Amylase fermentation with Bacillus amyloliquefaciens in an aqueous

distributed almost evenly in both phases, and -amylase generally preferred the bottom phase by a small margin. A continuous fermentor with bottom-phas...
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Bbtechnol. h g . 1001, 7, 439-444

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a-Amylase Fermentation with Bacillus amyloliquefaciens in an Aqueous Two-Phase System Kyung-Moon Park and Nam Sun Wang' Department of Chemical Engineering, University of Maryland, College Park, Maryland 20742

a-Amylase production from Bacillus amyloliquefaciens in an aqueous two-phase system of poly(ethy1ene glycol) and dextran was investigated. In both batch and continuous fermentations, cells preferentially partitioned to the dextran-rich bottom phase, glucose distributed almost evenly in both phases, and a-amylase generally preferred the bottom phase by a small margin. A continuous fermentor with bottom-phase recycle is proposed to achieve a significantly improved volumetric productivity of a-amylase. Continuous a-amylase fermentation with simultaneous product removal was demonstrated in 84 g/L poly(ethyleneglyco1) (MW 8000) and 84 g/L dextran (MW 71 500) at various dilution rates. Low overall cell densities economized glucose consumption. Unlike the polymerfree fermentation, a-amylase synthesis was not repressed even at high glucose concentrations. A properly designed continuous system with cell recycle can be considered as a nonconventional, but highly effective, method of cell immobilization.

Introduction A two-phase system can be obtained by simply mixing incompatible polymers in an aqueous solution. Specifically, a mixture of poly(ethy1ene glycol) (PEG) and dextran becomes turbid above certain polymer concentrations and subsequently separates into two distinct phases when left undisturbed. While both phases have a high water content of typically over 90%, the top phase is rich in PEG and the bottom phase is rich in dextran (Albertsson, 1986). Compared to conventional extractive systemsbased on organic solvents, an aqueous two-phase system has a low liquid-liquid interfacial tension and is highly biocompatible and nontoxic. For the last 30 years, there have been many developments in aqueous two-phase systems. A wide variety of biomaterials, such as proteins, nucleic acids, mitochondria, fragile subcellular particles, microorganisms, and animal and plant cells, have been successfullyseparated (Walter et al., 1985). In addition, there is a wide range of applications of aqueous two-phase systems in bioconversion (Andersson and Hahn-Hagerdal, 1990). Among these are toxin production by Clostridium tetani (Puziss and Heden, 1965), conversion of cellulose to ethanol (Hahn-Hagerdal et al., 1981a,b),deacylation of benzylpenicillinto 6-aminopenicillanicacid with penicillin acylase (Andersson et al., 1984),enzymatic hydrolysis of cellulose with cellulases (Tjerneld et al., 1985a,b), and alcoholic fermentation (Kuhn, 1980). Most past applications emphasized the partition of the product as applied to product removal and purification. As such, the partition behavior of agiven material between two phases had been critical in choosing a suitable system. In general, it was essential that the partition coefficient, defined as the ratio of a component's concentration in the top phase to that in the bottom phase, be removed as much as possible from unity. Given the material to be partitioned, one has few options in drastically altering the partition coefficient within practical operational constraints, although it depends on, among other properties, the characteristics of the two phases and temperature. This paper describes a novel way of using an aqueous twophase system in the context of cell immobilization. It

* To whom correspondence should be addressed. 8756-7938191/3007-0439$02.50/0

demonstrates that a two-phase system can be highly effective in extracting the desired product from the fermentation broth even when the product is distributed evenly between the two phases-a common occurrence. As a model system, a-amylase fermentation in a glucoselimited chemostat culture of Bacillus amyloliquefaciens with simultaneous product removal is studied. This industrially important fermentation has recently been examined (Rutten and Daugulis, 1987;Yo0 et al., 1988a,b; Alam et al.,1989;Roychoudhury et al., 19891,and aclosely related culture of Bacillus subtilis has been studied in a conventional system (Toda et al., 1979) as well as in an aqueous two-phase system operated in a batch or repeatedbatch mode (Andersson et al., 1985). a-Amylasesynthesis is repressed in excess glucose, and the production rate in a continuous fermentor rapidly declines when the dilution rate is increased (Heineken and O'Connor, 1972). First, the effect of phase-forming polymers on fermentation kinetics will be examined. Subsequently, the feasibility of continuous a-amylase production in an aqueous twophase system with cell recycle will be investigated.

Materials and Methods Aqueous Two-Phase System. Practical-grade PEG (average MW 8000) (Baker) and industrial-grade dextran (MW 71500) (Sigma) were the phase-forming polymers in all experiments reported in this paper. The binodal curve of the aqueous two-phase system formed with these two polymers was generated on the basis of composition measurements taken with gel-permeation chromatography (Gilson) equipped with a refractive index detector (Shodex). The curve was also independently checked by carefully recording the volumes of the polymer solutions of known concentrations required to cause the incipient of a two-phase system. Fermentation. Unless noted otherwise,B. amyloliquefaciens strain F (ATCC no. 23350) was cultured at 37 "C in a 2-L fermentor (New Brunswick Scientific) with a working volume of 1 L. Agitation was provided by two Rushton turbine impellers 5.0 cm in diameter operated at 500 rpm, and the aeration rate was set at 1 volume of air (volume of medium)-' min-l (wm). The fermentor was maintained at pH 7.0 with a steam-sterilizable pH electrode

0 1991 American Chemical Society and American InstAute of Chemical Engineers

Biotechnol. Prog., 1991, Vol. 7, No. 5

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(Ingold) and a pH controller (Cole-Parmer) by the combination of 3 N KOH or 3 N H2S04 additions. The culture media contained 10 g/L glucose, 5 g/L NH4H2PO4, 1 g/L yeast extract, 1 g/L K2HP04, 0.5 g/L MgS0~7H20,0.5g/L sodium citrate, 0.1 g/L CaC12,O.l g/L FeS0~7Hz0,and 0.1 g/L MnSOqH20. The schematic diagram of the continuous fermentation with cell recycle is shown in Figure 1. The fermentor feed consisted of the fresh cell-free nutrient solution and the cell-rich bottom phase recycled from the settler. The outflow from the overall system consisted of only the top phase from the settler. The 50-mL settler provided at least 15 min of residence time for a complete phase separation at the highest dilution rate (0.35h-l) encountered in this study. Oxygen depletion in the settler is a possible point for concern, but no attempt was made to study rigorously its effect on cell growth or product formation in this paper, nor was any extra effort made to provide oxygen through other means without disturbing the phase separation, e.g., the tubing method. To reduce the gradual composition change in each phase, which causes a drift in the volume ratio during a prolonged continuous steady-state operation, the phase-forming polymers taken out from the overall system must be replenished at the same rate. Thus, the polymer composition in the fresh feed must match that of the top phase in the fermentor. To achieve this reliably in a laboratory setting, an aqueous two-phase system of a given overall composition was first formed, its bottom phase was subsequently removed, and nutrient components were added to the top phase to form the feed media. Except for the extra settler and pumps needed to effect cell recycle, the fermentor and cultivation conditions were otherwise identical for both batch and continuous operations. Assays. Samples were periodically withdrawn from the fermentor and analyzed for cell density, glucose concentration, and extracellular a-amylase activity. The sample absorbance at 595 nm was measured with a spectrophotometer (Spectronic 21, Milton Roy) and converted into cell density via a calibration curve based on the cell dry weight. The glucose concentration was determined with a glucose analyzer (Beckman) based on the enzymatic conversion of glucose to gluconic acid and H202 by glucose oxidase. The a-amylase assay was performed according to Park and Wang (1991). Starch dyed with Remazol Brilliant Blue (RBB) by the method of Rinderknecht et al. (1967) was subjected to hydrolysis by the a-amylase contained in the sample. After the sample and the substrate solution (2 g of RBB-starch suspended in 100 mL of phosphate buffer at pH 7.0) were separately preincubated at 37 O C for 10 min, 0.3 mL of sample and 2.7 mL of substrate were

allowed to react for 10min. Starch hydrolysis was stopped with 1.2 mL of 18% (v/v) acetic acid. The remaining insoluble RBB-starch was removed with a syringe filter, and the color intensity of the filtrate at 595 nm was directly used as a measure of the enzyme activity (in units per milliliter) according to ( A - A&f(lO) (1) where A and A0 are the absorbances of the sample and the blank, respectively, f is the dilution factor of the sample before assay, and 10 is the dilution factor with the working substrate solution. Despite the lack of a uniform standard for expressing the a-amylase activity, the above definition is among the commonly used ones and can be translated into other units (Yo0 et al., 1987).

Results and Discussion Batch Fermentationin Single-phasePolymer Solutions. The effect of the phase-forming polymers on B. amyloliquefuciens cell growth, glucose consumption, and a-amylaseformation was examined. As a reference, Figure 2 shows a typical batch fermentation in the absence of polymers. For comparison, Figure 3 shows the otherwise identical fermentations in PEG at concentrations of 40 and 80 g/L, and Figure 4 shows the counterparts in dextrin at concentrations of 40 and 80 g/L. From Table I, it is clear that compared to the polymer-free fermentation the maximum cell density decreased by up to 25% in the presence of either polymer. Fermentation dynamics is considerably modified by the polymers. Notably, in the polymer-free fermentation, there was no stationary period due to the immediate massive sporulation when glucose was exhausted and the a-amylase activity ceased to increase after the cell density reached the maximum level, whereas for fermentations conducted in polymer solutions, longer stationary phases, lower maximum cell densities, and slightly continued accumulations of a-amylase were observed. As shown in Table I, the maximum a-amylase activity and the specific enzyme activity (per gram of biomass) at the point of maximum cell density were both generallyhigher in the presence of either polymer, although each polymer exerted different degrees of influence. Increased viscosity, reduced oxygen solubility, substrate/ oxygen diffusional limitation resulting from both the altered hydrodynamics and the extra phase boundary, and interaction of the polymer with the cellular surface are suspected of being responsible for this observation; however, the exact mechanism remains unknown. In extending the results of single-phase polymer studies to predicting the cell behavior in an aqeous two-phase system, the effect of dextran is more pertinent compared to that of PEG because most cells reside in the dextran-

Bbtechml. Rq., 1991, Vol. 7, No. 5 7-m

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Table 1. Effect of Phase-Forming Polymers in Single-Phase Batch Fermentation polymer concentration (g/L) dextran PEG 0 40 80 40 80 max. cell density (g/L) 2.20 1.84 1.74 1.97 1.64 max. amylase activity (units/ 1.40 1.90 1.70 1.85 2.00 mL) sp. amylase activity (units/mg 0.61 0.90 0.86 0.91 1.01 of cell)

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rich bottom phase. For example, the dextran-rich phase is much more viscous than the PEG-rich phase; thus, cells are likely to feel the increased viscosity and its manifestations.

Batch Fermentation in Aqueous Two-Phase Systems. An aqueous two-phase system of nearly equal top and bottom volumes was realized with PEG and dextran concentrationsat 50 and lOOg/L, respectively. The results of a batch shaker flask fermentation in this two-phase system are shown in Figure 5 for each phase, and Table I1 summarizes the partition coefficients for biomass, glucose, and a-amylase at three distinct stages (KO,before reaching the maximum cell density; K,, at the maximum cell density; and Kf,at the end of the run). Throughout the entire fermentation, there were few cells in the top phase. The optical density of the top phase was low, except toward the end of the run when spores started to appear after glucose exhaustion. At that point, opticalmicroscopic examination confirmed that the optical density in the top phase resulted not from vegetative cells but mainly from spores that distributed evenly between the two phases. Thus, the change in the apparent biomass partition coefficient was caused by sporulation. During the initial cell growth phase, the a-amylase activity in the bottom phase was higher than that in the top phase and increased in step with that in the top phase. However, during spore formation, the a-amylase partition shifted from the bottom phase to the top phase. The biomass and a-amylase partitions appear to be coupled. A certain degree of shift in the a-amylase partition is expected because of ita sensitivity to the broth environment, e.g., ionic composition, that continually changed during fermentation. The partition coefficient of glucose was only slightly less than unity, and the glucose concentrations in the two phases changed proportionately. Unfortunately, the factors that determine partition remain largely unknown, and predicting partition behavior is difficult. Although the partition coefficient of a-amylase is less than 1, the total amount of a-amylase in the top phase, the cell-free phase that can be readily withdrawn for product purification, can be increased by adjusting the volume ratio. It is noted that this argument for employing an aqueous two-phase system is somewhat misleading, because the critical factor is neither the amount of product in the cell-free phase nor the product formation rate per cell-containingphase volume but a judicious combination of these factors as well as the product concentration in the broth to be processed downstream and the production rate per fermentor volume. Continuous Fermentation in a Polymer-Free System. After inoculation, fermentation commenced in a batch mode to build up the biomass content, and then nutrient feed and broth withdrawal were simultaneously introduced at equal rates. Since the cell concentration in the discharged solution was the same as that in the fermentor, a significant quantity of cells was withdrawn and wasted. A time equal to at least three time constants, defined as the ratio of the total working fermentor volume t o the fresh nutrient feed rate, or equivalently the inverse of the dilution rate D, was allowed for each steady state

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442 50 g/L PEG 100 g/L DEXTRAN

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to be established in the bioreactor. The steady-state results are tabulated in Table 111. Glucose concentration strongly affects the steady-state a-amylase activity, and there is clearly an optimum dilution rate for a-amylase production. A t D = 0.07 h-l, the low glucose conce2tration limited the a-amylase activity to nearly zero; at D = 0.21h-l, the enzyme activity increased to 1.1 units/mL; further increasing D to 0.35h-l, however, suppressed the enzyme activity to 0.2 unit/mL due to the high residual glucose concentration (3.25 g/L). This observation agrees with the findings by Heineken and O'Connor (1972)and Toda et al. (1979). Continuous Fermentation in an Aqueous TwoPhase System. Equal overall concentrations of PEG and dextran at 84 g/L were employed in the fermentor. This combination gave a top/bottom volume ratio of approximately 7/3. The same volume ratio was maintained for each dilution rate by keeping a constant overall polymer composition in the fermentor. The fresh nutrient, which had the top-phase polymer composition by design, and the recycle, which had the bottom-phase polymer composition by default, were combined at the same ratio as the volume ratio. The dilution rate, D, in this recycled system is defined as the fresh nutrient feed rate for the entire working volume, both the top and bottom phases. The results of the continuous fermentation in an aqueous two-phase system with cell recycle are summarized in Table IV. As in the batch fermentation, most cells favored the dextran-rich bottom phase. The cell density of the bottom phase was approximately 3 g/L for all dilution rates, and that of the top phase exhibited a maximum at D = 0.21 h-l, where some vegetative cells were observed in the top phase. The overall cell concentration in the aqueous twophase system based on the total working volume was consistently much less than that operated conventionally without the polymers. The low overall cell density combined with the insignificant loss of cells in the exit stream enabled efficient utilization of the substrate. Glucose distributed evenly between the phases. A significant portion of the glucose partition coefficient's deviation from unity was probably due to experimental error. Because only a lower rate of glucose consumption

Table 111. Continuous Single-phase Fermentation without Polymers dilution rate (h-1) 0.07 0.21 0.35 cell concn (g/L) 3.08 2.11 1.68 glucose concn (g/L) 0.20 1.40 3.25 a-amylase activity (units/mL) 0.40 1.10 0.20 Table IV. Continuous Fermentation in an Aqueous Two-Phase System. dilution rate (h-1) 0.07 0.21 0.35 cell concn, top (g/L) 0.23 0.84 0.27 cell concn, bottom (g/L) 3.28 2.80 2.88 cell partition coeff 0.070 0.30 0.094 ratio of amount of cells, top/ 0.16 0.70 0.22 bottom overall cell density (g/L) 1.14 1.47 1.05 glucose concn, top (g/L) glucose concn, bottom (g/L) glucose partition coeff

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was needed to sustain cell growth, the glucose concentration in the two-phase system at each dilution rate was consistently higher than the corresponding value in the conventional single-phase system. During the initial batch growth before commencing continuous operation, the partition coefficient of a-amylase was near 0.7. At a steady state, it approached unity at D = 0.07 h-' and decreased to 0.8 at D = 0.35 h-l. In contrast to the continuous counterpart conducted in a polymer-free system, the residual glucose concentration did not affect the a-amylase activity as much. Enzyme production was not repressed even at high concentrations of residual glucose. For example, in approximately 5 g/L glucose at D = 0.35 h-l in the two-phase system an a-amylase activity of over 1.5 units/mL was achieved, whereas in less than 3.5 g/L glucose at the same dilution rate in the single-phase system, an activity of only 0.2 unit/mL was realized. This apparent lack of glucose repression is probably caused by glucose mass transfer limitation between the phase boundaries and within the highly viscous dextran-rich bottom phase where the more efficient mode of mass transfer via turbulence is not as active. Comparisonof Volumetric Productivity of a-Amylase. One of the more meaningful measures in judging the effectiveness of the proposed approach is the volumetric productivity of the enzyme. In Figure 6, a-amylase productivities in three operational modes are compared: batch fermentation, polymer-free single-phase continuous fermentation, and two-phase continuous fermentation. For the batch fermentation, the productivities were based on harvesting either at the point of maximum cell density or at the point of glucose exhaustion. In the conventionally operated continuous fermentor, as the dilution rate is increased, the a-amylase productivity increased to approximately 0.3 unit/(mL.h) at D = 0.21 h-' and then decreased to less than 0.1unit/(mL.h) at D = 0.35 h-1. For the two-phase continuous fermentation, the volumetric productivity is calculated on the basis of the top-

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phase a-amylase concentration, for the top phase is withdrawn from the overall system. The comparison in Figure 6 is fair because the dilution rate is based on (a) the flow rate of the fresh feed, not the combined flow rate with the recycle stream, and (b) the entire working volume, not just one of the phases. Significantly, the a-amylase productivity in the two-phase continuous fermentation, increasing to higher than 0.5 unit/(mL.h) at 0.35 h-' and continuing upward, is consistently higher than that obtained in the batch or single-phase continuous fermentation. In Figure 6, the numbers for the two-phase continuous fermentation should be multiplied by 10/3, the total/bottom volume ratio, if they were calculated on the basis of the volume in which fermentation takes place, i.e., the bottom phase, instead of the total working volume. The operating conditions can be modified to achieve an even higher productivity. First, the volume ratio used in this study (7/3) can be altered. In contrast to batch fermentation, where it is desirable to increase the top/ bottom volume ratio so that most of the desired product is present in the top phase and separated from the cellrich bottom phase, a small top/bottom volume ratio actually favors a-amylase production in a continuous operation. A higher bottom-phase volume fraction translates into additional volume available for a-amylase production for a given bioreactor, thus a higher productivity. Conceptually, the bottom phase immobilizes cells within the bioreactor, and the top phase ferries substrate into the bioreactor for bioconversion and the product out for downstream purification. The fractional amount of the product present in the top phase is meaningless in a continuous operation. Second, from the trend shown in Figure 6, it is possible that an even higher volumetric productivity can be realized at a higher dilution rate. However, a larger settler must be used to provide a long enough residence time for a complete phase separation. The size of the settler may become significant compared to that of the fermentor and should be properly included in the productivity calculation. Furthermore, a gain in the volumetric productivity a t a higher dilution rate is countered by a decrease in the product concentration, as shown in Table IV, which indicates a decline in the a-amylase activity in the top phase as D increases from 0.21 to 0.35 h-l. Thus, there exists an optimal dilution rate in balancing productivity against efficiency in product recovery. Optimizing the a-amylase productivity through volume ratio manipulation or studying the effect of a partial phase separation is outside the scope of this paper. Advantages of the Proposed Concept. In short, it is demonstrated that an aqueous two-phase system can be designed to effect cell immobilization via cell recycle,albeit

unconventionally, in the continuous production of a-amylase. The advantages of the proposed continuous fermentation with an aqueous two-phase system are (a) high volumetric productivity, (b) no apparent a-amylase repression by glucose even without controlled feed of glucose, (c) simultaneous cell-free product removal, and (d) cell retention through recycle leading to economical glucose utilization. Although not directly applicable to a-amylase, if the bioconversion product has a negative/inhibitory effect on the process, the continual product removal will also facilitate further conversion. In addition, because the partition coefficient is generally independent of the volume ratio of the phases as well as the concentration of the partitioned material, an aqueous two-phase system can be scaled up easily. The major disadvantages are (a) the presence of polymers in the product stream, which, to be sure, can be overcome, (b) the extra effort required for recycling the celhich phase, and (c) the relatively poor understanding of aqueous two-phase systems. Nevertheless, it can be concluded that an aqueous two-phase system can be used as a means of immobilizing cells without some of the problems that confront the traditional methods, e.g., cell loading, steric hindrance, surface cell growth, cell death, severe diffusional limitation, etc. Many other arguments favoring cell immobilization on solid supports or in gel matrices should also carry over to this fluid-based immobilization medium. Conclusions The effect of phase-forming polymers on batch fermentation kinetics is studied. In single-phase batch fermentation, a lower maximum cell density, longer stationary phase, and generally higher a-amylase activity are observed in the presence of PEG or dextran. The rate of a-amylase synthesis decreased at high concentrations of dextran but not PEG. The dextran-rich bottom phase's affinity for cells is exploited in the design of a continuous cell recycle system in which the bottom phase is returned to the fermentor and the virtually cell-free top phase is withdrawn for further product purification. Continuous fermentation in which cells are functionally and effectively immobilized in an aqueous two-phase system is compared to the conventional single-phase continuous fermentation. The overall cell density and the residual glucose concentration are both lower in the two-phase system; however, the volumetric productivity of a-amylase is significantly higher in a continuous aqueous two-phase system than in the conventional single-phase counterpart. Repression of a-amylase synthesis in excess glucose is observed in the conventional continuous fermentation but not in an aqueous two-phase system, even at high glucose concentrations of up to 5 g/L. Acknowledgment This work is supported in part by a DuPont Young Faculty Award, Allied-Signal Corporation Foundation Faculty Support Grant, and a Biomedical Research Support Award (NIH Grant RR07042-24). L i t e r a t u r e Cited Alam, S.; Hong, J.; Weigand, W. A. Effect of Yeast Extract on a-Amylase Synthesis by Bacillus amyloliquefaciens. Biotechnol. Bioeng. 1989, 33, 780-785.

Albertsson,P. A. Partition of Cell Particles and Macromolecules, 3rd ed.; John Wiley: New York, 1986. Andersson, E.; Mattiasson, B.; Hahn-Hagerdal, B. Enzymatic Conversion in Aqueous Two-Phase Systems: Deacylation of

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Benzylpenicillin to 6-aminopenicillanic acid with Penicillin Acylase. Enzyme Microb. Technol. 1984, 6, 301-306. Andersson, E.; Johansson, AX.; Hahn-Hagerdal, B. a-Amylase Production in Aqueous Two-Phase Systemswith Bacillus subtilis. Enzyme Microb. Technol. 1985, 7, 333-338. Andersson, E.; Hahn-Hagerdal, B. Bioconversions in Aqueous Two-Phase Systems. Enzyme Microb. Technol. 1990,12,242254. Hahn-Hagerdal, B.; Mattiasson, B.; Albertsson, P.-A. Extractive Bioconversion in Aqueous Two-PhaseSystems: A Model Study on the Conversion of Cellulose to Ethanol, Biotechnol. Lett. 1981a, 3,53-58. Hahn-Hagerdal, B.; Andersson, E.; Lopez-Leiva, M.; Mattiasson, B. Membrane Biotechnology, Co-Immobilization, and Aqueous Two-Phase Systems: Alternatives in Bioconversion of Cellulose. Biotechnol. Bioeng. Symp. 1981b, 11,651-661. Heineken, F. G.; O’Connor, R. J. Continuous Culture Studies on the Biosynthesis of Alkaline Protease, Neutral Protease and a-Amylase by Bacillus subtilis NRRL-B3411. J . Gen. Microbiol. 1972, 73, 35-44. Kuhn, I. Alcoholic Fermentation in an Aqueous Two-Phase System. Biotechnol. Bioeng. 1980,22, 2393-2398. Park, K. M.; Wang, N. S. Alpha-Amylase Assay with Dyed-Starch in Polyethylene Glycol and Dextran Solutions. Biotechnol. Tech. 1991,5,205-208. Puziss, M.; Heden, C.-G. Toxin Production by Clostridium tetani in Biophasic Alcoholic Fermentation in an Aqueous TwoPhase System. Biotechnol. Bioeng. 1965, 7, 355-366. Rinderknecht, H.; Wilding, P.; Haverback, B. J. A New Method for the Determination of a-Amylase. Erperientia 1967, 23, 805-805. Roychoudhury, S.; Parulekar, S. J.; Weigand, W. A. Cell Growth and a-Amylase Production Characteristics of Bacillus amyloliquefaciens. Biotechnol. Bioeng. 1989, 33, 197-206.

Rutten, R.; Daugulis,A. J. Continuous Production of a-Amylase by Bacillus amyloliquefaciens in aTwo-StageFermentor. Biotechnol. Lett. 1987, 9, 505-510. Tjerneld, F.; Persson, I.; Albertsson, P.-A.; Hahn-Hagerdal, B. Enzymatic Hydrolysis of Cellulose in Aqueous Two-Phase Systems. I. Partition of Cellulasesfrom Trichoderma reesei. Biotechnol. Bioeng. 1985a, 27, 1036-1043. Tjerneld, F.; Persson, I.; Albertsson, P.-A.; Hahn-Hagerdal, B. Enzymatic Hydrolysis of Cellulose in Aqueous Two-Phase Systems. 11. Semicontinuous Conversion of a Model Substrate, Solka Floc BW 200. BiotechnoL Bioeng. 1985b, 27, 1044-1050. Toda, K.; Takeuchi, T.; Sano, H. Growth Rate Dependence of Enzyme Synthesis in Chemostat Cultures: a-Amylase, &Galactosidase, Acid Phosphate and b-Fructosidase. J. Chem. Technol. Biotechnol. 1979, 29, 747-754. Walter, H.; Brooks, D. E.; Fisher, D. Partitioning in Aqueous Two-Phase Systems; Academic Press: New York, 1985. Yoo, Y. J.; Hong, J.; Hatch, R. T. Comparison of a-Amylase Activities from Different Assay Methods. Biotechnol. Bioeng. 1987,30, 147-151. Yoo, Y. J.; Cadman, T. W.; Hong, J.; Hatch, R. T. Kinetics of a-Amylase Synthesis from Bacillus amyloliquefaciens. Biotechnol. Bioeng. 1988a, 31, 357-365. Yoo, Y. J.; Cadman, T. W.; Hong, J.; Hatch, R. T. Fed-Batch Fermentation for the Production of a-Amylase by Bacillus amyloliquefaciens. Biotechnol. Bioeng. 1988b,31, 426-432. Accepted June 21, 1991.

Registry No. a-amylase, 9000-90-2; poly(ethy1ene glycol), 25322-68-3; dextran, 9004-54-0.