Nitrogenase Activity of Immobilized Azotubacter vinelandii E. SEYHAN* and D. J. KIRWAN, Department of Chemical Engineering, University of Virginia, Charlottesville, Virginia 22901
Summary As part of a program to investigate the use of biological nitrogen fixation for fertilizer ammonia production, an investigation into the immobilization of the aerobic, nitrogen-fixing bacterium, Azotohucter vinelmdii was undertaken. Immobilization was accomplished by adsorption onto an anionic exchange cellulose (Cellex E ) with loadings as high as 10" cellsig resin. Immobilized cell preparations were tested under both batch and continuous-flow conditions. Nitrogenase activities as high as 4200 nmollmin g resin were observed as measured by the acetylene reduction assay. Immobilized cells retained their activity for as long as 117 hr in a continuous-flow reactor. Activity loss appeared to be related to the development of a variant strain.
INTRODUCTION The use of biological processes involving either whole cells o r enzymes has the potential to contribute to solutions of problems involving energy, food, and waste treatment generally by the utilization of renewable rather than nonrenewable resources. In particular, we are concerned with the production of fertilizer ammonia. The commercial Haber Process efficiently produces ammonia from N2 and H,. However, the cost of the ammonia is directly related to H, costs and, consequently, to the rising costs of natural gas or other fossil fuels. Furthermore, the distribution costs of ammonia, which are nearly equal to the manufacturing costs, will also rise with increasing fuel costs. * Such a situation can have a very deleterious effect upon world cereal grain production, which has grown nearly in proportion to fertilizer nitrogen application. The concept of engineered biological fixation processes, which operate on a local level, may have the potential to maintain fertilizer ammonia supplies at reasonable costs by utilizing either photosynthesis or agricultural waste products as energy sources. * Present address: Merck and Co., Inc., Rahway, New Jersey 07065. Biotechnology and Bioengineering, Vol. XXI, Pp. 271-281 (1979) @ 1979 John Wiley & Sons, Inc. OoO6-3592/79/oo21-0271$01
.oo
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Biological nitrogen fixation, which is carried out by certain freeliving and symbiotic, anaerobic, and aerobic bacteria and algae, accounts for well over 50% of all fixed nitrogen.2 Hence, there are a number of potential organisms and systems to be investigated for their usefulness to fixed-nitrogen production. This project is concerned with the development of technology for using immobilized cells for nitrogen fixation for comparison to more conventional fermentation schemes. In order for such systems to become feasible a number of significant problems must be solved. One of these is the development of mutants that will excrete large quantities of NH,' into the A second problem is the development of suitable methods of immobilization of nitrogen-fixing organisms and the proper design of whole-cell reactors that can allow high nitrogenfixation rates for extended periods of time. Third, biological fixation of nitrogen requires large quantities of chemical energy (ATP) derived from carbohydrates. * For biological fixation to become an economic process a cheap carbon source must become available. Investigations into the conversion of waste cellulosic materials to utilizable carbohydrates or the use of photosynthetic, nitrogen-fixing organisms are underway in a number of laboratories. In our investigation we were concerned with the development of a whole-cell reactor system for nitrogen fixation. We chose to investigate the aerobic bacterium, Azotobacter vineiandii, a free-living, nitrogen-fixing organism, that grows on simple sugars. In particular, we were concerned with the development of simple, inexpensive methods of immobilization of A . vinelandii that would allow it to maintain its nitrogen-fixing capabilities. Since successful nitrogen fixation requires not only that nitrogenase (the enzyme that catalyzes the reduction of N,) be active, but also that cell metabolism be functioning, e.g., glycolysis and respiration for ATP synthesis; it is necessary that the immobilized cell maintain its viability. This may be in contrast to some applications of immobilized whole cells in which only a single enzyme or enzyme system must be intact. Thus, the development of a successful whole-cell immobilization technique could have general application to systems in which viability of the cell is required to effect the desired reaction( s). Whole-cell immobilization methods previously investigated include entrapment in polymeric gels and in collagen, adsorption onto various surfaces, and containment by hollow-fiber membranes. Many studies have reported on the activity of whole cells immobilized within a polyacrylamide gel lattice9-16 and industrial applica-
NITROGENASE ACTIVITY OF IMMOBILIZED A . vinrlut~lii
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tions have been noted for L-malic acid and L-aspartic acid production.15 In many of these studies the cells have apparently lysed within the gel, but the desired enzyme activity was retained. However, Mosbach and LarssonYand Franks'O retained viability for a number of days in acrylamide gels and Kierstan and Bucke17 maintained viability of Saccharomyces cerevisiue in calcium alginate gels. Hollow-fiber ultrafiltration membranes have been employed for enzyme immobilization 18-21 and more recently for retention of whole cells." Such a method has the advantage of having little effect on the environment of the cells since they can be suspended in an appropriate medium within the fibers. Marshall et al.23examined the adsorption of Achrornohac~rrrsp. and Pseudomonas sp. onto a glass surface. Studies at Oak Ridge National L a b ~ r a t o r y ?involved ~ , ~ ~ the adsorption of a wide variety of waste-treatment bacteria to coal particles and the use of such systems in immobilized cell reactors for denitrification and phenol degradation of waste liquids. A number of investigations into the use of ion-exchange resins for whole-cell adsorption have been conducted. Daniels and KempeP6characterized the adsorption of a number of microorganisms onto ion-exchange resins. Because of the preponderance of negative charges on cell surfaces, anionic exchange resins were generally effective for adsorption. Zuyagint ~ e valso ? ~ found that microorganisms (including Azotobacrer chroococcum) were adsorbed on positively charged resins. Hattori and FunenkaPnstudied changes in the physiological state of Escherichiu coli when adsorbed on Dowex I resin as compared to free suspension. Lowered oxidation rates for a variety of carbohydrate substrates were observed for the adsorbed cells. Johnson and Ciegler" immobilized spores of Aspergillus and Penicilliiim , which exhibited invertase activity, onto several ion-exchange celluloses. The preparations were packed into columns and operated continuously for a number of hours. The column could be reused provided care was taken to prevent germination of the spores. Based upon the available literature information, we chose to immobilize A . vinelandii onto ion-exchange resins and observe the nitrogenase activity of the preparation in a continuously operating reactor. EXPERIMENTAL Details of the experimental procedure may be found in the dissertation of Seyhan.30
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Organism, Media and Culture Procedures Azotobacter vinelandii, strain UW 590, was obtained from Professor w. J. Brill, Center for Nitrogen Fixation Research, University of Wisconsin, Madison. This derepressed mutant of the A . vinelandii OP strain is capable of synthesizing nitrogenase in the presence of NH,+ ion but is not known to excrete NH,+. Growth media was modified Burk's31 without fixed nitrogen and containing 1-20 g/liter sucrose. The culture was maintained on room-temperature agar slants of the above media and transferred every two weeks. Four hundred mi batch cultures for immobilization were grown in 1 liter flasks on a rotary shaker at 200 rpm and 30°C. The lag period was decreased by not agitating for the first 1-2 hr after inoculation. The cultures grew up in 15-30 hr. A typical batch curve is shown in Figure 1. Immobilization Procedures A number of ion-exchange resins were obtained from Bio-Rad Laboratories: anionic: Cellex E, Bio-Rex 9, Bio-Rex 5 , AG-3, AG21; cationic: Bio-Rex 70. The organism was grown in batch culture to the desired density, usually middle to late log phase. The cells were centrifuged, washed
220 -
- 2.0
- 1.0
0" -
Time (hours)
Fig. 1 . Batch growth and nitrogenase activity of UW 590 cells in Burk's media with 20 g/liter sucrose. (A) Activity; (0) OD.
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twice with fresh media, and resuspended in media at a concentration of lo8 to lo9 cells/ml. Typically, 1 g resin was added per 100 ml cell suspension and the mixture agitated for 0.5- 1 hr. The microorganisms adsorbed were determined by difference between the starting concentration and that remaining in solution after adsorption. Organism concentrations were determined by filtering the resin particles from the solution and then reading the optical density (OD) at 420 nm and by serial dilution onto plates containing Burk’s media with agar. The resin preparations were then washed with fresh media and stored as a suspension in media at 4°C. Results of tests of the adsorptive capacity of the various resins indicated that only Cellex E and Bio-Rex 9 resins adsorbed appreciable organisms (lo9 to 10” cells/resin), with Cellex E being superior in both adsorption rate and capacity. Cellex E is an anionic exchange cellulose made up of epichlorohydrin-methanolaminecellulose (ECTEOLA) whose exchange groups are readily accessible to macromolecules. Its superior performance may be due to its higher surface area per unit mass or to additional physicochemical properties. It became the resin of choice for our experiments. Assay Methods Since neither the wild strain nor the U W 590 mutant of A . vinelandii excretes ammonia into the medium, it was not possible to monitor the ammonia synthesis rate. Rather, the activity of the nitrogenase enzyme present was monitored with the acetylene reduction test. 32,33 Since reduction of acetylene also requires ATP, the test essentially monitors cell viability as well as the presence and activity of nitrogenase. The nitrogenase activity of free or immobilized cells was determined by incubating 5 ml cell suspension in a 25 ml flask containing an atmosphere of 5% C,H, in air or in argon plus 20% oxygen for 1-3 hr. A sample of the gas atmosphere was then analyzed chromatographically (Carbosieve B column, flame ionization detector) for the amount of ethylene produced. The number of viable cells adsorbed to the resin was determined by conducting a “desorption” experiment and measuring the viable cell concentration in the supernatant. It was found that Burk’s media (PH 7) containing 0.5M KCI and 1 gfiiter sucrose would desorb the cells from the resin and allow retention of their viability. Desorption of freshly immobilized cells indicated viable cell loadings 50% or greater of those adsorbed. The viability of cells desorbed was very sensitive to salt concentrations so that, in general, the
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technique should be considered to provide a lower bound to the number of viable cells on the resin.
Continuous Reactor As shown schematically in Figure 2 a well-agitated, continuousflow reactor (200 ml working volume) containing a slurry of cells immobilized on resin particles was employed. This was preferable to a column because of the poor hydraulic permeability of a packed column of resin particles. The desired constant flow rate of media was provided to the reactor by a syringe pump and solution was withdrawn at the same rate through a glass frit of a porosity small enough to prevent withdrawal of the particles. Thus, immobilized cells were retained within the reactor. Stirring was with a Tefioncoated magnetic stirring bar, and pH and dissolved oxygen were continuously monitored. Air was filtered through glass wool prior to entering the vessel. Slurry samples were withdrawn into a hypodermic needle through a three-way , sterilizable, stainless-steel valve and used to determined nitrogenase activity or viable organism counts in solution or on the resin as described above.
RESULTS AND DISCUSSION
Batch Tests The initial experiments to test the nitrogenase activity of immobilized A . vinelandii cells were under batch conditions in which the Air inlet,
Analyzer
Fig. 2.
Continuous-stirred reactor for immobilized cells
NITROGENASE ACTIVITY OF IMMOBILIZED A . vinelandii
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preparation was suspended in various NH,+-free media in Erlenmeyer flasks containing an atmosphere of air plus 5% C,H,. The purpose of these experiments was to demonstrate that immobilized cells did possess nitrogenase activity and to determine an optimum media in which to suspend them. After a number of experiments in which the phosphate and sucrose levels were varied, it appeared that Burk’s NH4+-free media with sucrose was preferred. It also appeared advisable to grow the cells at high aeration rates (200 rpm) to obtain higher nitrogenase activities of the free cells prior to immobilization. Analysis of a typical experiment indicated that the nitrogenase activity of the cells, prior to immobilization, was 116 nmol C,H4/ loacells min, while the initial activity of the immobilized preparation was 2400 nmol/g min, which corresponded to about 24 nmol/lO* cells min. Thus, immobilization reduced the activity by approximately a factor of five. The activity of this preparation dropped off rapidly in the batch system being below detectable limits at 34 hr. The preparation was then washed and supplied with fresh media and gas and activity was restored. After 70 hr the process was repeated but no activity was restored. The observed activities were due primarily to immobilized cells because the concentration of viable organisms in solution could not account for more than 10% of the observed acetylene reduction. These batch tests demonstrated that immobilized A . vinelandii cells do have nitrogenase activity, but that retention of activity was quite sensitive to the medium composition or the cell environment. Continuous-Flow Experiments
The continuous-flow reactor described above was used to study the nitrogenase activity of the immobilized preparations under constant (controlled) environmental conditions. All experiments were conducted at a dilution rate, D (reciprocal of hydraulic residence time), equal to 0.05 hr-’. It was not possible to operate at higher flow rates because the porous frit in the exit line tended to plug with the small resin particles. Preliminary experiments with the continuous reactor indicated that sucrose concentrations in the range of 1-20 gfliter in Burk’s media appeared to have little influence upon the level of nitrogenase activity or the stability of the preparations. It was found that excess aeration rates were detrimental to maintaining nitrogenase activity. Consequently, in the experiments described below, air was not sparged into the solution but was merely blown into the gas space above the solution. It is well known
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that the enzyme nitrogenase is inactivated by 0, and that the nitrogenase activity of Azotobacter is affected by dissolved oxygen concentrations. 34,35 Figure 3 presents a typical run showing the duration of the nitrogenase (acetylene reducing) activity of the immobilized cells. The specific activity of the preparations was initially quite comparable to that of free cells and was generally maintained for 50-100 hr at which time the nitrogenase activity rapidly decayed. It appeared that the duration of nitrogenase activity was directly related to the initial concentration of viable cells absorbed onto the resin.30 In order to further elucidate the reasons for loss of activity, a series of runs was conducted in which the viable cell count on the resin was determined by desorption of a small sample and the concentration of free cells in the solution was also measured. Figure 4 shows the results of such a run. It is clear from these results that, although the nitrogenase activity drops dramatically after about 80 hr, the cell concentration on the resin is increasing. Note that the concentration of cells in solution and on the resin appear to be following one another, indicating that there is an adsorption equilibrium between bound and free cells. Nevertheless, it is clear that activity loss is not due to loss of cells or cell viability. During these experiments it was noted that activity loss coincided with the dominance of a somewhat different appearing cell. These cells were designated “unpigmented” because their colonies did not exhibit the characteristic creamy yellow color of nitrogenasecontaining Azotobacter when grown on Burk’s media with agar. The organisms, when examined under the microscope, were smaller
-.-
I
I
c‘
I
I
1
I
I
I
I
50
I
E
2
- 40
10-
P
3 x: is E
A
-A
A \-‘A
-
30
A A
- 20
A
Time (hours)
Fig. 3. Nitrogenase activity of immobilized UW 590 cells in a continuous-flow reactor ( 5 g/liter sucrose; initial cell loading: 2 x loLocelldg Cellex E). (A) Activity.
NITROGENASE ACTIVITY OF IMMOBILIZED A . vinrlandii
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Time (hours)
Fig. 4. Nitrogenase activity of immobilized UW 590 cells in continuous-flow reactor (5 g/liter sucrose: initial cell loading: 4 x 10'' cells/g Cellex E). (A) Activity; (m) number of celUml; ( 0 )number of cellsig.
than normal A . vinelandii cells. They grew much faster than normal A . vinelandii on media containing amino acids. They were not able to grow significantly in liquid cultures with Burk's nitrogen-free media with sucrose unless UW 590 cells were also present in the culture. These organisms were never detectable in the beginning of a run, yet they regularly appeared and were found to dominate the culture when nitrogenase activity was observed to have greatly decreased. We believe that the most likely explanation of these cells is that they are variants of the UW 590 strain, possessing little nitrogenase activity, which are the result of adaptation of the cells to their environment. These cell types were also found in a continuousculture (D = 0.05 hr-') of "free" UW 590 A . vinelandii cells after about 30 hr and so are probably not caused by immobilization. It should be noted that if the second cell type were a contaminant rather than a variant of UW 590, it could only take over the culture in 50-100 hr if it were able to reproduce on the surface as well as in solution with a doubling time of approximately 5 hr. It would also be necessary for all of the UW 590 cells present to have lysed in this time period. Thus, it appears that a variant is appearing that is responsible for the loss of nitrogenase activity although the possibility of a contaminant cannot be completely ruled out. Further studies are underway with both free and immobilized cells to elucidate conditions necessary to maintain nitrogen fixation activity.
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CONCLUSIONS 1 ) Adsorption onto anionic exchange resins appears to be a simple method for immobilizing whole cells. There is a dynamic equilibrium between adsorbed and free cells that is affected by pH and ionic strength. 2 ) Viability of immobilized A . vinelandii cells was demonstrated by retention of nitrogenase (acetylene-reducing) activity for over 100 hr of continuous operation. 3) Loss of nitrogenase activity was postulated to be due to the development of a variant of A . vinelandii possessing little nitrogenase activity, rather than desorption of immobilized cells or the loss of viability of the immobilized cells. Partial support of this work by NSF/RANN grant No. APR 72-03442 A04 is gratefully acknowledged.
References 1. D. F. Safrany, Scientific American. 231, 64 (1974). 2. R. W. F. Hardy and U. P. Havelka, Science. 188, 633 (1975). 3. J. K. Gordon and W. J . Brill, Proc. Nut/. Acnd. Sci.. 69, 3501 (1972). 4. J. K. Gordon and W. J. Brill, Biochem. Biophys. Res. Commun., 59, 967 (1974). 5. K. T. Shanmugam and R. C. Valentine, Proc. N u f l . Acad. Sci., 72, 136 (1975). 6. R. C. Valentine, Proceedings of an NSF/RANN sponsored conference on “Enzyme Technology and Renewable Resources,” University of Virginia, May, 1976, p. 19. 7. P. E. Bishop, J. K. Gordon, V. K. Shan, and W. J. Brill, Genefic Engineering for Nifrogen Fixarion, A. Hollaender, Ed. (Plenum, New York, 1977), p. 67. 8. K. Andersen, K. T. Shanmugam, and R. C. Valentine, in Ref. 7, p. 95. 9. K. Mosbach and P. Larsson, Biofechnol. Bioeng., 12, 19 (1970). 10. N. E. Franks, Biotechnol. Bioeng. Svmp.. 3, 327 (1972). 1 1 . I. Chibati, T. Tosa, T. Sato, T. Mori, and K. Yamamoto, Biofechnol. Bioeng. S y m p . , 3, 303 (1972). 12. C. K. A. Martin and D. Perlman, Biotechnol. Bioeng.. 18, 217 (1976). 13. K. Yamamoto, T. Sato, T. Tosa, and I. Chibata, Biofechnol. Biorng., 16, 1589, 1601 (1974). 14. I. Chibata, T. Tosa, and T. Sato, Appl. Microbid., 27, 878 (1974). 15. K. Yamamoto, T. Tosa, K. Yamashita, and I. Chibata, Biotechnol. Bioeng., 19, 1101 (1977). 16. W. Slowinski and S . Charm, Biorechnol. Bioeng., 15, 973 (1973). 17. M. Kierstan and C. Bucke, Biofechnol. Bioeng., 19, 387 (1977). 18. P. R. Rony, Biotechnol. Bioeng.. 13, 431 (1971). 19. L. R. Waterland, A. S. Michaels, and C. R. Robertson, AIChE J . . 20, 50 (1974). 20. L . R. Waterland, C. R. Robertson, and A. S. Michaels, Chern. Eng. Commun., 2, 37 (1975).
NITROGENASE ACTIVITY OF IMMOBILIZED A . Linelandii
28 1
21. K. L. Smiley, D. E. Hensley, and H. J . Gardorf, A p p l . Environ. Mic,robiul., 31, 615 (1976). 22. J . Kan and M. L . Shuler, "An immobilized whole cell hollow fiber reactor for urocanic acid production," presented at 69th AIChE Annual Meeting, Chicago, Nov. 28-Dec. 2, 1976. 23. K. C. Marshall, R. Stout, and R. Mitchell, J . G r n . Microbiol.. 68, 337 (1971). 24. C. D. Scott and C. W. Hancher, Biorechnol. Bioeng., 18, 1393 (1976). 25. D. W. Holladay, C. W. Hancher, D. D. Chilcote, and C. D. Scott, "Biodegradation of phenolic waste liquors in stirred-tank, columnar and fluidized bed bioreactors," presented at 69th AIChE Annual Meeting, Chicago, Nov. 28-Dec. 2, 1976. 26. S . L. Daniels and L. L . Kempe, C . E . P . S y m p . S e r . , 62, 142 (1966). 27. D. G. Zuyagintsev, Microbiology ( U S S R ) , 31(2), 275 (1962). 28. T. Hattori and C. Funenka, Biochim. Biophys. A c t a . 31, 581 (1959). 29. D. E. Johnson and A . Ciegler, Arc,h. Biochrm. Biophys., 130, 384 (1969). 30. E. Seyhan. "Immobilized whole cell catalysis," Ph.D. dissertation, University of Virginia, May, 1977. 31. G. W. Strandberg and P. W. Wilson, Can. J . Microhiol.. 14, 25 (1968). 32. M. J . Dilworth, Biochini. Biopliys. Actu. 127, 285 (1966). 33. R. C. Burns and R. W. F. Hardy. Nitrogen Fixation in Bacteria arid Higher Plants (Springer-Verlag. Berlin, (1975). 34. H . Dalton m d J . R. Postgate, J . Gen. Microbiol.. 56, (1969). 35. J. R. Postgate, Tlie Clie/nistrya11d Biochemistry of'Nitrogen Fixation (Plenum, New York, 1971). Chap. 5.
Accepted for Publication May 20, 1978
Summary As part of a program to investigate the use of biological nitrogen fixation for fertilizer ammonia production, an investigation into the immobilization of the aerobic, nitrogen-fixing bacterium, Azotohucter vinelmdii was undertaken. Immobilization was accomplished by adsorption onto an anionic exchange cellulose (Cellex E ) with loadings as high as 10" cellsig resin. Immobilized cell preparations were tested under both batch and continuous-flow conditions. Nitrogenase activities as high as 4200 nmollmin g resin were observed as measured by the acetylene reduction assay. Immobilized cells retained their activity for as long as 117 hr in a continuous-flow reactor. Activity loss appeared to be related to the development of a variant strain.
INTRODUCTION The use of biological processes involving either whole cells o r enzymes has the potential to contribute to solutions of problems involving energy, food, and waste treatment generally by the utilization of renewable rather than nonrenewable resources. In particular, we are concerned with the production of fertilizer ammonia. The commercial Haber Process efficiently produces ammonia from N2 and H,. However, the cost of the ammonia is directly related to H, costs and, consequently, to the rising costs of natural gas or other fossil fuels. Furthermore, the distribution costs of ammonia, which are nearly equal to the manufacturing costs, will also rise with increasing fuel costs. * Such a situation can have a very deleterious effect upon world cereal grain production, which has grown nearly in proportion to fertilizer nitrogen application. The concept of engineered biological fixation processes, which operate on a local level, may have the potential to maintain fertilizer ammonia supplies at reasonable costs by utilizing either photosynthesis or agricultural waste products as energy sources. * Present address: Merck and Co., Inc., Rahway, New Jersey 07065. Biotechnology and Bioengineering, Vol. XXI, Pp. 271-281 (1979) @ 1979 John Wiley & Sons, Inc. OoO6-3592/79/oo21-0271$01
.oo
272
SEYHAN A N D KIRWAN
Biological nitrogen fixation, which is carried out by certain freeliving and symbiotic, anaerobic, and aerobic bacteria and algae, accounts for well over 50% of all fixed nitrogen.2 Hence, there are a number of potential organisms and systems to be investigated for their usefulness to fixed-nitrogen production. This project is concerned with the development of technology for using immobilized cells for nitrogen fixation for comparison to more conventional fermentation schemes. In order for such systems to become feasible a number of significant problems must be solved. One of these is the development of mutants that will excrete large quantities of NH,' into the A second problem is the development of suitable methods of immobilization of nitrogen-fixing organisms and the proper design of whole-cell reactors that can allow high nitrogenfixation rates for extended periods of time. Third, biological fixation of nitrogen requires large quantities of chemical energy (ATP) derived from carbohydrates. * For biological fixation to become an economic process a cheap carbon source must become available. Investigations into the conversion of waste cellulosic materials to utilizable carbohydrates or the use of photosynthetic, nitrogen-fixing organisms are underway in a number of laboratories. In our investigation we were concerned with the development of a whole-cell reactor system for nitrogen fixation. We chose to investigate the aerobic bacterium, Azotobacter vineiandii, a free-living, nitrogen-fixing organism, that grows on simple sugars. In particular, we were concerned with the development of simple, inexpensive methods of immobilization of A . vinelandii that would allow it to maintain its nitrogen-fixing capabilities. Since successful nitrogen fixation requires not only that nitrogenase (the enzyme that catalyzes the reduction of N,) be active, but also that cell metabolism be functioning, e.g., glycolysis and respiration for ATP synthesis; it is necessary that the immobilized cell maintain its viability. This may be in contrast to some applications of immobilized whole cells in which only a single enzyme or enzyme system must be intact. Thus, the development of a successful whole-cell immobilization technique could have general application to systems in which viability of the cell is required to effect the desired reaction( s). Whole-cell immobilization methods previously investigated include entrapment in polymeric gels and in collagen, adsorption onto various surfaces, and containment by hollow-fiber membranes. Many studies have reported on the activity of whole cells immobilized within a polyacrylamide gel lattice9-16 and industrial applica-
NITROGENASE ACTIVITY OF IMMOBILIZED A . vinrlut~lii
273
tions have been noted for L-malic acid and L-aspartic acid production.15 In many of these studies the cells have apparently lysed within the gel, but the desired enzyme activity was retained. However, Mosbach and LarssonYand Franks'O retained viability for a number of days in acrylamide gels and Kierstan and Bucke17 maintained viability of Saccharomyces cerevisiue in calcium alginate gels. Hollow-fiber ultrafiltration membranes have been employed for enzyme immobilization 18-21 and more recently for retention of whole cells." Such a method has the advantage of having little effect on the environment of the cells since they can be suspended in an appropriate medium within the fibers. Marshall et al.23examined the adsorption of Achrornohac~rrrsp. and Pseudomonas sp. onto a glass surface. Studies at Oak Ridge National L a b ~ r a t o r y ?involved ~ , ~ ~ the adsorption of a wide variety of waste-treatment bacteria to coal particles and the use of such systems in immobilized cell reactors for denitrification and phenol degradation of waste liquids. A number of investigations into the use of ion-exchange resins for whole-cell adsorption have been conducted. Daniels and KempeP6characterized the adsorption of a number of microorganisms onto ion-exchange resins. Because of the preponderance of negative charges on cell surfaces, anionic exchange resins were generally effective for adsorption. Zuyagint ~ e valso ? ~ found that microorganisms (including Azotobacrer chroococcum) were adsorbed on positively charged resins. Hattori and FunenkaPnstudied changes in the physiological state of Escherichiu coli when adsorbed on Dowex I resin as compared to free suspension. Lowered oxidation rates for a variety of carbohydrate substrates were observed for the adsorbed cells. Johnson and Ciegler" immobilized spores of Aspergillus and Penicilliiim , which exhibited invertase activity, onto several ion-exchange celluloses. The preparations were packed into columns and operated continuously for a number of hours. The column could be reused provided care was taken to prevent germination of the spores. Based upon the available literature information, we chose to immobilize A . vinelandii onto ion-exchange resins and observe the nitrogenase activity of the preparation in a continuously operating reactor. EXPERIMENTAL Details of the experimental procedure may be found in the dissertation of Seyhan.30
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Organism, Media and Culture Procedures Azotobacter vinelandii, strain UW 590, was obtained from Professor w. J. Brill, Center for Nitrogen Fixation Research, University of Wisconsin, Madison. This derepressed mutant of the A . vinelandii OP strain is capable of synthesizing nitrogenase in the presence of NH,+ ion but is not known to excrete NH,+. Growth media was modified Burk's31 without fixed nitrogen and containing 1-20 g/liter sucrose. The culture was maintained on room-temperature agar slants of the above media and transferred every two weeks. Four hundred mi batch cultures for immobilization were grown in 1 liter flasks on a rotary shaker at 200 rpm and 30°C. The lag period was decreased by not agitating for the first 1-2 hr after inoculation. The cultures grew up in 15-30 hr. A typical batch curve is shown in Figure 1. Immobilization Procedures A number of ion-exchange resins were obtained from Bio-Rad Laboratories: anionic: Cellex E, Bio-Rex 9, Bio-Rex 5 , AG-3, AG21; cationic: Bio-Rex 70. The organism was grown in batch culture to the desired density, usually middle to late log phase. The cells were centrifuged, washed
220 -
- 2.0
- 1.0
0" -
Time (hours)
Fig. 1 . Batch growth and nitrogenase activity of UW 590 cells in Burk's media with 20 g/liter sucrose. (A) Activity; (0) OD.
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twice with fresh media, and resuspended in media at a concentration of lo8 to lo9 cells/ml. Typically, 1 g resin was added per 100 ml cell suspension and the mixture agitated for 0.5- 1 hr. The microorganisms adsorbed were determined by difference between the starting concentration and that remaining in solution after adsorption. Organism concentrations were determined by filtering the resin particles from the solution and then reading the optical density (OD) at 420 nm and by serial dilution onto plates containing Burk’s media with agar. The resin preparations were then washed with fresh media and stored as a suspension in media at 4°C. Results of tests of the adsorptive capacity of the various resins indicated that only Cellex E and Bio-Rex 9 resins adsorbed appreciable organisms (lo9 to 10” cells/resin), with Cellex E being superior in both adsorption rate and capacity. Cellex E is an anionic exchange cellulose made up of epichlorohydrin-methanolaminecellulose (ECTEOLA) whose exchange groups are readily accessible to macromolecules. Its superior performance may be due to its higher surface area per unit mass or to additional physicochemical properties. It became the resin of choice for our experiments. Assay Methods Since neither the wild strain nor the U W 590 mutant of A . vinelandii excretes ammonia into the medium, it was not possible to monitor the ammonia synthesis rate. Rather, the activity of the nitrogenase enzyme present was monitored with the acetylene reduction test. 32,33 Since reduction of acetylene also requires ATP, the test essentially monitors cell viability as well as the presence and activity of nitrogenase. The nitrogenase activity of free or immobilized cells was determined by incubating 5 ml cell suspension in a 25 ml flask containing an atmosphere of 5% C,H, in air or in argon plus 20% oxygen for 1-3 hr. A sample of the gas atmosphere was then analyzed chromatographically (Carbosieve B column, flame ionization detector) for the amount of ethylene produced. The number of viable cells adsorbed to the resin was determined by conducting a “desorption” experiment and measuring the viable cell concentration in the supernatant. It was found that Burk’s media (PH 7) containing 0.5M KCI and 1 gfiiter sucrose would desorb the cells from the resin and allow retention of their viability. Desorption of freshly immobilized cells indicated viable cell loadings 50% or greater of those adsorbed. The viability of cells desorbed was very sensitive to salt concentrations so that, in general, the
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technique should be considered to provide a lower bound to the number of viable cells on the resin.
Continuous Reactor As shown schematically in Figure 2 a well-agitated, continuousflow reactor (200 ml working volume) containing a slurry of cells immobilized on resin particles was employed. This was preferable to a column because of the poor hydraulic permeability of a packed column of resin particles. The desired constant flow rate of media was provided to the reactor by a syringe pump and solution was withdrawn at the same rate through a glass frit of a porosity small enough to prevent withdrawal of the particles. Thus, immobilized cells were retained within the reactor. Stirring was with a Tefioncoated magnetic stirring bar, and pH and dissolved oxygen were continuously monitored. Air was filtered through glass wool prior to entering the vessel. Slurry samples were withdrawn into a hypodermic needle through a three-way , sterilizable, stainless-steel valve and used to determined nitrogenase activity or viable organism counts in solution or on the resin as described above.
RESULTS AND DISCUSSION
Batch Tests The initial experiments to test the nitrogenase activity of immobilized A . vinelandii cells were under batch conditions in which the Air inlet,
Analyzer
Fig. 2.
Continuous-stirred reactor for immobilized cells
NITROGENASE ACTIVITY OF IMMOBILIZED A . vinelandii
277
preparation was suspended in various NH,+-free media in Erlenmeyer flasks containing an atmosphere of air plus 5% C,H,. The purpose of these experiments was to demonstrate that immobilized cells did possess nitrogenase activity and to determine an optimum media in which to suspend them. After a number of experiments in which the phosphate and sucrose levels were varied, it appeared that Burk’s NH4+-free media with sucrose was preferred. It also appeared advisable to grow the cells at high aeration rates (200 rpm) to obtain higher nitrogenase activities of the free cells prior to immobilization. Analysis of a typical experiment indicated that the nitrogenase activity of the cells, prior to immobilization, was 116 nmol C,H4/ loacells min, while the initial activity of the immobilized preparation was 2400 nmol/g min, which corresponded to about 24 nmol/lO* cells min. Thus, immobilization reduced the activity by approximately a factor of five. The activity of this preparation dropped off rapidly in the batch system being below detectable limits at 34 hr. The preparation was then washed and supplied with fresh media and gas and activity was restored. After 70 hr the process was repeated but no activity was restored. The observed activities were due primarily to immobilized cells because the concentration of viable organisms in solution could not account for more than 10% of the observed acetylene reduction. These batch tests demonstrated that immobilized A . vinelandii cells do have nitrogenase activity, but that retention of activity was quite sensitive to the medium composition or the cell environment. Continuous-Flow Experiments
The continuous-flow reactor described above was used to study the nitrogenase activity of the immobilized preparations under constant (controlled) environmental conditions. All experiments were conducted at a dilution rate, D (reciprocal of hydraulic residence time), equal to 0.05 hr-’. It was not possible to operate at higher flow rates because the porous frit in the exit line tended to plug with the small resin particles. Preliminary experiments with the continuous reactor indicated that sucrose concentrations in the range of 1-20 gfliter in Burk’s media appeared to have little influence upon the level of nitrogenase activity or the stability of the preparations. It was found that excess aeration rates were detrimental to maintaining nitrogenase activity. Consequently, in the experiments described below, air was not sparged into the solution but was merely blown into the gas space above the solution. It is well known
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SEYHAN A N D KIRWAN
that the enzyme nitrogenase is inactivated by 0, and that the nitrogenase activity of Azotobacter is affected by dissolved oxygen concentrations. 34,35 Figure 3 presents a typical run showing the duration of the nitrogenase (acetylene reducing) activity of the immobilized cells. The specific activity of the preparations was initially quite comparable to that of free cells and was generally maintained for 50-100 hr at which time the nitrogenase activity rapidly decayed. It appeared that the duration of nitrogenase activity was directly related to the initial concentration of viable cells absorbed onto the resin.30 In order to further elucidate the reasons for loss of activity, a series of runs was conducted in which the viable cell count on the resin was determined by desorption of a small sample and the concentration of free cells in the solution was also measured. Figure 4 shows the results of such a run. It is clear from these results that, although the nitrogenase activity drops dramatically after about 80 hr, the cell concentration on the resin is increasing. Note that the concentration of cells in solution and on the resin appear to be following one another, indicating that there is an adsorption equilibrium between bound and free cells. Nevertheless, it is clear that activity loss is not due to loss of cells or cell viability. During these experiments it was noted that activity loss coincided with the dominance of a somewhat different appearing cell. These cells were designated “unpigmented” because their colonies did not exhibit the characteristic creamy yellow color of nitrogenasecontaining Azotobacter when grown on Burk’s media with agar. The organisms, when examined under the microscope, were smaller
-.-
I
I
c‘
I
I
1
I
I
I
I
50
I
E
2
- 40
10-
P
3 x: is E
A
-A
A \-‘A
-
30
A A
- 20
A
Time (hours)
Fig. 3. Nitrogenase activity of immobilized UW 590 cells in a continuous-flow reactor ( 5 g/liter sucrose; initial cell loading: 2 x loLocelldg Cellex E). (A) Activity.
NITROGENASE ACTIVITY OF IMMOBILIZED A . vinrlandii
279
Time (hours)
Fig. 4. Nitrogenase activity of immobilized UW 590 cells in continuous-flow reactor (5 g/liter sucrose: initial cell loading: 4 x 10'' cells/g Cellex E). (A) Activity; (m) number of celUml; ( 0 )number of cellsig.
than normal A . vinelandii cells. They grew much faster than normal A . vinelandii on media containing amino acids. They were not able to grow significantly in liquid cultures with Burk's nitrogen-free media with sucrose unless UW 590 cells were also present in the culture. These organisms were never detectable in the beginning of a run, yet they regularly appeared and were found to dominate the culture when nitrogenase activity was observed to have greatly decreased. We believe that the most likely explanation of these cells is that they are variants of the UW 590 strain, possessing little nitrogenase activity, which are the result of adaptation of the cells to their environment. These cell types were also found in a continuousculture (D = 0.05 hr-') of "free" UW 590 A . vinelandii cells after about 30 hr and so are probably not caused by immobilization. It should be noted that if the second cell type were a contaminant rather than a variant of UW 590, it could only take over the culture in 50-100 hr if it were able to reproduce on the surface as well as in solution with a doubling time of approximately 5 hr. It would also be necessary for all of the UW 590 cells present to have lysed in this time period. Thus, it appears that a variant is appearing that is responsible for the loss of nitrogenase activity although the possibility of a contaminant cannot be completely ruled out. Further studies are underway with both free and immobilized cells to elucidate conditions necessary to maintain nitrogen fixation activity.
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SEYHAN AND KIRWAN
CONCLUSIONS 1 ) Adsorption onto anionic exchange resins appears to be a simple method for immobilizing whole cells. There is a dynamic equilibrium between adsorbed and free cells that is affected by pH and ionic strength. 2 ) Viability of immobilized A . vinelandii cells was demonstrated by retention of nitrogenase (acetylene-reducing) activity for over 100 hr of continuous operation. 3) Loss of nitrogenase activity was postulated to be due to the development of a variant of A . vinelandii possessing little nitrogenase activity, rather than desorption of immobilized cells or the loss of viability of the immobilized cells. Partial support of this work by NSF/RANN grant No. APR 72-03442 A04 is gratefully acknowledged.
References 1. D. F. Safrany, Scientific American. 231, 64 (1974). 2. R. W. F. Hardy and U. P. Havelka, Science. 188, 633 (1975). 3. J. K. Gordon and W. J . Brill, Proc. Nut/. Acnd. Sci.. 69, 3501 (1972). 4. J. K. Gordon and W. J. Brill, Biochem. Biophys. Res. Commun., 59, 967 (1974). 5. K. T. Shanmugam and R. C. Valentine, Proc. N u f l . Acad. Sci., 72, 136 (1975). 6. R. C. Valentine, Proceedings of an NSF/RANN sponsored conference on “Enzyme Technology and Renewable Resources,” University of Virginia, May, 1976, p. 19. 7. P. E. Bishop, J. K. Gordon, V. K. Shan, and W. J. Brill, Genefic Engineering for Nifrogen Fixarion, A. Hollaender, Ed. (Plenum, New York, 1977), p. 67. 8. K. Andersen, K. T. Shanmugam, and R. C. Valentine, in Ref. 7, p. 95. 9. K. Mosbach and P. Larsson, Biofechnol. Bioeng., 12, 19 (1970). 10. N. E. Franks, Biotechnol. Bioeng. Svmp.. 3, 327 (1972). 1 1 . I. Chibati, T. Tosa, T. Sato, T. Mori, and K. Yamamoto, Biofechnol. Bioeng. S y m p . , 3, 303 (1972). 12. C. K. A. Martin and D. Perlman, Biotechnol. Bioeng.. 18, 217 (1976). 13. K. Yamamoto, T. Sato, T. Tosa, and I. Chibata, Biofechnol. Biorng., 16, 1589, 1601 (1974). 14. I. Chibata, T. Tosa, and T. Sato, Appl. Microbid., 27, 878 (1974). 15. K. Yamamoto, T. Tosa, K. Yamashita, and I. Chibata, Biotechnol. Bioeng., 19, 1101 (1977). 16. W. Slowinski and S . Charm, Biorechnol. Bioeng., 15, 973 (1973). 17. M. Kierstan and C. Bucke, Biofechnol. Bioeng., 19, 387 (1977). 18. P. R. Rony, Biotechnol. Bioeng.. 13, 431 (1971). 19. L. R. Waterland, A. S. Michaels, and C. R. Robertson, AIChE J . . 20, 50 (1974). 20. L . R. Waterland, C. R. Robertson, and A. S. Michaels, Chern. Eng. Commun., 2, 37 (1975).
NITROGENASE ACTIVITY OF IMMOBILIZED A . Linelandii
28 1
21. K. L. Smiley, D. E. Hensley, and H. J . Gardorf, A p p l . Environ. Mic,robiul., 31, 615 (1976). 22. J . Kan and M. L . Shuler, "An immobilized whole cell hollow fiber reactor for urocanic acid production," presented at 69th AIChE Annual Meeting, Chicago, Nov. 28-Dec. 2, 1976. 23. K. C. Marshall, R. Stout, and R. Mitchell, J . G r n . Microbiol.. 68, 337 (1971). 24. C. D. Scott and C. W. Hancher, Biorechnol. Bioeng., 18, 1393 (1976). 25. D. W. Holladay, C. W. Hancher, D. D. Chilcote, and C. D. Scott, "Biodegradation of phenolic waste liquors in stirred-tank, columnar and fluidized bed bioreactors," presented at 69th AIChE Annual Meeting, Chicago, Nov. 28-Dec. 2, 1976. 26. S . L. Daniels and L. L . Kempe, C . E . P . S y m p . S e r . , 62, 142 (1966). 27. D. G. Zuyagintsev, Microbiology ( U S S R ) , 31(2), 275 (1962). 28. T. Hattori and C. Funenka, Biochim. Biophys. A c t a . 31, 581 (1959). 29. D. E. Johnson and A . Ciegler, Arc,h. Biochrm. Biophys., 130, 384 (1969). 30. E. Seyhan. "Immobilized whole cell catalysis," Ph.D. dissertation, University of Virginia, May, 1977. 31. G. W. Strandberg and P. W. Wilson, Can. J . Microhiol.. 14, 25 (1968). 32. M. J . Dilworth, Biochini. Biopliys. Actu. 127, 285 (1966). 33. R. C. Burns and R. W. F. Hardy. Nitrogen Fixation in Bacteria arid Higher Plants (Springer-Verlag. Berlin, (1975). 34. H . Dalton m d J . R. Postgate, J . Gen. Microbiol.. 56, (1969). 35. J. R. Postgate, Tlie Clie/nistrya11d Biochemistry of'Nitrogen Fixation (Plenum, New York, 1971). Chap. 5.
Accepted for Publication May 20, 1978
