402
Ind. Eng. Chem. Fundam. 1982, 21, 402-406
Disruption of Protein Precipitates during Shear in Couette Flow and in Pumps Mlchael Hoare,* Thrlnayanl J. Narendranathan, Jonathan R. Fllnt, Deborah Heywood-Waddington, Donald J. Bell, and Peter Dunnill Department of Chemical and Biochemical Engineering, University College London, Torrington Place, London WC 1E 7JE, United Kingdom
The particle size distribution of soya protein isoelectric precipitates was found to be strongly dependent on the rate of shear strain and the time of exposure during laminar Couette shear. Similar rates of breakup to those observed for exposure to rates of shear strain greater than 33 000 s-’were also recorded for precipitates subjected to shear in various positive displacement and centrifugal pumps. Only the peristaltic pump with its much gentler action showed no disruptive effect on the precipitate particles.
The recovery and purification of proteins by precipitation is often a difficult and costly process. The generally small size of the protein precipitate aggregates and the small density differences between precipitate and supernatant mother liquor requires the use of high speed centrifugation equipment operating at very low liquid throughput. Protein precipitation involves both collision-controlled growth and shear-controlled breakup (Hoare, 1982a,b; Virkar et al., 1982a). Therefore, a basic understanding of protein precipitate disruption caused by shear is required to optimize precipitate characteristics during preparation, ageing, pumping, and feeding to the centrifuge. Protein precipitate preparation and growth has been studied for the sdting-out of casein in a stirred tank reactor (Hoare, 1982a,b) and for the isoelectric precipitation of soya protein in a tubular reactor (Virkar et al., 1982a). In neither case was it possible to predict precipitate growth rates on the basis of Smoluchowski’s orthokinetic aggregation theory (Levich, 19621, ignoring hydrodynamic interactions at close particle-to-particle distances and in conjunction with a simple maximum size break-up model. This was due to the complex nature of precipitate breakup during shear. The average ranges of rates of shear strain to which the precipitate was exposed were 12 to 640 s-l in the stirred tank reactor and 340 and 600 in the tubular reactor, although localized shear rates will exceed these values. Mean particle diameters of the precipitate extended from 5 to 15 pm, this being mainly a function of the shear conditions in the reactor and also of the protein concentration and time of ageing. Density differences as low as 31 kg m-3 were recorded between the casein precipitate and mother liquor, while for acid precipitated soya protein in batch reactors density differences ranged from 80 to 250 kg m-3 as a function of the precipitate size. Particles of diameter greater than 5 pm are of uniform density and therefore precipitate recovery should be greatly enhanced by increase in size above this level (Bell et al., 1982). Studies on acid precipitated soya protein showed that shear conditions during preparation in a turbine stirred batch reactor strongly controlled the breakup characteristics of the precipitate. Maximum stability of the precipitate aggregate is achieved when the product of the ageing time and average rate of shear strain in the preparation vessel exceeds lo5. Aggregate breakup during capillary shear (9000 s-l to 90 000 s-l) is due to the removal of 3-9 pm diameter fragments from the surface of the aggegates whose initial mean diameters ranged from 9 t o
50 pm. The fragment size is a weak function of the rate of shear strain. The rate of breakup is dependent on the rate of shear strain and time of exposure to shear (Bell and Dunnill, 1982). Similar time dependency was noted for casein precipitates over much longer periods of shear (up to lo5 s) at a rate of shear strain of 250 s-l (Hoare, 1982a,b). Mechanisms of precipitate breakup may include fragmentation due to particle-particle or particle-wall collisions or hydrodynamic shear, bulgy deformations arising from temporary pressure gradients across aggregates and erosion of primary particles from the surface of the parent aggregate (Glasgow and Luecke, 1980). The relatively compact structure of protein aggregates (Bell et al., 1982) with the wide range of available molecular binding mechanisms (Melander and Horvath, 1977) calls for different models from those used to predict the breakup of large (-lo00 pm) loose polymeric structures (Parker et al., 1972) or brittle particles such as coal dust (Karabelas, 1976) and crystals (Conti and Nienow, 1980).
Materials and Methods Precipitate Preparation. Soya protein total water extract was prepared at a concentration of 25 kg m-3 protein (Bell and Dunnill, 1982). This extract was contacted with 34% by wt H2S04 to pH 4.8 in a turbine stirred, baffled, reactor for a period of 600 s, precipitating 84% to 87% of the protein. Precautions were taken to avoid localized high concentrations of acid and to ensure reproducible mixing (Bell and Dunnill, 1982). Except where stated otherwise, precipitate was prepared at 4.17 rps in a 0.67 X m3 reactor ( E N 20 W m-3) and at 5.83 rps in a 0.27 X m3 reactor ( E N 36 W mT3).For high concentration runs the precipitate was concentrated by gravity settling. For low concentration runs the precipitate was diluted with 0.07 M sodium acetate buffer, pH 4.8. Shear in Couette Flow. Protein precipitate was exposed to rates of shear strain ranging from 400 s-l to 90 OOO using two Couette flow devices. Low rates of shear strain were achieved using a nylon cylinder (0.032 m diameter, 0.146 m long) rotated in a Perspex, water cooled, m concentric container with an annular gap of 4.75 X between the cylinder and container. The whole device was mounted horizontally to minimize settling of the precipitate during shear. The gap was filled with protein precipitate (prepared in reactor 2-see inset Figure 1). The inner cylinder was rotated at 1.86 rps and 9.32 rps giving averate rates of shear strain of 400 s-l and 2000 s-l, respectively, in Couette laminar flow. Taylor numbers equalled 8 and 38, respectively (Van Wazer et al., 1963).
0196-4313/82/1021-0402$01.25/0 0 1982 American Chemical Society
Ind. Eng. Chern. Fundarn., Vol. 21, No. 4, 1982 403
Table I. Description of Pumps Used for Shearing Protein Precipitates
Pump type centrifugal Mono screw gear peristaltic d
impeller radius, m
gap width, m
0.05 0.03 0.025
0.005 0.0005
rot. speed of impeller, rps io0
4.17 1.67
0.0001
no. of impeller blades
vol of pump head, L f
6
0.074 0.013
2 x 36
0.007
length of connecting pipe, m g 0.20 0.60 0.20 0.20
a Totton Electrical Sales Ltd., Southampton, U.K. Magnetically driven with plastic impeller. Model No. SB 14R, Mono Pumps Ltd., London U.K. Stainless steel rotor; Neoprene rubber stator. Model no. SPL 22, Slack and Parr (U.K.) Ltd., Derby, U.K. cl Hiloflo Metering Pump, Mono Pumps Ltd., London, U.K. Fitted with silicone rubber tubing. e At maximum flow rate. f Not including inlet and outlet ports. g 0,010m diameter tubing. Variable gap width.
Average rate of shear strain, s-l 601
100
200
I00
300
500
8
5
Reactor
Reactor
Reactor
Impeller
a?lume
0.070
0.070
0.029
O.Z7xIO-?
YO.
50,
LO
(h f l a t bladed turbine. baffled v e s s e l , baffle w i d t h = 0.1 x r e a c t o r diameter)
T
0
0
20
3c
C'
50 *,me h
Figure 2. Change in soya protein precipitate particle size during exposure to a rate of shear strain of 2000 s d in Couette flow. Total protein concentration in suspension = 2.5% by weight. Temperature = 23 OC.
Ol
0
100
200
300
LOO
500
600
Power per unit volume.( E I, w m-3
Figure 1. Effect of reactor preparation conditions on precipitate particle size distribution. Ageing time in reactor = 600 s. Total protein concentration in suspension = 2.5% by weight. Temperature = 23 "C. Precipitate suspension density = 1008 kg m-3, Viscosity = 0.002 N s m-2. Power derived from power number-Reynolds number curves of Rushton et al. (1950). Average rate of shear strain = (power per unit volume/viscosity)0~5.
Sodium acetate buffer (0.07M) in a graduated pipet fitted above one end of the annular device was used to displace 0.1-mL samples of precipitate from a 0.005 m diameter port sited a t the bottom center of the annulus. The device was not stopped during this operation. Shear was applied for up to 7200 s except where stated otherwise, the number of samples taken being limited to avoid more than 10% dilution of the precipitate suspension. A stainless steel concentric cylinder device was designed to produce rates of shear strain up to 80000 while maintaining laminar Couette flow (Virkar et al., 1981). The outer cylinder, of inside diameter 0.10084 m, was rotated at speeds of up to 833 rps. The annular gap width was 3.17 X lo-* m, and the precipitate suspension was prevented from overheating by cooling of the inner cylinder. The device was operated horizontally to minimize sedimentation of precipitate during shear. Precipitate, prepared in reactor 1 (see Figure l),was sheared in the Couette device for periods ranging from 120 to 6000 s. Because of the very small annular volume, each experiment produced only one sample of sheared precipitate. Start-up and shut-down times were insignificant compared with periods of exposure to shear. Shear in Pumps. The pumps used and their dimensions are detailed in Table I. The flow rates and volumes for each pumping system are given in the appropriate
figure legends. The pump inlet and outlet ports were linked by 0.01 m inside diameter flexible PVC tubing, the system being completely filled with protein precipitate, prepared in reactor 1,excluding all air bubbles. No back pressure was applied as valves to do so would have created their own particular shear effects. Single pass experiments were not possible due to intrinsic shear effects in the sample ports. Where appropriate, temperature rise was minimized by immersing the pump head in a cooling bath. Precipitate Particle Size Analysis. Precipitate samples were immediately diluted 1 in 100 in clean, filtered, 0.07 M sodium acetate buffer (pH 4.8)to prevent aggregation. Particle size distributions were monitored using a Coulter Counter, Model TA,Harpenden, Herts., U.K. Full details of experimental techniques used are published elsewhere (Virkar et al., 1982). Mean diameter (d,) and the diameter of 90% by weight oversize (d,) are used to represent the precipitate particle size distribution. The index dw is valuable in indicating the effectiveness of fines collection by centrifugation.
Results and Discussion Protein Precipitation in Turbine Stirred Batch Reactors. Studies of precipitate formation in the two reactors are summarized in Figure 1. For a 2.5-fold increase in reactor volume, there is poor correlation of precipitate size with power per unit volume or impeller tip speed, especially at higher stirrer speeds. A t low power inputs per unit volume closer agreement is reached for the two reactors. However, the scatter in results due to difficulties in analysis of wide particle sue distributions limits the comparisons which can be usefully made. Shear in Couette Flow. A range of experiments was conducted at shear rates of 2000 s-l to study the effects of a period of ageing and of precipitate concentration on particle size. Even after periods of ageing of 50 h the precipitate particle size distribution had not reached an
404
Ind. Eng. Chem. Fundam., Vol. 21, No. 4, 1982
8
\
8
i
A
o L - -
2i:o
-0::
500C
'23:
I
Tne 5
Figure 3. Change in soya protein precipitate particle size during exposure to a rate of shear strain of 2000 8-l in Couette flow. Total protein concentration in suspension = 10% by weight (V,V);2.5% by weight (0,0 ) ;0.25% by weight (A, A). Temperature = 23 "C.
I3
2000
LOX
L---J
5350
7200 Tne
85000
5
Figure 5. Change in soya protein precipitate particle size distribution during exposure to a rate of shear strain of 400 in Couette flow. Total protein concentration in suspension = 2.5% by weight. Temperature = 23 OC.
e e ,-/
d2
'
t
. I
I
030
?COO
3330
.GO@
@O& T-e
F r ~ l e i - c o i i e r t allor '+ by weg-t Figure 4. Effect of total protein concentration of precipitate suspension on particle size attained after exposure to a rate of shear strain of 2000 s-l in Couette flow for 7200 s: (0, 0 ) precipitate prepared and aged for 600 s in reactor 2 operating at 5.83 rps (e = 36 W m-3) (see Figure 1); (A, A) precipitate prepared as above and then aged at a rate of shear strain of 400 s-l in Couette flow for 3000 s; (v,V) precipitate prepared in continuous flow tubular reactor at an average rate of shear strain of 341 s-l (Virkar et al., 1982).
equilibrium (Figure 2). However, most of the change occurred over the first 2 h of exposure to shear. A range of studies carried out over this period for varying protein precipitate concentrations is summarized in Figure 3. At higher concentrations slower rates of breakdown were apparent and this evidently reflects the increase in aggregation rates. Sizes reached after 2 h ageing are shown as a function of protein concentration in Figure 4. Large precipitate particles with different preparation or shear histories were reduced to similar sizes after exposure to a rate of shear strain of 2000 s-l (Figure 4). Precipitate exposed to a rate of shear strain of 400 5-l breaks up at a slower rate, giving a larger final particle size (Figure 5). Experimental scatter occurred due to the need to use at least two Coulter Counter orifices to characterize the wide particle size distributions. The results for precipitate exposed to high rates of shear strain ranging from 19 700 to 82 200 s-l are summarized in Figure 6. In this case, for shear rates greater than 32 900 s-l, almost complete disruption is achieved within 900 s.
5
Figure 6. Change in soya protein precipitate particle size during exposure to high rates of shear strain in Couette flow. Rates of shear strain used: (V,V) 1.97 X IO4 8; (A, A) 3.29 X IO4 s-'; (0, 0 ) 4.93 X IO4 s-l; (0, ). 6.58 X lo4 s-l; ( 0 , +) 8.22 X lo4 s?. Total protein concentration in suspension = 2.5% by weight. Temperature = 23 "C.
The range of experimental scatter makes it difficult to analyze any long term trends in exposure to high rates of shear strain. The results of shear on particle size distribution after 300 s and 1800 s are summarized in Figure 7 . As expected, there is a marked decrease in the maximum size, as represented by the 5 % by weight oversize point in the distribution, and in the mean diameter with increasing rate of shear strain. This trend is probably retained, although not to such a pronounced degree, for longer periods of shear. Within experimental error the diameter representing the fine end of the precipitate size distribution (d,) appears to be largely independent of shear rate and time of shearing for periods greater than 300 s, though there is some evidence that for long periods of shear (1800 s) the d, value tends to increase with increasing shear rate. In this case higher shear rates evidently promote aggregation at a greater rate than precipitate breakup. The breakup mechanisms of the precipitate may be examined further in cases where the same precipitate sample is sheared and analyzed repeatedly over the period of study as in the low shear rate experiments. The number of particles in each band of the size distribution is evaluated from the cumulative weight distribution, and the change in number of particles per channel is plotted vs.
Ind. Eng. Chem. Fundam., Vol. 21, No. 4, 1982
‘
O
O
-
405
i
e--------
I
t
i
’t 1
5.d
03
5 ~ 1 0 ~ 105
10L
Rote of shear strain , 5‘‘
Figure 7. Effect of rate of shear strain on particle size. Time of shearing = 300 s (e);= 1800 s (0). Ranges of dsovalues for times of shearing from 300 s to 7200 s are shown. Total protein concentration in suspension = 2.5% by weight. Temperature = 23 O C .
0
lc00
2000
3WO
LOO0 Time.
5WO
s
Figure 8. Effect of ageing time at 2000 s-l in Couette flow on mean fragment size. See Figure 3 for change in particle size distribution and for conditions of experiment.
channel mean size. For any particular channel of width Ai, the change in particle numbers, Ani is given by nit - n;O Ani = NToAi where n equals the number of particles per unit volume in channel i, superscripts t and 0 refer to distributions at time t and the initial sample, respectively, and NTois the total number of particles in the initial sample. The fragment size is taken as the median value of the positive channel data. Fragment size data is presented in Figure 8 for the 2.5% by weight precipitate size distribution exposed to 2000 .& (see Figure 3). Initially large fragments of diameter -8 pm are produced, rapidly reducing to fragment sizes of -2.5 pm. This analysis does not take account of the growth of precipitate by aggregation, but studies of breakup at high shear rates show that fragment size data can give some indication of breakup mechanisms (Bell and Dunnill, 1982).
0 0
LOG
800 hrnber of
‘200 passes
Figure 9. Flow of protein precipitates through various pumps. Total protein concentration = 2.5% by weight. Mono pump-flow rate = 1.67 X m3 s-l, pump and pipe volume = 0.275 L, 0.061 flow rate = 2.95 X m3 s-l, pump and pipe passes s-l (-0-); volume = 0.290 L, 0.101 passes s-l (-O-). (Precipitate prepared in reactor 1 (see Figure 1)at higher stirrer speeds (6.0 rps or c LV 63 W m-3, and 6.7 rps or e N 84 W m-3) also tended to the same particle size after -700 passes.) Gear pump-flow rate = 2.5 X 10” m3 s-l, pump and pipe volume = 0.045 L, 0.067 passes s-l. (Precipitate prepared in reactor 1 (see Figure 1) at higher stirrer speed (6.0 rps or e N 63 W m-3) also tended to the same particle size after -600 passes.) Centrifugal pump-flow rate = 5 X lo4 m3 s-l, pump and pipe volume = 0.10 L, 5.0 passes s-’. (Precipitate prepared in reactor 1 (Figure 1)at higher stirrer speeds (6.7 rps at e E 84 W m-3), also tended to the same particle size after -100 passes.) Peristaltic pump-flow rate = 2.5 X 10” m3 s-l, pump and pipe volume = 0.05 L, 0.05 passes s-l. (Precipitate prepared in reactor 1 (see Figure 1) at higher stirrer speeds (6.0 rps or c N 63 W m-3); remained at same particle size (dw = 8 rm) for 100 passes).
Exposure of Precipitates to Shear in Pumps. The results of the studies of particle size changes in pumps are summarized in Figure 9. Exposure to shear in the Monoand gear-positive displacement pumps follow a similar breakup pattern, the Mono pump causing the lesser precipitate breakup. No precipitate breakup was observed for the peristaltic pump. Due to the high rates of flow it was not possible to obtain data for a low number of passes in a centrifugal pump. For the gear and Mono pumps, the fluid and precipitate experiences a maximum rate of shear strain in the gap between the impeller and the housing and, assuming no fluid back-flow in the gap and that the gap width is smaller than the boundary layer thickness, the maximum rate of shear strain ( Y ~ may ~ ) be approximated as follows (Schlichting, 1968) Ymax
= rND/6
where (rND)is the angular velocity of the impeller tip and 6 is the gap width. The maximum rates of shear strain obtained are of the order of 1500 for the Mono pump and 2600 s-l for the gear pump. The flow in the connecting pipe was laminar (Re = 160 for the gear pump and Re = 1100-1900 for the Mono pump), the maximum rates of shear strain being 25 and 300 s-l, respectively, on the basis of Ymax = 8 v / d For the centrifugal pump the flow of fluid in the connecting pipe was turbulent with a Reynolds number of
406
Ind. Eng. Chem. Fundam., Vol. 21, No. 4,
1982
32000 and an average shear rate (7)of 7700 s-l (Davies, 1972). The rates of shear strain as calculated above are obviously only approximations as no account is taken of the geometry of the tubing and entry and exit effects. However, only for the centrifugal pump is the rate of shear strain in the connecting tube of significance. For the Mono, gear, and centrifugal pumps using the mean residence time in the pump head for comparative purposes, the rate of precipitate breakdown is much the same as for precipitate exposure to high rates of shear strain (32900 to 82200 in the Couette flow device (Figure 6), only the Mono and centrifugal pumps giving significantly larger final particle size distributions. It is very probable that extensional rates of shear strain (van der Tempel, 1977) and particle-wall and particleimpeller impacts play a dominant role in precipitate breakup in the pumps. The breakup of precipitates in the centrifugal pump is difficult to distinguish from breakup in the connecting tubing. However, the centrifugal pump, operated under maximum head of 50 kN m-2 (zero flow) showed a similarly rapid rate of breakup. In the peristaltic pump with its much gentler action (maximum rate of shear strain of 70 s-l in laminar flow) no breakup was observed, and there was even evidence for particle growth.
Conclusions During laminar Couette shear, the particle size distribution of soya protein precipitates was found to be strongly dependent on the time of exposure to shear and the rate of shear strain. For exposure times of 300 s the degree of precipitate breakup ranged from a reduction to 70% of the original size at relatively low rates of shear strain of 2000 s-l to disruption to 30% of the original size at rates of shear strain greater than 33 000 s-l. Analysis of the changes in particle size distribution indicated that protein precipitate breakup occurs by fragmentation of the larger particles ultimately leading to erosion of primary particles making up the precipitate. Increased protein precipitate concentrations decreased the overall rate of precipitate breakup due to the greater rates of aggregation-controlled growth. Very rapid breakup of the precipitate was observed in the centrifugal, Mono, and gear pumps over the first few passes. Normally these pumps would be operated on a once-through basis only but with a significant back pressure. This would increase the backflow through the pump head and probably would have a similar effect to increasing the number of passes in the pump head operating at low back pressure. The pressure drop required to feed most centrifugal separators is generally quite small and the main pumping and feed requirement is for good flow control. The objective, in the m e of protein precipitate recovery, must be to minimize the production of fines by minimal
exposure to shear. A peristaltic pump or other positive displacement pumps (gear or Mono), operating under low back pressure and on a single pass basis only, should be suitable for this purpose. Alternatively, pumping systems may be avoided by developing sufficient pressure prior to precipitation in a continuous reactor or by using gravity feed or gas pressure for precipitate displacement. Acknowledgment The financial support given to J. R. Flint, D. Heywood-Waddington, and T. J. Narendranathan by the Science Research Council, United Kingdom, is gratefully acknowledged, as is the support of the National Research Advisory Council, New Zealand, to D. J. Bell. Nomenclature d = pipe diameter, m d, = mean particle diameter, Mm d, = particle diameter at x % cumulative weight oversize, Mm D = diameter of pump impeller, m f = Fanning friction factor N = impeller rotation speed, rev s-l LI = average linear pipe velocity, m s-l Greek Letters 6 = pump gap width, m y = rate of shear strain, = power per unit volume, W m-3 p = viscosity, N s m-2 p = density, kg m-3 Literature Cited Bell, D. J.; Dunnill, P. Biotech. Bioeng. in press, 1981. Bell, D. J.; Heywood-Waddington, D.; Hoare, M.; Dunnill, P. Biotech. Bioeng. 1982, 24, 127-141. Conti, R.; Nienow, A. W. Chem. Eng. Sci. 1980, 3 5 , 543-547. Davies, J. T. "Turbulence Phenomena". Academic Press: London and New York, 1972; p 54. Glasgow, L. A.; Luecke, R. H. Ind. Eng. Chem. Fundam. 1980, 19, 148- 156
Hoare, M.-?rans. Inst. Chem. Eng. 1982a, 6 0 , 79-87. Hoare, M. Trans. Inst. Chem. Eng. In press, 1982b. Karabelas, A. J. AIChE J . 1978. 22, 765-771. Levich, V. G. "Physic0 Chemical Hydrodynamics", Prentice-Hall, 1962; Chap ter 5. Melander, W.; Horvath, C. Arch. Biochem. Biophys. 1977, 183, 200-215. Parker, D. S.; Kaufman, W. J.; Jenkins, D. J. Sanit. Eng. Div. Am. SOC. Civ. Eng. 1972, 98 (No. SAl), 79-99. Rushton, J. H.; Costick, E. W.; Everett, H. J. Chem. Eng. Prog. 1950, 46, 467-476. Schlichting, H. "Boundary Layer Theory"; McGraw-Hill; Engelwood Cliffs. NJ. 1968. van der Tempel, M. The Chem. Eng. 1977, No. 317, 95-97. Van Wazer, J. R.; Lyons, J. W.; Kim, K. Y.; Colwell, R. E. "Viscosity and Flow Measurement", Interscience; New York, 1963. Virkar, P. D.; Hoare, M.; Chan, M. Y. Y.; Dunnill, P. Biotech. Bioeng. 1982, 24. 871-887. Virkar; P. D.; Narendranathan, T.J.; Hoare, M.; Dunnill, P. Biotech. Bioeng. 1981, 23, 425-429.
Receiued for review July 13, 1981 Accepted March 1, 1982
