Reverse Osmosis and Ultrafiltration - American Chemical Society

18 Membrane Bioreactors for High-Performance Fermentations Downloaded by STANFORD UNIV GREEN LIBR on July 2, 2012 | http://pubs.acs.org Publication Date: January 1, 1985 | doi: 10.1021/bk-1985-0281.ch018

MUNIR CHERYAN and MOHAMED A. MEHAIA University of Illinois, Urbana,IL61801 Membrane bioreactors provide an opportunity to vastly improve the productivity of fermentations by (a) converting batch processes into continuous processes, (b) maintaining very high cell concentrations in the system, (c) allowing high dilution (i.e., flow) rates with complete substrate conversion, and (d) removing inhibitory end-products continuously. The Membrane Recycle Fermentor, where the membrane module forms a semi-closed loop with a conventional fermentation vessel, appears to give much better performance than the Hollow Fiber bioreactors, where the microbial cells are loaded onto the shell-side and the feed is pumped through the tube side. Further advantages of this system are that the product stream is free of cells and particulates and capital expenditure for fermentation equipment can be dramatically reduced. Microbial conversion processes (fermentations) are commonly used in the production of foods, pharmaceuticals, organic chemicals, liquid fuels and other biological products. The traditional, and still the most common, method of fermentation is the use of "free" cultures of microbial cells in a batch reactor. Batch fermentors have several problems, including (a) their inherent inefficiency due to their start-up and shutdown nature, (b) large capital costs for equipment, due to the low productivity, (c) batch-to-batch variation in the product, (d) the need to separate out the microbial cells at the end of the fermentation, and (e) the long times needed for the fermentation, sometimes measured in days. The low productivity of batch fermentations (expressed as amount of product produced per unit volume per unit time) is also due to low concentration of the biocatalyst (i.e., the microbial cells), end-product inhibition and substrate depletion. There is now great

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In Reverse Osmosis and Ultrafiltration; Sourirajan, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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interest in high-performance fermentations, especially with the new breed of genetically-engineered microorganisms. Thus any high-performance fermentation system should have the following characteristics built into it: — It should be a continuous process — It should operate at high dilution rates (i.e., high flow rate) — High cell densities should be maintained at all times — Inhibitory end-products should be removed continuously from the system The advantages of conducting fermentations in a continuous mode instead of a batch mode include reduction in capital cost, better process control and usually improved productivity; in the case of ethanol production, it is usually about three times better. The simple continuous stirred tank reactor (CSTR) has one major disadvantage. Dilution rate cannot exceed the maximum growth rate of the microorganism, or there will be cell "washout11. Recycling the cells, typically with a centrifugal separator to separate and recycle the cells, results in much higher productivity. Although modern centrifuges are fairly efficient for cell separation, they are expensive to buy, to operate and maintain. An obvious choice for replacement of centrifuges and mechanical settlers would be membrane separation devices. One way to overcome these disadvantages and to achieve the efficiency of a continuous process has been to "immobilize" these biocatalysts. However, immobilization in the conventional manner, e.g., by fixing on to solid supports or entrapping the cells in a gel matrix, creates its own problems, such as diffusional restrictions and steric hindrance, loss in activity upon immobilization, possibility of contaminating the culture during immobilization and the expense of the immobilization step. An alternate approach involves the use of synthetic semi-permeable membranes of the appropriate chemical nature and physical configuration to either trap or confine the biocatalyst within the bioreactor, or to continuously separate the biocatalyst from the reaction mixture and recycle it back to the main reaction vessel for further reaction. These membrane bioreactors take advantage of the size differences that exist between the biocatalyst (whether an enzyme or microbial cell) and the product. The idea of using "semipermeable" membranes with microorganisms actually dates back to 1896 when Metchnikoff et. al. (1) showed the existence of diffusable cholera toxin in cultures of Cholera vibrios contained in collodion sacs. In the 1960s and 70s, Gerhardt and coworkers performed several pioneering experiments on in vitro dialysis culture systems for a variety of applications (2-4). Although they showed that dialysis culture was superior to the nondialysis process, it may not be practical on an industrial scale since the reaction rate will be limited by the rate at which substrate and product can diffuse through the membrane. Pressure-activated membrane processes, such as ultrafiltration and microfiltration should be more efficient. Two approaches have been used: the membrane recycle bioreactor, operated essentially as a continuous stirred-tank reactor (CSTR), and the hollow fiber bioreactor, operated essentially as a plug-flow reactor.


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Membrane Recycle Fermentor In this configuration, the main reaction vessel in which the fermentation occurs is coupled in a semi closed-loop configuration to a membrane module (Figure 1 ) . The membrane module is serving its intended purpose, i.e., as a separation device, to separate the microbial cells from the product and recycle the cells back to the fermentation vessel. In operation, the reaction vessel is initially filled with the culture with the required cell concentration, and feed pumped into the vessel at a controlled rate. The vessel contents are pumped through the membrane module and recycled back to the reaction vessel. The permeate, containing product molecules, and unconverted substrate molecules that are small enough to pass through the pores of the membrane, will be removed at the same volumetric flow rate as the feed rate, to keep the total reaction volume constant. The membrane should be selected to retain the biocatalyst while allowing free passage of product molecules. Considering the size differences between microbial cells and most products of fermentation, microfliters or large-pore ultrafliters can be used, in the appropriate module configuration. Compared to enzymatic conversions, little work has been done with fermentations in membrane bioreactors (5). Until recently, recycle of microorganisms was rarely practised with the notable exception of activated sludge processes for wastewater treatment, where recycling is essential for process stability and high performance (18). To the authors best knowledge, Budd and Okey (6) were the first to study this concept, primarily for sewage treatment. This work is apparently the basis for the MARS process (Membrane Anaerobic Reactor System) marketed by Dorr-Oliver (Figure 2 ) . Others (_7>J0 also suggested using the membrane recycle fermentor (MRF) for conducting enzymatic and microbial conversion processes. Little hard experimental data was provided until about 1970, when Wang et. al., (9) studied the production of extracellular proteases during continuous cultivation of Clostridium histolyticum (Figure 3). The cell concentration and the product (enzyme) concentration were higher in the continuous membrane system than in the batch system. In this particular case, since neither the cells nor the enzyme were permeable, the only possible explanation for their higher yields is the removal of toxic metabolites during fermentation. Ethanol Fermentation The most attention has been given to the production of ethanol by fermentation. Margaritis and Wilke (10,11) and Haroldsen and Rosen (12) suggested using "Rotofermentors", a schematic of which is shown in Figure 4. The membrane is fixed to a rotating module, which apparently helped to minimize fouling and concentration polarization due to the high turbulence at the rotating membrane surface. Productivity of 36.5 g/L/hr was obtained at cell concentrations of 51 g/L. The rotofermentors, however, appear to be too complex and expensive to be practical, and a much simpler system has been developed at the University of Illinois, Urbana by the authors, (13-15) and at the University of New South Wales, Australia (16). Figure 5 is a schematic of the University of Illinois process. Al-



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Figure 1. Schematic of membrane recycle bioreactor. The drawing is not drawn to scale. The membrane unit on the right is much smaller in volume than the fermentation vessel on the left.


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Figure 3. Comparison of batch and continuous membrane fermentor for the culture of Clostridium histolyticum. Solid lines are batch data, and broken lines are membrane fermentor data. (Reproduced from Ref. 8. Copyright 1972, American Chemical Society.)

Figure 4. Schematic of Rotofermentor. (Reproduced with permission from Ref. 35. Copyright 1981, Springer.)



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Figure 5. Process schematic for conversion of cheese whey into fermentation products with a membrane recycle fermentor.


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though the general process scheme can be applied to almost any fermentation process, the fermentation of cheese whey to ethanol will be used as a model to show the applicability of the membrane recycle concept. Cheese whey is a by-product of the cheese manufacturing industry. Over 40 billion kg are produced annually, over half in the U.S. alone, of which about half is not utilized in any way. This not only creates a potential pollution problem, but is also a waste of good quality protein and a renewable, easily fermentable carbohydrate source. The scheme in Figure 5 shows a method for utilizing both major components of cheese whey. The cheese whey, containing about 5.6% total solids (4.5% lactose, 0.4-0.6% true protein) undergoes the necessary pretreatment prior to ultrafiltration, (such as pasteurization, clarification and/or microfiltration), to maximize UF performance (17). The retentate from the UF unit, after diafiltration if necessary to improve the protein content of the retained solids, can be marketed as a highly functional and nutritious protein ingredient. The permeate contains a solution of lactose, non-protein nitrogen and dissolved salts. After addition of the appropriate additives/nutrients to maintain optimum growth of the microorganisms, it is then concentrated by reverse osmosis (if necessary) to the appropriate sugar concentration. The feed is then sterilized by heat or by microfiltration through a 0.22-micron cross-flow module, before being stored in a sterile balance tank. This prepared feed is then pumped into the MRF, which contains a highly concentrated suspension of the yeast, Kluyveromyces fragilis. The entire fermentation vessel contents are continuously pumped through the appropriate membrane module and the membrane operating parameters adjusted to give the required flux. The MRF is operated as a CSTR, as determined by residence time distribution studies. Table I is a comparison of the performance of the MRF with conventional systems for lactose-to-ethanol fermentations. Table II shows a comparison of ethanol productivities of different bioreactors for the fermentation of glucose to ethanol. The apparent superiority of the membrane recycle bioreactor is obvious. Similar process schemes have been used for the production of ethanol from glucose using the yeast Saccharomyces cerevisiae and the bacteria Zymomonas mobilis by our research group. Figure 6 shows the productivity as a function of dilution rate and cell concentration. Increasing the cell concentration allows a higher dilution rate (i.e., throughput rate) for the same conversions. These productivities are 20-60 times higher than traditional batch processes and better than immobilized whole cell reactors. In comparing the data in Table II, it should be borne in mind that the organism Zymomonas mobilis has a higher growth rate and is reputed to be a better ethanol producer than the yeasts we studied. Rogers et_ al. (16) used a Millipore Pellicon flat-sheet microfiltration system for cell recycle while we used hollow (fat ) fibers of Romicon and Amicon for our studies. Other Fermentations Bull and Young (19) have recently described partial cell recycle using tubular ceramic filter elements as the recycle device for the


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conversion of D-sorbitol to L-sorbose by Gluconobacter oxydans subspecies suboxydans 1916B and for the conversion of glucose to 2keto-gluconic acid by Serratia marcescens NRRL B-486. Cell recycle improved productivity of the former fermentation from 48 g sorbose per liter per hour to 76 g/L/hr, with only a partial cell recycle of 0.49. The productivity doubled for the latter fermentation. In our opinion, however, both fermentations were operated far from the optimum conditions and much greater improvements could have been obtained if the fermentation and the membrane system had been optimized viz-a-viz their operating parameters. The production of lactic acid in membrane recycle fermentars has been studied by Vick Roy et_ al. (20) . Their data is shown in Table III. Similar improvements in lactic acid productivity using different microorganisms and different substrates have been obtained by our own research group (21). Minier et al. (22) have reported an improvement in acetone-butanol fermentation productivity using cell recycle with Carbosep tubular membranes. Hollow Fiber Fermentor Hollow fiber bioreactors, where the microbial cells are trapped within the shell-side of the cartridge and the feed is pumped in through the tube side (Figure 7 ) , have not fared as well as membrane recycle fermentors (Tables I and II). Either productivities have been much lower than with membrane recycle fermentors, or the substrate utilization has been poor. For example, substrate conversions of only 3-4% were obtained for the production of lactic acid in holiow fiber bioreactors by Vick Roy et_ ad., (23) and our group (21^). The relatively poor performance is due to several practical problems that arise during the operation of hollow fiber fermentors. Because the biocatalyst (the microbial cells) is separated from the substrate and product stream by a physical barrier (the membrane), the rate-limiting step becomes the diffusion of substrate into, and the diffusion of the product out of, the shell-side of the hollow fiber cartridge. In our experiments, visual inspection of the shellside indicated that most of the microbial cells were attached to the

Table III. Conversion of Glucose (45 g/L) to Lactic Acid by Lactobacillus delbreuckei in a Membrane Recycle Fermentor Dilution Rate (hr-1) 0.26 0.54 1.14 2.16

Cell Concentration (g/L) 2-5 5-25 25-38 38-54

Lactic Acid (g/L)

Productivity (g/L/hr)

24 33 34 35

6.2 17.8 38.7 76.0

Source: Adapted from Ref. 20. Note: For comparison, batch fermentors generally have cell concentration of 7-11 g/L and productivity of 1-3 g/L/hr.



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Figure 6. Effect of dilution rate and cell concentration on ethanol productivity. The feed was glucose (100 g/L) and the microorganism was Sj_ cerevisiae. Cell concentration (x): • , 14 g/L; A, 60 g/L; and D, 100 g/L.

Figure 7. Schematic of hollow fiber bioreactor. The biocatalyst is packed into the shellside and feed pumped through the tubes.


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fibers or were localized to an annular volume just around the fibers. Thus the cells far from the fibers were probably starved of nutrients and dying, and so a means must be found of removing these dead cells continuously. If not, the growing mass of cells may even rupture the relatively sensitive fibers. One of the biggest problems is to purge the gas that is formed in the shell-side as a by-product of the fermentation. Michaels and Robertson et. al., have recently been awarded patents for applications of the hollow fiber concept (24,25); it appears to be most useful for reactions involving nongrowing cells. Conclusions Membrane bioreactors provide an opportunity to vastly improve the performance and productivity of fermentations. Specific advantages of membrane recycle fermentors include: — The inherently more efficient continuous process can be used — High cell densities can be obtained, exceeding 100 g/L of dry weight of cells, if necessary. This usually implies cell concentrations of per mL, close to theoretical loadings. The upper limit to cell concentrations is the physical properties of the fermentation mixture, which will affect the pumpability, polarization and fouling problems with the membrane module. — High dilution rates can be used, as a direct result of the high cell concentrations. For ethanol fermentation, dilurates of 1-2 hr can be used with complete substrate conversion, provided the substrate concentration is less than 15% w/v and the cell concentration is above 90 g/L, Higher substrate concentrations will lead to high ethanol concentrations and consequent lower fermentation rates, which will necessitate slowing down the flow rate to obtain complete conversion. A considerable amount of optimization work remains to be done to determine kinetic parameters in the high product concentration operating regions, — Control of the environment is easy with the membrane recycle fermentor. If the membrane module is properly designed, the bulk of the fermentation or reaction should occur in the reaction vessel. Thus control of temperature, pH, dissolved oxygen, etc., can be done with the usual methods as currently practised. However, we suspect that hardware design aspects such as heat transfer and mass transfer will be the limiting factors in these high-rate fermentations, rather than the microbiology or the membrane separations per se. For example, membrane bioreactors used for ethanol production will be producing at least 50 times as much heat and gas per unit volume of bioreactor than conventional fermentors, which will necessitate drastic redesign of the hardware. — Membrane recycle bioreactors can process both soluble and insoluble substrates, unlike the hollow fiber and most immobilized whole cell bioreactors. — The product stream from membrane bioreactors is free from microbial cells and other particulate material, which should reduce downstream processing costs.


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—

There are no gas hold-up problems as with packed-bed, immobilized whole cell reactors or hollow fiber reactors. This concept is probably the easiest to incorporate into an existing plant. At the very least the membrane modules can replace conventional centrifuges used for separating microbial cells from the fermentation broth which are expensive to buy, operate and maintain.

Some potential limitations should also be mentioned: the membrane recycle bioreactor is operating essentially as a continuous stirred tank reactor (CSTR). The outlet (permeate) stream will have the same concentration as the reaction mixture, which means the system will be ideally operating with a high product concentration at all times. This means a low fermentation rate, since most fermentations are severely product-inhibited, sometimes at very low product concentrations (for ethanol, it is usually 12-15% v/v). The long-term effects of continuous contact of microbial cells with high product concentrations has not yet been evaluated. In addition, as already alluded to, the key factor in successful long-term operation of a membrane recycle bioreactor is the performance of the membrane module. Several studies undertaken by our group (21) have indicated that the flux and "fouling" is controlled not only by the expected operating parameters, such as pressure, temperature, flow velocity and feed concentration, but also by specific interactions between the membrane and feed-stream components. Thus it is important not only to have the appropriate physical configuration of the module, but the chemical nature of the membrane should be such so as to minimize these interactions. Fermentation broths are highly complex mixtures of macromolecules, salts, vitamins, etc., and it is important to understand and minimize their interactions with the membrane in order to minimize membrane-related problems during long-term operation. Some membranes completely inactivate some enzymes by mere contact (5, 26). Finally, sterilizability of the membranes is also critical to its application in continuous bioreactors. Ideally, the membrane module should be repeatedly steam-sterilizable so as to fit in with the routine steam sterilization cycles of the fermentors and associated apparatus. Unfortunately, the membranes that have the desired properties as far as flux and fouling are concerned tend to have very poor temperature stability and vice versa. Perhaps a new generation of "biocompatible" membranes need to be developed for these specific applications. Literature Cited 1. 2. 3. 4. 5. 6.

Schultz, J. S.; Gerhardt, P. Bacteriol. Rev. 1969, 33, 1. Abbott, B. J.; Gerhardt, P. Biotechnol. Bioeng. 1970, 12, 577. Abbott, B. J.; Gerhardt, P. Biotechnol. Bioeng. 1970, 12, 601. Steiber, R. W.; Gerhardt, P. Biotechnol. Bioeng. 1981, 12, 535. Cheryan, M.; Mehaia, M. A. In "Membrane Separations in Biotechnology"; McGregor, W. C., Ed.; Marcel-Dekker, NY, 1985. Budd, W. E.; Okey, R. W. U.S. Patent 3 472 765, 1969.


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7. 8. 9.

10. 11.


12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35.

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Michaels, A. S. In "Progress in Purification and Separation"; Perry, E. S., Ed; Interscience, New York, 1968. p. 297. Porter, M. C.; Michaels, A. S. Chem. Tech. 1972, 2, 5. Wang, D. I. C.; Sinskey, A. J.; Butterworth, T. A. In "Membrane Science and Technology", Flinn, E. D., Ed.; Plenun, New York, 1970. Margaritis, A.; Wilke, C. R. Biotechnol. Bioeng. 1978, 20, 709. Margaritis, A.; Wilke, C. R. Biotechnol. Bioeng. 1978, 20, 727. Haroldsen, A.; Rosen, C. G. Eur. J. Appl. Microbiol. Biotechnol. 1982, 14, 216. Cheryan, M.; Mehaia, M. A. Biotechnol. Lett. 1983, 5, 519. Mehaia, M. A.;Cheryan, M.; Argoudelis, C. A. Cult. Dairy Prod. J., 1984, in press. Cheryan, M,; Mehaia, M. A. Process Biochem. 1984, 19(Dec)., Rogers, P. L.; Lee, K. J.; Tribe, D. E. Process Biochem. 1980, 15, 7. Kuo, K. P.; Cheryan, M. J. Food Sci. 1983, 48, 113. Hamer, G. Biotechnol. Bioeng. 1982, 24, 511. Bull, D. N.; Young, M. D. Biotechnol. Bioeng. 1981, 22, 373. Vick Roy, T. B.; Mandel, D. K.; Dea, D. K.; Blanch, H. W.; Wilke, C. R. Biotechnol. Lett. 1983, 10, 665. Cheryan, M. Unpublished results. Minier, M.; Ferras, E.; Goma, G.; Soucaille, P. 7th International Biotechnology Symposium, New Delhi, India, 1984. Vick Roy, T. B.; Blanch, H. W.; Wilke, C. R. Biotechnol. Lett. 1982, 4, 483. Michaels, A. S.; Robertson, C. R.; Cohen, S. N.; Inloes, D. S.; Smith, W. J. U.S. Patent 4 442 206, 1984. Michaels, A. S.; Robertson, C. R.; Cohen, S. N. U.S. Patent 4 440 853, 1984. Kohlwey, D. K.; Cheryan, M. Enzyme Microb. Technol. 1981, 3, 64. Janssens, J. H.; Bernard, A.; Bailey, R. B. Biotechnol. Bioeng. 1984, 26, 1. Mehaia, M. A.; Cheryan, M. Enzyme Microbial Technol. 1984, 6, 117. Mehaia, M. A.; Cheryan, M. Applied Microbiol. Biotechnol. 1984, 20, 100. Ghose, T. K.; Tyagi, R. D. Biotechnol. Bioeng. 1979, 21, 1387. Ghose, T. K.; Bandyopadhyay, K. K. Biotechnol. Bioeng. 1980, 22, 1489. Maragritis, A.; Bajpai, P. K., Wallace, J. B. Biotechnol. Lett. 1981, 3, 616. Klein, J.; Kressdorf, B. Biotechnol. Lett. 1983, 5, 497. Inloes, D. S.; Taylor, D. P.; Cohen, S. N.; Michaels, A. S.; Robertson, C. R. Appl. Environ. Microbiol. 1983, 46, 264. Maiorella,B; Wilke,C.R.;Blanch,H.W. Adv.Biochem.Engr. 1981, 20, 43.

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Reverse Osmosis and Ultrafiltration - American Chemical Society

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