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Fouling of microporous membranes in biological applications
Journal of Membrane Science, 40 (1989)
231-242 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands
FOULING OF MICROPOROUS APPLICATIONS*
MS.
MEMBRANES
231
IN BIOLOGICAL
LE** and K.L. GOLLAN
Domnick Hunter Filters Limited, Durham Road, Birtley, Co. Durham, DH3 2SF (Great Britain)
Summary The fouling of microporous of surface-modified
membranes
membranes,
in biological systems, leading to the recent development
is briefly reviewed. The use of a microporous
membrane
with a
galactopyranose coating in the separation of cr-amylase from a Bacillus broth and malate dehydrogenase from a yeast broth is reported. Moderate fluxes and enzyme transmissions of 100% could be sustained for broths with solid levels up to 10 g/l dry wt. The level of soluble protein in the broth appears to have little effect on either flux or enzyme transmission. Scanning electron microscopy shows that the fouling deposit comprises material presumed to be mostly protein precipitates.
a series of submicron
layers of a very fine
Introduction
Microporous membranes were first developed during World War II for detecting bacteria in water supplies for military uses. The first commercial microporous membranes were marketed by Millipore in the early 1950s for isolating and culturing single-celled micro-organisms. Since then microporous membranes have established themselves as an indispensable tool for the sterilisation and clarification of process fluids that contain not more than lo6 particles of suspended solid per ml. The emergence of a new generation of biotechnology in the late 1970s found a major new role for these membranes in downstream processing and, more generally, in the separation of suspended particulate matter from soluble substances. It would appear upon first consideration that microporous membranes, with their greatest resolving power for particles in the submicron to micron size range, are ideal for the separation of biomolecules from cells and cell debris. Indeed, the advantages of cross-flow microfiltration (MF) over centrifugation and other techniques, including ultrafiltration (UF), in biological applications *Paper presented at the Workshop
on Concentration
versity of Twente, The Netherlands, May 18-19,1987. **To whom correspondence should be addressed.
Polarization
and Membrane
Fouling, Uni-
232
have been widely reported [l-4]. The key features of an MF process that distinguish it from all other processes and make it attractive for such applications can be summarised as follows: 1. Good resolving power for macromolecules from suspended solid. 2. Gentle action (no phase change, low shear). 3. Ease of process containment. 4. Low operating pressure (100-300 kPa). 5. Continuous operation. Interest in this new process has been very significant both academically and commercially, as reflected by a large number of publications and new MF devices on the market in recent years. Unfortunately, however, fouling has proved to be particularly severe with microporous membranes. In addition to causing a drastic reduction in flux, fouling also causes impairment of the membrane fractionating capability, which is regarded as a primary function of the membranes in the present applications. For these reasons fouling has occupied many researchers throughout the world. This paper reviews some of the past work on the fouling of membranes and how it brought about our current understanding of the process. Some new data from a recent study will also be discussed. Studies of microfiltration in biological applications Some of the earliest studies of MF were concerned with the recovery of bacterial cells [ 5-71 using isotropic microporous membranes. The first reported attempt at cell debris removal using various types of membranes was conducted by Quirk and Woodrow [ 11. They showed that, UF membranes with very high molecular weight cut-off became fouled very quickly and began to reject solutes an order of magnitude smaller than their rated size. When fouled, even microporous membranes exhibited a very high solute rejection tendency. Quirk and Woodrow found, however, that certain highly asymmetric microporous membranes were less inclined to reject the solute when used with the more open side against the feed. Using the same type of membranes, Le et al. [ 2,3] showed that it was not possible to predict flux from a knowledge of particle size and membrane pore size. Flux and protein transmission increased with increasing feed velocity. However, although an increase in the applied pressure increased the protein transmission, fouling was also accelerated. Le and Atkinson [8] also found that the pH, ionic strength and other processing conditions all have significant influence on the fouling behaviour. Studies of membrane fouling Studies of membrane fouling in biological systems were first conducted with UF membranes [9,10]. Amongst the earliest reported attempts to combat fouling by a surface treatment was the work of Howell and Velicangil [ 111. They
233
pointed out that initial fouling occurred as a result of membrane surface protein binding in a non-specific adsorption process. Subsequent fouling was ascribed to protein “polymerisation” onto the first layer. However, Le suggested [ 121 that the responsible mechanism was more likely to be crosslinking through sulphide/disulphide interchange between proteins. The phenomenon of protein binding to surfaces was known to biochemists much earlier, as revealed by Dillman and Miller [ 131. In general, biochemists uphold the view that the major forces involved in surface adsorption include ionic interactions and interactions of hydrophobic groups. Since the majority of available polymers suitable for membrane fabrication are hydrophobic, many commercial membranes contain ionic surfactants as a wetting agent. Such combination of materials would clearly result in the worst possible membrane as far as protein fouling is concerned. The first reported attempt at membrane surface modification that took account of the material behaviour was made by Le and Howell [ 141. They showed that a coating of polyethylene glycol (PEG 20,000) on a membrane of polyacrylonitrile copolymer significantly reduced protein binding when the membrane was used for cheese whey ultrafiltration. Recent membrane
development
Although any work on membrane fouling by membrane manufacturers is jealously guarded by commercial secrecy, an examination of the patent literature revealed that industrial research on this subject has been very active. There appear to be two different approaches taken by manufacturers to solve the problem of membrane fouling. The first approach involves surface modification by coating the membrane with a material that exhibits no non-specific binding. Steuck showed [15] that, by coating various types of membranes, including polysulphone, PVDF and PTFE membranes, with a layer of hydroxypropyl acrylate, the resultant membranes exhibited little or no protein binding. The second approach involves the use of materials in the membrane formation process that do not cause protein binding. Parham and Milligan [ 161 used this approach and produced a non-adsorptive polyurethane membrane. Such membranes are not yet commercially available; however, the authors were able to evaluate similar membranes for some typical biological applications, as reported in the following paragraphs. Materials
and methods
Microporous membranes (1.2 pm, cellulosic base) with galactopyranose copolymer coating were prepared by Domnick Hunter Filters Ltd. The copolymer used in the preparation was a proprietary material. The suffixes in the membrane designations indicate an arbitrary degree of polymer crosslinking. All the membranes were shown to be stable at a temperature over 100” C, and
234
chemical compatibility was considered to be superior to that of ordinary cellulose membranes. a-Amylase-producing Bacillus megaterium broth was supplied by the North East Biotechnology Centre (Teesside Polytechnic). Yeast paste (25% dry wt.) was obtained from International Yeast Company. Yeast broth was prepared by adding water to the paste. Yeast enzyme concentrate (YEC) and bovine serum albumin (BSA) were supplied by Sigma Chemicals. The filtration unit comprised a 50 ml stirred cell with 15 cm2 of membrane area operated at approximately 800 rpm. Enzyme transmission
=
filtrate enzyme activity x loo feed enzyme activity
The feed activity was the activity of the feed samples with all cells removed by centrifugation. Results and discussion 1. Membrane
characterisation
The hydraulic permeability of the membranes was determined before subsequent use in the separation studies. Figure 1 shows the water flux for four different membranes up to a pressure of 410 kPa. All the membranes showed a slight degree of compressibility under pressure, as indicated by the deviation from linearity in the profiles. Figure 2 shows that any compaction in the membrane appeared to take place very quickly upon the application of pressure and 800 700
500 5
400
f = 300 i? z 200 100 0
0
50
100
150
Pressure CkPa) Fig. 1. Hydraulic permeability
200
250
300
350
400
450
235
250
1
,, A13XL120
I 0
0
10
5 Time
15
20
25
30
(mid
Fig. 2. Water flux stability.
no sign of creep was observed after the first 5 minutes of pressurisation. Since the permeability of the membranes has been shown to be stable with time at a constant pressure, any transient effect during subsequent filtration experiments was ascribed to other factors. From this point of view, type AI3XL60 membrane was employed throughout the study.
2. Separation of cu-amylase from Bacillus broth a-Amylase (mol.wt. 50,000) is an extracellular enzyme produced from a defined medium. As supplied, the broth contained approximately 1 g/l cells (dry wt. ) . Figure 3 shows the result of a typical run of this separation. The biomass was concentrated by a factor of 10 over a period of under 30 minutes with practically no cells or cell debris appearing in the permeate. The actual dry weight of the biomass in the stirred cell increased exponentially as liquid was reduced to a small volume. Flux decreased sharply in the first 10 minutes, partly because of the compression and presumably also, in part, owing to the formation of a layer of debris on the membrane surface. Somewhat surprisingly, no flux decline was observable during the second half of the run. The transmission of cr-amylase activity was practically 100% throughout the run. Some of the transmission values were over 100% due to substances (of starch origin) that were present in the feed and appeared in the initial permeate samples, causing interference with the assays.
. 120
10
w
Transmission
\
rt
100
-
60
-
40
-
20
r--------60 -k
4-
‘;;
0
5
Time
10
15
20
25
38
0
(min)
Fig. 3. Separation of cu-amylase from Bacillus broth. 137.88 kPa. membrane: A13~60.)
(Feed: 0.96 g/l wamylase
broth: pressure:
Transmission 1
e 16
Time
(mid
Fig. 4. Separation
of malate
g/l YEC;
137.88 kPa, membrane
3.
pressure
dehydrogenase
from yeast broth.
(Feed: 0.9 g/l yeast in H,O +0.05
Al3 X 60.)
Separation of malate dehydrogenase
from yeast broth
Malate dehydrogenase (M, 70,000) is released into solution when the yeast cells lyse. In fact, the broth contained a substantial proportion of lysed cells.
237
200 ,
11% 25
20
15 A
10
g
%l : L,
.d
m
s
Transmission
P
0
0
\
I,--------q 0
40
20 Time
Fig. 5. Separation
60
100
80
1 120
. 140
160
(mind
of malate dehydrogenase
10
from yeast broth (high initial cell concentration,
120
20 Transmission
100
r,
i c s-4
l4
40
J L”
Q 12 -
7 0 co
--I 20
(
p 0 10 0 Gi
60
10
5 Time
(mind
Fig. 6. Separation of malate dehydrogenase Fig. 4, BSA added to l%.)
15
20
25
30
0
from yeast broth with added BSA. (Conditions
as in
The broth was also spiked with YEC in order to enhance the enzyme level. Figure 4 shows the separation of the dehydrogenase from a broth with an initial concentration of 1 g/l yeast (dry wt. ). Flux and concentration rate similar to
238
Fig. 7. Standard
microporous
membrane.
those of the bacterial broth were achieved. After about 30 minutes of filtration, the yeast concentration reached 10 g/l, and at this point a second volume of the same broth was charged into the stirred cell. The upsurge in the flux on introduction of the second broth volume is presumably due to the temporary relaxation of the pressure on the membrane. This effect was also observed after the first 20 minutes when the stirred cell was opened for a feed sample. The transmission of the dehydrogenase activity remained constant at 100% with an increase of 20-fold in the yeast concentration. In a further run, the same membrane after rinsing in water was used to separate the enzyme from a broth with an initial yeast concentration of 10 g/l (dry wt.). Figure 5 shows that both flux and enzyme transmission were poor compared to the previous result. The regular surges in the flux and transmission were again due to pressure relaxation during sampling. The transmission of enzyme over the entire length of the run averaged only 6.5%. The average flux in this case was roughly a third of the value obtained previously. Surprisingly, the flux changed very little during a period in which the total yeast concentration increased about 20-fold. Even more puzzling is the fact that in the first 20 minutes of the run the yeast concentration was not much greater than
239
Fig. 8. Microporous membrane with a surface coating.
10 g/l, compared with about 15 g/l during the last 10 minutes of the previous run, and yet this time both flux and transmission were minimal. In yet a further experiment, the same membrane after rinsing with water was used to separate the enzyme from a broth with 1 g/l yeast (dry wt.) plus 10 g/l BSA. The high level of protein appeared to have no effect at all on either the transmission of the enzyme or the flux. Figure 6 indicates that 100% enzyme activity transmission was achieved with a high flux value, as in the first yeast run. 4. Scanning
electron microscopy
The morphology of the membrane and the fouling layer was explored with scanning electron microscopy. Figures 7 and 8 show a comparison between an unmodified and a surface-coated membrane, respectively. The membrane matrix comprises an integral network of fibres approximately 1 pm in diameter, which are clearly visible from the plate. The coating appears extremely uniform, with a thickness probably in the submicron range. Figures 9 and 10 show the upstream side of the membrane sample used in the amylase experiments. The fouling deposit appears to consist, not of a single layer, but of as series of at least two submicron layers (Fig. 9, arrowed). In
240
Fig. 9. Surface deposit of a-amylase
Bacillus broth showing multilayers
Fig. 10. Surface deposit of the Bacillus broth showing the membrane fouling layers.
of protein precipitate
structure
underneath
the
241
areas where the fouling layer has broken off, the outline of the membrane underneath can clearly be recognised (Fig. 10, arrowed). From the plates the total thickness of the deposit is estimated to be about 1 ,um. Bacillus is rod-shaped with dimensions of 1.2 x 5 pm approximately. Few whole cells seem discernible from the plates. The bulk of the deposit appears to consist of a very fine material, presumably a protein precipitate (probably much less than 0.1 pm in size ) . Conclusion
The result of this work so far has shown that microporous membranes with a coating of a hydrophilic substance could sustain practically 100% transmission of proteins in whole broth processing with some improvement in flux performance over the unmodified membranes [ 21. However, at solid levels greater than 10 g/l, the fouling layer severely reduced both flux and protein transmission. The fouling deposit appears to comprise a series of submicron layers of a very fine material, presumed to be protein precipitates. The amount of soluble protein in the total solid has little effect on either the flux or the enzyme transmission. Some uncertainty about the relationship between the solid content and the fouling behaviour still remains. It seems that a high initial solid level has a significant effect on fouling, but this does not apply if the solid content is raised to the same level by filtration.
References 1 2 3 4 5 6 7 8 9
10
A.V. Quirk and J.R. Woodrow, Tangential flow filtration - new method for the separation of bacterial enzymes from cell debris, Biotechnol. Lett., 5 (1983) 277. MS. Le, L.B. Spark and P.S. Ward, The separation of aryl acylamidase by crossflow microfiltration, J. Membrane Sci., 21 (1984) 219. M.S. Le, L.B. Spark, P.S. Ward and N. Ladwa, Microbial asparaginase recovery by membrane processes, J. Membrane Sci., 21 (1984) 307. W. Hanisch, Cell harvesting, in: WC. McGregor (Ed.), Membrane Separation in Biotechnology, Marcel Dekker, New York, NY, 1986. J.D. Henry and R.C. Allred, Concentration of bacterial cells by crossflow filtration, Dev. Ind. Microbial., 13 (1972) 177. G.B. Tanny, D. Mirelman and T. Pistole, Improved filtration technique for concentration and harvesting of bacteria, Appl. Environ. Microbial., 40 (1980) 269. D.E. Reid and C. Adlam, Large scale harvesting and concentration of bacteria by tangential flow filtration, J. Appl. Bacterial., 41 (1974) 321. M.S. Le and T. Atkinson, Microfiltration for intracellular product recovery, Process Biochem., 21 (1985) 26. W.F. Blatt, A. Dravid, A.S. Michaels and L. Nelsen, Solute polarization and cake formation in membrane ultrafiltration: causes, consequences and controlling techniques, in: J.E. Flinn (Ed.), Membrane Science and Technology, Plenum Press, New York, NY, 1970, p. 47. D.N. Lee and R.L. Merson, Chemical treatment of cottage cheese whey to reduce fouling of ultrafiltration membranes, J. Food Sci., 41 (1976) 778.
242 11
12 13 14
15 16
J.A. Howell and 0. Velicangil, Theory of membrane fouling and its treatment with immobilized proteases, in: A.R. Cooper (Ed.), Ultrafiltration Membranes and Applications, Plenum Press, New York, NY, 1980, pp. 217-230. M.S. Le, Membrane ultrafiltration fouling and treatment, Ph.D. Thesis, University of Wales, 1982. W.J. Dillman and I.F. Miller, Adsorption of serum proteins on polymer membrane surfaces, J. Colloid Interface Sci., 44 (1973) 221. M.S. Le and J.A. Howell, The fouling of ultrafiltration membranes and its treatment, in: C. Cantarelli and C. Peri (Eds.), Symposium of Progress in Food Engineering, Forster-Verlag AG/Forster Publishing Ltd., Kiisnacht, Switzerland, 1983, pp. 321-326. M.J. Steuck, Porous membrane having hydrophilic surface and process of its manufacture, European Patent Application 186,758, 1986. M.E. Parham and K.E. Milligan, Non-adsorptive semipermeable filtration membrane, Brit. Patent Application 2,174,641, 1986.
231-242 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands
FOULING OF MICROPOROUS APPLICATIONS*
MS.
MEMBRANES
231
IN BIOLOGICAL
LE** and K.L. GOLLAN
Domnick Hunter Filters Limited, Durham Road, Birtley, Co. Durham, DH3 2SF (Great Britain)
Summary The fouling of microporous of surface-modified
membranes
membranes,
in biological systems, leading to the recent development
is briefly reviewed. The use of a microporous
membrane
with a
galactopyranose coating in the separation of cr-amylase from a Bacillus broth and malate dehydrogenase from a yeast broth is reported. Moderate fluxes and enzyme transmissions of 100% could be sustained for broths with solid levels up to 10 g/l dry wt. The level of soluble protein in the broth appears to have little effect on either flux or enzyme transmission. Scanning electron microscopy shows that the fouling deposit comprises material presumed to be mostly protein precipitates.
a series of submicron
layers of a very fine
Introduction
Microporous membranes were first developed during World War II for detecting bacteria in water supplies for military uses. The first commercial microporous membranes were marketed by Millipore in the early 1950s for isolating and culturing single-celled micro-organisms. Since then microporous membranes have established themselves as an indispensable tool for the sterilisation and clarification of process fluids that contain not more than lo6 particles of suspended solid per ml. The emergence of a new generation of biotechnology in the late 1970s found a major new role for these membranes in downstream processing and, more generally, in the separation of suspended particulate matter from soluble substances. It would appear upon first consideration that microporous membranes, with their greatest resolving power for particles in the submicron to micron size range, are ideal for the separation of biomolecules from cells and cell debris. Indeed, the advantages of cross-flow microfiltration (MF) over centrifugation and other techniques, including ultrafiltration (UF), in biological applications *Paper presented at the Workshop
on Concentration
versity of Twente, The Netherlands, May 18-19,1987. **To whom correspondence should be addressed.
Polarization
and Membrane
Fouling, Uni-
232
have been widely reported [l-4]. The key features of an MF process that distinguish it from all other processes and make it attractive for such applications can be summarised as follows: 1. Good resolving power for macromolecules from suspended solid. 2. Gentle action (no phase change, low shear). 3. Ease of process containment. 4. Low operating pressure (100-300 kPa). 5. Continuous operation. Interest in this new process has been very significant both academically and commercially, as reflected by a large number of publications and new MF devices on the market in recent years. Unfortunately, however, fouling has proved to be particularly severe with microporous membranes. In addition to causing a drastic reduction in flux, fouling also causes impairment of the membrane fractionating capability, which is regarded as a primary function of the membranes in the present applications. For these reasons fouling has occupied many researchers throughout the world. This paper reviews some of the past work on the fouling of membranes and how it brought about our current understanding of the process. Some new data from a recent study will also be discussed. Studies of microfiltration in biological applications Some of the earliest studies of MF were concerned with the recovery of bacterial cells [ 5-71 using isotropic microporous membranes. The first reported attempt at cell debris removal using various types of membranes was conducted by Quirk and Woodrow [ 11. They showed that, UF membranes with very high molecular weight cut-off became fouled very quickly and began to reject solutes an order of magnitude smaller than their rated size. When fouled, even microporous membranes exhibited a very high solute rejection tendency. Quirk and Woodrow found, however, that certain highly asymmetric microporous membranes were less inclined to reject the solute when used with the more open side against the feed. Using the same type of membranes, Le et al. [ 2,3] showed that it was not possible to predict flux from a knowledge of particle size and membrane pore size. Flux and protein transmission increased with increasing feed velocity. However, although an increase in the applied pressure increased the protein transmission, fouling was also accelerated. Le and Atkinson [8] also found that the pH, ionic strength and other processing conditions all have significant influence on the fouling behaviour. Studies of membrane fouling Studies of membrane fouling in biological systems were first conducted with UF membranes [9,10]. Amongst the earliest reported attempts to combat fouling by a surface treatment was the work of Howell and Velicangil [ 111. They
233
pointed out that initial fouling occurred as a result of membrane surface protein binding in a non-specific adsorption process. Subsequent fouling was ascribed to protein “polymerisation” onto the first layer. However, Le suggested [ 121 that the responsible mechanism was more likely to be crosslinking through sulphide/disulphide interchange between proteins. The phenomenon of protein binding to surfaces was known to biochemists much earlier, as revealed by Dillman and Miller [ 131. In general, biochemists uphold the view that the major forces involved in surface adsorption include ionic interactions and interactions of hydrophobic groups. Since the majority of available polymers suitable for membrane fabrication are hydrophobic, many commercial membranes contain ionic surfactants as a wetting agent. Such combination of materials would clearly result in the worst possible membrane as far as protein fouling is concerned. The first reported attempt at membrane surface modification that took account of the material behaviour was made by Le and Howell [ 141. They showed that a coating of polyethylene glycol (PEG 20,000) on a membrane of polyacrylonitrile copolymer significantly reduced protein binding when the membrane was used for cheese whey ultrafiltration. Recent membrane
development
Although any work on membrane fouling by membrane manufacturers is jealously guarded by commercial secrecy, an examination of the patent literature revealed that industrial research on this subject has been very active. There appear to be two different approaches taken by manufacturers to solve the problem of membrane fouling. The first approach involves surface modification by coating the membrane with a material that exhibits no non-specific binding. Steuck showed [15] that, by coating various types of membranes, including polysulphone, PVDF and PTFE membranes, with a layer of hydroxypropyl acrylate, the resultant membranes exhibited little or no protein binding. The second approach involves the use of materials in the membrane formation process that do not cause protein binding. Parham and Milligan [ 161 used this approach and produced a non-adsorptive polyurethane membrane. Such membranes are not yet commercially available; however, the authors were able to evaluate similar membranes for some typical biological applications, as reported in the following paragraphs. Materials
and methods
Microporous membranes (1.2 pm, cellulosic base) with galactopyranose copolymer coating were prepared by Domnick Hunter Filters Ltd. The copolymer used in the preparation was a proprietary material. The suffixes in the membrane designations indicate an arbitrary degree of polymer crosslinking. All the membranes were shown to be stable at a temperature over 100” C, and
234
chemical compatibility was considered to be superior to that of ordinary cellulose membranes. a-Amylase-producing Bacillus megaterium broth was supplied by the North East Biotechnology Centre (Teesside Polytechnic). Yeast paste (25% dry wt.) was obtained from International Yeast Company. Yeast broth was prepared by adding water to the paste. Yeast enzyme concentrate (YEC) and bovine serum albumin (BSA) were supplied by Sigma Chemicals. The filtration unit comprised a 50 ml stirred cell with 15 cm2 of membrane area operated at approximately 800 rpm. Enzyme transmission
=
filtrate enzyme activity x loo feed enzyme activity
The feed activity was the activity of the feed samples with all cells removed by centrifugation. Results and discussion 1. Membrane
characterisation
The hydraulic permeability of the membranes was determined before subsequent use in the separation studies. Figure 1 shows the water flux for four different membranes up to a pressure of 410 kPa. All the membranes showed a slight degree of compressibility under pressure, as indicated by the deviation from linearity in the profiles. Figure 2 shows that any compaction in the membrane appeared to take place very quickly upon the application of pressure and 800 700
500 5
400
f = 300 i? z 200 100 0
0
50
100
150
Pressure CkPa) Fig. 1. Hydraulic permeability
200
250
300
350
400
450
235
250
1
,, A13XL120
I 0
0
10
5 Time
15
20
25
30
(mid
Fig. 2. Water flux stability.
no sign of creep was observed after the first 5 minutes of pressurisation. Since the permeability of the membranes has been shown to be stable with time at a constant pressure, any transient effect during subsequent filtration experiments was ascribed to other factors. From this point of view, type AI3XL60 membrane was employed throughout the study.
2. Separation of cu-amylase from Bacillus broth a-Amylase (mol.wt. 50,000) is an extracellular enzyme produced from a defined medium. As supplied, the broth contained approximately 1 g/l cells (dry wt. ) . Figure 3 shows the result of a typical run of this separation. The biomass was concentrated by a factor of 10 over a period of under 30 minutes with practically no cells or cell debris appearing in the permeate. The actual dry weight of the biomass in the stirred cell increased exponentially as liquid was reduced to a small volume. Flux decreased sharply in the first 10 minutes, partly because of the compression and presumably also, in part, owing to the formation of a layer of debris on the membrane surface. Somewhat surprisingly, no flux decline was observable during the second half of the run. The transmission of cr-amylase activity was practically 100% throughout the run. Some of the transmission values were over 100% due to substances (of starch origin) that were present in the feed and appeared in the initial permeate samples, causing interference with the assays.
. 120
10
w
Transmission
\
rt
100
-
60
-
40
-
20
r--------60 -k
4-
‘;;
0
5
Time
10
15
20
25
38
0
(min)
Fig. 3. Separation of cu-amylase from Bacillus broth. 137.88 kPa. membrane: A13~60.)
(Feed: 0.96 g/l wamylase
broth: pressure:
Transmission 1
e 16
Time
(mid
Fig. 4. Separation
of malate
g/l YEC;
137.88 kPa, membrane
3.
pressure
dehydrogenase
from yeast broth.
(Feed: 0.9 g/l yeast in H,O +0.05
Al3 X 60.)
Separation of malate dehydrogenase
from yeast broth
Malate dehydrogenase (M, 70,000) is released into solution when the yeast cells lyse. In fact, the broth contained a substantial proportion of lysed cells.
237
200 ,
11% 25
20
15 A
10
g
%l : L,
.d
m
s
Transmission
P
0
0
\
I,--------q 0
40
20 Time
Fig. 5. Separation
60
100
80
1 120
. 140
160
(mind
of malate dehydrogenase
10
from yeast broth (high initial cell concentration,
120
20 Transmission
100
r,
i c s-4
l4
40
J L”
Q 12 -
7 0 co
--I 20
(
p 0 10 0 Gi
60
10
5 Time
(mind
Fig. 6. Separation of malate dehydrogenase Fig. 4, BSA added to l%.)
15
20
25
30
0
from yeast broth with added BSA. (Conditions
as in
The broth was also spiked with YEC in order to enhance the enzyme level. Figure 4 shows the separation of the dehydrogenase from a broth with an initial concentration of 1 g/l yeast (dry wt. ). Flux and concentration rate similar to
238
Fig. 7. Standard
microporous
membrane.
those of the bacterial broth were achieved. After about 30 minutes of filtration, the yeast concentration reached 10 g/l, and at this point a second volume of the same broth was charged into the stirred cell. The upsurge in the flux on introduction of the second broth volume is presumably due to the temporary relaxation of the pressure on the membrane. This effect was also observed after the first 20 minutes when the stirred cell was opened for a feed sample. The transmission of the dehydrogenase activity remained constant at 100% with an increase of 20-fold in the yeast concentration. In a further run, the same membrane after rinsing in water was used to separate the enzyme from a broth with an initial yeast concentration of 10 g/l (dry wt.). Figure 5 shows that both flux and enzyme transmission were poor compared to the previous result. The regular surges in the flux and transmission were again due to pressure relaxation during sampling. The transmission of enzyme over the entire length of the run averaged only 6.5%. The average flux in this case was roughly a third of the value obtained previously. Surprisingly, the flux changed very little during a period in which the total yeast concentration increased about 20-fold. Even more puzzling is the fact that in the first 20 minutes of the run the yeast concentration was not much greater than
239
Fig. 8. Microporous membrane with a surface coating.
10 g/l, compared with about 15 g/l during the last 10 minutes of the previous run, and yet this time both flux and transmission were minimal. In yet a further experiment, the same membrane after rinsing with water was used to separate the enzyme from a broth with 1 g/l yeast (dry wt.) plus 10 g/l BSA. The high level of protein appeared to have no effect at all on either the transmission of the enzyme or the flux. Figure 6 indicates that 100% enzyme activity transmission was achieved with a high flux value, as in the first yeast run. 4. Scanning
electron microscopy
The morphology of the membrane and the fouling layer was explored with scanning electron microscopy. Figures 7 and 8 show a comparison between an unmodified and a surface-coated membrane, respectively. The membrane matrix comprises an integral network of fibres approximately 1 pm in diameter, which are clearly visible from the plate. The coating appears extremely uniform, with a thickness probably in the submicron range. Figures 9 and 10 show the upstream side of the membrane sample used in the amylase experiments. The fouling deposit appears to consist, not of a single layer, but of as series of at least two submicron layers (Fig. 9, arrowed). In
240
Fig. 9. Surface deposit of a-amylase
Bacillus broth showing multilayers
Fig. 10. Surface deposit of the Bacillus broth showing the membrane fouling layers.
of protein precipitate
structure
underneath
the
241
areas where the fouling layer has broken off, the outline of the membrane underneath can clearly be recognised (Fig. 10, arrowed). From the plates the total thickness of the deposit is estimated to be about 1 ,um. Bacillus is rod-shaped with dimensions of 1.2 x 5 pm approximately. Few whole cells seem discernible from the plates. The bulk of the deposit appears to consist of a very fine material, presumably a protein precipitate (probably much less than 0.1 pm in size ) . Conclusion
The result of this work so far has shown that microporous membranes with a coating of a hydrophilic substance could sustain practically 100% transmission of proteins in whole broth processing with some improvement in flux performance over the unmodified membranes [ 21. However, at solid levels greater than 10 g/l, the fouling layer severely reduced both flux and protein transmission. The fouling deposit appears to comprise a series of submicron layers of a very fine material, presumed to be protein precipitates. The amount of soluble protein in the total solid has little effect on either the flux or the enzyme transmission. Some uncertainty about the relationship between the solid content and the fouling behaviour still remains. It seems that a high initial solid level has a significant effect on fouling, but this does not apply if the solid content is raised to the same level by filtration.
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