252 IIUM Engineering Journal, Vol. 14, No. 1, 2013 Amara 15 OPTIMIZING PHB AND PROTEASE PRODUCTION BY BOX BEHNKEN DESIGN AMRO ABD-AL-FATTAH AMARA 1,2 1 Protein Research Department, Genetic Engineering and Biotechnology Research Institute, Mubarak City for Scientific Research and Technological Applications, Alexandria, Egypt. 2 Microbiology Division, Pharmaceutical Department, College of Pharmacy, King Saud University, Riyadh, Kingdom of Saudi Arabia. amroamara@web.de ABSTRACT: Mixed culture is more suitable to adapt more flexible fermentation process and also to produce different product simultaneously. In this study, a mixed Bacillus culture was investigated for their ability to produce the bioplastic "Polyhydroxybutyrate" and both of the mesophilic and the thermophilic proteases, in one flask. Box-Behnken experimental design was used in this investigation. The produced amount of PHB has been increased significantly. Meanwhile there is a competition between PHB and proteases. The maximum produced amount of PHB using Box-Behnken design was 2.82 g/l/48 h with protease activity equal to 41.9 Units/ml/48 h for thermophilic proteases and 99.65 Units/ml/48 h for mesophilic proteases. Excel solver was used for extra- optimization for the optimum conditions obtained from Box-Behnken experiments and its model. The maximum PHB obtained after using Excel solver was 2.88 g/l/48 h. The maximum mesophilic and thermophilic activities obtained at the same PHB production conditions were 175.68 and 243.38 Units/ml respectively. The model accuracy obtained from Excel solver was 118.8%, which prove that the power of the experimental design in optimizing such complicated process. The strategies used in this study are recommended for the production of PHB and different proteases simultaneously, using Bacillus mixed culture. ABSTRAK: Kultur campuran adalah lebih sesuai bagi proses penapaian yang fleksibel dan ia boleh menghasilkan produk yang berbeza secara serentak. Dalam kajian ini keupayaan menghasilkan "Polyhydroxybutyrate" bioplastik serta mesofilik dan termofilik protease dalam satu flask oleh kultur Bacillus campuran telah disiasat. Eksperimen rekabentuk Box-Behnken telah digunakan. Jumlah PHB yang dikeluarkan meningkat dengan ketara dan terdapat persaingan antara PHB dan protease. Jumlah keluaran PHB maksima menggunakan rekabentuk Box-Behnken adalah 2.82 g/l/48 jam dengan aktiviti protease sama dengan 41.9 Unit/ml/48 jam untuk protease termofilik dan 99.65 Unit/ml/48 h untuk protease mesofilik. Solver Excel telah digunakan untuk memperolehi kadar optimum tambahan dari keadaan optimum yang diperolehi dari experimen dan model Box-Behnken. PHB maksimum diperolehi setelah menggunakan solver Excel adalah 2.88 g/l/48 jam. Aktiviti mesofilik dan termofilik maksima diperolehi daripada keadaan pengeluaran PHB yang sama adalah 175.68 dan 243.38 Unit / ml. Ketepatan model diperolehi daripada solver Excel adalah 118.8%, membuktikan kekuatan eksperimen tersebut bagi mengoptimumkan proses yang rumit. Strategi yang digunakan dalam kajian ini adalah disyorkan bagi pengeluaran PHB dan proteas berbeza secara serentak menggunakan kultur Bacillus campuran. KEYWORDS: PHB; proteases; mixed culture; Box-Behnken design. IIUM Engineering Journal, Vol. 14, No. 1, 2013 Amara 16 1. INTRODUCTION Bacteria-produced PHB is approximately five to ten times more expensive than its competitors (e.g. polypropylene or polyethylene). Although this natural product is promising, its price at the early production time is rather expensive [1]. The cheapest substrate cost is $0.22/kg (sugar) of PHA while the cost of polypropylene is $0.185/kg [2]. The substrate cost affects the overall cost. When the PHB productivity was increased from 1.98 to 3.2 g/h, the PHB production cost was decreased from $5.37/kg to $4.91/kg [2]. In a laboratory fed-batch system using Alcaligenes latus, the highest reported productivity was 4.94 g/h with cost about $2.6 kg [3]. Thermostable enzymes can be produced by both of thermophilic and mesophilic microbes [4, 5]. Bacilli and particularly Bacillus licheniformis, Bacillus subtilis and Bacillus pumilus are the most used species [6]. Molecular biology tools were used to increase the PHB production. As an example random in vivo and in vitro mutageneses were performed [7-11]. Experimental design is another versatile tool could be used for optimizing different parameters and conditions. In this study Box-Behnken design and Excel solver were used to optimize the medium constituents[12]. PHB and the proteases were subjected to the optimization processes. The different obtained results from the conducted experiments were analyzed statistically using Excel 2000 and Essential Exp., version 2.205 software [13]. The results which have been obtained from Box-Behnken optimization in this study are promising. 2. MATERIALS AND METHODS 2.1 Microorganism Five Bacillus species were used in this study. They were isolated from the Egyptian ecosystem and identified using standard criteria as Bacillus subtilius, Bacillus pumilus, Bacillus thuringiensis, Bacillus licheniformis and Geobacillus stearothermophilus. They are grown routinely in LB medium (Luria-Bertani) [14] at 40-37 o C and maintained at - 70 o C. The Bacillus strains as mixed culture have been adjusted to have equal OD600 (OD600 = 0.025) (Sterilize distilled water has been used to adjust the OD). The used amount of each in the Box-Behnken design were: B. subtilius 100 µ L; B. pumilus 100 µ l; B. thuringiensis 10 µl; B. lichenifermis 10 µ l and Geobacillus stearothermophilus 10 µ l. 2.2 Cultivation Medium The used medium has the following constituents: tryptone soy bean 10 mg; skim milk 10, 5.1 or 0.2 mg; lactose 2 g; glucose 2, 1.2 or 0.4 g; yeast extract 0.4, 0.24 or 0.08 g; KH2PO4 0.01 g; FeSO4 solution 5 µl (12 mg/l); trace elements solution 5 µl/ml as described by Pfenning [15] and KCl2 0.03 g (skim milk, glucose and yeast extract have been used in three different values represented as +1, 0 and -1 following Box-Behnken design). The trace elements and FeSO4 solution were sterilized each separately using 0.22 µm sterilize filter system. 2.3 Preparation of Equal Cultures Bacillus strains were pre-cultivated on LB agar plate for overnight at 37 o C. One loop from the fresh colonies from each Bacillus strain was taken and inoculated to test tube contains 5 ml LB broth medium. The cultures incubated overnight at shaker incubator at 200 rpm and 37 o C. The OD600 for each species was adjusted to 0.025 by adding sterilize distilled water to the culture under aseptic condition. IIUM Engineering Journal, Vol. 14, No. 1, 2013 Amara 17 2.4 Shake Flask Fermentation Condition PHB and proteases production under the different experimental conditions were conducted using 250 ml Erlenmeyer-Flasks each containing 100 ml medium. The shaking rate was 250 rpm at 37 o C. The medium constituents were changed when randomized according to Box-Behnken design [12] as described in Table 1 (see section 3.1). 2.5 Proteases Activities The protease activities of mesophilic and thermophilic proteases have been determined at 37 and 60 o C respectively by the method described by Amara and Serour [16]. 2.6 PHB Characterization The assay was spectrophotometrically (PerkinElmer-UV/VIS Spectrometer Lambda) performed after modification as described by Law and Slepecky to determine PHB as crotonic acid [17]. Two milliliters from each cultivation was centrifuged at 13000 rpm for 15 min. One milliliter of H2SO4 then added to the precipitate, which contain the cells. The solution was incubated in 70 o C for 20 min. After cooling, 10 µl was taken and added to 990 µl of H2O and the absorbance of the solution was measured at 235 nm. The PHB amount for each experiment was determined by calculating the amount of crotonic acid against crotonic acid standard curve reproduced from standard PHB authentic sample. 2.7 Box-Behnken Design The Box-Behnken experimental design, the model and ANOVA analysis were carried out using the Excel 2000 and Essential Exp., Version 2.205 software [13]. Box-Behnken experimental design was used to optimize three variables represented at three levels high, medium and low, which are donated by +1, 0 and -1, respectively. They represent Glucose (X1), Skim milk (X2) and Yeast extract (X3) g/l. The design contain fifteen experiments as in Table 1. The PHB produced from the different experiments were analyzed by multiple regression analysis. The created model was generated using the coefficient of each variable as described in detailed by Amara and Salem [18]. The various interactions between responses have represented in the surface plots. 2.8 Excel Solver Optimization Considering that PHB is the main product, glucose (X1), skim milk (X2) and yeast extract (X3) were further optimized using Microsoft excel 2000 solver to calculate the best YPHB value. The experiment under mathematically calculated optimum glucose (X1), skim milk (X2) and yeast extract (X3) was practically conducted and the model accuracy was calculated using the following formula: �������� % �� � � � � ���������������� !����" # $ 100 (1) 3. RESULTS AND DISCUSSION 3.1 Box-Behnken Design Glucose, skim milk and yeast extract have been randomized according to Box- Behnken design while the other medium constituents as well as the amount of the used of each of the five Bacilli are constant as described above. The amount of the produced PHB, mesophilic and thermophilic proteases for each of the fifteen experiments were summarized in Table 1. IIUM Engineering Journal, Vol. 14, No. 1, 2013 Amara 18 Table 1: Box-Behnken experiments. Exp. no. Glucose Skim milk Yeast extract PHB g/100 ml/48h Measophilic Protease Units/ml/48hr Thermophilic protease Units/ml/48/hr 1 0 (1.2) 0 (5.1) 0 (0.24) 1.25 96.42 50.26 2 0 (1.2) 1 (10) -1 (0.08) 1.45 80.24 29.16 3 1 (2) -1 (0.2) 0 (0.24) 0.81 107.74 43.31 4 0 (1.2) 0 (5.1) 0 (0.24) 1.94 86.71 48.03 5 1 (2) 0 (5.1) -1 (0.08) 2.82 99.65 41.90 6 1 (2) 1 (10) 0 (0.24) 1.25 98.03 43.31 7 0 (1.2) -1 (0.2) 1 (0.4) 0.70 120.68 35.97 8 -1 (0.4) 1 (10) 0 (0.24) 1.49 102.89 27.14 9 0 (1.2) 1 (10) 1 (0.4) 1.01 115.83 21.27 10 1 (2) 0 (5.1) 1 (0.4) 0.99 114.21 25.99 11 -1 (0.4) 0 (5.1) -1 (0.08) 1.56 175.68 243.38 12 -1 (0.4) 0 (5.1) 1 (0.4) 1.12 151.42 74.13 13 0 (1.2) 0 (5.1) 0 (0.24) 1.21 112.59 194.45 14 0 (1.2) -1 (0.2) -1 (0.08) 1.13 135.24 219.12 15 -1 (0.4) -1 (0.2) 0 (0.24) 1.58 119.06 93.34 The best PHB produced amount was in experiment number five and was equal to 2.82 g/10 µ l/48 hr with mesophilic and thermophilic proteases activities were 99.65 and 41.90 Units/ml/48 hr respectively. In case of proteases production, experiment number eleven shows the highest mesophilic and thermophilic activities which are equal to 175.68 and 243.38 Units/ml/48 hr respectively and the produced PHB amount was equal to 1.56 g/100 ml/48 hr. Each response was analyzed statistically using the multiple regression analysis test and the estimate, standard error, T statistic, P-value and Coefficient % for each parameter was summarized (for each response) in Tables 2 (PHB), Table 4 (mesophilic proteases) and Table 6 (thermophilic protease). The model for each response was generated. The ANOVA test for PHB, mesophilic and thermophilic proteases were generated and the obtained results were summarized in Tables 3, 5 and 7, respectively. 3.2 PHB Model PHB = 1.46667 + 0.015* Glucose + 0.182917* Glucose* Glucose + 0.1325* Glucose * Skim milk - 0.3475* Glucose* Yeast extract + 0.1225* Skim milk - 0.367083* Skim milk* Skim milk - 0.0025* Skim milk* Yeast extract 3 - 0.3925* Yeast extract - 0.0270833* Yeast extract* Yeast extract. The P-value in the ANOVA table is greater or equal to 0.10. There is no a statistically significant relationship between the variables at the 90% or higher confidence level as in Table 3. IIUM Engineering Journal, Vol. 14, No. 1, 2013 Amara 19 Table 2: Linear Multiple regression analysis of the interaction between variables on PHB response. Parameter Estimate Standard Error T Statistic P-Value Confidence% CONSTANT 1.46667 0.278582 5.26476 0.0033 99.67 Glucose 0.015 0.170596 0.087927 0.9333 6.67 Glucose* Glucose 0.182917 0.25111 0.728432 0.499 50.1 Glucose*Skim milk 0.1325 0.241259 0.549203 0.6065 39.35 Glucose*Yeast extract -0.3475 0.241259 -1.44036 0.2093 79.07 Skim milk 0.1225 0.170596 0.718072 0.5049 49.51 Skim milk* Skim milk -0.36708 0.25111 -1.46184 0.2036 79.64 Skim milk* Yeast extract -0.0025 0.241259 -0.01036 0.9921 0.79 Yeast extract -0.3925 0.170596 -2.30076 0.0697 93.03 Yeast extract* Yeast extract -0.02708 0.25111 -0.10785 0.9183 8.17 Table 3: ANOVA test for PHB response. Source Sum of Squares Df Mean Square F-Ratio P-Value Model 2.57144 9 0.285716 1.23 0.4321 Residual 1.16412 5 0.232823 Total (Corr.) 3.73556 14 R-squared = 68.8369 percent; R-squared (adjusted for d. f.) = 12.7433 percent; Standard Error of Est. = 0.482518; Mean absolute error = 0.252444 Table 4: Linear Multiple regression analysis of the interaction between variables on measophilic protease response. Parameter Estimate Standard Error T Statistic P-Value Confidence % CONSTANT 98.5722 10.8994 9.04385 0.0003 99.97 Glucose -16.177 6.67446 -2.42372 0.0598 94.02 Glucose* Glucose 15.3008 9.82454 1.55741 0.1801 81.99 Glucose * Skim milk 1.6177 9.43912 0.171383 0.8706 12.94 Glucose * Yeast extract 9.70623 9.43912 1.0283 0.351 64.9 Skim milk -10.7173 6.67446 -1.60572 0.1692 83.08 Skim milk* Skim milk -6.94265 9.82454 -0.70666 0.5113 48.87 Skim milk * Yeast extract 12.5372 9.43912 1.32822 0.2415 75.85 Yeast extract 1.41549 6.67446 0.212076 0.8404 15.96 Yeast extract* Yeast extract 21.3672 9.82454 2.17488 0.0816 91.84 IIUM Engineering Journal, Vol. 14, No. 1, 2013 Amara 20 Table 5: ANOVA test for mesophilic protease response. Source Sum of Squares Df Mean Square F-Ratio P-Value Model 6764.39 9 751.598 2.11 0.2128 Residual 1781.94 5 356.388 Total (Corr.) 8546.32 14 R-squared = 79.1497 percent; R-squared (adjusted for d.f.) = 41.619 percent; Standard Error of Est. = 18.8782; Mean absolute error = 9.47256 3.3 Mesophilic Protease Model Mesophilic protease = 98.5722 - 16.177* Glucose + 15.3008* Glucose* Glucose + 1.6177* Glucose* Skim milk + 9.70623* Glucose* Yeast extract - 10.7173* Skim milk - 6.94265* Skim milk* Skim milk + 12.5372* Skim milk * Yeast extract + 1.41549* Yeast extract + 21.3672* Yeast extract* Yeast extract. The P-value from the ANOVA analysis is greater or equal to 0.10, there is not a statistically significant relationship between the variables at the 90% or higher confidence level as in Table 5. 3.4 Thermophilic Protease Model Thermophilic proteases = 97.58 - 35.435* Glucose - 12.9175* Glucose* Glucose + 16.55* Glucose* Skim milk + 38.335* Glucose - 33.8575* Skim milk - 32.8875* Skim milk* Skim milk + 43.815* Skim milk* Yeast extract - 47.025* Yeast extract + 11.6875* Yeast extract* Yeast extract. The P-value in the ANOVA table is greater or equal to 0.10. There is not a statistically significant relationship between the variables at the 90% or higher confidence level as in Table 7. Table 6: Linear Multiple regression analysis of the interaction between variables on thermophilic protease. Parameter Estimate Standard Error T Statistic P-Value Confidence% CONSTANT 97.58 38.5555 2.53089 0.0525 94.75 Glucose -35.435 23.6104 -1.50082 0.1937 80.63 Glucose * Glucose -12.9175 34.7535 -0.37169 0.7254 27.46 Glucose * Skim milk 16.55 33.3901 0.495656 0.6412 35.88 Glucose * Yeast extract 38.335 33.3901 1.1481 0.3029 69.71 Skim milk -33.8575 23.6104 -1.43401 0.211 78.9 Skim milk* Skim milk -32.8875 34.7535 -0.94631 0.3874 61.26 Skim milk * Yeast extract 43.815 33.3901 1.31222 0.2465 75.35 Yeast extract -47.025 23.6104 -1.99171 0.103 89.7 Yeast extract* Yeast extract 11.6875 34.7535 0.336297 0.7503 24.97 IIUM Engineering Journal, Vol. 14, No. 1, 2013 Amara 21 Table 7: ANOVA test for thermophilic protease response. Source Sum of Squares Df Mean Square F-Ratio P-Value Model 56789.3 9 6309.92 1.41 0.3669 Residual 22297.9 5 4459.59 Total (Corr.) 79087.2 14 R-squared = 71.8059 percent; R-squared (adjusted for d.f.) = 21.0565 percent; Standard Error of Est. = 66.7802; Mean absolute error = 31.9153 Fig. 1: Response surface for skim milk and glucose effect in PHB production. Fig. 2: Response surface for yeast extract and glucose effect in PHB production. IIUM Engineering Journal, Vol. 14, No. 1, 2013 Amara 22 Fig. 3: Response surface for yeast extract and skim milk effect in PHB production. Fig. 4: Response surface for skim milk and glucose effect in mesophilic protease production. IIUM Engineering Journal, Vol. 14, No. 1, 2013 Amara 23 Fig. 5: Response surface for yeast extract and glucose effect in mesophilic protease production. Fig. 6: Response surface for yeast extract and skim milk effect in mesophilic protease production. IIUM Engineering Journal, Vol. 14, No. 1, 2013 Amara 24 Fig. 7: Response surface for skim milk and glucose effect in thermophilic protease production. Fig. 8: Response surface for yeast extract and glucose effect in thermophilic protease production. IIUM Engineering Journal, Vol. 14, No. 1, 2013 Amara 25 Fig. 9: Response surface for yeast extract and skim milk effect in thermophilic protease production. 3.5 Excel Solver Optimization The best level of the three variable as obtained from the maximum point of polynomial PHB model was estimated using the solver function of Microsoft Excel 2000 tools and found to be for glucose = 2 g/100 ml, skim milk = 6.818 mg/100 ml and yeast extract = 0.08 g/100 ml, with a prediction calculated PHB equal to 24.2 g/l/48 h. Table 8: Excel 2002 Solver optimization for X1, X2 and X3. Term Glucose (X1) g/100 ml Skim milk (X2) mg/100 ml Yeast extract (X3) g/100 ml Data Minimum -1 (0.4) -1 (0.2) -1 (0.08) Data Average 0 (1.2) 0 (5.1) 0 (0.24) Data Maximum 1 (2) 1 (10) 1 (0.4) Data Solver 1 (2) 0.353571 (6.818) -1 (0.08) 3.6 Confirming accuracy of model The Y value which was calculated using Microsoft Excel is equal to 24.2 g/l/48 h. The in vivo experiments show that Y value (PHB) is 28.8 g/l/48 h. By calculating the model accuracy from the formula in material and methods section, the model accuracy % was 118.8%. The measophilic and thermophilic proteases activities determined to be 175.68% and 243.38 Units/ml respectively. 4. DISCUSSION Amara [7] suggests directing the research concerning the PHB production to the medicinal applications. This will greatly encourage the investors to give PHB researches IIUM Engineering Journal, Vol. 14, No. 1, 2013 Amara 26 more concern. However studies which were concerning with the reduction of the PHB cost should not be stopped. This study introduces a new concept. It concern with the use of heterogeneous substrates and mixed Bacillus culture to produce valuable products beside PHB (the main product). mixed bacterial culture were used. Thermophilic and mesophilic proteases were additionally produced under mesophilic condition. PHB production is rather sensitive process usually conducted under nitrogen limitation and excess of carbon sources [7]. Proteases, which are protein in nature require considerable amount of carbon and nitrogen sources to fit its amino acids' monomeric structure [4-6]. PHB formed mainly from O2, H and C [7]. To minimize the conflict between the PHB and proteases production conditions, Box-Behnken experimental design was used to map the points where PHB and the proteases could be produced simultaneously. Box-Behnken design was used to optimize three of the used medium constituents. Fifteen experiments were conducted according to the design. The three optimized medium constituents are glucose, skim milk and yeast extract. These three variables were selected based on literature review, experiments (data not shown) and basic science about PHB and proteases productions [4- 7]. The maximum PHB amount was 2.82 g/100 ml/48 h as shown in experiment no 5 (Table 1). In experiment no. 5, glucose was in its +1 value, skim milk was in its 0 value and yeast extract was in its -1 value. The amount of measophilic proteases was 99.65 Units/ml and for thermophilic proteases was 41.90 Units/ml in the same experiment. Comparing the amount of proteases in experiment no 5 with that in experiments no 11 and 14 as well as the related amount of the produced PHB prove that there is a conflict between the PHB and Proteases production. The linear multiple regression analysis of the data as in Table 2 shows that under the experimental conditions, yeast extract was significant with confidence level equal to 93.03. The interaction between glucose and yeast extract as well as between skim milk and skim milk were effective with confidence level equal to 79.07 and 79.64 respectively. The glucose, which is the main carbon source for PHB accumulation, was insignificant with confidence % equal to 6.67. This is due to the existence of substrates contain nitrogen such as the skim milk suppress the PHB production. In the case of yeast extract which effect negatively on the PHB production, this is agree with a well-proved criteria that PHB produced under nitrogen limitation [7]. Moreover, the amounts of the used glucose (0.4, 1.2 and 2g/l) is sufficient for PHB production. In contrast, glucose gives confidence level % equal to 94.02 in case of mesophilic proteases. However, it is affect negatively on the mesophilic protease production. By analysis experiment number 11 which give the maximum mesophilic proteases activity {175.68 Units/ml} the amount of the used Glucose was in its minimal used amount (-1 value). It is concluded that glucose has a negative role in measophilic protease production and that the cell give a priority to proteases production over PHB production. Skim milk even it is not statistically significant but it is consider being effective and giving confidence level % equal to 83.08. However, skim milk is also negatively affect on the mesophilic protease production. It can be concluded that glucose which give a very low confidance level with PHB production (6.67%) was concumed by the cell for producing proteases and that mixed culture is more able to produc proteases in low level of skim milk. High quantity of skim milk might interfere with the ability of the cells to produce proteases. Nearly the same result obtained from statistical analysis of the themophilic proteases data where glucose gives confidence level % equal to 89.7. The maximum amount of Thermophilic proteases was in experiment no 14 where the amount of glucose was at its 0 value. The ANOVA test of each of PHB, measophilic and thermophilic proteases which were insignificant prove that there is a chance for further optimization and that it is still a conflict between the used substrates. The Response surface for the three calculated responces show different levels of interactions between the IIUM Engineering Journal, Vol. 14, No. 1, 2013 Amara 27 responces and agree with the statistical analysis of the Box-Behnken results. For extra- optimization, Excel solver was used to optimize the model obtained from Box-Behnken experiments. The solver calculated Y value was 2.42 g/100 ml/48 h. Experimentally; the obtained Y value was equal to 2.88 g/100 ml/48 h. This gives a model accuracy % equal to 118.8%. The amounts of mesophilic and thermophilic proteases have been improved and the produced amount equal to 144.94 and 158 Units/ml respectively. It is important here to highlight that Excel solver which optimize the model from Blacket-Burman design gives less calculated PHB amount than that produced in experiment number five in Box-Behnken design. However experimentally it gives more PHB. This might be explained that regression analysis could dilute the optimal result during fitting the best line pass through all the results. This problem should be considered by the mathematicians and perhaps can be solved by using manual fitting. The responses of various interactions between the variables and responses have been visualized as response surface as in Figures from 1 to 9. The different response surface analysis results were agreed with the data obtained from the regression analysis and their models. 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