Results The results presented in Table 1 describe the initial and final THC and TPH values of the experimental samples during the treatment for pollutant removal. The control sample (S14) day zero value represents the initial THC (9641 mg kg-1) and TPH (9744 mg kg-1) values prior to treatment. All other Day zero samples values for both THC and TPH were taken after the application of the bioremediation cocktail. The treated samples' TPH measurement are all less than the control sample likely due to the biosurfactant leachate effect of the cocktail applied. However, for the purpose of modelling all day zero measurement were used as initial values. Table 1. THC and TPH results of the experimental sample as measured by UV-Vis Spectrophotometer and GC-FID. Total Hydrocarbon Content (THC) Total Petroleum Hydrocarbon (TPH) Day 0 Day 7 Day 14 Day 21 Day 56 Day 0 Day 21 Day 56 Sample Order mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg S1 8626 7495 5927 3276 1193 8856 2926 1315 S2 8286 5573 2971 1579 629 8247 1794 498 S3 8423 6295 3682 1685 1251 8390 1960 641 S4 8214 4833 2160 1192 487 8193 1599 548 S5 8564 7159 5384 2903 1052 8639 2809 1105 S6 8240 5059 2564 1314 417 8236 1650 428 S7 8465 6521 4199 1928 619 8495 2007 604 S8 8187 4502 1984 1059 360 8157 1466 1167 S9 8515 7026 4921 2617 895 8523 2546 938 S10 8153 4341 1640 952 848 8098 1299 835 S11 8358 5906 3105 1606 724 8256 1879 762 S12 8489 6877 4511 2190 1065 8516 2179 986 S13 8074 4168 1397 890 268 8030 832 299 S14(control) 9641* 9448 9240 9038 8766 9744* 9134 8860 The control sample (S14) day zero value represents the initial THC and TPH value prior to treatment. All other Day zero samples for both THC (Total Hydrocarbon content) and TPH (Total Petroleum Hydrocarbon) were taken after the application of the bioremediation cocktail. The treated samples' values are all less than the control sample likely due to the biosurfactant leachate effect of the cocktail. For the purpose of modelling all day zero value was treated as initial values. UV-Vis = Ultraviolet Visible and GC-FID = Gas Chromatography - Flame Ionization Detector. Table 2. Optimization of the Cocktail parameters for Optimum Degradation of Hydrocarbon using Box-Behnken Response Surface Model. 21 Days Treatment 56 Days Treatment Variable 1 Variable 2 Variable 3 Response 1 Response 2 Response 1 Response 2 Std Order Run Order A:Nitrate B:Phosphate C:Inoculum size TPH Removal THC TPH Removal THC mg/kg mg/kg % % % % % 5 1 0.331 118.41 5 66.96 62.02 85.15 86.16 6 2 0.662 118.41 5 78.24 80.95 93.96 92.41 11 3 0.4965 39.47 7 76.64 80.00 92.36 85.46 12 4 0.4965 197.35 7 80.48 85.49 93.32 94.08 1 5 0.331 39.47 6 67.48 66.10 87.21 87.72 8 6 0.662 118.41 7 79.97 84.05 94.81 94.93 2 7 0.662 39.47 6 76.38 77.22 92.89 92.69 3 8 0.331 197.35 6 82.03 87.07 85.75 95.61 10 9 0.4965 197.35 5 70.12 69.27 88.99 89.49 7 10 0.331 118.41 7 83.96 88.33 89.66 89.62 4 11 0.662 197.35 6 77.24 80.79 90.77 91.47 13 12 0.4965 118.41 6 89.64 88.97 96.28 96.69 14 13 0.4965 118.41 6 89.64 88.97 96.28 96.69 9 14 0.4965 39.47 5 74.41 74.20 88.48 87.48 15 15 0.4965 118.41 6 89.64 88.97 96.28 96.69 THC = Total Hydrocarbon Content; TPH = Total Petroleum Hydrocarbon; Potassium (plantain peels char) was model in the cocktail ratio with a value of 22mg/kg The results of Table 2 describe the design matrix for the optimization of the bioremediation cocktail for responses on % THC and TPH removal at both 21st and 56th days of the monitoring. The experiment was designed with 15 runs. The order suggests an order for permutations of nutrients in the bioremediation cocktail. The centre points at runs 12, 13 and 15 had the highest bioremediation with 88.97 and 89.64% removal for 21 days monitoring and 96.69 and 96.28% removal for 56 days monitoring for THC and TPH respectively. The result presented in Table 3 shows the robust data analysis for the responses for being significant and the lack of fit for being non-significant, of the data tested, p-value 21st - 56th days of 0.031 – 0.028 and 0.009 – 0.002 was reported for THC and TPH removal rate respectively. Table 4 presents the model’s predictable values for THC and TPH removal at 21st and 56th days of monitoring using the experimental data as model input. Table 3. ANOVA result for TPH and THC loss for the quadratic model. 21 Days Cocktail Treatment 56 Days Cocktail Treatment Terms TPH Removal THC Removal TPH Removal THC Removal F-value p-value Coefficient Estimate F-value p-value Coefficient Estimate F-value p-value Coefficient Estimate F-value p-value Coefficient Estimate Intercept 89.64 88.97 96.28 96.69 Model 10.87 0.009 significant 6.06 0.0307 significant 19.82 0.0021 significant 6.32 0.0281 significant A-Nitrate 2.17 0.201 1.42 2.62 0.1666 2.44 68.73 0.0004 3.08 5.41 0.0676 1.55 B-Phosphate 3.74 0.111 1.87 4.33 0.0919 3.14 0.5058 0.5088 -0.2643 10.53 0.0228 2.16 C-Inoculum size 16.37 0.01 3.91 18.22 0.008 6.43 20.84 0.006 1.7 2.57 0.1698 1.07 AB 6.25 0.055 -3.42 4.17 0.0967 -4.35 0.0996 0.7651 -0.1659 5.84 0.0604 -2.28 AC 7.79 0.038 -3.82 7.41 0.0416 -5.8 3.04 0.1416 -0.9168 0.0609 0.8149 -0.2326 BC 2.21 0.198 2.03 1.5 0.2759 2.6 0.0451 0.8402 0.1116 3.07 0.14 1.65 A² 17.73 0.008 -5.99 4.67 0.0831 -4.79 41.16 0.0014 -3.51 2.59 0.1684 -1.58 B² 30.5 0.003 -7.86 8.3 0.0345 -6.39 43.69 0.0012 -3.62 10.88 0.0215 -3.24 C² 19.98 0.007 -6.36 5.81 0.0608 -5.35 11.77 0.0186 -1.88 19.44 0.007 -4.33 THC = Total Hydrocarbon Content; TPH = Total Petroleum Hydrocarbon Table 4. The predicted and actual values for TPH and THC loss as determined by RSM. 21 Days Cocktail Treatment 56 Days Cocktail Treatment TPH Removal (%) THC Removal (%) TPH Removal (%) THC Removal (%) Standard Order Run Order Actual Value Predicted Value Actual Value Predicted Value Actual Value Predicted Value Actual Value Predicted Value 5 1 66.96 68.13 62.02 64.17 85.15 85.2 86.16 87.93 6 2 78.24 78.61 80.95 80.65 93.96 93.19 92.41 91.5 11 3 76.64 75.42 80 77.93 92.36 92.64 85.46 86.38 12 4 80.48 83.23 85.49 89.41 93.32 92.33 94.08 94.01 1 5 67.48 69.07 66.1 67.87 87.21 86.17 87.72 85.88 8 6 79.97 78.8 84.05 81.9 94.81 94.75 94.93 93.17 2 7 76.38 78.76 77.22 81.44 92.89 92.66 92.69 93.54 3 8 82.03 79.65 87.07 82.84 85.75 85.97 95.61 94.76 10 9 70.12 71.34 69.27 71.34 88.99 88.71 89.49 88.57 7 10 83.96 83.59 88.33 88.63 89.66 90.42 89.62 90.53 4 11 77.24 75.66 80.79 79.02 90.77 91.8 91.47 93.31 13 12 89.64 89.64 88.97 88.97 96.28 96.28 96.69 96.69 14 13 89.64 89.64 88.97 88.97 96.28 96.28 96.69 96.69 9 14 74.41 71.66 74.2 70.28 88.48 89.47 87.48 87.55 15 15 89.64 89.64 88.97 88.97 96.28 96.28 96.69 96.69 THC = Total Hydrocarbon Content; TPH = Total Petroleum Hydrocarbon The values in Table 3 provides the statistical relevance of the RSM model results. The model F-value of 10.87 for 21 days treatment and 19.82 for the 56 days treatment implies the model is significant in terms of predicting TPH removal. Also, there is only a 0.86% and 0.21% probably chance that the obtained F-value from the 26 and 56 days model could occur due to noise. The model p-values are both less than the model reference p-value of 0.05 (5%) indicating that the model variable terms are significant in predicting the removal of TPH. The following variable terms of C, AC, A², B², and C² are significant variable terms in the 21 days model while the A, C, A², B², and C² are significant variable terms in the 56 days model for TPH removal. The other variable terms in both models are considered insignificant since their p-values are greater than 0.10 (10%). If there are many insignificant terms in the model (not counting those required to support hierarchy), then the elimination of these terms may improve the model. Similarly, as shown in Table 3 for THC removal. The model F-value of 6.06 for 21 days treatment and 6.32 for the 56 days treatment implies the model is significant in terms of predicting TPH removal. Also, there is only a 3.07% and 2.81% probably chance that the obtained F-value from the 26 and 56 day model could occur due to noise. The model p-values are both less than the model reference p-value of 0.05 (5%) indicating that the model variable terms are significant in predicting the removal of THC. The following variable terms of C, AC, and B² are significant variable terms in the 21 days model while the B, B², and C² are significant variable terms in the 56 days model for THC removal. The other variable terms in both models are considered insignificant since their p-values are greater than 0.10 (10%). If there are many insignificant terms in the model (not counting those required to support hierarchy), then the elimination of these terms may improve the model. Table 3 also shows the values of the coefficient estimate which is the intercept in an orthogonal design of the overall average response of all the runs for TPH and THC removal for days 21 and 56 models. The values as shown in Table 3 all indicate the entire runs response are significant. Figure 1 shows that an increase in both phosphate and nitrate concentration increases the TPH removal rate up to a certain point. Precisely TPH removal decreases at both the low and high concentration limit of phosphate and nitrate. But optimal TPH removal is around the mid-level leaning more toward the high concentration limit of both phosphate and nitrate. Figure 1. Surface 3D plot of total petroleum hydrocarbon (TPH) removal (%) as a function of phosphate and nitrate concentration at a 6% inoculum size after 21 days of cocktail treatment. Figure 2 shows the interactive effects of inoculum size and nitrate on TPH removal at a constant phosphate concentration. It further shows that the THP removal rate would improve with an increase in the inoculum size. While TPH removal rate increases as the nitrate increase to a certain point, where further, increase in nitrate decrease the TPH removal rate. Optimal TPH removal is at high inoculum size and mid-level leaning toward high nitrate concentration. Figure 2. Surface 3D plot of total petroleum hydrocarbon (TPH) removal (%) as a function of inoculum size and nitrate concentration at constant phosphate concentration of 118.41 mg/kg after 21 days of cocktail treatment. Figure 3 shows the interactive effects of inoculum size and phosphate on TPH removal at a constant nitrate concentration. Both increases in inoculum size and phosphate increased TPH removal. However, at low phosphate concentration, high inoculum size does not result to increase TPH removal. Optimal TPH removal at constant nitrate is at high phosphate concentration and inoculum size. Figure 3. Surface 3D of total petroleum hydrocarbon (TPH) removal (%) as a function of inoculum size and phosphate concentration at a constant nitrate concentration of 0.497 mg/kg after 21 days of cocktail treatment. Figure 4 shows the interactive of nitrate and phosphate on THC removal at constant inoculum size. The increase in both nitrate and phosphate concentration increases THC removal. But the further increase of nitrate beyond certain points decreased THC removal. Figure 4. Surface 3D Plot of total hydrocarbon content (THC) Removal (%) as a function of Nitrate and Phosphate concentration at a constant 6% Inoculum size after 21 days of Cocktail treatment. Figure 5 shows the interactive effects of inoculum size and nitrate on THC removal at a constant phosphate concentration. It further shows that THC removal rate would improve with an increase in inoculum size. The THC removal rate would improve as nitrate increase to a certain point where further increase in nitrate decrease THC removal rate. Optimal THC removal is at high inoculum size and mid-level leaning toward high nitrate concentration. Figure 5. Surface 3D Plot of total hydrocarbon content (THC) Removal (%) as a function of Nitrate concentration and Inoculum size at a constant Phosphate concentration of 118.41 mg/kg after 21 days of Cocktail treatment. Figure 6 shows the interactive effect of inoculum size and phosphate concentration at constant nitrate concentration. Increase in both inoculum size and phosphate concentration increased THC removal. Further shows that optimal THC removal at the high-level inoculum size and phosphate concentration. Figure 6. Surface 3D Plot of total hydrocarbon content (THC) Removal (%) as a function of Inoculum size and Phosphate Concentration at constant Nitrate concentration of 0.497 mg/kg after 21 days of Cocktail treatment. Figure 7 shows that the TPH removal rate improves with the increase in phosphate and nitrate at constant inoculum size. However, further, increase in phosphate to a certain level of concentration decrease the TPH removal rate. Optimal TPH removal occurs at a mid-level concentration of phosphate and between mid-level to high-level concentration of nitrate. It also depicts the interactive effects of both phosphate and nitrate on TPH removal. Figure 7. Surface 3D plot of Total Petroleum Hydrocarbon (TPH) Removal (%) as a function of Phosphate and Nitrate concentration at a 6% Inoculum size after 56 days of Cocktail treatment. Figure 8 shows the significance of the interactive effect of inoculum size and nitrate concentration at constant phosphate concentration. The increase in both inoculum size and nitrate concentration increased TPH removal. Further shows that optimal TPH removal at the high-level inoculum size and between mid-level to a high level of nitrate concentration. Figure 8. Surface 3D Plot of Total Petroleum Hydrocarbon (TPH) Removal (%) as a function of Inoculum size and Nitrate concentration at constant Phosphate concentration of 118.41 mg/kg after 56 days of Cocktail treatment. Figure 9 shows that the TPH removal rate improves with the increase in phosphate and inoculum size at constant nitrate concentration. However, further, increase in phosphate to a certain level of concentration decrease the TPH removal rate. Optimal TPH removal occurs at a mid-level concentration of phosphate and between mid-level to high-level inoculum size. It also depicts the interactive effects of both phosphate and inoculum size on TPH removal. Figure 9. Surface 3D of Total Petroleum Hydrocarbon (TPH) Removal (%) as a function of Inoculum size and Phosphate concentration at a constant Nitrate concentration of 0.497 mg/kg after 56 days of Cocktail treatment. Figure 10 shows the interactive effects of phosphate and nitrate on the THC removal rate at a constant inoculum size. It further shows that THC removal increased with increase in phosphate. While THC removal increases as nitrate increases to a certain point. Further, the increase in nitrate towards high-level concentration decrease THC removal. Optimal THC removal is at high-level concentration of phosphate and mid-level leaning toward high nitrate concentration. Figure 10. Surface 3D Plot of total hydrocarbon content (THC) Removal (%) as a function of Nitrate and Phosphate concentration at a constant 6% Inoculum size after 56 days of Cocktail treatment. Figure 11 shows the significance of the interactive effect of inoculum size and nitrate concentration at constant phosphate concentration. The increase in nitrate concentration increased the THC removal rate. While THC removal increased with inoculum size to a certain point. Further shows that optimal THC removal is at mid-level inoculum size and mid to high level of the phosphate concentration. Figure 11. Surface 3D Plot of total hydrocarbon content (THC) Removal (%) as a function of Nitrate concentration and Inoculum size at a constant Phosphate concentration of 118.41 mg/kg after 56 days of Cocktail treatment. Figure 12 shows the interactive effects of inoculum size and phosphate on THC removal at a constant nitrate concentration. Both increases in inoculum size and phosphate increased THC removal. However, at low phosphate concentration, high inoculum size does not result to increase THC removal. Optimal THC removal at constant nitrate is at high phosphate concentration and inoculum size. Figure 12. Surface 3D Plot of total hydrocarbon content (THC) Removal (%) as a function of Inoculum size and Phosphate Concentration at constant Nitrate concentration of 0.497 mg/kg after 56 days of Cocktail treatment.