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Biological Aspects of Continuous Cultivation of Microorganisms
Biological Aspects of Continuous Cultivation of Microorganisms T. HOLME Department of Bacteriology, Karolinska lnstltutet, Stockholm, Sweden
.............................................. ...................................... Yield Measurements .................................... Morphology and Chemical Composition of Cells ............
I. Introduction
II. One-Stage Cultivation
A. B. C. Production of Antigenic Components ...................... D. Population Genetics in Continuous Culture ................ E. Mixed Populations ..................................... 111. Two-Stage Cultivation ...................................... IV. Design of Laboratory Scale Units ............................ A. Continuous Liquid Feed Devices ......................... B. Overflow Devices ...................................... C. Aeration-Agitation ..................................... D. Two-Stage Cultivation Equipment ........................ References ................................................
101 102 102 105 106 107 109 110 111 111 111 113 114 115
1. Introduction In the extensive review of continuous fermentation, which appeared in the first volume of this series, the theoretical as well as the practical aspects of the continuous culture methods were discussed and references to pertinent literature up to 1958 were also given (Gerhardt and Bartlett, 1959). During the last three years, a rapidly increasing number of reports have appeared in which the aid of continuous culture had been enlisted in tackling biological problems. Most of these studies have been made possible, or greatly facilitated, by the use of continuous culture methods and the experiments to be reviewed here all belong to this class. The main purpose of this article will be to present the possibilities afforded by continuous culture methods and the writer has attempted to do this by analyzing and summarizing some experimental work. The design of laboratory scale units will also be touched upon, No present-day microbiologist needs to hesitate to use the continuous culture technique on a laboratory scale because of technical difficulties, but the great variety of units described might cause Some confusion. Therefore, a description of useful features of design 101
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of laboratory scale units for the cultivation of bacteria and filamentous molds would seem to be of value.
It. One-Stage Cultivation In the study of the influence of environmental conditions on growth or on the synthesizing capabilities of growing cells, the one-stage cultivation offers great advantages over batch processes. Many workers have utilized the possibility of changing various environmental factors of cells growing at a fixed rate in the steady state to evaluate optimal conditions for the propagation of cells or the development of cell products. In these studies, yield measurements, analysis of chemical and antigenic composition of cells and of genetic stability of cell populations are of fundamental importance.
A. YIELDMEASWMENTS For yield measurements, the continuous culture method has proven to offer distinct advantages over batch experiments. In batch cultures cells might degrade nutrients during the lag period and transform them into other products which may be metabolized during the period of active growth. Thus, if measurements are performed on samples taken from the logarithmic phase, these products may be utilized simultaneously with the nutrients originally supplied. This will give rise to an error in the value of the yield constant. If the culture is allowed to enter the stationary phase, difficulties can again arise owing to an altered metabolic pattern of the celIs. When facing the problem of cultivating an organism with known nutritional requirements but with no information about optimal growth conditions in the continuous culture, it is frequently necessary to obtain specific information concerning the consumption of different nutrients. This is accomplished by decreasing the concentration of an essential nutrient in the medium until it limits the population density. It is then possible to determine the “yield constant,” which is usually expressed as grams of organisms formed per gram of nutrient consumed. At a certain cell density, determined by the concentration of the limiting factor, the optimum ranges for different environmental factors, such as temperature and pH, can be determined. Then a
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gradual increase in the concentration of the limiting factor can be started, and continued until oxygen deficiency or the accumulation of toxic metabolites prevent an increased total yield. When cultivating bacteria with complex nutritional requirements, the total yield is often limited by accumulation of toxic metabolites. In batch cultures, this has been partly overcome in dialysis bag culture (Gladstone, 1948) or by using two-phase systems (Tyrell et al., 1958). Dialyzing out toxic metabolites can also be successfully applied in continuous culture. An increase in the yield of Brucelln abortus has been reported by Hauschild and Pivnick (196l), when they included a dialysis tube in the fermentor. The culture, to which fresh medium was continuously added, was grown outside the dialysis tube. A continuous stream of fresh nutrient medium was also conducted through the dialysis tube enabling low-molecular metabolites to diffuse out of the culture fluid. The maximum output of cells in a continuous culture can usually be predicted from measurements of the doubling time of cells in batch cultures (Herbert et al., 1956). A close agreement with the theoretical value has been observed for many different enteric bacteria [Aerobacter spp. (Herbert, 1958; Herbert et al., 1956), Escherichia coli (Holme, 1957),] streptococci (Karush et al., 1956), yeasts [Torula (Herbert, 1958), Saccharomyces (Dawson, 1960)1, and €or molds [Gibberella fujikuroi (Holme and Zacharias, in press)]. If the population density decreases earlier than expected when the dilution rate is increased, oxygen deficiency should be suspected. In the study of Brucelkz abortus (Hauschild and Pivnick, 1961), maximum output was obtained at a much lower dilution rate than expected. It was claimed that aeration was in excess, although evidence in support of this was inconclusive, since the oxygen demand of the culture had not been determined. Chemical antifoam was also used, which may have caused a much lower oxygen transfer rate than that estimated by the sulfite oxidation method. An interesting application of continuous culture to studies of growth yields in relation to the energy metabolism has been made by Rosenberger and Elsden (1980) and Bauchop and Elsden (1960). Their work is presented in detail by Gunsalus and Shuster (1961) and will only be mentioned briefly. Streptococcus fueculis, which has an anaerobic metabolism, was the organism used in
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these investigations. It offers great advantages when energy-yield relationships are studied, since the cell carbon is furnished by compounds not participating in the energy release. Less than 1% of the fermented glucose (the energy source) is found in the cellular components. It was found that the glucose yield constant was greater when growth was limited by the glucose supply than when tryptophan was limiting. Under conditions of tryptophan limitation the amount of glucose used per unit weight of cells per unit time remained roughly constant, irrespective of the growth rate. This means that the requirements of cell synthesis in S . faecalis do not control the rate of the energy-yielding metabolism. When factors other than the energy source are limiting growth, the yield calculation can frequently be very simple. In nitrogenlimited growth of Escherichia coli practically all of the nitrogen is assimilated, except at dilution rates proximate to the critical rate. At low growth rates, however, polysaccharides accumulate in the cells to a great extent giving an apparent increase in the growth yield ( Holme, 1957). Parallel to polysaccharide synthesis, several low-molecular-weight substances such as a-ketoglutaric acid appeared in the culture fluid (Holme, 1958). In agreement with these experiments, Dawson (1960), cultivating Saccharomyces rouxii, an osmophilic yeast that produces glycerol and arabitol, found that nitrogen-limited growth resulted in an accumulation of glycerol in the culture fluid in fully aerobic cultures. The continuous culture technique has also been successfully applied to the study of nitrogen fixation by Axotobacter vinelandii (Zacharias, in press). Cells were grown in synthetic medium containing no nitrogen source. The population density was about 2.5gm. dry weight per liter of culture, at a dilution rate of about 0.3 hr.-l. Air and gaseous nitrogen was added from separate sources. When the additional nitrogen gas was subjected to irradiation with p-rays from a 10 pC. Srm source before entering the culture, an increased nitrogen fixation, measured as milligrams nitrogen fixed per gram glucose consumed, was observed. The population density remained constant, and, as a result of the decrease in glucose consumption, the glucose yield constant increased from approximately 0.4 to 0.5. Similar results were obtained with ultraviolet irradiation of the nitrogen gas.
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B. MORPHOLOGY AND CHEMICAL COMPOSITION OF CELLS There is, in general, a good agreement between data provided by continuous culture methods and by static cultures concerning the iduences of nutrient limitations and growth rate on the chemical composition of cells. In unrestricted, “balanced growth the ribonucleic acid (RNA) content of the cells increased with increased growth rate and the mean weight per cell also increased (Maalpre, 1960). The same observations were made in continuous cultures with the energy source constituting the limiting factor (Herbert, 1959). It was also found that the deoxyribonucleic acid (DNA) content per cell increased considerably with increased growth rate. However, staining for chromatinic bodies revealed that the rapidly growing, large cells were multinucleate, and that the amount of DNA per nucleus was practically constant. Munson and Maclean (1961) exploited the possibility of maintaining the steady-state growth of Escherichia coli cells having a certain length distribution. Data of survival after X-ray radiation combined with nuclear staining of cells from cultures with different cell length distribution provided evidence to suggest that radiosensitive sites were located in each nuclear body. Pirt and Callow (1959~)have performed a study on the influence of pH on the morphology of Penicillium chrysogenum in submerged culture. The mold was grown in a 2-liter, stirred fennentor at a density of about l o p . dry weight per liter. The dilution rate was 0.05 hr.-l. Glucose was limiting and aeration was in excess. At steady-state growth with a pH around 7, aberrant morphology was noted and, in these experiments, pellet formation was dependent on pH. At pH 6 a norma1 filamentous growth was obtained. The authors suggested that the resistance of the cell walls of the hyphae decreased with an increase in pH from 6.0 to 7.4. Studies of the influences of different limitations on the chemical composition of cells have given many results from which practical implications may be deduced. As mentioned in connection with the studies of Rosenberger and Elsden (1960), limitation by essential nutrients other than the energy source does not control the rate of oxidation of the energy source. In Streptococcus faecalis, tryptophan limitation resulted mainly in the production of large amounts of lactic acid; in Escherichia coli and Torula utilis, ammonia limitation resulted in a large increase in the polysaccharide content of the cells (Herbert, 1958;Holme, 1957).
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Isolation of specific, biosynthetic enzymes from bacterial cells might, in the near future, constitute a field of considerable interest. The mechanism governing the regulation of levels of such enzymes in the cells has been studied by Gorini (1960), who utilized the possibility of maintaining a stabile and very low concentration of an essential nutrient by making it the limiting factor in chemostat experiments. Certain arginine-deficient mutants of Escherichia coli have a barely detectable ornithine transcarbamylase activity when grown in the presence of exogenous arginine. This has been shown to depend on a repression by arginine on the production of this enzyme, which catalyzes an intermediate step in arginine biosynthesis. It could be shown, however, that slow-growing, argininelimited cells in the steady state had enzyme levels which were 25 to 50 times larger than those levels found in the wild type growing on minimal medium. An increase in flow rate, and, as a consequence, a small increase in the steady-state concentration of the limiting factor, led to a decrease in the rate of enzyme production. Maximum production of the enzyme was obtained at a dilution rate of 0.46 hr.-l, intermediate levels giving a lower output, being obtained at higher dilution rates. A selection for mutants with a high P-galactosidase activity was obtained in lactose-limited continuous cultures of a strain of Escherichia coli K-12, which originally formed the enzyme only in the presence of an inducer (Novick, 1961). The selected strain synthesized the enzyme constitutively. The maximum activity obtained in the continuous culture was about five times that usually found in constitutive strains. The influence of different limitations on the cytochrome content of a strictly aerobic pseudomonad was investigated by Rosenberger and Kogut (1958). It was observed that organisms grown with air as the growth-limiting factor always possessed about double the cytochrome content of cells grown at the same rates but with succinate as the growth-limiting factor.
C. PRODUCTION OF ANTIGENICCOMPONENTS For the production of bacterial vaccines the cultivation method can be highly critical. In the past, attention has been paid mainly to medium studies in batch cultures or using surface growth on solid media, and very few reports on variations in the immunizing capacity of bacterial cells grown in continuous culture have ap-
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peared. One investigation, however, may serve as an example of the usefulness of the continuous culture methods in this field. Pirt et al. (1961) cultivated Pasteurellu pestis on a 2-liter scale with a dilution rate of 0.1 hr.--l. The yield of three different antigens was determined quantitatively over a range of temperature and pH values. One of these antigens could be shown to be produced only at pH values below 6.9. Another interesting observation was that maximum values for the synthesis of this antigen were obtained during the period of stabilization at 37" of a culture originally maintained at 28°C. It could also be shown that selection occurred against virulent types at 37" but not at 28°C. However, a temperature of 37°C. was essential for the production of several important antigenic components. The authors concluded that a continuous flow process for producing cells synthesizing the desired antigens would require a two-stage process, where the organisms were first grown at 28°C. to prevent degeneration and then transferred to a second stage at a higher temperature.
D. POPULATION GENETICS IN CONTINUOUS CULTURE The phenomenon of periodic selection was observed in chemostat experiments by Novick and Szilard (1950). In the steady state, under constant environmental conditions, a linear increase in the frequency of T6 resistant mutants was observed in their strain of Escherichia coli B. This was expected as a consequence of the spontaneous mutation to TBresistance in this strain. The slope of the line was proportional to the mutation rate. However, the expected equilibrium that should eventually have been established was never obtained; instead there was a sporadic decrease followed again by an increase in the number of TBresistant mutants. The explanation to this behaviour could be as follows. In a growing population of bacteria, mutants are likely to arise which are better fitted to the environment than the parent type. In the chemostat, this can consist of an ability to grow faster than the parental strain at low concentrations of the limiting factor. The observed advantage in growth rate can be confined to these conditions; at high concentrations there might be no detectable difference. The T5 resistant mutants serve as indicators of new types replacing the existing in the steady-state culture. The phenomenon of periodic selection does not, however, appear to be specific for conditions of limited growth. Atwood et aZ. (1951)
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has described this behavior in a histidine-requiring mutant of Escherichia coli, which was serially transferred in a minimal medium supplemented with histidine. He also pointed out that the repeated replacement of an existing population by fitter types is a mechanism of homeostasis. The mutant content of the culture is continually suppressed. As it is usually not possible to reveal the nature of the phenotypical change giving a better fitted strain, the observed characteristics of the bacterial culture are maintained during the evolutionary process. Apart from the basic observations, there are very few systematic studies of the genetic stability of continuous cultures. Herbert et al. (1956), however, working with Aerobacter cloacae, were unable to observe any differences in growth characteristics in batch cultivation when comparing the original strain with an inoculum from a culture that had been operated continuously for 6 weeks. No rough colonies were observed in daily plate counts from Salmonella typhimurium, grown continuously for 3 weeks in synthetic medium at different growth rates (Holme and Edebo, in press ) , Rough colonies were easily isolated from aged batch cultures of the strain used. Formal et al. (1956) cultivated Salmonella typhi for 24 days using the nitrogen source as the limiting factor. No decrease in immunizing capacity or virulence was observed, but after 2 weeks’ cultivation the strain showed a change in the surface antigens, detectable by serological techniques. A comparison between serial transfer and continuous culture of Clostridium saccharobutylicum was made by Finn and Nowrey (1959). No decrease in glucose utilization and solvent production was noted during a continuous run of 2 weeks comprising 650 cell generations. After 4 serial transfers, during which 19 generations occurred, the cells had retained practically no capacity for solvent production. One of the main objections to the use of continuous cultivation in production is the hazard of degeneration. Degeneration is here defined as decreased ability to produce a desired product, this being a cellular component or an extracellular compound. Obviously no important degenerative changes occurred in the specific instances already mentioned. Furthermore, in a few cases where undesired variation occurred, this difficulty could be overcome by proper adjustment of environmental conditions. In continuous cultures of Brucellu abortus rough variants tended to replace the
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CULTIVATION
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smooth culture at high dilution rates (Hauschild and Pivnick, 1961). When the dilution rate was again reduced to a lower value, the smooth variants became reestablished. Selected smooth clones showed the same tendency to degenerate as nonselected strains. The situation was shown to be very complex in this case, in that chelating agents suppressed the establishment of nonsmooth variants in cultures grown at high dilution rates. Obviously some factor in the medium influenced the s'elective advantage of either variant. In experiments, which have already been mentioned, concerned with vaccine production from Pasteurella pestis, a selection against virulent types was avoided by growing the cultures at 28" instead of 37°C. (Pirt et al., 1961).
E. MIXEDPOPULATIONS The attractive possibilities of imitating continuous processes occurring in nature afforded by the use of in vitro continuous culture has been mentioned on several occasions. The idea has been realized in studies of interrelationships among members of the fecal flora and in rumen fermentation. Ransom et al. (1961) found that the growth of Vibrio cholerae was markedly suppressed by the presence of growing enterococci and lactobacilli. The growth of Shigella flexneri was not suppressed under similar conditions. Other studies in progress have been reported (Gavin and Boger, 1961; Zubrzycki and Spaulding, 1958), indicating a growing interest in this field. In a study of rumen fermentation, using continuous culture, it was possible to maintain the essential activities within the ranges normally found in the rumen contents (Stewart et al., M I ) . This implies that the possibilities of analyzing many of the problems of rumen activities, on the biochemical and cellular level, have been greatly improved. Another type of experiment, in which mixed populations were active, dealt with the biooxidation of wastes from forest industries (Rennerfelt, 1961). Shifts between wastes from hardwood and softwood always resulted in a marked reduction of the oxidation efficiency. By mixing the two types of waste water, it was possible to effect, in laboratory experiments, an evaluation of optimum conditions for the biooxidation.
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111. Two-Stage Cultivation Many of the activities of bacterial cells are not dependent on growth and division of the cells. In certain instances, the requirements for active growth may compete for th.e substrate which is utilized for the synthesis of a desired product (Holme, 1957). During the last five years, two-stage continuous culture has been used or suggested for a number of experiments on such systems, and the advantages of being able to control the environmental conditions independently in the two stages have been pointed out. This is of special importance if the optimum conditions for growth and for product formation are not identical. A direct comparison in productivity between batch, single-stage, and two-stage cultivation has been carried out on the formation of 2iSbutanedio1, using Aerobacter aerogenes grown on a m'edium with sucrose as the carbon source (Pirt and Callow, 1959a,b). The yield obtained in a one-stage continuous process was lower than in the corresponding batch cultivation, partly because of thee fact that the outflowing cells from the continuous culture were still capable of converting sucrose to 2,3-butanediol for a further long period. However, from one-stage experiments certain optimum conditions for product formation were elicited: a temperature of 30°C. and a pH of 5 to 6. In two-stage cultivation experiments it was found that a limited oxygen supply was also essential. Since these conditions obviously are unfavorable to the growth of the bacteria, a two-stage process appeared to present the best solution. The first stage was utilized for cell growth and the second stage was fed with the cells produced, the environmental conditions in this stage being adjusted for product formation. Accordingly, it could be shown that, in comparison with other methods, this method gave the best yield. Another case where a two-stage cultivation process has been suggested, is in the production of antigenic components from Pastewella pestis (Pirt et al., 1961) As mentioned previously, selection occurred against virulent types at 37" but not at 28"C., whereas a temperature of 37°C. was essential for the production of the desired antigens. There are a few further examples where the use of two-stage continuous cultivation has greatly improved the possibilities of producing certain specific compounds. In these cases micro-
.
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organisms carry out a transformation of a substance which has an inhibitory effect on the growth of the microorganisms. One example of this is the transformation carried out by Escherichia coli of azauracil to azauracil riboside, which is a cancerostatic compound (Mhlek, 1961). In batch culture the yield was low because of poor growth. In one-stage cultivation, however, it was possible to obtain better yields, but cells resistant to azauracil developed, and, since these cells did not perform the transformation, the duration of the productive period was limited. A two-stage system, in which the azauracil was continuously added to the second stage, was found to increase stability and gave a yield about three times greater than that obtained with the single-stage process. Two reports on the use of a two-stage process for steroid transformation have also appeared (Mateles and Fuld, 1961; Reusser et al., 1961). The situation in the latt,er case is similar to the state described above, in that the steroids inhibit the growth of the microorganisms.
IV. Design of laboratory Scale Units A. CONTINUOUS LIQUIDFEED DEVICES Metering pumps, chiefly of the hose-pressure type, have been found to be the most reliable feed devices. The great advantage of the hose-pressure pumps is that a rubber tube connection between medium reservoir and culture vessel can be sterilized, and part of the tube can be used in the pump without any risk of breaking sterility. Finger pumps have been used extensively and, provided with a good quality silicone or nylon tubing, they are good for a long period of continuous operation. However, the tubing becomes both worn and stretched and is therefore very susceptible to breakage. Another type of hose-pressure pump, which causes less strain on the tube, is shown in Fig. 1. By using a series of cog-wheels for transmission it is very simple to regulate the speed. B. OVERFLOW DEVICES Different arrangements for overflow are represented in Fig. 2. An improved type “B” is described by Callow and Pirt ( 1961), in which the culture volume can be varied during the continuous run by causing the outlet tube, sealed by a rubber ring, to pass through the bottom of the vessel. Part of the tube below the bottom is
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protected by rubber bellows to maintain sterility. However, there is one drawback with this type of overflow, as illustrated by the following experiment ( Holme, unpublished) : Using Escherichia coli B growing at different dilution rates in a glucose-salts medium at a population density of about 0.6 gm. dry weight per liter, it was found that surface overflow sampling gave
FIG.1. Hose-pressure pump, It has been possible to avoid stretching the silicone tubing by applying the pressure on it with the aid of an outer metal ring. (Available from the firm “Meyer’s Patents,” Stockholm, Sweden.)
a 20% lower dry weight than a sample taken from the bulk of the culture, In this experiment air was distributed through a sintered glass filter giving a foam layer of less than 10mm. depth on the top of the culture. Occasionally, backward growth from the culture in the feed line presents a problem. When growing filamentous molds the orifice also may become clogged with growth. In this event, the orifice of the feed line should be protected by enclosing it in an outer tube.
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If an air stream is introduced into this tube, it will remain dry and no backward growth will occur. C. AERATION-AG~ATION In this particular sphere, there are no special problems confined to the continuous culture devices. It should be pointed out, however, that in aeration-agitation investigations the continuous culture technique offers some advantages. Foaming is one of the great problems in the later stages of growth in aerated fermentors. Chemical defoamers may not be desirable in many instances, partly
A C 0 E FIG.2. Types of overflow devices used (schematic), A rapid transfer to a
cooled sampling flask is best achieved by permitting the air to leave the culture vessel through the overflow tube. If foam sampling is undesirable, the tubes C-E are to be preferred. For type E, a hose-pressure pump is used having a speed giving a flow rate below the feed rate of the culture. Samples withdrawn direct from the culture should be compared to the samples taken continuously as a control of the efficiency of the overflow device.
because of their influence on the oxygen transfer rate and partly because of the fact that they may interfere with fractionation procedures applied to the product. In continuous culture it is possible to operate at excess oxygen, and to limit the cell concentration by an essential nutrient. During operation, the cell concentration can be increased gradually by increasing the concentration of the limiting nutrient until the point is reached where foaming becomes a technical obstacle. In this manner it was possible to increase the yield of SuZmoneZZu typhimurium in a glucose-salts medium with glucose limiting the population density to about 7 gm. dry weight per liter in a pH-controlled continuous culture without using chemical defoamers or mechanical foam-breaking (Holme and Edebo, in press). Another type of equipment, based on the pulse-aerator system,
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can also be successfully used for continuous culture when strongly foam-producing media are used (Hed6n and Holme, 1961; Holmstrom and HedBn, in press). Callow and Pirt ( 1961 ) have carried out experiments with different fermentors for growing filamentous molds. They describe in detail a type of stirred fermentor supplied with additional devices for continuous operation. Nonbaffled fermentors of not less than a 2-liter working volume were recommended. D. TWO-STAGE CULTIVATION EQUIPMENT Two-stage operation has often been found to be very useful in continuous culture work. It allows independent control over dif-
FIG.3. Operation of a two-stage continuous culture device (schematic) with independent control of dilution rate of the two stages. Agitation-aeration is only indicated by a tube. The working volume of stage 2 should be chosen so that the output of stage 1 is equal to that of stage 2 at the mean of the dilution rates applicable in stage 2. The transfer of culture fluid from stage 1 to stage 2 is effected by a hose-pressure pump, the speed of which determines the dilution rate of stage 2. When operated according to the example given in the tabulation below simultaneous sampling is effected from both stages. Dilution rate Flow rate Volume Sampling rate
(hr.-l)
( ml./hr. )
(d.1
(ml./h.1
Stage 1 0.8 800 1000 200-600
Stage 2 0.2-0.6
20&600
lo00 200-600
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ferent metabolic stages in a fermentation cycle. Temperature, pH, supply of different gases, and nutritional environment can be independently varied in two culture vessels operating in a series. The possibility of varying the dilution rate of each stage independently is, of course, also desirable. This can be arrived at by varying the culture volume of the vessels (Callow and Pirt, 1961). Another possibility is shown in Fig. 3. In the present article an attempt has been made to elucidate, by reviewing some experimental work, the importance of continuous culture as an aid toward the solution of many vital problems in microbiology. A greater understanding of the physiological characteristics of microorganisms is the best approach to improving our possibilities to make use of their capabilities for research and production.
REFERENCES Atwood, K. C., Schneider, L. K., and Ryan, F. J. (1951). Proc. Natl. A d . Scf. U S . 91, 146. Bauchop, T., and Elsden, S. R. (1900). J. Gen. Microbiol. 23, 457. Callow, D. S., and Pirt, S. J. (1961). J Appl. Bacterbl. 24, 12. Dawson, P. S. S. (1960). J. Blochem. Microbiol. Technol. Eng. 2, 227. Finn, R. K., and Nowrey, J. E. (1959). Appl. Microbiol. 7, 29. Formal, S. B., Baron, L. S.,and Spilman, W. (1956). J . Bacterial. 72, 168. Gavin, J. J., and Boger, W. D. ( 1961). Bacterbl. Proc. ( SOC. Am. Bacteriologists), p. 52. Gerhardt, P., and Bartlett, M. C. (1959). Advances in Appl. Microbiol. 1, 215. Gladstone, G . D. (1948). Brit. J. Exptl. Pathol. 29, 379. Gorini, L. (1960). Proc. Nutl. Acad. Sci. U.S. 46, 682. Gunsalus, I. C.,and Shuster, C. W. (1961). In “The Bacteria” (I. C. Gunsalus and R. Y. Stanier, eds.), Vol. 11, p. 1. Academic Press, New York. Hauschild, A. H. W., and Pivnick, H. (1961). Can. J. Microblol. 7 , 491. Hedhn, C.-G., and Holme, T. (1961). In “Continuous Culture of Microorganisms,” SOC. Chem. Ind. Monograph No. 12,p. 118.London. Herbert, D. ( 1958). Continuous Cultivation of Microorganisms Prague, p. 45. Herbert, D. (1959). In “Recent Progress in Microbiology” p. 381, Stockholm. Herbert, D., Elsworth, R., and Telling, R. C. (1956). J. Gen. Microbiol. 14, 601. Holme, T. (1957). Acta Chem. Scand. 11, 763. Holme, T. ( 1958). Continuous Cultivation of Microorganisms Prague, p. 67. Holme, T.,and Edebo, L. Acta Pathol. Microbiol. Scand. In press. Holme, T., and Zacharias, B. Blotechnol. Bbeng. In press.
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Holmstrom, B., and Hedhn, C. G. Bbtechnol. Bioeng. In press. Karush, F., Iacocca, V., and Hams, T. N. (1958). J. Bactertol. 72, 283. MaalZe, 0. (1960). Symposia SOC. Gen. Microbiol. 10, 272. MBlek, I. (1981). In “Continuous Culture of Microorganisms,” SOC.Chem. lnd. Monograph No. 12, p. 3. London. Mateles, R. I., and Fuld, G. J. (1981). Antonle uan Leeuwenhoek J . Microbid. Serol. 27, 34. Munson, R. J., and Maclean, F. I. ( 1981). J. Gen. a4lcr0biol. 2S, 29. Novick, A. ( 1961). In “Growth in Living Systems” (M. X. Zarrow et al., eds.), p. 93. Basic Books, New York. Novick, A., and Szilard, L. (1950). Proc. Natl. Acad. Sci. U S . 86, 708. Pirt, S. J., and Callow, D. S . (1QSea). J . Appl. Bocterlol. 21, 188. Pirt, S . J., and Callow, D. S. (195913). Selected Sci. Papers 1st. Super. Sanitd 2, 292. Pirt, S . J., and Callow, D. S . (1959~).Nature 184, 307. Pirt, S. J., Thackeray, E. J., and Harris-Smith, R. (1981). J . Gen. Microbfol. as, 119. Ransom, J. P., Finkelstein, R. A., Ceder, R. E., and Formal, S. B. (1961). Proc. SOC. Exptl. Btol. Med. 107, 332. Rennerfelt, J. (lS6l). Thesis, Royal Institute of Technology, Stockholm. Reusser, F., Koepsell, H. J., and Savage, G. M. (1061). Appl. Microblol. 9, 348. Rosenberger, R. F., and Elsden, S. R. (1960). J . Gen. Microbbl. aZ, 726. Rosenberger, R. F., and Kogut, M. (1958). J . Gen. Mtcrobbl. 19, 228. Stewart, D. G., Warner, R. G., and Seeley, H.W. (1961) Appl. Microbbl. 9,150. Tyrell, E. A., MacDonald, R. E., and Gerhardt, P. (1958). J. Bacteriol. 7s, 1. Zacharias, B. Bfotechnol. Bfoeng. In press. Zubrzycki, L., and Spaulding, E. H. (1958). J . Bacteriol. 711, 278.
.............................................. ...................................... Yield Measurements .................................... Morphology and Chemical Composition of Cells ............
I. Introduction
II. One-Stage Cultivation
A. B. C. Production of Antigenic Components ...................... D. Population Genetics in Continuous Culture ................ E. Mixed Populations ..................................... 111. Two-Stage Cultivation ...................................... IV. Design of Laboratory Scale Units ............................ A. Continuous Liquid Feed Devices ......................... B. Overflow Devices ...................................... C. Aeration-Agitation ..................................... D. Two-Stage Cultivation Equipment ........................ References ................................................
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1. Introduction In the extensive review of continuous fermentation, which appeared in the first volume of this series, the theoretical as well as the practical aspects of the continuous culture methods were discussed and references to pertinent literature up to 1958 were also given (Gerhardt and Bartlett, 1959). During the last three years, a rapidly increasing number of reports have appeared in which the aid of continuous culture had been enlisted in tackling biological problems. Most of these studies have been made possible, or greatly facilitated, by the use of continuous culture methods and the experiments to be reviewed here all belong to this class. The main purpose of this article will be to present the possibilities afforded by continuous culture methods and the writer has attempted to do this by analyzing and summarizing some experimental work. The design of laboratory scale units will also be touched upon, No present-day microbiologist needs to hesitate to use the continuous culture technique on a laboratory scale because of technical difficulties, but the great variety of units described might cause Some confusion. Therefore, a description of useful features of design 101
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of laboratory scale units for the cultivation of bacteria and filamentous molds would seem to be of value.
It. One-Stage Cultivation In the study of the influence of environmental conditions on growth or on the synthesizing capabilities of growing cells, the one-stage cultivation offers great advantages over batch processes. Many workers have utilized the possibility of changing various environmental factors of cells growing at a fixed rate in the steady state to evaluate optimal conditions for the propagation of cells or the development of cell products. In these studies, yield measurements, analysis of chemical and antigenic composition of cells and of genetic stability of cell populations are of fundamental importance.
A. YIELDMEASWMENTS For yield measurements, the continuous culture method has proven to offer distinct advantages over batch experiments. In batch cultures cells might degrade nutrients during the lag period and transform them into other products which may be metabolized during the period of active growth. Thus, if measurements are performed on samples taken from the logarithmic phase, these products may be utilized simultaneously with the nutrients originally supplied. This will give rise to an error in the value of the yield constant. If the culture is allowed to enter the stationary phase, difficulties can again arise owing to an altered metabolic pattern of the celIs. When facing the problem of cultivating an organism with known nutritional requirements but with no information about optimal growth conditions in the continuous culture, it is frequently necessary to obtain specific information concerning the consumption of different nutrients. This is accomplished by decreasing the concentration of an essential nutrient in the medium until it limits the population density. It is then possible to determine the “yield constant,” which is usually expressed as grams of organisms formed per gram of nutrient consumed. At a certain cell density, determined by the concentration of the limiting factor, the optimum ranges for different environmental factors, such as temperature and pH, can be determined. Then a
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gradual increase in the concentration of the limiting factor can be started, and continued until oxygen deficiency or the accumulation of toxic metabolites prevent an increased total yield. When cultivating bacteria with complex nutritional requirements, the total yield is often limited by accumulation of toxic metabolites. In batch cultures, this has been partly overcome in dialysis bag culture (Gladstone, 1948) or by using two-phase systems (Tyrell et al., 1958). Dialyzing out toxic metabolites can also be successfully applied in continuous culture. An increase in the yield of Brucelln abortus has been reported by Hauschild and Pivnick (196l), when they included a dialysis tube in the fermentor. The culture, to which fresh medium was continuously added, was grown outside the dialysis tube. A continuous stream of fresh nutrient medium was also conducted through the dialysis tube enabling low-molecular metabolites to diffuse out of the culture fluid. The maximum output of cells in a continuous culture can usually be predicted from measurements of the doubling time of cells in batch cultures (Herbert et al., 1956). A close agreement with the theoretical value has been observed for many different enteric bacteria [Aerobacter spp. (Herbert, 1958; Herbert et al., 1956), Escherichia coli (Holme, 1957),] streptococci (Karush et al., 1956), yeasts [Torula (Herbert, 1958), Saccharomyces (Dawson, 1960)1, and €or molds [Gibberella fujikuroi (Holme and Zacharias, in press)]. If the population density decreases earlier than expected when the dilution rate is increased, oxygen deficiency should be suspected. In the study of Brucelkz abortus (Hauschild and Pivnick, 1961), maximum output was obtained at a much lower dilution rate than expected. It was claimed that aeration was in excess, although evidence in support of this was inconclusive, since the oxygen demand of the culture had not been determined. Chemical antifoam was also used, which may have caused a much lower oxygen transfer rate than that estimated by the sulfite oxidation method. An interesting application of continuous culture to studies of growth yields in relation to the energy metabolism has been made by Rosenberger and Elsden (1980) and Bauchop and Elsden (1960). Their work is presented in detail by Gunsalus and Shuster (1961) and will only be mentioned briefly. Streptococcus fueculis, which has an anaerobic metabolism, was the organism used in
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these investigations. It offers great advantages when energy-yield relationships are studied, since the cell carbon is furnished by compounds not participating in the energy release. Less than 1% of the fermented glucose (the energy source) is found in the cellular components. It was found that the glucose yield constant was greater when growth was limited by the glucose supply than when tryptophan was limiting. Under conditions of tryptophan limitation the amount of glucose used per unit weight of cells per unit time remained roughly constant, irrespective of the growth rate. This means that the requirements of cell synthesis in S . faecalis do not control the rate of the energy-yielding metabolism. When factors other than the energy source are limiting growth, the yield calculation can frequently be very simple. In nitrogenlimited growth of Escherichia coli practically all of the nitrogen is assimilated, except at dilution rates proximate to the critical rate. At low growth rates, however, polysaccharides accumulate in the cells to a great extent giving an apparent increase in the growth yield ( Holme, 1957). Parallel to polysaccharide synthesis, several low-molecular-weight substances such as a-ketoglutaric acid appeared in the culture fluid (Holme, 1958). In agreement with these experiments, Dawson (1960), cultivating Saccharomyces rouxii, an osmophilic yeast that produces glycerol and arabitol, found that nitrogen-limited growth resulted in an accumulation of glycerol in the culture fluid in fully aerobic cultures. The continuous culture technique has also been successfully applied to the study of nitrogen fixation by Axotobacter vinelandii (Zacharias, in press). Cells were grown in synthetic medium containing no nitrogen source. The population density was about 2.5gm. dry weight per liter of culture, at a dilution rate of about 0.3 hr.-l. Air and gaseous nitrogen was added from separate sources. When the additional nitrogen gas was subjected to irradiation with p-rays from a 10 pC. Srm source before entering the culture, an increased nitrogen fixation, measured as milligrams nitrogen fixed per gram glucose consumed, was observed. The population density remained constant, and, as a result of the decrease in glucose consumption, the glucose yield constant increased from approximately 0.4 to 0.5. Similar results were obtained with ultraviolet irradiation of the nitrogen gas.
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B. MORPHOLOGY AND CHEMICAL COMPOSITION OF CELLS There is, in general, a good agreement between data provided by continuous culture methods and by static cultures concerning the iduences of nutrient limitations and growth rate on the chemical composition of cells. In unrestricted, “balanced growth the ribonucleic acid (RNA) content of the cells increased with increased growth rate and the mean weight per cell also increased (Maalpre, 1960). The same observations were made in continuous cultures with the energy source constituting the limiting factor (Herbert, 1959). It was also found that the deoxyribonucleic acid (DNA) content per cell increased considerably with increased growth rate. However, staining for chromatinic bodies revealed that the rapidly growing, large cells were multinucleate, and that the amount of DNA per nucleus was practically constant. Munson and Maclean (1961) exploited the possibility of maintaining the steady-state growth of Escherichia coli cells having a certain length distribution. Data of survival after X-ray radiation combined with nuclear staining of cells from cultures with different cell length distribution provided evidence to suggest that radiosensitive sites were located in each nuclear body. Pirt and Callow (1959~)have performed a study on the influence of pH on the morphology of Penicillium chrysogenum in submerged culture. The mold was grown in a 2-liter, stirred fennentor at a density of about l o p . dry weight per liter. The dilution rate was 0.05 hr.-l. Glucose was limiting and aeration was in excess. At steady-state growth with a pH around 7, aberrant morphology was noted and, in these experiments, pellet formation was dependent on pH. At pH 6 a norma1 filamentous growth was obtained. The authors suggested that the resistance of the cell walls of the hyphae decreased with an increase in pH from 6.0 to 7.4. Studies of the influences of different limitations on the chemical composition of cells have given many results from which practical implications may be deduced. As mentioned in connection with the studies of Rosenberger and Elsden (1960), limitation by essential nutrients other than the energy source does not control the rate of oxidation of the energy source. In Streptococcus faecalis, tryptophan limitation resulted mainly in the production of large amounts of lactic acid; in Escherichia coli and Torula utilis, ammonia limitation resulted in a large increase in the polysaccharide content of the cells (Herbert, 1958;Holme, 1957).
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Isolation of specific, biosynthetic enzymes from bacterial cells might, in the near future, constitute a field of considerable interest. The mechanism governing the regulation of levels of such enzymes in the cells has been studied by Gorini (1960), who utilized the possibility of maintaining a stabile and very low concentration of an essential nutrient by making it the limiting factor in chemostat experiments. Certain arginine-deficient mutants of Escherichia coli have a barely detectable ornithine transcarbamylase activity when grown in the presence of exogenous arginine. This has been shown to depend on a repression by arginine on the production of this enzyme, which catalyzes an intermediate step in arginine biosynthesis. It could be shown, however, that slow-growing, argininelimited cells in the steady state had enzyme levels which were 25 to 50 times larger than those levels found in the wild type growing on minimal medium. An increase in flow rate, and, as a consequence, a small increase in the steady-state concentration of the limiting factor, led to a decrease in the rate of enzyme production. Maximum production of the enzyme was obtained at a dilution rate of 0.46 hr.-l, intermediate levels giving a lower output, being obtained at higher dilution rates. A selection for mutants with a high P-galactosidase activity was obtained in lactose-limited continuous cultures of a strain of Escherichia coli K-12, which originally formed the enzyme only in the presence of an inducer (Novick, 1961). The selected strain synthesized the enzyme constitutively. The maximum activity obtained in the continuous culture was about five times that usually found in constitutive strains. The influence of different limitations on the cytochrome content of a strictly aerobic pseudomonad was investigated by Rosenberger and Kogut (1958). It was observed that organisms grown with air as the growth-limiting factor always possessed about double the cytochrome content of cells grown at the same rates but with succinate as the growth-limiting factor.
C. PRODUCTION OF ANTIGENICCOMPONENTS For the production of bacterial vaccines the cultivation method can be highly critical. In the past, attention has been paid mainly to medium studies in batch cultures or using surface growth on solid media, and very few reports on variations in the immunizing capacity of bacterial cells grown in continuous culture have ap-
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peared. One investigation, however, may serve as an example of the usefulness of the continuous culture methods in this field. Pirt et al. (1961) cultivated Pasteurellu pestis on a 2-liter scale with a dilution rate of 0.1 hr.--l. The yield of three different antigens was determined quantitatively over a range of temperature and pH values. One of these antigens could be shown to be produced only at pH values below 6.9. Another interesting observation was that maximum values for the synthesis of this antigen were obtained during the period of stabilization at 37" of a culture originally maintained at 28°C. It could also be shown that selection occurred against virulent types at 37" but not at 28°C. However, a temperature of 37°C. was essential for the production of several important antigenic components. The authors concluded that a continuous flow process for producing cells synthesizing the desired antigens would require a two-stage process, where the organisms were first grown at 28°C. to prevent degeneration and then transferred to a second stage at a higher temperature.
D. POPULATION GENETICS IN CONTINUOUS CULTURE The phenomenon of periodic selection was observed in chemostat experiments by Novick and Szilard (1950). In the steady state, under constant environmental conditions, a linear increase in the frequency of T6 resistant mutants was observed in their strain of Escherichia coli B. This was expected as a consequence of the spontaneous mutation to TBresistance in this strain. The slope of the line was proportional to the mutation rate. However, the expected equilibrium that should eventually have been established was never obtained; instead there was a sporadic decrease followed again by an increase in the number of TBresistant mutants. The explanation to this behaviour could be as follows. In a growing population of bacteria, mutants are likely to arise which are better fitted to the environment than the parent type. In the chemostat, this can consist of an ability to grow faster than the parental strain at low concentrations of the limiting factor. The observed advantage in growth rate can be confined to these conditions; at high concentrations there might be no detectable difference. The T5 resistant mutants serve as indicators of new types replacing the existing in the steady-state culture. The phenomenon of periodic selection does not, however, appear to be specific for conditions of limited growth. Atwood et aZ. (1951)
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has described this behavior in a histidine-requiring mutant of Escherichia coli, which was serially transferred in a minimal medium supplemented with histidine. He also pointed out that the repeated replacement of an existing population by fitter types is a mechanism of homeostasis. The mutant content of the culture is continually suppressed. As it is usually not possible to reveal the nature of the phenotypical change giving a better fitted strain, the observed characteristics of the bacterial culture are maintained during the evolutionary process. Apart from the basic observations, there are very few systematic studies of the genetic stability of continuous cultures. Herbert et al. (1956), however, working with Aerobacter cloacae, were unable to observe any differences in growth characteristics in batch cultivation when comparing the original strain with an inoculum from a culture that had been operated continuously for 6 weeks. No rough colonies were observed in daily plate counts from Salmonella typhimurium, grown continuously for 3 weeks in synthetic medium at different growth rates (Holme and Edebo, in press ) , Rough colonies were easily isolated from aged batch cultures of the strain used. Formal et al. (1956) cultivated Salmonella typhi for 24 days using the nitrogen source as the limiting factor. No decrease in immunizing capacity or virulence was observed, but after 2 weeks’ cultivation the strain showed a change in the surface antigens, detectable by serological techniques. A comparison between serial transfer and continuous culture of Clostridium saccharobutylicum was made by Finn and Nowrey (1959). No decrease in glucose utilization and solvent production was noted during a continuous run of 2 weeks comprising 650 cell generations. After 4 serial transfers, during which 19 generations occurred, the cells had retained practically no capacity for solvent production. One of the main objections to the use of continuous cultivation in production is the hazard of degeneration. Degeneration is here defined as decreased ability to produce a desired product, this being a cellular component or an extracellular compound. Obviously no important degenerative changes occurred in the specific instances already mentioned. Furthermore, in a few cases where undesired variation occurred, this difficulty could be overcome by proper adjustment of environmental conditions. In continuous cultures of Brucellu abortus rough variants tended to replace the
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CULTIVATION
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smooth culture at high dilution rates (Hauschild and Pivnick, 1961). When the dilution rate was again reduced to a lower value, the smooth variants became reestablished. Selected smooth clones showed the same tendency to degenerate as nonselected strains. The situation was shown to be very complex in this case, in that chelating agents suppressed the establishment of nonsmooth variants in cultures grown at high dilution rates. Obviously some factor in the medium influenced the s'elective advantage of either variant. In experiments, which have already been mentioned, concerned with vaccine production from Pasteurella pestis, a selection against virulent types was avoided by growing the cultures at 28" instead of 37°C. (Pirt et al., 1961).
E. MIXEDPOPULATIONS The attractive possibilities of imitating continuous processes occurring in nature afforded by the use of in vitro continuous culture has been mentioned on several occasions. The idea has been realized in studies of interrelationships among members of the fecal flora and in rumen fermentation. Ransom et al. (1961) found that the growth of Vibrio cholerae was markedly suppressed by the presence of growing enterococci and lactobacilli. The growth of Shigella flexneri was not suppressed under similar conditions. Other studies in progress have been reported (Gavin and Boger, 1961; Zubrzycki and Spaulding, 1958), indicating a growing interest in this field. In a study of rumen fermentation, using continuous culture, it was possible to maintain the essential activities within the ranges normally found in the rumen contents (Stewart et al., M I ) . This implies that the possibilities of analyzing many of the problems of rumen activities, on the biochemical and cellular level, have been greatly improved. Another type of experiment, in which mixed populations were active, dealt with the biooxidation of wastes from forest industries (Rennerfelt, 1961). Shifts between wastes from hardwood and softwood always resulted in a marked reduction of the oxidation efficiency. By mixing the two types of waste water, it was possible to effect, in laboratory experiments, an evaluation of optimum conditions for the biooxidation.
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111. Two-Stage Cultivation Many of the activities of bacterial cells are not dependent on growth and division of the cells. In certain instances, the requirements for active growth may compete for th.e substrate which is utilized for the synthesis of a desired product (Holme, 1957). During the last five years, two-stage continuous culture has been used or suggested for a number of experiments on such systems, and the advantages of being able to control the environmental conditions independently in the two stages have been pointed out. This is of special importance if the optimum conditions for growth and for product formation are not identical. A direct comparison in productivity between batch, single-stage, and two-stage cultivation has been carried out on the formation of 2iSbutanedio1, using Aerobacter aerogenes grown on a m'edium with sucrose as the carbon source (Pirt and Callow, 1959a,b). The yield obtained in a one-stage continuous process was lower than in the corresponding batch cultivation, partly because of thee fact that the outflowing cells from the continuous culture were still capable of converting sucrose to 2,3-butanediol for a further long period. However, from one-stage experiments certain optimum conditions for product formation were elicited: a temperature of 30°C. and a pH of 5 to 6. In two-stage cultivation experiments it was found that a limited oxygen supply was also essential. Since these conditions obviously are unfavorable to the growth of the bacteria, a two-stage process appeared to present the best solution. The first stage was utilized for cell growth and the second stage was fed with the cells produced, the environmental conditions in this stage being adjusted for product formation. Accordingly, it could be shown that, in comparison with other methods, this method gave the best yield. Another case where a two-stage cultivation process has been suggested, is in the production of antigenic components from Pastewella pestis (Pirt et al., 1961) As mentioned previously, selection occurred against virulent types at 37" but not at 28"C., whereas a temperature of 37°C. was essential for the production of the desired antigens. There are a few further examples where the use of two-stage continuous cultivation has greatly improved the possibilities of producing certain specific compounds. In these cases micro-
.
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organisms carry out a transformation of a substance which has an inhibitory effect on the growth of the microorganisms. One example of this is the transformation carried out by Escherichia coli of azauracil to azauracil riboside, which is a cancerostatic compound (Mhlek, 1961). In batch culture the yield was low because of poor growth. In one-stage cultivation, however, it was possible to obtain better yields, but cells resistant to azauracil developed, and, since these cells did not perform the transformation, the duration of the productive period was limited. A two-stage system, in which the azauracil was continuously added to the second stage, was found to increase stability and gave a yield about three times greater than that obtained with the single-stage process. Two reports on the use of a two-stage process for steroid transformation have also appeared (Mateles and Fuld, 1961; Reusser et al., 1961). The situation in the latt,er case is similar to the state described above, in that the steroids inhibit the growth of the microorganisms.
IV. Design of laboratory Scale Units A. CONTINUOUS LIQUIDFEED DEVICES Metering pumps, chiefly of the hose-pressure type, have been found to be the most reliable feed devices. The great advantage of the hose-pressure pumps is that a rubber tube connection between medium reservoir and culture vessel can be sterilized, and part of the tube can be used in the pump without any risk of breaking sterility. Finger pumps have been used extensively and, provided with a good quality silicone or nylon tubing, they are good for a long period of continuous operation. However, the tubing becomes both worn and stretched and is therefore very susceptible to breakage. Another type of hose-pressure pump, which causes less strain on the tube, is shown in Fig. 1. By using a series of cog-wheels for transmission it is very simple to regulate the speed. B. OVERFLOW DEVICES Different arrangements for overflow are represented in Fig. 2. An improved type “B” is described by Callow and Pirt ( 1961), in which the culture volume can be varied during the continuous run by causing the outlet tube, sealed by a rubber ring, to pass through the bottom of the vessel. Part of the tube below the bottom is
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protected by rubber bellows to maintain sterility. However, there is one drawback with this type of overflow, as illustrated by the following experiment ( Holme, unpublished) : Using Escherichia coli B growing at different dilution rates in a glucose-salts medium at a population density of about 0.6 gm. dry weight per liter, it was found that surface overflow sampling gave
FIG.1. Hose-pressure pump, It has been possible to avoid stretching the silicone tubing by applying the pressure on it with the aid of an outer metal ring. (Available from the firm “Meyer’s Patents,” Stockholm, Sweden.)
a 20% lower dry weight than a sample taken from the bulk of the culture, In this experiment air was distributed through a sintered glass filter giving a foam layer of less than 10mm. depth on the top of the culture. Occasionally, backward growth from the culture in the feed line presents a problem. When growing filamentous molds the orifice also may become clogged with growth. In this event, the orifice of the feed line should be protected by enclosing it in an outer tube.
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If an air stream is introduced into this tube, it will remain dry and no backward growth will occur. C. AERATION-AG~ATION In this particular sphere, there are no special problems confined to the continuous culture devices. It should be pointed out, however, that in aeration-agitation investigations the continuous culture technique offers some advantages. Foaming is one of the great problems in the later stages of growth in aerated fermentors. Chemical defoamers may not be desirable in many instances, partly
A C 0 E FIG.2. Types of overflow devices used (schematic), A rapid transfer to a
cooled sampling flask is best achieved by permitting the air to leave the culture vessel through the overflow tube. If foam sampling is undesirable, the tubes C-E are to be preferred. For type E, a hose-pressure pump is used having a speed giving a flow rate below the feed rate of the culture. Samples withdrawn direct from the culture should be compared to the samples taken continuously as a control of the efficiency of the overflow device.
because of their influence on the oxygen transfer rate and partly because of the fact that they may interfere with fractionation procedures applied to the product. In continuous culture it is possible to operate at excess oxygen, and to limit the cell concentration by an essential nutrient. During operation, the cell concentration can be increased gradually by increasing the concentration of the limiting nutrient until the point is reached where foaming becomes a technical obstacle. In this manner it was possible to increase the yield of SuZmoneZZu typhimurium in a glucose-salts medium with glucose limiting the population density to about 7 gm. dry weight per liter in a pH-controlled continuous culture without using chemical defoamers or mechanical foam-breaking (Holme and Edebo, in press). Another type of equipment, based on the pulse-aerator system,
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can also be successfully used for continuous culture when strongly foam-producing media are used (Hed6n and Holme, 1961; Holmstrom and HedBn, in press). Callow and Pirt ( 1961 ) have carried out experiments with different fermentors for growing filamentous molds. They describe in detail a type of stirred fermentor supplied with additional devices for continuous operation. Nonbaffled fermentors of not less than a 2-liter working volume were recommended. D. TWO-STAGE CULTIVATION EQUIPMENT Two-stage operation has often been found to be very useful in continuous culture work. It allows independent control over dif-
FIG.3. Operation of a two-stage continuous culture device (schematic) with independent control of dilution rate of the two stages. Agitation-aeration is only indicated by a tube. The working volume of stage 2 should be chosen so that the output of stage 1 is equal to that of stage 2 at the mean of the dilution rates applicable in stage 2. The transfer of culture fluid from stage 1 to stage 2 is effected by a hose-pressure pump, the speed of which determines the dilution rate of stage 2. When operated according to the example given in the tabulation below simultaneous sampling is effected from both stages. Dilution rate Flow rate Volume Sampling rate
(hr.-l)
( ml./hr. )
(d.1
(ml./h.1
Stage 1 0.8 800 1000 200-600
Stage 2 0.2-0.6
20&600
lo00 200-600
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ferent metabolic stages in a fermentation cycle. Temperature, pH, supply of different gases, and nutritional environment can be independently varied in two culture vessels operating in a series. The possibility of varying the dilution rate of each stage independently is, of course, also desirable. This can be arrived at by varying the culture volume of the vessels (Callow and Pirt, 1961). Another possibility is shown in Fig. 3. In the present article an attempt has been made to elucidate, by reviewing some experimental work, the importance of continuous culture as an aid toward the solution of many vital problems in microbiology. A greater understanding of the physiological characteristics of microorganisms is the best approach to improving our possibilities to make use of their capabilities for research and production.
REFERENCES Atwood, K. C., Schneider, L. K., and Ryan, F. J. (1951). Proc. Natl. A d . Scf. U S . 91, 146. Bauchop, T., and Elsden, S. R. (1900). J. Gen. Microbiol. 23, 457. Callow, D. S., and Pirt, S. J. (1961). J Appl. Bacterbl. 24, 12. Dawson, P. S. S. (1960). J. Blochem. Microbiol. Technol. Eng. 2, 227. Finn, R. K., and Nowrey, J. E. (1959). Appl. Microbiol. 7, 29. Formal, S. B., Baron, L. S.,and Spilman, W. (1956). J . Bacterial. 72, 168. Gavin, J. J., and Boger, W. D. ( 1961). Bacterbl. Proc. ( SOC. Am. Bacteriologists), p. 52. Gerhardt, P., and Bartlett, M. C. (1959). Advances in Appl. Microbiol. 1, 215. Gladstone, G . D. (1948). Brit. J. Exptl. Pathol. 29, 379. Gorini, L. (1960). Proc. Nutl. Acad. Sci. U.S. 46, 682. Gunsalus, I. C.,and Shuster, C. W. (1961). In “The Bacteria” (I. C. Gunsalus and R. Y. Stanier, eds.), Vol. 11, p. 1. Academic Press, New York. Hauschild, A. H. W., and Pivnick, H. (1961). Can. J. Microblol. 7 , 491. Hedhn, C.-G., and Holme, T. (1961). In “Continuous Culture of Microorganisms,” SOC. Chem. Ind. Monograph No. 12,p. 118.London. Herbert, D. ( 1958). Continuous Cultivation of Microorganisms Prague, p. 45. Herbert, D. (1959). In “Recent Progress in Microbiology” p. 381, Stockholm. Herbert, D., Elsworth, R., and Telling, R. C. (1956). J. Gen. Microbiol. 14, 601. Holme, T. (1957). Acta Chem. Scand. 11, 763. Holme, T. ( 1958). Continuous Cultivation of Microorganisms Prague, p. 67. Holme, T.,and Edebo, L. Acta Pathol. Microbiol. Scand. In press. Holme, T., and Zacharias, B. Blotechnol. Bbeng. In press.
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Holmstrom, B., and Hedhn, C. G. Bbtechnol. Bioeng. In press. Karush, F., Iacocca, V., and Hams, T. N. (1958). J. Bactertol. 72, 283. MaalZe, 0. (1960). Symposia SOC. Gen. Microbiol. 10, 272. MBlek, I. (1981). In “Continuous Culture of Microorganisms,” SOC.Chem. lnd. Monograph No. 12, p. 3. London. Mateles, R. I., and Fuld, G. J. (1981). Antonle uan Leeuwenhoek J . Microbid. Serol. 27, 34. Munson, R. J., and Maclean, F. I. ( 1981). J. Gen. a4lcr0biol. 2S, 29. Novick, A. ( 1961). In “Growth in Living Systems” (M. X. Zarrow et al., eds.), p. 93. Basic Books, New York. Novick, A., and Szilard, L. (1950). Proc. Natl. Acad. Sci. U S . 86, 708. Pirt, S. J., and Callow, D. S . (1QSea). J . Appl. Bocterlol. 21, 188. Pirt, S . J., and Callow, D. S. (195913). Selected Sci. Papers 1st. Super. Sanitd 2, 292. Pirt, S . J., and Callow, D. S . (1959~).Nature 184, 307. Pirt, S. J., Thackeray, E. J., and Harris-Smith, R. (1981). J . Gen. Microbfol. as, 119. Ransom, J. P., Finkelstein, R. A., Ceder, R. E., and Formal, S. B. (1961). Proc. SOC. Exptl. Btol. Med. 107, 332. Rennerfelt, J. (lS6l). Thesis, Royal Institute of Technology, Stockholm. Reusser, F., Koepsell, H. J., and Savage, G. M. (1061). Appl. Microblol. 9, 348. Rosenberger, R. F., and Elsden, S. R. (1960). J . Gen. Microbbl. aZ, 726. Rosenberger, R. F., and Kogut, M. (1958). J . Gen. Mtcrobbl. 19, 228. Stewart, D. G., Warner, R. G., and Seeley, H.W. (1961) Appl. Microbbl. 9,150. Tyrell, E. A., MacDonald, R. E., and Gerhardt, P. (1958). J. Bacteriol. 7s, 1. Zacharias, B. Bfotechnol. Bfoeng. In press. Zubrzycki, L., and Spaulding, E. H. (1958). J . Bacteriol. 711, 278.