# Assessment of _Bacillus pumilus_ Isolated from Fresh Water Milieu for Bioflocculant Production
## 3 Results and Discussion
### Time Course of Bioflocculant Production  
The time course profile of biofloculant production by the bacterial strain was investigated over a growth period of 120 h and the results are depicted in Figure 6.  
Figure 6: Time course profile for biofloculant production by _Bacillus pumilus._ Bacterial count (CFU/mL)—Green line, pH—Black line, Optical density measured at 660 nm (OD)—Red line, and Flocculating activity (%)—Blue line. There was a sharp increase in flocculating activity between 0 and 24 h of fermentation after which a steady decline was noticeable up to 96 h, beyond which a very sharp drop in flocculating activity was observed. Maximum flocculating activity of 89.7% (Figure 6) was attained at early stationary phase (24 h) and 2.5 g of biofoculant was recovered from 1 L of fermentation broth. The reason why the flocculating activity was higher during the time course of biofoculant production was because an optimal culture conditions were used in this experiment compared to the previous study. The biofoculant was produced at optimal fermentation conditions and hence, an increase in flocculating activity was observed with the flocculating activity. The decline in flocculating activity observed between 96 and 120 h could be associated with the production of biofoculant-degrading enzymes by the microorganism and cell autolysis [57]. There was a corresponding increase in bacterial growth with flocculating activity up to 24 h after which a sharp decline was observed. These results suggest that the biofoculant was as a result of biosynthesis during its growth and not by cell autolysis [46]. Based on these results, a culturing period of 24 h was adapted for subsequent experiments using the test strain. The short time (24 h) recorded for optimum biofoculant production by _Bacillus pumilus_ strain suggest that bulk production of the biofoculant may be achieved over a short period of time, which implies cost-containing possibilities on an industrial production scale. Similarly, _Bacillus majuensis_ reached its maximum flocculating efficiency in the early stationary phase which was also recorded at 24 h, while the cell production reached its maximum in the stationary phase (at 72 h) suggesting that the produced biofoculant was as a result of biosynthesis [58]. The optical density curve which may also be representative of cell growth also showed a similar trend to that of flocculating activity curve until 72 h of fermentation where it started to increase. During the first 24 h, the OD had a similar behavior as the flocculating activity because as the bacteria utilize the nutrient in the medium for cell growth, the bacterial cells increase in population and also produce biofoculant gradually in to the medium in the presence of abundant nutrient. This process increases the turbidity of the culture with increase in cultivation as it was noticed in this study. When the nutrient was depleted, the metabolic waste products accumulate as well as the bacterial cells (bacterial count) decreases in the presence of low nutrient. On the other hand, the metabolic waste products and dead bacterial cells increases the turbidity of the culture and OD only show the turbidity of the culture which cannot differentiate between the viable cells and dead cells whereas, the bacterial counts expressed as CFU/mL only count the number of viable cells, this is the reason why the cell count and OD are not concordant [58]. At 70 h, the nutrient has been depleted and bacteria have reached the death phase of growth and viable cells declined drastically whereas a sharp increase in OD was observed which might be the a result of an increase in metabolic waste products and dead cells that increases the turbidity of the medium. The initial pH of the medium is reported to be a very important factor in biofoculation process. The pre-determined optimum initial pH of the medium was pH 7. In contrast to flocculating activity, the pH decreased from 7.0 to 5.39 within 24 h, followed by a slow decline until it reached a pH of 4.57 at 120 h of incubation (Figure 6).