Yeast cake layers as secondary membranes in dead-end microfiltration of bovine serum albumin

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journal of

MEEzFE ELSEVIER Journal of Membrane Science 92 ( 1994) 247-256

Yeast cake layers as secondary membranes in dead-end microfiltration of bovine serum albumin

Naveen Arora, Robert H. Davis’ Department of Chemical Engineering, University of Colorado, Boulder, CO 80309-0424, USA

(Received September 23, 1993; accepted in revised form March 8, 1994)

Abstract

Deadend filtration of bovine serum albumin was performed in a phosphate buffer through a yeast cake layer deposited on an asymmetric, polysulfone microtiltration membrane having a nominal pore size of 0.2 pm. Yeast cake layers of varying thicknesses were preformed in two ways - by gravity settling prior to the onset of filtration and by vacuum filtration. The role of the cake layer of rejected ceils as a secondary membrane for reducing protein transmission and affecting permeate flux was studied. The steady-state flux was found to be higher in the presence of a thin cake layer than in the absence of a cake layer. This is thought to result from the yeast cake serving as a filter-aid to remove membrane foulants from the protein solution. Due to the micro-channeling effects, higher fluxes were observed for filtration through a cake layer preformed by vacuum filtration, as compared to the corresponding values for a cake layer of equal dry mass preformed by gravity settling. The protein transmission studies revealed three types of fouling - membrane fouling due to the internal pore deposition of protein, protein adsorption through the entire cake depth, and surface filtration leading to protein rejection at the cake surface.

Keyworuk Biotechnology; Dynamically formed membranes; Fouling; Microfiltration

1. Introduction

An ideal membrane process for recovering proteins from a suspension containing cells or cell debris is characterized by a high steady flux and 100% protein transmission, where flux is de- fined as the volume of permeate collected per unit time per unit membrane area, and protein transmission represents the ratio of the concentration of protein in the permeate to that in the feed. However, a major drawback associated with a typical membrane filtration process is a largely

*Corresponding author. Tel.: 303-492-7314; Fax: 303-492- 4341; E-mail: [email protected].

irreversible decline in flux which is usually attributed to solute adsorption to the membrane or pore occlusion by suspended solids. This phenomenon is called fouling. The irreversibility re- fers to the absence of a flux increase upon increasing the mass-transfer coefficient by increasing the cross-flow velocity or stirring speed. Chemical treatment, backflushing or other cleaning techniques are needed to restore the initial membrane permeability. Microfilters, which are expected to transmit proteins and other ma- cromolecules, are especially susceptible to intra- pore fouling. In addition to causing a drastic re- duction in flux, fouling also causes impairment of the membrane fractionating capability. This

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248 N. Arora. R.H. Davis /Journal of Membrane Science 92 (1994) 247-256

effect can be more critical than the effect on flux because it cannot be compensated for by merely increasing the membrane area or altering the hydrodynamics.

Over the past few years, investigations have been carried out by several researchers to study microfiltration of protein solutions. Heinemann et al. [ 1 ] studied microfiltration of whey protein solutions of different pH, in the presence and absence of salt, through modified hydrophilic hol- low fiber membranes and found the protein- membrane interaction to be a consequence of charge effects in the double-layer of the membrane and the charged ions. The adsorption of bovine serum albumin (BSA) on polymeric and inorganic microfiltration membranes has been studied as a function of protein concentration, pH, and flow rate through the membrane [ 2,3]. The adsorption was found to be rapid initially, the rate depending on the protein concentration and the permeation rate. The pH dependence of the extent of adsorption indicated the signifi- cance of both electrostatic and hydration effects. Le and Gollan [ 41 used a cellulosic base microporous membrane with a galactopyranose coat- ing in the separation of a-amylase from a Bacil- lus broth and malate dehydrogenase from a yeast broth. Using scanning electron microscopy, they found that the fouling deposit consisted of a se- ries of submicron layers of a very line material, presumed to be protein precipitates.

There have been few studies of the role of protein aggregation in membrane fouling. Kelly et al. [ 51 obtained experimental data for the flux decline during the stirred-cell filtration of different commercial preparations of BSA through asymmetric polyethersulfone microfiltration membranes. They observed that BSA fouling of the microporous membranes was associated with the deposition of trace quantities of aggregated and/or denatured BSA which served as initiation sites for the continued deposition of bulk protein. Also, Howell and Velicangil [ 6 ] pointed out that initial fouling occurred as a result of membrane surface protein binding in a non-spe- cific adsorption process. Subsequent fouling was ascribed to protein polymerization onto the first layer. Suki et al. [ 71 developed a theoretical

model of flux decrease based on the flocculation of protein in the polarized zone. Using a quasi- elastic light scattering technique, Chandavarkar [ 81 demonstrated that prolonged peristaltic pumping during cross-flow microfiltration of BSA led to the formation of relatively large protein aggregates in the bulk solution, with the observed fouling behavior attributed to the convec- tive deposition of these aggregates leading to pore occlusion followed by particle polarization.

Unfortunately, besides protein adsorption on the membranes, considerable loss of protein product of interest occurs due to its low transmission through the dynamic cake layer of rejected biomass which forms on the membrane surface during the filtration of fermentation broths and cell debris. However, quantitative information is lacking on the effects of biomass/ cake formation on permeate flux and protein transmission during membrane filtration. In order to provide such information, a study was performed on the microfiltration of BSA suspension in a phosphate buffer through an asymmetric polysulfone membrane with 0.2 pm nominal pore size in the presence of a yeast cake layer deposited on the membrane prior to the onset of filtration. Such an arrangement provides an idealized situation analogous to that normally occurring when microtiltration membranes are used to separate proteins from cells. The focus is on investigating the role of the cake layer of rejected cells as a secondary membrane for reducing protein transmission and affecting the tiltrate flux profiles.

2. Materials and methods

The bench-scale dead-end filter used for BSA microfiltration experiments was manufactured by Millipore. The equipment consisted of a borosilicate glass funnel and a ground-glass base with a coarse-frit glass support for the membrane. The ground-glass base was equipped with a neoprene stopper which fitted exactly into a 1000 ml collection flask. A rubber gasket was used to provide an air-tight contact between the funnel and the filter base surfaces. Two alumi-

N. Arora, R.H. Davis /Journal of Membrane Science 92 (1994) 247-256 249

num split rings were fabricated and used, instead of the anodized aluminum spring clamp sup- plied by Millipore, to provide a more uniform and effective sealing in order to minimize the chances of an overflow/leakage occurring at the funnel-filter base contact surfaces.

The microfilters used were modified polysulfone membranes (manufactured by Gelman Sci- ences Inc.) in the form of circular discs of di- ameter 47 mm, nominal pore size (NPS) 0.2 p, and thickness 150 m. These modified polysulfone membranes, coated with a layer of hydrox- yethyl methacrylate, offer higher flow rates, lower extractables and greater strength than competi- tive cellulose acetate or cellulose nitrate membranes. The modified membranes are hydrophilic, and their low protein binding characteristic minimizes loss of critical media components. A new membrane was used for each experiment conducted. The collection flask was subjected to a vacuum of 18 in Hg (6.1 x lo4 Pa) in order to effect the permeate flow through the microfiltration membrane. The pressure was measured with the help of an on-line vacuum gauge (Fisher Scientific) having a range of 0 to 30 in Hg. The transmembrane pressure drop was kept constant at 8.8 psi in all the experiments.

The suspension used for yeast cake formation was Saccharomyces cerevisiae (commercially available Fleischmann’s active dry yeast) suspended in deionized water having a resistivity of 18.2 MSZ cm. Prior to being used for cake formation, the suspension containing a known dry mass of yeast cells was subjected to three runs of centrifugation in a Beckman GPR centrifuge for washing. Each centrifugation run was carried out at 2000 rpm, for a period of 5 min. The super- natant containing the yeast cell fragments, and the surface-active proteins separated from the yeast cells, was discarded at the end of each centrifugation run.

Phosphate buffer solution (PBS) served as the suspending medium for BSA crystals and was also used to determine the membrane resistance in these experiments. The buffer solution was prepared by dissolving 8.0 g NaCl, 0.2 g KCl, 1.44 g Na,HPO,, and 0.24 g RI&PO4 in a final volume of 1000 ml deionized water. BSA solution

was prepared by suspending BSA crystals (Sigma Chemical Co., grade A6793, fractionated by heat shock, 98-99% albumin) in the buffer solution having a pH of 7.4. BSA is a well-characterized globular protein of molecular weight 67,000, size 11.6x 2.7x 2.7 nm, and with the isoelectric point at pH 4.7-4.9 [ 21. The concentration of BSA solution used in all filtrations was 0.1 mg/ml. All experiments were conducted at room tempera- ture (2352°C).

Prior to the yeast experiments, the membrane resistance was determined by measuring the flow of the buffer solution through the membrane, as a function of time. The flow rate (or flux) was measured by recording the time required for a known volume of feed solution to pass through the microporous membrane. The volume of solution filtered through the membrane was moni- tored visually, using a graduated cylinder (Pyrex, No. 3022). Similar flux measurements were also conducted for BSA microfiltration through the membrane (in the absence of any yeast cake layer).

Each yeast cake layer experiment consisted of filtering BSA in the phosphate buffer through the preformed yeast cake layer and the microporous membrane for a certain period of time (typically 5 h) until the flux became steady or nearly steady. The borosilicate glass funnel had a capacity of 250 ml, and had to be resupplied with the feed solution from the reservoir several times during the experiment. This was done in a gradual manner, so as to not disturb the yeast cake layer. For the cake preformation, the washed yeast cells were suspended in 100 ml deionized water (obtained using Barnstead NANOpure II filtration system) and then subjected to gravity settling or vacuum filtration. It took N 15 min to form the yeast cake by vacuum filtration, whereas gravity settling extended over a period of lo-12 h for the deposition of the yeast cake layer. The permeation rate for BSA solution filtration through the yeast cake layer and the polymeric membrane was measured as a function of time, in a manner similar to that used for PBS flux measurement in the membrane resistance experiment. Similar flux measurement experiments were also performed to study PBS filtration

250 N. Arora, R.H. Davis /Journal of Membrane Science 92 (1994) 24 7-256

(without BSA ) through the yeast cake layer and the microfiltration membrane.

The flux measurements were followed by protein assays, performed to determine the ratio of absorbance of permeate samples to that of feed samples, at different times during the experiment, thus obtaining the protein transmission variation with time or with the permeate volume collected. The absorbance measurements were carried out in a Beckman DU-50 spectrophoto- meter at a wavelength of 280 nm. A calibration of absorbance at 280 nm versus BSA concentration (mg/ml) in the buffer solution was made. The results of the calibration and further details of the experimental protocols are given in ref. 9.

3. Results and discussion

Fig. 1 shows the flux results for the experiment in which PBS was filtered through the microporous membrane. Each data point represents the arithmetic mean of the corresponding values obtained from the three runs of the experiment performed with three different membranes, and the

I@+++++

7TTm * ” 0 II ” 1 1 ” 1 I’ 0 “’ g ” ‘I ,- 500 1000 1500 2000 2:

time (set)

Fig. 1. Variation with time of the permeation rate of phosphate buffer solution (PBS) through a 0.2 c polysulfone membrane at 6.1 x lo4 Pa transmembrane pressure. The error bars represent the 90% confidence intervals for three experiments.

error bars on the data represent the 90% confidence intervals. The 90% confidence limits for the mean were calculated using the standard de- viation, the student-t value and the degrees of freedom [ lo]. A rapid but modest decrease in the PBS flux is observed. Since PBS contains no fouling species, it is thought that this decrease is due to membrane compaction upon the imposi- tion of the pressure drop. The membrane compaction was found to be partly reversible, since PBS flux could be partially restored by depres- surization of the membrane for several minutes.

The permeation rate for BSA filtration through the membrane (in the absence of a cake layer) was found to decrease continuously with time, over a period of N 3 h (Fig. 2 ). The continual decrease in the filtrate flux with time, under constant applied pressure, is much greater than can be explained by protein adsorption, which is generally limited to monolayer coverage. Such decreases have been interpreted in terms of protein deposition resulting from shear-induced dis- tortion of the molecules on passage through the micropores. Circular dichroism (CD) and Four- ier transform infrared spectroscopy (FIIS) have shown that changes in secondary structure of

0.20 ,

0 5000 10000 15000 20

time (set)

Fig. 2. Variation with time of the permeation rate of 0.1 mg/ ml BSA solution through a 0.2 pm polysulfone membrane at 6.1 x lo4 Pa transmembrane pressure. The error bars represent the 90% confidence intervals for three experiments.

N. Arora, R.H. Davis /Journal of Membrane Science 92 (1994) 247-256 2.51

protein can occur on passage through microfiltration membranes [ 111. The decrease in flux with time can be quantitatively described as a decrease in pore volume or number due to internal fouling of the membrane caused by protein deposition at the walls or mouths of the pores. Tracey and Davis [ 121 have found that proteins accumulate near the entrance of the pores and on the membrane surface, rather than accumulating uniformly along the pore walls. Kelly et al. [ 5 ] have explained the flux decline in terms of denatured protein aggregates fouling the membrane and serving as initiation sites for the continued deposition of bulk (native) protein.

Protein transmission exhibited an initial decrease and then increased to unity within N 6 min after the onset of filtration (Fig. 3 ). The mini- mum level of protein transmission for BSA microfiltration was observed N 2 min after the onset of the experiment. This time period possibly accounts for the growth of denatured protein ag- glomerates which promote further adsorption of bulk (native) protein on the pores of the membrane. The subsequent increase in protein transmission can be attributed to saturation coverage.

OX m 2000 3000 4000 time (set)

Fig. 3. Protein transmission as a function of time, for 0.1 mg/ ml BSA solution microtiltration through a 0.2 pm polysulfone membrane at 6.1 x 1 O4 Pa transmembrane pressure. The error bars represent the 90% confidence intervals for three experiments.

The total protein deposition corresponds to monolayer coverage of the internal structure of the membrane [ 91, although it is expected that this coverage is nonuniform [ 12 1.

Since BSA is the only foulant in these experiments, it seems surprising that substantial flux decline is observed (Fig. 2 ) while the BSA transmission remains high (Fig. 3). A possible expla- nation is that the flux decline is caused by aggregates which constitute a very small fraction of the protein which block many of the pores while leaving a small percentage at least partially open for the protein-containing permeate to pass through. Substantial flux decline through microfiltration membranes without significant reduc- tion in protein transmissions is more the excep- tion than the rule, but it has been observed previously by Bowen and Gan [ 13 ] for alcohol dehydrogenase filtered through thin-film composite polysulfone microfiltration membranes and by Palecek and Zydney [ 141 for lysozyme filtered through polyethersulfone microfiltration membranes.

Figs. 4 and 5 depict the time-dependent tiltrate flux profiles obtained for BSA microfiltra-

0 020

- 0.014 g/sq.cm. yeast cake *s 0.028 g/sq.cm. yeast cake l z 0.056 g/sq.cm. yeast cake

f ““” t

O.Ooo 6”“““sbb’o” time (set)

too

Fig. 4. Filtrate flux variation with time, for 0.1 mg/ml BSA solution filtration through a 0.2 pm polysulfone membrane in the presence of different thicknesses of vacuum filtered yeast cake layers at 6.1 x 1 O4 Pa transmembrane pressure. The error bars represent the 90% confidence intervals for three experiments.

252 N. Arora, R.H. Davis /Journal of Membrane Science 92 (1994) 24 7-256

q oooo 0.014 g/sq.cm yeast cake *- 0.026 g/sq.cm. yeast cake au&e 0.056 g/sq.cm. yeast cake

2 E +: 0.005

;;:

O.Ooo ~00 time (set)

Fig. 5. Permeate flux variation with time, for 0.1 mg/ml BSA solution filtration through a 0.2 ,um polysulfone membrane in the presence of different thicknesses of gravity settled yeast cake layers at 6.1 x 1 O4 Pa transmembrane pressure. The error bars represent the 90% confidence intervals for three experiments.

tion through the polymeric membranes in the presence of yeast cake layers of varying thicknesses preformed by vacuum filtration and by gravity settling, respectively, prior to the onset of microfiltration. Cake layers of 0.2,0.4, and 0.8 g (dry mass) yeast were formed in these experiments. These led to the formation of 0.0 14,0.028, and 0.056 g/cm2 yeast cake layers, respectively, with the conversion based on the external surface area of the membrane. The corresponding cake thicknesses, calculated using the appropri- ate conversion factor correlating the yeast cake dry mass with the cake volume [ 15 1, were found to be 0.05 1,O. 102, and 0.204 cm, respectively.

As depicted in Figs. 2 and 4, the steady-state flux for BSA solution filtration in the absence of a cake layer is found to be 2.60~ 10m3 cm/s, whereas the steady-state flux values in the presence of 0.014, 0.028, and 0.056 g/cm2 vacuum preformed yeast cake layers are found to be 1.34x 10p2, 5.58x 10e3, and 2.49x 10e3 cm/s, respectively. Similarly, from Fig. 5, the steady- state flux values for BSA solution filtration in the presence of the above three masses of gravity settled yeast cakes are found to be 9.94x 10-3, 3.77~ 10e3, and 1.63x 10m3 cm/s, respectively. Comparing these flux values leads to the conclu- sion that the long-time flux in the presence of a thin cake layer (0.014 and 0.028 g/cm2 yeast cakes, for example) is greater than the long-time flux in the absence of a cake layer, thus illustrat- ing the role of the cake layer as a pre-filter which removes membrane foulants from the BSA solution. The cake resistance offered by the 0.056 g/ cm2 yeast cake layer, however, predominates its role as a pre-filter, thereby leading to no im- provement in the long-time flux for BSA microfiltration.

As illustrated in Figs. 4 and 5, lower long-time Figs. 6 and 7 show the filtrate flux behavior fluxes were observed for filtration through a cake observed for PBS filtered through the micropo- layer preformed by gravity settling, as compared rous membranes in the presence of the vacuum to the corresponding values for a cake layer of filtered and gravity settled yeast cake layers, re- equal dry mass preformed by vacuum filtration. spectively. The motivation behind conducting This can be ascribed to the fact that the vacuum PBS filtrations was to investigate whether or not filtration cake layer, which is formed under suc- the cake resistance for PBS filtration was propor- tion, has microchannels incorporated in its tional to the cake thickness. These experiments structure which provide reduced resistance to confirmed the fact that, unlike the non-linear de- flow, thereby leading to higher permeation rates pendence of the cake resistance on the cake than those for the more uniform cake layer pre- thickness observed earlier for BSA filtration

formed by overnight gravity settling. Also, the fact that the initial flux values associated with a gravity cake were found to be approximately the same or slightly higher than those for a vacuum cake of the same thickness leads to the conclu- sion that the vacuum filtration cake layer is less compressible or more compact to begin with, as compared to a cake layer of equal dry mass preformed by gravity settling. Thus, initially, the looser structure of the gravity settled cake layer counterbalances the micro-channeling associated with the vacuum filtration cake layer, in terms of affecting the tiltrate flux.

N. Arora, R.H. Davis /Journal of Membrane Science 92 (I 994) 247-256 253

'!! 0.014 g/sq.cm. yeast cake *u 0.026 g/sq.cm. yeast cake A%&&% 0.056 g/sq.cm. yeast cake

1 I O.Ooo -00

time (set)

Fig. 6. Time-dependent filtrate flux profiles for PBS ftltra- tion through a 0.2 pm polysulfone membrane in the presence of different thicknesses of vacuum filtered yeast cake layers at 6.1 x 1 O4 Pa transmembrane pressure. The error bars rep resent the 90% confidence intervals for three experiments.

0.020

- 0.014 g/sq.cm. yeast cake •a%~ 0.026 g/aq.cm. yeast cake -- 0.056 g/sq.cm. yeaat cake

5 0.010

G

8

2 2 0.005

z

1 I 0.000 1 I I I I I t I, 1111111,11!11 III

0 2000 4000 6000

time (set)

Fig. 7. Filtrate flux variation with time for PBS filtration through a 0.2 p polysulfone membrane in the presence of different thicknesses of gravity settled yeast cake layers at 6.1 x lo4 Pa transmembrane pressure. The error bars represent the 90% confidence intervals for three experiments.

through these membranes in the presence of a cake layer (see Figs. 4 and 5 ) , the filtrate flux for PBS microfiltration varied inversely with the

cake thickness, in accordance with the conven- tional Darcy-law predictions when the cake resistance dominates the membrane resistance. It is significant to note that the membrane resistance for PBS filtration is an order of magnitude lower than the cake resistance offered by the thinnest cake layer (i.e., 0.014 g/cm’ yeast cake) preformed in these experiments. This implies that the membrane resistance is not of much sig- nificance in determining the total resistance in PBS filtration experiments. The difference in the two experimental findings can be ascribed to the difference in the fouling characteristics of BSA and PBS. While PBS causes no fouling, BSA lil- tration leads to gradual changes in the original cake layer structure due to the formation of a protein deposit around the yeast cells, which renders the cake layer gradually more resistant to the fluid flow. Thus, it is hypothesized that during BSA microfiltration, the observed non- linear dependence must be due to the fact that the protein fouling of yeast does not occur uniformly throughout the depth of the yeast cake. In thick cakes, the protein fouling is primarily con- fined to the top surface layers of yeast cells, which act as a pre-filter for the layers underneath and for the membrane.

Figs. 8 and 9 illustrate the protein transmission profiles obtained for BSA microfiltration in the presence of the vacuum filtered and gravity settled yeast cake layers of varying thicknesses. Each data point represents the arithmetic mean of the corresponding protein transmission values obtained from the three runs of the experiment performed with three different membranes, and the error bars represent the average 90% confidence intervals for the three experiments, calculated using the F-test procedure for averaging the standard deviations of a popula- tion of means [ lo]. Since the microfiltration membranes used in these filtrations exhibit es- sentially 100% protein transmission (in the absence of a cake layer), except during the first few minutes of filtration, the low levels of protein transmission observed initially in the preformed cake layer experiments can be attributed to protein adsorption through the entire cake depth. Gradually, the protein transmission was found

254 N. Arora, R. H. Davis /Journal of Membrane Science 92 (1994) 24 7-256

0.00 l,l,“,,~i,ln~,i,r~,,,,~,,,ll,l,,il,ll,,,llll,,ll, 0 200 400 600 600 1000

permeate volume (ml)

-E 0.014 g/sq.cm. yeast cake *S 0.028 g/sq.cm. yeast cake *z 0.056 g/sq.cm. yeast cake

Fig. 8. Protein transmission variation with the permeate volume collected, for 0.1 mg/ml BSA solution microfiltration in the presence of different thicknesses of vacuum filtered yeast cake layers at 6.1 x lo4 Pa transmembrane pressure. The error bars represent the average 90% confidence intervals for three experiments, calculated for each of the three cake thicknesses.

to level off to a constant value of N 9596, inde- pendent of the cake thickness (or dry mass) and the cake formation pattern. As anticipated, the rise in protein transmission was found to be more gradual for a thicker yeast cake as compared to that observed for a thinner cake layer. The small offset from 100% transmission, observed even at large times, is possibly due to protein rejection at the cake surface. This phenomenon of depth filtration followed by surface filtration was observed for the three thicknesses of both the vacuum filtered and gravity settled yeast cakes, in- vestigated in the present study.

Fig. 10 shows the mass of BSA rejected (excluding protein rejection due to surface filtration) per unit external surface area of the cake versus the cake dry mass per unit external surface area for the vacuum filtered cakes. Nearly identical behavior was observed for the gravity settled cakes [ 9 1. The mass of BSA rejected was calculated based on the data obtained from the protein transmission measurements conducted for different thicknesses of yeast cake layers. For

c 0 60

0 ._

2 ._ E o 0 60

c

=I

ALLLG 0 056 g/sq cm yeast cake *LLCU 0 026 g/sq.cm yeast cake EL!C 0014 g/sq.cm. yeast cake

000 ~,,,~,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,~ 0 200 400 600 600 1c

permeate volume (ml) 10

Fig. 9. Protein transmission as a function of the permeate volume collected, for 0.1 mg/ml BSA solution microfiltration in the presence of different thicknesses of gravity settled yeast cake layers at 6.1 x lo4 Pa transmembrane pressure. The error bars represent the average 90% confidence intervals for three experiments, calculated for each of the three cake thicknesses.

000 I,# I /~~~,I,, 1,111111

0 00 0.02 0.04 0.1

cake dry mass (g/sq.cm.) 6

Fig. 10. Plot of the mass of BSA rejected per unit external cake surface area (excluding BSA rejection due to the surface filtration) versus the cake dry mass per unit external surface area, for the vacuum filtered yeast cakes. The dashed line represents the best-tit of the data with the case of no cake layer excluded. The error bars represent the 90% confidence intervals for three experiments.

N. Arora, R.H. Davis /Journal of Membrane Science 92 (1994) 247-256 255

excluding the protein losses due to surface rejeo tion, the arithmetic mean of the last few data points in the horizontal region (which corresponds to the surface filtration) of each curve was calculated, and the mass of BSA rejected corresponding to this average offset from 100% protein transmission was subtracted from all the data points in each of the three curves. The summa- tion of all the mass rejections (corrected for protein losses due to surface rejection) yields the total mass of BSA rejected due to protein adsorption through the entire cake depth. The mass of BSA rejected appears to have varied lin- early with the cake dry mass, within the experimental error limits. A closer look reveals that the best-fit line intersects the y-axis (the BSA rejection axis) at a point lower than what corresponds to the mass of BSA rejected by the membrane alone (in the absence of a cake layer). This clearly establishes the role of the cake layer as a pre-filter for removing aggregated or denatured proteins which would otherwise foul the membrane. The dashed line in Fig. 10 is the best lit of the data with the case of no cake layer excluded. Instead of the best-lit linear plot, one could also draw a concave-down curve, which passes through the three data points, within experimental error limits. This non-linear variation of the mass of BSA rejected as a function of the cake dry mass can be considered a consequence of the fact that the protein fouling of yeast does not occur uniformly throughout the depth of the yeast cake. The non-uniform protein fouling can be appropriately attributed to the top surface layers of yeast cells acting as a pre-ftlter for the under- lying cells. A concave-up curve could also be drawn through the data, which would indicate more protein deposition per unit mass for the thicker cake layers. This may result, for example, from the lower flux through the thicker cakes giving more time for protein deposition and less shear to remove or prevent protein deposits.

4. Conclusions

Experiments performed to study dead-end microliltration of BSA through asymmetric, 0.2 pm

polysulfone membranes in the presence of yeast cake layers confirmed that the time-dependent permeate flux profiles were appreciably influ- enced by the cake formation pattern. Owing to the micro-channeling effects associated with the vacuum filtration cake layer, lower long-time fluxes were observed for filtration through a cake layer preformed by gravity settling, as compared to the corresponding values for a similar cake layer preformed by vacuum filtration. The fact that the long-time flux in the presence of a thin cake layer was found to be greater than that in the absence of a cake layer led to the inference that the cake layer acted as a pre-filter for the protein solution, most likely sieving protein aggregates which otherwise would foul the membrane. Also, the protein transmission studies revealed three types of fouling - membrane fouling due to the internal pore deposition of protein, protein adsorption through the entire cake depth, and protein rejection at the cake surface. These protein losses may have an important influence on the overall yield of a small volume, high value product of interest like BSA. In summary, the experimental methods outlined above provide a simple and effective means of investigating the effect of cake fouling on the capacity and selec- tivity of the desired membrane separation.

Acknowledgements

This work was supported by Grant CTS 9 107703 from the National Science Foundation and by the Center for Separations Using Thin Films at the University of Colorado. Assistance with the experiments by Paul Suding and Larry Gallegos is gratefully acknowledged, as is the support of the University of Colorado’s Summer Minority Access to Research Training program and the National Science Foundation’s Research Experiences for Undergraduates and Coalition to Increase Minority Degrees programs.

256 N. Arora, R.H. Davis /Journal of Membrane Science 92 (1994) 247-256

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Yeast cake layers as secondary membranes in dead-end microfiltration of bovine serum albumin