Blotechnol. hog. 1992, 8, 479-485
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ARTICLES Recovery of Propionic and Acetic Acids from Fermentation Broth by Electrodialysis Anthony J. Weier,+Bonita A. Glatz,f and Charles E. Glatz*J Departments of Food Science and Human Nutrition and Chemical Engineering, Center for Crops Utilization Research, Iowa State University, Ames, Iowa 50011
Acetic and propionic acids can be produced by fermentation. In this process, acid recovery is important both for producing a concentrated product from the broth and for removing acid from the broth to reduce product inhibition during the fermentation. The purpose of this work was to characterize the performance of an electrodialysis unit for carrying out both tasks. Current efficiencies, water transport, and product concentrations were determined both for model solutions of the acids and for fermentation broths to determine the limiting factors in using this separation method. In addition, observations were made of fouling behavior of the membranes in this use. The highest concentrations of product were obtained by contacting with the most concentrated feed and doing so in the absence of competing ions. For the range studied here, a maximum concentration of 13.8% (w/v) sodium propionate was obtained. In the batch recycle mode, final concentrations in the concentrate were reduced, but acid recoveries were improved by the presence of other ions because the low-current shut-off was delayed. The use of electrodialysis for producing a concentrated acid product is not promising unless salt levels in the medium can be reduced and acid levels increased. However, electrodialysis can be used to maintain low acid levels in the fermentor during extractive fermentation.
Introduction Astumbling block in the use of fermentation to produce volatile organic acids, such as acetic and propionic, is the difficultyof their recovery from the generally dilute broths in which they are produced. A typical fermentation using Propionibacterium to produce the mixed acids results in acid concentrations in the range of 20-40 kg/m3. Distillation encounters an azeotrope at atmospheric pressure (Horsley, 19731, and the 141 “C boiling point of propionic acid is above that of water. Drying of the nonvolatile salts of the acid would be feasible only after some prior concentration step. Electrodialysis offers a potential means of concentrating the salts while at the same time selectivelyremoving them from the nonionic components of the broth. Nishiwaki (1972) included sodium acetate among a number of salts studied for concentration by electrodialysis. The feed was not described, but a final concentration of 20.8% (w/v) was reported paired with a 7.4% depleted product. Since the fermentation is usually performed at neutral pH (e.g., Boyaval and Corre, 1987), the ionized forms of the acids predominate among the products obtained. After removal of the cells from the both, typical contaminants would be residual substrate sugar and salts from the original medium. Electrodialysis could be employed at that point simply for product recovery. In an extractive
* Author to whom correspondence should be addressed.
+ Department of Food Science and Human Nutrition. f
Department of Chemical Engineering. 8756-7938/92/300&0479$03.00/0
fermentation, on the other hand, electrodialysis would be used throughout the course of the fermentation to keep acid levels low and control the pH. Substrate levels would be higher in this system. In either case, the broth fed to the electrodialysisunit would be separated into an enriched acid product (concentrate) and an acid-depleted broth. Previous studies of the electrodialysis of fermentation broths have included amino acid removal to increase the productivity of an L-lysine fermentation (Nomura et al., 1987a)and extraction of itaconic (Playne, 1985)and malic acids (Sridhar, 1988). Perhaps the most studied case is that of fermentation to produce lactic acid. Hongo et al. (1986) extracted lactic acid during the course of the fermentation for product recovery and pH control. Adhesion of microbial cells to the anion-exchange membranes impaired performance, but acid recovery was 90 % . These and other similar studies (Czytko et al., 1987; Nomura et al., 1987b) all report significant enhancements in the productivity of fermentation when the fermentation is carried out in the extractive mode. Boyaval et al. (1987) preceded electrodialysis with ultrafiltration to reduce membrane fouling (cellswere removed in the ultrafiltration step and recycled to the fermentor). This work was extended to recovery of propionic acid by Boyaval and Come (1987). They reported depletion of the broth concentration of propionic acid from 20 to 3 kg/m3while obtaining a “concentrate” of 7 kg/m3. A coupling of microfiltration and electrodialysis to a continuous immobilized cell fermentation (von Eysmondt and Wandrey,
0 1992 American Chemlcai Society and American Institute of Chemical Engineers
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1990) led to a several-fold increase in productivity and product concentration of acetic acid. A measure of the efficiency of acid transport is the fraction of the total current passing through the device accounted for by transport of acid to the concentrate. Sugar would enter the product only as a result of concentrationdriven diffusion across the membrane. Other salts would also be driven across by the electropotential gradient and thus reduce the current efficiency. Current efficiency can be calculated as (Meller, 1984)
Table I. Compositions of Feed Solutions
feed 1 2
3
4
F z ( V J j ~ f-j ViZjC,)lOO E=
I
(1)
NJ)
at
where V and c can be based on either the feed or concentrate side and the summation is over the concentrated (ionic) components. In addition to cotransport of salts, concentration-driven back-diffusion of propionate and acetate from the concentrate to the feed side of the membrane and current leakages bypassing the membranes also act to reduce current efficiency. A second measure of performance is the level of concentration that can be achieved. Several factors combine to limit the final concentration. One is that the net driving force for transport will be zero when the electropotential gradient across the membrane is offset by the counteracting concentration gradient. From the thermodynamic expression for electrochemical potential, this equilibrium concentration difference is related to the electropotential difference by
R T In
C
= FZiAO
cd
If all the electropotential gradient were confined to the membrane, this would provide for a very large concentration of product. However, in operation, a large portion of the potential gradient results from cell resistance and concentration polarization. Countercurrent staging of electrodialysis such that the final concentrate exits opposite the incoming broth will then result in the highest concentration. Since electrodialysis units operate with recycle circulation loops to maintain high crossflow for better mass transfer (which reduces concentration polarization), true countercurrent operation is not achieved, but it can be approximated by the use of multiple units in series. A second limit to concentration is dilution by the electrolyte introduced on the concentrate side to complete the current path and to carry away the transferred ions. The amount of diluent can be reduced essentially to zero, but water can still enter the product stream by two other routes. One is the cotransport of water with the ions. The amount of such water transport depends on both the membranes and the ion. Because of this electroosmotic water flux, a maximum concentration, independent of applied potential, is that given by the ratio of the ion to the cotransported water. Ponomarev et al. (1984) have determined that this is an important limitation in the concentration of brine. Additional water can enter the concentrate side as a result of osmosis since the osmotic pressure will be higher on the concentrate side. Hence, the mode by which dilution of the concentrated product can best be avoided is the use of only the electroosmotic water flux to displace concentrate from the cell once the unit is initially filled. This strategy has been used for producing brine from sea water (Nishiwaki, 1972). It is a different strategy from that used in the more
A
B UB B1.5/1 B3 B3/2
Components sodium propionate sodium propionate sodium acetate sodium propionate sodium acetate NaCl KzHP04 sodium propionate sodium acetate NaCl KzHP04 glucose sodium propionate sodium acetate sodium propionate sodium acetate sodium propionate sodium acetate sodium propionate sodium acetate sodium propionate sodium acetate sodium propionate sodium acetate
concentration M 1.50 0.156 1.50 0.156 1.00 0.122 1.50 0.156 1.00 0.122 0.17 0.029 0.08 0.005 1.50 0.156 1.00 0.122 0.17 0.029 0.08 0.005 1.00 0.055 0.50 0.052 0.60 0.073 0.90 0.094 0.70 0.085 0.90 0.094 0.70 0.085 1.500 0.156" l.w 0.122" 3.w 0.312" 0.70 0.085 3.000 0.312" 2.w 0.244"
% (w/v)
a Sodium propionate and/or sodium acetate were added to these preparations to adjust their concentrations to the stated levels.
common application of electrodialysis for desalination of sea water. In the latter application, the product of interest is the desalinated water. The concentrate from desalination is a brine only slightly more concentrated than the sea water. It was the purpose of this work to characterize the performance of an electrodialysis unit for producing a concentrated solution of the salts of acetic and propionic acids from a fermentation broth. Current efficiencies, water transport, and product concentrations were determined for model solutions of the acids and fermentation broths to determine the limiting factors in using this separation method. In addition, observations were made of the fouling behavior of the membranes in this use.
Materials and Methods Materials. Table I summarizes the compositions of the model acid solutions and the fermentation broths. Model solutions were prepared from analytical grade salts dissolved in distilled, deionized water with the pH adjusted as necessary by the addition of 4 N HC1or 5 N NaOH. The broths were from the fermentation by Propionibacteria acidipropionici of a medium consisting of 6 kg/m3of yeast extract (Difco Laboratories, Detroit, MI), 2.87 kg/m3 of trypticase peptone (Baltimore Biological Laboratories, Inc., Cockeysville,MD), 18kg/m3of sodiumlactate (Fisher Scientific Co., Chicago, IL), 1.67 kg/m3of NaC1, and 0.83 kg/m3of KzHPO4. Two batches of broth were used. One was from a 10 dm3 fermentation that produced 0.05 M propionic acid (broth A). The other was a pooling of seven 0.5-1 dm3 fermentations that produced 0.09-0.16 M propionic acid (broth B). Cells had been removed by centrifugation at 8700 G for 25 min at 5 OC. Broths not used immediately were frozen at -70 "C. Some portions were ultrafiltered in a stirred-cell system (Model 2000, Amicon Co., Lexington, MA) equipped with a 20000 MWCO polysulfone membrane (broth UBI, while others hadtheacidlevels increased (brothsB1.5/1,B3, andB3/2). Electrodialysis Unit. The laboratory-scale electrodialysis unit (Medimat 5, Ionics, Inc., Watertown, MA) consisted of a fixed-voltage power supply (11V, 800 mA
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maximum current, low-current shut-off a t 100mA),a fixedrate peristaltic pump (flow rate 0.017-0.020 dm3/min), and a membrane stack. The stack consisted of alternating cation- and anion-exchange membranes (Ionics CR61CZL386 and AR103QZL386, respectively) separated by tortuous-path polyethylene spacers to constitute two-cell pairs, each with one concentrating and one depleting channel. The two-cell pair provided a total effective cross m2. Electrolyte solution (0.05 M Na2section of 3 X SO4) flowed through the channels adjacent to each electrode. Operation was at room temperature. ElectrodialysisRuns. Electrolyte and product streams were always recirculated. Two feed side modes of operation were used. In the (Lone-passnmode, the feed solution makes only a single pass through the depleting channel. The acid level in the concentrate then rises to a plateau (for that feed) which simulates the concentration that would be achieved in purely countercurrent operation. In the recycle mode, feed solution is continually recycled, i.e., single-stage batch recovery. At the start of a run, the product side solution volume was 0.01 dm3 (the minimum amount required to fill the unit and have a workable volume in the reservoir), and the electrolyte volume was 0.1 dm3. The feed volume was 0.1 dm3in the recycle mode and varied between 1.5 and 3 dm3 for one-pass operation. The product side was initiallyfiied with feed to minimize osmotic effects. The product reservoir was a 0.1 dm3 graduated cylinder to allow monitoring of the product volume. Voltage was applied 2 min after circulating flow had begun. In the recycle mode, runs were terminated when the current dropped to 100mA. One-pass runs lasted for approximately 150 min for the model solutions and 90 min for the broths, sufficient time for the conductivity of the product stream to stop rising. Except where noted, all run conditions were dm3) were obtained from replicated. Samples (3 X both reservoirs a t the beginning and at the end of the experiment and from the product reservoir every 30 min during the runs, and they were stored in screw-capped glass vials a t 4 "C before preparation for gas chromatography. All electrodialysis runs with the model solutions were carried out twice, except for those using solution 4, which were done three times in the one-pass mode and five times in the recycle mode. With the broths, only B1.5/1 was replicated in the one-pass mode, while all but A were replicated in the recycle mode. The required feed volume and broth supply limited the number of replications possible in the one-pass mode. Before and after each run, the system was cleaned by circulating (2-5 min) and draining, in the following order, distilled water, 0.1 N NaOH, distilled water, 0.1 N HC1, and distilled water. The membrane stack was stored in 15% (v/v) aqueous ethanol. Intermittently, a standard batch desalting run of a 0.2 M NaCl feed was run to check for any deterioration in membrane performance. The time to desalt was the measure of performance. Analyses. During the run, product volume and conductivity (Fisher Model 09-327 digital conductivity meter) were monitored, as conductivity provides a convenient indirect measure of ionic concentration. Current was monitored on a strip-chart recorder (Recordall Series 500, Fisher Scientific Co.), and cumulative charge transported was calculated as the area under the current vs time plot by cutting out and weighing the chart paper traces. Propionic and acetic acid concentrations of the samples were determined by gas chromatography (Series 3700, Varian Associates, Inc., Walnut Creek, CA) using a
40 1
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Figure 1. (A) Concentration of propionic acid (as sodium propionate) in the concentrate stream for one-pass operation at , solution 1;A, solution 2; pH 7 for the four model solutions: . 0, solution 3; solution 4. Standard errors of means: 0.29 (with time for given solution) and 0.25 (with solution for a given time). (B) Concentration of acetic acid (as sodium acetate) in the concentrate stream for one-pass operation at pH 7 for the three model solutions: A, solution 2; 0, solution 3; 0, solution 4. Standard errors of means: 0.26 (with time for given solution) and 0.18 (with solution for a given time).
+,
prepacked '/*-in. stainless steel column with 15% FFAP on Chromosorb W-AW, 80/100 mesh packing (Chemical Research Supplies, Addison, IL). The column, injector, and detector temperatures were 140, 220, and 250 "C, respectively. For injection, the sample was acidified by the addition of 4 N HC1, and pure valeric acid was added as an internal standard. Volume ratios of the three components were 1:0.3:0.25. Ratios of peak areas (peak height times peak width at ' 1 2 peak height) were correlated to acid concentration using standard curves prepared by the analysis of known samples of 1,2, and 3% (w/v)sodium acetate and sodium propionate. All samples were assayed in triplicate. Statistical analyses for comparisons of electrodialysis performance were based on the Waller-Dunken K-ratio t-test, carried out using the SAS program WALLER (SAS Institute, Inc., Cary, NC). Results Model Solutions. Figure 1A,B shows the rise in product concentration with time for one-pass runs with the four model solutions a t pH 7; the forms are typical of those a t all pH values. Since the propionate concentration is the same for all four feeds, what we are observing in Figure 1A is a lowering of the concentration in the recovery stream as a result of cotransport of acetic acid and salt
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Table 11. Concentration and Efficiency Results for the One-Pass Mode Electrodialysis of Model Solutions final concentrationa NaP NaA final water current efficiency ( % ) solution pH % (w/v) M % M NaP/NaAb trans.no.C NaP NaA NaP+NaA 1 1
1 2 2 2 3 3 3 4
7.0 6.0 4.9 7.0 6.0 4.9 7.0 6.0 4.9 7.0
13Bd 12.P 5.7f 9.18 7.7h 3.6' 6.lf 5.v 2.9 5.3
1.436d 1.249 0.593f 0.9478 0" 0.374' 0.635f 0.572f 0.239 0.552
5.0" 4.gd 4.2d 4.3d 4.od 1.8O 5.8
0.609 0.585d 0.512d 0.524d 0.4Nd 0.219 0.707
21.6 29.8 26.5 30.4 28.9 21.6 21.6 21.5 19.7 18.6
1.56 1.37 0.730 1.21 1.17 1.09 0.781
71.0" 74.9 30.7fJ 58.8d.0 47.9f 16.2h 30.dJ 29.58 8.8h 29.4
71.0" 74.9 30.7O 97.4d 83Ad 31.1e 53.41 49.g 17.68 83.9
38.6d 35.6d 149f 23.4O 20.3ef
8.V 54.5
NaP is sodium propionate and NaA is sodium acetate. Initial ratio = 1.28. Water transportnumber is moles of water transferred/current equivalents transferred;moles of water were estimated from volume change of recovery solution. d-j Numbers with the same superscript me not different baaed upon the Waller-Dunken K-ratio t-test ( K = 100).
and the electroosmotic water flow associated with each. The addition of the nonionic glucose is seen to have little effect. Table I1shows the final concentrations taken from such runs for both acids at three pH values. The runs at pH 4.9 are close to the pKa values for the acids (4.87 and 4.75 for propionic and acetic acids, respectively),and the lower concentrations reflect the fact that half of the acids will be in the free acid form and, hence, are not transported by the potential gradient. In most cases, no significant selectivity of transport is evidenced between the two ions as the ratios of final concentrations of the two acids do not differ greatly from the value of 1.28in the feed. There are differences at pH 4.9 and in the glucose-containing feed. The difference at pH 4.9 is partly accounted for by propionic acid's lower degree of dissociation (0.51 vs 0.54 for acetic acid); the latter discrepancy results from the unexplained continuing rise in acetate concentration in this run (see Figure 1B). Glucose may be influential by increasing solution viscosity or fouling of the membranes; however, the effects are not clear and we have not pursued the mechanism. The transport numbers for carboxylic acid anions have been reported to decreasewith an increase in carbon number (Dohno et al., 19751, with propionate being about 3% lower than acetate. The current efficienciesof Table I1show that the highest efficiencies (based on acid transferred) are realized when totalacid concentrations are high and competing salts are absent (e.g., solution 2). The effect of salts on current efficiencies for acid recovery is magnified by the higher mobilities of the inorganic salts. Hence, the relatively small mole % of inorganic salts (11% ) brings about a 55 5% reduction in current efficiency for transport of the acid ions. A similar picture emerges from electrodialysis of the model solutions in the recycle mode. Figure 2A,B shows the concentrationin the concentratereservoir as a function of time up to the point at which the current has dropped to 100 mA. As expected, the final concentrations are not as high as those in the one-pass mode. The influence of competing ions on the achievable concentration is not statistically significant for propionate, but the presence of glucose does lower the final concentration. For acetate, the presence of the salts does significantly lower the final concentration (see Table 111). As in the one-pass runs, water transport numbers are in the mid to upper 20s. Current efficiencies are also similar to those in the onepass runs except in the case of solution 3, where efficiency remains high as a reflection of the continued high transfer of propionate. Product recoveries are seen to be in the neighborhood of 80% in all cases.
0
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-E. 3 a? e
.z
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Figure 2. (A) Concentration of propionic acid (as sodium propionate) in the concentrate stream for recycle operation at pH 7 for the four model solutions: U, solution 1; A, solution 2; 0 , solution 3; solution 4. Standard errors of means: 0.29 (with time for given solution) and 0.25 (with solution for a given time). (B) Concentration of acetic acid (as sodium acetate) in the concentrate stream for recycle operation at pH 7 for the three model solutions: A, solution 2; 0, solution 3; 0 , solution 4. Standard errors of means: 0.26 (withtime for given solution) and 0.18 (with solution for a given time).
+,
The ratio of final to initial total acid concentrations at pH 7 ranges from 9.2 to 4.1 for the one-pass runs (and from 4.1 to 2.4 for the recycle mode), representing an important degree of concentration. In absolute terms, the final concentration is limited by the electroosmotic water flux. Water transport numbers reported here are moles of water transported to equivalents of charge. A water transport number of 25 for propionate would mean that propionate cannot be recovered in a concentration greater
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Table 111. Concentration and Efficiency Results for Electrodialysis of Model Solutions at pH 7 in the Recycle Mode final concentration” NaP
NaA
solution
%
M
1 2 3 4
6.2d 6.1dve 6.5d 4.2e
0.645d 0.635dpe 0.677d 0.437e
%
M
recovery ( % ) NaP NaA
waterb trans.no.
current efficiency ( % ) c NaA NaP+NaA 64.2d 64.9 NaP
~
6.od 4.1e 3.6e
0.731d 0.5We 0.439O
74.9 81.5 89.5 75.0
27.2d 28.5d 26.6d 24.7d
83.2 77.0 83.2
62.3d 36.2O 27.d
44.8‘ 47.2e 28.9f
~
~~~
97.1’ 83.4= 56.g
NaP is sodium propionate and NaA is sodium acetate in 5% (w/v) and molar. Water transport number is moles of water estimated from recovery solution volume change. Current efficienciesof sodium propionate and sodium acetate only. d-f Numbers with the same superscript are not different based on the Waller-Dunken K-ratio t-test ( K = 100).
than 1mol of propionate in 25 mol of water (less than 2.2 M). The uncertainties in the transport numbers reported here are large because of the errors in measuring the volume of water transferred. However, it is clear that the water transport number is a t the high end of typical ranges (525) and about twice the value of NaCl (i.e., 8 for Na+ and 4 for C1-) (Shaffer and Mintz, 1980). A check was made to determine whether simple osmosis might account for some of the water transport. Equal volumes (0.05dm3)of distilled water and 12 % (w/v)sodium propionate (representing the most dilute and concentrated streams to occur) were circulated in the feed and product sides of the stack for 2 h with no applied voltage. The net transfer of water, assuming all of the volume increase to be water, in this time was 8 X dm3 or 0.22 mol/h compared to the 0.86 moVh transferred in the recyclemode or the 1.0 mol/h transferred in the one-pass mode. Hence, the osmotic contribution, while not negligible in this worst case scenario, is certainly smaller than the electroosmotic transport. A final effect, back-diffusion, was also assessed in this same worst case experiment. About 22% of the propionate was found to have diffused from product to feed side in the 2 h for an average flux that was 21 and 415% of the average net forward fluxes of propionate during the one-pass and recycle runs, respectively. Such backdiffusion would be responsible for the lower current efficiencies shown by solution 1. Fermentation Broths. The fermentation broths correspond most closely to model solutions 3 and 4. Only relatively low product concentrations are obtained from broths with the lowest initial acid concentrations. A comparison of the 1.5%propionate broth and solution 4 is seen in Figure 3A,B and Table IV. Comparison of the transfer rates of B1.5/1 and solution 4 shows the fluxes for the broth to be only about one-half of those for the model solution, while the current efficienciesare lower still. Given that the final product conductivities are comparable, one concludes that other ions are playing a larger role in the broth than they were in solution 4. While the level of such interference is largely determined by the medium composition and hence might be remedied to a degree, the interference would appear to be more limiting for this application than was the water transport limitation observed with the model solutions. In fact, water transport is seen to be lower with the broth feeds. Only by increasing the level of propionate in the feed (e.g., B3) can the efficiency and product concentration be improved. For the 3% initial propionate concentration, the final concentrations in the recycle and one-pass modes were quite similar. Significant additional concentration with longer operation seems unlikely since the concentration appears to be reaching a plateau. Also, not much additional concentration occurred with the model solutions after 90 min. To use electrodialysis in the extractive mode would require operation at low acid concentration where its use
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Figure 3. (A) Concentration of propionic and acetic acids (as the sodium salts)in the concentrate stream for one-paw operation at pH 7 for the model solution 4 and broth B1.5/1: A, acetate in broth; 0, acetate in solution 4; A, propionate in broth; propionate in solution 4. Standard errors of means for propionate: 0.40 (with time for given solution) and 0.67 (with solution for a given time). Standard errors of means for acetate 0.31 (with time for given solution) and 0.54 (with solution for a given time). (B) Concentration of propionic and acetic acids (as the sodium salts) in the concentrate stream for recycle operation at pH 7 for the model solution 4 and broth B1.5/1: A, acetate in broth; 0, acetate in solution 4; A, propionate in broth; propionate in solution 4. Standard errors of means for propionate: 0.29 (with time for given solution) and 0.61 (with solution for a given time). Standard errors of means for acetate 0.26 (with time for given solution) and 0.43 (with solution for a given time).
+,
+,
to produce a concentrated product does not seem appropriate unless other electrolytes can be reduced in the medium. Operation in the recycle mode (Figure 3B)is actually somewhat better, indicating that once competing electrolytes are removed (during the early portion of the batch recovery) the acid ions can be concentrated. Prolonged operation leads to a slight decrease in the concentration,
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Table IV. Comparison of Electrodialysis Performance at pH 7 for Broth Bl.S/l with That for Model Solution 4
operation
one-pass recycle
solution type B1.5/1 4 B1.5/1 4
finala concentration ( % ) (w/v) NaP NaA 2.6d 5.3d 3.4d 4.2d
final conductivity (mmho/cm)
2.ld 5.8d 2.2d 3.6d
65.8 59.0 46.1 39.4
rate of removal (10-4mol/min) NaP NaA 0.9 2.0 nd
0.9 2.4 nd nd
recovery (%)
current efficiency ( % )
waterb
NaP
NaA trans.no. NaP
NaA
NaP+NaA
ndc nd 72.8 75.0
nd nd 67.1 83.2
12.1d 29.4e 15.8d 27.4d
24.W 54.bd 36.6d 56.3d
20.6d 25.6d 24.Ie
ll.gd 25.1d 20.8d 28.90
nd NaP is sodium propionate; NaA is sodium acetate. * Water transport number is moles of water transferred/current equivalentstransferred; moles of water were estimated from volume change of recovery solution. e Not determined. d,e Numbers with the same superscripts ate not different based on the Waller-Dunken K-ratio t-test (K = 100); recycle and continuous mode data were compared separately.
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0.1
0.2
0.3
Initial Concentration, M
Figure 4. Rate of propionate removal from fermentation broth as a function of the initial propionate concentrationin the broth. The two higher concentrations were obtained by adding propionic acid to the low concentration broth.
0'05 0.04
based on albumin, which will have a different color yield/ mass than small peptides. Use of fermentation broth did result in discoloration and deposition of a slimy layer on the membranes, especially the anion-exchange membrane. Acid and base cleaning removed the slime and lightened the discoloration somewhat. Membrane performance was also satisfactorily restored as judged by the time to complete the desalting of 0.05 dm3 of a 0.2 M NaCl solution. Checks at various times throughout the course of this study (25 testa distributed among the 96 experiments) only once showed a desalting time longer than the manufacturer's criterion of 25 min (run 23); this run was performed directly after a period of storage and an acidtbase rinse reduced the desalting time to 20.5 min. For these cell-free broths, the additional ultrafiltration step did not improve performance. For use in extractive fermentation, where one must remove acid a t the rate at which it is being produced and do so at the desired broth concentration, the most useful design data are seen in Figure 4. This shows the propionic acid fluxes observed for the fermentation broths tested here. These removal rates, combined with a typical fermentor productivity in our laboratory of 3.42 X mol/dm3/h,lead to the membrane area requirements shown in Figure 5(assuming the same operating conditions as in our laboratory unit). Propionic acid levels begin to inhibit growth above 0.1 M.
>
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E m'
$ 0.03 .-lo -z 0
e
2
0.02
Conclusions
B w 0.01
0
0
4
8
12
16
20
Fermentor Size, dm3
Figure 5. Required membrane area to match removal rate to acid production rate as a function of fermentor size and the propionate concentration at which the broth acid concentration is to be maintained U, 0.32 M, 0 , 0.13 M, A, 0.10 M. possibly indicating transfer of an even less mobile ion. Recoveries were quite comparable to those for the model solutions, and concentrations approached those of solution 4. For one of the recycle runs, the protein/peptide contents of feed and products were determined. The initial filtered broth contained 0.8 kg/dm3 of protein, while the final concentrated and depleted streams contained 1.09 and 0.65 kg/dm3, respectively. Since the concentrate stream also doubled in volume (initially 10 mL) during the run, there was significant transfer of peptides. These are likely to be of low molecular weight since the nominal MWCO of the membranes is 300. These numbers should be viewed as estimates since the standard curve for the assay was
The highest concentrations of product are obtained by contacting with the most concentrated feed and doing so in the absence of competing ions. For the range studied here, a maximum concentration of 13.8% (w/v) sodium propionate was obtained. In the batch recycle mode, final concentrations were lower but acid recoveries were improved by the presence of other ions because the lowcurrent shut-off was delayed. The use of electrodialysis for producing a concentrated acid product is not promising unless salt levels in the medium can be reduced and acid levels increased. However, electrodialysis can be used to maintain low acid levels during the fermentation.
Notation Symbols C
E
F Z N t V R T
concentration (mol/L3) current efficiency (%) Faraday's constant, 1608 A-slequiv current (A) number of cell pairs time volume (L3) gas constant temperature
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2 A0
charge number (equiv/mol) electropotential difference (V)
Subscripts concentrated solution d depleted solution i initial solution f final solution i component C
Acknowledgment Our thanks t o Steve Woskow and Sei-Kyoung Park, who provided the fermentation broths, to Earl Hammond, for helpful comments, and to the Iowa State University Biotechnology Council and the Iowa Corn Promotion Board, who funded this work. Literature Cited Boyaval, P.; Corre, C. Continuous Fermentation of Sweet Whey Permeate for Propionic Acid Production in CSTR with UF Recycle. Biotechnol. Lett. 1987,9,801-806. Boyaval, P.; Corre, C.; Terre, S. Continuous Lactic Acid Fermentation with Concentrated Product Recovery by Ultrafiltration and Electrodialysis. Biotechnol. Lett. 1987,9,207-
212. Czytko, M.; Ishii, K.; Kawai, K. ContinuousGlucoseFermentation for Lactic Acid Production: Recovery of Acid by Electrodialysis. Chem. Eng. Technol. 1987,59,952-954;Chem. Abstr. 1988,108,130014s. Dohno, R.;Azumi, T.; Takashima, S. Permeability of MonoCarboxylate Ions Across an Anion Exchange Membrane. Desalination 1975, 16,55-64.
Hongo, M.; Nomura, Y.; Iwahara, M. Novel Method of Lactic Acid Production by Electrodialysis Fermentation. Appl. Environ. Microbiol. 1986,52,314-319. Horsley, L. H. In Advances in Chemistry Series; Gould, R. F., Ed.; American Chemical Society: Washington, D. C., 1973; Vol. 116,p 17. Meller, F. H. Electrodialysis and Electrodialysis Reversal Technoloev: Ionics: Watertown. MA. 1984. Nishiwaki,":In Industrial Processing with Membranes; Lacey, R. E., Loeb, S., Eds.; Wiley Interscience: New York, 1972;pp
83-105. Nomura, Y.; Iwahara, M.; Hongo, M. Application of Electrodialysis Fermentation with L-lysine Fermentation. Nippon Nogei Kagaku Kaishi 1987a,61,1293-1296;Chem. Abstr. 1988,
108,20486g. Nomura, Y.; Iwahara, M.; Hongo, M. Lactic Acid Production by Electrodialysis Fermentation Using Immobilized Growing Cells. Biotechnol. Bioeng. 1987b,30, 788-793. Playne, M. J. In ComprehensiveBiotechnology-the principles, applications and regulations of biotechnology in industry, agriculture and medicine; Moo-Young, M., Ed.; Pergamon: New York, 1985;Vol. 2,pp 731-759. Pomonarev, M. I.; Lokota-Fabulyak, Ya., G.; Grebenyuk, V. D. Concentration of Electrol*s by Electrodialysis. J. Appl. Chem. USSR 1984,56,2416-2418;Zh. Prikl. Khim. 1983,56,
2601-2603. Shaffer, L. H.; Mintz, M. S. In Principles of Desalination; Spiegler, K. S., Laird, A. D. K., Eds.; Academic: New York, 1980; pp 257-357. Sridhar, S. Applications of Electrodialysis in the Production of Malic Acid. J. Membr. Sci. 1988,36,489-495. Von Eysmondt, J.; Wandrey, C. Integrated Product Work-up by Electrodialysis in the Microbial Production of Organic Acids. Chem. Eng. Technol. 1990,62,134-135. Accepted August 5, 1992. Registry No. Acetic acid, 64-19-7; propionic acid, 79-09-4.
