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Use of colloid chitin and diatomaceous earth in continuous cake-filtration fermentation to produce creatinase
Proces.s Biochemistry Vol. 33, No. 5, pp. 519-526, 1998 ~'~ 19qN Publishcd by Elsevier Scicncc Ltd. All rights rcscrvcd Printed in Grcat Brilain 0(132-9592/98 $19.00+0.110
.~4~ .~j~,ll ELSEVIER
PI I:
S0032-9592~98j00017-X
Use of colloid chitin and diatomaceous earth in continuous cake-filtration fermentation to produce creatinase Shiue-Cheng Tang, '** Ming-Chung Chang b and Chu-Yuan Cheng ~' "Department of Chemical Engineering, National Cheng Kung University, Tainan, Taiwan ~'Department of Biochemistry, Medical College, National Cheng Kung University, Tainan, Taiwan (Received 28 July 1997: revised version received 5 January 1998: accepted 26 January 1998)
Abstract
A recombinant Escherichia coli M15(pQE3208) producing creatinase was cultured in a cake-filtration fermentor containing colloid chitin and diatomaceous earth. The filter medium of this cake filtration was a hollow cylinder made of a 20 1tin stainless steel sieve located in the centre of the fermentor. During filtration, colloid chitin, diatomaceous earth, and E. coli cells formed a film of filter cake on the 20 Itm sieve. The filter cake impeded the outward flow of cells from the fermentor. By controlling the concentration of colloid chitin (3 g/litre), diatomaceous earth (6 g/litre), and the interval of air sparging (0'5 h), continuous cake-filtration fermentation achieved a cell density in the reactor three times higher and specific creatinase activity 35% higher than in ordinary continuous fermentation. An operation mode has been proposed for continuous cake-filtration fermentation to implement the production of intracellular protein which is inversely related to the growth rate of microorganisms. © 1998 Published by Elsevier Science Ltd. All rights reserved Keywords: filtration fermentation, cake filtration, colloid chitin, diatomaceous earth, stainless steel sieve,
creatinase.
the reactor, however, cells arc exposed to an inadequate growth environment with problems such as oxygen limitation, pH shift, and nutrient deficiency. Internal separation systems using a ceramic membrane incorporated into the fermentors were developed to avoid these problems [6-8]. Cells and cell broth can be separated inside the fermentor by membrane filtration. However, a ceramic membrane lacks mechanical stability [9]: it is fragile and has difficulty in resisting the shear force from agitation when it is applied to large-scale fermentation. It is also difficult to increase its surface area in the reactor to improve the separation efficiency. To overcome these limitations, the present researchers tried to find an alternative way to perform filtration fermentation. Cake filtration and fermentation were combined in a bioreactor. Colloid chitin and diatomaceous earth were added in the reactor and fer-
Introduction Many useful products from microorganisms are produced or over-produced when microorganisms are in a state of low growth rate. Under these conditions, continuous fermentation and high-cell-density cultures are difficult to obtain simultaneously. Cell retention systems have been developed to overcome this difficulty and these systems can maintain a high cell density in the reactor to increase volumetric productivity and decrease the growth rate of microorganisms to achieve high specific productivity. External separation systems based on m e m b r a n e filtration [1-3], sedimentation [4], or centrifugation [5] were first developed to achieve cell retention. Outside *To whom all correspondence should be addressed. Present address: Chung Yuan Christian University, Department of Chemical Engineering, Chung-Li, 32023, Taiwan. 519
S.-C. Tang et al.
520
mented with the cells. When the cells with the colloid chitin and diatomaceous earth flowed through the filter medium, they formed a film of filter cake. The cake impeded the outward flow of cells from the fermentor. By accumulation, the cell concentration increased in the fermentor. By controlling the property of the filter cake, cell concentration can be adjusted in the output. A stainless steel sieve was chosen as the filter medium. It is as durable as a ceramic membrane when applied in fermentation. Moreover, unlike a ceramic membrane, a stainless steel sieve is malleable. It can be further shaped to increase its surface area for filtration, if necessary. From the cake-filtration equation [10] flux =
(dp/dL)gc~? Kp( 1 l:)2(Sp/Vp)2 -
( 1)
-
where dp is the pressure drop, dL is the thickness of the cake, g~ is Newton's law proportionality factor, c is the porosity, K is a constant, It is the viscosity of the filtrate, sp is the surface area of a single particle and Ve is the volume of a single particle; all the factors in this equation influence the process of cake filtration. However, only 'porosity' and 'thickness of cake' were used in this study to manipulate the flux because of their convenience in being controlled. During filtration, filter cake is composed of colloid chitin, diatomaceous earth and Escherichia coil cells. The particle size of colloid chitin and diatomaceous earth is 25/~m in this study. The size of E. coli cells is about 0"5 ltm in diameter and 2.5 ltm in length. By controlling the concentration of colloid chitin and diatomaceous earth in the fermentor, the composition and the porosity of the filter cake can be controlled. On the other hand, by controlling the interval of air sparging, the average thickness of the filter cake can be controlled. Creatinase fermentation was used as the model system in this study. Creatinase is an intracellular enzyme which is over-produced by the host at a low growth rate. The objective of this study was to use continuous cake-filtration fermentation to achieve cell retention and increase cell density in the fermentor. The growth rate of the host could be reduced because the nutrients allocated for each cell decreased. Thus, the specific productivity of creatinase can be increased while the host maintains a low growth rate. In this study, specific enzyme activity and cell density inside and outside the fermentor were measured with different colloid chitin concentrations and air sparging intervals to obtain the optimal cultivation condition. Materials and methods
Creatinase production E. coli M15 carried a protein expression and purification system, QIAexpress [11]. QIAexpress includes two
plasmids: pQE and pREP4. Piasmid pQE carried the gene of creatinase from Pseudomonas putida [12] and permitted the expression of creatinase under the control of lac promoter. Plasmid pREP4 provides lac repressor. Plasmid pQE is an ampicillin-resistant vector, whereas plasmid pREP4 confers kanamycinresistance.
Preparation of colloid chitin 10 g of chitin powder (Sigma, Practical Grade, C7170, with a particle size between 150 and 25/tm) was added into 250 ml of concentrated hydrochloric acid and vigorously stirred at room temperature for 13 min. 500 ml of deionized water was added to the solution and gently stirred for l min. The solution was maintained for 4 min and then 2000 ml of deionized water was poured into it. The colloid chitin particles were screened using 20 l~m and 30 lira stainless steel sieves and washed with deionized water until the washings became neutral.
Medium Two media were used in this study: (1) a complex medium based on an LB medium containing 10 g/litre tryptone, 5 g/litre yeast extract, and 5 g/litre NaCI; (2) a semi-synthetic medium based on an M9 medium comprised of 12"8 g/litre Na2HPO4"7H20, 3 g/litre KHEPO4, 0.5g/litre NaCI, l g/litre NH4CI, 10g/litre glucose, 5 g/litre tryptone, 1.0 mM MgSO4, and 0.1 mM CaCI2. LB medium was used for the preculture of cells. A semi-synthetic medium was used for continuous culture and a continuous cake-filtration fermentation system. Isopropyl-fl-D-thiogalactopyranoside (IPTG) was added to the semi-synthetic medium to a final concentration of 100 mg/litre to induce the expression of creatinase. 50 mg/litre of ampicillin and 25 mg/litre of kanamycin were added to both the complex and the semi-synthetic media to ensure plasmid stability. The concentration of colloid chitin depended on the requirement of each experiment. Diatomaceous earth (Wako Pure Chemical Industries: 045-00875) was the filter aid. The amount of diatomaceous earth was twice the concentration of colloid chitin [13].
Assays The cell concentration was measured from the absorbance of the culture broth at 600 nm using a spectrophotometer (model UV-2000, Hitachi Ltd., Tokyo, Japan) and also from the dry cell weight per unit volume. One absorbance unit ( 1 0 . D . unit) corresponds to about 0.4 g/litre dry cell weight. The samples were diluted until the measured absorbance ranged from 0"2 to 0"8. If colloid chitin particles coexisted with cells in the
Crea tin ase production using colloid chitin and dia tom aceous earth
sample, the optical density could not be measured directly. Cells were separated from the mixture by the principle of extraction as follows. (a) A 1.5 ml samplc was withdrawn from the culture and centrifuged at 6000 rpm for 10 s. All of the colloid chitin particles and some cells settled, whereas other cells remained suspended in the supernatant; (b) 1 ml of the supernatant was withdrawn without disturbing the pellet and preserving the supernatant and 1 ml of 0.1 M Na2HPO4 (pH 8'9) solution was added to the tube with thc remaining solution and pellet. The tube was mixed by vortexing and centrifuging at 6000rpm for 10s. (c) Step (b) was repeated nine times to obtain 9 ml of
521
supernatant. (d) The optical density of the 9 ml supernatant was measured using a spectrophotometer. The rcal O.D. in the original 1.5 ml sample was obtained by multiplying the O.D. of the 9 ml supernatant by 6. (The 9 ml supernatant included over 90% of the cells in the original 1.5 ml sample. This was demonstrated by testing the mixture of colloid chitin and cell suspension. The optical density of the cell suspension had already bccn measured by the spectrophotometer.) A total enzymic analysis was done using the supernatant of the disrupted culture after centrifugation at 17(100g for 20min. The cells wcrc disrupted using a sonicator (model 2020, Heat System-Ultrasonics, Inc.,
[ D(
1
OFF
p[
I
"Level
Controller
ON
sensor and "Pump
M
I
Temp. 37 °C
Agit. 200 rpm
Air
Fermentor
]
It
Fresh medium
Broth
.....
Air sparging
eve
amc
Fig. 1. (a) The experimental apparatus for continuous cake-filtration fermentation. Diameter of the filter-medium cylinder: 4 cm, height: 4 cm, surface area: 50 cm 2. *The pump reduces the pressure inside the filter-medium cylinder. #The level sensor controls the on-off switch of the pump. (b) The detailed structure of the fermentor for continuous cake-filtration fermentation. The rubber fastens the tubes connecting the filter-medium cylinder; the steel frame supports the stainless steel sieve and connects with the fcrmcntor.
522
S.-C. Tang et al.
New York, USA). Sonication was implemented in a pulsar cycle for 6 min with a pulse rate of 0"5 s. The creatinase assay was based on the methods proposed by Watt and Chrisp [14] and Koyama et al. [15]. A reaction mixture consisting of 0.1 ml of enzyme solution, 0.1 ml of 0 ' 3 m potassium phosphate buffer (pH 7"7), and 0.8 ml of 0.1 m creatine was incubated at 37°C for 10 min. The reaction was stopped by the addition of 2 m l of a colour reagent containing 50% ethanol, 1% p-dimethlyaminobenzaldehyde, and l m HCI. The optical density was measured at 435 nm. One
Table 1. Comparison of the cell density and the ability of E. coli M15(pQE3208) to express creatinase at different dilution rates Dilution rate (I/h) Cell density (O.D.6,,0 Specific enzyme activity (U/mg cell)
0.05 6.1 95.7
0.10 4.0 69-2
0.20 4.0 25'5
0.30 washout 22-0
unit of creatinase was defined as the amount of enzyme which liberates 1/~mol of urea under these conditions. Cultivation methods
For continuous fermentation without colloid chitin and diatomaceous earth in the reactor, a chemostat with a working volume of 1000 ml was maintained in continuous cultivation with a 2 litre mini-jar fermentor (type CTB-31 from Taitec Co., Tokyo, Japan). The cells were grown at 37°C, and maintained at pH 8 by the addition of HC1 and NaOH. The aeration rate and the agitation speed were maintained at 0.5vvm and 2000 rpm respectively. The apparatus used for continuous cake filtration fermentation is shown in Fig. l(a) and (b). In this set-up, the centre of the fermentor was a hollow cylinder. Outside the cylinder, colloid chitin and diatomaceous earth were added and fermented with the cells. Inside the cylinder, a tube connected to a pump was used to withdraw air (flow rate 100 ml/min) and
D=0.3 (l/h) E
]D=0.2 (l/h) E
D=0 Batch cultivation
]
D=0.1 (l/h) I
•
]
15--
A
o o ¢.D
12--
ci
ci C
.o_
9--
C o C 0 0 m m
0 0
I 5
1 10
I 15
Time
I 20
I 25
I 30
(hr)
Fig. 2. Increase in cell concentration during continuous cake-filtration fermentation. Colloid chitin concentration 7 g/litre, diatomaceous earth concentration 14 g/litre, D is the dilution rate (I/h).
Creatinase production using colloid chitin and diatomaceous earth
523
Table 2. Comparison of the average cell density in the output when different colloid chitin concentrations and intervals of air sparging were employed" Average cell density in the output (O.D.~,,,) Interval of air sparging (h)
Colloid chitin concentration (g/litre) 3 5
1
0"25 0'50 0"75 1"00 1"25
6.9 h 6.1 b 5.6 h 4"9b impermeable
5"8I' 4.2 3-U 2"3~ impermeable
7
4.0 2-2~ 2-5~ 1"0~ impermeable
2.4 ~" I .U 0.7" 11'2c impermeable
" Data were obtained within 2 h of starting filtration. Dilution rate 0'1 (l/h); O.D. = 12 in the fermentor: diatomaceous earth concentratkm 2 x colloid chitin concentration. b O.D. gradually decreased in the fermentor and decreased from 12 when the cultivation time increased, since more cells left the fermentor than were produced. O.D. gradually increased in the fermentor and increased from 12 when the cultivation time increased, since less cells left the fermentor than were produced.
1.25 h, was used to control the thickness o f filter cake. T h e time for each air sparging was 60 s, and the flow rate was 2(100ml/min. T h e c o n c e n t r a t i o n o f colloid chitin in the f e r m e n t o r , varied from 1 g/litre to 7 g/litre, was used to control the c o m p o s i t i o n and the p o r o s i t y of filter cake. T h e o t h e r cultivation c o n d i t i o n s for c o n t i n u o u s cake-filtration f e r m e n t a t i o n were the s a m e as those for ordinary continuous fermentation.
r e d u c e the p r e s s u r e in it. A level s e n s o r in the cylinder was used to c o n t r o l the o n - o f f switch on the p u m p to m a i n t a i n a s t e a d y - s t a t e o p e r a t i o n . Because the pressure was lower in the cylinder, the cell b r o t h o u t s i d e the cylinder flowed t h r o u g h the filter cake a n d was then p u m p e d out. W h e n the filter cake b e c a m e i m p e r m e a b l e , air sparging was used to b r e a k up the filter cake and r e m o v e it f r o m the filter m e d i u m . T h e air sparging interval, v a r i e d from 0.25 h to
.5
A
O O ¢,D
1.2
-
d O t-
0.9-
O •m
,I,,a
t-
¢J
0.6
e.-
O u
(J
0.3
0.0
'
0
I
20
'
I
'
I
40 60 Time (min)
'
r
80
'
1
100
Fig. 3. Cell concentration in the output during the first 90 rain when the dilution rate was adjusted to 0"3 (l/h) in the system described in Fig. 2.
524
S.-C. Tang et al.
earth was 25 Fm. The size of E. coli cells is about 0.5 l~m in diameter and 2.5 F~m in length. During filtration, the filter cake is composed of colloid chitin, diatomaceous earth and E. coli cells. When cell density increased in the fermentor, the percentage of E. coli cells in the filter cake also increased and the porosity of filter cake decreased because of the small particle size of E. coli cells. From the cake-filtration equation (eqn (1)), porosity is the major factor in the equation. If the porosity of the filter cake decreased, the flux decreased greatly. As shown in Fig. 2, at the beginning of the cake-filtration fermentation, the culture could be operated at a dilution rate of 0'3 (l/h) because the cell concentration in the fermentor was low. As the cell concentration increased, the porosity of the cake decreased. It was necessary to reduce the dilution rate to 0.1 (l/h) and increase the frequency of air sparging to prevent overflowing and maintain continuous cultivation. The effects of various colloid chitin concentrations and intervals of air sparging on continuous cake-filtration fermentation are compared in Table 2. When the concentration of colloid chitin was high, a filter cake formed quickly and cells could not flow out. However,
Results and discussion
For ordinary continuous fermentation, the dilution rate is the specific growth rate of E. coli. As shown in Table 1, the rate was inversely related to the production of creatinase. The dilution rate also influenced cell density. Washout occurred when the dilution rate was 0.3 (I/h). For continuous cake-filtration fermentation (Fig. 2), the cell concentration in the fermentor could be increased when colloid chitin and diatomaceous earth were added and fermented with the cells. Figure 3 shows the time course of the cell concentration in the output during the first 90 min, when the dilution rate was 0.3 (l/h). The filter cake formed gradually as the filtration time increased. The cells were impeded by the filter cake and could not flow out from the fermentor. When the cell broth continued flowing through the filter cake, the thickness of the filter cake gradually increased and became impermeable. At this time, the cake should be broken-up by air sparging. The higher the cell concentration in the fermentor, the smaller the porosity of the filter cake. In this study, the particle size of colloid chitin and diatomaceous
T h e 2 n d st age of cult ivat ion
T h e 1st s t a g e of c u l t i v a t i o n I Batch
E J
A
14--
140
12
120
o o cD
N
"-"
U
01
lo
100
c
8
8o
~U
c
6
- G0
m
u c 0 u
4
d mom
ffl
=
U
--
-
40
u U
2O
I 0
'
I 10
'
I 20
'
I 30
'
I 40
'
I 60
'
( 60
~
0
' 70
Time (hr) Fig. 4. Cultivation of continuous cake-filtration fermentation: D, cell concentration in the fermentor; e, cell concentration in the output; zx, specific creatinase activity in the output. Colloid chitin concentration 3 g/litre, diatomaceous earth concentration 6 g/litre, dilution rate 0-1 (l/h). During the first stage of cultivation air sparging was applied only when the filter cake was totally impermeable. During the second stage of cultivation, air sparging was applied every ()'5 h.
(~eatinase production using colloid chitin and diatomaceous earth Table 3. Comparison of continuous cake filtration fermentation and ordinary continuous fermentation;' Comparative items
Continuous cake-filtration fermentation
Ordinary continuous fermentation
12.(I
4.0
4.2
4.[t
93.8
69.2 ~'
Cell density in the fermenter (O.D.,,I,,,) Cell density in the output (O.D.,,,,,.) Specific enzyme activity (U/rag cell)
~' For continuous cake-filtration fermentation, colloid chitin concentration was 3 g/litre, diatomaceous earth concentration was 6 g/litre, interval of air sparging was 0"5 hour, dilution rate was 0.1 (l/h); for ordinary continuous fermentation, dilution rate was (~'1 (l/h).
when the concentration of colloid chitin was low, the filter cake formed slowly and the cells had more opportunities to flow out from the fermenter. The interval of air sparging also affected the average cell density in the output. The longer the interval, the thicker the film of the filter cake, and the more difficult it was for cells to flow out from the fermenter. The shorter the interval, the thinner the film of the filter cake, and the cells flowed out from the fermenter more easily. On the precondition of maintaining a high cell density in the fermenter, the optimal cultivation condition was to obtain the maximal cell density in the output to achieve the highest productivity. As shown in Table 2, when the colloid chitin concentration was 3 g/litre and the interval was 0.5 h, or when the colloid chitin concentration was 5 g/litre and the interval was 0.25 h. the maximal cell concentration in the output was achieved. The former condition is superior because it consumes less colloid chitin for each fermentation. A cultivation method for cake-filtration fermentation has been proposed (Fig. 4). In the batch cultivation, the cell density increased first, then, at the end of the batch cultivation when nutrients were depleted, fresh medium was continuously fed into the fermenter (the first stage of cultivation). At this stage, the cells continued growing, and the metabolic inhibitors were removed from the fermenter. Because of the immobilization effect ,ff the filter cake, the cells could not flow
Table 4. Characteristics of different filtration methods to perform filtration fermentation Comparative items Sterilization Thermal, chemical stability Mechanical stability Flux Cost
Hollow fbre
Ceramic membrane
Cake filtration
hard poor
easy good
easy good
malleable low high
brittle high high
malleable high low
525
out from the fermenter and the cell density increased further. When the O.D. in the fermenter increased to 12, the cultivation reached the second stage. At this stage, air sparging was used to adjust the cell density in the output. If the O.D. in the output was maintained at 4. the results obtained were similar to that from ordinary continuous fermentation. By controlling the concentration of colloid chitin in the fermenter (3 g/litre) and the interval used for air sparging (0.5 h) in the continuous cell cake fermentation. the cell density in the reactor became three times higher and specific creatinase activity 35% higher than in ordinary continuous fermentation (Table 3). The cultivation condition in Fig. 4 is suitable for intracellular products whose productivity can be improved at a low growth rate, e.g. the production of creatinasc by E. coli M15(pQE3208). As the cell concentration increased in the fermenter, the nutrients distributed for each cell decreased and the growth rate of the cells was also reduced. In ordinary continuous fermentation, E. coli M15(pQE3208) expressed creatinasc better at low specific growth rates, as shown in Table 1. Using colloid chitin and diatomaceous earth to perform continuous cake-filtration fermentation can increase the cell density in the fermenter and decrease the growth rate of the cells, as well as improve the expression of creatinasc. Thus. the culture may be grown at a higher dilution rate and the productivity of creatinasc increased. The choice of an appropriate method to implement large-scale filtration fermentation requires a balance among several factors (Table 4) [c~,16]. These data suggest that using cake filtration to perform filtration fermentation is a promising method fi)r the future.
Conclusion
The continuous cake-filtration fermentation system reported in this article is an alternative way to perform continuous cell-retention fermentation. Controlling the concentration of colloid chitin and the interval of air sparging enabled continued filtering of the culture and cell retention in the fermenter to be achieved. The benefit of this system is its potential of scale-up. Unlike ceramic membrane and hollow fibre modules, stainless steel sieves arc malleable and can be easily sterilized. The cell-retention system performed by stainless steel sieves as the filter medium is more suitable for application in large-scale fermentations.
References
1. Taniguchi, M., Kotani, N. and Kobayashi, T., Highconcentration cultivation of lactic acid bacteria in fermenter with cross filtration. Journal ~1"Fermentation Technology 1987, 65, 179-184. 2. Imasaka, T., Kanekuni, N., So, H. and Yoshino, S.,
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3.
4. 5.
6.
7.
8.
9.
S.-C. Tanget al. Cross-flow filtration of methane fermentation broth by ceramic membrane. Journal of Fermentation and Bioengineering 1989, 68, 200-206. Nakano, K., Matsumura, M. and Kataoka, H., Application of a rotating ceramic membrane to dense cell culture. Journal of Fermentation and Bioengineering 1993, 76, 49-54. Cysewski, G. R. and Wike, C. R., Rapid ethanol fermentations using vacuum and cell recycle. Biotechnology and Bioengineering 1978, 19, 1125-1143. Cysewski, G. R. and Wike, C. R., Process design and economic studies of alternative fermentation methods for the production of ethanol. Biotechnology and Bioengineering 1978, 20, 1421-1444. Kang, B. C., Lee, S. Y. and Chang, H. N., Production of Bacillus thuringiensis spores in total cell retention culture and two-stage continuous culture using an internal ceramic filter system. Biotechnology and Bioengineering 1993, 42, 1107-1112. Suzuki, T., Sato, T. and Kominami, M., A dense cell retention culture system using a stirred ceramic membrane reactor. Biotechnology and Bioengineering 1994, 44, 1186-1192. Suzuki, T., A dense cell culture system for microorganisms using a stirred ceramic membrane reactor incorporating asymmetric porous ceramic filters. Journal of Fermentation and Bioengineering 1996, 82, 264-271. Mulder, M. Basic Principles of Membrane Tech-
nology. Kluwer, 1991, pp. 47-48. 10. McCabe, W. L., Smith, J. C. & Harriott, P. Unit Operation of Chemical Engineering. McGraw-Hill, New York, 1985, p. 871. 11. Hoffmann, A. and Roeder, R. G., Purification of Histagged proteins in non-denaturing conditions suggests a convenient method for protein interaction studies. Nucleic Acids Research 1991, 19, 6337-6338. 12. Chang, M. C., Chang, C. C. and Chang, J. C., Cloning of creatinase gene from Pseudomonas putida in Escherichia coli by using an indicator plate. Applied and Environmental Microbiology 1992, 58, 3437-3440. 13. Perry, R. H. & Green, D., Perry's Chemical Engineers' Handbook. McGraw-Hill, New York, 1984, pp. 19-72-19-73. 14. Watt, G. W. and Chrisp, J. D., Spectrophotometric method for determination of urea. Analytical Chemistry 1954, 26, 452-453. 15. Koyama, Y., Kitao, S., Yamamoto-Otake, H., Suzuki, M. and Nakano, E., Cloning and expression of the creatinase gene from Flavobacterium sp. U-188 in Escherichia coli. Agricultural and Biological Chemistry 1990, 54, 1453-1457. 16. Baker, R. W., Cussler, E. L., Eykamp, W., Koros, W. J., Riley, R. L. & Strathmann, H. Membrane Separation Systems. Noyes Data Corporation, New Jersey, 1991, pp. 136-145.
.~4~ .~j~,ll ELSEVIER
PI I:
S0032-9592~98j00017-X
Use of colloid chitin and diatomaceous earth in continuous cake-filtration fermentation to produce creatinase Shiue-Cheng Tang, '** Ming-Chung Chang b and Chu-Yuan Cheng ~' "Department of Chemical Engineering, National Cheng Kung University, Tainan, Taiwan ~'Department of Biochemistry, Medical College, National Cheng Kung University, Tainan, Taiwan (Received 28 July 1997: revised version received 5 January 1998: accepted 26 January 1998)
Abstract
A recombinant Escherichia coli M15(pQE3208) producing creatinase was cultured in a cake-filtration fermentor containing colloid chitin and diatomaceous earth. The filter medium of this cake filtration was a hollow cylinder made of a 20 1tin stainless steel sieve located in the centre of the fermentor. During filtration, colloid chitin, diatomaceous earth, and E. coli cells formed a film of filter cake on the 20 Itm sieve. The filter cake impeded the outward flow of cells from the fermentor. By controlling the concentration of colloid chitin (3 g/litre), diatomaceous earth (6 g/litre), and the interval of air sparging (0'5 h), continuous cake-filtration fermentation achieved a cell density in the reactor three times higher and specific creatinase activity 35% higher than in ordinary continuous fermentation. An operation mode has been proposed for continuous cake-filtration fermentation to implement the production of intracellular protein which is inversely related to the growth rate of microorganisms. © 1998 Published by Elsevier Science Ltd. All rights reserved Keywords: filtration fermentation, cake filtration, colloid chitin, diatomaceous earth, stainless steel sieve,
creatinase.
the reactor, however, cells arc exposed to an inadequate growth environment with problems such as oxygen limitation, pH shift, and nutrient deficiency. Internal separation systems using a ceramic membrane incorporated into the fermentors were developed to avoid these problems [6-8]. Cells and cell broth can be separated inside the fermentor by membrane filtration. However, a ceramic membrane lacks mechanical stability [9]: it is fragile and has difficulty in resisting the shear force from agitation when it is applied to large-scale fermentation. It is also difficult to increase its surface area in the reactor to improve the separation efficiency. To overcome these limitations, the present researchers tried to find an alternative way to perform filtration fermentation. Cake filtration and fermentation were combined in a bioreactor. Colloid chitin and diatomaceous earth were added in the reactor and fer-
Introduction Many useful products from microorganisms are produced or over-produced when microorganisms are in a state of low growth rate. Under these conditions, continuous fermentation and high-cell-density cultures are difficult to obtain simultaneously. Cell retention systems have been developed to overcome this difficulty and these systems can maintain a high cell density in the reactor to increase volumetric productivity and decrease the growth rate of microorganisms to achieve high specific productivity. External separation systems based on m e m b r a n e filtration [1-3], sedimentation [4], or centrifugation [5] were first developed to achieve cell retention. Outside *To whom all correspondence should be addressed. Present address: Chung Yuan Christian University, Department of Chemical Engineering, Chung-Li, 32023, Taiwan. 519
S.-C. Tang et al.
520
mented with the cells. When the cells with the colloid chitin and diatomaceous earth flowed through the filter medium, they formed a film of filter cake. The cake impeded the outward flow of cells from the fermentor. By accumulation, the cell concentration increased in the fermentor. By controlling the property of the filter cake, cell concentration can be adjusted in the output. A stainless steel sieve was chosen as the filter medium. It is as durable as a ceramic membrane when applied in fermentation. Moreover, unlike a ceramic membrane, a stainless steel sieve is malleable. It can be further shaped to increase its surface area for filtration, if necessary. From the cake-filtration equation [10] flux =
(dp/dL)gc~? Kp( 1 l:)2(Sp/Vp)2 -
( 1)
-
where dp is the pressure drop, dL is the thickness of the cake, g~ is Newton's law proportionality factor, c is the porosity, K is a constant, It is the viscosity of the filtrate, sp is the surface area of a single particle and Ve is the volume of a single particle; all the factors in this equation influence the process of cake filtration. However, only 'porosity' and 'thickness of cake' were used in this study to manipulate the flux because of their convenience in being controlled. During filtration, filter cake is composed of colloid chitin, diatomaceous earth and Escherichia coil cells. The particle size of colloid chitin and diatomaceous earth is 25/~m in this study. The size of E. coli cells is about 0"5 ltm in diameter and 2.5 ltm in length. By controlling the concentration of colloid chitin and diatomaceous earth in the fermentor, the composition and the porosity of the filter cake can be controlled. On the other hand, by controlling the interval of air sparging, the average thickness of the filter cake can be controlled. Creatinase fermentation was used as the model system in this study. Creatinase is an intracellular enzyme which is over-produced by the host at a low growth rate. The objective of this study was to use continuous cake-filtration fermentation to achieve cell retention and increase cell density in the fermentor. The growth rate of the host could be reduced because the nutrients allocated for each cell decreased. Thus, the specific productivity of creatinase can be increased while the host maintains a low growth rate. In this study, specific enzyme activity and cell density inside and outside the fermentor were measured with different colloid chitin concentrations and air sparging intervals to obtain the optimal cultivation condition. Materials and methods
Creatinase production E. coli M15 carried a protein expression and purification system, QIAexpress [11]. QIAexpress includes two
plasmids: pQE and pREP4. Piasmid pQE carried the gene of creatinase from Pseudomonas putida [12] and permitted the expression of creatinase under the control of lac promoter. Plasmid pREP4 provides lac repressor. Plasmid pQE is an ampicillin-resistant vector, whereas plasmid pREP4 confers kanamycinresistance.
Preparation of colloid chitin 10 g of chitin powder (Sigma, Practical Grade, C7170, with a particle size between 150 and 25/tm) was added into 250 ml of concentrated hydrochloric acid and vigorously stirred at room temperature for 13 min. 500 ml of deionized water was added to the solution and gently stirred for l min. The solution was maintained for 4 min and then 2000 ml of deionized water was poured into it. The colloid chitin particles were screened using 20 l~m and 30 lira stainless steel sieves and washed with deionized water until the washings became neutral.
Medium Two media were used in this study: (1) a complex medium based on an LB medium containing 10 g/litre tryptone, 5 g/litre yeast extract, and 5 g/litre NaCI; (2) a semi-synthetic medium based on an M9 medium comprised of 12"8 g/litre Na2HPO4"7H20, 3 g/litre KHEPO4, 0.5g/litre NaCI, l g/litre NH4CI, 10g/litre glucose, 5 g/litre tryptone, 1.0 mM MgSO4, and 0.1 mM CaCI2. LB medium was used for the preculture of cells. A semi-synthetic medium was used for continuous culture and a continuous cake-filtration fermentation system. Isopropyl-fl-D-thiogalactopyranoside (IPTG) was added to the semi-synthetic medium to a final concentration of 100 mg/litre to induce the expression of creatinase. 50 mg/litre of ampicillin and 25 mg/litre of kanamycin were added to both the complex and the semi-synthetic media to ensure plasmid stability. The concentration of colloid chitin depended on the requirement of each experiment. Diatomaceous earth (Wako Pure Chemical Industries: 045-00875) was the filter aid. The amount of diatomaceous earth was twice the concentration of colloid chitin [13].
Assays The cell concentration was measured from the absorbance of the culture broth at 600 nm using a spectrophotometer (model UV-2000, Hitachi Ltd., Tokyo, Japan) and also from the dry cell weight per unit volume. One absorbance unit ( 1 0 . D . unit) corresponds to about 0.4 g/litre dry cell weight. The samples were diluted until the measured absorbance ranged from 0"2 to 0"8. If colloid chitin particles coexisted with cells in the
Crea tin ase production using colloid chitin and dia tom aceous earth
sample, the optical density could not be measured directly. Cells were separated from the mixture by the principle of extraction as follows. (a) A 1.5 ml samplc was withdrawn from the culture and centrifuged at 6000 rpm for 10 s. All of the colloid chitin particles and some cells settled, whereas other cells remained suspended in the supernatant; (b) 1 ml of the supernatant was withdrawn without disturbing the pellet and preserving the supernatant and 1 ml of 0.1 M Na2HPO4 (pH 8'9) solution was added to the tube with thc remaining solution and pellet. The tube was mixed by vortexing and centrifuging at 6000rpm for 10s. (c) Step (b) was repeated nine times to obtain 9 ml of
521
supernatant. (d) The optical density of the 9 ml supernatant was measured using a spectrophotometer. The rcal O.D. in the original 1.5 ml sample was obtained by multiplying the O.D. of the 9 ml supernatant by 6. (The 9 ml supernatant included over 90% of the cells in the original 1.5 ml sample. This was demonstrated by testing the mixture of colloid chitin and cell suspension. The optical density of the cell suspension had already bccn measured by the spectrophotometer.) A total enzymic analysis was done using the supernatant of the disrupted culture after centrifugation at 17(100g for 20min. The cells wcrc disrupted using a sonicator (model 2020, Heat System-Ultrasonics, Inc.,
[ D(
1
OFF
p[
I
"Level
Controller
ON
sensor and "Pump
M
I
Temp. 37 °C
Agit. 200 rpm
Air
Fermentor
]
It
Fresh medium
Broth
.....
Air sparging
eve
amc
Fig. 1. (a) The experimental apparatus for continuous cake-filtration fermentation. Diameter of the filter-medium cylinder: 4 cm, height: 4 cm, surface area: 50 cm 2. *The pump reduces the pressure inside the filter-medium cylinder. #The level sensor controls the on-off switch of the pump. (b) The detailed structure of the fermentor for continuous cake-filtration fermentation. The rubber fastens the tubes connecting the filter-medium cylinder; the steel frame supports the stainless steel sieve and connects with the fcrmcntor.
522
S.-C. Tang et al.
New York, USA). Sonication was implemented in a pulsar cycle for 6 min with a pulse rate of 0"5 s. The creatinase assay was based on the methods proposed by Watt and Chrisp [14] and Koyama et al. [15]. A reaction mixture consisting of 0.1 ml of enzyme solution, 0.1 ml of 0 ' 3 m potassium phosphate buffer (pH 7"7), and 0.8 ml of 0.1 m creatine was incubated at 37°C for 10 min. The reaction was stopped by the addition of 2 m l of a colour reagent containing 50% ethanol, 1% p-dimethlyaminobenzaldehyde, and l m HCI. The optical density was measured at 435 nm. One
Table 1. Comparison of the cell density and the ability of E. coli M15(pQE3208) to express creatinase at different dilution rates Dilution rate (I/h) Cell density (O.D.6,,0 Specific enzyme activity (U/mg cell)
0.05 6.1 95.7
0.10 4.0 69-2
0.20 4.0 25'5
0.30 washout 22-0
unit of creatinase was defined as the amount of enzyme which liberates 1/~mol of urea under these conditions. Cultivation methods
For continuous fermentation without colloid chitin and diatomaceous earth in the reactor, a chemostat with a working volume of 1000 ml was maintained in continuous cultivation with a 2 litre mini-jar fermentor (type CTB-31 from Taitec Co., Tokyo, Japan). The cells were grown at 37°C, and maintained at pH 8 by the addition of HC1 and NaOH. The aeration rate and the agitation speed were maintained at 0.5vvm and 2000 rpm respectively. The apparatus used for continuous cake filtration fermentation is shown in Fig. l(a) and (b). In this set-up, the centre of the fermentor was a hollow cylinder. Outside the cylinder, colloid chitin and diatomaceous earth were added and fermented with the cells. Inside the cylinder, a tube connected to a pump was used to withdraw air (flow rate 100 ml/min) and
D=0.3 (l/h) E
]D=0.2 (l/h) E
D=0 Batch cultivation
]
D=0.1 (l/h) I
•
]
15--
A
o o ¢.D
12--
ci
ci C
.o_
9--
C o C 0 0 m m
0 0
I 5
1 10
I 15
Time
I 20
I 25
I 30
(hr)
Fig. 2. Increase in cell concentration during continuous cake-filtration fermentation. Colloid chitin concentration 7 g/litre, diatomaceous earth concentration 14 g/litre, D is the dilution rate (I/h).
Creatinase production using colloid chitin and diatomaceous earth
523
Table 2. Comparison of the average cell density in the output when different colloid chitin concentrations and intervals of air sparging were employed" Average cell density in the output (O.D.~,,,) Interval of air sparging (h)
Colloid chitin concentration (g/litre) 3 5
1
0"25 0'50 0"75 1"00 1"25
6.9 h 6.1 b 5.6 h 4"9b impermeable
5"8I' 4.2 3-U 2"3~ impermeable
7
4.0 2-2~ 2-5~ 1"0~ impermeable
2.4 ~" I .U 0.7" 11'2c impermeable
" Data were obtained within 2 h of starting filtration. Dilution rate 0'1 (l/h); O.D. = 12 in the fermentor: diatomaceous earth concentratkm 2 x colloid chitin concentration. b O.D. gradually decreased in the fermentor and decreased from 12 when the cultivation time increased, since more cells left the fermentor than were produced. O.D. gradually increased in the fermentor and increased from 12 when the cultivation time increased, since less cells left the fermentor than were produced.
1.25 h, was used to control the thickness o f filter cake. T h e time for each air sparging was 60 s, and the flow rate was 2(100ml/min. T h e c o n c e n t r a t i o n o f colloid chitin in the f e r m e n t o r , varied from 1 g/litre to 7 g/litre, was used to control the c o m p o s i t i o n and the p o r o s i t y of filter cake. T h e o t h e r cultivation c o n d i t i o n s for c o n t i n u o u s cake-filtration f e r m e n t a t i o n were the s a m e as those for ordinary continuous fermentation.
r e d u c e the p r e s s u r e in it. A level s e n s o r in the cylinder was used to c o n t r o l the o n - o f f switch on the p u m p to m a i n t a i n a s t e a d y - s t a t e o p e r a t i o n . Because the pressure was lower in the cylinder, the cell b r o t h o u t s i d e the cylinder flowed t h r o u g h the filter cake a n d was then p u m p e d out. W h e n the filter cake b e c a m e i m p e r m e a b l e , air sparging was used to b r e a k up the filter cake and r e m o v e it f r o m the filter m e d i u m . T h e air sparging interval, v a r i e d from 0.25 h to
.5
A
O O ¢,D
1.2
-
d O t-
0.9-
O •m
,I,,a
t-
¢J
0.6
e.-
O u
(J
0.3
0.0
'
0
I
20
'
I
'
I
40 60 Time (min)
'
r
80
'
1
100
Fig. 3. Cell concentration in the output during the first 90 rain when the dilution rate was adjusted to 0"3 (l/h) in the system described in Fig. 2.
524
S.-C. Tang et al.
earth was 25 Fm. The size of E. coli cells is about 0.5 l~m in diameter and 2.5 F~m in length. During filtration, the filter cake is composed of colloid chitin, diatomaceous earth and E. coli cells. When cell density increased in the fermentor, the percentage of E. coli cells in the filter cake also increased and the porosity of filter cake decreased because of the small particle size of E. coli cells. From the cake-filtration equation (eqn (1)), porosity is the major factor in the equation. If the porosity of the filter cake decreased, the flux decreased greatly. As shown in Fig. 2, at the beginning of the cake-filtration fermentation, the culture could be operated at a dilution rate of 0'3 (l/h) because the cell concentration in the fermentor was low. As the cell concentration increased, the porosity of the cake decreased. It was necessary to reduce the dilution rate to 0.1 (l/h) and increase the frequency of air sparging to prevent overflowing and maintain continuous cultivation. The effects of various colloid chitin concentrations and intervals of air sparging on continuous cake-filtration fermentation are compared in Table 2. When the concentration of colloid chitin was high, a filter cake formed quickly and cells could not flow out. However,
Results and discussion
For ordinary continuous fermentation, the dilution rate is the specific growth rate of E. coli. As shown in Table 1, the rate was inversely related to the production of creatinase. The dilution rate also influenced cell density. Washout occurred when the dilution rate was 0.3 (I/h). For continuous cake-filtration fermentation (Fig. 2), the cell concentration in the fermentor could be increased when colloid chitin and diatomaceous earth were added and fermented with the cells. Figure 3 shows the time course of the cell concentration in the output during the first 90 min, when the dilution rate was 0.3 (l/h). The filter cake formed gradually as the filtration time increased. The cells were impeded by the filter cake and could not flow out from the fermentor. When the cell broth continued flowing through the filter cake, the thickness of the filter cake gradually increased and became impermeable. At this time, the cake should be broken-up by air sparging. The higher the cell concentration in the fermentor, the smaller the porosity of the filter cake. In this study, the particle size of colloid chitin and diatomaceous
T h e 2 n d st age of cult ivat ion
T h e 1st s t a g e of c u l t i v a t i o n I Batch
E J
A
14--
140
12
120
o o cD
N
"-"
U
01
lo
100
c
8
8o
~U
c
6
- G0
m
u c 0 u
4
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ffl
=
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--
-
40
u U
2O
I 0
'
I 10
'
I 20
'
I 30
'
I 40
'
I 60
'
( 60
~
0
' 70
Time (hr) Fig. 4. Cultivation of continuous cake-filtration fermentation: D, cell concentration in the fermentor; e, cell concentration in the output; zx, specific creatinase activity in the output. Colloid chitin concentration 3 g/litre, diatomaceous earth concentration 6 g/litre, dilution rate 0-1 (l/h). During the first stage of cultivation air sparging was applied only when the filter cake was totally impermeable. During the second stage of cultivation, air sparging was applied every ()'5 h.
(~eatinase production using colloid chitin and diatomaceous earth Table 3. Comparison of continuous cake filtration fermentation and ordinary continuous fermentation;' Comparative items
Continuous cake-filtration fermentation
Ordinary continuous fermentation
12.(I
4.0
4.2
4.[t
93.8
69.2 ~'
Cell density in the fermenter (O.D.,,I,,,) Cell density in the output (O.D.,,,,,.) Specific enzyme activity (U/rag cell)
~' For continuous cake-filtration fermentation, colloid chitin concentration was 3 g/litre, diatomaceous earth concentration was 6 g/litre, interval of air sparging was 0"5 hour, dilution rate was 0.1 (l/h); for ordinary continuous fermentation, dilution rate was (~'1 (l/h).
when the concentration of colloid chitin was low, the filter cake formed slowly and the cells had more opportunities to flow out from the fermenter. The interval of air sparging also affected the average cell density in the output. The longer the interval, the thicker the film of the filter cake, and the more difficult it was for cells to flow out from the fermenter. The shorter the interval, the thinner the film of the filter cake, and the cells flowed out from the fermenter more easily. On the precondition of maintaining a high cell density in the fermenter, the optimal cultivation condition was to obtain the maximal cell density in the output to achieve the highest productivity. As shown in Table 2, when the colloid chitin concentration was 3 g/litre and the interval was 0.5 h, or when the colloid chitin concentration was 5 g/litre and the interval was 0.25 h. the maximal cell concentration in the output was achieved. The former condition is superior because it consumes less colloid chitin for each fermentation. A cultivation method for cake-filtration fermentation has been proposed (Fig. 4). In the batch cultivation, the cell density increased first, then, at the end of the batch cultivation when nutrients were depleted, fresh medium was continuously fed into the fermenter (the first stage of cultivation). At this stage, the cells continued growing, and the metabolic inhibitors were removed from the fermenter. Because of the immobilization effect ,ff the filter cake, the cells could not flow
Table 4. Characteristics of different filtration methods to perform filtration fermentation Comparative items Sterilization Thermal, chemical stability Mechanical stability Flux Cost
Hollow fbre
Ceramic membrane
Cake filtration
hard poor
easy good
easy good
malleable low high
brittle high high
malleable high low
525
out from the fermenter and the cell density increased further. When the O.D. in the fermenter increased to 12, the cultivation reached the second stage. At this stage, air sparging was used to adjust the cell density in the output. If the O.D. in the output was maintained at 4. the results obtained were similar to that from ordinary continuous fermentation. By controlling the concentration of colloid chitin in the fermenter (3 g/litre) and the interval used for air sparging (0.5 h) in the continuous cell cake fermentation. the cell density in the reactor became three times higher and specific creatinase activity 35% higher than in ordinary continuous fermentation (Table 3). The cultivation condition in Fig. 4 is suitable for intracellular products whose productivity can be improved at a low growth rate, e.g. the production of creatinasc by E. coli M15(pQE3208). As the cell concentration increased in the fermenter, the nutrients distributed for each cell decreased and the growth rate of the cells was also reduced. In ordinary continuous fermentation, E. coli M15(pQE3208) expressed creatinasc better at low specific growth rates, as shown in Table 1. Using colloid chitin and diatomaceous earth to perform continuous cake-filtration fermentation can increase the cell density in the fermenter and decrease the growth rate of the cells, as well as improve the expression of creatinasc. Thus. the culture may be grown at a higher dilution rate and the productivity of creatinasc increased. The choice of an appropriate method to implement large-scale filtration fermentation requires a balance among several factors (Table 4) [c~,16]. These data suggest that using cake filtration to perform filtration fermentation is a promising method fi)r the future.
Conclusion
The continuous cake-filtration fermentation system reported in this article is an alternative way to perform continuous cell-retention fermentation. Controlling the concentration of colloid chitin and the interval of air sparging enabled continued filtering of the culture and cell retention in the fermenter to be achieved. The benefit of this system is its potential of scale-up. Unlike ceramic membrane and hollow fibre modules, stainless steel sieves arc malleable and can be easily sterilized. The cell-retention system performed by stainless steel sieves as the filter medium is more suitable for application in large-scale fermentations.
References
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3.
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9.
S.-C. Tanget al. Cross-flow filtration of methane fermentation broth by ceramic membrane. Journal of Fermentation and Bioengineering 1989, 68, 200-206. Nakano, K., Matsumura, M. and Kataoka, H., Application of a rotating ceramic membrane to dense cell culture. Journal of Fermentation and Bioengineering 1993, 76, 49-54. Cysewski, G. R. and Wike, C. R., Rapid ethanol fermentations using vacuum and cell recycle. Biotechnology and Bioengineering 1978, 19, 1125-1143. Cysewski, G. R. and Wike, C. R., Process design and economic studies of alternative fermentation methods for the production of ethanol. Biotechnology and Bioengineering 1978, 20, 1421-1444. Kang, B. C., Lee, S. Y. and Chang, H. N., Production of Bacillus thuringiensis spores in total cell retention culture and two-stage continuous culture using an internal ceramic filter system. Biotechnology and Bioengineering 1993, 42, 1107-1112. Suzuki, T., Sato, T. and Kominami, M., A dense cell retention culture system using a stirred ceramic membrane reactor. Biotechnology and Bioengineering 1994, 44, 1186-1192. Suzuki, T., A dense cell culture system for microorganisms using a stirred ceramic membrane reactor incorporating asymmetric porous ceramic filters. Journal of Fermentation and Bioengineering 1996, 82, 264-271. Mulder, M. Basic Principles of Membrane Tech-
nology. Kluwer, 1991, pp. 47-48. 10. McCabe, W. L., Smith, J. C. & Harriott, P. Unit Operation of Chemical Engineering. McGraw-Hill, New York, 1985, p. 871. 11. Hoffmann, A. and Roeder, R. G., Purification of Histagged proteins in non-denaturing conditions suggests a convenient method for protein interaction studies. Nucleic Acids Research 1991, 19, 6337-6338. 12. Chang, M. C., Chang, C. C. and Chang, J. C., Cloning of creatinase gene from Pseudomonas putida in Escherichia coli by using an indicator plate. Applied and Environmental Microbiology 1992, 58, 3437-3440. 13. Perry, R. H. & Green, D., Perry's Chemical Engineers' Handbook. McGraw-Hill, New York, 1984, pp. 19-72-19-73. 14. Watt, G. W. and Chrisp, J. D., Spectrophotometric method for determination of urea. Analytical Chemistry 1954, 26, 452-453. 15. Koyama, Y., Kitao, S., Yamamoto-Otake, H., Suzuki, M. and Nakano, E., Cloning and expression of the creatinase gene from Flavobacterium sp. U-188 in Escherichia coli. Agricultural and Biological Chemistry 1990, 54, 1453-1457. 16. Baker, R. W., Cussler, E. L., Eykamp, W., Koros, W. J., Riley, R. L. & Strathmann, H. Membrane Separation Systems. Noyes Data Corporation, New Jersey, 1991, pp. 136-145.