In this section will be discusses on the two important part based on objective to be achieve. First part will be discusses about the effectiveness of culturing bioflocculant produced by Bacillus sphaericus UPMB10 and the second part will be discuss about the comparison between bioflocculant and chemical flocculant to remove suspended solid in river water. Some of the results were tested using statistical analysis.
4.1 Culturing of flocculant produced by bacteria
For the culturing new flocculant producing bacteria, were culture from Bacillus sphaericus UPMB10. Bacterial strain with highly mucous and ropy on the agar plate was culture in the nutrient broth. The culture broth was tested for its ability to flocculate kaolin clay suspension.
Many study on bioflocculant reported microorganism producing bioflocculant during their growth. Figure 1 shows the bacterial growth and bioflocculant production for five days. As can be seen, there is a clear trend upward movement of bacterial growth from first day until five day. For trend of flocculating activity appears downward starting from second day until reach negative flocculating activity at five day. The production of flocculant reached a maximum at second day while the bacteria cell growth reached at maximum after fourth day. Therefore flocculating activity increased with cell growth and then decreased as well as viscosity.
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Figure 1: Bioflocculant production
Figure 3: Bioflocculant production and flocculating activity
This result may be explained by the fact that flocculating activity was reached during at the second phase of growth called the exponential or log phase. This is the period in which the cells grow most rapidly and doubling time. When cell growth come to third phase (stationary phase), flocculant production was gradually decrease in third day. In this phase, cells still growth but metabolism is slow caused by lack of nutrient. They may divide slowly for a time. But soon stop dividing completely. They are still alive and maintain a slow metabolic activity. In this phase, depletion of nutrient and accumulation of waste product caused cell changes to slow growth.
At the last phase, flocculant production continually decrease caused by the growth cycle is in the death phase. In this phase the cell quickly lose the ability to divide even if they are placed in fresh medium. Like the phase of rapid growth, the death phase is also exponential; therefore, cells die quickly and within hours a culture may have no living cells.
The present findings did not seem to be consistent with other research findings that described on the flocculating activity production. However, there have similarities between the attitudes express in this study. Gong et al, (2003) found flocculating activity increased rapidly with cell growth at exponential phase, and reached climax at 48 h and then decreased as well as viscosity. Gong et el (2007) described flocculating activity of Serratia ficaria is in early stationary phase (at 72 h) and the flocculating activity started decreased slowly after 84 h. In contrast to these findings, the flocculation production in Alcaligenes cupidus (Toeda et al, 1991), Bacillus licheniformis (Shih et al, 2007), Bacillus mucilaginosus (Deng et al, 2002), and Vogococcus sp, (Jie, 2005), and Bacillus firmus (Salehizadeh & Shojaosadati 2001) has been found to parallel cell growth.
In cultures of Alcaligenes cupidus, flocculating activity increased for three days and leveled off, and followed increased in viscosity (Toeda et al, 1991). For Bacillus licheniformis showed the production of flocculant and the relative viscosity of the medium reached a maximum after 96 h incubation (Shih et al, 2007). The flocculant production by Bacillus mucilaginosus began rapidly increased during the logarithmic growth period (from 24 h to 60 h) and when viscosity increase will increasing the flocculating activity (Deng et al, 2002). For Vogococcus sp, flocculating activity reached maximum at 60 h and decreased slowly thereafter (Jie, 2005). The maximum flocculating activity for Bacillus firmus reached maximum at the stationary phase and decrease after 33 h (Salehizadeh & Shojaosadati, 2001).
Table 2 : Comparison of bioflocculant production with other bioflocculants
Toeda et al, 1991
Gong et al, 2007
Shih et al, 2001
Deng et al, 2002
Gong et al, 2003
Always on Time
Marked to Standard
Salehizadeh & Shojaosadati, 2001
Note: h =hour
4.2 Cation effect on bioflocculant.
The flocculation of kaolin clay particle by the bioflocculant was studied in the presence of CaCl2 and in the kaolin suspension 45 mg/l. As shown in figure 2, when Cacl2 in the absence of bioflocculant the flocculating rate was too low. Bioflocculant can cause flocculation respectively as expected, but when both of them were added into kaolin suspension, the flocculating activity increased from 78.5%.to 94% Based on the result, it can be concluded obviously that the flocculating rate of kaolin clay was significantly increased by the addition CaCl2. (P <0.01)
Figure 4 : Cation effect on bioflocculant
The results may be explained by the fact that suspended solid in water carry the same negative charge, and repulsion prevents them from combining into large particulate to settle. When cation and microbial flocculant with having an opposite charge was added, they will neutralizing the charge and allowing the particles to combine and form large particles, and finally settle down. These findings in agreement with Gong et al, (2007) that found in addition to achieve high flocculating activity, metal cation are often added. Apparently, divalent cations (Ca2+ and Mg 2+) were more effective than monovalent (Na+) and trivalent cation (Al3+ and Fe3+). Ca2+ and Mg2+ could destabilizing the negatively charged kaolin particle by neutralizing and bridging.
Table 3, shows selected bioflooculants from previous study indicated that flocculating rate and ion added to achieve maximum flocculating activity. From the Table 1, the study conducted by Deng et al, (2002) did not support the finding described by other studies. Bioflocculant Bacillus mucilaginosus has high flocculating rate without adding Ca2+ because the carboxyl groups present on the molecular chain of bioflocculant make the chain stretched out because of electrostatic repulsion and the stretched molecular chains provide more effective sites for particle attachment.
Table 3: Different bioflocculant and flocculating rate with ions added
Flocculating Rate (%)
Hyun et al, 1997
Bacillus coagulants As101
Ca2+, Fe3+, Al3+
Salehizadeh et al, 1999
Salehizadeh and Shojaosadati, 2001
Deng et al, 2002
Paenibacillus polumyxa BY-28
Gong et al, 2003
Gao et al, 2005
Li et al, 2008
Bacillus sphaericus UPMB10
4.3 Chemical flocculant
Figure 3 shows the optimum concentration can be apply to conduct comparison test between bioflocculant and chemical flocculant. The aim of this experiment is to get optimum concentration of PAM can be applied to compare with same dosage of bioflocculant. The result from the analysis show 5mg/l is optimum concentration of PAM when 0.5ml was added in 45ml kaolin suspension. At 5mg/l the rate of flocculating activity was higher at 98% in contrast at 10 mg/l and 1 mg/l the rate of flocculating was fall to 91%.
Figure 5: Optimum concentration of PAM
A possible explanation for this might be that the flocculating activity initially increased with increasing flocculant dosage, but then decreased as the adsorption of excess flocculant restabilized the particle. Because of incomplete dispersion of excess flocculant, only particles around flocculant participated in the flocculating reaction in a moment. Therefore, other particles did not participate in the flocculating reaction and the flocculating activity decreased (Hyun et al, 1997).
4.4 Comparison Test
4.4.1 Comparison test on kaolin suspension 50 g/l
Figure 6 show the comparison result between bioflocculant and chemical flocculant. Both flocculant with same dosage was added into 100 ml kaolin suspension. As can be seen, the average rate of flocculating activity for bioflocculant (bacteria + cation) from three replicate is 94.8% and chemical flocculant (PAM) is 98.3%. From the statiscal analysis, bioflocculant and chemical flocculant has not significant different (P <0.01) when apply in kaolin suspension. Therefore bioflocculant performed slightly better flocculating efficiency to floc kaolin suspension. These finding did not support previous research but it similar to the finding conducted by Suh et al, (1997) and Salehizadeh and Shojaosadati, (2001) that found bioflocculant has high flocculating activity than polyacrylamide (table 4).
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Figure 6: Comparison between bioflocculant and chemical flocculant on kaolin suspension 50g/l
Table 4: Comparison of flocculating activity on previous study
Suh et al, 1997
Salehizadeh & Shojaosadati, 2001
Floculating activity (%)
4.4.2 Comparison test on water sample
Figure 5 shows a comparison of experimental results obtained using bioflocculant (bacteria + cation) and chemical flocculant (PAM) on water sample taken from Belatop river. The results present that PAM was higher than bioflocculant were removal rate of PAM is 81.3% while bacteria + cation is 63.2%. From the statiscal analysis, PAM and bacteria + cation have significant different at P >0.01
Figure 7: Comparison test using water sample
It is difficult to explain this result, but it might be related to production of bioflocculant. A possible explanation for this might be that the bacterial growths not achieve the optimum production during experiment conducted. As mentioned in literature review many factor influence the production of bioflocculant such as temperature and the agitation speed used in the fermentor need to be optimized for efficiency production. This optimization is essential because productivity and distribution of bioflocculant depend on the culture condition.
Table 5: Flocculation of kaolin and river water
In general, it can be seen in Table 5 that comparison between two flocculant applied in two water sample. The most important finding appear from the table is that both flocculant could effectively aggregate floc on tested sample even though having some differences in flocculating activity. The result also showed that bioflocculant possesses high flocculating activity in kaolin suspension than in river water.
It seems possible that these results are related to more negative electrical charge is present on the surface river water than kaolin particle. River water has brief opportunity to increasing negative electrical charge caused by dissolved oxygen and mineral carry runoff from the land surface. When high negative electrical charge, more opposite charge are needed to neutralize the charge and combine the particle to settle down. In contrast, for kaolin suspension, the result is consistent with those of the other studies which found that bioflocculant is effective to aggregate floc on kaolin particle.
Another possible explanation for this is that the situation of floc formation by the flocculants is dependent on the suspended solid tested. According to Kurane et al, (1986), kaolin clay was flocculated immediately after the addition of the flocculant, and sedimented in an aggregate. By this fact, have possibility river water needs more time to aggregate and settle compared to kaolin suspension.
Suspended solid (mg/l)
Removal rate (%)
76.3Table 6: comparison of suspended solid removal
Table 6 indicates that comparison of suspended removal between bioflocculant and chemical flocculant. As can be seen, removal rate of bioflocculant is 68.6% while chemical flocculant is 76.3% and this is significant different at the P <0.01.
The most important finding to appear from the table is that bioflocculant investigated in this study were able to reduce suspended solid in river water more than 50% from the initial rate. These results of this study seemed to confirmed the finding of a study by Deng et al, 2002 who found bioflocculant Bacillus mucilaginosus can reduce 85% suspended solid in wastewater while Hyun et el, 1997 reported bioflocculant Bacillus sp. DP 152 can reduce 90% suspended solid from wastewater. (Table 7)
Table 7: Comparison of suspended removal with other bioflocculant