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Enhancement of α-amylase production by immobilized Bacillus subtilis in an airlift fermenter
Enhancement of a-amylase production by immobilized Bacillus subtilis in an airlift fermenter Pierre Chevalier* and Joifl de la Noiie*' t * Centre de Recherche en Nutrition and f D~partement de Biologie, Universitb Laval, Qubbec, Canada G I K 7P4
(Received 16 May 1986; revised 28 July 1986)
Bacillus subtiilis A TCC 21770 was entrapped in a carrageenan gel, especially formulated for immobilization. Bacterial growth and ¢t-amylase (1,4-Ot-D-olucan 91ueanohydrolase EC 3.2.1.1) production were tested. The bead suspensions were submitted to two aeration modes, one consistin9 of bubbling air into a round flask, the other involvin9 sparoin9 of air into an airlift fermenter. The latter system, which produces microbubbles, 9ave 40-70% increase in enzyme production over the former and a doublin9 of bacterial density within the beads was measured. The use of CaCl 2 instead of K Cl as polymerization aoent led to a better yield of ~-amylase.
Keywords:Bacillus subtilis; or-amylase;carrageenan; airlift fermenter;immobilization
Introduction
Test organism
Enzyme production by immobilized bacteria is an active research area despite the fact that industrial applications are still limited. 1 Research on a-amylase production by immobilized Bacillus sp, for example, was initiated a few years ago first using polyacrylamide 2 and carrageenan gels 3 in shaken flasks. Immobilization was shown to be beneficial 3' 4 even though mass transfer problems were identified. For example, the production of metabolites or enzymes like at-amylase by strictly aerobic bacteria such as Bacillus subtilis raises serious oxygen diffusion problems inside the beads. This aspect of oxygen diffusion has been studied for several years by a Swedish research team which tried to improve oxygen diffusion and availability in calcium alginate beads by physical and chemical means, s-8 Unfortunately, the use of these often toxic substances places limits on the utility of such a process. The purpose of the present work was to compare the efficacy of an airlift fermenter to that of aerated round flasks for the growth of immobilized B. subtilis and the production of amylase and to assess the potential advantages of a carrageenan especially formulated for immobilization procedures over a standard food grade carrageenan commonly used to entrap microorganisms.
The bacterial strain used was Bacillus subtilis ATCC 21770 known to produce or-amylase. G r o w t h and p r o d u c t i o n m e d i a
The growth medium consisted of (g l-X): N H 4 N O 3, 0.5; KzHPO,,, 0.03; CaC12 • 2H20, 0.05; yeast extract, 0.5, cassitone, 1.8. The mixture was buffered at pH 7.2 with a Tris-HC1 solution (20 mM). This medium was used for the maintenance (on agar slants) and the preculture of bacteria. The production medium was as above, with the addition of 12 g 1-1 starch. Immobilization
Immobilization procedure involved preculturing the cells in 25 ml growth medium for 18 h. This broth was then added to 125 ml sterile carrageenan solution, the final concentration being 3%. The mixture was aseptically transferred with a specially designed multineedle bead maker fitted with no. 18 syringe needles to a gently stirred 0.3 M KCI or 0.3 M CaC12 solution to produce spherical beads of about 3 4 mm. The particles were soaked in the solutions for 30 min. E n z y m e p r o d u c t i o n b y f r e e and i m m o b i l i z e d cells
The carrageenan used was NJAL 798 (batch E4 13-60) especially developed for immobilization procedures and kindly provided by Marine Colloids Corporation (Rockland, ME). All the chemicals used were reagent grade.
For experiments with free cells, 25 ml precultured broth (18 h) was inoculated into 500 ml production medium. For experiments with immobilized bacteria, 150 g gel beads (containing 25 ml precultured broth) were incubated in 500 ml of the same medium and a semicontinuous mode was used: after 24 h growth, the beads were removed, washed four times with sterile 0.3 M KCI or CaC12 solution and reincubated in fresh production medium. Incubations were performed in
0141-0229/87/010053-04 $03.00 ~) 1987 Butterworth & Co. (Publishers) Ltd
Enzyme Microb. Technol., 1987, vol. 9, January
Materials and methods Chemicals
53
Papers 1 litre round flasks with an aeration rate of |/3 vvm through a 2 m m (i.d.) glass tube and in a BRL Airlift Fermenter (Bethesda Research Laboratory, MD) under similar aeration through a 5 #m pore sparger. Incubation temperature was 30°C.
Analytical methods To measure cell density inside the gel, aliquots of beads were taken out, gently melted and poured into spectrophotometer cuvettes (1 cm pathlength). Absorbance was measured at 660 nm (ref. 9) with an LKB spectrophotometer model Ultrospec II. Viability of free cells was determined by agar plate counts using production medium after 48 h incubation at 30°C. Viability of bacteria released into the medium, in the case of CaC12 utilization, was measured by taking 25 ml culture broth at t = 24 h and inoculating it into 500 ml fresh production medium. Growth was monitored as described above. The ~-amylase concentration of the medium was measured by the Sigma (St Louis, MO) diagnostic kit no. 575 U.V. based on enzymatic breakdown of maltotetraose resulting, as a final step, in the generation of N A D H which was measured at 340 nm. One enzymatic unit of amylase is defined as the amount of enzyme that results in the production of 7 pmol of maltose per minute at 30°C. Dissolved oxygen was measured with a YSI oxygen meter (model 58).
Results Effects of aeration on free cells Bacterial growth and enzyme production with the two aeration modes used are shown in Figure 1. In round flasks the early growth of B. subtilis was slower than in the fermenter but m a x i m u m biomass concentrations were identical after 24 h (A660 of 1.60 VS 1.65). The maximum growth rates (Pro.x) were 0.55 h -a for the airlift and 0.37 h -1 for round flasks. A more striking difference was observed for amylase production: in the airlift fermenter peak production was observed after 12 h (18 units) as compared to 24 h in the flasks (15.7 units). For the remainder of the fermentation time the pattern was similar for both. At the beginning of fermentation dissolved oxygen was 7.09 mg l -x (99.4% saturation) for the airlift fermenter and 5.20 mg 1-1 (71% saturation) in flasks.
-E
1.8
18
1.6
16 ~"
14
14
1.2
12
1.0
10
~o.8
"E
8
o0.6
6
0.4
4 <
0.2
2
0.0
0 0
10
20
30
40
Time (h)
Figure 1
Growth of free Bacillus subtilis and amylase production. 17, m, Growth; A, A, amylase production; I-7, A, flask; I , A, fermenter
54
Enzyme Microb. Technol., 1987, vol. 9, January
18 16
1.4 1.2
14 ~E= 1.0
12 .~
o= 0.8
10 g
0.6
8 6
o 0.4 L9
4
0.2
<
2 0
0.0 0
5
10
15
20
25
30
35
40
Time (h)
Figure 2 Growth and amylase production by Bacillus subtilis immobilized in NJAL carrageenan in round flask under a semicontinuous mode, starting at 24 h . . , Cells inside beads; [], cells released into medium; A, amylase production
Growth and production in round flasks Fioure 2 shows the results obtained under a semicontinuous mode (starting after 24 h). Immobilized B. subtilis grew within the gel, with a #m,~ half that of free cells (0.19 h -1) up to 9 h after which a significant decrease in the bacterial biomass density in the gel occurred up to 24 h. U p o n the addition of fresh medium, at t = 24 h, a#max of 0.15 h -1 was measured as growth resumed but decreased rapidly and reached a plateau phase at t = 33 h with A66 o of 0.66. Five hours after the start of the fermentation, free cells appeared in the medium and showed a high /~m,x (0.62 h 1). Their appearance was more rapid during the second part of the fermentation, after only a few minutes, and their growth showed a pattern broadly similar to that observed in the first part. Amylase could be detected in the medium after 6 h and a peak of 17 units was observed after 24 h. During the second part of the cycle amylase appeared after a shorter interval. After this additional period of 12 h (t = 36 h) 13.0 units of enzyme activity were measured.
Growth and production followin 9 use of CaCI 2 to polymerize N J A L carraoeenan Figure 3 shows results obtained in the airlift fermenter with N J A L carrageenan which was soaked in CaC12 instead of KC1. A high bacterial density was obtained after 12 h but no decrease was observed between 12 and 24 h; the addition of fresh medium did not stimulate growth inside beads. As previously observed with fermentation performed in the airlift fermenter, using KCI (data not shown), the release of cells from beads and their subsequent growth was low but in this particular case it was still lower. Amylase production was good: it could be detected in the medium after only 3 h, during the first cycle, and attained nearly 30 units in each cycle. A viability test performed on released cells showed linear, rather than logarithmic growth. After 24 h, cell density reached 0.6 (data not shown) compared to 1.6 for healthy free cells in the airlift system (Figure 1). However this cell biomass was twice that observed at t = 24 h for released cells which was 0.3 (Fioure 3).
Amylase production by immobilized B. subtilis: P. Chevalier and J. de la NoOe 2.5
30 25 ~" L E 2O "E
~=E 2,0 o 1.5 1.o
10
~0.5
5 <
0.0
0 0
5
10
15
20
25
30
35
40
Time (h)
Figure 3
Growth and amylase production by B. subtili$ in carrageenan, with CaCI 2 for hardening, in the airlift fermenter with a semicontinuous mode at 24 h. Refer to Figure 2 for symbol explanations
Discussion Some points must be clarified at the outset. It was decided to stop the second cycle of the semicontinuous fermentation after 12 h because in some cases further incubation resulted in such a release of cells into the medium that one could not really distinguish between enzyme production by immobilized or free cells. Because our aim was to determine the amylase production by immobilized bacteria, only the relevant part of each experiment was therefore monitored. M a n y attempts were made to solubilize carrageenan beads in 1% sodium citrate solution 1° or in saline solution 1~ but we were unable to obtain a suitable breakdown of the gels polymerized in KC1 solution. This explains the methodology chosen for measuring bacterial biomass. The results obtained with free cells in fermentation flasks and in the airlift fermenter (Figure 1) suggest that oxygen limitation occurred in the former although the air flow rate was the same in both cases. The presence of air microbubbles in the airlift system resulted in a higher oxygen solubility and could lead to a better transfer of oxygen. As shown by Fogarty and Bourke x2 a higher biomass gives a higher amylase concentration and a similar pattern was observed here. Thereafter, when nutrients, including starch, are exhausted and metabolites begin to accumulate in the medium (stationary phase), oxygen does not seem to be the limiting factor and a-amylase is no longer synthesized. Similar results were obtained with bacteria immobilized and grown in flasks (Figure 2). The significant decrease of bacterial density in gel, after 9 h, could be attributed to unfavourable oxygen conditions for immobilized cells, thus leaving nutrients available for free cells which grew rapidly and could significantly contribute to amylase synthesis. Shynmyo et al. 3 noted the same pattern with Bacillus amyloliquefaciens immobilized in carrageenan; from 8 to 32 h they observed a diminution of cell mass in beads while free bacteria continued to grow exponentially. This pattern seems to be typical of a batch fermentation in flasks and correlates with our results. The relationship between bacterial biomass and enzyme production is probably influenced by diffusion problems in the carragccnan beads, aAmylase is produced during the decrease in bacterial density in the beads and during the noticeable growth of free bacteria in the medium suggesting that the later
ones are those which contribute to enzyme synthesis; this indicates the occurrence of some impairments which could be due to nutrient or oxygen shortage in gels as noted by others. 13' 14 Figure 3 indicates interesting results. First, there was almost no loss of bacteria from the gel, resulting in few free bacteria in the medium; this could facilitate amylase recovery. We observed that beads formed with CaCI 2 instead of KCl were more brittle (data not shown); after ~ 9 h some beads began to break apart and some fragments of gel began to appear in the culture medium, thus increasing the surface available for colonization and for secretion of amylase which was released more rapidly and in larger quantities. The results of viability tests indicated that cells released into the medium were not healthy but their growth in the absence of colonized beads was better: this indicated that they could not sustain competition with immobilized cells, hence a lesser growth. It is worth noting that a-amylase would also benefit from the presence of calcium in the medium because it requires this metal for catalytic activity) 5 In brief, Figure 1 shows that after 24 h the aeration mode was without effect on bacterial growth or amylase synthesis. Figure 2 indicates that during the second cycle it became difficult to differentiate between amylase production from free or immobilized cells, a fact often neglected by m a n y authors. However, results of Figure 3 leave no doubt that a-amylase comes from immobilized bacteria. In conclusion, it was demonstrated that an airlift fermenter can increase biomass and a-amylase production not only by free bacteria but also by immobilized ones; the increase of the enzyme reached a maximum of 70% in the case of immobilized cells polymerized with CaC12 . The use of an airlift fermenter may bypass the addition of chemicals likely to interfere with the purity of the recovered product or the use of costly physical means such as supplying pure oxygen. The N J A L carrageenan specially developed for immobilization procedures is attractive because it is usable at room temperature in solution.
Acknowledgements We are grateful to the Fonds F C A R of the Ministry of Education of Quebec and the Centre de Recherche en Nutrition for financial support. We thank Dr J. Goulet for reviewing the manuscript and Dr Ni Eidhin for improving it.
References 1 Chibata, I. and Tosa, T. Immobilized Microbial Cells Academic Press, 1983 2 Kokubu, T., Karube, I. and Suzuki, S. Eur. J. Appl. Microbiol. BiotechnoL 1978, 5, 233-240 3 Shinmyo, A., Kimura, H. and Okoda, H. Eur. J. Appl. Microbiol. BiotechnoL 1982, 14, 7-12 4 Mattiasson, B. and Hahn-H~igerdal, B. Eur. J. Appl. Microbiol. Biotechnol. 1982, 16, 52-55 5 Adlercreutz, P., Hoist, O. and Mattiasson, B. Enzyme Microb. Technol. 1982, 4, 395-400 6 Adlercreutz, P. and Mattiasson, B. Appl. Microbiol. Biotechnol. 1984, 20, 296-302 7 Adlercreutz, P., Holst, O. and Mattiasson, B. Appl. Microbiol. Biotechnol. 1985, 22, 1-7
Enzyme Microb. Technol., 1987, vol. 9, January
55
Papers 8
Hoist, O., Lunbiick, H. and Mattiasson, B. Appl. Microbiol. Bioteehnol. 1985, 22, 383-388 9 Koch, A.L. in Manual of Methods for General Bacteriolooy (Costilow, R.N. et al., eds), American Society for Microbiology, Washington DC, 1981, pp. 179-207 10 Baudet, C., Barbotin, J.N. and Guespin-Michel, J. Appl. Environ. Mierobiol. 1983, 45, 297-301 11 Wada, M., Kato, J. and Chibata, I. Eur. J. Appl. Microbiol. Bio-
56
Enzyme Microb. Technol., 1987, vol. 9, January
technol. 1980, 10, 275-287 12 Fogarty, W.M. and Bourke, E.J.J. Chem. Technol. Biotechnol. 1983, 338, 145-154 13 Toda, K. and Sato, K. J. Ferment. Technol. 1984, 19, 79-84 14 Fujimura, M., Kato, J., Tosa, T. and Chibata, I. Appl. Microbiol. Biotechnol. 1984, 19, 79-84 15 Fogarty, W.M. and Kelley, C.T. Pro#. Ind. Microbiol. 1979, 15, 89-142
(Received 16 May 1986; revised 28 July 1986)
Bacillus subtiilis A TCC 21770 was entrapped in a carrageenan gel, especially formulated for immobilization. Bacterial growth and ¢t-amylase (1,4-Ot-D-olucan 91ueanohydrolase EC 3.2.1.1) production were tested. The bead suspensions were submitted to two aeration modes, one consistin9 of bubbling air into a round flask, the other involvin9 sparoin9 of air into an airlift fermenter. The latter system, which produces microbubbles, 9ave 40-70% increase in enzyme production over the former and a doublin9 of bacterial density within the beads was measured. The use of CaCl 2 instead of K Cl as polymerization aoent led to a better yield of ~-amylase.
Keywords:Bacillus subtilis; or-amylase;carrageenan; airlift fermenter;immobilization
Introduction
Test organism
Enzyme production by immobilized bacteria is an active research area despite the fact that industrial applications are still limited. 1 Research on a-amylase production by immobilized Bacillus sp, for example, was initiated a few years ago first using polyacrylamide 2 and carrageenan gels 3 in shaken flasks. Immobilization was shown to be beneficial 3' 4 even though mass transfer problems were identified. For example, the production of metabolites or enzymes like at-amylase by strictly aerobic bacteria such as Bacillus subtilis raises serious oxygen diffusion problems inside the beads. This aspect of oxygen diffusion has been studied for several years by a Swedish research team which tried to improve oxygen diffusion and availability in calcium alginate beads by physical and chemical means, s-8 Unfortunately, the use of these often toxic substances places limits on the utility of such a process. The purpose of the present work was to compare the efficacy of an airlift fermenter to that of aerated round flasks for the growth of immobilized B. subtilis and the production of amylase and to assess the potential advantages of a carrageenan especially formulated for immobilization procedures over a standard food grade carrageenan commonly used to entrap microorganisms.
The bacterial strain used was Bacillus subtilis ATCC 21770 known to produce or-amylase. G r o w t h and p r o d u c t i o n m e d i a
The growth medium consisted of (g l-X): N H 4 N O 3, 0.5; KzHPO,,, 0.03; CaC12 • 2H20, 0.05; yeast extract, 0.5, cassitone, 1.8. The mixture was buffered at pH 7.2 with a Tris-HC1 solution (20 mM). This medium was used for the maintenance (on agar slants) and the preculture of bacteria. The production medium was as above, with the addition of 12 g 1-1 starch. Immobilization
Immobilization procedure involved preculturing the cells in 25 ml growth medium for 18 h. This broth was then added to 125 ml sterile carrageenan solution, the final concentration being 3%. The mixture was aseptically transferred with a specially designed multineedle bead maker fitted with no. 18 syringe needles to a gently stirred 0.3 M KCI or 0.3 M CaC12 solution to produce spherical beads of about 3 4 mm. The particles were soaked in the solutions for 30 min. E n z y m e p r o d u c t i o n b y f r e e and i m m o b i l i z e d cells
The carrageenan used was NJAL 798 (batch E4 13-60) especially developed for immobilization procedures and kindly provided by Marine Colloids Corporation (Rockland, ME). All the chemicals used were reagent grade.
For experiments with free cells, 25 ml precultured broth (18 h) was inoculated into 500 ml production medium. For experiments with immobilized bacteria, 150 g gel beads (containing 25 ml precultured broth) were incubated in 500 ml of the same medium and a semicontinuous mode was used: after 24 h growth, the beads were removed, washed four times with sterile 0.3 M KCI or CaC12 solution and reincubated in fresh production medium. Incubations were performed in
0141-0229/87/010053-04 $03.00 ~) 1987 Butterworth & Co. (Publishers) Ltd
Enzyme Microb. Technol., 1987, vol. 9, January
Materials and methods Chemicals
53
Papers 1 litre round flasks with an aeration rate of |/3 vvm through a 2 m m (i.d.) glass tube and in a BRL Airlift Fermenter (Bethesda Research Laboratory, MD) under similar aeration through a 5 #m pore sparger. Incubation temperature was 30°C.
Analytical methods To measure cell density inside the gel, aliquots of beads were taken out, gently melted and poured into spectrophotometer cuvettes (1 cm pathlength). Absorbance was measured at 660 nm (ref. 9) with an LKB spectrophotometer model Ultrospec II. Viability of free cells was determined by agar plate counts using production medium after 48 h incubation at 30°C. Viability of bacteria released into the medium, in the case of CaC12 utilization, was measured by taking 25 ml culture broth at t = 24 h and inoculating it into 500 ml fresh production medium. Growth was monitored as described above. The ~-amylase concentration of the medium was measured by the Sigma (St Louis, MO) diagnostic kit no. 575 U.V. based on enzymatic breakdown of maltotetraose resulting, as a final step, in the generation of N A D H which was measured at 340 nm. One enzymatic unit of amylase is defined as the amount of enzyme that results in the production of 7 pmol of maltose per minute at 30°C. Dissolved oxygen was measured with a YSI oxygen meter (model 58).
Results Effects of aeration on free cells Bacterial growth and enzyme production with the two aeration modes used are shown in Figure 1. In round flasks the early growth of B. subtilis was slower than in the fermenter but m a x i m u m biomass concentrations were identical after 24 h (A660 of 1.60 VS 1.65). The maximum growth rates (Pro.x) were 0.55 h -a for the airlift and 0.37 h -1 for round flasks. A more striking difference was observed for amylase production: in the airlift fermenter peak production was observed after 12 h (18 units) as compared to 24 h in the flasks (15.7 units). For the remainder of the fermentation time the pattern was similar for both. At the beginning of fermentation dissolved oxygen was 7.09 mg l -x (99.4% saturation) for the airlift fermenter and 5.20 mg 1-1 (71% saturation) in flasks.
-E
1.8
18
1.6
16 ~"
14
14
1.2
12
1.0
10
~o.8
"E
8
o0.6
6
0.4
4 <
0.2
2
0.0
0 0
10
20
30
40
Time (h)
Figure 1
Growth of free Bacillus subtilis and amylase production. 17, m, Growth; A, A, amylase production; I-7, A, flask; I , A, fermenter
54
Enzyme Microb. Technol., 1987, vol. 9, January
18 16
1.4 1.2
14 ~E= 1.0
12 .~
o= 0.8
10 g
0.6
8 6
o 0.4 L9
4
0.2
<
2 0
0.0 0
5
10
15
20
25
30
35
40
Time (h)
Figure 2 Growth and amylase production by Bacillus subtilis immobilized in NJAL carrageenan in round flask under a semicontinuous mode, starting at 24 h . . , Cells inside beads; [], cells released into medium; A, amylase production
Growth and production in round flasks Fioure 2 shows the results obtained under a semicontinuous mode (starting after 24 h). Immobilized B. subtilis grew within the gel, with a #m,~ half that of free cells (0.19 h -1) up to 9 h after which a significant decrease in the bacterial biomass density in the gel occurred up to 24 h. U p o n the addition of fresh medium, at t = 24 h, a#max of 0.15 h -1 was measured as growth resumed but decreased rapidly and reached a plateau phase at t = 33 h with A66 o of 0.66. Five hours after the start of the fermentation, free cells appeared in the medium and showed a high /~m,x (0.62 h 1). Their appearance was more rapid during the second part of the fermentation, after only a few minutes, and their growth showed a pattern broadly similar to that observed in the first part. Amylase could be detected in the medium after 6 h and a peak of 17 units was observed after 24 h. During the second part of the cycle amylase appeared after a shorter interval. After this additional period of 12 h (t = 36 h) 13.0 units of enzyme activity were measured.
Growth and production followin 9 use of CaCI 2 to polymerize N J A L carraoeenan Figure 3 shows results obtained in the airlift fermenter with N J A L carrageenan which was soaked in CaC12 instead of KC1. A high bacterial density was obtained after 12 h but no decrease was observed between 12 and 24 h; the addition of fresh medium did not stimulate growth inside beads. As previously observed with fermentation performed in the airlift fermenter, using KCI (data not shown), the release of cells from beads and their subsequent growth was low but in this particular case it was still lower. Amylase production was good: it could be detected in the medium after only 3 h, during the first cycle, and attained nearly 30 units in each cycle. A viability test performed on released cells showed linear, rather than logarithmic growth. After 24 h, cell density reached 0.6 (data not shown) compared to 1.6 for healthy free cells in the airlift system (Figure 1). However this cell biomass was twice that observed at t = 24 h for released cells which was 0.3 (Fioure 3).
Amylase production by immobilized B. subtilis: P. Chevalier and J. de la NoOe 2.5
30 25 ~" L E 2O "E
~=E 2,0 o 1.5 1.o
10
~0.5
5 <
0.0
0 0
5
10
15
20
25
30
35
40
Time (h)
Figure 3
Growth and amylase production by B. subtili$ in carrageenan, with CaCI 2 for hardening, in the airlift fermenter with a semicontinuous mode at 24 h. Refer to Figure 2 for symbol explanations
Discussion Some points must be clarified at the outset. It was decided to stop the second cycle of the semicontinuous fermentation after 12 h because in some cases further incubation resulted in such a release of cells into the medium that one could not really distinguish between enzyme production by immobilized or free cells. Because our aim was to determine the amylase production by immobilized bacteria, only the relevant part of each experiment was therefore monitored. M a n y attempts were made to solubilize carrageenan beads in 1% sodium citrate solution 1° or in saline solution 1~ but we were unable to obtain a suitable breakdown of the gels polymerized in KC1 solution. This explains the methodology chosen for measuring bacterial biomass. The results obtained with free cells in fermentation flasks and in the airlift fermenter (Figure 1) suggest that oxygen limitation occurred in the former although the air flow rate was the same in both cases. The presence of air microbubbles in the airlift system resulted in a higher oxygen solubility and could lead to a better transfer of oxygen. As shown by Fogarty and Bourke x2 a higher biomass gives a higher amylase concentration and a similar pattern was observed here. Thereafter, when nutrients, including starch, are exhausted and metabolites begin to accumulate in the medium (stationary phase), oxygen does not seem to be the limiting factor and a-amylase is no longer synthesized. Similar results were obtained with bacteria immobilized and grown in flasks (Figure 2). The significant decrease of bacterial density in gel, after 9 h, could be attributed to unfavourable oxygen conditions for immobilized cells, thus leaving nutrients available for free cells which grew rapidly and could significantly contribute to amylase synthesis. Shynmyo et al. 3 noted the same pattern with Bacillus amyloliquefaciens immobilized in carrageenan; from 8 to 32 h they observed a diminution of cell mass in beads while free bacteria continued to grow exponentially. This pattern seems to be typical of a batch fermentation in flasks and correlates with our results. The relationship between bacterial biomass and enzyme production is probably influenced by diffusion problems in the carragccnan beads, aAmylase is produced during the decrease in bacterial density in the beads and during the noticeable growth of free bacteria in the medium suggesting that the later
ones are those which contribute to enzyme synthesis; this indicates the occurrence of some impairments which could be due to nutrient or oxygen shortage in gels as noted by others. 13' 14 Figure 3 indicates interesting results. First, there was almost no loss of bacteria from the gel, resulting in few free bacteria in the medium; this could facilitate amylase recovery. We observed that beads formed with CaCI 2 instead of KCl were more brittle (data not shown); after ~ 9 h some beads began to break apart and some fragments of gel began to appear in the culture medium, thus increasing the surface available for colonization and for secretion of amylase which was released more rapidly and in larger quantities. The results of viability tests indicated that cells released into the medium were not healthy but their growth in the absence of colonized beads was better: this indicated that they could not sustain competition with immobilized cells, hence a lesser growth. It is worth noting that a-amylase would also benefit from the presence of calcium in the medium because it requires this metal for catalytic activity) 5 In brief, Figure 1 shows that after 24 h the aeration mode was without effect on bacterial growth or amylase synthesis. Figure 2 indicates that during the second cycle it became difficult to differentiate between amylase production from free or immobilized cells, a fact often neglected by m a n y authors. However, results of Figure 3 leave no doubt that a-amylase comes from immobilized bacteria. In conclusion, it was demonstrated that an airlift fermenter can increase biomass and a-amylase production not only by free bacteria but also by immobilized ones; the increase of the enzyme reached a maximum of 70% in the case of immobilized cells polymerized with CaC12 . The use of an airlift fermenter may bypass the addition of chemicals likely to interfere with the purity of the recovered product or the use of costly physical means such as supplying pure oxygen. The N J A L carrageenan specially developed for immobilization procedures is attractive because it is usable at room temperature in solution.
Acknowledgements We are grateful to the Fonds F C A R of the Ministry of Education of Quebec and the Centre de Recherche en Nutrition for financial support. We thank Dr J. Goulet for reviewing the manuscript and Dr Ni Eidhin for improving it.
References 1 Chibata, I. and Tosa, T. Immobilized Microbial Cells Academic Press, 1983 2 Kokubu, T., Karube, I. and Suzuki, S. Eur. J. Appl. Microbiol. BiotechnoL 1978, 5, 233-240 3 Shinmyo, A., Kimura, H. and Okoda, H. Eur. J. Appl. Microbiol. BiotechnoL 1982, 14, 7-12 4 Mattiasson, B. and Hahn-H~igerdal, B. Eur. J. Appl. Microbiol. Biotechnol. 1982, 16, 52-55 5 Adlercreutz, P., Hoist, O. and Mattiasson, B. Enzyme Microb. Technol. 1982, 4, 395-400 6 Adlercreutz, P. and Mattiasson, B. Appl. Microbiol. Biotechnol. 1984, 20, 296-302 7 Adlercreutz, P., Holst, O. and Mattiasson, B. Appl. Microbiol. Biotechnol. 1985, 22, 1-7
Enzyme Microb. Technol., 1987, vol. 9, January
55
Papers 8
Hoist, O., Lunbiick, H. and Mattiasson, B. Appl. Microbiol. Bioteehnol. 1985, 22, 383-388 9 Koch, A.L. in Manual of Methods for General Bacteriolooy (Costilow, R.N. et al., eds), American Society for Microbiology, Washington DC, 1981, pp. 179-207 10 Baudet, C., Barbotin, J.N. and Guespin-Michel, J. Appl. Environ. Mierobiol. 1983, 45, 297-301 11 Wada, M., Kato, J. and Chibata, I. Eur. J. Appl. Microbiol. Bio-
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