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Oxygen uptake by κ-carrageenan entrapped Streptomyces clavuligerus
Oxygen uptake by K-carrageenan entrapped Streptomyces clavuligerus R. I. Scott, S. J. Wills and C. Bucke School o f Biotechnology, Polytechnic o f Central London, 115 N e w Cavendish Street, L o n d o n W 1 M 8JS, U K
(Received 8 May 1987; revised 2 October 1987)
Oxygen uptake rates have been measured in free and r-carrageenan immobilized cells of Streptomyces clavuligerus. Specific oxygen uptake rates of immobilized cells were only about 50% of the rates of free cells. The factors contributing to this reduction in oxygen uptake include the presence of carrageenan itself, internal oxygen diffusion limitations, the temperature of preparation of the immobilized cell beads and the presence of ions, particularly K ÷. Measurement of the hardness of individual gel beads showed that maximal hardness was reached after about 40 min exposure to 1 M KCl. However, during this exposure the specific oxygen uptake rate had fallen to only 20% of the rate of immobilized cells exposed to KCl for 2 rain.
Keywords: Oxygenuptake; r-carrageenan; immobilizedcells; Streptomyces clauuligerus Introduction Microorganisms and other cell types can be immobilized in a variety of different ways.l The advantages of using immobilized cells instead of free cells can be great and have been reviewed many times. 2,3 Entrapment in polymers, such as alginate or carrageenan, has been commonly carried out, mainly because the method is easy to perform and is believed to have few deleterious effects on the immobilized cells. Oxygen is required by cells to carry out respiratory and oxygenase functions. However, at high cell densities, diffusion of substrates, such as oxygen, into gelimmobilizing matrices may be rate limiting.4-6 For aerobic microorganisms, the supply of sufficient oxygen to gel immobilized cells may be problematic. 7,8 Various methods have been studied to try to increase the supply of oxygen to immobilized cells. These methods include the use of oxygen instead of air; 7 the use of coimmobilized algae and bacteria for in situ oxygen generation; 9 supplying extra oxygen via oxygen carders such as hemoglobin or emulsions of perfluorochemicals; ~° the decomposition of hydrogen peroxide using catalase. H In addition, the use of alternative electron acceptors, such as p-benzoquinone, has been studied and has resulted in a reduced oxygen demand.12 Simulation studies 13 have suggested that to minimize the resistance of oxygen diffusion, cells should be immobilized in particles less than 0.2 mm in diameter. In spite of this effort, little is known about the physiology of immobilized microorganisms. Such knowledge will be required if the use of immobilized cells are © 1988 Butterworth Publishers
going to make a significant impact on industrial product formation. The Streptomycetes are a group of relatively little studied, industrially important microorganisms the physiology of which we have chosen to study as a model system, r-Carrageenan is believed to be one of the gentler methods for cell immobilization. In this paper we investigate the factors that affect oxygen uptake in carrageenan entrapped cells of the filamentous bacterium Streptomyces clavuligerus and show that, in this case at least, immobilization has deleterious effects on oxygen uptake by cells.
Materials and methods O r g a n i s m a n d culture m e t h o d s Streptomyces claouligerus (ATCC 27064) was grown in liquid cultures in a medium of the following composition: Malt Extract (Oxoid) 10 g 1-1; Bactopeptone (Oxoid) 10 g 1-1; glycerol 20 g 1-1. The pH of the medium was initially 7.2. The organism was maintained on 1.5% w/v agar slopes of the same composition as the growth medium. Seed cultures were inoculated with mycelium from a slope and grown in 100 ml Erlenmeyer flasks containing 25 ml of medium. Growth was for 50-60 h at 26°C on an orbital shaker (200 revolutions min-1). These cultures were used to inoculate 1000 ml Erlenmeyer flasks containing 250 ml medium: these experimental cultures were grown under the same conditions as the seed culture for 36-40 h. Cells were used directly from the culture or concentrated by centrifugation where necessary. Enzyme Microb. Technol., 1988, vol. 10, March
151
Papers Determination o f biomass
Deformation o f carrageenan beads
Biomass in cell suspensions used for immobilization was determined from optical density measurements at 650 nm using an LKB Novaspec 4049 spectrophotometer (1 cm path length, 3 ml capacity cuvettes) and relating optical density to dry weight using a calibration curve.
The hardness of individual carrageenan beads was assessed by measurement of the distance that a 3 mm diameter fiat ended probe could penetrate into the bead when a force equivalent to a mass of 60 g was applied to the probe. Further details of the method will appear elsewhere.17
Results and discussion
Cell immobilization Cells were immobilized in carrageenan 14,~5 (type 1, Sigma, Poole, UK). Use of the commercially available carrageenan was problematic as gels were formed, even in the absence of added K ÷, at 55°C or below. Exposing cells to this elevated temperature had deleterious effects on cell respiration (see Results). The commercial product contains high concentrations ~6 (9.15%) of K+; this was removed by dialyzing 200 ml aliquots of 4% w/v solutions of carrageenan in water against 1% EDTA/I% NaC1 for 12 h at 60°C. The solution was then further dialyzed three times against distilled water at 25°C for 3 h. The dialyzed solution was then mixed with undialyzed carrageenan solution in the ratio of 2:1 by volume. This mixture gelled instantly at 35°C when added to KC1 solution and was used to immobilize cells: the mixture could be kept for at least 3 h at 35°C in the absence of KCI without gelling. The solution at 35°C was mixed with the cell suspension (also at 35°C) and the mixture dropped into a solution of 1M KCI from a height of 10 cm, using a 5 ml syringe without an attached needle. The internal diameter of the nozzle of the syringe was 1.0 mm. The KCI solution was slowly stirred using a magnetic stirrer. The final carrageenan concentration was 2% and the beads were left to harden for 10 min in KCI, unless otherwise stated. Beads were washed twice in distilled water before use. Beads were of a spherical nature, although slightly flattened in shape. The mean diameter of the beads (largest axis) was 4.32 mm (SD 0.517 mm). The axis at 90° to this was mean diameter 3.69 mm (SD 0.34 mm) and the shortest axis was 2.98 mm (SD 0.164 mm). (35 Determinations).
Suspensions of free cells were characterized by showing linear rates of oxygen uptake over a wide range of dissolved oxygen concentrations in the medium (Figure la). It was only at low dissolved oxygen concentrations (below about 3/ZM)that the oxygen uptake rate was reduced. All measurements of oxygen uptake rate were carded out well above this critical oxygen concentration. Immobilized cell preparations, containing the same amount of biomass in the electrode vessel as the free cells (Figure lb), showed non-linear rates of oxygen uptake over a wide range of oxygen concentrations. The initial rate of oxygen consumption was lower than in the free cells. Presumably the immobilized cells used up the oxygen in the beads: the rate of external diffusion of oxygen into the beads from the surrounding medium, together with the rate of internal diffusion, may not be rapid enough to give a sufficiently high oxygen concentration in the bead to allow oxygen uptake to occur at its maximum rate. This is shown as a continuous reduction in oxygen uptake rate in Figure lb. In experiments involving oxygen uptake of immobilized cells, the initial linear rate was measured. Similarly shaped responses have been reported for Gluconobacter oxydans immobilized in calcium alginate 18 and Candida lipolytica immobilized in agar. 19 The rates of oxygen diffusion have been measured in various gel immobilized systems. Adlercreutz2° found the rate of oxygen diffusivity in calcium alginate gels
a
Measurement o f oxygen uptake rates Oxygen uptake rates were measured at 26°C using a Rank Brothers polarographic oxygen electrode (Bottisham, UK). Samples, of either free or immobilized cells, were added to prewarmed, aerated medium in the electrode vessel to give a final volume of 2 ml. Rates of oxygen uptake were measured in the closed vessel after the vessel lid had been put in place. Measurements were made on immobilized cells immediately after the gel beads had been prepared, i.e., before any growth of microorganisms could occur. The effect of various ions on oxygen uptake in free cells was measured by repeated addition of small aliquots (up to 150/~1 at a time) of 2M solutions through an addition port in the lid of the vessel.
152
Enzyme Microb. Technol., 1988, vol. 10, March
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Time (rain) Figure 1
Oxygen uptake of free and carrageenan immobilized
S. clavuligerus. In (a) cells were harvested after 36 h and 0.4 ml of culture added to 1.6 ml medium in the electrode vessel. The temperature was 26°C. In (b) the same amount of biomass as used in (a) (0.36 mg dry weight) was immobilized in 2% w/v carrageenan beads and added to fresh medium, to give a final volume of 2 ml. Immobilized cells were used after 10 min hardening in KCI
Oxygen uptake and Streptomyces clavuligerus:/7. I. Scott et al. to be only 77% of that in water. Hiemstra et al. 21 found the rate in barium alginate gels to be only 25% and Sata and Toda 19 showed the rate in a g a r t o be reduced to 70%. An investigation was made of other factors that could account for the reduction in oxygen uptake in immobilized cells. As the carrageenan preparation used gelled at 35°C, it was necessary to expose the cells to elevated temperatures during immobilization. Oxygen uptake of cells (at 26°C) was measured after exposure for various time periods to increased temperatures. Figure 2 shows that 30 rain exposure of cells to a temperature of 50°C caused the specific oxygen uptake rate to fall to zero. After exposure to 45°C for 90 min, cells could still utilize oxygen although the rate was only 30% of the rate in cells that had not been exposed to increased temperatures. Exposure of cells to 35°C (the temperature used for immobilization) for 90 min caused the oxygen uptake rate to decrease to about 65% of the original level. During the time that would normally be taken for immobilization (about 10 rain) the oxygen uptake rate showed a decrease of only about 15 to 85% of the original level. Obviously, exposure of S. claouligerus to elevated temperatures during immobilization should be reduced as far as possible. The use of lower temperatures than 35°C is not possible due to the premature gelling that occurs with this blend of carrageenan. The specific oxygen uptake rate of free and immobilized cells was dependent on the amount of biomass present in the vessel (Figure 3). The specific oxygen uptake rate of free cells was over twice that of immobilized cells, at the same biomass concerttration. With
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Time of exposure Imin) Rgure 2 The effect of exposure to increased temperature on oxygen uptake by free cells of S. clavuligerus. Samples (10 ml) of culture were incubated at various temperatures and 2 ml of sample transferred to the electrode vessel. The rate of oxygen uptake at 26°C was then measured. The temperature of incubation was: ©, 350C; 0 , 45°C; II, 50°C
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Eell concenfrafion (mg m1-11 Figure :3 The effect of cell concentration on specific oxygen uptake rate by free and immobilized S. clavuligerus. Cells were harvested and different concentrations were added to prewarmed medium in the vessel to give a final volume of 2 ml (O). A similar sample of cells was harvested and different biomass concentrations immobilized in 10 2% carrageenan beads (11). At high cell concentrations cells were concentrated 10-fold by centrifugation before use. Immobilized cells were allowed to harden in 1M KCI for 10 min before use
both free and immobilized cells the specific oxygen uptake rate decreased with increasing biomass. The effect of biomass concentrations on oxygen uptake in gel entrapped microorganisms has been studied. 7,22 The Gosman and Rehms study of calcium alginate entrapped microorganisms showed decreasing specific oxygen uptake rates with increasing concentrations of immobilized cells. Perhaps surprisingly the specific oxygen rate of free cells also decreased at much lower cell concentrations than the entrapped cells. 22 The reduction in specific oxygen uptake rate in the immobilized cells was thought to be due to increased diffusion limitations around the entrapped cells. The K-carrageenan layer will have the same effect in the present study but other factors may also contribute to the reduction in oxygen uptake (see later). Fujimura et al. 7 showed that the specific oxygen uptake rate of free and carrageenan immobilized Serratia marcescens were similar with increasing biomass concentrations until a level of about 101° cells/(ml gel) was reached. Above this concentration the specific oxygen uptake rate of the entrapped cells decreased. The effect of different carrageenan concentrations on oxygen uptake by free and immobilized cells has been examined (Figure 4). The specific oxygen uptake rate of immobilized cells was only about 40% of the rate in free cells; the rate decreased slightly at higher carrageenan concentrations. This reduction may be
Enzyme Microb. Technol., 1988, vol. 10, March
153
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various carrageenan concentrations and the oxygen uptake rate of the immobilized preparations measured, 0. A similar amount of free cells was added to the vessel and carrageenan beads of different concentration containing no cells were added, ©. Various concentrations of carrageenan solution were added to free cells, I . Immobilized cells were allowed to harden in 1M KCI for 10 min before use
due, in part, to the problems of oxygen diffusion already discussed. Decreases in specific oxygen uptake rates of Pseudomonas putida at increasing calcium alginate concentrations have been observed. 22 The addition of carrageenan solutions to free cells also caused a decrease in the specific oxygen uptake rate, although the effect was not as great as in immobilized cells. These viscous solutions of carrageenan did not influence the speed of the stirrer in the electrode vessel; oxygen uptake rates were independent of stirrer speed, at least at the speeds (>360 rev min -~) used in this study (results not shown). The addition of empty carrageenan beads to free cells also caused some inhibition, up to a level of 37% when 3% carrageenan beads were added. This inhibition could be due to the carrageenan or ions used in making the beads. Measurement of the "hardness" of individual carrageenan beads was carried out by following the deformation and penetration of beads by a 3 mm diameter flat ended probe. 17 Beads reached their maximum hardness after about 40 min exposure to IM KC1. However, prolonged exposure (up to 1 h) of immobilized cells to KC1 resulted in a decrease in the specific oxygen uptake rate to virtually zero (Figure 5). This inhibition may be due either to the high concentrations of ions present or to the decreased level of oxygen available to the cells in the KCI solution. The solubility of oxygen in 1M KC1 is only 74% of that in distilled water. 23 Gentle stirring of the beads in the KCI solution results in low rates of oxygen transfer from the gas phase to the liquid and hence to the cells in the beads. 154
E n z y m e M i c r o b . T e c h n o l . , 1988, v o l . 10, M a r c h
This reduced oxygen concentration may have deleterious effects on aerobic microorganisms, such as S. claouligerus. The effect of the addition of various concentrations of ions on oxygen uptake in free cells of S. claouligerus is shown in Figure 6. KCI and KNO3 showed the greatest inhibitory effects. Fifty percent inhibition of oxygen uptake was shown by 0.70 M KCI, by 0.76 M KNO3 and by 0.90 M NaC1. This inhibitory effect will be important in cell immobilization as beads are exposed to 1 M KC1. Presumably cells at the outer layers of the bead will experience higher concentrations of KC1, during the early stages of bead hardening, than cells at the interior of the bead. The addition of similar volumes of water to those used in Figure 6 to the cell suspensions did not show any inhibitory effect (results not shown). Indeed, when relatively high volumes (1 ml) of water were added, the specific oxygen uptake rate was increased by about 20%. Slight increases (about 10%) in oxygen uptake rate were observed when ions were added at low concentrations to free cells (Figure 6). The nature of the reduction in oxygen uptake rates in carrageenan immobilized cells compared with free cells is thus complex and is the result of many factors. These include problems with internal diffusion of oxygen, exposure to relatively high temperatures during bead preparation, exposure to high concentrations of ions and the presence of carrageenan itself. Further work is necessary to elucidate the importance of each of these factors and to see if these problems can be overcome. Possibly of most interest is the
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Oxygen uptake and S t r e p t o m y c e s c l a v u l i g e r u s : R. L Scott et al.
References
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Ion concentration (H) Figure 6 The effect of various ions on oxygen uptake in free cells of S. c/avuligerus. Cells were harvested, added to medium in the vessel and the initial rate of oxygen uptake determined. Various volumes of 2 M solutions were sequentially added to the vessel and the oxygen uptake rate remeasured. ©, NaCI; 0, KNOa; II, KCI
effect that ions (particularly K ÷, Na ÷ or C1- ) can have on metabolism, causing complete cessation of oxygen uptake during bead preparation (Figure 5). The physiological affects of these ions on Streptomycetes are not known. Similarly, the finding that carrageenan itself can cause some slight inhibition of oxygen by free cells (perhaps due to ions present in the carrageenan?) requires further investigation. In summary, the effect of all of these factors on cellular metabolism should be studied before immobilized microorganisms can be used to better advantage in industrial product formation.
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
Scott, C..D.:Enzy~e Microb. Technol. 1987, 9, 66-73 Bucke, C. Proe. Roy. Soc. Lond. Series B. 1983, 300, 369389 Cheetham, P. S. J. in Topics in Enzyme and Fermentation Technology (Wiseman, A., ed.) Ellis Horwood, Chichester, UK, 1980, vol 4, pp. 189-238 Nguyen, A.-L. and Luong, J. H. T. Biotechnol. Bioeng. 1986, 28, 1261-1267 Radovich, J. M. Enzyme Microb. Technol. 1985, 7, 2-10 Venkatasubramanian, K., Karkare, S. B. and Vieth, W. R. in Applied Biochemistry and Bioengineering (Chibata, I., Wingard, L. B. Jr., eds.) Academic Press, NY and London, 1983, vol. 4, pp. 311-349 Fujimura, M. et al. Appl. Microbiol. Biotechnol. 1984, 19, 79-84 Enfors, S. O. and Mattiasson, B. in Immobilized Cells and Organelles (Mattiasson, B., ed.) CRC Press, London, 1983, pp. 41-60 Adlercreutz, P., Holst, O., and Mattiasson, B. Enzyme Microb. Technol. 1982, 4, 395-400 Adlercreutz, P. and Mattiasson, B. Eur. J. Appl. Microbiol. Biotechnol. 1982, 16, 165-170 Hoist, O., Enfors, S. O., Mattiasson, B. Eur. J. Appl. Microbiol. Biotechnol. 1982, 14, 64-68 Adlercreutz, P., Mattiasson, B. Appl. Microbiol. Biotechnol. 1984, 20, 296-302 Toda, K. and Sato, K. J. Ferment. Technol. 1985, 63, 251258 Koshcheyenko, K. A. and Sukhodolskaya, G. V. in lmmobilised Cells and Enzymes--A Practical Approach (Woodward, J., ed.) IRL Press, Oxford, 1985, pp. 91-125 Chibata, I. American Chem. Soc. Syrup., 1979, Series 106, 187-202 Anonymous. Sigma Technical Bulletin C1013. Scott, R. I. et al. J. Chem. Tech. Biotech. In press. Adlercreutz, P., Holst, P. and Mattiasson, B. Appl. Microbiol. Biotechnol. 1985, 22, 1-7 Sato, K. and Toda, K. J. Ferment. Technol. 1983, 61, 239-245 Adlercreutz, P. Biotechnol. Bioeng. 1986, 28, 223-232 Hiemstra, H., Dijkhuizen, L. and Harder, W. Eur. J. Appl. Microbiol. Biotech. 1983, 18, 189-196 Gosmann, B. and Rehm, H. J. Appl. Microbiol. Biotechnol. 1986, 23, 163-167 Seidell, A. in Solubilities of Inorganic and Metal Organic Compounds. American Chemical Society, Washington, DC, 4th edn, 1965, vol. 2
E n z y m e M i c r o b . T e c h n o l . , 1988, v o l . 10, M a r c h
155
(Received 8 May 1987; revised 2 October 1987)
Oxygen uptake rates have been measured in free and r-carrageenan immobilized cells of Streptomyces clavuligerus. Specific oxygen uptake rates of immobilized cells were only about 50% of the rates of free cells. The factors contributing to this reduction in oxygen uptake include the presence of carrageenan itself, internal oxygen diffusion limitations, the temperature of preparation of the immobilized cell beads and the presence of ions, particularly K ÷. Measurement of the hardness of individual gel beads showed that maximal hardness was reached after about 40 min exposure to 1 M KCl. However, during this exposure the specific oxygen uptake rate had fallen to only 20% of the rate of immobilized cells exposed to KCl for 2 rain.
Keywords: Oxygenuptake; r-carrageenan; immobilizedcells; Streptomyces clauuligerus Introduction Microorganisms and other cell types can be immobilized in a variety of different ways.l The advantages of using immobilized cells instead of free cells can be great and have been reviewed many times. 2,3 Entrapment in polymers, such as alginate or carrageenan, has been commonly carried out, mainly because the method is easy to perform and is believed to have few deleterious effects on the immobilized cells. Oxygen is required by cells to carry out respiratory and oxygenase functions. However, at high cell densities, diffusion of substrates, such as oxygen, into gelimmobilizing matrices may be rate limiting.4-6 For aerobic microorganisms, the supply of sufficient oxygen to gel immobilized cells may be problematic. 7,8 Various methods have been studied to try to increase the supply of oxygen to immobilized cells. These methods include the use of oxygen instead of air; 7 the use of coimmobilized algae and bacteria for in situ oxygen generation; 9 supplying extra oxygen via oxygen carders such as hemoglobin or emulsions of perfluorochemicals; ~° the decomposition of hydrogen peroxide using catalase. H In addition, the use of alternative electron acceptors, such as p-benzoquinone, has been studied and has resulted in a reduced oxygen demand.12 Simulation studies 13 have suggested that to minimize the resistance of oxygen diffusion, cells should be immobilized in particles less than 0.2 mm in diameter. In spite of this effort, little is known about the physiology of immobilized microorganisms. Such knowledge will be required if the use of immobilized cells are © 1988 Butterworth Publishers
going to make a significant impact on industrial product formation. The Streptomycetes are a group of relatively little studied, industrially important microorganisms the physiology of which we have chosen to study as a model system, r-Carrageenan is believed to be one of the gentler methods for cell immobilization. In this paper we investigate the factors that affect oxygen uptake in carrageenan entrapped cells of the filamentous bacterium Streptomyces clavuligerus and show that, in this case at least, immobilization has deleterious effects on oxygen uptake by cells.
Materials and methods O r g a n i s m a n d culture m e t h o d s Streptomyces claouligerus (ATCC 27064) was grown in liquid cultures in a medium of the following composition: Malt Extract (Oxoid) 10 g 1-1; Bactopeptone (Oxoid) 10 g 1-1; glycerol 20 g 1-1. The pH of the medium was initially 7.2. The organism was maintained on 1.5% w/v agar slopes of the same composition as the growth medium. Seed cultures were inoculated with mycelium from a slope and grown in 100 ml Erlenmeyer flasks containing 25 ml of medium. Growth was for 50-60 h at 26°C on an orbital shaker (200 revolutions min-1). These cultures were used to inoculate 1000 ml Erlenmeyer flasks containing 250 ml medium: these experimental cultures were grown under the same conditions as the seed culture for 36-40 h. Cells were used directly from the culture or concentrated by centrifugation where necessary. Enzyme Microb. Technol., 1988, vol. 10, March
151
Papers Determination o f biomass
Deformation o f carrageenan beads
Biomass in cell suspensions used for immobilization was determined from optical density measurements at 650 nm using an LKB Novaspec 4049 spectrophotometer (1 cm path length, 3 ml capacity cuvettes) and relating optical density to dry weight using a calibration curve.
The hardness of individual carrageenan beads was assessed by measurement of the distance that a 3 mm diameter fiat ended probe could penetrate into the bead when a force equivalent to a mass of 60 g was applied to the probe. Further details of the method will appear elsewhere.17
Results and discussion
Cell immobilization Cells were immobilized in carrageenan 14,~5 (type 1, Sigma, Poole, UK). Use of the commercially available carrageenan was problematic as gels were formed, even in the absence of added K ÷, at 55°C or below. Exposing cells to this elevated temperature had deleterious effects on cell respiration (see Results). The commercial product contains high concentrations ~6 (9.15%) of K+; this was removed by dialyzing 200 ml aliquots of 4% w/v solutions of carrageenan in water against 1% EDTA/I% NaC1 for 12 h at 60°C. The solution was then further dialyzed three times against distilled water at 25°C for 3 h. The dialyzed solution was then mixed with undialyzed carrageenan solution in the ratio of 2:1 by volume. This mixture gelled instantly at 35°C when added to KC1 solution and was used to immobilize cells: the mixture could be kept for at least 3 h at 35°C in the absence of KCI without gelling. The solution at 35°C was mixed with the cell suspension (also at 35°C) and the mixture dropped into a solution of 1M KCI from a height of 10 cm, using a 5 ml syringe without an attached needle. The internal diameter of the nozzle of the syringe was 1.0 mm. The KCI solution was slowly stirred using a magnetic stirrer. The final carrageenan concentration was 2% and the beads were left to harden for 10 min in KCI, unless otherwise stated. Beads were washed twice in distilled water before use. Beads were of a spherical nature, although slightly flattened in shape. The mean diameter of the beads (largest axis) was 4.32 mm (SD 0.517 mm). The axis at 90° to this was mean diameter 3.69 mm (SD 0.34 mm) and the shortest axis was 2.98 mm (SD 0.164 mm). (35 Determinations).
Suspensions of free cells were characterized by showing linear rates of oxygen uptake over a wide range of dissolved oxygen concentrations in the medium (Figure la). It was only at low dissolved oxygen concentrations (below about 3/ZM)that the oxygen uptake rate was reduced. All measurements of oxygen uptake rate were carded out well above this critical oxygen concentration. Immobilized cell preparations, containing the same amount of biomass in the electrode vessel as the free cells (Figure lb), showed non-linear rates of oxygen uptake over a wide range of oxygen concentrations. The initial rate of oxygen consumption was lower than in the free cells. Presumably the immobilized cells used up the oxygen in the beads: the rate of external diffusion of oxygen into the beads from the surrounding medium, together with the rate of internal diffusion, may not be rapid enough to give a sufficiently high oxygen concentration in the bead to allow oxygen uptake to occur at its maximum rate. This is shown as a continuous reduction in oxygen uptake rate in Figure lb. In experiments involving oxygen uptake of immobilized cells, the initial linear rate was measured. Similarly shaped responses have been reported for Gluconobacter oxydans immobilized in calcium alginate 18 and Candida lipolytica immobilized in agar. 19 The rates of oxygen diffusion have been measured in various gel immobilized systems. Adlercreutz2° found the rate of oxygen diffusivity in calcium alginate gels
a
Measurement o f oxygen uptake rates Oxygen uptake rates were measured at 26°C using a Rank Brothers polarographic oxygen electrode (Bottisham, UK). Samples, of either free or immobilized cells, were added to prewarmed, aerated medium in the electrode vessel to give a final volume of 2 ml. Rates of oxygen uptake were measured in the closed vessel after the vessel lid had been put in place. Measurements were made on immobilized cells immediately after the gel beads had been prepared, i.e., before any growth of microorganisms could occur. The effect of various ions on oxygen uptake in free cells was measured by repeated addition of small aliquots (up to 150/~1 at a time) of 2M solutions through an addition port in the lid of the vessel.
152
Enzyme Microb. Technol., 1988, vol. 10, March
0
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Time (rain) Figure 1
Oxygen uptake of free and carrageenan immobilized
S. clavuligerus. In (a) cells were harvested after 36 h and 0.4 ml of culture added to 1.6 ml medium in the electrode vessel. The temperature was 26°C. In (b) the same amount of biomass as used in (a) (0.36 mg dry weight) was immobilized in 2% w/v carrageenan beads and added to fresh medium, to give a final volume of 2 ml. Immobilized cells were used after 10 min hardening in KCI
Oxygen uptake and Streptomyces clavuligerus:/7. I. Scott et al. to be only 77% of that in water. Hiemstra et al. 21 found the rate in barium alginate gels to be only 25% and Sata and Toda 19 showed the rate in a g a r t o be reduced to 70%. An investigation was made of other factors that could account for the reduction in oxygen uptake in immobilized cells. As the carrageenan preparation used gelled at 35°C, it was necessary to expose the cells to elevated temperatures during immobilization. Oxygen uptake of cells (at 26°C) was measured after exposure for various time periods to increased temperatures. Figure 2 shows that 30 rain exposure of cells to a temperature of 50°C caused the specific oxygen uptake rate to fall to zero. After exposure to 45°C for 90 min, cells could still utilize oxygen although the rate was only 30% of the rate in cells that had not been exposed to increased temperatures. Exposure of cells to 35°C (the temperature used for immobilization) for 90 min caused the oxygen uptake rate to decrease to about 65% of the original level. During the time that would normally be taken for immobilization (about 10 rain) the oxygen uptake rate showed a decrease of only about 15 to 85% of the original level. Obviously, exposure of S. claouligerus to elevated temperatures during immobilization should be reduced as far as possible. The use of lower temperatures than 35°C is not possible due to the premature gelling that occurs with this blend of carrageenan. The specific oxygen uptake rate of free and immobilized cells was dependent on the amount of biomass present in the vessel (Figure 3). The specific oxygen uptake rate of free cells was over twice that of immobilized cells, at the same biomass concerttration. With
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Eell concenfrafion (mg m1-11 Figure :3 The effect of cell concentration on specific oxygen uptake rate by free and immobilized S. clavuligerus. Cells were harvested and different concentrations were added to prewarmed medium in the vessel to give a final volume of 2 ml (O). A similar sample of cells was harvested and different biomass concentrations immobilized in 10 2% carrageenan beads (11). At high cell concentrations cells were concentrated 10-fold by centrifugation before use. Immobilized cells were allowed to harden in 1M KCI for 10 min before use
both free and immobilized cells the specific oxygen uptake rate decreased with increasing biomass. The effect of biomass concentrations on oxygen uptake in gel entrapped microorganisms has been studied. 7,22 The Gosman and Rehms study of calcium alginate entrapped microorganisms showed decreasing specific oxygen uptake rates with increasing concentrations of immobilized cells. Perhaps surprisingly the specific oxygen rate of free cells also decreased at much lower cell concentrations than the entrapped cells. 22 The reduction in specific oxygen uptake rate in the immobilized cells was thought to be due to increased diffusion limitations around the entrapped cells. The K-carrageenan layer will have the same effect in the present study but other factors may also contribute to the reduction in oxygen uptake (see later). Fujimura et al. 7 showed that the specific oxygen uptake rate of free and carrageenan immobilized Serratia marcescens were similar with increasing biomass concentrations until a level of about 101° cells/(ml gel) was reached. Above this concentration the specific oxygen uptake rate of the entrapped cells decreased. The effect of different carrageenan concentrations on oxygen uptake by free and immobilized cells has been examined (Figure 4). The specific oxygen uptake rate of immobilized cells was only about 40% of the rate in free cells; the rate decreased slightly at higher carrageenan concentrations. This reduction may be
Enzyme Microb. Technol., 1988, vol. 10, March
153
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due, in part, to the problems of oxygen diffusion already discussed. Decreases in specific oxygen uptake rates of Pseudomonas putida at increasing calcium alginate concentrations have been observed. 22 The addition of carrageenan solutions to free cells also caused a decrease in the specific oxygen uptake rate, although the effect was not as great as in immobilized cells. These viscous solutions of carrageenan did not influence the speed of the stirrer in the electrode vessel; oxygen uptake rates were independent of stirrer speed, at least at the speeds (>360 rev min -~) used in this study (results not shown). The addition of empty carrageenan beads to free cells also caused some inhibition, up to a level of 37% when 3% carrageenan beads were added. This inhibition could be due to the carrageenan or ions used in making the beads. Measurement of the "hardness" of individual carrageenan beads was carried out by following the deformation and penetration of beads by a 3 mm diameter flat ended probe. 17 Beads reached their maximum hardness after about 40 min exposure to IM KC1. However, prolonged exposure (up to 1 h) of immobilized cells to KC1 resulted in a decrease in the specific oxygen uptake rate to virtually zero (Figure 5). This inhibition may be due either to the high concentrations of ions present or to the decreased level of oxygen available to the cells in the KCI solution. The solubility of oxygen in 1M KC1 is only 74% of that in distilled water. 23 Gentle stirring of the beads in the KCI solution results in low rates of oxygen transfer from the gas phase to the liquid and hence to the cells in the beads. 154
E n z y m e M i c r o b . T e c h n o l . , 1988, v o l . 10, M a r c h
This reduced oxygen concentration may have deleterious effects on aerobic microorganisms, such as S. claouligerus. The effect of the addition of various concentrations of ions on oxygen uptake in free cells of S. claouligerus is shown in Figure 6. KCI and KNO3 showed the greatest inhibitory effects. Fifty percent inhibition of oxygen uptake was shown by 0.70 M KCI, by 0.76 M KNO3 and by 0.90 M NaC1. This inhibitory effect will be important in cell immobilization as beads are exposed to 1 M KC1. Presumably cells at the outer layers of the bead will experience higher concentrations of KC1, during the early stages of bead hardening, than cells at the interior of the bead. The addition of similar volumes of water to those used in Figure 6 to the cell suspensions did not show any inhibitory effect (results not shown). Indeed, when relatively high volumes (1 ml) of water were added, the specific oxygen uptake rate was increased by about 20%. Slight increases (about 10%) in oxygen uptake rate were observed when ions were added at low concentrations to free cells (Figure 6). The nature of the reduction in oxygen uptake rates in carrageenan immobilized cells compared with free cells is thus complex and is the result of many factors. These include problems with internal diffusion of oxygen, exposure to relatively high temperatures during bead preparation, exposure to high concentrations of ions and the presence of carrageenan itself. Further work is necessary to elucidate the importance of each of these factors and to see if these problems can be overcome. Possibly of most interest is the
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Oxygen uptake and S t r e p t o m y c e s c l a v u l i g e r u s : R. L Scott et al.
References
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Ion concentration (H) Figure 6 The effect of various ions on oxygen uptake in free cells of S. c/avuligerus. Cells were harvested, added to medium in the vessel and the initial rate of oxygen uptake determined. Various volumes of 2 M solutions were sequentially added to the vessel and the oxygen uptake rate remeasured. ©, NaCI; 0, KNOa; II, KCI
effect that ions (particularly K ÷, Na ÷ or C1- ) can have on metabolism, causing complete cessation of oxygen uptake during bead preparation (Figure 5). The physiological affects of these ions on Streptomycetes are not known. Similarly, the finding that carrageenan itself can cause some slight inhibition of oxygen by free cells (perhaps due to ions present in the carrageenan?) requires further investigation. In summary, the effect of all of these factors on cellular metabolism should be studied before immobilized microorganisms can be used to better advantage in industrial product formation.
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
Scott, C..D.:Enzy~e Microb. Technol. 1987, 9, 66-73 Bucke, C. Proe. Roy. Soc. Lond. Series B. 1983, 300, 369389 Cheetham, P. S. J. in Topics in Enzyme and Fermentation Technology (Wiseman, A., ed.) Ellis Horwood, Chichester, UK, 1980, vol 4, pp. 189-238 Nguyen, A.-L. and Luong, J. H. T. Biotechnol. Bioeng. 1986, 28, 1261-1267 Radovich, J. M. Enzyme Microb. Technol. 1985, 7, 2-10 Venkatasubramanian, K., Karkare, S. B. and Vieth, W. R. in Applied Biochemistry and Bioengineering (Chibata, I., Wingard, L. B. Jr., eds.) Academic Press, NY and London, 1983, vol. 4, pp. 311-349 Fujimura, M. et al. Appl. Microbiol. Biotechnol. 1984, 19, 79-84 Enfors, S. O. and Mattiasson, B. in Immobilized Cells and Organelles (Mattiasson, B., ed.) CRC Press, London, 1983, pp. 41-60 Adlercreutz, P., Holst, O., and Mattiasson, B. Enzyme Microb. Technol. 1982, 4, 395-400 Adlercreutz, P. and Mattiasson, B. Eur. J. Appl. Microbiol. Biotechnol. 1982, 16, 165-170 Hoist, O., Enfors, S. O., Mattiasson, B. Eur. J. Appl. Microbiol. Biotechnol. 1982, 14, 64-68 Adlercreutz, P., Mattiasson, B. Appl. Microbiol. Biotechnol. 1984, 20, 296-302 Toda, K. and Sato, K. J. Ferment. Technol. 1985, 63, 251258 Koshcheyenko, K. A. and Sukhodolskaya, G. V. in lmmobilised Cells and Enzymes--A Practical Approach (Woodward, J., ed.) IRL Press, Oxford, 1985, pp. 91-125 Chibata, I. American Chem. Soc. Syrup., 1979, Series 106, 187-202 Anonymous. Sigma Technical Bulletin C1013. Scott, R. I. et al. J. Chem. Tech. Biotech. In press. Adlercreutz, P., Holst, P. and Mattiasson, B. Appl. Microbiol. Biotechnol. 1985, 22, 1-7 Sato, K. and Toda, K. J. Ferment. Technol. 1983, 61, 239-245 Adlercreutz, P. Biotechnol. Bioeng. 1986, 28, 223-232 Hiemstra, H., Dijkhuizen, L. and Harder, W. Eur. J. Appl. Microbiol. Biotech. 1983, 18, 189-196 Gosmann, B. and Rehm, H. J. Appl. Microbiol. Biotechnol. 1986, 23, 163-167 Seidell, A. in Solubilities of Inorganic and Metal Organic Compounds. American Chemical Society, Washington, DC, 4th edn, 1965, vol. 2
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