World Journal of Microbiology and Biotechnology 8, 21-23
Effect of carbon source and dissolved oxygen level on cell growth and pullulanase production by Bacillus stearothermophilus G-82 E.I. Emanuilova and M.S. Kambourova* Cell growth and extracellular pullulanase production of Bacillus sfearothermophilus G - 8 2 were investigated in batch cultures using a defined medium with glucose, maltose, pullulan or amylopectin as carbon source. Maximum enzyme activity was with pullulan or amylopectin. Cell g r o w t h in batch culture was better under o x y g e n unlimited conditions, while higher total and specific enzyme activities, using pullulan or amylopectin, were obtained in oxygen-limited conditions. Enzyme accumulation t o o k place in the late g r o w t h phase. The highest enzyme production of 300 U/I was reached when pullulan was used as carbon source in conditions of o x y g e n limitation.
Key words: Pullulanase production, Bacillus stearothermophilus, carbon source, dissolved oxygen.
Microbial pullulanases (pullulan 1--* 0 glucan hydrolase, EC 3.2.1.41) which catalyse the hydrolysis of 0r 1 ~ 6 glucosidic bonds in pullulan, amylopectin, d-limit dextrins and glycogen are of industrial importance for maltose and maltose syrup production. There is an increased interest in producing thermostable pullulanase to function above 60~ (Ng & Kenealy I986). Previously we have reported that a thermophilic strain of Bacillus stearothermophilus G-g2 produced a thermostable pullulanase (Kambourova & Emanuilova 1987) and the present paper deals with the effect of different carbon sources and of dissolved oxygen on cell growth and pullulanase production.
Materials and Methods Strain Bacillus stearothermopkdus G-82 isolated from a Bulgarian hot spring was used. The strain was deposited in the National Collection for Industrial Microorganisms and Cell Cultures, Sofia, Bulgaria.
Media and Cultivation The stock culture of the strain was maintained on peptone ~gar containing (g/l): Bactopeptone, 3; yeast extract, 3; starch, I, pH The authors are at the Institute of Microbiology, Department of Enzyme Biosynthesis, Acad. G. Bonchev str. B1.26, 1113 Sofia, Bulgaria. *Corresponding author.
7.8. The basal liquid medium contained (g/l): Bactopeptone, 3; yeast extract, 3, pH 7.8. Glucose, maltose, amylopectin or pullulan (1.0 g/l) were added and used as carbon sources. Peptone and pullulan (1 g) contained, respectively, 0.68 and 1.3 mg maltose. Medium was sterilized at 120~C for 30 rain. An inoculum of 3.5 ml (from a 3-h flask-shake culture) was used. Fermentation was carried out in a 'Bioflo' stirred fermenter (Model C-30, New Brunswick Co., Edison, New Jersey, USA) with a working volume of 0.350 litre at 55~ and an air-flow rate of 1.0 vol/vol/min, pH was not controlled. The dissolved oxygen was recorded by a dissolved oxygen probe (New Brunswick Scientific, Model DO-50).
Assays Biomass was determined turbidometricaUy. An optical density of 1.0 at 660 n m = 0.9 g cell dry wt/l. Pullulanase activity of the cu2ture supernatants was assayed at 65~ and pH 7.0 by the method of Suzuki & Chishiro (1983). Residual reducing sugars in culture supematants were determined by the method of Somogyi (1952). Total residual carbohydrates were determined by mixing 2 ml of the supematant with I ml of 3 M HCI, boiled for 3 h and then neutralized with NaHCO 3. Released reducing sugars were measured according to the method of Somogyi (1952).
Chemicals Glucose was obtained from Reachira (USSR); maltose and amylopectin from Fluka (Buchs, Switzerland); pullulan from Hayashibara Biochem. Lab. Inc. (Japan); and sugar standards for thin-layer chromatography from Boehringer (Mannheim, GmbH, Germany). All other reagents used were of analytical grade.
9 1992 Rapid Communications of Oxford Ltd l~orld ]ot~rrlal of Microbiology and BW~eclmoloo~y,Vol 8, .7992
~1
E.I, Emanuilova and M.S. Kambourova pullulanase production by Bacillus cereus var. mycoides (Takasaki 1976) and pullulan had no effect on the enzyme synthesized by B. stearothermophilus KP 1064 (Suzuki & Chishiro 1983). High concentrations of pullulanase could be detected in the culture of Clostridium sp. EM1 (Madi et al. 1987) when pullulan and maltose served as substrates and thus these results resemble our own experiments. The function of pullulanase in maltose-utilizing cells is not clear. Abdullah & French (1966) reported that besides hydrolysis of oligosaccharides, pullulanase catalyses the synthesis of 1,6 dimer or trimer from maltose. Dean et al. (1972) supposed a possible function of the enzyme in the synthesis of polysaccharides from maltose in maltose utilizing cells. This could explain the inducible effect of maltose and malto-oligosaccharides in pullulanase synthesis by B. stearothermophilus G-82. To improve enzyme yield, the effect of dissolved oxygen concentration on cell growth and pullulanase production was studied when amylopectin or pullulan were used as carbon source. The air-flow rate in these experiments was I vol/vol/min and agitation speed was applied to maintain a minimum dissolved oxygen level of 30% (over 450 rev/min) or 0% saturation for 1 to 2 h (between 350 and 450 rev/min). The results of these experiments are presented in Figures 1 and 2. Enzyme production of the strain, which paralleled the growth, resembled that in B. cereus var. mycoides (Takasaki 1976) and Clostridium sp. EM1 (Madi et al. I987). The maximum cell concentration of B. stearothermophilus was lower and maximum enzyme activity was higher for oxygen-limited cultures than for oxygen unlimited. Analysis of spent culture filtrates revealed that none of the experiments were carbon limited. The results in Figures 1 and 2 provide convincing evidence that to obtain higher
Table 1. Effect of different carbon sources on growth and pullulanase production in culture filtrates. Medium*
Cell dry weight
t~ (h)
Pullulanase activity
(Off) 1 2 3 4 5
(U/I)
0.85 0.76 1.22 1.00 0.95
5.0 4.0 4.5 6.5 5.5
25 30 120 200 250
* Basal medium (1) alone or with glucose (2), maltose (3), amylopectin (4) or pullulan (5) grown as given in Materials and Methods. t~, Cultivation time of maximum enzyme concentration.
Results and Discussion Bacillus stearothermophilus grew and produced pullulanase in all media used (Table 1). The maximum biomass (1.22 g/l) was achieved after 6.5 h of cultivation in maltose-containing medium. Although the strain produced pullulanase (25 U/l) in the basal nutrient medium, highest activity was obtained using a medium to which pullulan had been added. The carbon source influenced not only the enzyme yield but the time course of the process. Pullulanase was first detected after 2 h of cultivation when glucose, maltose, amylopectin or pullulan were used but when grown in the basal medium not until about 4 h. The activity of pullulanase remained constant when the culture was in stationary phase. There is much controversy over the regulation of pullulanase synthesis in different bacterial strains and the effect of carbon source. For example, unlike the results of our experiments, glucose was an effective carbon source for
A
B
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Figure 1. Cell growth (X) O---O; pullulanase activity (E) H ;
($2) A---z~; dissolved oxygen level (DO) ment, (B) oxygen-limited experiment.
1
2
3
l,
Time
Timelh}
9.2
t
x-.-x;
World Journal of Microbiology and Biotechnology, Vol 8, 1992
5
6
7
8
[ h I
total residual carbohydrates ($1) A - - A ; residual reducing sugars using amylopectin as carbon source. (A) Oxygen-unlimited experi-
Pullulanase production by B. stearothermophilus A $I
$1
9
E
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x
1,0I !2~
li0o i~
/ ',. \./
o
1
2
3
/, 5 Time [hi
6
7
8
I
2
3
4
5
6
7
8
Time (h)
Figure 2. Cell growth (X) O - - O ; pullulanase activity (E) H ; ($2) A---z~; dissolved oxygen level (DO) x - . - x ; oxygen-limited experiment.
total residual carbohydrates ($1) i F i ; residual reducing sugars using pullulan as carbon source. (A) oxygen-unlimited experiment, (B)
pullulanase yields using pullulan or amylopectin as carbon source the culture has to be grown under oxygen limitation.
References Abdullah, M. & French, D. 1966 Reversible action of pullulanase. Nature 210, 200. Dean, A.C.R., Gray, S.C., Hope, G.C. & Johnson, I.M. 1972 Pullulanase synthesis in Klebsiella (Aerobacter) aerogenes. Biochemical Journal 126, 15P-16P. Kambourova, M. & Emanuilova, E. 1987 Biosynthesis and properties of extracellular pullulanase from Bacillus stearothermophilus G-82. In ExtracellularEnzymes of Microorganisms, eds Chaloupka, J. & Krumphanzl M.V., pp. 195-199. New York: Plenum Press. Madi, E., Antranikian, G., Ohmiya, K. & Gottschalk, G. 1987 Thermostable amylolytic enzymes from a new Clostridium isolate. Applied and Environmental Microbiology 53, I661-1667.
Ng, T.K. & Kenealy, W.F. 1986 Industrial applications of thermostable enzymes. In Thermophiles: general, molecular and applied microbiology, ed. Brock, T.D. pp. I97-215. New York: John Wiley. Somogyi, M. 1952 Notes on sugar determination. Journal of Biological Chemistry 195, 19-23. Suzuki, Y. & Chishiro, M. 1983 Production of extracellular thermostable pullulanase by an amylolytic obligately thermophilic soil bacterium, Bacillus stearothermophilus KP 1064. European Journal of Applied Microbiology and Biotechnology 17, 24-29. Takasaki, Y. 1976 Productions and utilizations of//-amylase and pullulanase from Bacillus cereus var. mycoides. Agricultural and Biological Chemistry 40, 1515-1522.
(Received in revised form 30 May 199I; accepted 2 June 199I)
World Journal of Microbiology and Biotechnology, Vol 8, 1992
2..~
Effect of carbon source and dissolved oxygen level on cell growth and pullulanase production by Bacillus stearothermophilus G-82 E.I. Emanuilova and M.S. Kambourova* Cell growth and extracellular pullulanase production of Bacillus sfearothermophilus G - 8 2 were investigated in batch cultures using a defined medium with glucose, maltose, pullulan or amylopectin as carbon source. Maximum enzyme activity was with pullulan or amylopectin. Cell g r o w t h in batch culture was better under o x y g e n unlimited conditions, while higher total and specific enzyme activities, using pullulan or amylopectin, were obtained in oxygen-limited conditions. Enzyme accumulation t o o k place in the late g r o w t h phase. The highest enzyme production of 300 U/I was reached when pullulan was used as carbon source in conditions of o x y g e n limitation.
Key words: Pullulanase production, Bacillus stearothermophilus, carbon source, dissolved oxygen.
Microbial pullulanases (pullulan 1--* 0 glucan hydrolase, EC 3.2.1.41) which catalyse the hydrolysis of 0r 1 ~ 6 glucosidic bonds in pullulan, amylopectin, d-limit dextrins and glycogen are of industrial importance for maltose and maltose syrup production. There is an increased interest in producing thermostable pullulanase to function above 60~ (Ng & Kenealy I986). Previously we have reported that a thermophilic strain of Bacillus stearothermophilus G-g2 produced a thermostable pullulanase (Kambourova & Emanuilova 1987) and the present paper deals with the effect of different carbon sources and of dissolved oxygen on cell growth and pullulanase production.
Materials and Methods Strain Bacillus stearothermopkdus G-82 isolated from a Bulgarian hot spring was used. The strain was deposited in the National Collection for Industrial Microorganisms and Cell Cultures, Sofia, Bulgaria.
Media and Cultivation The stock culture of the strain was maintained on peptone ~gar containing (g/l): Bactopeptone, 3; yeast extract, 3; starch, I, pH The authors are at the Institute of Microbiology, Department of Enzyme Biosynthesis, Acad. G. Bonchev str. B1.26, 1113 Sofia, Bulgaria. *Corresponding author.
7.8. The basal liquid medium contained (g/l): Bactopeptone, 3; yeast extract, 3, pH 7.8. Glucose, maltose, amylopectin or pullulan (1.0 g/l) were added and used as carbon sources. Peptone and pullulan (1 g) contained, respectively, 0.68 and 1.3 mg maltose. Medium was sterilized at 120~C for 30 rain. An inoculum of 3.5 ml (from a 3-h flask-shake culture) was used. Fermentation was carried out in a 'Bioflo' stirred fermenter (Model C-30, New Brunswick Co., Edison, New Jersey, USA) with a working volume of 0.350 litre at 55~ and an air-flow rate of 1.0 vol/vol/min, pH was not controlled. The dissolved oxygen was recorded by a dissolved oxygen probe (New Brunswick Scientific, Model DO-50).
Assays Biomass was determined turbidometricaUy. An optical density of 1.0 at 660 n m = 0.9 g cell dry wt/l. Pullulanase activity of the cu2ture supernatants was assayed at 65~ and pH 7.0 by the method of Suzuki & Chishiro (1983). Residual reducing sugars in culture supematants were determined by the method of Somogyi (1952). Total residual carbohydrates were determined by mixing 2 ml of the supematant with I ml of 3 M HCI, boiled for 3 h and then neutralized with NaHCO 3. Released reducing sugars were measured according to the method of Somogyi (1952).
Chemicals Glucose was obtained from Reachira (USSR); maltose and amylopectin from Fluka (Buchs, Switzerland); pullulan from Hayashibara Biochem. Lab. Inc. (Japan); and sugar standards for thin-layer chromatography from Boehringer (Mannheim, GmbH, Germany). All other reagents used were of analytical grade.
9 1992 Rapid Communications of Oxford Ltd l~orld ]ot~rrlal of Microbiology and BW~eclmoloo~y,Vol 8, .7992
~1
E.I, Emanuilova and M.S. Kambourova pullulanase production by Bacillus cereus var. mycoides (Takasaki 1976) and pullulan had no effect on the enzyme synthesized by B. stearothermophilus KP 1064 (Suzuki & Chishiro 1983). High concentrations of pullulanase could be detected in the culture of Clostridium sp. EM1 (Madi et al. 1987) when pullulan and maltose served as substrates and thus these results resemble our own experiments. The function of pullulanase in maltose-utilizing cells is not clear. Abdullah & French (1966) reported that besides hydrolysis of oligosaccharides, pullulanase catalyses the synthesis of 1,6 dimer or trimer from maltose. Dean et al. (1972) supposed a possible function of the enzyme in the synthesis of polysaccharides from maltose in maltose utilizing cells. This could explain the inducible effect of maltose and malto-oligosaccharides in pullulanase synthesis by B. stearothermophilus G-82. To improve enzyme yield, the effect of dissolved oxygen concentration on cell growth and pullulanase production was studied when amylopectin or pullulan were used as carbon source. The air-flow rate in these experiments was I vol/vol/min and agitation speed was applied to maintain a minimum dissolved oxygen level of 30% (over 450 rev/min) or 0% saturation for 1 to 2 h (between 350 and 450 rev/min). The results of these experiments are presented in Figures 1 and 2. Enzyme production of the strain, which paralleled the growth, resembled that in B. cereus var. mycoides (Takasaki 1976) and Clostridium sp. EM1 (Madi et al. I987). The maximum cell concentration of B. stearothermophilus was lower and maximum enzyme activity was higher for oxygen-limited cultures than for oxygen unlimited. Analysis of spent culture filtrates revealed that none of the experiments were carbon limited. The results in Figures 1 and 2 provide convincing evidence that to obtain higher
Table 1. Effect of different carbon sources on growth and pullulanase production in culture filtrates. Medium*
Cell dry weight
t~ (h)
Pullulanase activity
(Off) 1 2 3 4 5
(U/I)
0.85 0.76 1.22 1.00 0.95
5.0 4.0 4.5 6.5 5.5
25 30 120 200 250
* Basal medium (1) alone or with glucose (2), maltose (3), amylopectin (4) or pullulan (5) grown as given in Materials and Methods. t~, Cultivation time of maximum enzyme concentration.
Results and Discussion Bacillus stearothermophilus grew and produced pullulanase in all media used (Table 1). The maximum biomass (1.22 g/l) was achieved after 6.5 h of cultivation in maltose-containing medium. Although the strain produced pullulanase (25 U/l) in the basal nutrient medium, highest activity was obtained using a medium to which pullulan had been added. The carbon source influenced not only the enzyme yield but the time course of the process. Pullulanase was first detected after 2 h of cultivation when glucose, maltose, amylopectin or pullulan were used but when grown in the basal medium not until about 4 h. The activity of pullulanase remained constant when the culture was in stationary phase. There is much controversy over the regulation of pullulanase synthesis in different bacterial strains and the effect of carbon source. For example, unlike the results of our experiments, glucose was an effective carbon source for
A
B
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m
$1 2
1,0
w O,S
[ i200
E
w
0O ---'x--x,.,,,.x
DO
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~
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,/
v
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/
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,=
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li,oo
3
~o~
x 1
2
3
/,
5
G
7
8
Figure 1. Cell growth (X) O---O; pullulanase activity (E) H ;
($2) A---z~; dissolved oxygen level (DO) ment, (B) oxygen-limited experiment.
1
2
3
l,
Time
Timelh}
9.2
t
x-.-x;
World Journal of Microbiology and Biotechnology, Vol 8, 1992
5
6
7
8
[ h I
total residual carbohydrates ($1) A - - A ; residual reducing sugars using amylopectin as carbon source. (A) Oxygen-unlimited experi-
Pullulanase production by B. stearothermophilus A $I
$1
9
E
\
x
1,0I !2~
li0o i~
/ ',. \./
o
1
2
3
/, 5 Time [hi
6
7
8
I
2
3
4
5
6
7
8
Time (h)
Figure 2. Cell growth (X) O - - O ; pullulanase activity (E) H ; ($2) A---z~; dissolved oxygen level (DO) x - . - x ; oxygen-limited experiment.
total residual carbohydrates ($1) i F i ; residual reducing sugars using pullulan as carbon source. (A) oxygen-unlimited experiment, (B)
pullulanase yields using pullulan or amylopectin as carbon source the culture has to be grown under oxygen limitation.
References Abdullah, M. & French, D. 1966 Reversible action of pullulanase. Nature 210, 200. Dean, A.C.R., Gray, S.C., Hope, G.C. & Johnson, I.M. 1972 Pullulanase synthesis in Klebsiella (Aerobacter) aerogenes. Biochemical Journal 126, 15P-16P. Kambourova, M. & Emanuilova, E. 1987 Biosynthesis and properties of extracellular pullulanase from Bacillus stearothermophilus G-82. In ExtracellularEnzymes of Microorganisms, eds Chaloupka, J. & Krumphanzl M.V., pp. 195-199. New York: Plenum Press. Madi, E., Antranikian, G., Ohmiya, K. & Gottschalk, G. 1987 Thermostable amylolytic enzymes from a new Clostridium isolate. Applied and Environmental Microbiology 53, I661-1667.
Ng, T.K. & Kenealy, W.F. 1986 Industrial applications of thermostable enzymes. In Thermophiles: general, molecular and applied microbiology, ed. Brock, T.D. pp. I97-215. New York: John Wiley. Somogyi, M. 1952 Notes on sugar determination. Journal of Biological Chemistry 195, 19-23. Suzuki, Y. & Chishiro, M. 1983 Production of extracellular thermostable pullulanase by an amylolytic obligately thermophilic soil bacterium, Bacillus stearothermophilus KP 1064. European Journal of Applied Microbiology and Biotechnology 17, 24-29. Takasaki, Y. 1976 Productions and utilizations of//-amylase and pullulanase from Bacillus cereus var. mycoides. Agricultural and Biological Chemistry 40, 1515-1522.
(Received in revised form 30 May 199I; accepted 2 June 199I)
World Journal of Microbiology and Biotechnology, Vol 8, 1992
2..~
