SQU Journal for Science, 2022, 27(2),77-83 DOI:10.53539/squjs.vol27iss2pp77-83 Sultan Qaboos University 77 Optimization of Independent Variables for the Production of Extracellular Alpha Amylase by Bacillus subtilis IMD34 Using Plackett- Burman Design Henshaw Effiom* and Josiah Lennox Antimicrobials and Bioactive Molecules Research Group, Microbiology Department, Faculty of Biological Sciences, University of Calabar, Nigeria. Email: henzefilo@yahoo.co.uk. ABSTRACT: Bacillus species are widely used for thermostable α-amylase production for various applications such as in the detergent and food industries. This research aimed to study the production of α-amylase by B. subtilis IMD34 in submerged fermentation conditions using Plackett-Burman (PB) design, along with the optimization of certain environmental factors for optimal growth of amylolytic bacteria. Plackett-Burman design was used to evaluate the effect of ten independent variables on α-amylase production. Maximum enzyme activity was recorded in a medium containing (g/l); starch (6.0), peptone (12.0), K2HPO4 (1.0), Na2HPO4 (2.0), NaCl (0.6), Na2SO4 (0.1), MgSO4.7H20 (1.0), FeCl3.6H2O (0.02), CaCl2.2H2O (0.02) and with an agitation speed (200 rpm). From the pareto chart it was observed that the most important independent variables for amylase production were starch, peptone, NaCl, Mg2SO4, CaCl2.2H2O and agitation. Optimization of environmental parameters to assess their effect on the growth of bacteria showed that optimal pH, incubation time and temperature were recorded at 6, 48h and 37˚C. Evaluating independent variables statistically using PB design revealed that some of the medium’s components had a positive impact on α- amylase production by B. subtilis IMD34. Keywords: Optimization; Independent variables; Alpha amylase; Bacillus subtilis IMD34; Plackett-Burman Design. ستخدام تصميم إب BACILLUS SUBTILIS IMD34بواسطة تحسين المتغيرات المستقلة إلنتاج أميلز ألفا خارج الخلية Burman-Plackett هينشو إفيوم و جوسيا لينوكس المستقر حراريا α-usacuBa ج إنزيم ألفاتستخدم على نطاق واسع في إنتا sullccaB البكتيريا العصويةمن بين البكتيريا ، من المعروف أن :صلخمال لتخمير في حالة ا 43DMI عصويةبواسطة بكتريا إنزيم ألفالتطبيقات مختلفة مثل المنظفات والصناعات الغذائية. كان الهدف من هذا البحث دراسة إنتاج بكتيريا. تم استخدام تصميم بالكيت بورمان لتقييم تأثير عشرة لبورمان مع تحسين بعض العوامل البيئية للنمو األمثل ل-المغمور باستخدام تصميم بالكيت OPHI. ( ، 6..0ببتون )( ، ال0.6تم تسجيل أقصى نشاط لإلنزيم في وسط يحتوي على )جم / لتر( ؛ النشا ) أنزيم ألفا.متغيرات مستقلة على إنتاج (0.6 ، )0u.OPHI )..6( كلوريد الصوديوم ، )0( ، )6.0uI( . HI )6.0( ،3) HI. O.6 )0.6( ، a2cM.0O.H )6.6.( ، 2u2c...O.H )6.6.( سرعة التحريك ، ).الببتون كانت النشا اإلنزيمدورة في الدقيقة(. من مخطط باريتو لوحظ أن أهم المتغيرات المستقلة إلنتاج 66 ، . أظهر تحسين المعلمات البيئية لتقييم تأثيرها على نمو البكتيريا أنه تم تسجيل درجة التحريض HI ،2u2c...O.H .(3، كلوريد الصوديوم ، أن Psدرجة مئوية. كشف تقييم المتغيرات المستقلة إحصائيًا باستخدام تصميم M 6ساعة و I4و 0الحموضة المثلى ووقت الحضانة ودرجة الحرارة عند .s. Ba slclB 43DMIبواسطة أنزيم ألفالى إنتاج بعض مكونات الوسيط كان لها تأثير إيجابي ع .، تصميم بالكيت بورمان sullccaB Ba slclB 43DMI، لمتغيرات المستقلة ، ألفا أميليز، االتحسين :مفتاحيةالكلمات ال HENSHAW EFFIOM and JOSIAH LENNOX 78 1. Introduction mylases are enzymes which hydrolyze starch molecules to give diverse products including dextrin and progressively smaller polymers composed of glucose units. They are a class of industrial enzymes having an approximately 25% share of the market for enzymes [1]. These enzymes are very useful in industrial processes such as making starch, paper and detergents, and in the brewing, textile and pharmaceutical industries. Although there are large number of microorganisms capable of producing useful enzymes, bacterial amylase is generally preferred for starch liquefaction due to its high stability [2] and neutral to alkaline pH. In the food industry, amylases are used in the production of glucose syrups, crystalline glucose, high fructose corn syrups, maltose syrups, and for the reduction of viscosity of sugar syrup and the reduction of haze formation in juices, as well as for solubilization and saccharification of starch for alcohol fermentation in the brewing industry and retardation of staling in the baking industry [2]. In the detergent industry amylases are used as an additive to remove starch based dirt, as well as forin warp sizing of textile fibers in the textile industry. Conventionally the production of alpha amylase has been carried out by submerged fermentation [3,4] of a number of bacterial species such as Bacillus subtilis, B. stearothermophilus, B. amyloliquefaciens, B. licheniformis, B. acidocaldarius and B. marcesens [3,5,6]. Designing an appropriate fermentation medium for highly efficient enzyme production is imperative and necessary as the medium’s constituents and incubation condition can significantly affect the yield of this product [7-9]. Screening of independent variables in the medium used can be carried out by PB experimental design [10]. It is a partial factorial design where a large number of independent variables (N) are studied in small number of experiments (N+1) [11-12]. There are reports on the use of PB design for medium composition for enzyme production by bacteria [3,4,5,7,8,13-17]; however information on the use of PB design to study the influences of independent variables on the production of α-amylase by B. subtilis IMD34 along with the optimization of environmental process parameters for the growth of this bacterial strain is not available. In this research, ten independent variables, namely starch, peptone, MgSO4, K2HPO4, NaHPO4, NaCl, Na2SO4, FeCl3.6HO, CaCl2.2H2O, and agitation speed were screened for their influences on α-amylase production by B. subtilis IMD34 under submerged fermentation using PB design. In addition, the optimization of certain environmental factors including pH, temperature and incubation time on the growth of this bacterial strain was studied. 2. Materials and method 2.1. Microorganism and cultivation Bacillus subtilis IMD34 was obtained from Department of the Microbiology, University of Ibadan, Ibadan- Nigeria. All chemicals used in the experiment were of analytical grade. The organism was maintained on a nutrient agar (Oxoid, Difco, USA) slant and preserved at 4˚C. 2.2. Plackett-Burman experimental design The importance of each independent variable including starch, peptone, MgSO4, K2HPO4, Na2HPO4, NaCl, Na2SO4, FeCl3.6H2O, CaCl2.2H2O, and agitation speed in the fermentation medium required for enzyme production in a shake flask culture using B. subtilis IMD34 was screened for. The PB experimental design of twelve runs (Table 2) for ten variables, nine nutritional factors and one environmental factor (Table 1), were used to evaluate the influence of each on α-amylase production. In Table 2, each row represents an experiment and each column represents an independent variable. For each variable two different concentrations, high and low, were tested (Table 1). 2.3. Submerged fermentation All experiments were carried out in triplicate using 250 ml Erlenmeyer flasks containing 30 ml fermentation media as per experimental design, with pH adjusted to 7.0. Flasks were autoclaved at 15psi and 121˚C for 15 minutes, after which they were inoculated with an 18h old culture with a cell concentration of 1.6x10 5 using aseptic procedures, and then incubated on an orbital incubator shaker (Model 10X400.XX2.C, Sanyo Gallenkamp PLC, UK) at 37˚C and 100 rpm for 24h [3,18]. 2.4. Enzyme extraction assay The culture fluids were centrifuged using a refrigerated high-speed ultra-centrifuge at 10,000xg (4˚C) for 5 minutes, after which the filtrates (supernatants) were assayed for extracellular 2-amylase using the DNSA reagent method [19]. Boiled culture served as a control. A standard curve was prepared with glucose, and the calibration curve so established was used to convert the spectrophotometer values to glucose equivalent, and activities were calculated [18, 20]. 2.5. Optimization of environmental process parameters Environmental process variables such as incubation time, temperature, and pH were varied to evaluate their effects on enzyme production and growth of B. subtilis IMB34. The fermentation medium as defined by PB design above was employed for the optimization process. The effect of incubation time on growth and amylase production A OPTIMIZATIOIN OF INDEPENDENT VARIABLES FOR THE PRODUCTION OF EXTRACELLULAR ALPHA AMYLASE 79 was evaluated at 12h, 24h, 36h, 48h, 60h, 72h, and at each 12 hourly interval growth (optical density) was measured at 610 nm using a Perkin-Elmer lambda UV/VIS spectrophotometer, and the enzyme activity was assayed as stated above. The incubation temperature was studied at (25˚C, 37˚C, 45˚C and 55˚C) and the pH of the fermentation medium was also varied (pH:4, pH:6, pH:7, pH:8 and pH:10). The procedure for measuring the growth (optical density) and enzyme assay as described above was adopted. The data were statistically evaluated adhering to the following formula a) The effect of each independent variable was evaluated with the equation below Exi = 2(∑Hxi-∑Lxi)/N [13], where, Exi is the concentration effect of the independent variable, Hxi and Lxi are the concentrations of amylase at high and low levels of the same variables, and N is the number of trials (12). When the sign is positive, the influence of the variable on enzyme production is greater at high concentrations, and when negative the influence is greater at low concentrations. b) The mean square of each variable was calculated as follows Vxi = ( ∑Hxi-∑Lxi)/N [13] where Vxi is the mean square of the variable. Factors showing larger effects were identified using F-test [13]. Percentage concentration was calculated from F-test. 3. Results and Discussion Amylases are ubiquitous enzymes produced by plants, animals and microorganisms, in which they play a dominant role in carbohydrate metabolism. Amongst the sources of amylase, microbes have been paid a great deal of attention because of their greater stability and safe use [21]. Among bacteria Bacillus species are known to be widely used for thermostable amylase production for various applications such as fermentation, detergents, food etc. [1, 22]. To enhance the production of amylase by B. subtilis IMD34 using PB design, ten independent variables were screened to evaluate their effects on enzyme production, and in addition, the optimization of certain environmental factors for the growth of B. subtilis IMD34 was investigated. The amylase activities from the twelve runs are presented in Table 2, where the results indicate a variation in amylase production with a range from 0.134-0.547Uml -1 by B. subtilis IMD34. These results have shown the need to screen the medium’s components for efficient amylase yield [7, 14, 15, 23, 24, 25]. Figure 1 shows that the most important independent variables for amylase production were starch, peptone, NaCl, Mg2SO4, CaCl2.2H2O and agitation.The highest amylase activity was recorded in trial number 9 containing (g/l); Starch (6.0), Peptone (12), K2HPO4 (1.0), NaHPO4 (2.0), NaCl (0.6), Na2SO4 (0.2), MgSO4.7H2O (1.0), FeCl3.6H2O (0.02), and CaCl2.2H2O (0.02), with an agitation speed of 200rpm. The data in Table 2 was statistically evaluated to ascertain the effect of each independent variable and its contribution to amylase production, as presented in Table 3.A positive effect indicates a direct relationship and the contribution of independent variables to the considered response (amylase production), and a negative effect indicates an inverse relationship between the variable and the response (Table 3). A positive sign for the effect means that by increasing the independent variable from low to high level leads to an increase in the response. An effect with a negative sign means that by changing the level of the independent variable from low to high, the response decreases. Results from Table 3 indicate that starch, peptone, NaCl, MgSO4.7H2O, CaCl2.2H2O and increased agitation speed each showed a positive effect on amylase production while K2HPO4, Na2HPO4, Na2SO4 and FeCl3.6H2O had negative effects. In this study, the positive effect shown by starch and peptone, CaCl2 and 2H2O is consistent with the research of Bansode [8], who reported the positive effect of the media components, starch, tryptone and CaCl2.2H2O, on amylase production using bacteria. A similar report by [7] revealed that peptone, agitation and MgSO4.7H2O directly affected the rate of amylase production from the fungus Aspergillus tubingensis 1. Agitation intensity influences the mixing and oxygen transfer rate during submerged fermentation, and thus product formation [25]. Starch has been found to be the best carbon source for the production of amylase by B. stearothermophilus [26]. The commercial production of amylase is carried out in stages, essentially because the environmental factors required for the optimum growth of bacteria being employed for production may differ from those required for the maximum production of enzymes. In this study three environmental factors including pH, temperature and incubation time were optimized to ascertain the optimal conditions for bacterial growth. Optimum temperature, pH and incubation time were determined by varying incubation temperature (25˚C, 37˚C, 45˚C, 55˚C) (Figure 3), adjusting the fermentation medium pH (4, 6, 7, 8, 10) (Figure 4), and incubating the medium for various periods of time (12h, 24h, 36h, 48h, 60h, 72h) (Figure 2). Results obtained showed the optimum temperature for growth of B. subtilis IMD34 to be 37˚C (OD600nm; 2.12) (Figure 3). Optimal growth of amylolytic B. amyloliquefaciens at 36˚C has been reported [27] and B. subtilis at a range of 35-37˚C [18]. The optimum pH for optimal bacterial growth (OD600nm; 1.89) was observed at pH 6. The pH of the growth medium plays an important role by inducing morphological change in the organism and in enzyme secretion. It has been suggested that the metabolic activity of bacteria is very sensitive to the pH level of their media [16]. The result obtained in this study is similar to the observation of [27] who reported pH 6.5 to be the most favourable for growth of B. marcescen. The pH dependent changes in the growth might have been due to pH control of metabolic gene expression [17]. The optimum incubation time for growth (OD600nm; 2.64) was observed at 48h (Figure 2). This result agrees with the earlier report by [18] who found that the optimum incubation time for optimal growth of Bacillus sp. was in the range 36-48h. The effect of incubation time on optimal growth decreases as its duration increases because the growth of the cells may reach their peak, move through the stationary phase without numerical increase and enter the decline phase [29]. HENSHAW EFFIOM and JOSIAH LENNOX 80 Table 1. Plackett-Burman design for studying the influences of ten variables on α-amylase production by B. subtilis IMD 34. Independent variables Units Low Level(-) High Level(+) Z1: Starch Z2: Peptone Z3: K2HPO4 Z4: Na2HPO4 Z5: Nacl Z6: Na2SO4 Z7: MgSO4.7H2O Z8: Fecl3.6H2O Z9: Cacl2.2H2O Z10: Agitation speed g/l g/l g/l g/l g/l g/l g/l g/l g/l rpm 3.0 6.0 1.0 2.0 0.6 0.1 0.5 0.02 0.01 100 6.0 12.0 2.0 4.0 1.2 0.2 1.0 0.04 0.02 200 Table 2. Plackett-Burman experimental design in 12 runs for studying the influences of independent variables on α- amylase production in shake culture and amylase activity. Trials No Z1 Z2 Z3 Z4 Z5 Z6 Z7 Z8 Z9 Z10 Amylase activity(Uml -1 ) 1 + + - + 2 - + + - 3 + - + + 4 - + - + 5 - - + - 6 - - - + 7 + - - - 8 + + - - 9 + + + - 10 - + + + 11 + - + + 12 - - - - + + - + + + - + + + - + + + - - + + + - + - + - - - + - - - + - - - - - - - + - - - + - - + + - + + + - + + + - + + + - - + + + - + - + - - - - 0.513 0.352 0.284 0.243 0.501 0.314 0.223 0.134 0.547 0.172 0.412 0.182 Table 3. Effects of independent variables on α-amylase production by B. subtilis IMD34 in shake culture. Independent H variables L MS Effect F-value % contribution Starch 3.243 Peptone 2.127 K2HPO4 1.587 Na2HPO4 1.702 NaCl 1.423 Na2SO4 1.413 MgSO4.7H2O 1.782 Fecl3.6H2O 1.524 Cacl2.2H2O 1.674 Agitation speed 1.824 1.124 0.212 1.520 0.141 1.810 0.012 1.791 0.131 1.645 0.045 1.522 0.061 1.325 0.064 1.487 0.071 1.534 0.052 1.642 0.021 0.216 989.12 0.084 201.41 -0.043 62.03 -0.024 44.01 0.052 32.41 -0.023 95.38 0.018 55.26 -0.017 22.41 0.041 8.49 0.032 5.92 65.23 13.28 4.09 2.90 2.14 6.29 3.64 1.48 0.56 0.39 OPTIMIZATIOIN OF INDEPENDENT VARIABLES FOR THE PRODUCTION OF EXTRACELLULAR ALPHA AMYLASE 81 Figure 1. Pareto chart of the influences of independent variables on the response variable by B. subtilis IMD34 Figure 2. Growth pattern of B. subtilis IMD34 for various incubation times. Figure 3. Growth pattern of B. subtilis IMD34 at different incubation temperatures. CaCl2.2H20 FeCl3.6H2O NaCl Na2HPO4 MgSO4.7H… K2HPO4 (Na4)2SO4 Peptone Starch 0.5 1 1.5 2 0 0.5 1 1.5 2 2.5 3 3.5 12 24 36 48 60 72 G ro w th (O D 6 0 0 n m ) 0 0.5 1 1.5 2 2.5 25 37 45 55 G ro w th (O D 6 0 0 n m ) (Response is B. subtilis IMD34 amylase, α = 0.1) Incubation Time Temperature Na2SO4 HENSHAW EFFIOM and JOSIAH LENNOX 82 Figure 4. Growth pattern of B. subtilisIMD34 at various pH. Conclusion This study revealed, using PB design, that certain independent variables in the composition of the growth medium used had a positive impact on α-amylase production by B. subtilis IMD34. Conflict of interest The authors declare no conflict of interest. Ethical Approval Not applicable Acknowledgment The corresponding author acknowledges the valuable contributions of Professor Josiah Asime Lennox to this study. This research was not funded by any funding agency. References 1. Das, S., Singh, S., Sharma, V. and Soriz, M.L. 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