- Email: [email protected]
Production of bacterial α-amylase by B. amyloliquefaciens under solid substrate fermentation
Biochemical Engineering Journal 37 (2007) 294–297
Production of bacterial ␣-amylase by B. amyloliquefaciens under solid substrate fermentation a , Murat Elibol b ¨ M. Saban Tanyildizi a,∗ , Dursun Ozer a
Department of Chemical Engineering, Faculty of Engineering, Fırat University, 23119 Elazig, Turkey b Department of Bioengineering, Ege University, 35100 Bornova, Izmir, Turkey Received 3 February 2006; received in revised form 8 May 2007; accepted 18 May 2007
Abstract Production of ␣-amylase by Bacillus amyloliquefaciens, under solid substrate fermentation (SSF) was investigated in shaken-culture. The maximum ␣-amylase activity was obtained under the following optimized conditions: corn gluten meal (CGM) 30 g/l, yeast extract (YE) 10 g/l, agitation rate 150 rpm and fermentation temperature 33 ◦ C. The results showed that ␣-amylase production in a medium with CGM was five times higher than that in the medium contained starch and other components. The temperature of fermentation was found to be most crucial factor in ␣-amylase production. © 2007 Elsevier B.V. All rights reserved. Keywords: Solid substrate fermentation; Corn gluten meal; B. amyloliquefaciens; ␣-amylase
1. Introduction ␣-Amylases are extracellular enzymes that randomly cleave the ␣-1,4 glucosidic bonding of linear amylose and branching amylopectin. They are the most important group of enzymes produced commercially. Bacterial ␣-amylases have several applications in many food and textile processes [1–3]. ␣-Amylase can be produced by different species of microorganisms using both submerged fermentation (SMF) and solid substrate fermentation (SSF). Most of the production has been carried out using SMF; however, SSF systems appear promising due to the natural potential and advantages they offer [4]. SSF is generally characterized by the growth of microorganism on and/or within particles of a solid substrate in the presence of varying amounts of water. The solid substrate acts as a source of carbon, nitrogen, minerals and growth factors, and has a capacity to absorb water, necessary for microbial growth. As the microorganisms in SSF are growing under conditions similar to their natural habitats, they may be able to produce certain enzymes and metabolites more efficiently than in submerged fermentation [5–7].
∗
Corresponding author. Tel.: +90 424 237 0000/5529; fax: +904242415526. E-mail address: [email protected] (M.S. Tanyildizi).
1369-703X/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2007.05.009
SSF has many advantages over SMF, including superior productivity, simple technique, low capital investment, low energy requirement and less water output, better product recovery and lack of foam build up and reported to be the most appropriate process for developing countries. A further advantage of SSF is that cheap and easily available substrates, such as agriculture and food industry by-products [8]. Crude or partially purified enzymes produced by SSF have industrial applications (e.g. pectinases used for fruit juice clarification, ␣-amylase for saccharification of starch) [9]. Inexpensive agriculture and agro-industrial residues represented one of the most energy-rich sources on the planet can be used as a substrate in SSF. These residues are in fact, one of the best reservoirs of fixed carbon in nature [10]. In the SSF, the solid substrate not only supplies the nutrients to the culture, but also serves as an anchorage for the microbial cells [11]. The composition and concentration of media and fermentation conditions greatly affect the growth and production of extracellular enzymes from microorganisms. Cost and availability are important considerations, and therefore the selection of an appropriate solid substrate plays an important role in the development of efficient SSF processes [12]. On preliminary cost analysis, a net savings of about 60 and 50% on fermentation medium cost and the expenditure on down-stream processing, respectively, as compared to the presently employed SMF technique was evident [13].
M.S. Tanyildizi et al. / Biochemical Engineering Journal 37 (2007) 294–297
Corn gluten meal (CGM), a by-product of corn wet milling, generated large quantities from starch industrial practice is relatively cheap substrate. Traditionally, CGM has been used for animal feed, and, therefore, it is desirable to find new uses for CGM [14]. It contains rich proteins (≥60%) and vitamin and other minerals. It is known that SSF is mainly confined to processes involving fungi and not suitable for bacterial cultures because of higher water activity requirements. However, successful bacterial growth and production ␣-amylase by using the SSF technique is known in many natural fermentations [11]. The genus Bacillus is major source of industrial enzymes and B. amyloliquefaciens one of the most widely used species for the bulk production of ␣-amylase [15]. The main objective of this study was to investigate into ␣-amylase production by B. amyloliquefaciens under SSF condition with CGM. 2. Materials and methods 2.1. Strain and medium The B. amyloliquefaciens NRRL B-645 was obtained from Agricultural Research Service Culture Collection in USA. The strain was maintained on agar slopes at 4 ◦ C. A standard inoculum medium containing (g/l) glucose 15, peptone 2.5, yeast extract (YE) 2.0, NaCl 1.5, KH2 PO4 0.5, MgSO4 0.5 and CaCl2 0.1 was inoculated into 250 ml. Erlenmayer flask which was then kept at 37 ◦ C and 150 rpm for 18 h. The initial pH of the medium was adjusted to 7.0. 1% (v/v) inoculum was transferred into 250 ml Erlenmayer flasks containing 50 ml production medium. The production medium contained CGM and tap water only. However, in order to determine the effect of different concentrations of medium constituents and process conditions on the production of ␣-amylase, enzyme production medium based on that described by Anderson et al. was also used [16]. The pH of medium was initially adjusted to 7 and allowed to follow its natural course throughout the fermentation. 2.2. Enzyme analysis
295
Fig. 1. Time course ␣-amylase production by B. amyloliquefaciens on CGM (Initial pH: 7.0, CGM concentration: 10 g/l, fermentation temperature: 37 ◦ C, agitation rate: 150 rpm).
the B. amyloliquefaciens utilized the CGM effectively, producing ␣-amylase. The physico-chemical parameters and amount of media components apparently influenced the production of the enzyme. The results reported here are the averages of three values. The production of ␣-amylase reached a peak of 663 IU at 24 h. The production of enzyme relatively decreased after 30 h for this basal medium. In order to follow the profile of enzyme production, the fermentation was run for a long time of period (72 h). In this study, two different sizes of CGM (50 and 100 mesh) were used. Commercially produced CGM has very small particles, so it cannot be ground more. The results show that there is no difference in the values of enzyme and time to reach maximum enzyme production in both experiments. Therefore, it was decided that the natural size of CGM was sufficient for ␣-amylase production. The Fig. 2 shows the effect of nitrogen source (peptone and YE) in fermentation medium on ␣-amylase production. In all concentrations, the higher enzyme activity was obtained when YE was used as a nitrogen source. The figure also shows that 10 g/L of YE is the optimum for maximum ␣-amylase production. However, the activity decreased higher and lower concentrations of YE. Bajpai and Santos also reported similar results in their works [20,21].
The fermented broth was taken after 30 h and centrifuged at 7000 rpm for 15 min, and then supernatant was used for estimation of enzyme activity. The activity was measured by decrease in iodine color reaction showing dextrinization of starch. The activity was measured against the control in which no enzyme was added. The detail of the method is given elsewhere [17]. One unit of enzyme activity is defined 0.0284 optical density reduction of blue color intensity of starch iodine solution at 37 ◦ C [18]. 3. Results and discussions In SSF, the selection of a suitable substrate for a fermentation process is a critical factor [19]. The change of ␣-amylase production with incubation time, in which medium contained CGM and tap water only, is shown in Fig. 1. The results showed that
Fig. 2. Effect of the YE and peptone concentration on ␣-amylase production. (Initial pH: 7.0, CGM concentration: 10 g/l, fermentation temperature: 37 ◦ C, agitation rate: 150 rpm).
296
M.S. Tanyildizi et al. / Biochemical Engineering Journal 37 (2007) 294–297
Fig. 3. Effect of CGM on ␣-amylase production. (Initial pH: 7.0, YE concentration: 10 g/l, fermentation temperature: 37 ◦ C, agitation rate: 150 rpm).
One of the most important parameters in fermentation systems is the level of substrate used. In this study, seven different CGM concentrations varying from 5 to 40 g/l were used (Fig. 3). Maximum ␣-amylase activity was achieved at 30 g/l CGM concentration. Enzyme activity increased with increasing substrate concentration until 30 g/l; any further increase in substrate concentration however, resulted in a decrease in enzyme production. It is well known that substrate concentration has a direct effect on water content in fermentation medium, because water level in the fermentation medium decreases with an increase in substrate concentration. When the quantity of the water becomes insufficient and does not allow a good diffusion of solutes and gas, the cell metabolism slows, or can stop, because of a lack of substrates or through too high concentration of inhibitive metabolites in or near the cell [22]. Likewise, the effects of MgSO4 and CaCl2 on ␣-amylase production were investigated in a medium containing CGM, YE and tap water. However, there was no significant effect in the use of these salts. The enzyme activity values obtained with and without these salts were 2687 and 2645 IU respectively, which are almost the same values. Therefore, it was decided that these salts had no effect on the enzyme production. Although there are many reports indicating an enhancement of ␣-amylase production by these salts [23–25], the salt requirement for production
Fig. 5. Effects of fermentation temperature on ␣-amylase production. (Initial pH: 7.0, CGM concentration: 30 g/l, YE concentration: 10 g/l, agitation rate:150 rpm, 25 ◦ C, 30 ◦ C, 33 ◦ C, × 37 ◦ C, ♦ 40 ◦ C, + 45 ◦ C).
of this particular enzyme was apparently provided by the nature of CGM and tap water. These are of great important in terms of the cost of production of enzyme. Enzymes are susceptible to mechanical force, which may disturb the elaborate shape of complex molecule to such a degree that denaturation occurs. ␣-amylase production was investigated at three different speeds, i.e., 100, 150 and 250 rpm. The highest ␣-amylase activity was obtained at 150 rpm (Fig. 4). Fermentation temperature is also very important for SSF since growth and production of enzymes or metabolites are usually sensitive to temperature [26]. In this study, six different incubation temperatures varying from 25 to 45 ◦ C were used. The results are shown in Fig. 5. The highest ␣-amylase production was recorded at 33 ◦ C. The enzyme production however decreased at higher temperatures. In a previous study, ␣-amylase production using complex medium containing starch, the optimum incubation temperature was 37 ◦ C (not shown here). The thermal characteristics of substrate and the low moisture content in SSF are especially difficult conditions for heat transfer. Heat removal is inadequate for dissipating metabolic heat due to the poor thermal conductivity of most solid substrates and result in unacceptable temperature gradients [27]. Microbial growth and metabolism inevitably lead to a change in the hydrogen ion balance and hence, the pH of the culture medium [12]. To study the effect of pH on enzyme production the initial pH was varied from 5.0 to 8.0 each at 1.0 interval. After the sterilization in the autoclave, all these media having different initial pH, were showed the same pH. It was then thought that CGM contained high proteins (about 60%) serve as a buffer. Therefore, in the subsequent experiments, the initial pH of the fermentation medium was adjusted to 7.0. 4. Conclusions
Fig. 4. Effects of rotary velocity of the shake flask on ␣-amylase production. (Initial pH: 7.0, CGM concentration: 30 g/l, YE concentration: 10 g/l fermentation temperature: 37 ◦ C).
Commercial ␣-amylase production is usually produced by submerged fermentation; however, SSF appear promising due to the natural potential and advantages they offer. In this study, CGM was found to be a suitable substrate for ␣-amylase production. The study of the CGM as solid substrate for ␣-amylase production by B. amyloliquefaciens is of interest because the
M.S. Tanyildizi et al. / Biochemical Engineering Journal 37 (2007) 294–297
substrate was sufficient enough with only a supplement of YE (10 g/l). The medium compounds, which are essential in submerged fermentation for maximum ␣-amylase production are not required in SSF. The knowledge about, obtained higher enzyme activity in case of using SSF than SMF are given in literature [6]. Activity of ␣-amylase, obtained in this study, is nearly five times higher than our previous study [28] related to using of SMF method. The CGM, which is a by-product of starch industries, is not expensive and readily available. It was also determined that one of the most important parameters in ␣-amylase production was the fermentation temperature. References [1] W.M. Fogarty, C.T. Kelly, Microbial enzymes and biotechnology, Elsevier Science Publishers, London, 1990, pp. 71–132. [2] C. Vishnu, B.J. Naveena, Md. Altaf, M. Venkateshwar, G. Reddy, Amylopullulanase A novel enzyme of L. amylophilus GV6 in direct fermentation of starch to L(+) lactic acid, Enzyme Microb Technol 38 (3–4) (2006) 545–550. [3] Lili Kandra, ␣-Amylases of medical and industrial importance, J Mol Struct: Theochem 666–667 (2003) 487–498. [4] S. Ramachandran, A.K. Patel, K.M. Nampoothiri, F. Francis, V. Nay, G. Szakacs, A. Pandey, Coconut oil cake-a potential raw material for production of ␣-amylase, Bioresour Technol 93 (2004) 169–174. [5] B. Han, J.L. Kiers, R.M.J. Nout, Solid-substrate fermentation of soybeans with Rhizopus spp.: Comparison of discontinuous rotation with stationary bed fermentation, J Biosci and Bioeng 88 (2) (1999) 205–209. [6] A. Pandey, Solid-state fermentation, Biochem Eng J 13 (2003) 81–84. [7] A.P. Goes, J.D. Sheppard, Effect of surfactants on ␣-amylase production in a solid substrate fermentation process, J Chem Technol Biotechnol 74 (7) (1999) 709–712. [8] M. Stredansky, E. Conti, L. Navarini, C. Bertocchi, Production of bacterial exopolysaccharides by solid substrate fermentation, Proc Biochem 34 (1999) 11–16. [9] V.H. Mulimani, G.N. Patil, ␣-Amylase production by solid state fermentation: an approach to biotechnology courses, Biochem Educ 28 (2000) 161–163. [10] F. Francis, A. Sabu, K.M. Nampoothiri, S. Ramachandran, S. Ghosh, G. Szakacs, A. Pandey, Use of response surface methodology for optimizing process parameters for the production of ␣-amylase by Aspergillus oryzae, Bioch Eng J 15 (2003) 107–115. [11] Z. Baysal, F. Uyar, C ¸ . Aytekin, Solid state fermentation for production of ␣-amylase by a thermotolerant Bacillus subtilis from hot-spring water, Proc Biochem 38 (2003) 1665–1668.
297
[12] M. Elibol, A.R. Moreira, Optimizing some factors affecting alkaline protease production by a marine bacterium Teredinobacter turnirae under solid substrate fermentation, Proc Biochem 40 (5) (2005) 1951–1956. [13] P.K.R. Kumar, B.K. Lonsane, Gibberellic acid by solid state fermentation: Consistent and improved yields, Biotechnol Bioeng 30 (2) (2004) 267–271. [14] H.J. Suh, J.H. Whang, Y.S. Kim, S.H. Bae, D.O. Noh, Preparation of angiotensin I converting enzyme inhibitor from corn gluten, Proc Biochem 38 (2003) 1239–1244. [15] M.A. Abate, G.R. Castro, F. Sineriz, D.A.S. Callieri, Production of amylolytic enzymes by Bacillus amyloliquefaciens in pure culture and in co-culture with Zymomonas mobilis, Biotechnol Lett 21 (1999) 249–252. [16] E. Andersson, A.C. Johanson, B. Hahn H¨ogerdal, Alpha amylase production in aqueous two phase systems with Bacillus subtilis, Enzyme Microb Technol 7 (1985) 333–338. [17] M.S. Tanyilidizi, D. Ozer, M. Elibol, Optimization of ␣-amylase production by Bacillus sp. using response surface methodology, Proc Biochem 40 (2005) 2291–2296. [18] S.L. Pfueller, W.H. Elliott, The intracellular Alpha-Amylase of Bacillus stearothermophilus, J Biol Chem 244 (1) (1969) 48–54. [19] A. Kunamneni, K. Permaul, S. Singh, Amylase production in solid state fermentation by the thermophilic Fungus Thermomyces Lanuginosus, Society Biotechnol 100 (2005) 2168–2171. [20] P. Bajpai, P. Bajpai, High-temperature alkaline ␣-amylase from Bacillus licheniformis TCRDC-B13, Biotechnol Bioeng 33 (1989) 72–78. [21] E.O. Santos, M.L.L. Martins, Effect of the medium Composition on formation of amylase by Bacillus sp., Braz Arch Biol Technol 46 (1) (2003) 129–134. [22] P. Gervais, P. Molin, The role of water in solid-state fermentation, Biochem Eng J 13 (2–3) (2003) 85–101. [23] W.X. Wu, J. Mabinadji, T.F. Betrand, Effect of growth culture conditions on production of an extracelluler thermostable alpha-amylase from an isolate Bacillus sp., Agric Life Sci 45 (1999) 404–408. [24] E.H. Fishcher, E.A. Stein, Methods in Enzymology, Academic Press, New York, 1961, 313. [25] M.V. Ramesh, B.K. Lonsane, Solid state fermentation for production of ␣-amylase by Bacillus megaterium 16 M, Biotechnol Lett 11 (1989) 49–50. [26] H.K. Sodhi, K. Sharma, J.K. Gupta, S.K. Soni, Production of a thermostable ␣-amylase from Bacillus sp. PS-7 by solid state fermentation and its synergistic use in the hydrolysis of malt starch for alcohol production, Process Biochem 40 (2005) 525–534. [27] M. Raimbault, General and microbiological aspects of solid substrate fermentation, Electron J Biotechnol 1 (3) (1998) 174–188. ¨ [28] M.S¸. Tanyıldızı, M. Elibol, D. Ozer, Optimization of ␣-amylase from Bacillus amyloliquefaciens growth medium for the production of using response surface methodology, J Chem Technol Biotechnol 81 (81) (2006) 618–622.
Production of bacterial ␣-amylase by B. amyloliquefaciens under solid substrate fermentation a , Murat Elibol b ¨ M. Saban Tanyildizi a,∗ , Dursun Ozer a
Department of Chemical Engineering, Faculty of Engineering, Fırat University, 23119 Elazig, Turkey b Department of Bioengineering, Ege University, 35100 Bornova, Izmir, Turkey Received 3 February 2006; received in revised form 8 May 2007; accepted 18 May 2007
Abstract Production of ␣-amylase by Bacillus amyloliquefaciens, under solid substrate fermentation (SSF) was investigated in shaken-culture. The maximum ␣-amylase activity was obtained under the following optimized conditions: corn gluten meal (CGM) 30 g/l, yeast extract (YE) 10 g/l, agitation rate 150 rpm and fermentation temperature 33 ◦ C. The results showed that ␣-amylase production in a medium with CGM was five times higher than that in the medium contained starch and other components. The temperature of fermentation was found to be most crucial factor in ␣-amylase production. © 2007 Elsevier B.V. All rights reserved. Keywords: Solid substrate fermentation; Corn gluten meal; B. amyloliquefaciens; ␣-amylase
1. Introduction ␣-Amylases are extracellular enzymes that randomly cleave the ␣-1,4 glucosidic bonding of linear amylose and branching amylopectin. They are the most important group of enzymes produced commercially. Bacterial ␣-amylases have several applications in many food and textile processes [1–3]. ␣-Amylase can be produced by different species of microorganisms using both submerged fermentation (SMF) and solid substrate fermentation (SSF). Most of the production has been carried out using SMF; however, SSF systems appear promising due to the natural potential and advantages they offer [4]. SSF is generally characterized by the growth of microorganism on and/or within particles of a solid substrate in the presence of varying amounts of water. The solid substrate acts as a source of carbon, nitrogen, minerals and growth factors, and has a capacity to absorb water, necessary for microbial growth. As the microorganisms in SSF are growing under conditions similar to their natural habitats, they may be able to produce certain enzymes and metabolites more efficiently than in submerged fermentation [5–7].
∗
Corresponding author. Tel.: +90 424 237 0000/5529; fax: +904242415526. E-mail address: [email protected] (M.S. Tanyildizi).
1369-703X/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2007.05.009
SSF has many advantages over SMF, including superior productivity, simple technique, low capital investment, low energy requirement and less water output, better product recovery and lack of foam build up and reported to be the most appropriate process for developing countries. A further advantage of SSF is that cheap and easily available substrates, such as agriculture and food industry by-products [8]. Crude or partially purified enzymes produced by SSF have industrial applications (e.g. pectinases used for fruit juice clarification, ␣-amylase for saccharification of starch) [9]. Inexpensive agriculture and agro-industrial residues represented one of the most energy-rich sources on the planet can be used as a substrate in SSF. These residues are in fact, one of the best reservoirs of fixed carbon in nature [10]. In the SSF, the solid substrate not only supplies the nutrients to the culture, but also serves as an anchorage for the microbial cells [11]. The composition and concentration of media and fermentation conditions greatly affect the growth and production of extracellular enzymes from microorganisms. Cost and availability are important considerations, and therefore the selection of an appropriate solid substrate plays an important role in the development of efficient SSF processes [12]. On preliminary cost analysis, a net savings of about 60 and 50% on fermentation medium cost and the expenditure on down-stream processing, respectively, as compared to the presently employed SMF technique was evident [13].
M.S. Tanyildizi et al. / Biochemical Engineering Journal 37 (2007) 294–297
Corn gluten meal (CGM), a by-product of corn wet milling, generated large quantities from starch industrial practice is relatively cheap substrate. Traditionally, CGM has been used for animal feed, and, therefore, it is desirable to find new uses for CGM [14]. It contains rich proteins (≥60%) and vitamin and other minerals. It is known that SSF is mainly confined to processes involving fungi and not suitable for bacterial cultures because of higher water activity requirements. However, successful bacterial growth and production ␣-amylase by using the SSF technique is known in many natural fermentations [11]. The genus Bacillus is major source of industrial enzymes and B. amyloliquefaciens one of the most widely used species for the bulk production of ␣-amylase [15]. The main objective of this study was to investigate into ␣-amylase production by B. amyloliquefaciens under SSF condition with CGM. 2. Materials and methods 2.1. Strain and medium The B. amyloliquefaciens NRRL B-645 was obtained from Agricultural Research Service Culture Collection in USA. The strain was maintained on agar slopes at 4 ◦ C. A standard inoculum medium containing (g/l) glucose 15, peptone 2.5, yeast extract (YE) 2.0, NaCl 1.5, KH2 PO4 0.5, MgSO4 0.5 and CaCl2 0.1 was inoculated into 250 ml. Erlenmayer flask which was then kept at 37 ◦ C and 150 rpm for 18 h. The initial pH of the medium was adjusted to 7.0. 1% (v/v) inoculum was transferred into 250 ml Erlenmayer flasks containing 50 ml production medium. The production medium contained CGM and tap water only. However, in order to determine the effect of different concentrations of medium constituents and process conditions on the production of ␣-amylase, enzyme production medium based on that described by Anderson et al. was also used [16]. The pH of medium was initially adjusted to 7 and allowed to follow its natural course throughout the fermentation. 2.2. Enzyme analysis
295
Fig. 1. Time course ␣-amylase production by B. amyloliquefaciens on CGM (Initial pH: 7.0, CGM concentration: 10 g/l, fermentation temperature: 37 ◦ C, agitation rate: 150 rpm).
the B. amyloliquefaciens utilized the CGM effectively, producing ␣-amylase. The physico-chemical parameters and amount of media components apparently influenced the production of the enzyme. The results reported here are the averages of three values. The production of ␣-amylase reached a peak of 663 IU at 24 h. The production of enzyme relatively decreased after 30 h for this basal medium. In order to follow the profile of enzyme production, the fermentation was run for a long time of period (72 h). In this study, two different sizes of CGM (50 and 100 mesh) were used. Commercially produced CGM has very small particles, so it cannot be ground more. The results show that there is no difference in the values of enzyme and time to reach maximum enzyme production in both experiments. Therefore, it was decided that the natural size of CGM was sufficient for ␣-amylase production. The Fig. 2 shows the effect of nitrogen source (peptone and YE) in fermentation medium on ␣-amylase production. In all concentrations, the higher enzyme activity was obtained when YE was used as a nitrogen source. The figure also shows that 10 g/L of YE is the optimum for maximum ␣-amylase production. However, the activity decreased higher and lower concentrations of YE. Bajpai and Santos also reported similar results in their works [20,21].
The fermented broth was taken after 30 h and centrifuged at 7000 rpm for 15 min, and then supernatant was used for estimation of enzyme activity. The activity was measured by decrease in iodine color reaction showing dextrinization of starch. The activity was measured against the control in which no enzyme was added. The detail of the method is given elsewhere [17]. One unit of enzyme activity is defined 0.0284 optical density reduction of blue color intensity of starch iodine solution at 37 ◦ C [18]. 3. Results and discussions In SSF, the selection of a suitable substrate for a fermentation process is a critical factor [19]. The change of ␣-amylase production with incubation time, in which medium contained CGM and tap water only, is shown in Fig. 1. The results showed that
Fig. 2. Effect of the YE and peptone concentration on ␣-amylase production. (Initial pH: 7.0, CGM concentration: 10 g/l, fermentation temperature: 37 ◦ C, agitation rate: 150 rpm).
296
M.S. Tanyildizi et al. / Biochemical Engineering Journal 37 (2007) 294–297
Fig. 3. Effect of CGM on ␣-amylase production. (Initial pH: 7.0, YE concentration: 10 g/l, fermentation temperature: 37 ◦ C, agitation rate: 150 rpm).
One of the most important parameters in fermentation systems is the level of substrate used. In this study, seven different CGM concentrations varying from 5 to 40 g/l were used (Fig. 3). Maximum ␣-amylase activity was achieved at 30 g/l CGM concentration. Enzyme activity increased with increasing substrate concentration until 30 g/l; any further increase in substrate concentration however, resulted in a decrease in enzyme production. It is well known that substrate concentration has a direct effect on water content in fermentation medium, because water level in the fermentation medium decreases with an increase in substrate concentration. When the quantity of the water becomes insufficient and does not allow a good diffusion of solutes and gas, the cell metabolism slows, or can stop, because of a lack of substrates or through too high concentration of inhibitive metabolites in or near the cell [22]. Likewise, the effects of MgSO4 and CaCl2 on ␣-amylase production were investigated in a medium containing CGM, YE and tap water. However, there was no significant effect in the use of these salts. The enzyme activity values obtained with and without these salts were 2687 and 2645 IU respectively, which are almost the same values. Therefore, it was decided that these salts had no effect on the enzyme production. Although there are many reports indicating an enhancement of ␣-amylase production by these salts [23–25], the salt requirement for production
Fig. 5. Effects of fermentation temperature on ␣-amylase production. (Initial pH: 7.0, CGM concentration: 30 g/l, YE concentration: 10 g/l, agitation rate:150 rpm, 25 ◦ C, 30 ◦ C, 33 ◦ C, × 37 ◦ C, ♦ 40 ◦ C, + 45 ◦ C).
of this particular enzyme was apparently provided by the nature of CGM and tap water. These are of great important in terms of the cost of production of enzyme. Enzymes are susceptible to mechanical force, which may disturb the elaborate shape of complex molecule to such a degree that denaturation occurs. ␣-amylase production was investigated at three different speeds, i.e., 100, 150 and 250 rpm. The highest ␣-amylase activity was obtained at 150 rpm (Fig. 4). Fermentation temperature is also very important for SSF since growth and production of enzymes or metabolites are usually sensitive to temperature [26]. In this study, six different incubation temperatures varying from 25 to 45 ◦ C were used. The results are shown in Fig. 5. The highest ␣-amylase production was recorded at 33 ◦ C. The enzyme production however decreased at higher temperatures. In a previous study, ␣-amylase production using complex medium containing starch, the optimum incubation temperature was 37 ◦ C (not shown here). The thermal characteristics of substrate and the low moisture content in SSF are especially difficult conditions for heat transfer. Heat removal is inadequate for dissipating metabolic heat due to the poor thermal conductivity of most solid substrates and result in unacceptable temperature gradients [27]. Microbial growth and metabolism inevitably lead to a change in the hydrogen ion balance and hence, the pH of the culture medium [12]. To study the effect of pH on enzyme production the initial pH was varied from 5.0 to 8.0 each at 1.0 interval. After the sterilization in the autoclave, all these media having different initial pH, were showed the same pH. It was then thought that CGM contained high proteins (about 60%) serve as a buffer. Therefore, in the subsequent experiments, the initial pH of the fermentation medium was adjusted to 7.0. 4. Conclusions
Fig. 4. Effects of rotary velocity of the shake flask on ␣-amylase production. (Initial pH: 7.0, CGM concentration: 30 g/l, YE concentration: 10 g/l fermentation temperature: 37 ◦ C).
Commercial ␣-amylase production is usually produced by submerged fermentation; however, SSF appear promising due to the natural potential and advantages they offer. In this study, CGM was found to be a suitable substrate for ␣-amylase production. The study of the CGM as solid substrate for ␣-amylase production by B. amyloliquefaciens is of interest because the
M.S. Tanyildizi et al. / Biochemical Engineering Journal 37 (2007) 294–297
substrate was sufficient enough with only a supplement of YE (10 g/l). The medium compounds, which are essential in submerged fermentation for maximum ␣-amylase production are not required in SSF. The knowledge about, obtained higher enzyme activity in case of using SSF than SMF are given in literature [6]. Activity of ␣-amylase, obtained in this study, is nearly five times higher than our previous study [28] related to using of SMF method. The CGM, which is a by-product of starch industries, is not expensive and readily available. It was also determined that one of the most important parameters in ␣-amylase production was the fermentation temperature. References [1] W.M. Fogarty, C.T. Kelly, Microbial enzymes and biotechnology, Elsevier Science Publishers, London, 1990, pp. 71–132. [2] C. Vishnu, B.J. Naveena, Md. Altaf, M. Venkateshwar, G. Reddy, Amylopullulanase A novel enzyme of L. amylophilus GV6 in direct fermentation of starch to L(+) lactic acid, Enzyme Microb Technol 38 (3–4) (2006) 545–550. [3] Lili Kandra, ␣-Amylases of medical and industrial importance, J Mol Struct: Theochem 666–667 (2003) 487–498. [4] S. Ramachandran, A.K. Patel, K.M. Nampoothiri, F. Francis, V. Nay, G. Szakacs, A. Pandey, Coconut oil cake-a potential raw material for production of ␣-amylase, Bioresour Technol 93 (2004) 169–174. [5] B. Han, J.L. Kiers, R.M.J. Nout, Solid-substrate fermentation of soybeans with Rhizopus spp.: Comparison of discontinuous rotation with stationary bed fermentation, J Biosci and Bioeng 88 (2) (1999) 205–209. [6] A. Pandey, Solid-state fermentation, Biochem Eng J 13 (2003) 81–84. [7] A.P. Goes, J.D. Sheppard, Effect of surfactants on ␣-amylase production in a solid substrate fermentation process, J Chem Technol Biotechnol 74 (7) (1999) 709–712. [8] M. Stredansky, E. Conti, L. Navarini, C. Bertocchi, Production of bacterial exopolysaccharides by solid substrate fermentation, Proc Biochem 34 (1999) 11–16. [9] V.H. Mulimani, G.N. Patil, ␣-Amylase production by solid state fermentation: an approach to biotechnology courses, Biochem Educ 28 (2000) 161–163. [10] F. Francis, A. Sabu, K.M. Nampoothiri, S. Ramachandran, S. Ghosh, G. Szakacs, A. Pandey, Use of response surface methodology for optimizing process parameters for the production of ␣-amylase by Aspergillus oryzae, Bioch Eng J 15 (2003) 107–115. [11] Z. Baysal, F. Uyar, C ¸ . Aytekin, Solid state fermentation for production of ␣-amylase by a thermotolerant Bacillus subtilis from hot-spring water, Proc Biochem 38 (2003) 1665–1668.
297
[12] M. Elibol, A.R. Moreira, Optimizing some factors affecting alkaline protease production by a marine bacterium Teredinobacter turnirae under solid substrate fermentation, Proc Biochem 40 (5) (2005) 1951–1956. [13] P.K.R. Kumar, B.K. Lonsane, Gibberellic acid by solid state fermentation: Consistent and improved yields, Biotechnol Bioeng 30 (2) (2004) 267–271. [14] H.J. Suh, J.H. Whang, Y.S. Kim, S.H. Bae, D.O. Noh, Preparation of angiotensin I converting enzyme inhibitor from corn gluten, Proc Biochem 38 (2003) 1239–1244. [15] M.A. Abate, G.R. Castro, F. Sineriz, D.A.S. Callieri, Production of amylolytic enzymes by Bacillus amyloliquefaciens in pure culture and in co-culture with Zymomonas mobilis, Biotechnol Lett 21 (1999) 249–252. [16] E. Andersson, A.C. Johanson, B. Hahn H¨ogerdal, Alpha amylase production in aqueous two phase systems with Bacillus subtilis, Enzyme Microb Technol 7 (1985) 333–338. [17] M.S. Tanyilidizi, D. Ozer, M. Elibol, Optimization of ␣-amylase production by Bacillus sp. using response surface methodology, Proc Biochem 40 (2005) 2291–2296. [18] S.L. Pfueller, W.H. Elliott, The intracellular Alpha-Amylase of Bacillus stearothermophilus, J Biol Chem 244 (1) (1969) 48–54. [19] A. Kunamneni, K. Permaul, S. Singh, Amylase production in solid state fermentation by the thermophilic Fungus Thermomyces Lanuginosus, Society Biotechnol 100 (2005) 2168–2171. [20] P. Bajpai, P. Bajpai, High-temperature alkaline ␣-amylase from Bacillus licheniformis TCRDC-B13, Biotechnol Bioeng 33 (1989) 72–78. [21] E.O. Santos, M.L.L. Martins, Effect of the medium Composition on formation of amylase by Bacillus sp., Braz Arch Biol Technol 46 (1) (2003) 129–134. [22] P. Gervais, P. Molin, The role of water in solid-state fermentation, Biochem Eng J 13 (2–3) (2003) 85–101. [23] W.X. Wu, J. Mabinadji, T.F. Betrand, Effect of growth culture conditions on production of an extracelluler thermostable alpha-amylase from an isolate Bacillus sp., Agric Life Sci 45 (1999) 404–408. [24] E.H. Fishcher, E.A. Stein, Methods in Enzymology, Academic Press, New York, 1961, 313. [25] M.V. Ramesh, B.K. Lonsane, Solid state fermentation for production of ␣-amylase by Bacillus megaterium 16 M, Biotechnol Lett 11 (1989) 49–50. [26] H.K. Sodhi, K. Sharma, J.K. Gupta, S.K. Soni, Production of a thermostable ␣-amylase from Bacillus sp. PS-7 by solid state fermentation and its synergistic use in the hydrolysis of malt starch for alcohol production, Process Biochem 40 (2005) 525–534. [27] M. Raimbault, General and microbiological aspects of solid substrate fermentation, Electron J Biotechnol 1 (3) (1998) 174–188. ¨ [28] M.S¸. Tanyıldızı, M. Elibol, D. Ozer, Optimization of ␣-amylase from Bacillus amyloliquefaciens growth medium for the production of using response surface methodology, J Chem Technol Biotechnol 81 (81) (2006) 618–622.