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ARTICLES Application of Continuous Substrate Feeding to the ABE Fermentation: Relief of Product Inhibition Using Extraction, Perstraction, Stripping, and Pervaporation Nasibuddin Qureshi, Ian S. Maddox,* and Anton Friedlt Biotechnology Department, Massey University, Palmerston North, New Zealand
The technique of continuous substrate feeding has been applied to the batch fermentation process using freely suspended cells, for ABE (acetone-butanol-ethanol) production. T o avoid the product inhibition which normally restricts ABE production to less than 20 g/L and sugar utilization to 60 g/L, a product removal technique has been integrated into the fermentation process. The techniques investigated were liquid-liquid extraction, perstraction, gas-stripping, and pervaporation. By using a substrate of whey permeate, the reactor productivity has been improved over that observed in a traditional batch fermentation, while equivalent lactose utilization and ABE production values of 180 g and 69 g, respectively, have been achieved in a 1-L culture volume.
Introduction The acetone-butanol-ethanol (ABE) fermentation process, using the anaerobic bacterium Clostridium ucetobutylicum, continues to attract attention as a potential source of feedstock chemicals and liquid fuels. During recent years, considerable work has been conducted toward improvement of the traditional batch fermentation process and the development of some novel fermentation technologies (Maddox, 1989). One of the aims of these studies has been to improve the productivity of the system, and substantial success has been achieved. Productivities in the range 0.1-0.5 g/(L.h) have been reported for batch fermentation, while values up to 1.0 g/(L.h) have been achieved during continuous fermentation using free cells (Maddox, 1989). The development of cell recycle systems (Afschar et al., 1985; Ennis and Maddox, 1989) and immobilized cell reactors (Ennis et al., 1986b;Qureshi and Maddox, 1988) has allowed productivities of up to 6.5 g/(L.h) to be achieved. However, these high productivity values are often associated with low product concentrations in the fermentation broth and low sugar utilization values. One approach for solving these problems is to recycle the fermentor effluent to the fermentor, thus allowing residual sugar to be converted to product. Unfortunately, the ABE fermentation suffers from severe product inhibition so that product concentrations rarely exceed 20 g/L. Thus, recycling will be successful only if it is coupled to an effective product recovery technique to remove the inhibitory products. On this basis, several integrated product removal techniques have been investigated for solvent removal during both batch and continuous ABE fermentations. These techniques include gas-stripping (Ennis et al., 1986a; Qureshi and Maddox, 1991),pervaporation (Groot and Luyben, 1987; Fried1 et al., 1991), liquid-liquid extraction (Eckert and Schugerl, 1987;
* Corresponding author.
Permanent address: Institut fur Verfahrenstechnik, Technical University Vienna, Getreidemarkt 9, A-1060 Vienna, Austria. t
Roffler et al., 1987; Wayman and Parekh, 19871, perstraction (Groot et al., 1987), reverse osmosis (Garcia et al., 1986), and the use of solid adsorbents (Ennis et al., 1987; Nielsen et al., 1988). Although considerable progress has been made in the integration of high-productivity continuous fermentation techniques with a product recovery technique, there is little commercial application of these newer fermentation technologies. In contrast, batch fermentation using freely suspended cells with continuous substrate feeding is a widely used industrial technique. In this case, a concentrated nutrient solution is continuously fed to a process which was commenced in the traditional batch mode. It differs from continuous fermentation in that there is no concomitant removal of culture, and hence the culture volume increases with time. Some of the advantages of continuous feeding over traditional batch culture include reductions in process volumes and reactor sizes and increases in reactor productivity, sugar utilization, and product concentration. The purpose of the present work was to investigate the application of the continuous feeding technique to the ABE batch fermentation process using freely suspended cells. To overcome the problem of product inhibition, the technique was integrated with several different product recovery techniques, in order that they might be compared with each other. The techniques studied were liquidliquid extraction, perstraction, gas-stripping, and pervaporation. Whey permeate was used as the raw material for the process because of its potential commercial application.
Materials and Methods Organism. Clostridium ucetobutylicum P262 was obtained from Prof. D. R. Woods (University of Cape Town, South Africa) and was stored as a spore suspension in sterile distilled water at 4 "C. Details of cell cultivation have been published elsewhere (Ennis and Maddox, 1985). Media. Spray-dried sulfuric acid casein whey permeate
8756-7938/92/3008-0382$03.00/00 1992 American Chemical Society and American Institute of Chemical Engineers
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Figure 1. Schematic diagram of ABE production coupled with product removal using liquid-liquid extraction. 1, reactor; 1.1, temperature controller; 1.2,balance; 1.3,feed tank;1.4,nitrogen gas inlet; 1.5, nitrogen gas outlet; 1.6, feed pump; 1.7, pH controller; 1.8,ammonia pump; 1.9,ammonia. 2,extractor; 2.1, pump t o return aqueous phase to the reactor; 2.2,pump to feed reaction mixture into the extractor; 2.3,pump; 2.4, nitrogen gas in; 2.5,nitrogen gas out. 3,separator; 3.1,transfer chamber; 3.2, pump; 3.3,pump to return oleyl alcohol; 3.4,nitrogen gas in; 3.5, nitrogen gas out; 3.6, oleyl alcohol for return; 3.7, oleyl alcohol recovery; 3.8,pump to return oleyl alcohol; 3.9,nitrogen gas out; 3.10,nitrogen gas in; 3.11,pump for oleyl alcohol.
powder, obtained from the New Zealand Dairy Research Institute (Palmerston North, New Zealand), was reconstituted using distilled water to 60 g/L or 350 g1L and supplemented with yeast extract (5 g/L; Difco Laboratories, Detroit, MI). These media contained lactose a t approximately 45 g/L and 300 g/L, respectively. The medium was adjusted to pH 5 using 1 M NaOH and was then autoclaved at 121"C for 15-20 min. The concentrated whey permeate feed medium was held at 35-40 "C to avoid lactose crystallization. During cooling, oxygen-free nitrogen gas was purged over the medium surface to maintain anaerobic conditions. Sterilization. The reactor, pump tubings, and extraction vessels were sterilized by autoclaving at 121 "C for 20 min followed by cooling under oxygen-free nitrogen gas to maintain anaerobiosis. Extraction solvent (oleyl alcohol) was sterilized a t 200 "C for 6 min followed by purging with oxygen-free nitrogen. The perstraction membrane module was sterilized at 121 "C for 20 min. The pervaporation membrane and all condensers were sterilized using 30% and 50% ethanol, respectively, overnight followed by washing with sterile distilled water. The ABE extraction vessel, a 4-L Pyrex glass conical flask, was sterilized by heating in an oven a t 165 "C for 2 h followed by cooling under nitrogen gas. Bioreactor and Cultivation. A 2-L glass bioreactor (New Brunswick Scientific Co., New Brunswick, NJ) was used for these studies. One liter of medium containing whey permeate (60g/L) and yeast extract (5g/L) was used to start the process. The medium, after being cooled to 35 "C under an anaerobic atmosphere, was inoculated with a 5% (v/v) inoculum of highly motile cells (Ennis and Maddox, 1985). Nitrogen gas was swept over the surface until the culture commenced production of its own gases (COz and Hz),and samples were withdrawn regularly for analysis. After 2-3 days of fermentation at 35 "C, i.e., when the ABE concentration was approaching 8 g/L, the product removal technique was applied, while lactose which had been consumed during ABE production was replaced by feeding with concentrated whey permeate medium (350 glL). During the entire process, the culture was maintained at pH 5.0 using 50% (v/v) ammonia solution. The reactor was provided with a foam control device, while the medium flow rate was controlled using a balance or an in-line flowmeter.
Table 1. Details and Operational Characteristics of the Perstraction and Pervaporation Membrane Modules Perstraction Membrane material silicone 3.92mm internal diameter wall thickness 0.40mm 0.215 m2 effective filtration area (based on internal diameter) length of membrane tube 8720 mm operational temperature 35 O C rate of recycle of reaction mixture 24 L/h total internal volume of the membrane 141 mL pump inlet pressure 8-10 psig 250 rpm agitation of organic phase Pervaporation Membrane material fiber internal diameter fiber external diameter length of fibers number of fibers pore diameter in the membrane filtration area based on internal diameter of the fiber liquid recycle rate during pervaporation liquid recycle rate when no pervaporation gas recycle rate 72-98 h of reaction 98-307 h of reaction liquid outlet temperature from the pervaporation module gas inlet temperature to the module gas outlet temperature from the module pressure P1
P2 condensation temperature
polypropylene 1.8mm 2.6 mm 470 mm 40
0.2 p m 0.1 m2 26 L/h 13 L/h 20 W h 10 L/h 30-33 O C 23-29 'C 28-31 O C 0.66-0.69 bar abs 1.2-1.3bar abs 10-12 "C
ABE Removal Systems Liquid-Liquid Extraction. Oleyl alcohol (BDH ChemicalsLtd., Poole, England) was used as the extractant (Roffler et al., 1987). Figure 1shows a schematic diagram of the integrated fermentation/product recovery system. After 2 days of fermentation in a batch mode, the entire reaction mixture containing ABE, cells, lactose, and minerals was pumped into an extractor to which oleyl alcohol (approximately 1L) was added. The mixture was agitated, using a magnetic stirrer bar, for 25 min at 35 "C. This short contact period was chosen to minimize any cell toxicity. After extraction, the mixture was transferred to a separating funnel and allowedto separate into two phases. The aqueous phase containing cells, residual solvents, and lactose was returned to the reactor to which concentrated whey permeate feed medium was added to replace the lactose which had been consumed. Fermentation was then allowed to continue, until ABE production again reached a maximum, and the extraction process was repeated. Oleyl alcohol was recovered and recycled after the ABE and water were distilled off. The pump tubing for oleyl alcohol and the reactor contents was made of Tygon, while all transfer tubings were of silicone. Perstraction. The membrane used for this technique was obtained from Elastomer Products Ltd (Auckland, New Zealand). Details of the perstraction membrane module are given in Table I, while Figure 2 shows a schematic diagram of the integrated fermentationlproduct recovery system. Oleyl alcohol was used as the extractant. The fermentation was commenced in a batch mode (60 g/L whey permeate, 5 g/L yeast extract) and was allowed to produce ABE for 67 h. At this time, circulation of the broth through the membrane was commenced, using a peristaltic pump, at a rate of 24 Llh, and the continuous feed of concentrated whey permeate medium was begun at a rate such that the lactose concentration in the reactor
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Figure 2. Schematic diagram of ABE production coupled with product removal using perstraction. 1,reactor; 1.1,temperature controller; 1.2,balance; 1.3,feed tank;1.4,nitrogen gas inlet; 1.5, nitrogen gas outlet; 1.6, feed pump; 1.7, pH controller; 1.8, ammonia pump; 1.9,ammonia. 2,membrane module; 2.1,pump for oleyl alcohol; 2.2,pump to feed reaction mixture to membrane module; 2.3,oleyl alcoholfor recovery; 2.4,pump for oleyl alcohol; 2.5, pump; 2.6,recovered oleyl alcohol; 2.7,nitrogen gas in; 2.8, nitrogen gas out; 2.9,recovered oleyl alcohol; 2.10,nitrogen gas in; 2.11, nitrogen gas out. 113 a
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Figure 4. Schematic diagram of ABE production coupled with product removal using pervaporation. 1, reactor; 1.1,concentrated feed medium pump; 1.2, sterile water addition; 1.3, pH control arrangement; 1.4,agitator; 1.5, condenser; 1.6,thermometer; 1.7,nitrogen gas in; 1.8, nitrogen gas out; 1.9,feed tank; 1.10,balance. 2,membrane module; 2.1, cooling machine; 2.2, condensate receiver; 2.3, condenser; 2.4, safety condensate collector;2.5,NOinlet; 2.6,gas recycle pump; 2.7,heat exchanger, to cool gas; 2.8,three-way valve; 2.9,gas flow measuring machine; 2.10,gas flow control valve. TI,liquid outlet temperature from the membrane; Tz, gas recycle inlet temperature; Ts,gas outlet temperature from the module; PI, gas pressure regulator 1;Pz, gas pressure regulator 2.
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Figure 3. Schematic diagram of ABE production coupled with product removal using gas-stripping. 1,reactor; 1.1,temperature controller; 1.2,balance; 1.3,feed tank; 1.4,nitrogen gas inlet; 1.5, nitrogen gas outlet; 1.6, feed pump; 1.7,pH controller; 1.8, ammonia pump; 1.9, ammonia; 1.10, foam controller; 1.11, magnetic stirrer; 1.12,antifoam reservoir; 1.13 antifoam pump. 2,condenser; 2.1,cooling machine; 2.2,pump for coolant; 2.3,gas recyclepump; 2.4,receiver; 2.5,pump for condensate;2.6,product; 2.7, cylinder for gas bleed; 2.8, COz and HZgases out.
was maintained between 30 and 60 g/L. The volume of the extractant was approximately 950 mL, and it was maintained anaerobic using oxygen-free nitrogen gas. Recovery of the extractant was as described above. Gas-Stripping. Batch fermentation was allowed to proceed for 46 h, after which time gas-stripping was commenced using the gases produced during fermentation ( ( 2 0 2 and H2). The gases were recycled (3.0-3.2 L/min) through the culture in the reactor using a twin-head peristaltic pump, and the vapors were cooled in a condenser (condenser volume 650 mL, length 660 mm, and cooling coil volume 220 mL) to 0-3 "C using chilled water circulated at a flow rate of 2.5 L/min through the condenser jacket. A schematic diagram of the process is shown in Figure 3. The gases from the reactor were bled from the condensate receiver and were bubbled into ice-cold water. During the fermentation, silicone oil (Dow Corning) was used as an antifoam agent and was added automatically using a peristaltic pump controlled by foam probes. Concentrated whey permeate medium was fed continuously to the reactor at a rate such that the lactose concentration was maintained between 30 and 60 g/L, and when needed, sterile distilled water was added to maintain a constant liquid level inside the vessel. Pervaporation. A pervaporation hollow fiber module (Enka, Wuppertal, FRG) was used to remove solvents from
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Figure 5. ABE production in a batch fermentation process, without product removal. Symbols: m, butanol; +, acetone; 0, ethanol; X, acetic acid; A, butyric acid.
the reaction mixture. Details of this module are given in Table I while a schematic diagram of production and recovery of ABE by pervaporation is given in Figure 4. Oxygen-free nitrogen gas at 10-20 L/min was used as the sweep gas and was recycled using a gas recycle pump (Gast MFG Corp., Benton Harbor, MI) and a flow measuring device (Electronic Flo-meters Ltd., Hounslow, England). The ABE which diffused through the membrane were subsequently recovered in a condenser (length of coiled double-cooling coil 250 mm, condenser length 325 mm, outer diameter 52 mm). Ethylene glycol (305% v/v in water) was circulated through the condenser at a rate of 6-8 L/min for cooling purposes. The temperature of the coolant in the cooling machine (Haake Instruments, FRG) was 0.20.4 "C. All the connecting tubings were made of silicone rubber. Thermometers and pressure gauges were used to measure the temperatures and pressures of various streams. Batch fermentation was allowed to proceed for 72 h, after which continuous product removal was commenced and concentrated whey permeate medium was fed to the reactor, at a rate such that the lactose
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Table 11. Product Concentrations in Aqueous Phase during Extraction into Oleyl Alcohol product concentration, g/L third extraction first extraction second extraction before after before after before after 2.73 2.53 3.60 3.16 1.03 1.04 acetone 1.59 4.51 1.52 1.16 4.42 3.75 butanol 0.22 0.27 0.26 0.29 0.10 0.15 ethanol 0.35 0.40 0.62 0.70 0.50 0.49 acetic acid trace trace 0.50 0.28 0.33 0.22 butyric acid culture volume, mL oleyl alcohol volume, mL ABE in oleyl alcohol, g/L
LO15 955 3.2
957 973 2.5
Table 111. Production Data for Experiment Using Extraction into Oleyl Alcohol products, g (total after four lactose extractions) yield, glg utilization, % 32.9 acetone 7.52 runA 0.28 30.7 butanol 15.63 runB 0.32 29.6 ethanol 0.66 runC 0.38 24.4 total ABE 23.81 runD 0.34 acetic acid 0.89 total lactose 68.6 butyric acid 0.62 used, g total acids 1.51
fourth extraction before after 4.18 2.70 5.34 1.80 0.31 0.27 0.75 0.32 0.59 0.44
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Figure 7. Diffusion of ABE through a silicone membrane into oleyl alcohol. Symbols: -, butanol flux; other symbols are a~for Figure 5.
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Figure 6. ABE production during substrate feeding coupled with product removal using liquid-liquid extraction. Symbols are as for Figure 5.
concentration in the reactor was maintained between 30 and 60 g/L. Analyses. Acetone, butanol, ethanol, and acids were determined by gas chromatography (Model G C - 8 4 Shimadzu Corporation,Kyoto, Japan) using a flame ionization detector and a column of Porapak Q. Lactose was determined by high-performanceliquid chromatography (Ennis and Maddox, 1985). All the reactors were monitored and controlled by the injection of samples for ABE and lactose analysis, simultaneously. If needed, flow rates of feed streams and gas streams were varied to control the systems. ABE or reactor productivity was calculated as the total ABE produced in the system (ABE produced per liter of the culture) divided by total reaction time.
Rssults Initially, a control batch fermentation experiment was run without product recovery or continuous feeding to determine ABE production and the kinetic parameters. This reactor produced 7 g/L total ABE and 0.6 g/L total acids (acetic acid and butyric acid), giving a reactor ABE
productivity of 0.07 g/(L.h) (Figure 5 ) . The ABE yield and lactose utilization were 0.32 and 45.1% of that available, respectively. Liquid-Liquid Extraction. A batch reactor was operated as above, for 48 h, at which time the total reaction mixture was pumped into the extractor to which oleyl alcohol was added. Samples were taken before and after the extraction. The amounts of reaction mixture and oleyl alcohol used and the concentrations of acetone, butanol, ethanol, acetic acid, and butyric acid are shown in Table 11. The progress of the fermentation is shown in Figure 6.
After the first extraction, the aqueous phase containing cells, lactose, minerals, nutrients, and residual products was returned to the reactor and was supplied with sufficient concentratedwhey permeate medium to replace the lactose which had been utilized. Fermentation then recommenced and was allowed to proceed for 31 h prior to a second extraction being performed (Figure 6 and Table 11). After extraction, the aqueous phase was returned to the reactor, and the procedure was repeated. In total, four extractions were performed, after which time no further fermentation was observed in the reactor. Table I1 and Figure 6 show the data for the extractions and fermentation, respectively. During the entire process, the total amount of ABE produced was 23.8 g, representing a productivity of 0.15 g/(L.h) (Table 111). The total amount of lactose utilized was 68.6 g, giving an ABE yield of 0.35. During the extractions, acids were also extracted into the oleyl alcohol (Tables I1 and 111), but there was little extraction of ethanol. No lactose was extracted. Perstraction. Initially, the membrane module was characterized for acetone, butanol, and ethanol flux. It was also of interest to determine whether any reaction intermediates (acids) or lactose diffused across the mem-
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Figure 8. ABE production during substrate feeding coupled with product removal using perstraction. Symbols;7,recycle of reaction mixture started through membrane; 4, oleyl alcohol replaced; 0,lactose; other symbols are as for Figure 5. Table IV. Product Concentrations in Oleyl Alcohol during Integrated Fermentation/Perstraction Experiment perstraction Droducta, g/L first second third 1.62 2.40 1.00 acetone 10.29 6.30 8.89 butanol 0.08 0.07 0.00 ethanol 10.51 12.77 7.37 total ABE 0.0 0.0 0.0 acetic acid 0.07 0.07 0.10 butyric acid 0.10 0.07 0.07 total acids 980 980 amount of oleyl alcohol, mL 950
brane. This was tested using an ABE model solution of the following composition: Acetone, 3 g/L; butanol, 6 g/L; ethanol, 0.8 g/L; acetic acid, 1.0 g/L; butyric acid, 1 g/L; sulfuric acid casein whey permeate 60 g/L; and yeast extract, 5 g/L; pH 5.1. The volume of this solution was 1000 mL, and the amount of oleyl alcohol (extractant on the transmembrane side) was 1793.3mL. Figure 7 shows
Table V. Total ABE and Acids Produced and ABE Productivity during Integrated Fermentation/Product Removal Experiments perstraction gas-stripping pervaporation acetone, g 18.6 25.2 13.2 37.4 butanol, g 38.6 26.8 ethanol, g 1.8 5.3 2.0 total ABE, g 57.8 69.1 42.0 acetic acid. e 1.3 0.4 3.3 .. butyric acid, g 0.8 0.3 6.6 total acids, g 2.1 0.7 9.9 total lactose used, g 157.5 182.5 123.4 ABE yield, g/g 0.37 0.38 0.34 ABE productivity, 0.24 0.26 0.14 g/(L.h) . O
the diffusion of ABE and the butanol flux across the membrane. In a butanol concentration range of 3.8-2.1 g/L, the rate of butanol diffusion was 0.13 g/(L-h) (total ABE diffusion rate 0.15 g/(L-h)),which is close to the ABE production rate typically achieved in the integrated
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Figure 9. ABE production during substrate feeding coupled with product removal by gas-stripping. Symbolsare as for Figure 5.
fermentation processes currently under study. Lactose did not diffuse across the membrane, while the rates for acetone and ethanol were rather low. An integrated fermentation/product recovery experiment was now performed. The reactor was started in batch mode, and when the ABE concentration reached 7.7 g/L, circulation of the culture through the membrane was commenced, as was the continuous feed of concentrated whey permeate medium. The process was controlled in such a way to maintain the concentration of lactose within the desired range (30-60 g/L, Figure 8). The oleyl alcohol was replaced with recovered extractant when it was observed that the ABE concentration in the culture was approaching 5 g/L (Figure 8). After about 240 h of operation, the rate of reaction was observed to slow considerably, and the process was stopped. The concentrations of ABE in the oleyl alcohol are given in Table IV, while the total amounts of ABE produced and lactose utilized are shown in Table V. The reactor was productive for 238 h, giving an overall ABE productivity of 0.24 g/ (L-h) a t a yield of 0.37. Gas-Stripping. A batch fermentation process was started in a manner similar to that described above. After 46 h of fermentation, gas-stripping was commenced and concentrated whey permeate medium was fed to the reactor. The process was continued for 267 h, after which ABE production slowed, and the process became acidogenic;i.e., acids, rather than ABE, accumulated. A total of 69 g of ABE were produced from 182.5 g of lactose. The ABE yield and productivity were 0.38 and 0.26 g/(L.h), respectively, while the total acid production was 0.7 g (Figure 9 and Table V). Only the volatile products acetone, butanol, and ethanol were removed from the culture. Acetic and butyric acids, and all nutrients, remained in the medium. Pervaporation. The reactor was started in the same manner as above and allowed to produce ABE for 72 h. Circulation of the culture through the membrane was then commenced, and concentrated whey permeate medium was fed to the reactor to maintain the lactose concentration in the range 30-60 g/L. After 23 h of product removal, the ABE concentration had dropped to0.42 g/L. The progress of the fermentation is shown in Figure 10. The removal of ABE was faster than its production, and this resulted in an ABE concentration in the reactor of 0.2 g/L after 141 h of operation. Since this product concentration is not inhibitory, ABE removal was discontinued to allow an increase of ABE. Hence, ABE were removed in a dis-
continuous mode rather than a continuous one, as shown in Figure 10. The reaction was stopped after 311 h of operation, when the culture had become highly acidogenic; i.e., acids, rather than ABE, accumulated. The totallactose utilization in the reactor was 123.4 g, while the total ABE produced was 42 g (corrected for the losses described below). Thus, the ABE yield was 0.34, a t an overall reactor productivity of 0.14 g/(L-h) (Table V). With regard to acid production, at the end of the batch reaction (72 h), the concentration of acids was 0.4 g/L. Subsequently, there was a slight trend upward until about 260 h, when the butyric acid concentration increased sharply (Figure 10). At the end of the process, 7.9 g/L of total acids had accumulated in the reactor, while the total amount produced was 9.9 g (Table VI. ABE flux and membrane selectivity are two important parameters which need to be characterized for a solvent removal process using a membrane. Figure llshows flux of ABE during this experiment. When ABE removal was started, a high flux was observed which was due to the high concentration of ABE in the reactor and a high gas flow rate (20 L/min). Since this flux was much higher than the rate of ABE production, the gas recycle rate was subsequently reduced to 10L/min. Membrane selectivity is defined as b / ( l - y)l/[x/(l - x)l, where y and x are weight fractions of ABE in the pervaporate and reaction mixture, respectively. It is a function of solvent concentration in the reactor, the membrane, and the sweep gas recycle rate. The higher the selectivity, the higher the concentration of solvents in the pervaporate. The selectivity of ABE observed in the present experiment varied between 2.8 and 9 (Figure 12). Removal of reaction intermediates, sugars, and nutrients should be avoided when ABE is separated from reaction mixtures. At low concentrations of acids in the reaction mixture, low acid fluxes were observed, while at high concentrations high fluxes were obvious (Figure 13). The increase in the flux of butyric acid was proportional to its concentration in the reactor. There was no diffusion of acetic acid through the membrane below a concentration of 0.8 g/L. Lactose did not pass through this membrane. To identify and quantify any losses of ABE under the experimental conditions employed, pervaporation experiments were performed using a model solution (acetone, 1.29 g/L; butanol, 4.65 g/L; ethanol, 0.26 g/L; acetic acid, 0.12 g/L; butyric acid, 0.13 g/L). From the results, it was ascertained that 10-18 96 of butanol and 4 2 4 4 % of acetone were lost in the integrated fermentationlpervaporation experiment. These losses probably occurred through the connecting silicone tubings, and all data have been corrected appropriately. The ABE flux from both the fermentation experiment and the model solution are given in Figure 14. The membrane selectivity observed with the model solution is shown in Figure 12.
Discussion Batch fermentation coupled with continuous feeding of substrate is a technique which allows higher reactor productivities and product concentrations to be achieved when compared with a conventional batch fermentation process. The ABE fermentation, however, suffers severely from product inhibition, so that a continuous feeding technique will be successful only if the fermentation is integrated with a product removal technique. Hence, the purpose of the present work was to investigate four product removal techniques for their application to this process and to determine whether a continuous feeding technique could be used successfully. The results ofthe experiments
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Figure 10. Concentration of ABE in the reactor during substrate feeding coupled with product removal using pervaporation. Boxes above sections of the graphs indicate that pervaporation was operational during this time. Symbols are as for Figure 5, are summarized in Table V. The control experiment recovery systems (Table VI. Hence, it appears that direct contact between the culture and the oleyl alcohol has led produced results which are typical of a batch fermentation to saturation of the aqueous phase with the extractant, process using a substrate of whey permeate. leading to cell toxicity. Other results in this laboratory Liquid-liquid extraction using oleyl alcohol has been support this conclusion (unpublished data). Another reported previously by Roffler et al. (1987), and this possible disadvantage of extraction with oleyl alcohol is extractant was chosen for the present study on the basis that it removes acetic and butyric acids from the culture, of its distribution coefficient for butanol (4.4)and its thus depleting some reaction intermediates. Other probrelatively low toxicity to bacterial cells. The criteria for lems include loss of cells at the interface, formation of a successful process have been described previously emulsions, and low concentrations of ABE in the extrac(Maddox, 1989). The results show that a continuous tant. feedingtechnique is possible and that there is improvement Perstraction may be viewed as a liquid-liquid extraction in all fermentation parameters compared to the control, technique in which a membrane is placed between the two but the fermentation ceased after four batch extractions. phases, thereby minimizing passage of extractant into the There are several possible reasons for this, including the aqueous phase (culture) and also alleviating some common presence of oxygen in the system, accumulation of minerals in the culture, depletion of nutrients from the culture, problems of the process, e.g., formation of emulsions. Any and toxicity of oleyl alcohol to the cells. The first reason membrane used should have a high selectivity for the reaction products but not for nutrients or reaction is unlikely, because of the stringent precautions taken, while the second and third are also considered unlikely intermediates. The membrane used in the present work because of the results achieved using other product allowed diffusion of butanol into the extractant, but
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Time, h Figure 11. Flux of ABE through the membrane during product removal by pervaporation. Symbols: open bars, total solvents; hatched bars, butanol.
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Figure 12. Membrane selectivityat variousABE concentrations
duringproduct removal by pervaporationand in a model aqueous model aqueous solution *, fermentation solution. Symbols: reaction mixture.
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Figure 13. Acid flux through the membrane at various acid concentrations during product removal by pervaporation. Symbols: A, butyric acid; X, acetic acid.
diffusion of acetone was poor. Diffusion of acetic and butyric acids was also poor, but their retention contributed to a high product yield (0.37, Table V) since these acids are reaction intermediates. Overall,the use of perstraction gave superior results to liquid-liquid extraction, suggesting that cell toxicity is a major problem in the latter. However,
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Figure 14. ABE flux through the pervaporation membrane. 0 , fermentation data, X, aqueous model solution data.
it is also likely to be a problem during perstraction since the oleyl alcohol will diffuse across the membrane until the aqueous phase is saturated with it. Hence, this may be the reason for the eventual failure of the fermentation, but it may also have been due to accumulation of mineral salts (estimated to be 20-22 g/L). Gas-stripping is a relatively simple technique which uses the gases produced during fermentation to remove the volatile products. The selectivity for ABE ranged from 6 to 23. Nutrients or reaction intermediates are not removed from the culture. In the present work, this system allowed the highest production rate of ABE and utilization of lactose, but the fermentation eventually became acidogenic. The reason for the cessation of solventogenic activity is not yet certain, but it is probably accumulation of mineral salts. One advantage of gas-stripping over some of the other techniques studied is that it provides a more concentrated ABE solution to be presented to the distillation column for further purification. Also, recycling of the gases produced during the fermentation may be beneficial to solvent production by retaining a source of reducing power (hydrogen) within the culture. However, there were foaming problems which necessitated control using antifoam. Pervaporation is a technique by which volatile chemicals pass across a membrane and are then removed using a sweep gas or a vacuum, followed by their recovery by
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condensation. In the present work, nitrogen was used as the sweep gas. As with the other product recovery techniques studied, this system allowed considerable improvement over the control experiment. A possible advantage of pervaporation is that the technique can be used as a replacement for distillation in the purification of ABE. Although acetic and butyric acids do diffuse across the membrane, they do so only at high concentrations which are not normally reached in the ABE fermentation process. With this technique, it might be interesting to speculate on whether any bacterial cells become immobilized in the hollow fiber pervaporation membrane, and if this would have any subsequent effect on the process. Such immobilization may help to explain the data shown in Figure 14, where the ABE flux is greater in the fermentation experiment than observed with the model solution, at least at low ABE concentrations. Modification of the membrane's properties following cell immobilization may also explain the lower selectivity observed in the fermentation experiment than that with the model solution (Figure 12). In conclusion, this work has demonstrated the feasibility of operating the ABE batch fermentation process with continuous substrate feeding, when integrated with a product recovery technique. This allows the use of concentrated sugar solutions in the process, thereby reducing process and waste stream volumes. The relatively high salt content of whey permeate (14% of total solids) appears to be a major reason for cessation of fermentation, and this conclusion is supported by other work in our laboratory (unpublished data). Pervaporation and gas-stripping appear to be the most promising product recovery techniques, but further evaluation awaits some costing data.
Acknowledgment We are grateful to Professor A. Schmidt, Head of the Institut f. Verfahrenstechnik, Technical University Vienna, Austria, for supplying the pervaporation membrane module. A.F. gratefully acknowledges the Bundesministerium f. Wissenschaft und Forschung, Vienna, Austria, for the award of a Research Fellowship to study the application of pervaporation to the ABE process.
Literature Cited Afschar, A. S.; Biebl, H.; Schaller, K.; Schugerl, K. Production of acetone and butanol by Clostridium acetobutylicum in continuous culture with cell recycle. Appl. Microbiol. Biotechnol. 1985,22, 394-398. Eckert, G.; Schugerl, K. Continuous acetone-butanol production with direct product removal. Appl. .~ Microbiol. Biotechnol, 1987,27,221-228. Ennis, B. M.; Maddox, I. S. Use of Clostridium acetobutylicum P262 for production of solvents from whey permeate. Biotechnol. Lett. 1985,7,601-606.
Ennis, B. M.; Maddox, I. S. Production of solvents (ABE fermentation) from whey permeate by continuousfermentation in a membrane bioreactor. Bioprocess Eng. 1989,4,27-34. Ennis, B. M.; Marshall, C. T.; Maddox, I. S.; Paterson, A. H. J. Continuous product recovery by in-situ gas strippinglcondensation during solventproduction from whey permeate using Clostridiumacetobutylicum.Biotechnol.Lett. 1986a,8,725730. Ennis, B. M.; Maddox, I. S.; Schoutens, G. H. Immobilized Clostridiumacetobutylicum for continuous butanol production from whey permeate. N. Z. J.Dairy Sci. Technol. 1986b,21, 99-109. Ennis, B. M.; Qureshi, N.; Maddox, I. S. In-line toxic product removal during solvent production by continuous fermentation using immobilized Clostridium acetobutylicum. Enzyme Microb. Technol. 1987,9,672-675. Friedl, A.; Qureshi, N.; Maddox, I. S. Continuous acetonebutanol-methanol (ABE)fermentation using immobilizedcells of Clostridium acetobutylicum in a packed bed reactor and integration with product removal by pervaporation. Biotechnol. Bioeng. 1991,38,518-527. Garcia, A.; Ianotti, E. L.; Fischer, J. L. Butanol fermentation liquor production and separation by reverse osmosis. Biotechnol. Bioeng. 1986,28,785-791. Groot, W. J.; Luyben, K. Ch. A. M. Continuous production of butanol from a glucose/xylose mixture with an immobilized cell system coupled to pervaporation. Biotechnol. Lett. 1987, 9,867-870. Groot, W. J.; Timmer, J. M. K.; Luyben, K. Ch. A. M. Membrane solvent extraction for in situ butanol recovery in fermentations. In Proceedings of the 4th European Congress on Biotechnology; Neijssel, 0.M., van der Meer, R. R., Luyben, K. Ch. A. M., Eds.; Elsevier: Amsterdam, 1987;Vol. 2,pp 564-566. Maddox, I. S.The acetone-butanol-ethanol fermentation: recent progress in technology. Biotechnol. Genet.Eng. Rev. 1989,7, 189-220. Nielsen, L.; Larsson, M.; Holst, 0.; Mattiasson, B. Adsorbenta for extractive bioconversion applied to the acetone-butanol fermentation. Appl. Microbiol. Biotechnol. 1988,28, 335339. Qureshi, N.; Maddox, I. S. Reactor design for ABE fermentation using cells of Clostridium acetobutylicum immobilized by adsorption onto bonechar. Bioproc. Eng. 1988,3,69-72. Qureshi, N.; Maddox, I. S. Integration of continuous production and recovery of solvents from whey permeate: use of i"obilized cells of Clostridium acetobutylicum in a fluidized bed reactor coupled with gas stripping. Bioproc. Eng. 1991,6, 63-69. Roffler, S. R.; Blanch, H. W.; Wilke, C. R. In-situ recovery of butanol during fermentation. Bioproc. Eng. 1987,2,1-12. Wayman, M.; Parekh, S. Production of acetone-butanol by extractive fermentation using dibutyl phthalate as extractant. J . Ferment. Technol. 1987,65, 295-300. Accepted May 19,1992.
Registry No. Acetone, 67-64-1; butanol, 71-36-3; ethanol, 6417-5.
