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Continuous Operation of a Pressure-Cycled Membrane Bioreactor D. E. Steinmeyert and M. L. Shuler* School of Chemical Engineering, Cornel1 University, Ithaca, New York 14853-5201
A membrane bioreactor system has been used to continuously ferment glucose t o ethanol with Saccharomyces cereuisiae and extract the product into a solvent, tributyl phosphate. T h e reactor cycles gas pressure t o force a convective flux of nutrient into the cell chamber and product out. The reactor has been run for over 3000 h with minimal deterioration of operating characteristics after the first hours of operation. A corn starch hydrolysate was also fermented, yielding similar results. Zymomonas mobilis was tested as a n alternative catalyst, but it formed filaments that reduce the effectiveness of the pressure cycle. 2. mobilis appears to be less attractive than S. cereuisiae for such a reactor.
Introduction The productivity of traditional biological reactors is limited in part by the rate of regeneration of the catalyst, since cell mass is removed from the reactor with the product. Immobilizing the cell mass within the reactor effectively decouples growth and dilution rates in continuous operation, allowing higher space velocities. Retaining the cells within the reactor also eliminates a cell removal step from the product recovery train. One method used to immobilize cells within a reactor has been to segregate them behind one side of a semipermeable membrane. Membranes have been used in this fashion in a variety of reactor designs [see the recent review by Cheryan and Mehaia (1986)], most of which may be classified as either stirred vessels with recycle or hollow fiber units. Many biological processes are inhibited by the end product of the pathway, a phenomenon known as feedback inhibition. The classic example of a feedback-inhibited process is ethanolic fermentation by yeasts. Under most circumstances, feedback inhibition hampers the development and commercial utility of bioprocesses affected by it. Feedback inhibition is exacerbated when the catalyst density within the reactor is increased. Though higher productivities may be obtained initially through cell immobilization, this improvement is short-lived unless the product is removed from the system as rapidly as it is made. Product removal schemes are highly product specific. Liquid-liquid extraction has been suggested to be the most attractive method for the removal of ethanol from aqueous solutions (Pye and Humphrey, 19791, but many of the most attractive solvents appear to be toxic when allowed to come into direct contact with the process organism, a phenomenon termed "phase" or physical toxicity (Cho and Shuler, 1986). If t h e solubilitylimited concentration of solvent in the fermentation medium is not harmful, the solvent may still be utilized if it is effectively segregated from the cells. Semipermeable membranes may also be used to separate an aqueous solution from a hydrophobic extractant. A
* Corresponding author. Current address: Kinetek Systems, 11802 Borman Drive, St. Louis, MO 63146. t
8756-7938/90/3006-0286$02.50/0
hydrophobic solvent will wet a hydrophobic membrane but may be prevented from passing through it by keeping the pressure on the aqueous side slightly higher than on the solvent side (Kiani et al., 1984). A hydrophilic membrane with the pressure higher on the solvent side would work equally well, and a hydrophilic/ hydrophobic laminate would remove the need for a pressure differential (Prasad and Sirkar, 1987). Cho and Shuler (1986) have suggested a bioreactor design, termed the multimembrane reactor, in which microporous membranes spatially segregate the reactor into gas, immobilized cell, nutrient, and extractant layers. This arrangement of compartments within the reactor differs from other membrane-based bioreactor systems. The cell suspension is immobilized at high density between hydrophilic and hydrophobic membranes, and a second hydrophobic membrane effectively segregates the solvent from the nutrient layer. Soluble gases enter into and exit from the cell layer through the gas/cell hydrophobic membrane, yet the flowthrough of the fermentation medium is prevented if the pressure differential across the membrane is kept below a critical value. The hydrophilic membrane separates the cells from a nutrient supply stream, across which substrates and products readily diffuse. Another hydrophobic membrane separates the nutrient and extractant layers. The solvent may be effectively immobilized in the pores of this membrane and prevented from forming an emulsion in the nutrient if a slightly higher pressure is maintained on the nutrient side. The multimembrane reactor concept is quite flexible and applicable to any of a variety of fermentations. The model system that has been studied in the reactor is the fermentation of glucose to ethanol by Saccharomyces cereuisiae, with tributyl phosphate (TBP) as the extractive solvent. Cho and Shuler (1986) have demonstrated with batch recycle operation that the toxic effects of this solvent may be minimized with this new design and that extraction greatly increases the rate and extent of reaction. Efthymiou and Shuler (1988) have improved reactor performance with a pressure swing operation, termed pressure cycling, in which the substrate- and productladen suspension medium is convectively forced into and out of the cell layer.
0 1990 American Chemical Society and American Institute of Chemical Engineers
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In the work about to be described, t h e previous experimental results have been extended by operating a modified reactor continuouslyfor long periods of time. The effects of more natural substrates and the alternative catalyst Zymomonus mobilis on reactor performance have also been investigated.
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Materials and Methods Organisms. S. cereuisiae ATCC 24858 (American Type Culture Collection, Rockville, MD) was maintained on slopes of YM agar at 4 "C. The organism was transferred to fresh medium every 6 months. 2. mobilis ATCC 10988 was maintained a t 30 "C on plates of agar containing 20 g of glucose, 10 g of yeast extract, 20 g of agar, and distilled water to 1L a t pH 5.0. The organism was transferred to fresh agar every week. Media and Propagation of Inocula. The composition of the inoculum propagation and fermentation medium for S. cereuisiae was as follows: glucose, 200 g; yeast extract, 10 g; (NH4)2S04, 5 g; KH2PO4, 1.4 g; MgS04.7H20, 1g; CaC1~2H20,0.7g; antifoam (food grade C emulsion, Dow Corning), 60 mg; deionized water to 1 L. A 1-L flask containing 250 mL of fermentation medium was inoculated from agar culture and incubated with mild agitation a t 35 "C for 20 h and then used as inoculum. The composition of the continuous reactor feed medium was as follows: glucose, 350 g; yeast extract, 10 g; (NH4)2S04, 5 g; KH2P04, 1.4 g; MgS04.7H20, 1 g; CaC12-2H20,0.7 g; antifoam (food grade C emulsion, Dow Corning) 60 mg; deionized water to 1 L. To test the effects of natural substrates on reactor operation, the media were modified. The fermentation and feed medium was derived from a solution of enzymatically hydrolyzed corn starch. A 420-g quantity of corn starch (Sigma Chemical Co., containing roughly 75 % amylopectin and 25% amylose) was brought to 1L with deionized water, and the pH of the solution was adjusted to 6.9. a-Amylase (0.75 g; Sigma Chemical Co., from Aspergillus oryzae) was added, and the solution was agitated for 2 h at room temperature. The temperature was increased to 55 "C and the pH dropped to 4.5, and 2.2 g of glucoamylase (Sigma Chemical Co., from Rhizopus mold) was added. The solution was then stirred for 20 h. The unhydrolyzed fraction was then removed by passing the solution through a Millipore AP prefilter. The resulting filtrate was somewhat hazy. The filtrate was autoclaved for 10 min a t 121 "C and prefiltered again. The final filtrate was clear and contained about 220 g of glucose/ L. To each liter of this solution were then added the following: (NH&S04,6.5 g; KHzPO4,1.5 g; MgS04-7H20, 1g; CaC12-2H20,0.7 g; yeast extract, 10 g; and antifoam, 60 mg. The pH was adjusted to 4.0 prior to autoclaving. This final solution was used as both the fermentation and feed medium. When 2. mobilis was used, modifications to the inoculum preparation procedures were made. The cells were transferred from a plate to a preseed medium containing 100 g of glucose/L and 10 g of yeast extract/L at pH 5.0. The preseed was incubated with mild agitation at 30 "C for 24 h. The inoculum medium, containing (per liter) 100 g of glucose, 10 g of yeast extract, 1g of KH2P04,l g of (NHJ2S04, and 0.5 g of MgS04-7H20 at pH 5.0, was then inoculated with a 10% inoculum of the preseed medium and incubated for 18 h a t 30 "C. The fermentation medium was the same as the inoculum medium, as was the feed, except the glucose concentration was increased to 350 g/L. Assays. Glucose was analyzed by an enzymatic method (Calbiochem-BehringS.V.R. glucose test kit). Ethanol was
Solvent
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Figure 1. (a) Overall schematic of reactor system. (b) Details of reactor system and placement of liquid-level sensing probes. (c) Details of nutrient recycle vessel and reactor support system. (d) Details of configuration of extraction loop.
also analyzed by an enzymatic method (Sigma Chemical Co., 322-UV EtOH assay kit). Biomass was determined by collecting the cells from a known volume of medium onto preweighed filters, washing with several volumes of deionized water, and reweighing after drying to constant mass. Reactor System. A diagram of the reactor system used in the experiments to establish long-term continuous operation is given as Figure la. Solvent extraction was performed outside the reactor by pumping the nutrient medium from the recycle vessel through an extraction unit and back to the reservoir. This procedure (when rapid nutrient recycle is used) is equivalent to the original design except that the external extraction membrane allows the
288
investigator to more easily alter the ratio of cell/nutrient membrane area:nutrient/TBP membrane area. Millipore's 0.22-pm Duropore membrane was used as the cell/nutrient membrane, and Celanese Celgard K442 was used for the nutrient/solvent membrane, as in previous experiments. T h e surface area of the cell/nutrient membrane was 100 cm2, and the total surface area of extraction membrane was 300 cm2. The cell/nutrient membrane was supported by nylon and steel mesh screens on both sides. These supports increased the rigidity of the membrane, preventing bulging and rupture during longterm continuous operation with pressure cycling. The gas/ cell membrane used previously (Cho and Shuler, 1986; Efthymiou and Shuler, 1988) was replaced in the current work with a liquid-level controller (Dyna-Sense LiquidLevel Controller, Model 7186, Cole-Parmer Instrument Co., Chicago, IL). This membrane had acted as a control device (preventing overflow of the cell layer during the fill phase) and as vent for produced carbon dioxide. Thus, in the current work there was no physical separation of the overhead gas and the cell suspension. The pressure cycle is implemented by selectivelyopening and closing the pinch valves (Trombatta Corp., Milwaukee, WI) shown in Figure lb. During the fill phase, the valves on the nutrient return line and on the gas supply line are closed and the valve on the gas outlet line is open. The positions are reversed during the empty phase. The movement of gas into and out of the gas layer was therefore limited to a few seconds immediately after the switch of the phase. Conversely, the flow of nutrient medium through the nutrient layer continued during the empty phase. The opening and closing of the valves is controlled by the liquid-level controller. The empty phase is started when the medium touches the tip of the high-level probe and continues until the liquid level drops below the tip of the low-level probe, at which point the cycle is reversed and the fill phase begins. The ground probe is always immersed in the medium. Assembled reactor systems were sterilized with 75% ethanol solution, pH 2, for at least 24 h. All other components were autoclaved at 121 "C for 20 min. The fermentation and feed media were autoclaved within their respective reservoirs. The temperature and pH of the nutrient recycle were maintained a t 35 OC and 5.0 for the 2. mobilis fermentation. Each fully assembled and completely sterilized system was operated with an active pressure cycle for at least 24 h prior to inoculation to verify sterility. Reactor Operation. In the experiments establishing long-term continuous operation, the reactor was inoculated with 150 mL of inoculum containing 1.0 g of S. cereuisiae. The initial volumes of fermentation medium and of fresh TBP were 1650 and 2000 mL, respectively. The feed was started 45 h after inoculation at a flow rate of 4.5 mL/h and was manipulated over the course of the experiment to maintain the concentration of glucose in the nutrient recycle vessel a t a constant level around 80 g/L. The average feed rate over the first half of the experiment was approximately 5.9 mL/h, and over the second half, 3.8 mL/ h. TBP was withdrawn and replaced with fresh solvent regularly, averaging about 1L of TBP every 2 days. Cells were withdrawn from the reactor periodically (when their concentration began to interfere with the operation of the liquid-level controller) by taking larger assay volumes from the cell layer. During extended operation, this interval was about 500 h. Samples were withdrawn from the cell layer during the empty phase. For the experiments testing the response to corn hydrolysate, the cell layer was inoculated with 250 mL of in-
Biotechnol. Prog., 1990, Vol. 6,No. 4 0
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Figure 2. Concentration of ethanol in the cell, nutrient, and
solvent layers in the long-term continuous experiment.
oculum, containing 1.2 g of cells. The initial volumes of fermentation medium and TBP were 1300 and 1000 mL, respectively. The feed was started at 3.75 mL/h at 71 h, and increased to 4.3 mL/h a t 142 h and 6.9 mL/h at 241 h. A 500-mL portion of TBP was removed at 21 h and replaced with 1500 mL of fresh solvent. TBP (1.5L) was changed approximately every 48 h thereafter for the duration of the experiment. The experiment was ended voluntarily at 260 h, at which point the cell layer contained 2.9 g of cells. For the experiment testing the use of 2. mobilis the cell layer was inoculated with 250 mL of inoculum, containing 0.22 g of cells. The initial volumes of fermentation medium and TBP were 1500 and 1000 mL, respectively. At 23 h, 500 mL of TBP was removed and 1.5 L of fresh solvent was added. TBP was changed again at 73 and 119 h (1.5-L portions) and at 167 h (1-L portion). The feed was started at 23 h at 9 mL/h, discontinued at 73 h, restarted at 95 h a t 5 m L / h , decreased to 3.5 m L / h a t 119 h , and discontinued at 148 h.
Results and Discussion The reactor was operated continuously a t steady state for over 3000 h. The residual concentrations of glucose and ethanol in the system over the course of one fermentation are illustrated in Figures 2 and 3. As is evident from Figure 2, the concentration of ethanol in the cell layer was always somewhat higher than in the nutrient layer. Conversely, the concentration of glucose was lower in the nutrient than in the cell layer (Figure 3). These data indicate that the reactor was to a degree mass transfer limited, increasingly so as the experiment continued. A gradient of some degree is unavoidable, but since it represents an operational disadvantage, the reactor is pressure cycled to minimize it. The effectiveness of the cycle in accomplishing this task is a function of the rate of flux across the membrane and the rate of reaction within the cell layer. The concentration of ethanol in the solvent was always lower than in the nutrient, as is mandated by the distribution coefficient of ethanol between an aqueous solution and TBP. The sawtooth pattern of the TBP data is the result of the batchwise removal of spent solvent and replacement with fresh TBP. The increasing concentration differences between the cell and nutrient layers over the course of the experiment were direct results of fouling of the cell/nutrient membrane. As the membrane fouled, less volume could be exchanged per unit time with the same pressure drop. Most of t h e fouling of t h e membrane took place during t h e period prior t o inoculation w h e n sterile m e d i u m was
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Biotechnol. Prog., 1990,Vol. 6, No. 4 ,
glvcorr In
cell loyer ~ I Y C O SinC nulrienl layer
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CELL U Y E R GLUCOSE. NUTRENT U Y E R GLUCOSE CELL LAYER E l W L NUTRIENT LAYER ETHANOL A SOLMNT ETHANOL.
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Figure. 3. Concentration of glucose in the cell and nutrient layers in the long-term continuous experiment.
Figure 4. Concentrations of glucose and ethanol in the cell, nutrient, and solvent layers in the corn starch hydrolysate continuous experiment.
pressure cycled. The rate of flow through the membrane when cycling first began was 168.2 mL/(cm2.h) and decreased to 7.2 mL/(cm2-h)by the time of inoculation but dropped much more slowly thereafter, to 2.1 mL/ (cm2-h)a t 172 h a n d 1.9 mL/(cm2.h) a t 1128 h. Given the rapid drop prior to inoculation, the drop caused by the addition and initial growth of the cells (partly a function of polarization rather than true fouling) was surprisingly small, since at 22 h the rate of flow was 3.9 mL/(cmZ.h). The fill phase of the cycle was typically twice as long as the empty phase, an unexpected result, since the pressure differential during the fill half (12 psi) was twice that of the empty phase (6 psi). Given twice the pressure drop during the fill phase and the polarizing effect of the cells during the empty phase, the fill would be expected to take less than half the time needed to empty, the reverse of the experimental observation. This effect was investigated in a series of test cell experiments (results not shown), which demonstrated that Millipore’s 0.22-pm Duropore membrane exhibited direction-dependent differences in flux, a not uncommon result of the method of manufacture of this type of membrane (Brock, 1983). However, we were unaware of this directionality until after several experiments were completed. Though fouling of the cell/nutrient membrane lowered the level of production, the productivity of the reactor in this experiment was ultimately limited by the rate a t which ethanol was extracted, a function of the rate a t which TBP was changed and of the transfer characteristics of the extractors used. These were chosen for experimental ease, and therefore the experimental results do not represent the optimal or maximal production capability of the reactor [see Steinmeyer and Shuler (1990)l. A t the end of the experiment, the cell/nutrient membrane was removed from the reactor and visually examined. It was not pleated or stretched and showed no other signs of physical damage. These results, along with the fact that no cells were detected in the nutrient recycle a t any point during 3000 h of operation, indicate t h a t membrane integrity may be maintained with the aid of a rigid support. This finding was confirmed in other experiments of shorter duration (1460 h). In these experiments, great emphasis was placed on excluding cells from the nutrient layer and recycle during long-term operation. Describing the operation of the system mathematically would be much more difficult if cells were allowed to pervade the system freely. Also, if the reaction were occurring in both the cell and nutrient layers, the experimental results would become ambiguous. Finally, it was feared that cells would adhere to and foul the extractive membrane, a phenomenon reported
elsewhere (Matsumura and Markl, 1986), diminishing extractive capability and reactor performance. However, sterility of the nutrient stream is probably not an absolute operating requirement in a practical commercial-scale system. In such a case, a membrane with a bigger pore size might be used, allowing more rapid volume exchange and better mixing of the cell and nutrient layers, decreasing or effectively eliminating the reactor’s mass transfer limitation and greatly improving reactor performance. Other experiments using a 0.45-pm pore size version of the Duropore membrane were unsuitable for our laboratory purposes due to poor retention of cells but did greatly facilitate exchange of fluid between compartments. Our initial experiments were with an idealized feed stream. A practical system would utilize less defined substrates. A medium derived from a hydrolysate of corn starch was tested. The concentrations of glucose and ethanol in the cell, nutrient, and solvent layers are given in Figure 4 as functions of time. In most respects, the experimental results were very similar to those of the longterm continuous experiment; the substrate seems to have had no deleterious effects on operation. The rate of volume exchange between the cell and nutrient layers was 2.7 mL/(cm2.h) a t 241 h, down from an initial value of 108 mL/(cm2.h) a t start-up but slightly better than that of the long-term experiment a t the same point in time [2.0 mL/(cm2-h)]. Again, most of the decrease occurred in the first 24 h after start-up. Since mixing was somewhat improved in this experiment, the concentration differences (glucoseand ethanol) between the cell and nutrient layers were smaller than in the longterm continuous experiment. On average, the concentration of glucose was 19.2 g/L higher in the nutrient than in the cell layer in the current experiment, compared to 24.9 g/L during the same period of time in the longterm experiment. Similarly, the concentration of ethanol was 6.3 g/L higher in the nutrient than in the cell layer in the current experiment and 12.7 g/L higher in the longterm experiment. One aspect of this experiment that differed somewhat from the long-term experiment was the slower development of the glucose consumption rate. In the current experiment, the inoculum was 20% greater than in the longterm experiment, but the glucose consumption rates were comparable over the first 2 days of operation of both reactors, both averaging about 1.5 g/h. Although the rate had increased in the long-term experiment to 2.7 g/h by 260 h, the rate had increased to about 2.1 g/h by the end of the experiment with the hydrolysate. The lower rate might be t h e result of t h e higher initial glucose
Biotechnol. Prog., 1990, Vol. 6, No. 4
290 Zymomonos mobllis Fermentation CELL U Y E R GLUCOSE, NUTRIENT U Y E R GLUCOSE 0 CELL U Y E R ETWNOL. NUTRIENT U Y E R ETHANOL A
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Figure 5. Concentrations of glucose and ethanol in the cell, nutrient, and solvent layers of the Z. mobilis continuous
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concentrations in this experiment, which in conjunction with other sugars not detected by the glucose assay would have produced an osmotic environment that would have slowed the rate of growth a t the start of the experiment. If this was the cause of the lower consumption rate, then the lower productivity is really more a function of initial conditions than the result of a limitation imposed by the new substrate. As in the long-term experiment, no cells were detected in the nutrient recycle at any point. A preliminary reactor experiment with 2. mobilis was conducted with the same conditions and reactor system as in the continuous experiments with S. cereuisiae, but the reaction stopped shortly after inoculation because, as was concluded after a series of characterization experiments (results not shown), glucose concentrations greater than 200 g/L significantly inhibit the growth of our strain, ATCC 10988. This difficulty was avoided in the subsequent experiments by reducing the initial concentration of glucose in the fermentation medium to 100 g/L. The concentrations of glucose and ethanol in the cell, nutrient, and solvent layers are given as functions of time in Figure 5. The concentration differences of glucose and ethanol across the celllnutrient membrane remained relatively constant over the course of the experiment, in spite of a gradual slowing of the pressure cycle rate. The concentration of glucose averaged 66 g/L higher in the nutrient layer than in the cell layer. Similarly, ethanol averaged 30 g/L higher in the cell layer. These differences are significantly higher than those observed in the longterm continuous experiment (24 g/L for glucose and 12 g/L for ethanol) during the 200 h after start-up. The cause of the greater differences than those observed in the long-term experiment was an increasingly slow volume exchange between the cell and nutrient layers. The average rate of flow through the cell/nutrient membrane over the course of a cycle decreased to 9.1 mL/(cm2.h) by 23 h but dropped further to 0.5 and 0.4 mL/(cm2-h)by 73 and 167 h, respectively. A t the same point in the longterm continuous experiment, the rate was 2.1 mL/ (cm2.h) (165 h), more than 5 times as high. The length of the fill phase of the cycle was comparable to that of the long-term experiment for a similar exchange, but the empty phase was 20 times longer. This great difference was probably caused by the tendency of Z y m o m o n a s to form long filaments. Z y momonas is a rod, usually 2-6 pm in length, occurring singly and in pairs, but in most strains, some of the cells become elongated to as much as 28 Fm in length (Swings and de Ley, 1977). At the end of the current experiment,
the cells also tended to be elongated. Though no direct measurements were taken, it was estimated that many of the cells were 5-10 times as long as a t the start-up. The low rate of volume exchange during the empty phase was likely due to the formation of a filamentous web of cells a t the surface of the membrane, causing a large pressure drop. Since the total drop across both the “fouling layer” (due to cell debris) and the membrane is constant, the rate of flow across the membrane would decrease as the pressure drop across the cells increased. Krug and Daugulis (1983) have also reported difficulties in operating a reactor packed with Zymomonas immobilized on resin beads because of filament formation and resultant pressure drop increases. The rate of glucose consumption over the first 48 h of the experiment averaged 3.5 g/ h, roughly two-thirds that of the long-term continuous experiment. The rate dropped dramatically thereafter, averaging about 1g/h. The low rate was mostly due to the difficulty in pressure cycling the medium. Since t h e exchange was low, the concentration of ethanol in the cell layer averaged 61 g/L from the second day of operation onward. Jobses and Roels (1986) report that at this concentration the specific glucose consumption rate of ATCC 10988 is reduced to onethird of the maximum rate. Despite the smaller size of this organism, it was retained within the cell layer, and the nutrient layer again remained sterile over the course of the experiment.
Summary The efficacy of the multimembrane concept has been demonstrated through long-term operation with sustained productivity. The reactor may be pressure cycled for long periods of time with minimal damage to the cell/nutrient membrane if the membrane is well supported. The membrane fouled quickly a t t h e beginning of t h e experiment-even in sterile media-but additional fouling of the same membrane (due to cellular growth) was minimal over the course of the experiment. It was concluded that reactor performance is essentially the same whether a corn starch hydrolysate or the more ideal glucose-salts-yeast extract medium is used as the fermentation and feed medium. The rate and degree of fouling are comparable in either situation, and the productivities were similar, though somewhat lower with starch hydrolysate. These results suggest that the results of future experiments with the glucose-salts-yeast extract medium should be comparable to those which would have been obtained if a corn starch hydrolysate had been used and that the reactor should be expected to be able to ferment the hydrolysate continuously for long periods of time in an actual process. Although potentially much more productive than S. cereuisiae, 2. mobilis was less productive in our reactor due to a lower rate of volume exchange between the cell and nutrient layers, resulting in a decreased ethanol removal rate and consequently higher feedback inhibition. Unless the formation of filamentous cells could be suppressed over the long term, either through environmental manipulation or with the use of a strain with a lower tendency to filament, 2. mobilis does not appear to be an attractive alternative to S. cereuisiae for this reactor.
Acknowledgment We gratefully acknowledge support, in part, for this work from a contract from DOE’S Energy Conversion and Utilization Technology Project in Biocatalysis managed by the Jet Propulsion Laboratory.
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Literature Cited Brock, T. D. Membrane Filtration: A User’s Guide and Reference Manual. Science Tech.: Madison, WI, 1983. Cheryan, M.; Mehaia, M. Membrane Bioreactors. In Membrane Separations in Biotechnology; McGregor, W. C., Ed.; Dekker: New York, 1986. Cho, T.; Shuler, M. L. Multimembrane bioreactor for extractive fermentation. BiotechnoL Prog. 1986, 2, 53-60. Efthymiou, G. S.; Shuler, M. L. Elimination of diffusional limitations in a membrane entrapped cell reactor by pressure cycling. Biotechnol. Prog. 1987,3, 259-264. Jobses, I. M. L.; Roels, J. A. The inhibition of the maximum specific growth and fermentation rate of Zymomonas mobilis by ethanol. Biotechnol. Bioeng. 1986, 28, 554-563. Jones, R. P.; Greenfield, P. F. Role of water activity in ethanol fermentations. Biotechnol. Bioeng. 1986, 28, 29-40. Kiani, A,; Bhave, R. R.; Sirkar, K. K. Solvent extraction with immobilized interfaces in a microporous hydrophobic membrane. J . Membr. Sci. 1984,20,125-145.
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Krug, T. A.; Daugulis, A. J. Ethanol production using Zymomonas mobilis immobilized on an ion exchange resin. Biotechnol. Lett. 1983,5, 159-164. Matsumura, M.; Markl, H. Elimination of ethanol inhibition by perstraction. Biotechnol. Bioeng. 1986,28, 534-541. Prasad, R.; Sirkar, K. K. Solvent extraction with microporous hydrophilic and composite membranes. AIChE J. 1987,33, 1057-1066. Steinmeyer, D. E.; Shuler, M. L. Mathematical modeling and simulations of membrane bioreactor extractive fermentations. Biotechnol. Prog. 1990, in press. Swings, J.; de Ley, J. The biology of Zymomonas. Bacteriol. Rev. 1977,41, 1-46. Accepted June 20, 1990.
Registry No. Ethanol, 64-17-5; tributyl phosphate, 126-738.
