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Aqueous-organic membrane bioreactors part II. Breakthrough pressure measurement
journal of M:~i%%E ELSEVIER
Journal of Membrane Science 97 ( 1994) 13-26
Aqueous-organic membrane bioreactors Part II. Breakthrough pressure measurement A.M. Vaidya”,*, G. Bell”, P.J. Hallingb ‘Department of Chemical Engineering, StrathclydeUniversity,75, Montrose Street, GlasgowGI IXJ, UK bDepartmentof Bioscience and Biotechnology, StrathclydeUniversity,George Street, GlasgowGl 1Xw UK
Received 28 March 1994; accepted in revised form 1July 1994
Abstract The effect of membrane type - structure and wettability - on the operation of two-phase, aqueous-organic, membrane bioreactors has been studied. The influence of surfactants on membrane wettability is reported. A simple, but highly sensitive, technique for the measurement of breakthrough pressures is described. Experimental measurements of the variation in break through pressures as the concentration of tenside in the system was changed are reported. On the basis of the results from these measurements it is concluded that: (i) hydrophilic and, highly retentive, amphiphilic ultrafiltration membranes may be used to operate two-phase bioreactors, (ii) amphiphilic microfiltration membranes should never be used in such reactors and (iii) PTFE membranes would always be a poor choice for use in such devices because they always have a low breakthrough resistance in two-liquid systems - breakthrough pressures as low as 100 mbar were observed for the system ethyl laurate-water-PTFE, which contains no surface-active component. It is shown that these results are in general agreement with rules of thumb for the selection of membranes, presented earlier. The influence of membrane history on its wetting behavior due to effects such as polymer surface restructuring - is highlighted. The limits on the utility of simple breakthrough pressure tests in determining suitable membranes, for use in two-phase bioreactors, owing to possible complications resulting from the exact mechanism of enzyme action is pointed out. Keywords:Two-phase biocatalysis; Membrane bioreactor; Surfactant-induced breakthrough; Membrane selection
1. Introduction
Membrane reactors using hydrophobic as well as hydrophilic polymeric membranes have been used to carry out two-phase, aqueous-organic, biocatalytic reactions [ l-9 1. In such devices the membrane, which is selectively wetted by one of the two phases, separates the aqueous phase from an immiscible organic phase. This is accomplished by applying a slight pressure to the non-
wetting liquid in order to create an interface between the two liquids in the plane of the membrane. If the applied pressure is made sufflciently large one can cause displacement of the wetting liquid from the pores in the membrane which is followed by flow of the non-wetting liquid through the membrane. This effect is known as breakthrough - and the pressure at which it occurs is known as the breakthrough pressure. The sequence of events that precedes breakthrough is described in detail in the Appendix.
* Corresponding author. 0376-7388/94/$07.00 0 1994 Elsevier Science B.V. All rights reserved SSDl0376-7388(94)00144-8
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A.M. Vaidya et al. /Journal
ofMembrane
The occurrence of breakthrough makes it impossible to use the reactor. A large number of two-phase biocatalytic reactions involve a surface-active product and/or reactant. The presence of such materials can lead to difficulties in reactor operation because of tenside-induced changes in the wetting behavior of the membrane - which in turn can lead to a decrease in the breakthrough pressure. The theory of such effects has been described in some detail [ lo]. Evidently, some care must be exercised in the selection of membranes for use in such reactors. A few rules of thumb, based on theoretical considerations, to assist this selection process have been given earlier [ lo]. The process of the selection of membranes for use in such reactors would be aided if one could devise a simple experiment to study the breakthrough behavior of the reaction system of interest. A few measurements of breakthrough pressure have been done to assist the evaluation of membranes for use in non-dispersive solvent extraction [ ll13 1. However, the systems studied are considerably simpler since they do not involve the strongly surface-active solutes which are commonly encountered in two-phase biocatalysis. Further, these measurements have been reported at only one concentration of the solute. In this paper a more sensitive experimental technique for the selection of suitable membranes will be described. The breakthrough effects in these experiments will be compared with those observed when the membranes are used in reactors for the lipase-catalyzed hydrolysis of ethyl laurate.
2. Experimental 2. I. Breakthrough pressure experiments Most of the breakthrough pressure experiments were done with pure water as the aqueous phase. However, a few experiments were done with the buffered aqueous phase (0.1 M NaH,POJNaOH, pH 8.0 at 25’C) - used for enzyme immobilization and for carrying out the reaction - since the distribution of the surfactant between the two phases could to be significantly
Science 97 (1994) 13-26
affected by the presence of the buffer species which in turn may have a bearing on the magnitude of the observed breakthrough pressures. Laurie acid ethyl ester (L4625, Sigma Chemical Co., Poole, UK) and the appropriate aqueous phase - buffer or single distilled water - were used as test liquids. The variation in surfactant composition during the course of the lipase-catalyzed hydrolysis of the ester was simulated by shaking different masses of lauric acid (L4250, Sigma) with known quantities of ester and water for 24 h at room temperature and then separating the aqueous and organic phases. To determine acid dissolved in the aqueous phase, a small amount of ester - 5 0.5 g - was added to a large volume - over 20 ml - of the aqueous phase and the acid was extracted into the ester by shaking the mixture for 60 min. The acid enriched ester was extracted from the resulting two-phase mixture by shaking it with an equal volume of hexane (H9379, Sigma) and then separating the two phases. The concentration of acid in the ester was determined on a gas chromatograph - using the technique described later in this section. The concentration of acid in the aqueous phase water as well as buffer - was found to be negligible. The types of membranes used and their properties are summarized in Table 1. The polypropylene and the asymmetric deacetylated cellulose acetate membranes were supplied in the form of A4 sheets. The remaining membranes were supplied in the form of 47 mm discs. Discs of the appropriate size - 25 mm for the breakthrough pressure tests and 47 mm for the reactor experiments - were punched out, where necessary. In order to obtain rapid detection of breakthrough it is vital to minimize the dead volume under the membrane. This is difficult to achieve if a large test area is used, since the membrane must then be supported in order to enable it to withstand the applied pressure. Polysulfone grids, commonly used as supports in ultrafiltration cells, were initially used for this purpose. However, it was found that holdup of liquid in the interstices of the support lead to a considerable delay in the detection of breakthrough. (A delay is also likely to occur if one relies on the obser-
A.M. Vaidya et al. /Journal ofMembraneScience 97 (1994) 13-26 Table I Membranes used in breakthrough
15
pressure tests
Product code
Supplier
Material
Wetting characteristic
Type (UForMF)
Pore size/MWCO
Cuprophan RC70PP Nylaflo BM-5 BM-1000 Celgard 2400d Goretex’
Enka Dow Gelman Sciences Berghof Berghof Hoechst Celanase W.L. Gore
Cellulose” Cellulose’ Nylon-66 Meta-aramid Meta-aramid Polypropylene PTFE
Hydrophilic Hydrophilic Amphiphilic Amphiphilic Amphiphilic Hydrophobic Hydrophobic
UF UF MF UF UF MF MF
2 nmb 10,000 Da 0.2 ,um 500 Da 10,000 Da 0.2x0.04pm 0.2 ,um
a Dense, renegerated cellulose membranes. b Estimated pore size [ 191 ’ Asymmetric membranes made by deacetylation of cellulose acetate reverse osmosis membranes d These membranes had rectangular slits rather than circular pores. ’ These membranes were thermally bonded onto a polypropylene support.
vation of a droplet of the non-wetting liquid appearing on the surface of a single hollow fiber membrane to obtain an estimate of the breakthrough pressure - which is the technique employed by Tompkins et al. [ 111. The experimental technique employed by Prasad and coworkers [ 12,131 in their breakthrough pressure measurements has not been clearly specified.) An accurate estimate of the breakthrough pressure can be obtained by having a very small area of unsupported membrane, as shown in Fig. 1. Any liquid passing through this small test area can immediately be observed in the 0.5 mm bore glass capillary attached to the base of the test cell. A 25 mm membrane disc - wetted with the appro-
Air pressure
Cell
connector
cover
with cavity for test liquid
10 mm
1 Silicone
rubber
gasket
Fig. 1.Test cell used for breakthrough
pressure experiments.
priate test liquid - was clamped in between the two halves of the test cell. A seal between the lowand high-pressure halves of the test cell was made by the silicone rubber seals embedded in the two parts of the test cell. The second test liquid - the non-wetting liquid - was carefully introduced into the cavity in the upper half of the test cell with the aid of a hypodermic syringe and a tine needle. Air pressure was applied to this liquid through the l/4 BSP - 6 mm tube adaptor shown in the figure. It was found necessary to remove all sharp corners from the test liquid cavity in order to prevent bubbles of residual air from being trapped inside the cell, since their presence was found to lead to spurious breakthrough pressure results. (This problem was more pronounced in experiments with amphiphilic and hydrophobic membranes since the air bubbles displayed a tendency to adhere to the surface of such membranes. On pressurization, the resulting air-organic liquid interface was displaced at extremely low pressures.) The cell was pressurized gradually until continuous flow of liquid in the glass capillary was observed - at which point breakthrough was deemed to have occurred. A precision pressure regulator (Negeretti Valve Co., UK) was used for this purpose and the cell pressure was monitored using a high precision transducer (Penny & Giles Transducers Ltd., UK). All the tests were carried out at 25 ‘C. The total duration of the tests was normally not longer
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A.M. Vaidya et al. /Journal of Membrane Science 97 (1994) 13-26
than 10 min. However, some of the tests with the Celgard (polypropylene) membranes were carried out over a period of 24 h with each test pressure being maintained for periods up to 12 h. This was done to investigate the possibility of delayed breakthrough effects arising due to gradual adsorption of the surfactant. Before being subjected to breakthrough pressure tests the membranes were pretreated. The nature of the pretreatment depended on the membrane type. 1. Microporous membranes: The membranes were wetted with the appropriate wetting liquid and any excess liquid was wiped off the surface with a lint-free tissue. It was observed that the Nylaflo (microporous nylon-6,6 ) membranes were more easily wetted by the organic phase than by the aqueous phase. 2. Ultrafiltration membranes: The Cuprophan (regenerated cellulose) membranes were thoroughly washed in distilled water before use. The BM5, BM 100 (meta-aramid) and RC70PP (deacetylated cellulose) membranes - the latter were supplied in a 50% aqueous glycerol solution containing sodium hydroxide and propionic acid were first soaked in distilled water overnight. Immediately prior to the test distilled water was filtered through the membrane at a pressure of 1 bar until a filtrate of N 1 ml had been collected. 2.2. Membrane reactor experiments The flat sheet membrane reactor used in all the experiments is shown in Fig. 2. It consisted of two flanged nylon-6,6 shells of circular cross section held together by three M4 bolts threaded through the flanges. A silicone rubber ‘0’ ring prevented leakage of the reactants when the reactor was pressurized. A polysulfone grid was placed in the low pressure half of the reactor to support the membrane. AlCoVar bar magnets (Merck Ltd., UK), encapsulated in a thin film of perfluoropolyether (Fomblin, Italy) were placed in the cylindrical cavities in each half of the reactor. A fine stainless steel wire mesh was placed above the membrane, on the high-pressure side of the reactor, to prevent damage to the membrane by the rotation of the magnet. Two
Fig. 2. Exploded view of flat sheet two-phase membrane bioreactor: ( 1) high pressure half cell, (2) magnetic stirrers, (3) ‘0’ ring, (4) stainless steel mesh, (5) membrane, (6) membrane support, (7) low pressure half cell, (8) header tubes, (9) sampling port.
PEEK (polyether-ether-ketone) header tubes (30 mm length and 1.6 mm bore) were glued to both the half-cells comprising the reactor to permit the introduction of reactants and/or purging of air. A PTFE-faced silicone rubber septum of 6 mm diameter, held in place by a l/4 UNF nut with a 1/ 16 fl hole drilled through it, was used to gain access to the reactant in the high-pressure half of the reactor. The contents in the two halves of the reactor were stirred magnetically after clamping it horizontally in a thermostated water bath (25°C). The reactor had an active membrane area of 4.9 cm*. The precision pressure regulator used in the breakthrough pressure experiments was employed to maintain a constant pressure of compressed air above the non-wetting liquid and the pressure in the head space was monitored by a digital manometer (Platon Flowbits Ltd., UK), 2.3. Enzyme immobilization Solutions of lipase from Candida rugosa (L1754, Sigma) were made up in phosphate buffer (2.7 mg/g). MF membranes: The enzyme was immobilized at the liquid-liquid interface by physical adsorption. The membrane was first wetted with a few drops of the ester and then mounted in the
A.M. Vaidya et al. /Journal
of Membrane Science 97 (1994) 13-26
reactor. The buffered aqueous enzyme solution was then introduced into the high-pressure side of the reactor. After pressurizing the high-pressure side of the reactor, the low-pressure side was filled with the ester. The liquids were left in contact with each other for 1 h after which they were removed from the reactor and the two halves of the reactor were washed with the appropriate liquid-ester or aqueous buffer. The activity of the enzyme solution was measured, before and after immobilization, to obtain an estimate of the quantity of immobilized enzyme. UF membranes: The membrane was pretreated in a manner similar to that described above for the breakthrough pressure tests. The membrane was then mounted in the reactor and 10 ml of distilled water was filtered through it. The filtrate was rejected and 4.5 ml of lipase solution was filtered through the membrane, at a pressure of 1 bar, followed by 8 ml of buffer. These operations were done without stirring. Finally, an additional 2 ml of buffer was filtered with the stirrer in operation. The entire filtrate was collected and assayed for lipase activity. 2.4. Assays The concentration of lauric acid in the organic phase was determined on a gas chromatograph, with 80-100 mesh Chromosorb WHP and 3% OV 1 as the stationary phase packed in a column of internal diameter 3 mm and length 2 m (Phase Separations Ltd., UK), at 180°C. The samples were dried and derivatized with MTBSTFA (Phase Separations) prior to analysis. The activity of the lipase was determined by titrating the butyric acid released during the lipase catalyzed hydrolysis of tributyrin at pH 8 (25°C). This was done on a pHstat (PHM 82, Radiometer, Denmark). The reaction was carried out with a 3% (w/v) emulsion of tributyrin (Aldrich, UK) made in a buffered gum arabic (G9752, Sigma) solution. The latter was made by stirring an excess of gum arabic in the buffer (0.1 M NaH2 PO,/NaOH) for 3 h at 40” C - and then filtering out the excess. A 100 mM aqueous solution of NaOH (Sigma) was used as the titrant and one unit of enzyme was defined as the
17
amount of enzyme required to release butyric acid at an initial rate of 1 pmol/min. The water content of the organic phase was determined by Karl-Fischer titration on a KF 653 coulometer (Metrohm, Switzerland). 2.5. Reactor operation Fresh reactants were loaded into the two halfcells of the reactor - after enzyme immobilization had been completed and the reactor had been washed, as described above - and the non-wetting liquid was pressurized. The contents of the reactor were stirred magnetically and the lipasecatalyzed hydrolysis of ethyl laurate in the buffered aqueous phase was allowed to continue until breakthrough was observed. Breakthrough was detected visually by observing the movement of liquid in a narrow bore silicone rubber tubing attached to one of the two header tubes on the lowpressure half of the reactor (of the two header tubes on the high-pressure side of the reactor, one was used to pressurize the high-pressure half-cell whilst the other was closed using a short length of silicone rubber tubing clamped at its open end). This process is complicated in the case of hydrophilic membranes since the hydrolysis reaction results in a net transfer of the system volume from the organic to the aqueous phase. In such cases it is necessary to collect a well-stirred sample of liquid from the low-pressure half-cell and check for the presence of two distinct phases in order to establish the occurrence of breakthrough. Samples of the organic phase were taken at frequent intervals and fresh ethyl laurate was added to the organic side of the reactor to make up for the resulting depletion. The water content of each of the collected organic phase samples was estimated by Karl-Fischer titration in order to ensure that they did not contain small quantities of finely emulsified aqueous phase. In a separate experiment the variation of the saturation solubility of water in the organic phase as the acid content of the latter is changed was determined. In experiments with the Celgard (polypropylene) membranes, breakthrough was deemed to have occurred when a sample was found to con-
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A.M. Vaid.vaet al. /Journal ofMembraneScience 97 (1994) 13-26
tain a higher proportion of water than the saturation solubility of water at the measured concentration of acid. This was done in order to get a more accurate estimate of the instant of breakthrough than was offered by the visual observation made during the experiment.
3. Results and discussion A two-phase membrane reactor can be operated with microfiltration (MF) or ultrafiltration (UF) membranes. One has the further choice of using either hydrophilic or hydrophobic membranes. In principle it should also be possible to use an amphiphilic polymer, such as a polyamide, if the adsorption of tenside at the two polymer-liquid interfaces occurs in such a manner as to keep the breakthrough pressure, dP, sufficiently high. The process by which this could happen has been outlined elsewhere [ lo]. The membranes tested in the experiments described in this paper cover the whole spectrum of possibilities. (In this paper the terms hydrophobic and hydrophilic have been used in a more rigorous sense than is the norm. Polypropylene and PTFE membranes have been considered to be hydrophobic and regenerated cellulose membranes have been treated as being hydrophilic. All other commercially available membranes, such as polysulfones, polyamides, etc., are - in the strict sense of the term - amphiphilic since they are made from polymers containing both polar and non-polar groups. ) The performance of all membranes which could be used in membrane reactors with some measure of success is given in Table 2. The timecourse curves for reactors operated with these membranes are shown in Figs. 3 and 4. The results from the breakthrough pressure tests on these membranes are shown in Figs. 5, 6 and 7. The breakthrough pressures reported in these figures are an average value obtained from a large number of experiments. The breakthrough pressure results reported here were obtained with pure water as the aqueous phase. In the presence of a pH 8 buffer there is an increase in the concentration of acid in the aqueous phase due to
some ionization of the fatty acid. However, this was found to have no significant effect on breakthrough pressures. 3.1. Nylajlo (microporous nylon-6,6) membranes Breakthrough pressure results for these membranes were obtained with the membranes used both as hydrophilic and as hydrophobic membranes. The breakthrough pressure curve in the hydrophilic mode is shown in Fig. 5. In the hydrophobic mode the breakthrough pressure was found to be constant at 280 mbar. An asymmetry in breakthrough pressures depending on the mode of operation - hydrophilic or hydrophobic - has been previously reported, without any explanations, by Prasad et al. [ 121 for a microporous membrane made from an unspecified polyamide. The existence of a positive breakthrough pressure in both modes of operation is extremely surprising. An examination of the LaplaceYoung equation [ lo] suggests that the displacement of one of the two liquids must occur spontaneously.
In other words, one of the two breakthrough pressures must be negative. It is possible to account for the occurrence of positive breakthrough pressures in both the modes of operation if one invokes the existence of restructuring effects at the surface of an amphiphilic polymer membrane. In Eq. ( 1 ), the difference da= a,, -a,,
(2)
must remain unchanged by the surfactant concentrations in the system when the membrane is used in its hydrophobic mode. On the other hand, in the hydrophilic mode, 60 undergoes a significant decrease, leading to the breakthrough pressure curve of Fig. 5. If one invokes restructuring phenomena, the explanation for this behavior of 60 is as follows. As Ruckenstein and Gourisankar [ 141 have noted, restructuring effects will lead to the emergence of buried polar groups from the bulk
A.M. Vaidya et al. /Journal of Membrane Science 97 (1994) 13-26
19
Table 2 Enzyme loadings, operating pressures and initial reaction rates of membrane reactors. The acid concentration at which breakthrough was first observed is also given. The reaction rate was found to decrease rapidly from its initial value for the Celgard 2400, RC70PP and BM5 membrane reactors. With the others it was constant at its initial value Membrane
Total adsorbed/ retained enzyme (Units)
Enzyme loading (Units/m*)
1.7 6.7 9.1 7.5 4.5
3,460 13,650 18,540 15,300 9,100
Celgard 2400 RC’IOPP Cuprophan BM5 BMlOO
Operating pressure (mbar)
Initial reaction rate
15 100 250-400” 250-400” 200
Interfacial (molhm’)
Enzyme (mol/s Kat )
0.05 0.31 0.02 0.08 0.04
0.22 0.37 0.02 0.09 0.06
Acid concentration at breakthrough (pmol/g)
No breakthrough 93.5 No breakthrough No breakthrough 116
a The pressure was increased to 400 mbar towards the end of the experiment.
Acid Concentration
Acid Concentration
[pmol/g]
[pmollg]
100
50
0’ 0
I
’
50
’
’
100
’
’
150
’
’
200
’
250
Time [min] Fig. 3. Time-course curve for the lipase-catalyzed hydrolysis of ethyl laurate in a membrane reactor with a deacetylated cellulose acetate membrane. The reactor was operated with the skinned side of the membrane facing the non-wetting, organic phase. Other experimental conditions are as shown in Table 2.
of the membrane polymer to the membraneaqueous interface. They have demonstrated that these effects are quite rapid (occurring within a few seconds), whilst the reverse effect - the subsidence of polar groups when the surface is exposed to a non-polar environment - is relatively slow (requiring a period of several days). In the light of their observations one can deduce the existence of four membrane-liquid interfacial ten-
0
0
500
1.ooo
1.500
2.;00
Time [min] Fig. 4. Time-course curves for the lipase-catalyzed hydrolysis of ethyl laurate. Both the meta-aramid membranes were used with their skinned side facing the non-wetting, organic, phase. Experimental conditions are shown in Table 2.
sions for an amphiphilic membrane. In the polar mode of operation the interfacial tensions are 0 am and @A,,- between the aqueous and organic phases and the membrane, respectively. Whilst the former will be low due to restructuring effects, the latter will be higher and there will be a tendency for the tenside to adsorb at the organic-membrane interface. If the extent of this adsorption displays a concentration dependence it follows that
A.M. Vaidyaet al. /Journal of Membrane Science 97 (1994) 13-26
20 Breakthrough
Pressure
[mbar]
Nylaflo - Hydrophilic
20’
0
100
200
300
RC70PP
400
Acid concentration
- Open
500
600
700
[pmol/g]
Fig. 5. Dependence of breakthrough pressure on acid concentration for the systemethyl laurate-lauric acid-water.
W=a&
-a*am
(3)
will decrease with increasing concentrations of the tenside. On the other hand, in the non-polar mode of operation the interfacial tension o,“&will have a relatively low value and the surfactant will have only a very limited propensity to adsorb at this interface. The interfacial tension at the aqueous-membrane interface, a:,$, will exist only momentarily since it will rapidly change to ai, as a result of restructuring effects. One would thus have two constant, and low, liquid-membrane interfacial tensions with the result that &P = crtm - a;&
(4)
will be constant, but positive. In offering these explanations we have assumed that reverse restructuring, i.e., the subsidence of polar groups in a non-polar environment is a relatively slow process - considerably slower than the adsorption of surfactant from the non-polar organic phase. We were unable to operate membrane reactors using this type of membrane in either of its modes of wetting since the maintenance of a stable liquid-liquid interface was found to be impossible. This result is not unexpected for the hydrophilic
mode of operation since the generation of fatty acid in the reactor would rapidly lead to a decrease in the breakthrough pressure. The magnitude of this effect may be considerably more severe than indicated by the breakthrough pressure curve of Fig. 5 since the concentration of the surfactant is likely to be significantly higher in the immediate vicinity of the membrane - where it is being produced during the operation of a membrane reactor. In light of the fact that these membranes gave a relatively high, and constant, breakthrough pressure in their hydrophobic mode of operation one would expect that it should be possible to operate a membrane reactor in this mode. However, it was found that there was virtually instantaneous breakthrough of the aqueous phase - even when the pressure applied to the latter was as low as 20 mbar. This effect could be caused by the formation of a composite hydrophilic membrane during the enzyme immobilization step of reactor operation. Absolom and Newmann [ 15 ] have noted such an effect in their experiments in which a number of proteins were adsorbed on to a range of polymeric surfaces. Schroen et al. [ 161 have also commented on the modification of membrane wettability induced by proteins.
3.2. Berghof (meta-aramid) membranes
The breakthrough pressure curve for the BMlOO membrane, shown in Fig. 6, used in its hydrophilic mode follow the same trend as the Nylaflo membranes in their hydrophilic mode. The breakthrough pressures are higher since these UF membranes have a considerably smaller pore size. The more retentive BM5 membranes were found to be completely resistant to breakthrough, at all the surfactant concentrations tested, for pressures as high as 2000 mbar. These membranes were not tested in their hydrophobic mode. The increase in surfactant concentration with time during the operation of reactors with these membranes is shown in Fig. 4. As in the breakthrough pressure experiment, the BM5 mem-
A.M. Vaidya et al. /Journal ofMembrane Science 97 (I 994) 13-26
Breakthrough 300 250
Pressure [mbar]
r c
200 150 100 50
t
OL 0
100
200
300
400
Acid Concentration
500
600
700
[pmollg]
Fig. 6. Dependence of breakthrough pressure on acid concentration for the systemethyl laurate-lauric acid-water.
brane was found to be completely resistant to breakthrough when operated with a pressure of 250 mbar on the aqueous phase. A maximum interfacial reaction rate of 0.083 mol/h m2 was obtained. Breakthrough of the aqueous phase through the BMlOO membrane occurred at a lauric acid concentration of 116 ,umol/g in the bulk of the organic phase.
21
laflo membranes, it is likely that the membrane was rendered more easily wettable by the adsorption of lipase at the interface. This improved wettability, combined with the applied pressure, could have resulted in the observed occurrence of breakthrough. In an attempt to verify this mechanism, a breakthrough pressure experiment was conducted with a Celgard membrane which had been subjected to enzyme adsorption -by allowing the ester-wet membrane to remain in contact with an aqueous lipase solution (a 1 mg/ml solution of the same lipase as was used for reactor operation) for 1 h. However, it was still found to be resistant to breakthrough for pressures as high as 1200 mbar. We have been unable to find a satisfactory explanation for these discrepancies. 3.4. GoreTex (PTFE) membranes The breakthrough pressure curve for a GoreTex membrane (pore size 0.2 pm) is shown in Fig. 7. The breakthrough behavior of these membranes was found to be different from that of the other membranes, described above. Some displacement of wetting liquid into the capillary was observed at pressures below the breakBreakthrough
Pressure [mbar]
3.3. Celgard (polypropylene) membranes The Celgard membranes were found to be resistant to breakthrough of the aqueous phase up to pressures as high as 2 100 mbar. (This was found to be the case even when the membrane was exposed to the surfactant for an extended period of time - as was done in some of the experiments.) This is in sharp contradiction with the observation by Hoq et al. [ 1 ] who found that during reactor operation with Juragard, polypropylene, membranes - similar in structure to Celgard - the pressure on the non-wetting, aqueous, phase had to be kept below 20 mbar. In our own reactor experiments with Celgard membranes - Fig. 4 - breakthrough was found to occur when the pressure applied to the aqueous phase was greater than 15 mbar. As with the Ny-
8o I 70 60
I 100
1
I 200
I
1 300
Acid concentration
I 400
, 500
[pmollg]
Fig. 7. Breakthrough behavior of GoreTex membranes for the system ethyl laurate-lauric acid-water.
A.M. Vaidya et al. /Journal of Membrane Science 97 (1994) 13-26
22
through pressure. The amount of displaced liquid increased with increasing pressure. However, continuous flow only occurred when the breakthrough pressure was reached. It was found impossible to operate membrane reactors with these membranes since breakthrough of the aqueous phase invariably occurred very early during their operation. The fact that the Celgard 2400 (polypropylene) membrane is resistant to surfactant-induced breakthrough whilst the GoreTex (PTFE) membrane is not is somewhat surprising. The difference can be easily explained when one takes into account the observation, first made by Bascorn and Singleterry [ 171, that the wetting behavior of PTFE in liquid-liquid systems is significantly different from its behavior in liquidgas systems. The changes in the contact angle, 8wm>for the system water-isopropylbiphenylpolymer (PTFE or polyethylene) as the liquidliquid interfacial tension is varied (by dissolving dinonylsulfonates of sodium, calcium, magnesium and barium at their CMC - in the organic phase) are shown in Fig. 8. At sufficiently low Contact angle eWrn 140 ,
/
I
PTFE Polyethylene -%+
100 80
~_ Preferentially Preferentially
~~
wetted by wetted by
60
Oil-water interfacial tzsion
[dynlcm]
(Increasing concentration of tenside) Fig. 8. Variation of contact angles in the system oil-waterPTFE/polyethylene. The contact angle is measured in the organic phase and its dependence on the concentration oftenside in the system is plotted.
liquid-liquid interfacial tensions there is a reversal in the preferential wettability of PTFE. This phenomenon is not observed with polyethylene. One expects the behavior of a polypropylene surface to resemble that of polyethylene. The dramatic increase in the contact angle of an organic liquid over PTFE in an aqueous-organic, two-liquid, system as the liquid-liquid interfacial tension is decreased has also been reported by Kim and Hariott [ 18 ] - for systems not containing any strongly surface-active components. As Bascom and Singleterry [ 171 have noted, this behavior is the very reverse of the wetting behavior of PTFE and polyethylene surfaces in air. In light of this information it is somewhat surprising that Goto et al. [ 93 have reported the successful operation of a hollow fiber membrane bioreactor using PTFE membranes for the lipase-catalyzed hydrolysis of glycerol trioleate. 3.5. Cuprophan (regenerated cellulose) membranes The Cuprophan membranes were found to be completely resistant to breakthrough, for all the surfactant concentrations tested, up to pressures as high as 2000 mbar. This high resistance to breakthrough is consistent with the observation by Pronk et al. [ 71 that reactors with such membranes could be successfully operated with a transmembrane pressure drop of 1 bar and is not unexpected when one takes into account the fact that these membranes are estimated to have pores with an average diameter of 2 nm [ 193. The increase in acid concentration during ester hydrolysis in a membrane reactor with a Cuprophan membrane is shown in the time-course curve of Fig. 4. This reactor was operated without any breakthrough effects for pressures as high as 400 mbar. However, the maximum interfacial reaction rate obtained was the lowest amongst all the membrane reactors despite the fact that the enzyme loading was the highest with Cuprophan membranes (see Table 2 ) . This could be due to the dense structure of these membranes, which are made by a regeneration process [20], and their low porosity.
A.M. Vaidya et al. /Journal ofMembrane Science 97 (1994) 13-26
3.6. RC70PP (deacetylated cellulose acetate) membranes The breakthrough pressure curve for RC70PP membranes which have MWCO of 10,000 Da only twice as much as Cuprophan - is shown in Fig. 6. From this figure, and the time course curve of Fig. 3, it can be seen that these membranes have an unexpectedly low breakthrough resistance. It is conceivable that the washing of the membranes prior to their use in the breakthrough experiment was insufficient to remove all traces of the humectant solution in which they were supplied. However, it is more probable that these membranes were susceptible to breakthrough since they were regenerated by first making an RO-type cellulose acetate membrane, by phase inversion, and then deacetylating it. It is unlikely that this process would remove all the acetate groups from the membrane surface. One expects such a partially deacetylated cellulose surface to have a significantly more hydrophobic character than pure cellulose - as can be seen from the surface tension data of Saito and Yabe [21], reproduced in Table 3, an increase in the degree of acetylation of cellulose results in a rapid decline in the polar component of its surface tension. This is accompanied by a simultaneous albeit small - increase in the non-polar component of the surface tension. The resulting increase in hydrophobicity would render a partially deacetylated cellulose membrane more susceptible to wetting by the organic phase. Table 3 Relationship between degree of acetylation of cellulose and the polar (1) and dispersive (s) components of its surface tension Degree of acetylation (%)
0 38.4 39.4 42.3 43.8
Surface tension and its component ( dyn/cm)q YIII
I Y*
Y:,
58.6 47 46.7 45.7 44.2
32.7 19 18.3 16.3 14.7
25.9 28 28.4 29.3 29.5
a Surface tension data from Saito and Yabe [ 2 11.
23
Breakthrough pressure tests on these membranes were also carried out with the non-wetting liquid placed on the open side of the membrane. As can be seen from Fig. 5, breakthrough in this mode of operation occurs at pressures significantly lower than when the non-wetting liquid (the organic phase) is placed on the skinned side of the membrane. In this configuration the liquid-liquid interface - which is initially located in the spongy matrix of the membrane will be set in motion at a relatively low pressure. As Rose and Heins [22] have noted, the phenomena which occur once motion of this interface begins are extremely complicated. We have not attempted to address these problems.
4. Conclusions A highly sensitive experimental technique for measuring breakthrough pressures -the pressure at which a liquid wetting the pores of a membrane is displaced by another, immiscible, nonwetting liquid - has been described in this paper. The change in breakthrough pressures for the system ethyl laurate-lauric acido-water as the composition of the tenside is varied has been reported for a number of membranes. An attempt has been made to explain the mechanisms which lead to the reported breakthrough behavior. The breakthrough behavior of the membranes has also been verified - where possible - by using them as separators in two-phase reactors for the lipase-catalyzed hydrolysis of ethyl laurate. On the basis of these discussions one can deduce the following guidelines for the selection of membranes for use in two-phase membrane bioreactors: 1. Highly hydrophilic ultrafiltration membranes can be used in two phase membrane bioreactors without incurring the risk of breakthrough - this is observed with the Cuprophan membranes used in this work. 2. Highly retentive ultrafiltration membranes can be successfully used in such reactors, even when they are amphiphilic, since the very small size of pores in such membranes assists in increasing the breakthrough pressure. The results
24
A.M. Vaidya et al. /Journal ofMembrane Science 97 (1994) 13-26
with the BM5 membrane support this conclusion. 3. Amphiphilic membranes can be used with the organic phase wetting the membrane since the liquid-liquid interface is stabilized in this mode of operation as a result of restructuring effects at the membrane-liquid interfaces. 4. Microporous polypropylene membranes too are resistant to surfactant-induced breakthrough when they are tested in a simple breakthrough pressure experiment. However, caution must be exercised in operating reactors with such membranes since there appear to be anomalies in their breakthrough behavior in such devices. 5. PTFE membranes would always be a poor choice as phase separators for a two-liquid phase reaction system. It must be noted that it may be necessary to reassess the validity of these guidelines when an enzyme with a different mode of action - bulk aqueous, as opposed to interfacial - is being used. It is instructive to compare these conclusions with the rules of thumb - based on purely theoretical discussions - for membrane selection presented by us previously [ lo]. It will be seen that all of the guidelines reported above are in general agreement with the rules presented therein. However, as we have noted there, a simple breakthrough pressure experiment can only be used as a guide to membrane selection since the presence of the biocatalyst and the other reaction components could have a profound effect on the behavior of the membrane in a membrane reactor. The importance of this limitation is best demonstrated by the anomalies in the breakthrough behavior of Celgard (polypropylene) membranes reported in this paper. A useful further analysis of breakthrough effects and interface stability in two-phase membrane bioreactors can be done if one carries out a reaction, in a reactor with amphiphilic or polypropylene membranes, using the same substrate but with two different enzymes - one interfacially active and the other acting in the bulk of the aqueous phase. The results of such an experimental study will be presented in a separate paper.
Appendix The sequence of events which precede the occurrence of breakthrough in a two-liquid phase membrane device is as follows: 1. The membrane, wetted with the appropriate liquid, is brought into contact with the non-wetting liquid. At this stage, there is a ‘pool’ of the wetting liquid covering the surface of the membrane - as shown in Fig. 9A. There is no contact between the non-wetting liquid and the membrane. Consequently, any pressure that is applied to the non-wetting liquid will result in drainage of liquid from the ‘pool’. This is not a breakthrough phenomenon, merely the flow of liquid under applied pressure. With a sufficiently sensitive device, such as the breakthrough pressure test cell described in this paper, this flow can be observed.
rl
Non-wetting
Phase
Membrane
(Cl Fig. 9. Sequence of events preceding the occurrence of breakthrough in a two-liquid phase membrane device. (A) Wetted membrane in contact with the non-wetting liquid. AP=O; Ab= 0. (B) Three-phase line of contact formed after drainage of wetting liquid from membrane surface O< AP< AP,. (C) Liquid-liquid interface in motion after the occurrence of breakthrough. AP> AP,.
A.M. Vaidya et al. /Journal of Membrane Science 97 (1994) 13-26
2. Eventually, a stage is reached when the coverage of the membrane surface by the wetting liquid is reduced to a thin wetting film. Capillary effects now come into play and a three phase line of contact is formed - as shown in Fig. 9B. Breakthrough of the non-wetting liquid will not occur as long as the applied pressure is less than the breakthrough pressure - which can be calculated using the relations described elsewhere
[lOI* 3. When the applied pressure becomes equal to the breakthrough pressure, movement of the liquid-liquid interface will commence. This results in conflicting demands being made on the shape of the interface. The thermodynamic requirement on the contact angle prescribes a certain interfacial shape. However, the no-slip boundary condition on the moving liquid at the pore walls requires an entirely different shape. This conflict results in rotational flows being setup which cause the various interfacial tensions, a, to change from their, ‘old’, static values. This in turn causes a change in the contact angle, f3,,, - as shown in Fig. 9C. One can no longer calculate a breakthrough pressure with the aid of an equilibrium relationship, such as the LaplaceYoung equation [ 10 1.
List of symbols
AP applied pressure (Pa) AP, breakthrough pressure (Pa) pore radius at three phase line of contact r (m) surface tension of partially acetylated cellulose (dyn/cm ) I9wm angle of contact between wetting liquid and membrane interfacial tension at membrane-liquid in0 terface (N/m) 6cJ difference between interfacial tensions at the membrane-non-wetting liquid and membrane-wetting liquid interfaces (N/ m)
Y
25
Superscripts 1 P nP S
polar component polar, or hydrophilic, mode of operation of an amphiphilic membrane non-polar, or hydrophobic, mode of operation of an amphiphilic membrane dispersive component
Subscripts a m n 0 W
aqueous phase membrane non-wetting phase organic phase wetting phase
References [ 1 ] M.M. Hoq, T. Yamane, S. Shim& ST. Funada and S. Ishida, Continuous synthesis of glycerides by lipase in a microporous membrane bioreactor, J. Am. Oil Chem. Sot., 61 (1984) 776. [ 2lM.M. Hoq, T. Yamane, S. Shimizu, T. Funada and S. Ishida, Continuous hydrolysis of olive oil by hpase in microporous hydrophobic membrane bioreactor, J. Am. Oil Chem. Sot., 62 (1985) 1016. [ 31 M.M. Hoq, M. Koike, T. Yamane and S. Shimizu, Continuous hydrolysis of olive oil by lipase in microporous hydrophobic hollow fiber bioreactor, Agric. Biol. Chem., 49 (1985) 3171. [4] M. Rucka, B. Turkiewicz and J.S. Zuk, Polymeric membranes for lipase immobilization. J. Am. Oil Chem. Sot., 67 ( 1990) 887. [ 5]M. Rucka, B. Turkiewicz, Ultrafiltration membranes as carriers for lipase immobilization, Enzym. Microb. Technol., 12 (1990) 52. [ 61 M. Rucka, B. Turkiewicz, Hydrolysis of sunflower oil by means of hydrophobic membrane with lipolytic activity, Biotechnol. Lett., 11 (1989) 167. [ 71 W. Prank, P.J.A.M. Kerkhof, C. van Helden and K. van? Riet, The hydrolysis of triglycerides by immobilized lipase in a hydrophilic membrane reactor, Biotechnol. Bioeng., 32 (1988) 512. [ 81 W. Pronk, G. Boswinkel and K. van? Riet, Parameters influencing hydrolysis kinetics of lipase in a hydrophilic membrane bioreactor, Enzym. Microb. Technol., 14 (1992) 214. [ 9]M. Goto, M. Goto, F. Nakashio, K. Yoshizuka and K. Inoue, Hydrolysis of triolein by lipase in a hollow fiber reactor, J. Membrane Sci., 74 ( 1992) 207. [lo] A.M. Vaidya, G. Bell and P.J. Halling., Aqueous-organic membrane bioreactors. Part 1. A guide to membrane selection, J. Membrane Sci., 7 1 ( 1992) 139.
26
[ 11 ]C.J. Tompkins,
A.M. Vaidya et al. /Journal of Membrane Science 9 7 (1994) 13-26
A.S. Michaels and SW. Peretti, Removal ofpnitrophenol from aqueous solution by membrane-supported solvent extraction, J. Membrane Sci., 75 (1992) 277. [ 12]R. Prasad: S. Khare, A. Sengupta and K.K. Sirkar, Novel liquid-in-pore configurations in membrane solvent extraction, Am. Inst. Chem. Eng. J., 36 ( 1990) 1592. [ 13]R. Prasad and K.K. Sirkar, Solvent extraction with microporous hydrophilic and composite membranes, Am. Inst. Chem. Eng. J., 33 (1987) 1057. [ 14]E. Ruckenstein and S.V. Gourisankar, Surface restructuring of polymeric solids and its effects on the stability ofthe polymer-water interface, J. Colloid Interface Sci., 109 (1986) 557. [ 15lD.R. Absolom and A.W. Neumann, Modification of substrate surface properties through protein adsorption, Colloids Surf., 30 ( 1988) 25. [ 16lC.G.P.H. Schroen, M.C. Wijers, M.A. Cohen-Stuart, A. van der Padt and K. van? Riet. Membrane modilication to avoid wettability changes due to protein adsorp-
tion in an emulsion membrane bioreactor, J. Membrane Sci., 80 (1993) 265. [ 171W.D. Bascom and C.R. Singleterry, The effect of polarnon-nolar solutes on the water wettabilitv of solid surfacessubmerged in oil, J. Phys. Chem., 68 ( 1962) 236. 181 B.S. Kim and P. Harriott, Critical entry pressure for liquids in hydrophobic membranes, J. Colloid Interface Sci., 115 (1987) 1. 191 K. Sakai, H. Chiba and A. Naitoh, Determination of the pore radius of regenerated cellulose membranes by a dyeing technique, J. Membrane Sci., 37 ( 1988) 101. 20lR.E. Kesting., Synthetic Polymeric Membranes - A Structural Perspective, Wiley, New York, NY, 1985, pp. 302-303. 2 I] M. Saito and A. Yabe, Dispersion and polar force components of surface tension of some polymerlilms, Textile Res. J., 1 (1983) 154. 221 W. Rose and R.H. Heins, Moving interfaces and contact angle rate dependency, J. Colloid Interface Sci., 17 (1962) 39.
Journal of Membrane Science 97 ( 1994) 13-26
Aqueous-organic membrane bioreactors Part II. Breakthrough pressure measurement A.M. Vaidya”,*, G. Bell”, P.J. Hallingb ‘Department of Chemical Engineering, StrathclydeUniversity,75, Montrose Street, GlasgowGI IXJ, UK bDepartmentof Bioscience and Biotechnology, StrathclydeUniversity,George Street, GlasgowGl 1Xw UK
Received 28 March 1994; accepted in revised form 1July 1994
Abstract The effect of membrane type - structure and wettability - on the operation of two-phase, aqueous-organic, membrane bioreactors has been studied. The influence of surfactants on membrane wettability is reported. A simple, but highly sensitive, technique for the measurement of breakthrough pressures is described. Experimental measurements of the variation in break through pressures as the concentration of tenside in the system was changed are reported. On the basis of the results from these measurements it is concluded that: (i) hydrophilic and, highly retentive, amphiphilic ultrafiltration membranes may be used to operate two-phase bioreactors, (ii) amphiphilic microfiltration membranes should never be used in such reactors and (iii) PTFE membranes would always be a poor choice for use in such devices because they always have a low breakthrough resistance in two-liquid systems - breakthrough pressures as low as 100 mbar were observed for the system ethyl laurate-water-PTFE, which contains no surface-active component. It is shown that these results are in general agreement with rules of thumb for the selection of membranes, presented earlier. The influence of membrane history on its wetting behavior due to effects such as polymer surface restructuring - is highlighted. The limits on the utility of simple breakthrough pressure tests in determining suitable membranes, for use in two-phase bioreactors, owing to possible complications resulting from the exact mechanism of enzyme action is pointed out. Keywords:Two-phase biocatalysis; Membrane bioreactor; Surfactant-induced breakthrough; Membrane selection
1. Introduction
Membrane reactors using hydrophobic as well as hydrophilic polymeric membranes have been used to carry out two-phase, aqueous-organic, biocatalytic reactions [ l-9 1. In such devices the membrane, which is selectively wetted by one of the two phases, separates the aqueous phase from an immiscible organic phase. This is accomplished by applying a slight pressure to the non-
wetting liquid in order to create an interface between the two liquids in the plane of the membrane. If the applied pressure is made sufflciently large one can cause displacement of the wetting liquid from the pores in the membrane which is followed by flow of the non-wetting liquid through the membrane. This effect is known as breakthrough - and the pressure at which it occurs is known as the breakthrough pressure. The sequence of events that precedes breakthrough is described in detail in the Appendix.
* Corresponding author. 0376-7388/94/$07.00 0 1994 Elsevier Science B.V. All rights reserved SSDl0376-7388(94)00144-8
14
A.M. Vaidya et al. /Journal
ofMembrane
The occurrence of breakthrough makes it impossible to use the reactor. A large number of two-phase biocatalytic reactions involve a surface-active product and/or reactant. The presence of such materials can lead to difficulties in reactor operation because of tenside-induced changes in the wetting behavior of the membrane - which in turn can lead to a decrease in the breakthrough pressure. The theory of such effects has been described in some detail [ lo]. Evidently, some care must be exercised in the selection of membranes for use in such reactors. A few rules of thumb, based on theoretical considerations, to assist this selection process have been given earlier [ lo]. The process of the selection of membranes for use in such reactors would be aided if one could devise a simple experiment to study the breakthrough behavior of the reaction system of interest. A few measurements of breakthrough pressure have been done to assist the evaluation of membranes for use in non-dispersive solvent extraction [ ll13 1. However, the systems studied are considerably simpler since they do not involve the strongly surface-active solutes which are commonly encountered in two-phase biocatalysis. Further, these measurements have been reported at only one concentration of the solute. In this paper a more sensitive experimental technique for the selection of suitable membranes will be described. The breakthrough effects in these experiments will be compared with those observed when the membranes are used in reactors for the lipase-catalyzed hydrolysis of ethyl laurate.
2. Experimental 2. I. Breakthrough pressure experiments Most of the breakthrough pressure experiments were done with pure water as the aqueous phase. However, a few experiments were done with the buffered aqueous phase (0.1 M NaH,POJNaOH, pH 8.0 at 25’C) - used for enzyme immobilization and for carrying out the reaction - since the distribution of the surfactant between the two phases could to be significantly
Science 97 (1994) 13-26
affected by the presence of the buffer species which in turn may have a bearing on the magnitude of the observed breakthrough pressures. Laurie acid ethyl ester (L4625, Sigma Chemical Co., Poole, UK) and the appropriate aqueous phase - buffer or single distilled water - were used as test liquids. The variation in surfactant composition during the course of the lipase-catalyzed hydrolysis of the ester was simulated by shaking different masses of lauric acid (L4250, Sigma) with known quantities of ester and water for 24 h at room temperature and then separating the aqueous and organic phases. To determine acid dissolved in the aqueous phase, a small amount of ester - 5 0.5 g - was added to a large volume - over 20 ml - of the aqueous phase and the acid was extracted into the ester by shaking the mixture for 60 min. The acid enriched ester was extracted from the resulting two-phase mixture by shaking it with an equal volume of hexane (H9379, Sigma) and then separating the two phases. The concentration of acid in the ester was determined on a gas chromatograph - using the technique described later in this section. The concentration of acid in the aqueous phase water as well as buffer - was found to be negligible. The types of membranes used and their properties are summarized in Table 1. The polypropylene and the asymmetric deacetylated cellulose acetate membranes were supplied in the form of A4 sheets. The remaining membranes were supplied in the form of 47 mm discs. Discs of the appropriate size - 25 mm for the breakthrough pressure tests and 47 mm for the reactor experiments - were punched out, where necessary. In order to obtain rapid detection of breakthrough it is vital to minimize the dead volume under the membrane. This is difficult to achieve if a large test area is used, since the membrane must then be supported in order to enable it to withstand the applied pressure. Polysulfone grids, commonly used as supports in ultrafiltration cells, were initially used for this purpose. However, it was found that holdup of liquid in the interstices of the support lead to a considerable delay in the detection of breakthrough. (A delay is also likely to occur if one relies on the obser-
A.M. Vaidya et al. /Journal ofMembraneScience 97 (1994) 13-26 Table I Membranes used in breakthrough
15
pressure tests
Product code
Supplier
Material
Wetting characteristic
Type (UForMF)
Pore size/MWCO
Cuprophan RC70PP Nylaflo BM-5 BM-1000 Celgard 2400d Goretex’
Enka Dow Gelman Sciences Berghof Berghof Hoechst Celanase W.L. Gore
Cellulose” Cellulose’ Nylon-66 Meta-aramid Meta-aramid Polypropylene PTFE
Hydrophilic Hydrophilic Amphiphilic Amphiphilic Amphiphilic Hydrophobic Hydrophobic
UF UF MF UF UF MF MF
2 nmb 10,000 Da 0.2 ,um 500 Da 10,000 Da 0.2x0.04pm 0.2 ,um
a Dense, renegerated cellulose membranes. b Estimated pore size [ 191 ’ Asymmetric membranes made by deacetylation of cellulose acetate reverse osmosis membranes d These membranes had rectangular slits rather than circular pores. ’ These membranes were thermally bonded onto a polypropylene support.
vation of a droplet of the non-wetting liquid appearing on the surface of a single hollow fiber membrane to obtain an estimate of the breakthrough pressure - which is the technique employed by Tompkins et al. [ 111. The experimental technique employed by Prasad and coworkers [ 12,131 in their breakthrough pressure measurements has not been clearly specified.) An accurate estimate of the breakthrough pressure can be obtained by having a very small area of unsupported membrane, as shown in Fig. 1. Any liquid passing through this small test area can immediately be observed in the 0.5 mm bore glass capillary attached to the base of the test cell. A 25 mm membrane disc - wetted with the appro-
Air pressure
Cell
connector
cover
with cavity for test liquid
10 mm
1 Silicone
rubber
gasket
Fig. 1.Test cell used for breakthrough
pressure experiments.
priate test liquid - was clamped in between the two halves of the test cell. A seal between the lowand high-pressure halves of the test cell was made by the silicone rubber seals embedded in the two parts of the test cell. The second test liquid - the non-wetting liquid - was carefully introduced into the cavity in the upper half of the test cell with the aid of a hypodermic syringe and a tine needle. Air pressure was applied to this liquid through the l/4 BSP - 6 mm tube adaptor shown in the figure. It was found necessary to remove all sharp corners from the test liquid cavity in order to prevent bubbles of residual air from being trapped inside the cell, since their presence was found to lead to spurious breakthrough pressure results. (This problem was more pronounced in experiments with amphiphilic and hydrophobic membranes since the air bubbles displayed a tendency to adhere to the surface of such membranes. On pressurization, the resulting air-organic liquid interface was displaced at extremely low pressures.) The cell was pressurized gradually until continuous flow of liquid in the glass capillary was observed - at which point breakthrough was deemed to have occurred. A precision pressure regulator (Negeretti Valve Co., UK) was used for this purpose and the cell pressure was monitored using a high precision transducer (Penny & Giles Transducers Ltd., UK). All the tests were carried out at 25 ‘C. The total duration of the tests was normally not longer
16
A.M. Vaidya et al. /Journal of Membrane Science 97 (1994) 13-26
than 10 min. However, some of the tests with the Celgard (polypropylene) membranes were carried out over a period of 24 h with each test pressure being maintained for periods up to 12 h. This was done to investigate the possibility of delayed breakthrough effects arising due to gradual adsorption of the surfactant. Before being subjected to breakthrough pressure tests the membranes were pretreated. The nature of the pretreatment depended on the membrane type. 1. Microporous membranes: The membranes were wetted with the appropriate wetting liquid and any excess liquid was wiped off the surface with a lint-free tissue. It was observed that the Nylaflo (microporous nylon-6,6 ) membranes were more easily wetted by the organic phase than by the aqueous phase. 2. Ultrafiltration membranes: The Cuprophan (regenerated cellulose) membranes were thoroughly washed in distilled water before use. The BM5, BM 100 (meta-aramid) and RC70PP (deacetylated cellulose) membranes - the latter were supplied in a 50% aqueous glycerol solution containing sodium hydroxide and propionic acid were first soaked in distilled water overnight. Immediately prior to the test distilled water was filtered through the membrane at a pressure of 1 bar until a filtrate of N 1 ml had been collected. 2.2. Membrane reactor experiments The flat sheet membrane reactor used in all the experiments is shown in Fig. 2. It consisted of two flanged nylon-6,6 shells of circular cross section held together by three M4 bolts threaded through the flanges. A silicone rubber ‘0’ ring prevented leakage of the reactants when the reactor was pressurized. A polysulfone grid was placed in the low pressure half of the reactor to support the membrane. AlCoVar bar magnets (Merck Ltd., UK), encapsulated in a thin film of perfluoropolyether (Fomblin, Italy) were placed in the cylindrical cavities in each half of the reactor. A fine stainless steel wire mesh was placed above the membrane, on the high-pressure side of the reactor, to prevent damage to the membrane by the rotation of the magnet. Two
Fig. 2. Exploded view of flat sheet two-phase membrane bioreactor: ( 1) high pressure half cell, (2) magnetic stirrers, (3) ‘0’ ring, (4) stainless steel mesh, (5) membrane, (6) membrane support, (7) low pressure half cell, (8) header tubes, (9) sampling port.
PEEK (polyether-ether-ketone) header tubes (30 mm length and 1.6 mm bore) were glued to both the half-cells comprising the reactor to permit the introduction of reactants and/or purging of air. A PTFE-faced silicone rubber septum of 6 mm diameter, held in place by a l/4 UNF nut with a 1/ 16 fl hole drilled through it, was used to gain access to the reactant in the high-pressure half of the reactor. The contents in the two halves of the reactor were stirred magnetically after clamping it horizontally in a thermostated water bath (25°C). The reactor had an active membrane area of 4.9 cm*. The precision pressure regulator used in the breakthrough pressure experiments was employed to maintain a constant pressure of compressed air above the non-wetting liquid and the pressure in the head space was monitored by a digital manometer (Platon Flowbits Ltd., UK), 2.3. Enzyme immobilization Solutions of lipase from Candida rugosa (L1754, Sigma) were made up in phosphate buffer (2.7 mg/g). MF membranes: The enzyme was immobilized at the liquid-liquid interface by physical adsorption. The membrane was first wetted with a few drops of the ester and then mounted in the
A.M. Vaidya et al. /Journal
of Membrane Science 97 (1994) 13-26
reactor. The buffered aqueous enzyme solution was then introduced into the high-pressure side of the reactor. After pressurizing the high-pressure side of the reactor, the low-pressure side was filled with the ester. The liquids were left in contact with each other for 1 h after which they were removed from the reactor and the two halves of the reactor were washed with the appropriate liquid-ester or aqueous buffer. The activity of the enzyme solution was measured, before and after immobilization, to obtain an estimate of the quantity of immobilized enzyme. UF membranes: The membrane was pretreated in a manner similar to that described above for the breakthrough pressure tests. The membrane was then mounted in the reactor and 10 ml of distilled water was filtered through it. The filtrate was rejected and 4.5 ml of lipase solution was filtered through the membrane, at a pressure of 1 bar, followed by 8 ml of buffer. These operations were done without stirring. Finally, an additional 2 ml of buffer was filtered with the stirrer in operation. The entire filtrate was collected and assayed for lipase activity. 2.4. Assays The concentration of lauric acid in the organic phase was determined on a gas chromatograph, with 80-100 mesh Chromosorb WHP and 3% OV 1 as the stationary phase packed in a column of internal diameter 3 mm and length 2 m (Phase Separations Ltd., UK), at 180°C. The samples were dried and derivatized with MTBSTFA (Phase Separations) prior to analysis. The activity of the lipase was determined by titrating the butyric acid released during the lipase catalyzed hydrolysis of tributyrin at pH 8 (25°C). This was done on a pHstat (PHM 82, Radiometer, Denmark). The reaction was carried out with a 3% (w/v) emulsion of tributyrin (Aldrich, UK) made in a buffered gum arabic (G9752, Sigma) solution. The latter was made by stirring an excess of gum arabic in the buffer (0.1 M NaH2 PO,/NaOH) for 3 h at 40” C - and then filtering out the excess. A 100 mM aqueous solution of NaOH (Sigma) was used as the titrant and one unit of enzyme was defined as the
17
amount of enzyme required to release butyric acid at an initial rate of 1 pmol/min. The water content of the organic phase was determined by Karl-Fischer titration on a KF 653 coulometer (Metrohm, Switzerland). 2.5. Reactor operation Fresh reactants were loaded into the two halfcells of the reactor - after enzyme immobilization had been completed and the reactor had been washed, as described above - and the non-wetting liquid was pressurized. The contents of the reactor were stirred magnetically and the lipasecatalyzed hydrolysis of ethyl laurate in the buffered aqueous phase was allowed to continue until breakthrough was observed. Breakthrough was detected visually by observing the movement of liquid in a narrow bore silicone rubber tubing attached to one of the two header tubes on the lowpressure half of the reactor (of the two header tubes on the high-pressure side of the reactor, one was used to pressurize the high-pressure half-cell whilst the other was closed using a short length of silicone rubber tubing clamped at its open end). This process is complicated in the case of hydrophilic membranes since the hydrolysis reaction results in a net transfer of the system volume from the organic to the aqueous phase. In such cases it is necessary to collect a well-stirred sample of liquid from the low-pressure half-cell and check for the presence of two distinct phases in order to establish the occurrence of breakthrough. Samples of the organic phase were taken at frequent intervals and fresh ethyl laurate was added to the organic side of the reactor to make up for the resulting depletion. The water content of each of the collected organic phase samples was estimated by Karl-Fischer titration in order to ensure that they did not contain small quantities of finely emulsified aqueous phase. In a separate experiment the variation of the saturation solubility of water in the organic phase as the acid content of the latter is changed was determined. In experiments with the Celgard (polypropylene) membranes, breakthrough was deemed to have occurred when a sample was found to con-
18
A.M. Vaid.vaet al. /Journal ofMembraneScience 97 (1994) 13-26
tain a higher proportion of water than the saturation solubility of water at the measured concentration of acid. This was done in order to get a more accurate estimate of the instant of breakthrough than was offered by the visual observation made during the experiment.
3. Results and discussion A two-phase membrane reactor can be operated with microfiltration (MF) or ultrafiltration (UF) membranes. One has the further choice of using either hydrophilic or hydrophobic membranes. In principle it should also be possible to use an amphiphilic polymer, such as a polyamide, if the adsorption of tenside at the two polymer-liquid interfaces occurs in such a manner as to keep the breakthrough pressure, dP, sufficiently high. The process by which this could happen has been outlined elsewhere [ lo]. The membranes tested in the experiments described in this paper cover the whole spectrum of possibilities. (In this paper the terms hydrophobic and hydrophilic have been used in a more rigorous sense than is the norm. Polypropylene and PTFE membranes have been considered to be hydrophobic and regenerated cellulose membranes have been treated as being hydrophilic. All other commercially available membranes, such as polysulfones, polyamides, etc., are - in the strict sense of the term - amphiphilic since they are made from polymers containing both polar and non-polar groups. ) The performance of all membranes which could be used in membrane reactors with some measure of success is given in Table 2. The timecourse curves for reactors operated with these membranes are shown in Figs. 3 and 4. The results from the breakthrough pressure tests on these membranes are shown in Figs. 5, 6 and 7. The breakthrough pressures reported in these figures are an average value obtained from a large number of experiments. The breakthrough pressure results reported here were obtained with pure water as the aqueous phase. In the presence of a pH 8 buffer there is an increase in the concentration of acid in the aqueous phase due to
some ionization of the fatty acid. However, this was found to have no significant effect on breakthrough pressures. 3.1. Nylajlo (microporous nylon-6,6) membranes Breakthrough pressure results for these membranes were obtained with the membranes used both as hydrophilic and as hydrophobic membranes. The breakthrough pressure curve in the hydrophilic mode is shown in Fig. 5. In the hydrophobic mode the breakthrough pressure was found to be constant at 280 mbar. An asymmetry in breakthrough pressures depending on the mode of operation - hydrophilic or hydrophobic - has been previously reported, without any explanations, by Prasad et al. [ 121 for a microporous membrane made from an unspecified polyamide. The existence of a positive breakthrough pressure in both modes of operation is extremely surprising. An examination of the LaplaceYoung equation [ lo] suggests that the displacement of one of the two liquids must occur spontaneously.
In other words, one of the two breakthrough pressures must be negative. It is possible to account for the occurrence of positive breakthrough pressures in both the modes of operation if one invokes the existence of restructuring effects at the surface of an amphiphilic polymer membrane. In Eq. ( 1 ), the difference da= a,, -a,,
(2)
must remain unchanged by the surfactant concentrations in the system when the membrane is used in its hydrophobic mode. On the other hand, in the hydrophilic mode, 60 undergoes a significant decrease, leading to the breakthrough pressure curve of Fig. 5. If one invokes restructuring phenomena, the explanation for this behavior of 60 is as follows. As Ruckenstein and Gourisankar [ 141 have noted, restructuring effects will lead to the emergence of buried polar groups from the bulk
A.M. Vaidya et al. /Journal of Membrane Science 97 (1994) 13-26
19
Table 2 Enzyme loadings, operating pressures and initial reaction rates of membrane reactors. The acid concentration at which breakthrough was first observed is also given. The reaction rate was found to decrease rapidly from its initial value for the Celgard 2400, RC70PP and BM5 membrane reactors. With the others it was constant at its initial value Membrane
Total adsorbed/ retained enzyme (Units)
Enzyme loading (Units/m*)
1.7 6.7 9.1 7.5 4.5
3,460 13,650 18,540 15,300 9,100
Celgard 2400 RC’IOPP Cuprophan BM5 BMlOO
Operating pressure (mbar)
Initial reaction rate
15 100 250-400” 250-400” 200
Interfacial (molhm’)
Enzyme (mol/s Kat )
0.05 0.31 0.02 0.08 0.04
0.22 0.37 0.02 0.09 0.06
Acid concentration at breakthrough (pmol/g)
No breakthrough 93.5 No breakthrough No breakthrough 116
a The pressure was increased to 400 mbar towards the end of the experiment.
Acid Concentration
Acid Concentration
[pmol/g]
[pmollg]
100
50
0’ 0
I
’
50
’
’
100
’
’
150
’
’
200
’
250
Time [min] Fig. 3. Time-course curve for the lipase-catalyzed hydrolysis of ethyl laurate in a membrane reactor with a deacetylated cellulose acetate membrane. The reactor was operated with the skinned side of the membrane facing the non-wetting, organic phase. Other experimental conditions are as shown in Table 2.
of the membrane polymer to the membraneaqueous interface. They have demonstrated that these effects are quite rapid (occurring within a few seconds), whilst the reverse effect - the subsidence of polar groups when the surface is exposed to a non-polar environment - is relatively slow (requiring a period of several days). In the light of their observations one can deduce the existence of four membrane-liquid interfacial ten-
0
0
500
1.ooo
1.500
2.;00
Time [min] Fig. 4. Time-course curves for the lipase-catalyzed hydrolysis of ethyl laurate. Both the meta-aramid membranes were used with their skinned side facing the non-wetting, organic, phase. Experimental conditions are shown in Table 2.
sions for an amphiphilic membrane. In the polar mode of operation the interfacial tensions are 0 am and @A,,- between the aqueous and organic phases and the membrane, respectively. Whilst the former will be low due to restructuring effects, the latter will be higher and there will be a tendency for the tenside to adsorb at the organic-membrane interface. If the extent of this adsorption displays a concentration dependence it follows that
A.M. Vaidyaet al. /Journal of Membrane Science 97 (1994) 13-26
20 Breakthrough
Pressure
[mbar]
Nylaflo - Hydrophilic
20’
0
100
200
300
RC70PP
400
Acid concentration
- Open
500
600
700
[pmol/g]
Fig. 5. Dependence of breakthrough pressure on acid concentration for the systemethyl laurate-lauric acid-water.
W=a&
-a*am
(3)
will decrease with increasing concentrations of the tenside. On the other hand, in the non-polar mode of operation the interfacial tension o,“&will have a relatively low value and the surfactant will have only a very limited propensity to adsorb at this interface. The interfacial tension at the aqueous-membrane interface, a:,$, will exist only momentarily since it will rapidly change to ai, as a result of restructuring effects. One would thus have two constant, and low, liquid-membrane interfacial tensions with the result that &P = crtm - a;&
(4)
will be constant, but positive. In offering these explanations we have assumed that reverse restructuring, i.e., the subsidence of polar groups in a non-polar environment is a relatively slow process - considerably slower than the adsorption of surfactant from the non-polar organic phase. We were unable to operate membrane reactors using this type of membrane in either of its modes of wetting since the maintenance of a stable liquid-liquid interface was found to be impossible. This result is not unexpected for the hydrophilic
mode of operation since the generation of fatty acid in the reactor would rapidly lead to a decrease in the breakthrough pressure. The magnitude of this effect may be considerably more severe than indicated by the breakthrough pressure curve of Fig. 5 since the concentration of the surfactant is likely to be significantly higher in the immediate vicinity of the membrane - where it is being produced during the operation of a membrane reactor. In light of the fact that these membranes gave a relatively high, and constant, breakthrough pressure in their hydrophobic mode of operation one would expect that it should be possible to operate a membrane reactor in this mode. However, it was found that there was virtually instantaneous breakthrough of the aqueous phase - even when the pressure applied to the latter was as low as 20 mbar. This effect could be caused by the formation of a composite hydrophilic membrane during the enzyme immobilization step of reactor operation. Absolom and Newmann [ 15 ] have noted such an effect in their experiments in which a number of proteins were adsorbed on to a range of polymeric surfaces. Schroen et al. [ 161 have also commented on the modification of membrane wettability induced by proteins.
3.2. Berghof (meta-aramid) membranes
The breakthrough pressure curve for the BMlOO membrane, shown in Fig. 6, used in its hydrophilic mode follow the same trend as the Nylaflo membranes in their hydrophilic mode. The breakthrough pressures are higher since these UF membranes have a considerably smaller pore size. The more retentive BM5 membranes were found to be completely resistant to breakthrough, at all the surfactant concentrations tested, for pressures as high as 2000 mbar. These membranes were not tested in their hydrophobic mode. The increase in surfactant concentration with time during the operation of reactors with these membranes is shown in Fig. 4. As in the breakthrough pressure experiment, the BM5 mem-
A.M. Vaidya et al. /Journal ofMembrane Science 97 (I 994) 13-26
Breakthrough 300 250
Pressure [mbar]
r c
200 150 100 50
t
OL 0
100
200
300
400
Acid Concentration
500
600
700
[pmollg]
Fig. 6. Dependence of breakthrough pressure on acid concentration for the systemethyl laurate-lauric acid-water.
brane was found to be completely resistant to breakthrough when operated with a pressure of 250 mbar on the aqueous phase. A maximum interfacial reaction rate of 0.083 mol/h m2 was obtained. Breakthrough of the aqueous phase through the BMlOO membrane occurred at a lauric acid concentration of 116 ,umol/g in the bulk of the organic phase.
21
laflo membranes, it is likely that the membrane was rendered more easily wettable by the adsorption of lipase at the interface. This improved wettability, combined with the applied pressure, could have resulted in the observed occurrence of breakthrough. In an attempt to verify this mechanism, a breakthrough pressure experiment was conducted with a Celgard membrane which had been subjected to enzyme adsorption -by allowing the ester-wet membrane to remain in contact with an aqueous lipase solution (a 1 mg/ml solution of the same lipase as was used for reactor operation) for 1 h. However, it was still found to be resistant to breakthrough for pressures as high as 1200 mbar. We have been unable to find a satisfactory explanation for these discrepancies. 3.4. GoreTex (PTFE) membranes The breakthrough pressure curve for a GoreTex membrane (pore size 0.2 pm) is shown in Fig. 7. The breakthrough behavior of these membranes was found to be different from that of the other membranes, described above. Some displacement of wetting liquid into the capillary was observed at pressures below the breakBreakthrough
Pressure [mbar]
3.3. Celgard (polypropylene) membranes The Celgard membranes were found to be resistant to breakthrough of the aqueous phase up to pressures as high as 2 100 mbar. (This was found to be the case even when the membrane was exposed to the surfactant for an extended period of time - as was done in some of the experiments.) This is in sharp contradiction with the observation by Hoq et al. [ 1 ] who found that during reactor operation with Juragard, polypropylene, membranes - similar in structure to Celgard - the pressure on the non-wetting, aqueous, phase had to be kept below 20 mbar. In our own reactor experiments with Celgard membranes - Fig. 4 - breakthrough was found to occur when the pressure applied to the aqueous phase was greater than 15 mbar. As with the Ny-
8o I 70 60
I 100
1
I 200
I
1 300
Acid concentration
I 400
, 500
[pmollg]
Fig. 7. Breakthrough behavior of GoreTex membranes for the system ethyl laurate-lauric acid-water.
A.M. Vaidya et al. /Journal of Membrane Science 97 (1994) 13-26
22
through pressure. The amount of displaced liquid increased with increasing pressure. However, continuous flow only occurred when the breakthrough pressure was reached. It was found impossible to operate membrane reactors with these membranes since breakthrough of the aqueous phase invariably occurred very early during their operation. The fact that the Celgard 2400 (polypropylene) membrane is resistant to surfactant-induced breakthrough whilst the GoreTex (PTFE) membrane is not is somewhat surprising. The difference can be easily explained when one takes into account the observation, first made by Bascorn and Singleterry [ 171, that the wetting behavior of PTFE in liquid-liquid systems is significantly different from its behavior in liquidgas systems. The changes in the contact angle, 8wm>for the system water-isopropylbiphenylpolymer (PTFE or polyethylene) as the liquidliquid interfacial tension is varied (by dissolving dinonylsulfonates of sodium, calcium, magnesium and barium at their CMC - in the organic phase) are shown in Fig. 8. At sufficiently low Contact angle eWrn 140 ,
/
I
PTFE Polyethylene -%+
100 80
~_ Preferentially Preferentially
~~
wetted by wetted by
60
Oil-water interfacial tzsion
[dynlcm]
(Increasing concentration of tenside) Fig. 8. Variation of contact angles in the system oil-waterPTFE/polyethylene. The contact angle is measured in the organic phase and its dependence on the concentration oftenside in the system is plotted.
liquid-liquid interfacial tensions there is a reversal in the preferential wettability of PTFE. This phenomenon is not observed with polyethylene. One expects the behavior of a polypropylene surface to resemble that of polyethylene. The dramatic increase in the contact angle of an organic liquid over PTFE in an aqueous-organic, two-liquid, system as the liquid-liquid interfacial tension is decreased has also been reported by Kim and Hariott [ 18 ] - for systems not containing any strongly surface-active components. As Bascom and Singleterry [ 171 have noted, this behavior is the very reverse of the wetting behavior of PTFE and polyethylene surfaces in air. In light of this information it is somewhat surprising that Goto et al. [ 93 have reported the successful operation of a hollow fiber membrane bioreactor using PTFE membranes for the lipase-catalyzed hydrolysis of glycerol trioleate. 3.5. Cuprophan (regenerated cellulose) membranes The Cuprophan membranes were found to be completely resistant to breakthrough, for all the surfactant concentrations tested, up to pressures as high as 2000 mbar. This high resistance to breakthrough is consistent with the observation by Pronk et al. [ 71 that reactors with such membranes could be successfully operated with a transmembrane pressure drop of 1 bar and is not unexpected when one takes into account the fact that these membranes are estimated to have pores with an average diameter of 2 nm [ 193. The increase in acid concentration during ester hydrolysis in a membrane reactor with a Cuprophan membrane is shown in the time-course curve of Fig. 4. This reactor was operated without any breakthrough effects for pressures as high as 400 mbar. However, the maximum interfacial reaction rate obtained was the lowest amongst all the membrane reactors despite the fact that the enzyme loading was the highest with Cuprophan membranes (see Table 2 ) . This could be due to the dense structure of these membranes, which are made by a regeneration process [20], and their low porosity.
A.M. Vaidya et al. /Journal ofMembrane Science 97 (1994) 13-26
3.6. RC70PP (deacetylated cellulose acetate) membranes The breakthrough pressure curve for RC70PP membranes which have MWCO of 10,000 Da only twice as much as Cuprophan - is shown in Fig. 6. From this figure, and the time course curve of Fig. 3, it can be seen that these membranes have an unexpectedly low breakthrough resistance. It is conceivable that the washing of the membranes prior to their use in the breakthrough experiment was insufficient to remove all traces of the humectant solution in which they were supplied. However, it is more probable that these membranes were susceptible to breakthrough since they were regenerated by first making an RO-type cellulose acetate membrane, by phase inversion, and then deacetylating it. It is unlikely that this process would remove all the acetate groups from the membrane surface. One expects such a partially deacetylated cellulose surface to have a significantly more hydrophobic character than pure cellulose - as can be seen from the surface tension data of Saito and Yabe [21], reproduced in Table 3, an increase in the degree of acetylation of cellulose results in a rapid decline in the polar component of its surface tension. This is accompanied by a simultaneous albeit small - increase in the non-polar component of the surface tension. The resulting increase in hydrophobicity would render a partially deacetylated cellulose membrane more susceptible to wetting by the organic phase. Table 3 Relationship between degree of acetylation of cellulose and the polar (1) and dispersive (s) components of its surface tension Degree of acetylation (%)
0 38.4 39.4 42.3 43.8
Surface tension and its component ( dyn/cm)q YIII
I Y*
Y:,
58.6 47 46.7 45.7 44.2
32.7 19 18.3 16.3 14.7
25.9 28 28.4 29.3 29.5
a Surface tension data from Saito and Yabe [ 2 11.
23
Breakthrough pressure tests on these membranes were also carried out with the non-wetting liquid placed on the open side of the membrane. As can be seen from Fig. 5, breakthrough in this mode of operation occurs at pressures significantly lower than when the non-wetting liquid (the organic phase) is placed on the skinned side of the membrane. In this configuration the liquid-liquid interface - which is initially located in the spongy matrix of the membrane will be set in motion at a relatively low pressure. As Rose and Heins [22] have noted, the phenomena which occur once motion of this interface begins are extremely complicated. We have not attempted to address these problems.
4. Conclusions A highly sensitive experimental technique for measuring breakthrough pressures -the pressure at which a liquid wetting the pores of a membrane is displaced by another, immiscible, nonwetting liquid - has been described in this paper. The change in breakthrough pressures for the system ethyl laurate-lauric acido-water as the composition of the tenside is varied has been reported for a number of membranes. An attempt has been made to explain the mechanisms which lead to the reported breakthrough behavior. The breakthrough behavior of the membranes has also been verified - where possible - by using them as separators in two-phase reactors for the lipase-catalyzed hydrolysis of ethyl laurate. On the basis of these discussions one can deduce the following guidelines for the selection of membranes for use in two-phase membrane bioreactors: 1. Highly hydrophilic ultrafiltration membranes can be used in two phase membrane bioreactors without incurring the risk of breakthrough - this is observed with the Cuprophan membranes used in this work. 2. Highly retentive ultrafiltration membranes can be successfully used in such reactors, even when they are amphiphilic, since the very small size of pores in such membranes assists in increasing the breakthrough pressure. The results
24
A.M. Vaidya et al. /Journal ofMembrane Science 97 (1994) 13-26
with the BM5 membrane support this conclusion. 3. Amphiphilic membranes can be used with the organic phase wetting the membrane since the liquid-liquid interface is stabilized in this mode of operation as a result of restructuring effects at the membrane-liquid interfaces. 4. Microporous polypropylene membranes too are resistant to surfactant-induced breakthrough when they are tested in a simple breakthrough pressure experiment. However, caution must be exercised in operating reactors with such membranes since there appear to be anomalies in their breakthrough behavior in such devices. 5. PTFE membranes would always be a poor choice as phase separators for a two-liquid phase reaction system. It must be noted that it may be necessary to reassess the validity of these guidelines when an enzyme with a different mode of action - bulk aqueous, as opposed to interfacial - is being used. It is instructive to compare these conclusions with the rules of thumb - based on purely theoretical discussions - for membrane selection presented by us previously [ lo]. It will be seen that all of the guidelines reported above are in general agreement with the rules presented therein. However, as we have noted there, a simple breakthrough pressure experiment can only be used as a guide to membrane selection since the presence of the biocatalyst and the other reaction components could have a profound effect on the behavior of the membrane in a membrane reactor. The importance of this limitation is best demonstrated by the anomalies in the breakthrough behavior of Celgard (polypropylene) membranes reported in this paper. A useful further analysis of breakthrough effects and interface stability in two-phase membrane bioreactors can be done if one carries out a reaction, in a reactor with amphiphilic or polypropylene membranes, using the same substrate but with two different enzymes - one interfacially active and the other acting in the bulk of the aqueous phase. The results of such an experimental study will be presented in a separate paper.
Appendix The sequence of events which precede the occurrence of breakthrough in a two-liquid phase membrane device is as follows: 1. The membrane, wetted with the appropriate liquid, is brought into contact with the non-wetting liquid. At this stage, there is a ‘pool’ of the wetting liquid covering the surface of the membrane - as shown in Fig. 9A. There is no contact between the non-wetting liquid and the membrane. Consequently, any pressure that is applied to the non-wetting liquid will result in drainage of liquid from the ‘pool’. This is not a breakthrough phenomenon, merely the flow of liquid under applied pressure. With a sufficiently sensitive device, such as the breakthrough pressure test cell described in this paper, this flow can be observed.
rl
Non-wetting
Phase
Membrane
(Cl Fig. 9. Sequence of events preceding the occurrence of breakthrough in a two-liquid phase membrane device. (A) Wetted membrane in contact with the non-wetting liquid. AP=O; Ab= 0. (B) Three-phase line of contact formed after drainage of wetting liquid from membrane surface O< AP< AP,. (C) Liquid-liquid interface in motion after the occurrence of breakthrough. AP> AP,.
A.M. Vaidya et al. /Journal of Membrane Science 97 (1994) 13-26
2. Eventually, a stage is reached when the coverage of the membrane surface by the wetting liquid is reduced to a thin wetting film. Capillary effects now come into play and a three phase line of contact is formed - as shown in Fig. 9B. Breakthrough of the non-wetting liquid will not occur as long as the applied pressure is less than the breakthrough pressure - which can be calculated using the relations described elsewhere
[lOI* 3. When the applied pressure becomes equal to the breakthrough pressure, movement of the liquid-liquid interface will commence. This results in conflicting demands being made on the shape of the interface. The thermodynamic requirement on the contact angle prescribes a certain interfacial shape. However, the no-slip boundary condition on the moving liquid at the pore walls requires an entirely different shape. This conflict results in rotational flows being setup which cause the various interfacial tensions, a, to change from their, ‘old’, static values. This in turn causes a change in the contact angle, f3,,, - as shown in Fig. 9C. One can no longer calculate a breakthrough pressure with the aid of an equilibrium relationship, such as the LaplaceYoung equation [ 10 1.
List of symbols
AP applied pressure (Pa) AP, breakthrough pressure (Pa) pore radius at three phase line of contact r (m) surface tension of partially acetylated cellulose (dyn/cm ) I9wm angle of contact between wetting liquid and membrane interfacial tension at membrane-liquid in0 terface (N/m) 6cJ difference between interfacial tensions at the membrane-non-wetting liquid and membrane-wetting liquid interfaces (N/ m)
Y
25
Superscripts 1 P nP S
polar component polar, or hydrophilic, mode of operation of an amphiphilic membrane non-polar, or hydrophobic, mode of operation of an amphiphilic membrane dispersive component
Subscripts a m n 0 W
aqueous phase membrane non-wetting phase organic phase wetting phase
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[ 11 ]C.J. Tompkins,
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tion in an emulsion membrane bioreactor, J. Membrane Sci., 80 (1993) 265. [ 171W.D. Bascom and C.R. Singleterry, The effect of polarnon-nolar solutes on the water wettabilitv of solid surfacessubmerged in oil, J. Phys. Chem., 68 ( 1962) 236. 181 B.S. Kim and P. Harriott, Critical entry pressure for liquids in hydrophobic membranes, J. Colloid Interface Sci., 115 (1987) 1. 191 K. Sakai, H. Chiba and A. Naitoh, Determination of the pore radius of regenerated cellulose membranes by a dyeing technique, J. Membrane Sci., 37 ( 1988) 101. 20lR.E. Kesting., Synthetic Polymeric Membranes - A Structural Perspective, Wiley, New York, NY, 1985, pp. 302-303. 2 I] M. Saito and A. Yabe, Dispersion and polar force components of surface tension of some polymerlilms, Textile Res. J., 1 (1983) 154. 221 W. Rose and R.H. Heins, Moving interfaces and contact angle rate dependency, J. Colloid Interface Sci., 17 (1962) 39.