Immobilization of Tolypocladium inflatum spores into porous celite beads for cyclosporine production

Journal of Bioteehnology, 9 (1989) 237-254

237

Elsevier JBT 00362

Immobilization of Tolypocladium inflatum spores into porous celite beads for cyclosporine production G . - T . C h u n I a n d S.N. A g a t h o s 1,2 1 Department of Chemical and Biochemical Engineering and 2 Waksman Institute of Microbiologt' Rutgers-The State University of New Jersey, Piseataway, NJ 08854, U.S.A.

(Received 1 November 1988; accepted 1 December 1988)

Summary Conidia of Tolypocladium inflatum were immobilized into porous celite beads for the production of the immunosuppressive agent cyclosporine (cyclosporin A, Cy). The strong enhancement in volumetric Cy production as well as specific production based on unit cell mass was demonstrated in immobilized cell culture as compared with freely suspended culture. Altered metabolic pattern in the immobilized fungus was indicated by the production of pink-colored pigments. In view of the diffusional resistance typically encountered in immobilized cell culture, bead size limitation was investigated. Cells entrapped into small beads (diameter in the range of 150/~m-207 /~m) showed markedly higher and sustained productivity compared with cells entrapped into middle-sized (208-294 /~m) or large-sized (295-500 ~tm) celite particles. The immunosuppressant yields on carbon or nitrogen source for the immobilized cell culture were up to three-fold higher than for the free cell culture, thus clearly indicating that the immobilized cell system provides more efficient utilization of substrates for the biosynthesis of Cy than the free cell system, especially when the fungal cells are entrapped into small sized celite particles.

Cyclosporine; Immunosuppressant; Tolypocladium inflatum; Immobilization; Celite bead

Correspondence to: S.N. Agathos, Department of Chemical and Biochemical Engineering, Rutgers University, P.O. Box 909, Piscataway, NJ 08855-0909, U.S.A.

0168-1656/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

238 Introduction

Over the last decade the successful application of immobilized cell biotechnology to processes based on secondary metabolism has generated considerable research interest in this area. This is justified by the potential operational advantages cited for immobilized whole cells (Venkatasubramanian and Vieth, 1979; Blanch, 1984; Karube et al., 1984), i.e., operation at nigh dilution rate, increased volumetric productivity, lower viscosity in filamentous fungal growth, separate optimization of growth phase and production phase, ease of product recovery, increased biocatalyst stability and possibly enhanced metabolic rates, etc. Additionally, the use of immobilized cells appears to be the only possible approach for the (semi-)continuous production of complex compounds through multistep reactions requiring cofactors. To date, the majority of research applying immobilized cell biotechnology has been directed towards the development of processes for the production of primary metabolites (Williams and Munnecke, 1981; Fujimura et al., 1984). Recently, however, due to the ever increasing commercial value of. antibiotics and related medicinal chemicals, concerted research efforts have been applied to the development of immobilized cell biotechnology for the production of secondary metabolites. Examples of such systems include the biosynthesis of five types of antibiotics: /~-lactams [penicillin G (Deo and Gaucher, 1984) and thienamycin (Arcuri et al., 1986)], macrolides [tylosin (Veelken and Pape, 1982) and candicidin (Karkare et al., 1986)], peptides [bacitracin (Morikawa et al., 1980)], nucleosides [nikkomycin (Veelken and Pape, 1982)], and polyketides [patulin (Berk et al., 1984)]. For mycelial organisms such as fungi, an immobilized cell system has several additional advantages compared with a conventional suspended culture system. A key feature of the growth of these mycelial organisms lies in the characteristic highly branched hyphal filaments. This pattern of cell growth results in high viscosities, thus leading to decreased mass transfer capacity in conventional suspended culture. To overcome such problems of filamentous fungal cultures, growth in pelleted form may be induced by manipulation of the growth medium or other environmental factors (Kim et al., 1983; K~Snig et al., 1982). However, due to the complex interactions among these factors, it would be extremely difficult to ensure reproducible conditions for regular pellet formation in these mycelial bioprocesses. The whole fungal culture fluid may be induced to assume a rheology similar to that of a mycelial pellet system by confining the mycelial cells into porous biosupport materials, such as celite (Gbewonyo and Wang, 1983a; 1983b). As an inert biosupport material, celite consists of highly porous diatomaceous beads composed of 90% silica (SiO2) plus some other inorganic oxides. Due to their chemical inertness and unique interconnected pore structure which is very suitable for the physical entrapment of mycelial cells, there is a growing interest in the use of celite beads as biosupport material in recent years (Baker et al., 1984; Arcuri et al., 1986; Jones et al., 1986). Cyclosporins are cyclic peptide antibiotics composed of eleven amino acids produced as secondary metabolites by strains of the filamentous fungus, Tolypo-

239

cladium inflatum. Due to its remarkable selective immunosuppressive effects, cyclosporin A (also spelled cyclosporine, ciclosporin and abbreviated as Cy hereafter) has been the focus of great interest in recent years. Cy is very effective in organ transplants as a key agent used to prevent foreign tissue rejection, and has considerable promise in the control of autoimmune and parasitic diseases (Borel, 1986). Nonetheless, the current availability of Cy is still limited and its cost remains high. Therefore, advances in understanding and control of the formation of Cy are necessary in order to arrive at more efficient processes for this unique drug. Despite the numerous clinical and basic immunological studies of Cy and its analogs (Borel, 1986, 1988; Kahan, 1988a, b), there is little published information on the basic biology and process engineering of this secondary metabolic bioprocess (Dreyfuss et al., 1976; Kobel and Traber, 1982). Progress in the understanding of Cy biosynthesis has been achieved recently, implicating a non-ribosomal, thiotemplate enzyme mechanism of peptide formation (Kobel et al., 1983; Zocher et al., 1986; Billich and Zocher, 1987). In our laboratory, Agathos et al. (1986, 1987) have addressed microbiological and process aspects of the fungal production of Cy aiming at a better understanding of the factors that may contribute to optimal drug production in freely suspended cultures. In this study, we are exploring the potential for process improvement by use of immobilized cells of T. inflatum entrapped into porous celite beads for the production of Cy. The morphological and physiological changes of immobilized cells of T. inflatum were examined based on bioprocess kinetic analysis and were also compared with the corresponding data from suspension cultures under identical nutritional and operational conditions at the shake flask level. Additionally, in view of the diffusional resistance typically encountered in immobilized systems, different sizes of celite beads were compared in order to assess the effect of particle size limitation.

Materials and Methods

Microorganism and culture development Suspension cultures of Tolypocladium inflatum (ATCC no. 34921), recently renamed Beauveria nivea were cultivated in our previously developed (Agathos et al., 1986) semisynthetic medium (SSM) composed of glucose (50 g 1-1), peptone (10 g 1 1), KHePO4 (5 g 1-1) and KC1 (2.5 g 1-1). Growth took place in 250 ml shake flasks at 200 rpm and 27 o C. The pH of SSM was initially adjusted to 5.7 with K O H (1 N) before sterilization. After 5 d of growth, 25 ml of fresh seed cultures were inoculated into about 1 inch thick agar slants in a 2 liter Fernbach flask in order to allow to sporulate. The composition of the solid medium was SSM containing 20 g 1 1 agar. Heavy sporulation of the mycelial cells occurred after 7 d of incubation at 27 ° C. The conidiospores were harvested by vigorously shaking the flask with aliquots of sterile distilled water. The concentrated spore suspension containing also fragments of mycelial cells was filtered through autoclaved Whatman No. 1 filter paper in order to obtain completely de-aggregated spores. After estimating the spore

240 concentration by use of a haemocytometer, the spore suspension was diluted in aliquots of sterile distilled water to give a concentration of 108-109 spores per ml. The spore suspension obtained was used to inoculate the celite beads or to seed freely suspended cultures for comparison.

Cell immobilization methodology Celite was selected as a suitable bead matrix for immobilization of Cy-producing

T. inflatum. The immobilization procedure developed by G b e w o n y o and Wang (1983a) with cells of P. chrysogenum was adapted to our mycelial cells with a number of modifications. Most of the batch experiments in shake flasks were carried out using the following protocol, unless mentioned otherwise: celite grade 560 obtained from Manville Corp. (Englewood Cliffs, N J) was classified on mesh screens into the following three different particle sizes: (large sized beads) 295-500 /Lm; (middle sized beads) 208-294 t~m; (small sized beads) 150--207/~m. The celite beads were pretreated by washing with distilled water several times and heated in a furnace overnight at 600 ° C to remove volatile materials. The particles were then steam-autoclaved for 1 h and allowed to dry at 121°C for 30 min. The prepared spore suspension was added directly to the dry autoclaved celite beads in the proportion 2 : 1 (v/v). The inoculated spores were adsorbed and immobilized into celite beads at 27 ° C and 200 r p m on a New Brunswick Scientific (Edison, N J) rotary shaker. After 2 h of incubation, the supernatant was decanted and the celite beads were washed with sterile distilled water to remove unentrapped spores. Finally synthetic medium that had been autoclaved at 121°C for 30 min was added aseptically to the immobilized celite beads in 250 ml flasks and the inoculated beads were incubated at 27 o C on a New Brunswick Scientific rotary shaker at 250 rpm for growth and Cy production. The synthetic medium (SM) modified from Kobel and Traber (1982) was composed as follows: 30 g 1 1 glucose, 10 g I 1 (NH4)2SO4, 0.5 g 1-1 MgSO 4, 0.1 g 1-1 CaC12, 0.75 g 1-1 K H 2 P O 4 and 0.1% ( v / v ) of a trace metal solution. The trace metal solution contained: 4400 mg 1-1 Z n S O 4 . 7 H 2 0 , 180 mg 1-1 M n C 1 2 . 4 H 2 0 , 25 m g 1 - 1 N a 2 M o O 4, 80 m g l 1 C u S O 4 . 5 H 2 0 , 5000 m g 1 - 1 FeSO 4 • 7H20, 2 ml H2SO 4 and 1 1 water. The p H of the SM medium was initially adjusted to 6.0. Modified synthetic medium was the same as SM, except that Mg and Ca salts were omitted.

Analytical methods Cyclosporine A 10 ml portion of whole immobilized culture (colonized beads plus medium) was ground in a cell homogenizer and was subsequently kept frozen at - 2 0 ° C until extraction time. Extraction of the thawed sample was performed by adding an equal volume of butyl acetate. Just before extracting the sample, a concentrated solution of N a O H was added to the sample to reach a concentration of 1 N and heated at 60 ° C for 30 min. The mixed sample was incubated at 27 o C in a rotary shaker at 250 rpm for 24 h. After centrifuging the extracted sample, a defined amount of the supernatant was removed and dried at room temperature by air blown through a multi-nozzle manifold. After drying the sample, an equal

241

amount of acetonitrile was added to redissolve the extracted Cy, which was then filtered through a microfilter and injected to HPLC. The HPLC pump (Spectroflow 400) and the UV detector (Spectroflow 783) were supplied from Kratos (Ramsey, N J). An integrator (C-R 3A) from Shimadzu (Kyoto, Japan) was used for data analysis. The 4.6 × 150 mm reversed phase column packed with 5/tm Spherisorb C8 (Phase Separations, Norwalk, CT) was kept at 81 ° C. The sample was injected via a Model 7010 injection valve (Rheodyne, Cotani, CA). The mobile phase consisted of acetonitrile/water (67/33) plus 0.1% trifluoroacetic acid (TFA) dissolved in the water phase, with a flow rate of 1.5 ml min-l. The quantification of Cy was based on authentic Cy standards, generously provided by Sandoz (Hanover, N J). Glucose and ammonia concentration Glucose was measured by use of the Beckman Glucose Analyzer 2 (Beckman Instruments Inc., Fullerton, CA) and ammonium was determined by use of an ammonium electrode (Model 95-12) supplied from Orion Research Inc. (Boston, MA). All samples were assayed twice and the average of the readings was taken for computing the glucose and ammonium concentrations of the samples. Biomass Three 10 ml samples consisting of culture fluid and solids (cells and beads) were taken from each flask sacrificed. After each of five consecutive centrifugations, the samples were washed with distilled water to completely remove the medium from deep inside the celite particles. The washed solids were dried at 85°C for 24 h to obtain the dry weight of dry cells plus celite particles. After heating the solids in a furnace at 600 °C for 5 h, the weight of celite particles alone was obtained. The dry cell concentration of each sample was calculated by deducting the weight of the celite particles from the weight of the solids. Finally the amount of cell growth obtained in immobilized cultures was determined by averaging the measured dry cell mass of the three samples.

Results and Discussion

Comparison between immobilized and free cells in culture Comparative studies were carried out to investigate the biological and engineering performances of the immobilized and free cell culture under the cultivation conditions described in Fig. 1. Cy production for both the immobilized and free cells appears consistently associated with cell growth. This suggests operating a reactor system in a way in which higher growth rate could be obtained for higher volumetric Cy production. As shown in Fig. 1, the immobilized system produced slightly lower cell density compared with the free cell culture. Only a minor fraction (approximately 5%) of the overall biomass in the immobilized system was found in the liquid suspension. This clearly indicates that in the immobilized system, the mycelial cells have grown predominantly entrapped into celite beads. However, even with lower cell growth, the immobilized culture produced about 2.3 times higher volumetric Cy production than the free cell culture. Thus the specific Cy production

242 12 •

FREE CELLS

" - ~

10

I:ELI~BIUZED CELLS

8 ¸ ¢JO .~

6 ¸

~o

4 2¸ O, 50

100

150

200

250

300

350

T I M E (h)

Fig. 1. Comparison of cell growth between freely suspended and celite-entrapped cells of T. inflatum (bead size: 295-500 ffm, 200 rpm, 27 o C, 30% v / v beads in 100 ml SM medium per 250 ml flask).

of the immobilized cells based on unit cell mass was remarkably higher (3.2-fold) compared with that of the free cells (Fig. 3). In Fig. 2, it is also worthwhile to note that not only the maximum production but also production rate in the immobilized cell cultivation was increased compared with the free cell system. Fig. 4 shows residual glucose concentration as a function of incubation time for immobilized and free cell cultivation. Most of the glucose was utilized before 300 h of incubation in the free cell system while the glucose in the immobilized system was not completely

200

150

1O0 '

o 50'

0 i

i

co

i

1oo

i

,co'2oo

"2;o"3oo

T I M E (h)

Fig. 2. Comparison of Cy production between freely suspended and celite-entrapped cells of 72 inflatum, and effect of grinding celite beads before extraction on Cy production (same conditions as Fig. 1). (O) Free cell culture, (m) immobilized cell culture (no celite grinding), (O) immobilized cell culture (celite grinding), (&) released cells in immobilized cell culture.

243 35 t

FREE CELLS IMMOBILIZED CELLS

30Z o

~

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f..,) O ~

20-

>"

15-

13. O0

D. 5-

100

150

200

250

300

350

TIME (h) Fig. 3. Comparison of specific production of Cy as a function of incubation time between freely suspended and celite-entrapped cells of T. inflatum. The specific production is defined as mg Cy produced per g ceils (same conditions as Fig. 1).

consumed until the end of cultivation, thus indicating more economic utilization of the carbon source in the immobilized system. Table 1 summarizes the performance of the immobilized mycelial culture in comparison with the free cell culture under identical nutritional and operational conditions at the shake flask level. The higher specific production of the immobilized cells might be related to the stability of the enzymes involved in the biosynthesis of Cy. Immobilized cells often show improved operational stability of intracellular biosynthetic enzymes, thus leading to higher net product formation (Chibata and Tosa, 1981). Gaucher and

TABLE 1 C O M P A R I S O N B E T W E E N F R E E A N D I M M O B I L I Z E D C U L T U R E S O F T. I N F L A T U M SYNTHETIC MEDIUM Parameter

Units

Free cells

Immobilized cells

Max. cyclosporin A production Max. cell mass Cyclosporin A production per cell b Glucose per cyclosporin A b Cells per glucose c

m g 1-1

69.6

g 1-a

10.9

161.6 (97.3) a 8.6

mg g- 1

6.4

g mg- 1

0.43

0.15

g g-1

0.37

0.33

a Cy titre obtained from the immobilized cells without grinding of solids. b Values corresponding to m a x i m u m concentration of Cy. c Values corresponding to m a x i m u m concentration of cell mass.

20.2

IN

244

40. •

FREE CELLS IMMOBILIZED CELLS

30

I.u (n 0 (J

20

..J 10

0

.

. 50

. 100

.

. 150 TIME

. 200

. 250

300

350

(h)

Fig. 4. Comparison of glucose consumption as a function of incubation time between freely suspended and celite-entrapped cells of T. inflatum (same conditions as Fig. 1).

co-workers (1981) have reported superior stability of the secondary metabolitesynthesizing apparatus in immobilized Penicillium urticae for the production of patulin. On the other hand, such higher specific production could be attributable to increased activities of secondary metabolic enzymes, at least partly due to an increase in enzyme synthesis in the immobilized cells in culture. As shown in Table 1, the cell yield on glucose was not much different in the two systems. However, the specific glucose consumption (the reverse of Cy yield on glucose) for the immobilized system was markedly lower (35%) as compared with the freely suspended cells (100%). This result clearly demonstrates that the immobilized cell system provides a more efficient utilization of carbon/energy source for biosynthesis of Cy than the free cell system. Superior carbon conversion efficiency was also observed by Karkare et al. (1986) for immobilized Streptomyces griseus producing candicidin. This was assumed to be due to the restricted growth of the immobilized cells. It is possible that the higher secondary metabolite productivity in the immobilized system is causally linked with the slower cell growth rate after 150 h of cultivation, a point beyond which glucose uptake rate appeared considerably lower than that of the free cell system (Fig. 4). Thus, further improvement in the production of Cy by immobilized cells could be possible if judicious manipulation of medium composition and physical cultivation environment is done in order to increase the conversion of carbon/energy source to intermediates leading to the immunosuppressant. Considerable increase in measurable Cy titre in the immobilized cell system, a result of an improvement in the extraction process, can be noted in Fig. 2. Indeed, by introducing the procedure of grinding the celite particles before extracting the samples (see Materials and Methods), and thus releasing the whole cells out of these particles, volumetric Cy production was increased about 1.6 times compared with a control (no grinding) system. In view of the fact that Cy is normally produced in a growth-associated mode (Agathos et al., 1987; this work), this result suggests that

245

Fig. 5. Photomicrographof mycelialcells of T. inflaturn entrapped into celite beads (incubation time: 10 d, magnification:400 x ). the mycelial cells have grown compactly inside the celite particles, thus imposing a diffusion barrier against the solvent during the extraction procedure. The immobilized cells in culture showed completely different physiological and morphological characteristics from the freely suspended cells in a parallel culture. As mentioned above, the immobilized cells grew mainly entrapped into the celite particles. As shown in the photomicrograph (Fig. 5), the filaments of the immobilized cells appear to extend radially outward from inside the beads, thus covering the whole spatial area of the celite particles. With increasing cultivation time, the immobilized cells produced pink-colored pigments in the media, which could be regarded as an indicator for altered metabolic pattern in the immobilized fungus. On the contrary, free cells in culture never produced such a kind of pigments during the whole cultivation period. In addition, free mycelia originated from conidia of T. inflatum showed very limited formation of mycelial pellets, resulting in apparently higher viscosities of the culture broth as compared with those of the immobilized cells in culture. Usually, during the growth of mycelial organisms in submerged liquid culture, the highly branched hyphae characteristic of filamentous fungi impose high viscosities and non-Newtonian behavior on the culture, thus leading to decreased mass transfer capacities. With respect to these phenomena, Gbewonyo and Wang (1983b) demonstrated approximately a 2-3 fold increase in oxygen mass transfer rates in a bubble column reactor when mycelial cells of P. chrysogenurn were grown confined into celite particles as compared with a free cell cultivation.

246

2o

II

• FREECELLS ---o--- SMALLBEADS 1 " LARGEBEADS I

"~

i

///~.~

~

10

0

0

50

100 150 TIME (h)

I

200

I

250

Fig. 6. Pattern of cell growth as a function of incubation time for freely suspended cells and immobilized cells entrapped into either small sized or large sized celite beads (small beads, 150-207 btm; large beads, 295-500/~m; 250 rpm, 27 o C, 50%, v/v, beads/80 ml modified SM medium in 250 ml flask).

Bead size effect on Cy synthesis In order to select optimum-sized celite beads, we assessed the effects of support particle size on Cy bioprocess kinetics. To this end, a set of batch experiments was carried out at the shake flask level using modified SM medium (SM medium without CaC12 and MgSO 4) under the cultivation conditions specified in Fig. 6. The respective range of bead diameters is also described in Fig. 6. For comparison, freely suspended cells were also cultivated under identical nutritional and environmental conditions. As shown in Fig. 6, growth rate as well as maximum cell growth of the immobilized fungus were much higher than those of freely suspended cells during the whole cultivation period. The cells in the suspension culture were somewhat restricted in their growth, possibly due to some limiting factor(s) in the simple modified SM medium as compared with that of the freely suspended cells grown in the previously used SM medium. It should be noted that the highest volumetric production of Cy was observed in the small bead system (max. Cy titres 2.2-fold and 5.4-fold higher than those of the large bead system and the free cell system, respectively), even with a relatively low level of cell growth. Accordingly, the small bead system showed the highest values of specific Cy production per unit cell mass among all the systems examined (Figs. 7 and 8). The possible reason for such higher specific production of the immobilized cells in comparison with the freely suspended cells could be attributable to enhanced operational stability or increased activities of the secondary metabolic enzymes involved in the biosynthesis of the immunosuppressant as already mentioned in the previous section. On the other hand, when considering the immobilized cell systems only, it should be noted that the same volume of celite particles was inoculated (50%, v / v ) for each system in this experiment, thus resulting in a higher collision frequency among the particles in the

247 3o0

•

FREE CELLS

2OO

Z

o,

100

0 0

|

|

|

50

100

150

=

|

200

250

TIME (h)

Fig. 7. Pattern of Cy production as as function of incubation time for freely suspended and immobilized cells of T. inflaturn (same conditions as Fig. 6).

small bead system. Therefore, the resulting high stress (shear) exerted over the cells immobilized in the small beads could possibly lead to a restriction of intra-particle growth but an enhancement of secondary metabolite production. This suggests that a higher portion of the substrates consumed would be utilized for biosynthesis of Cy rather than for cell growth in the small bead system. Consequently, a much higher specific production per unit cell mass for the small bead system made it possible to compensate for the somewhat restricted cell growth, thus leading to the highest volumetric Cy production observed. These conditions can be regarded as compatible

30

z

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~

20

_.9. ° u. ~ a.

10

0

i

i

i

|

i

50

100

150

200

250

TIME (h)

Fig. 8. Pattern of specific production as a function of incubation time for freely suspended and immobilized cells of T. inflatum (same conditions as Fig. 6).

248 T ABLE 2 C O M P A R I S O N OF Cy BIOPROCESS P E R F O R M A N C E F R O M I M M O B I L I Z E D T 1 N F L A T U M (BEAD SIZE EFFECT) U T I L I Z I N G SIMPLE M O D I F I E D SM M E D I U M Parameter

Units

Max. cyclosporin A production Max. cell growth Cyclosporin A production per cell a Cells per glucose b Cyclosporin A per glucose a Cells per ammonium sulfate ~' Cyclosporin A per ammonium sulfate a

Free cells

Immobilized cells Large bead

Small bead

52.2

128.2

281.0

g 1- 1

6.8

14.6

10.4

mg g 1

7.7

13.1

27.7

gg 1

0.31

0.43

0.27

mg g 1

2.35

4.17

7.37

gg ]

3.4

4.8

3.0

26.2

46.3

82.2

mg 1 1

mg g - 1

a Values corresponding to m a x i m u m concentration of Cy. b Values corresponding to maximum concentration of cell mass.

with currently used general strategies for enhancement of secondary metabolic productivity based on maintaining active conversion of substrates preferentially for idiolite production and not for cell growth. Further analysis of this phenomenon is given below by use of systematic analytical data from Table 2. Fig. 9 indicates that the consumption rate of glucose in the small bead system was much faster than that in the large bead system during the exponential growth

40 •

FREE CELLS SMALL BEADS

A

UJ

u) O

3O

20

.J

10

0

-

.

so

,

-

100

,

150

-

|

200

-

i

2so

TIME {h)

Fig. 9. Pattern of glucose consumption as a function of incubation time for freely suspended and immobilized cells of T. infiatum (same conditions as Fig. 6).

249

12 • ~

FREE CELLS SMALL BEADS LARGE BEADS

UJ

,,=,1 ,,.=

u'~ 9

0 ~

8'

i

i

*

|

i

50

100

150

200

250

TIME (h) Fig. 10. Pattern of ammonium sulfate as a function of incubation time for freely suspended and immobilized cells of T. inflatum (same conditions as Fig. 6).

phase. Furthermore the consumption rate of ammonium sulfate also exhibited a similar trend as the consumption rate of glucose, although in both immobilized cell cultures ammonium sulfate was not completely utilized (Fig. 10). The observed results indicate that the cells immobilized in the small beads may be more active in their metabolic functions supplying sufficient amounts of intermediates, thus leading to higher production of secondary metabolites. On the other hand, mass transfer limitation against the substrates, which might be caused by higher cell density within the beads as well as by large particle size, could be functionally related to the lower consumption rate for both substrates in the large bead system. Diffusional resistance associated with immobilized cell culture has been repeatedly recognized as a rate-limiting step (Hiemstra et al., 1983; Black et al., 1984), thus leading to lower efficiency of immobilized biocatalysts due to substrate limitation in the interior of the particles. Table 2 summarizes the bioprocess results of the three systems obtained under identical nutritional and operational conditions at the shake flask level. The cell yields and Cy yields on glucose and ammonium sulfate for the culture of freely suspended cells showed the lowest values, clearly revealing very limited activities of the free cells, possibly due to some limiting factor(s) which remained uncontrolled in both primary and secondary metabolism. When comparing the data obtained with the immobilized cell culture by use of different sized beads, markedly higher Cy yields on glucose and ammonium sulfate (both 1.8 times) were observed in the small bead system compared with the corresponding parameters in the large bead system. However, the cell yields on each substrate in the small bead system were shown to be about 1.6 times lower than in the large bead system. These results, once again, obviously demonstrate a most efficient utilization of both carbon and nitrogen sources for the biosynthesis of Cy by the T. inflatum immobilized in the small sized celite particles. Therefore a critical combination of immobilized cell

250 0.3 •

LARGE BEADS, 295-500 pm MEDIUM BEADS, 208-294 #m SMALL BEADS,

O o

0.2'

~ u

0.1

0.0 0

150-207 ~.m

!

i

i

20

30

40

50

CELITE BEAD CONCENTRATION (% V/V) Fig. 11. Effect of bead concentration on cell loading into variously sized celite beads in batch shake flask cultivation (initial pH 6.0, 200 rpm, 27 ° C, 80 ml medium per 250 rrd flask, modified SM medium, 10 d cultivation).

growth with optimum sized beads in order to ensure sufficient amount of mycelium with high activities of secondary metabolic enzymes seems likely to be a promising strategy for the enhancement of Cy production in immobilized cell culture. Figs. 11 and 12 depict experimental data obtained in order to evaluate the theoretical maximum capacity for Cy production by immobilized cells of T. inflatum. For this purpose, various sizes of celite beads as well as various celite concentrations were tested using modified SM medium under the cultivation conditions described in Fig. 11. As expected, the cell loading capacity of the celite beads (based on unit celite mass) which was highest at 10% (v/v) bead concentration

28 26' Z O~"

24

"'1 ~

22

~. O¢..~

20

w v ~ffl

16

•

LARGE BEADS,

295-500 l~m

MEDIUM BEADS, 208-294 ~m

14

*-

12 0

SMALL BEADS,

150-207 p.m

i

i

i

20

30

40

50

CELITE BEAD CONCENTRATION (% V/V)

Fig. 12. Effect of the concentration of variously sized beads on specific production of Cy by celite entrapped cells of T. inflatum in batch shake flask cultivation (same conditions as Fig. 11).

251 (volume of celite per votume of liquid medium) declined to lower levels with increased celite concentration. However, at each celite concentration, practically no difference was observed in cell loading capacity amongst variously sized celite beads (Fig. 11). Differences in specific Cy production for the corresponding preparations was clearly manifested as a function of celite concentration (Fig. 12). For example, at 50% ( v / v ) bead concentration, the small sized bead system showed higher specific Cy production (100%) compared with the middle sized (78%) or large sized (61%) bead system respectively. Assuming that the respective celite bead types could accommodate the highest amount of immobilized cells and also that these cells could maintain the capacity for maximum specific production, then the hypothetical maximum amount of Cy produced based on unit reactor volume could be given by: m = PQC

where m, hypothetical maximum amount of Cy produced (mg Cy per 1); P, maximum celite capacity for cell growth (g cell per g celite); Q, maximum Cy specific production (mg Cy per g cell); C, inoculated amount of celite beads per unit volume of reactor (g celite per 1). Based on the experimental values shown in Figs. 11 and 12, the resulting hypothetical values of Cy production for the respective celite beads at 50% (v/v) bead concentration are as follows: (large beads) 611 mg Cy per 1; (middle beads) 745 mg Cy per 1; (small beads) 960 mg Cy per 1. Although these values were derived based on celite capacity only, without considering the cells' physiological characteristics and possible regulatory mechanisms involved in the biosynthesis of Cy, we may conclude that the small sized celite particles are the most productive based on unit reactor volume. Indeed these results suggest that the immobilized cell culture of T. i n f l a t u m could be a promising alternative to the conventional free cell bioprocess, possibly leading to remarkably higher volumetric Cy production if the engineering aspects of the immobilized cell bioculturing on a large scale could be handled judiciously. Furthermore, when physiological characteristics as well as regulatory mechanisms of the immobilized cells involved in the biosynthesis of Cy are considered together with the engineering aspects, then probably striking enhancement of Cy production could be expected.

Acknowledgements We gratefully acknowledge the generous assistance of J. Lee in the course of this experimental study. This project was supported in part by grant No. CBT 87-09083 from the National Science Foundation and by Biomedical Research Support Grant No. PHS RR 07058-21 to S.N.A. The initiation of these investigations was made possible by a Rutgers University Research Council grant to S.N.A. The gift of Cy standards from Sandoz, Inc. (Hanover, N J) is gratefully acknowledged.

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