Separation, Recovery, and Purification in Biotechnology - American

3 Dual Hollow-Fiber Bioreactor for Aerobic Whole-Cell Immobilization 1

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Ho Nam Chang , Bong Hyun Chung , and In Ho Kim

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Department of Chemical Engineering, Korea Advanced Institute of Science and Technology, Dongdaemun, Seoul, Korea Lucky Central Research Institute, Daeduk, Korea

Downloaded by CORNELL UNIV on October 5, 2016 | http://pubs.acs.org Publication Date: July 11, 1986 | doi: 10.1021/bk-1986-0314.ch003

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Aerobically growing Escherichia coli, Aspergillus niger and Norcardia mediterranei were immobilized in the interstitial space of a dual hollow-fiber bioreactor formed by a parallel arrangement of three microporous polypropylene hollow fibers contained within a silicone tubule. All three types of cells grew well and attain ed high densities to reach 550-600 g dry cell weight per liter of the cell containing volume. In the culti vation of E. coli, cell growth among the fibers was not uniform and leakage of cells through the fiber walls was observed. The unlimited growth of A. niger expand ed the silicone fiber and compressed the inner fibers to reduce the substrate flow rates gradually to zero. Only Nocardia mediterranei was grown successfully to make possible long term operation of 50 days or more, producing antibiotics rifamycin Β with a volumetric productivity of 125 μg/mL/h based on the volume occupi ed by the immobilized cells. This corresponds to a 30fold increase over the productivity of a comparable batch system. For the past f i f t e e n years hollow-fiber membrane bioreactors have been extensively used for immobilizing enzymes (1,2.), animal c e l l s (3), microbial c e l l s (4,5) and plant c e l l s (6). Immobilization of microbial c e l l s i n a hollow-fiber reactor offers some d i s t i n c t advan tages over other methods: c e l l s can be e a s i l y immobilized without much preparation; primary separation of products i s carried out; very l i t t l e energy w i l l be consumed i n the scale-up operation. How ever, there are some disadvantages as well. Insoluble substrates can not be used and usually substrate pretreatment i s required to prevent the f i b e r s from being blocked. In general, polymer-based hollow f i b e r s can not be repeatedly h e a t - s t e r i l i z e d . Inherently the transport of gas i s d i f f i c u l t and thus the c u l t i v a t i o n of aerobic c e l l s with high oxygen demand becomes d i f f i c u l t . Robertson and h i s colleagues at Stanford University have exam ined hollow-fiber membrane bioreactors as a means for continuous 0097-6156/ 86/ 0314-0032506.00/ 0 © 1986 American Chemical Society

Asenjo and Hong; Separation, Recovery, and Purification in Biotechnology ACS Symposium Series; American Chemical Society: Washington, DC, 1986.


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Dual Hollow-Fiber Bioreactor

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production of ^-lactamase and ethanol using IS. c o l i and S_. c e r e v i siae immobilized i n the sponge layer of asymmetric hollow f i b e r s (7,8). In the _E. c o l i culture c e l l s leaked through the f i b e r walls, and the production of carbon dioxide was a problem i n the ethanol production. Recently Robertson and Kim (9) developed a dual hollowf i b e r bioreactor consisting of s i l i c o n e tubules for oxygen transport and microporous polypropylene hollow f i b e r s for substrate transport to study the production of tetracycline using Streptomyces aureofaciens. Higher productivity as compared to that of the batch fermentation was achieved. The production remained at high l e v e l s for three days and declined sharply after that for unknown reasons. The present study reports the f i r s t successful long-term c u l t i v a t i o n of rifamycin-B producing Nocardia mediterranei and the problems encountered i n growing IS. c o l i and A_. niger c e l l s i n the dual hollowf i b e r bioreactor. Materials and Methods Materials and s t r a i n s . Yeast extract, bactopeptone, malt extract and trypton were products of Difco Laboratories and glucose was from Hayashi Pure Ind. (Tokyo, Japan). E. c o l i (Sigma EC-1, a l k a l i n e phosphatase-rich mutant) was from Sigma Chemical Co. (St. Louis, MO) and Nocardia mediterranei (ATCC 21789) was from American Type Culture C o l l e c t i o n . Aspergillus niger B-60 was obtained from Kubicek at Technical University Wien (Austria) who used t h i s s t r a i n for c i t r i c acid production studies (10,11). Bioreactor system. The reactor used i n t h i s study was constructed d i f f e r e n t l y from that of Robertson and Kim (9). The reactor was a glass tubing of 30cm length (0.8 cm i.d.) i n which ten dual hollowf i b e r units were bundled together i n a p a r a l l e l assemblage. Each unit had one s i l i c o n e tubule (Dow Corning, 0.147 cm i . d . , 0.196 cm o.d.) that contained three microporous polypropylene hollow f i b e r s (Enka, West Germany, 0.03 cm i . d . , 0.065 cm o.d.) inside. Robertson and Kim used one polypropylene hollow f i b e r i n which three s i l i c o n e tubules were placed to make one polypropylene/silicone f i b e r assemblage. Thus the order i n s i l i c o n e and polypropylene f i b e r s was oppos i t e to the o r i g i n a l l y developed bioreactor. The cross section of a dual hollow f i b e r unit i s shown i n Figure 1 wherein microbial c e l l s are supposed to grow i n the r e s t r i c t e d i n t e r s t i t i a l space between the two f i b e r walls. The detailed dimensions of the reactor are shown i n Figure 2. The t o t a l volume of the glass tube based on the 16-cm e f f e c t i v e length was 8.04 cm^ and that of the i n t e r s t i c e for c e l l growth was 1.12 cm^. The c e l l inoculum port was covered with rubber through which inoculation could be made with a syringe needle. Reactor operation. The polypropylene hollow f i b e r s i n the reactor were prewetted prior to inoculation with r e c i r c u l a t i o n of 50% ethanol and s t e r i l i z e d chemically with 5% formalin solution. Then the reactor was washed by u l t r a f i l t r a t i o n of one l i t e r of autoclaved d i s t i l l e d water. The reactor was placed i n a water bath maintained at a d e s i r ed temperature. C e l l s were inoculated through the inoculation port using a syringe needle. The detailed experimental setup i s shown i n Figure 3.



SEPARATION, RECOVERY, AND PURIFICATION IN BIOTECHNOLOGY

NUTRIENT

CELL NUTRIENT INOCULUM OUT

EPOXY SEAL

\SILICONE RUBBER SEAL 235mm 300mm

F i g u r e 2. D e t a i l e d bioreactor.

specification

o fa dual

hollow-fiber


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CHANG ET AL.

Dual Hollow-Fiber

Bioreactor

35

IS. c o l i seed culture from the l y o p h i l i z e d c e l l s was grown i n a 250 mL flask with a LB medium (yeast extract, 10 g/L, trypton, 10 g/L , pH 7 adjusted with 1 Ν NaOH) placed on a rotary shaker (250 rpm). When the flask culture reached exponential growth phase, i t was d i l u ted 20 times and inoculated into the reactor. The medium and a i r flow rates were maintained at 2 mL/h and 100 mL/min, respectively. In order to see nutrient consumption during the c e l l growth a LB medium with 5% glucose was used. The temperature for the seed c u l t ure and the reactor operation was 37°C. For the A_. niger culture, spores grown i n sugar agar slant were diluted with s t e r i l i z e d d i s t i l l e d water to a concentration of 10-10 spores/L and inoculated into the reactor. The medium for the reac tor operation consisted of sucrose, 60 g/L; ΥΆ^Ο 1 g/L; MgSO,· 7H 0, 0.25 g/L; NH^NO^, 2.5 g/L and the pH was adjusted to 3.1 with 2N HC1. The flow rates for the medium and a i r were the same as i n the IS. c o l i case. The temperature was maintained at 30°C. The medium for If. mediterranei seed culture were: glucose, 20 g/L; yeast extract, 5 g/L; bactopeptone, 5 g/L; malt extract, 5 g/L (pH 7.3 adjusted with 1 Ν NaOH). The seed culture was carried out as i n the IS. c o l i culture except the temperature (30°C). When the glucose l e v e l dropped to 9 - 11 g/L, the seed culture was diluted 10 times and used i n the inoculation. For comparison, a batch c u l ture was performed with the following medium composition: glucose, 110 g/L; yeast extract, 10 g/L; bactopeptone, 10 g/L; sodium barbi t a l , 0.7 g/L. The batch fermentation was carried out i n a 500 mL flask at 30°C and at 250 rpm on a rotary shaker. For the hollowf i b e r reactor the medium flow rate was 1.7 mL/h and the a i r flow rate was 100 mL/min. The medium composition for the reactor operation was: glucose, 20 g/L; yeast extract, 5 g/L; malt extract, 5 g/L; sodium b a r b i t a l , 0.5 g/L.


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A n a l y t i c a l methods. After the reactor operation the f i b e r s were cut into 10 cm segments and dried i n an oven at 90°C for 72 hours. The dry mass density was obtained by taking the difference between the dry mass of the cut f i b e r and that of an empty one of equivalent length. This difference corresponds to the biomass accumulated i n the i n t e r s t i t i a l space between the inner and outer f i b e r s . Glucose was determined by glucose analyzer (YSI model 23 A, Yellow Springs, OH) and rifamycin Β was measured spectrophotometrically at 425 nm. Microscopic techniques. Morphological examination of JE. c o l i and N_. mediterranei contained i n the reactor was done i n a Jeol trans mission electron microscope (model 100CX). The sample was prepared according to the method by Robertson and Kim (9). For the picture of _A. niger reactor the f i b e r was cut into 1 mm pieces, which were photographed with a l i g h t microscope. Results and Discussion Cultivation of E. c o l i . Figure 4 shows glucose concentration and pH h i s t o r i e s during the course of IS. c o l i c u l t i v a t i o n i n the reactor. After 4 days IS. c o l i c e l l s began to appear i n the effluent, which means that the c e l l s i n the reactor leaked through the pores of the polypropylene membrane which were supposed to be smaller than the



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Figure 3· Schematic diagram of experimental setup: 1. medium reservoir, 2. p e r i s t a l t i c pump, 3. dual hollow-fiber bioreactor, 4. water bath, 5, a i r or pure oxygen bombe, 6. rotameter, 7. humidifier, 8. inoculum syringe, 9. sampling bottle, 10. effluent reservoir.

DAYS Figure 4. course of ; glucose triangles

Glucose and pH h i s t o r i e s i n the effluent during the the dual hollow-fiber reactor operation. - ο -, - · cone, pH. The f i l l e d c i r c l e s and represent the effluents containing E. c o l i c e l l s .


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size of IS. c o l i c e l l s . Figures 5(a) and 5(b) show the electron micr ographs of the JE. c o l i c e l l s at the boundary of the polypropylene f i b e r and i n the middle region between the two f i b e r s . The c e l l s were packed l i k e tissue and some of the c e l l s penetrated into the i s o t r o p i c membrane structure. The dry c e l l mass was 550 g/L, which i s the highest c e l l mass ever reported i n the l i t e r a t u r e as shown i n Table 1. This high c e l l mass compares well with 1 0 ^ E. c o l i cells/mL achieved by Inloes et a l . (7) i n the sponge region of a hollow-fiber reactor i f we assume that the mass of a single IS. c o l i i s roughly 10~12g Leakage of c e l l s and high c e l l densities seem common i n t h i s type of reactors i n the case of IS. c o l i . After the experiment the reactor was dismantled and each of ten dual hollow-fiber units was v i s u a l l y examined. Only i n 4 out of the ten f i b e r s c e l l s were densely packed, which suggests that the med ium was not adequately supplied to many of these f i b e r s . Probably the medium was not equally d i s t r i b u t e d among the f i b e r s . In other words, i n some of f i b e r s the medium flow was not adequate to support the c e l l growth i n the f i b e r . The nonuniform flow d i s t r i b u t i o n among the f i b e r s of a hollow f i b e r device i s an i n t r i n s i c problem, which was studied i n depth i n the authors* laboratory (16). The work of E, c o l i immobilization i n the dual hollow f i b e r reactor was reported previously from the authors laboratory (17).


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Culivation of A_. niger. In the culture of A_. niger B-60, the c e l l s did not leak through the f i b e r s . In other words, no mycelia were detected i n the e f f l u e n t . In a l l the f i b e r s the c e l l s grew well and appeared uniform along the f i b e r s , but the s i l i c o n e tubes were expan ded and the polypropylene tubes were contracted. Figure 6(a) i s the photograph of an empty s i l i c o n e tube and Figure 6(b) shows the cross section of the f i b e r after 15 days of the A_. niger growth. It was observed that the flow rate decreased gradually to zero meaning that the pumping head of a p e r i t a l t i c pump was not s u f f i c i e n t to overcome the flow resistance exerted by the growing fungi. Thus the continu ous operation of the reactor was not f e a s i b l e . It i s suggested that the control of the c e l l growth be needed after a growth period by switching the culture to a nitrogen d e f i c i e n t medium or by some other means. Production of rifamycin Β by N. mediterranei. Figure 7 shows the results of shake flask culture for rifamycin Β production. After 8 days of fermentation 60 g/L of glucose was consumed and 820 pg/mL of rifamycin Β was produced. This gives a volumetric productivity of 4.3 pg of rifamycin/mL/h. The continuous production of rifamycin Β i n the dual hollow-fiber bioreactor i s shown i n Figure 8. The succ essful production of the a n t i b i o t i c s continued more than 50 days without showing signs of decreased production. This i s i n contrast to the t e t r a c y c l i n e production by Robertson and Kim that lasted for a few days. The cause of t h i s decline i n Robertson and Kim s work has not been understood. S t a b i l i t i e s i n the a n t i b i o t i c s production i n the dual hollow- f i b e r bioreactor are speculated to be associated with reactor design or producing organism or both. Two d i s t i n c t differences are noted between the f l a s k culture and the present reactor system. F i r s t , the effluent concentration of rifamycin Β was ca. l/10th of that obtained i n the f l a s k culture. 1




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Figure 5. Electron micrographs of densely packed E. c o l i K-12 cells, (a). The c e l l s at the boundary of the polypropylene f i b e r (pp). Magnification, ΙΟ,ΟΟΟΧ. (b). The c e l l s i n the middle space between the s i l i c o n e tube and the polypropylene f i b e r . Maginification, 20,000X.


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CHANG ET AL.

Dual Hollow-Fiber

Table 1.

Bioreactor

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Comparison of c e l l mass of IS. c o l i i n various fermentations.

System ο Shake-flask culture

C e l l density

Reference

1 - 2 g/L


0 Submerged-culture under

controlled conditions

10 g/L

(12,13)

ο Submerged-culture with pure oxygen supply and semi-continuous feeding of glucose at 22°C.

55 g/L

(14)

0 Immobilization

carrageenan

with beads

A.5 χ 1 0 cells/mL

1 0

viable (15)

0 Immobilization i n a

hollow-fiber bioreactor

10

12

cells/mL

(7)

0 Immobilization i n a dual

hollow-fiber bioreactor

550 g/L

This work

Figure 6. Expansion of s i l i c o n e tube and contraction of poly propylene tubes by growing A. niger B-60. The length scale shown i n the pictures i s 250 pm. ( a ) . Cross section of an empty s i l i c o n e tube. (b). Deformed bioreactor.





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CHANG ET AL.


This i s not small at a l l i f we consider that the residence time i n the hollow-fiber reactor was 12 minutes based on the t o t a l f i b e r lumen voume of 0.339 cm while that i n the flask culture was 8 days. The volumetric productivity of the reactor was 125 pg/mL/h based on the void voulme of the reactor where the c e l l s were a c t u a l l y immobil i z e d . This was about t h i r t y - f o l d as compared to that of the flask culture. This number drops to 15 or 10 i f we include the reactor volume or the volume of the glass tubing used. This high p r o d u c t i v i ty comes essentially from a highly dense c e l l mass i n the reactor shown i n the electron micrograph ( F i g . 9). The measured dry c e l l mass was 600 g/L. The c e l l s neither penetrated into the propylene f i b e r s nor expanded the tubes. This growth c h a r a c t e r i s t i c s i s i n good contrast to that of _E. c o l i or A_. niger c e l l s . The c e l l s grew uniformly along the f i b e r s , which made possible the successful longterm operation of the reactor. The main advantages of a hollow-fiber reactor system are: very l i t t l e energy w i l l be consumed i n the aeration; primary p u r i f i c a t i o n i s accomplished concurrently with the production. In conventional a n t i b i o t i c s or c i t r i c acid fermentation with A_. niger much energy i s consumed i n several days of continuous aeration and mixing of viscous fermentation broths which adds up to a substantial portion of f i n a l production costs. If t h i s membrane bioreactor i s ever successful i n a scale-up operation, there w i l l be a tremendous savings in aeration and mixing costs. Also the benefit of primary separation can never be underestimated because simpler downstream processing i s a key to production cost reduction. However, as yet, much work needs to be done for t h i s reactor to become a t t r a c t i v e for 3


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Figure 9. Electron micrograph of densely packed Nocardia mediterranei (ATCC 21789) c e l l s near the polypropylene hollow f i b e r (magnification, 25,000X).



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i n d u s t r i a l production of valuable materials. Achieving higher volu metric productivity i s c e r t a i n l y an advantage, but the f i n a l product concentration i s too low for any r e a l recovery process to be c o n s i dered as compared to that i n batch system. Substrate d i f f u s i o n l i m i t a t i o n through the membrane can be blamed f o r t h i s low product con centration, but t h i s i s not the case considering that more than 80% of glucose i s consumed during the 10 minutes residence time i n the IS. c o l i reactor. Oxygen l i m i t a t i o n can be a cause f o r t h i s . Perhaps the most important reason i s that c e l l s i n a distressed state can not function as well as the c e l l s i n suspension. The water content of the c e l l s i n the hollow-fiber would be around 40% f o r the c e l l s to a t t a i n such a high dry c e l l weight. Currently we are working on ways of increasing the f i n a l product concentration of rifamycin comparable to that i n the batch system and are trying to improve the s t a b i l i t y i n the operation of reactor f o r A. niger c e l l s .


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Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

Rony, P. R. Biotechnol. Bioeng. 1971, 13, 431. Waterland, L. R.; Michaels, A. S.; Robertson, C. R. AIChE J. 1974, 20, 50. Knazek, R. Α.; Gullino, R. M.; Kohler, P. O.; Dedrick, R. L. Science, 1972, 178, 65. Kan, J. K.; Shuler, M. L. Biotechnol. Bioeng. 1978, 20, 217. Vick Roy, T. B.; Blanch, H. W.; Wilke, C. R. Biotechnol. Lett. 1982, 4, 483. Shuler, M. L. Ann. N.Y. Acad. Sci. 1981, 369, 65. Inloes, D. S.; Smith, W. J.; Taylor, D. P.; Cohen, S. N.; Michaels, A. S.; Robertson, C. R. Biotechnol. Bioeng. 1983, 25, 2653. Inloes, D. S.; Taylor, D. P.; Cohen, S. N.; Michaels, A. S.; Robertson, C. R. Appl. Environ. Microbiol. 1983, 46, 264. Robertson, C. R.; Kim, I. H. Biotechnol. Bioeng. 1985, 27, 1012. Habison, Α.; Kubicek, C. P.; Röhr, M. FEMS Microbial Lett. 1979, 5, 39. Mischak, H.; Kubicek, C. P.; Röhr, M. Biotechnol. Lett. 1984, 6, 425. Elsworth, R.; Miller, G. Α.; Whitaker, A. R.; Kitching, D.; Sayer, P. D. J. Appl. Chem. 1968, 17, 157. Phares, E. F. In "Methods in Enzymology"; Colowick, S. P.; Kaplan, N. O., Ed; Academic Press: New York, 1971; vol. 22 p. 157. Shiloach, J.; Bauer, S. Biotechnol. Bioeng. 1975, 17, 227. Wada, M; Kato, J.; Chibata, I. Eur. J. Appl. Microbiol. Biotech. 1979, 8, 241. Park, J. K.; Chang, H. N. AIChE J. (accepted). Chung, Β. H.; Chang, H. N.; Kim, I. H. Korean J. Appl. Microbiol. Biotechnol. 1985, 13, 209.

Received March 26, 1986


Separation, Recovery, and Purification in Biotechnology - American

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