid conditions, as the chilling temperature used was 0 \({}^{\circ}\)C or during thawing. The results indicate that crude biofoculant can be retained for future use under frozen conditions because it does not lose much of its effectiveness.