Appl Microbiol Biotechnol (1991) 35:14-18 017575989100081E
App//ed
Microbiology Biotechnology © Springer-Verlag 1991
Lysosomal enzymes produced by immobilized Tetrahymena thermophila Thomas Kiy and Arno Tiedtke Zoologisches Institut der Universit~it MOnster, Schlossplatz 5, D-4400 MOnster, Federal Republic of Germany Received 8 November 1990/Accepted 12 December 1990
Summary. The ciliated protozoon Tetrahymena thermophila was immobilized for production of secreted lysosomal enzymes in two ways. Cells entrapped in solid Ca-alginate spheres survived but were unable to grow and multiply. However, when encapsulated in hollow Ca-alginate spheres Tetrahymena multiplied well, reaching 0.9 x 107 cells/ml. These immobilized cells secreted large amounts of lysosomal enzymes when the medium was changed daily. This system was transferred to a reactor scale using a conical bubble column reactor for semicontinuous cultivation of the encapsulated cells. Under these conditions a-glucosidase, fl-glucosidase, fl-hexosaminidase and acid phosphatase were produced for at least 4 weeks. The hollow spheres were stable for 3 months and contained living and secreting Tetrahymena cells during this time. Immobilized T. thermophila cells can thus serve as a good source for production of commercially interesting enzymes.
Introduction Many lysosomal enzymes are used in diagnostic and other analytical tests. Animals and plants have often served as sources for many of these enzymes, but as the scope of enzymatic tests has increased, the proportion of diagnostic enzymes made by fermentation with microorganisms has also increased (Frost and Moss 1987). In all these organisms the relevant enzymes are intracellularly located and require cumbersome procedures for isolation. Some lower eukaryotes, especially protozoa, secrete lysosomal enzymes by a normal, physiological mechanism (Munro 1985). Thus the ciliate Tetrahyrnena therrnophila constitutively releases large amounts of lysosomal enzymes into the culture medium (Mtiller 1972). This makes it easy to produce cell-free supernatants containing valuable enzymes in high concentrations using immobilized cells. So far, work with immobilized Tetrahyrnena in biotechnology has not been reported.
Offprint requests to: A. Tiedtke
Here we describe a method fo the immobilization o f
Tetrahymena. We also present a method for semicontinuous cultivation of immobilized Tetrahymena cells using a conical bubble column reactor for the production of a-glucosidase, fl-glucosidase, fl-hexosaminidase and acid phosphatase, enzymes well characterized by Banno and Nozawa (1984, 1985) and Tiedtke (1983). Employed the right way this may provide us with a wide range of cheap enzymes.
Materials and methods
Microoryanism. All investigations were carried out with T. thermophila originally obtained from Dr. P. Bruns, Cornell University, Ithaca, NY.
Medium. The cells were propagated under aerobic conditions in a solution containing: 1% proteose peptone, 0.1% yeast extract, 0.003% Sequestrene (Tiedtke 1983); CaC12.2H20 was added to cultures of immobilized cells at a concentration of 0.15% in erlenmeyer flasks and 0.2% in the reactor. We found that these concentrations were necessary to stabilize the alginate beads. The nutrient medium used in fermentations with a bubble column reactor contained 0.005% silicone oil to prevent foaming.
Immobilization techniques. Living Tetrahymena cells were immobilized both in solid and in hollow spheres made of alginate. Cells for immobilization were harvested in the late exponential phase by centrifugation. For entrapment in solid spheres the cell pellet of a 10-ml preculture was resuspended in 200 ~tl of 10 mM TRIS/HC1 buffer, pH 7.4, and mixed with 10 ml of 3% Na-alginate solution. The mixture was pressed through a thin cannula (0.8 × 35 mm) into 100 ml of a stirred 2% CaC12-2H20 solution and left to harden for 20 min. Then the gel beads were washed twice in 10 mr~ TRIS/ HCI buffer and transferred into the fermentation medium. Tetrahymena cells were encapsulated in hollow spheres according to Spiekermann et ai. (1987). One part of a cell suspension (4 × 105 cells/ml) was mixed with three parts of a sterile 1.5% carboxymethylcellulose solution containing 1.5% CaC12.2HzO. This mixture was dropped into a sterile 0.875% Na-alginate solution. Ca-alginate hollow spheres are formed by cross-linking from inside to outside (Spiekermann et al. 1987). The cells were encapsulated in the liquid centre of the hollow spheres. The remaining Na-alginate was removed by three washings with distilled water and then the hollow spheres were hardened in 0.9% CaC12-2H20 solution for 15 min.
15
Semicontinuous cultivation. Tetrahymena immobilized in solid
8xlO 5
spheres was incubated in 1000-ml erlenmeyer flasks in 35 ml medium at 30 ° C. The flasks were shaken (Gyrotory Incubator Shaker, New Brunswick Scientific, Edison, NJ, USA) at 120 strokes/ min. The supernatant was replaced by fresh medium every 24 h. Semicontinuous cultivation of cells immobilized in hollow spheres was carried out by incubating 50 ml beads in a 1000-ml erlenmeyer flask containing 200 ml nutrient medium. Cells immobilized in 50 ml hollow spheres were also cultivated in a 500-ml bubble-column reactor in 200 ml medium at 28° C. The medium was changed every 24 h.
Cell counts. For assessing cell density in the solid spheres a defined volume of beads was incubated in 0.05 M sodium hexametaphosphate and stirred for 10 min. After solubilization of the spheres the cell density was determined using a Fuchs-Rosenthal counting chamber. To determine cell density in the cavity of the hollow spheres, the diameter of the liquid centre of a single sphere was measured using a binocular microscope. From this value the volume of the cavity was calculated. Then the hollow sphere was dissolved in a defined volume of 0.2 M citrate buffer by stirring and the released cells were counted using a Fuchs-Rosenthal counting chamber. This procedure was repeated four times per time point. Enzyme assays. The extracellular activities of acid phosphatase, fl-hexosaminidase, fl-glucosidase, and a-glucosidase were assayed at 37°C, using appropriate p-nitrophenyl substrates (Tiedtke 1983). A sample of 100 ~xl was added to 100 ~tl of 10 mM p-nitrophenyl substrate dissolved in 0.1 M citrate buffer, pH 4.6, supplemented with 0.04% sodium azide and 0.2% bovine serum albumin. After incubation, 1 ml of 0.4 M glycine/NaOH buffer, pH 10.4, was added to stop the reaction. The liberated p-nitrophenol was determined photometrically at 405 nm. One unit (U) was defined as the the amount of enzyme that release~d 1 ~tmol p-nitrophenol/ min at 37 ° C.
Scanning electron microscopic(SEM) investigations. Scanning electron micrographs of spheres and immobilized cells were made using an Electron Microscope Model S-530 (Hitachi, Tokyo, Japan).
Chemicals. Proteose peptone and yeast extract were obtained from Difco (Detroit, Mich., USA) and Sequestrene from CibaGeigy (Greensboro, NC, USA). Sodium alginate (Manugel DJX) for production of solid, spheres was from Alginate Industries (Hamburg, FRG) and sodium alginate (Protonal LF 20/60) for hollow spheres from Protan (Norderstedt, FRG). Sodium carboxymethylcellulose (7HF-CMC) was obtained from Aqualon (Dtisseldorf, FRG). All p-nitrophenyl substrates for the enzyme assays were purchased from Serva (Heidelberg, FRG).
6xlO 5
4xlO 5 0
i
i
~
i
~
~
~
i
2
4
6
8
10
12
14
16
Time
[d]
18
Fig. 1. Concentration of Tetrahymena thermophila entrapped in solid Ca-alginate spheres during semicontinuous cultivation
Secretion of lysosomal enzymes by entrapped Tetrahymena cells Both cell density a n d the a m o u n t o f secreted e n z y m e s decreased during fermentation. As an example, acid p h o s p h a t a s e secretion is s h o w n in Fig. 2. The highest c o n c e n t r a t i o n o f acid p h o s p h a t a s e was m e a s u r e d after the first 24 h o f cultivation. As m u c h as 2 U / 3 5 ml h a d b e e n secreted in this time period. O n l y 0.11 U / 2 4 h p e r 35 ml was secreted after 19 days. The kinetics o f flh e x o s a m i n i d a s e secretion s h o w e d the same general course. C o m p a r e d to the first 24 h the rates o f secretion o f acid p h o s p h a t a s e and fl-hexosaminidase d r o p p e d to a value o f 5%. I n the same time period a decline o f only 32% in cell n u m b e r t o o k place (Fig. 1). Thus the decreases in cell density a n d extracellular e n z y m e activity were disproportional.
Growth of Tetrahymena in hollow spheres Tetrahymena cells encapsulated in Ca-alginate h o l l o w spheres s h o w e d excellent g r o w t h (Fig. 3). Initially, the cell density was 2.1 x 105 c e l l s / m l h o l l o w space. T w o days later the cells h a d r e a c h e d the stationary p h a s e with cell densities o f up to 0.9 x 107 c e l l s / m l h o l l o w space. F o r comparison~ the average cell density o f a
Results 2,5
Growth of Tetrahymena in solid spheres T w o i m m o b i l i z a t i o n m e t h o d s were c o m p a r e d , the first b a s e d o n i m m o b i l i z a t i o n o f cells by e n t r a p m e n t in solid Ca-alginate b e a d s a n d the s e c o n d on e n c a p s u l a t i o n o f cells in Ca-alginate h o l l o w spheres. W h e n Tetrahymena was immobilized in solid spheres no g r o w t h c o u l d be observed. Instead, a decrease in cell density over a time p e r i o d o f 18 days was detected (Fig. 1). At zero time the cell density in the gel b e a d s was 7.8 x 10 5 c e l l s / m l ; after 18 days o f fermentation 5.3 x 10 5 c e l l s / m l Ca-alginate gel were present. The remaining cells were still alive, evident f r o m the activity o f the contractile v a c u o l e a n d f r o m the fact that cells released f r o m the gel regained the ability to divide.
[
~Acid
~
2
¢~
1,5
~
i
~
phosphatase
0,5
0 ~ 1
2
3
4
5
6
7
8
9 Time
10 11
12
13
14
15
16
17
18
19
[d 1
Fig. 2. Secretion of acid phosphatase by T. thermophila entrapped in solid Ca-alginate spheres during semicontinuous cultivation in erlenmeyer flasks
16
~o~
enzymes for 56 days. After 3 weeks suspended cells appeared in the medium due to disintegration of some of the hollow spheres.
© © ~
Semicontinuous cultivation in a bubble column reactor I0 °
10 0
2
1
3
4
5
6
7
8
9
10
11
12
13
14
Time [d] Fig. 3. Multiplication of T. thermophila encapsulated in hollow Ca-alginate spheres during semicontinuous cultivation in erlenmeyer flasks
suspension culture was about 1 x 106 cells/ml. Scanning electron micrographs demonstrate dense cell packing within the hollow spheres (Fig. 5B). The cell density remained more or less constant over a time period of 10 days (Fig. 3). The generation time for immobilized Tetrahymena cells was 6.9 h, compared to 2.5 h in suspension culture under similar conditions.
Secretion of lysosomal enzymes by encapsulated Tetrahymena cells The activities of released acid phosphatase, fl-hexosaminidase and a-glucosidase were measured during the cultivation period. Parallel with the increase in cell density during the first few days we observed a clear increase in extracellular enzyme concentration (Fig. 4). In 3 weeks we harvested 235 U fl-hexosaminidase, 278 U acid phosphatase and 36 U a-glucosidase. During this time a population with high density and cells in g o o d condition was observed using SEM (Fig. 5C and D). Every 24 h, spent medium was replaced by fresh broth. Under such conditions we produced lysosomal
20
o o
~, 1o
.~ ~
5-
.~ 1
2
3
4
5
6
7
8
9
10 11 12 13 14 15 16 17 18 19 20 21 Time [d]
Fig. 4. Secretion of a-glucosidase, fl-hexosaminidase, and acid phosphatase by encapsulated T. thermophila cells during semicontinuous cultivation in erlenmeyer flasks. The supernatant was harvested and replaced by fresh medium every 24 h
The culture experiments with Tetrahymena immobilized in hollow spheres in erlenmeyer flasks showed that production of lysosomal enzymes for a longer time period was possible. Further investigations were made to examine if it was possible to transfer this system to a reactor scale. A conical bubble column reactor with a capacity of 500 ml was chosen. The medium containing additional silicone oil (0.005%) to prevent foaming, was replaced every 24 h. A CaC12.2H20 concentration of 0.2% was necessary to stabilize the spheres. Preliminary experiments using suspension cultures showed that silicone oil had no adverse effect, whereas CaC12.2H20 (0.2%) lowered both growth and secretion rates (U/ 5 x 105 cells) of non-adapted cells (Kiy 1990). During the first experimental run, free cells appeared in the medium of the bubble column reactor after only 10 days. This was due to the disintegration of the spheres. Scanning electron micrographs showed that the outside of the alginate mantle of the hollow spheres was damaged (Fig. 5A). We thought that this damage was due to the rough surface of the fritted glass filter. Therefore it was overlaid with a thin layer of smooth glass beads of 5 mm diameter. Now suspended cells no longer appeared during the first few weeks and the hollow spheres were stable for 12 weeks. We measured the extracellular activities of acid phosphatase, flhexosaminidase, fl-glucosidase and a-glucosidase. The results of this semicontinuous fermentation are shown in Fig. 6.
Discussion
Tetrahymena cells entrapped in solid Ca-alginate spheres did not multiply. During microscopic observations of immobilized Tetrahymena we never observed dividing cells. We assume that the gel structure of the Ca-alginate is too close-meshed to allow the growth necessary for cell multiplication. Klein et al. (1983) detected pore sizes of up to 16.6 nm when investigating structures of Ca-alginate carriers. For comparison, the size of a single Tetrahymena cell is in the range of about 50 ~tm. The gel structure may also have compressed and damaged the living cells and might explain why the secretion of enzymes decreased more than the cell density. The experiments with entrapped Tetrahymena cells, however, showed that, in principle, Ca-alginate is suitable as a carrier because the gel matrix allowed diffusion of the enzymes and survival of the cells. Tetrahymena cells encapsulated in Ca-alginate hollow spheres grew well and reached cell concentrations one order of magnitude higher than those in suspension cultures. The reason for the increase in cell concentra-
17
Fig. 5 A-D. Scanning electron micrographs of Ca-alginate hollow spheres and immobilized T. thermophila. A Hollow sphere after 14 days of cultivation in a bubble column reactor. B A halved hollow sphere filled with T. thermophila. C View into a fenestrated hollow sphere. D Detailed view of cells encapsulated in Ca-alginate hollow spheres
2,5
~
2
~
1,5
~
0,5
2
3
4
5
6
7
5
9
10111213141516171819202122232425 T i m e [d]
1 2 3 4 5 6 7 8
9 10111213141516171619202122232425 T i m e [d]
Fig. 6. Secretion of a-glucosidase, fl-glucosidase, fl-hexosaminidase, and acid phosphatase by encapsulated T. thermophila during semicontinuous cultivation in a conical bubble column reactor. The supernatant was harvested and replaced by fresh medium every 24 h
18
tion remains to be explained. The long generation time of encapsulated Tetrahymena cells (6.9 h) may be due to an adaptation phase of the encapsulated cells and/or diffusion limitations for nutrients and oxygen. The increase in enzyme secretion during the first 4 days can be explained by the increase in cell density during this time period. The little variations in enzyme secretion from the 5th day onwards may be due to population dynamics in the hollow spheres. The different enzyme secretion pattern in the bubble column reactor compared to that in edenmeyer flasks is a result of a higher CaC12 concentration. We propose that the cells need a longer adaptation phase and reach the stationary phase later. This is evident from the amounts of secreted enzymes. As much as 10 U acid phosphatase were already produced after 3 days in erlenmeyer flasks but 10 days passed before this amount was secreted in the bubble column reactor. However, the disadvantage of initially lower secretion rates at higher CaCI2 concentrations is more than compensated for by the long lasting stability of the spheres. Thus we produced enzymes using the same hollow spheres over a period of 3 months. We were able to show that the initially low secretion rates can be avoided when cells that were adapted to 0.2% CaCI2 in suspension culture were encapsulated (unpublished results). Evidently immobilized Tetrahymena cells can serve as good sources for commercially interesting production of lysosomal enzymes. Acknowled#ements. We thank Prof. H. J. Rehm and Dr. K. D. Vorlop for advice and constructive discussion and Prof. L. Rasmussen for critical reading and helpful comments on the manuscript. This study was supported by a grant of the Deutsche Forschungsgemeinschaft (SFB 310) to A. T.
References Banno Y, Nozawa Y (1984) Purification and characterization of extracellular acid phosphatase of Tetrahymena pyriformis. Biochim Biophys Acta 799:20-28 Banno Y, Nozawa Y (1985) Purification and characterization of lysosomal c~-glucosidase secreted by eukaryote Tetrahymena. J Biochem 97: 409-418 Frost GM, Moss DA (1987) Production of enzymes by fermentation. In: Rehm HJ, Reed G (eds) Biotechnoiogy 7a: enzyme technology. VCH, Weinheim, pp 65-211 Kiy T (1990) Sekretion der sauren Phosphatase und der fl-Hexosaminidase und anderer Enzyme durch immobilisierte Tetrahymena Zellen. Diploma thesis, Institut for Mikrobiologie,
University of MOnster Klein J, Stock J, Vorlop KD (1983) Pore size and properties of spherical Ca-alginate biocatalysts. Eur J Appl Microbiol Biotechnol 18:86-91 MOiler M (1972) Secretion of acid hydrolases and its intracellular source in Tetrahymena pyriformis. J Cell Biol 52:478-487 Munro IG (1985) Protozoa as sources of commercially produced enzymes - a review. Process Biochem 20:139-144 Spiekermann P, Vorlop KD, Klein J (1987) Animal cell encapsulation within Ca-alginate hollow spheres. In: Neissel RR, Meer KC von der, Luyben AM (eds~ Proceedings of the 4th European Congress on Biotechnology 1987, vol 3. Elsevier Science Publishers, Amsterdam, pp 590~593 Tiedtke A (1983) Purification and properties of secreted N-acetylfl-D-hexosaminidase of Tetrahyrnena thermophila. Comp Biochem Physiol 75B:239-243
App//ed
Microbiology Biotechnology © Springer-Verlag 1991
Lysosomal enzymes produced by immobilized Tetrahymena thermophila Thomas Kiy and Arno Tiedtke Zoologisches Institut der Universit~it MOnster, Schlossplatz 5, D-4400 MOnster, Federal Republic of Germany Received 8 November 1990/Accepted 12 December 1990
Summary. The ciliated protozoon Tetrahymena thermophila was immobilized for production of secreted lysosomal enzymes in two ways. Cells entrapped in solid Ca-alginate spheres survived but were unable to grow and multiply. However, when encapsulated in hollow Ca-alginate spheres Tetrahymena multiplied well, reaching 0.9 x 107 cells/ml. These immobilized cells secreted large amounts of lysosomal enzymes when the medium was changed daily. This system was transferred to a reactor scale using a conical bubble column reactor for semicontinuous cultivation of the encapsulated cells. Under these conditions a-glucosidase, fl-glucosidase, fl-hexosaminidase and acid phosphatase were produced for at least 4 weeks. The hollow spheres were stable for 3 months and contained living and secreting Tetrahymena cells during this time. Immobilized T. thermophila cells can thus serve as a good source for production of commercially interesting enzymes.
Introduction Many lysosomal enzymes are used in diagnostic and other analytical tests. Animals and plants have often served as sources for many of these enzymes, but as the scope of enzymatic tests has increased, the proportion of diagnostic enzymes made by fermentation with microorganisms has also increased (Frost and Moss 1987). In all these organisms the relevant enzymes are intracellularly located and require cumbersome procedures for isolation. Some lower eukaryotes, especially protozoa, secrete lysosomal enzymes by a normal, physiological mechanism (Munro 1985). Thus the ciliate Tetrahyrnena therrnophila constitutively releases large amounts of lysosomal enzymes into the culture medium (Mtiller 1972). This makes it easy to produce cell-free supernatants containing valuable enzymes in high concentrations using immobilized cells. So far, work with immobilized Tetrahyrnena in biotechnology has not been reported.
Offprint requests to: A. Tiedtke
Here we describe a method fo the immobilization o f
Tetrahymena. We also present a method for semicontinuous cultivation of immobilized Tetrahymena cells using a conical bubble column reactor for the production of a-glucosidase, fl-glucosidase, fl-hexosaminidase and acid phosphatase, enzymes well characterized by Banno and Nozawa (1984, 1985) and Tiedtke (1983). Employed the right way this may provide us with a wide range of cheap enzymes.
Materials and methods
Microoryanism. All investigations were carried out with T. thermophila originally obtained from Dr. P. Bruns, Cornell University, Ithaca, NY.
Medium. The cells were propagated under aerobic conditions in a solution containing: 1% proteose peptone, 0.1% yeast extract, 0.003% Sequestrene (Tiedtke 1983); CaC12.2H20 was added to cultures of immobilized cells at a concentration of 0.15% in erlenmeyer flasks and 0.2% in the reactor. We found that these concentrations were necessary to stabilize the alginate beads. The nutrient medium used in fermentations with a bubble column reactor contained 0.005% silicone oil to prevent foaming.
Immobilization techniques. Living Tetrahymena cells were immobilized both in solid and in hollow spheres made of alginate. Cells for immobilization were harvested in the late exponential phase by centrifugation. For entrapment in solid spheres the cell pellet of a 10-ml preculture was resuspended in 200 ~tl of 10 mM TRIS/HC1 buffer, pH 7.4, and mixed with 10 ml of 3% Na-alginate solution. The mixture was pressed through a thin cannula (0.8 × 35 mm) into 100 ml of a stirred 2% CaC12-2H20 solution and left to harden for 20 min. Then the gel beads were washed twice in 10 mr~ TRIS/ HCI buffer and transferred into the fermentation medium. Tetrahymena cells were encapsulated in hollow spheres according to Spiekermann et ai. (1987). One part of a cell suspension (4 × 105 cells/ml) was mixed with three parts of a sterile 1.5% carboxymethylcellulose solution containing 1.5% CaC12.2HzO. This mixture was dropped into a sterile 0.875% Na-alginate solution. Ca-alginate hollow spheres are formed by cross-linking from inside to outside (Spiekermann et al. 1987). The cells were encapsulated in the liquid centre of the hollow spheres. The remaining Na-alginate was removed by three washings with distilled water and then the hollow spheres were hardened in 0.9% CaC12-2H20 solution for 15 min.
15
Semicontinuous cultivation. Tetrahymena immobilized in solid
8xlO 5
spheres was incubated in 1000-ml erlenmeyer flasks in 35 ml medium at 30 ° C. The flasks were shaken (Gyrotory Incubator Shaker, New Brunswick Scientific, Edison, NJ, USA) at 120 strokes/ min. The supernatant was replaced by fresh medium every 24 h. Semicontinuous cultivation of cells immobilized in hollow spheres was carried out by incubating 50 ml beads in a 1000-ml erlenmeyer flask containing 200 ml nutrient medium. Cells immobilized in 50 ml hollow spheres were also cultivated in a 500-ml bubble-column reactor in 200 ml medium at 28° C. The medium was changed every 24 h.
Cell counts. For assessing cell density in the solid spheres a defined volume of beads was incubated in 0.05 M sodium hexametaphosphate and stirred for 10 min. After solubilization of the spheres the cell density was determined using a Fuchs-Rosenthal counting chamber. To determine cell density in the cavity of the hollow spheres, the diameter of the liquid centre of a single sphere was measured using a binocular microscope. From this value the volume of the cavity was calculated. Then the hollow sphere was dissolved in a defined volume of 0.2 M citrate buffer by stirring and the released cells were counted using a Fuchs-Rosenthal counting chamber. This procedure was repeated four times per time point. Enzyme assays. The extracellular activities of acid phosphatase, fl-hexosaminidase, fl-glucosidase, and a-glucosidase were assayed at 37°C, using appropriate p-nitrophenyl substrates (Tiedtke 1983). A sample of 100 ~xl was added to 100 ~tl of 10 mM p-nitrophenyl substrate dissolved in 0.1 M citrate buffer, pH 4.6, supplemented with 0.04% sodium azide and 0.2% bovine serum albumin. After incubation, 1 ml of 0.4 M glycine/NaOH buffer, pH 10.4, was added to stop the reaction. The liberated p-nitrophenol was determined photometrically at 405 nm. One unit (U) was defined as the the amount of enzyme that release~d 1 ~tmol p-nitrophenol/ min at 37 ° C.
Scanning electron microscopic(SEM) investigations. Scanning electron micrographs of spheres and immobilized cells were made using an Electron Microscope Model S-530 (Hitachi, Tokyo, Japan).
Chemicals. Proteose peptone and yeast extract were obtained from Difco (Detroit, Mich., USA) and Sequestrene from CibaGeigy (Greensboro, NC, USA). Sodium alginate (Manugel DJX) for production of solid, spheres was from Alginate Industries (Hamburg, FRG) and sodium alginate (Protonal LF 20/60) for hollow spheres from Protan (Norderstedt, FRG). Sodium carboxymethylcellulose (7HF-CMC) was obtained from Aqualon (Dtisseldorf, FRG). All p-nitrophenyl substrates for the enzyme assays were purchased from Serva (Heidelberg, FRG).
6xlO 5
4xlO 5 0
i
i
~
i
~
~
~
i
2
4
6
8
10
12
14
16
Time
[d]
18
Fig. 1. Concentration of Tetrahymena thermophila entrapped in solid Ca-alginate spheres during semicontinuous cultivation
Secretion of lysosomal enzymes by entrapped Tetrahymena cells Both cell density a n d the a m o u n t o f secreted e n z y m e s decreased during fermentation. As an example, acid p h o s p h a t a s e secretion is s h o w n in Fig. 2. The highest c o n c e n t r a t i o n o f acid p h o s p h a t a s e was m e a s u r e d after the first 24 h o f cultivation. As m u c h as 2 U / 3 5 ml h a d b e e n secreted in this time period. O n l y 0.11 U / 2 4 h p e r 35 ml was secreted after 19 days. The kinetics o f flh e x o s a m i n i d a s e secretion s h o w e d the same general course. C o m p a r e d to the first 24 h the rates o f secretion o f acid p h o s p h a t a s e and fl-hexosaminidase d r o p p e d to a value o f 5%. I n the same time period a decline o f only 32% in cell n u m b e r t o o k place (Fig. 1). Thus the decreases in cell density a n d extracellular e n z y m e activity were disproportional.
Growth of Tetrahymena in hollow spheres Tetrahymena cells encapsulated in Ca-alginate h o l l o w spheres s h o w e d excellent g r o w t h (Fig. 3). Initially, the cell density was 2.1 x 105 c e l l s / m l h o l l o w space. T w o days later the cells h a d r e a c h e d the stationary p h a s e with cell densities o f up to 0.9 x 107 c e l l s / m l h o l l o w space. F o r comparison~ the average cell density o f a
Results 2,5
Growth of Tetrahymena in solid spheres T w o i m m o b i l i z a t i o n m e t h o d s were c o m p a r e d , the first b a s e d o n i m m o b i l i z a t i o n o f cells by e n t r a p m e n t in solid Ca-alginate b e a d s a n d the s e c o n d on e n c a p s u l a t i o n o f cells in Ca-alginate h o l l o w spheres. W h e n Tetrahymena was immobilized in solid spheres no g r o w t h c o u l d be observed. Instead, a decrease in cell density over a time p e r i o d o f 18 days was detected (Fig. 1). At zero time the cell density in the gel b e a d s was 7.8 x 10 5 c e l l s / m l ; after 18 days o f fermentation 5.3 x 10 5 c e l l s / m l Ca-alginate gel were present. The remaining cells were still alive, evident f r o m the activity o f the contractile v a c u o l e a n d f r o m the fact that cells released f r o m the gel regained the ability to divide.
[
~Acid
~
2
¢~
1,5
~
i
~
phosphatase
0,5
0 ~ 1
2
3
4
5
6
7
8
9 Time
10 11
12
13
14
15
16
17
18
19
[d 1
Fig. 2. Secretion of acid phosphatase by T. thermophila entrapped in solid Ca-alginate spheres during semicontinuous cultivation in erlenmeyer flasks
16
~o~
enzymes for 56 days. After 3 weeks suspended cells appeared in the medium due to disintegration of some of the hollow spheres.
© © ~
Semicontinuous cultivation in a bubble column reactor I0 °
10 0
2
1
3
4
5
6
7
8
9
10
11
12
13
14
Time [d] Fig. 3. Multiplication of T. thermophila encapsulated in hollow Ca-alginate spheres during semicontinuous cultivation in erlenmeyer flasks
suspension culture was about 1 x 106 cells/ml. Scanning electron micrographs demonstrate dense cell packing within the hollow spheres (Fig. 5B). The cell density remained more or less constant over a time period of 10 days (Fig. 3). The generation time for immobilized Tetrahymena cells was 6.9 h, compared to 2.5 h in suspension culture under similar conditions.
Secretion of lysosomal enzymes by encapsulated Tetrahymena cells The activities of released acid phosphatase, fl-hexosaminidase and a-glucosidase were measured during the cultivation period. Parallel with the increase in cell density during the first few days we observed a clear increase in extracellular enzyme concentration (Fig. 4). In 3 weeks we harvested 235 U fl-hexosaminidase, 278 U acid phosphatase and 36 U a-glucosidase. During this time a population with high density and cells in g o o d condition was observed using SEM (Fig. 5C and D). Every 24 h, spent medium was replaced by fresh broth. Under such conditions we produced lysosomal
20
o o
~, 1o
.~ ~
5-
.~ 1
2
3
4
5
6
7
8
9
10 11 12 13 14 15 16 17 18 19 20 21 Time [d]
Fig. 4. Secretion of a-glucosidase, fl-hexosaminidase, and acid phosphatase by encapsulated T. thermophila cells during semicontinuous cultivation in erlenmeyer flasks. The supernatant was harvested and replaced by fresh medium every 24 h
The culture experiments with Tetrahymena immobilized in hollow spheres in erlenmeyer flasks showed that production of lysosomal enzymes for a longer time period was possible. Further investigations were made to examine if it was possible to transfer this system to a reactor scale. A conical bubble column reactor with a capacity of 500 ml was chosen. The medium containing additional silicone oil (0.005%) to prevent foaming, was replaced every 24 h. A CaC12.2H20 concentration of 0.2% was necessary to stabilize the spheres. Preliminary experiments using suspension cultures showed that silicone oil had no adverse effect, whereas CaC12.2H20 (0.2%) lowered both growth and secretion rates (U/ 5 x 105 cells) of non-adapted cells (Kiy 1990). During the first experimental run, free cells appeared in the medium of the bubble column reactor after only 10 days. This was due to the disintegration of the spheres. Scanning electron micrographs showed that the outside of the alginate mantle of the hollow spheres was damaged (Fig. 5A). We thought that this damage was due to the rough surface of the fritted glass filter. Therefore it was overlaid with a thin layer of smooth glass beads of 5 mm diameter. Now suspended cells no longer appeared during the first few weeks and the hollow spheres were stable for 12 weeks. We measured the extracellular activities of acid phosphatase, flhexosaminidase, fl-glucosidase and a-glucosidase. The results of this semicontinuous fermentation are shown in Fig. 6.
Discussion
Tetrahymena cells entrapped in solid Ca-alginate spheres did not multiply. During microscopic observations of immobilized Tetrahymena we never observed dividing cells. We assume that the gel structure of the Ca-alginate is too close-meshed to allow the growth necessary for cell multiplication. Klein et al. (1983) detected pore sizes of up to 16.6 nm when investigating structures of Ca-alginate carriers. For comparison, the size of a single Tetrahymena cell is in the range of about 50 ~tm. The gel structure may also have compressed and damaged the living cells and might explain why the secretion of enzymes decreased more than the cell density. The experiments with entrapped Tetrahymena cells, however, showed that, in principle, Ca-alginate is suitable as a carrier because the gel matrix allowed diffusion of the enzymes and survival of the cells. Tetrahymena cells encapsulated in Ca-alginate hollow spheres grew well and reached cell concentrations one order of magnitude higher than those in suspension cultures. The reason for the increase in cell concentra-
17
Fig. 5 A-D. Scanning electron micrographs of Ca-alginate hollow spheres and immobilized T. thermophila. A Hollow sphere after 14 days of cultivation in a bubble column reactor. B A halved hollow sphere filled with T. thermophila. C View into a fenestrated hollow sphere. D Detailed view of cells encapsulated in Ca-alginate hollow spheres
2,5
~
2
~
1,5
~
0,5
2
3
4
5
6
7
5
9
10111213141516171819202122232425 T i m e [d]
1 2 3 4 5 6 7 8
9 10111213141516171619202122232425 T i m e [d]
Fig. 6. Secretion of a-glucosidase, fl-glucosidase, fl-hexosaminidase, and acid phosphatase by encapsulated T. thermophila during semicontinuous cultivation in a conical bubble column reactor. The supernatant was harvested and replaced by fresh medium every 24 h
18
tion remains to be explained. The long generation time of encapsulated Tetrahymena cells (6.9 h) may be due to an adaptation phase of the encapsulated cells and/or diffusion limitations for nutrients and oxygen. The increase in enzyme secretion during the first 4 days can be explained by the increase in cell density during this time period. The little variations in enzyme secretion from the 5th day onwards may be due to population dynamics in the hollow spheres. The different enzyme secretion pattern in the bubble column reactor compared to that in edenmeyer flasks is a result of a higher CaC12 concentration. We propose that the cells need a longer adaptation phase and reach the stationary phase later. This is evident from the amounts of secreted enzymes. As much as 10 U acid phosphatase were already produced after 3 days in erlenmeyer flasks but 10 days passed before this amount was secreted in the bubble column reactor. However, the disadvantage of initially lower secretion rates at higher CaCI2 concentrations is more than compensated for by the long lasting stability of the spheres. Thus we produced enzymes using the same hollow spheres over a period of 3 months. We were able to show that the initially low secretion rates can be avoided when cells that were adapted to 0.2% CaCI2 in suspension culture were encapsulated (unpublished results). Evidently immobilized Tetrahymena cells can serve as good sources for commercially interesting production of lysosomal enzymes. Acknowled#ements. We thank Prof. H. J. Rehm and Dr. K. D. Vorlop for advice and constructive discussion and Prof. L. Rasmussen for critical reading and helpful comments on the manuscript. This study was supported by a grant of the Deutsche Forschungsgemeinschaft (SFB 310) to A. T.
References Banno Y, Nozawa Y (1984) Purification and characterization of extracellular acid phosphatase of Tetrahymena pyriformis. Biochim Biophys Acta 799:20-28 Banno Y, Nozawa Y (1985) Purification and characterization of lysosomal c~-glucosidase secreted by eukaryote Tetrahymena. J Biochem 97: 409-418 Frost GM, Moss DA (1987) Production of enzymes by fermentation. In: Rehm HJ, Reed G (eds) Biotechnoiogy 7a: enzyme technology. VCH, Weinheim, pp 65-211 Kiy T (1990) Sekretion der sauren Phosphatase und der fl-Hexosaminidase und anderer Enzyme durch immobilisierte Tetrahymena Zellen. Diploma thesis, Institut for Mikrobiologie,
University of MOnster Klein J, Stock J, Vorlop KD (1983) Pore size and properties of spherical Ca-alginate biocatalysts. Eur J Appl Microbiol Biotechnol 18:86-91 MOiler M (1972) Secretion of acid hydrolases and its intracellular source in Tetrahymena pyriformis. J Cell Biol 52:478-487 Munro IG (1985) Protozoa as sources of commercially produced enzymes - a review. Process Biochem 20:139-144 Spiekermann P, Vorlop KD, Klein J (1987) Animal cell encapsulation within Ca-alginate hollow spheres. In: Neissel RR, Meer KC von der, Luyben AM (eds~ Proceedings of the 4th European Congress on Biotechnology 1987, vol 3. Elsevier Science Publishers, Amsterdam, pp 590~593 Tiedtke A (1983) Purification and properties of secreted N-acetylfl-D-hexosaminidase of Tetrahyrnena thermophila. Comp Biochem Physiol 75B:239-243
