rapid growth, nonsporulation, apparently stable strain characteristics, and

Transcription

1 BRUCELLA SUIS IN AERATED BROTH CULTURE III. CONTINUOUS CULTURE STUDIES' PHILIPP GERHARDT Camp Detrick, Frederick, Maryland Received for publication February, The maintenance of a given bacterial population by the continuous addition of fresh medium and the withdrawal of culture presumably represents the ultimate in efficiency for the production of the comparatively large amounts of organisms that are normally required for immunochemical investigations. The rapid growth, nonsporulation, apparently stable strain characteristics, and aerobic nature of Brucella suis made it seem particularly adaptable to such a process. Consequently, this study was undertaken to devise a laboratory process for continuous culture of the organism that might be translated to the production of larger quantities for chemical and immunological studies. The literature contains several reports regarding similar techniques, although none was directly applicable. A continuous vinegar fermentation process has been described and applied industrially, but it seemed to have limited usefulness in this study. Similarly, laboratory apparatus and experimental uses have been described by several workers (Moyer, ; Haddon, ), but in each case the method was not adapted to the requirements of this investigation. A system originated by Rogers (Rogers and Whittier, 0) and adapted by Cleary (Cleary, Beard, and Clifton, ) suggested a means of attaining the results desired. Modifications were necessary, however, for its use with a highly pathogenic organism, for more accurate flow control, and for carefully controlled aeration. Probably the most applicable studies have been reported by Kolachov and coworkers (Unger et al., ; Bilford et al., ), who have developed an apparently successful continuous aerobic process for the production of distillers' yeast on a pilot plant scale. They were able to reduce the allover probessing time from 0 to 0 hours to hours for an equivalent volume of product, at the same time increasing yields from 0,000,000 to 00,000,000 cells per ml and obtaining yeast of high quality, uniformity, purity, and fermentability. It is to be noted, however, that in no case reported in the literature were highly virulent organisms employed extensively. APPARATUS AND MErHODS Preliminary experiments on the regeneration time (i.e., the time necessary to reproduce a given population) and growth characteristics of the organism were conducted in shaker flasks, but subsequent studies employed modifications of l Studies conducted at Camp Detrick, Frederick, Maryland, January to September,. lst Lt., Infantry. Downloaded from on January, 0 by guest

2 , PHILIPP GERHARDT [VOL. aeration bottles (Gee and Gerhardt, ). In the first technique, cultures were grown in ml of medium in a 0-ml Erlenmeyer flask, and aerated by continuous agitation on a reciprocating shaker at approximately 0 rpm; by this method a high, constant, and easily reproducible rate of air supply was obtained, providing accurately controlled cultural conditions. In the second technique, ordinary l-quart jars were fitted with a rubber stopper and a coarse grade sintered glass sparger and partially filled with 00 ml of medium; filtered air was supplied by negative pressure. The following medium (McCullough et al., ) was used: per cent bacto tryptose, 0. per cent sodium chloride, 0.7 or.0 per cent glucose, 0.,ug thiamine hydrochloride per ml, and distilled water. A single strain of Brucella suis was employed throughout (received through the courtesy of Dr. I. F. Huddleson, his strain no. 7-A). Incubation was at 7 C. Plate counts were made by the usual poured plate methods using tryptose agar (Difco). Light transmittance was determined by use of the Coleman universal spectrophotometer, using matched -mm pyrex test tubes as cuvettes. Evaluations were made on the :0 dilution of the culture, using a :0 dilution of the original medium as the reference standard. Initially, two basic concepts of continuous culture were considered: () a so-called "cyclic" continuous system, in which the addition of fresh medium and the withdrawal of culture were made at periodic intervals, a portion of the product being retained as starter for the ensuing cycle; and () a completely continuous system, in which the addition of fresh medium and the withdrawal of product were accomplished continuously and simultaneously. A descriptionof the laboratory apparatus devised for these systems follows: () The "cyclic" system was applied to either one or a number of culture vessels in series. When operated as a unit, the culture was grown for a given period of time, at which point 0 or 0 per cent of the product was withdrawn to a receiving flask, an equivalent amount of fresh sterile medium was added to the vessel, and the culture was allowed to regenerate again to the given level to complete the cycle. Fundamentally, this amounted to a serial transfer in a closed system employing large inocula. The same system, operated with a number of vessels, was so arranged as to employ one vessel to provide inoculum for two or more culture vessels; for instance, the inoculum vessel might regenerate.in a -hour cycle to balance three culture vessels, each maintaining a -hour cycle. Either modification of the cyclic system was- operated in a completely closed apparatus; addition of medium, withdrawal of culture, and aeration were accomplished by negative pressure through manifold flow tubes. () A schematic diagram of the apparatus devised for the completely continuous system is presented in figure. A supply of sterile medium was contained in the storage flask and was replenished infrequently by aseptic addition through a sampling tube. The medium was drawn from this flask to the culture vessel through capillary tubing and a standard orifice to regulate the supply. The change in flow that would be encountered as the supply of medium was depleted (due to the change in hydrostatic pressure) was obviated by the compensating effect of the air inlet capillary and the medium outlet capillary reaching Downloaded from on January, 0 by guest

3 ] BRUCELLA SUIS IN AERATED BROTH CULTURE to the bottom of the flask. In the culture vessel, a given population was maintained, depending upon the rate of medium supply and product removal. This rate of operation was regulated by the capillary and standard orifice restrictions and by the amount of negative pressure. Air was supplied at a constant and previously determined optimal rate of 00 ml per minute. Foaming was controlled by the addition of an equal mixture of lard and tributyl citrate. Samples were withdrawn through the sampling tube. Twenty-four-hour intervals were allowed for the system to reach equilibrium. Culture was withdrawn through the vacuum line, maintaining a constant level of 00 ml in the reactor. The rate L ~~~~~~~~~~~~~~~~~~~0 CSTANT TMPERAT$ E IA AtR NOTAMETER L~~~~~~~~ CL= MEDIUM STORAGE CONTINUOUS EFFLUENT RECEIVING VACUUM FLASK CULTURE METER FLASK PUMP VESSEL KEY I -FILTER. CLAMP. SPARGER - STANDARD ORIFICE -CAPILLARY TUBING, FIG.. DIAGRAM OF COMPLETELY CONTINUOUS CULTURE APPARATUS of product withdrawal (and therefore medium supply as well) was measured by by-passing the effluent into the meter and tinming the passage of a given amount. The product was collected in a large receiving flask. A constant vacuum was maintained in the system by the use of a portable HP rotary pump. Sterilization of the air entering the culture and medium storage vessels, as well as the effluent air, was effected by cotton filters. The efficiency of operation of the various systems was expressed as the increase in cell count per hour. Although calculated somewhat differently for each system, the various efficiency data are on a comparable basis. EXPERIMENTAL Preliminary experiments were conducted to determine the time necessary for the regeneration of cultures, using different levels of inocula taken from various Downloaded from on January, 0 by guest

4 PHIILIPP GERHARDT periods in the growth phase. Thus, samples were removed from a parent culture when it had reached light transmittance levels of 0, 0, and 0 per cent, and were used to inoculate subcultures; the time necessary to regenerate these levels was then determined. Theoretically, a cyclic continuous system would merely represent a continued repetition of this process. Composite results of such an experiment are given in table. Although a 0 per cent inoculum level apparently gave a somewhat comparable efficiency of production, the 0 per cent level was used in subsequent work by reason of its more ready adaptation to larger scale processes. Regeneration to a near-peak turbidity was also found to be more efficient under the conditions of the experiment. An apparatus was assembled having one inoculum and three culture vessels, for operation in a cyclic continuous system. The inoculum vessel was trans- TABLE Regeneration time of aerated (shaker) cultures of Brucella Ui PARENT CULTURE SUBCULTURE Lgtanet Viable cells Amount of Regenera- Ltrangt Viable cells Eficiency NO 0. trnmt X cy transmit X 0' inoculum tion time tance x- % per ml % hours % per ml * Efficiency = final count - initial count - regeneration time [VOL. ferred at -, -, and -hour intervals, with respective generation periods for each reactor of,, and hours. Each schedule was maintained for at least nine transfers, or three complete cycles. Results are given in table. These results indicate that for a given schedule, an equilibrium which was rather constant was attained in the system. It appeared that a -hour interval between harvests was feasible; a -hour schedule apparently resulted in a disproportionally decreased count in the product. Obviously, employment of one inoculum and three culture vessels represented but one of several methods of obtaining maximum utilization of a given number of vessels; in practice, this factor would be adjusted to specifications required for yield and modified to available facilities. As mentioned before, the cyclic continuous system may be applied to a unit operation; i.e., a single culture vessel may be used for culture, merely retaining sufficient product after each phase to start the ensuing one. Such a system was examined in some detail, as it afforded a readily adaptable, simple system for larger scale operations. Duplicate sets of apparatus were made for testing Downloaded from on January, 0 by guest

5 ] BRUCELLA SUIS IN AERATED BROTH CULTURE 7 TABLE Operation of multiple* cyclic continfuous culture system with 0 per cent inoculum INOCULUPL VESSEL SERIES NO. Aeo ih culture tance CULTURE VESSELS Xculture tancmite X 0- c re of transmit- Viable cels No. Age of transmit- Viable cells hours S B 0.Q per ml * One inoculum vessel and three culture vessels. Sours per ml Downloaded from on January, 0 by guest 0 and 0 per cent inoculum operations. Results of the experiments are given in tables and. The efficiency of generation was expressed as increase in cell

6 PRIIJPP GERHARDT [VOL. TABLE Operation of unit cyclic continuous culture system with 0 per cent inoculum URRtS NO. AC E O LIGHT TRANSMITTANCE VIABLE. CELLS X O' TICIENcy CULTUR hours avg. % var.t per ml 0 0 * Efficienpv ' final count - initial count x, - age of culture t Var.: no calculations possible as culture failed to reach equilibrium avg. per ml 0 var.t GIg..... var.t Downloaded from on January, 0 by guest

7 ] BRUCELLA SUIS IN AERATED BROTH CULTURE count per hour. It may be seen that higher efficiency figures were obtained for the 0 per cent system; this served to substantiate the preference for a 0 per cent level. Furthermore, the highest efficiency wag obtained when transfers were TABLE Operation of unit cyclic continuous culture ystem with 0 per cent inoculum.ltur SERIES NO. CUELTUR LIGHT TRANSMITTANCE VIABLE CELLS X 0-' EFFICIENCY* _., atg t 7 hours * Efficiency - final count-initial count X 0- age of cultre t Air supply accidentally cut off at this point. par ml avg per ml made at -hour intervals. Highest counts were, of course, found at the longest interval. In practice, the balance of efficiency vs. cell count per unit volume of medium would be established by the requirements of the product. It was of interest to note that when a -hour transfer schedule was maintained, the culture failed to regenerate to the previous level and was merely being diluted out. Thus, it would appear that the generation time of the organism is greater than Downloaded from on January, 0 by guest

8 0 PHILIPP GERHARDT hours; previous calculations from growth curves indicated it to be approximately. hours. This unit system was operated continuously for as many as days; in every case contamination arose in the medium storage flasks, where it was necessary to open the system infrequently to replenish the medium supply. As a consequence of these preliminary studies, an apparatus was developed in which the addition of fresh medium and the withdrawal of product were accomplished continuously and simultaneously; its operation has previously been described in detail and a diagram is given in figure. Although most of the time available was devoted to designing the apparatus and modifying it for efficiency, a few satisfactory runs were made. The results of one experiment are given in table. Samples were taken at -hour intervals, in which period it was presumed that the system had reached equilibrium. Although the results TABLE Operation of completely continuous culture system with varying rates of operation CALCULATED RATE OF OPERATION AGE OF CULTURE TRANSHTTAC CIBELSX' EFFICIENCY' Replacement Time for complete T ITE CELLSX 0: per hour replacement hours % hours % per ml average count *Efficiency=.X 0- time for complete replacement [VOL. are limited, it would appear that an excellent efficiency can be obtained by the process, comparable or better than those from previous cyclic continuous systems. Thus, employing the "optimum" schedule in each case, this completely continuous system gave a harvest of an equivalent full-vessel volume (00 ml) every 7.7 hours with a count of X 0, whereas the best previous system gave an equal amount of product every hours with a count of X 0. Expressed as efficiency (cells per ml increase per hour, X 0-), they were. and.7, respectively. The experiment was stopped when contamination appeared in the medium storage vessel, after a replenishment of the medium became necessary. The apparatus was then modified to include a second medium storage carboy (approximately 0 liters), from which medium could be replenished through a, completely closed system. With this modification, the apparatus was successfully operated for more than weeks, with a complete turnover in the culture vessel every hours, at most, and with a product having a count in excess of 0 X 0 cells per ml. Preliminary strain studies had shown that strain mutation was minimal after as many as serial transfers, and the same strain constancy apparently occurred in these experiments. No virulence tests were conducted. Downloaded from on January, 0 by guest

9 ] BRUCELLA SUIS IN AERATED BROTH CULTURE DISCUSSION Although maintenance of bacterial cultures by serial transfers is a routine and accepted procedure, few efforts have been made to study serial transfer in a continuous phase, beyond theoretical considerations. Such a procedure, however, presumably represents the ultimate in efficiency when production of comparatively large quantities of microorganisms or their by-products is desired, and thus assumes practical importance. Application of this principle to the culture of Brucella suis resulted in the development of a laboratory apparatus in which the addition of fresh medium and the withdrawal of product were accomplished continuously and simultaneously. It was operated safely and aseptically for prolonged periods of time with little manual attention, and gave a product of very high cell concentration, uniformity, and purity. The advantages of such a continuous process are primarily an increase in production efficiency and reduction in manual attention. With the customary "batch" methods, it had previously been possible to produce a given large unit of Brucella suis in approximately hours over-all time; whereas, using a completely continuous system, it is believed that an equivalent product could be produced in approximately hours, not including time which may be gained from the elimination of frequent sterilization of equipment and handling. Once started and adjusted, the continuous system required only infrequent attention to replenish the medium supply and to remove the product. Operated under negative pressure, the system minimized the hazards of producing a highly virulent organism in quantity. The product contained a very high concentration of cells, was free from contamination, and contained practically no morphological strain variants. The disadvantages of the process are also evident. Strain mutation, particularly from the standpoint of virulence, has not been fully studied and might possibly arise as an objection.' More extensive use of the apparatus might introduce increased problems of contamination control. It might be necessary to convert the process to positive pressure, with accompanying difficulties. Foaming of the medium constantly provides a difficult problem of control. These problems will, of course, require further study. It is believed that, going beyond the scope of this investigation, the process might find successful application to the experimental preparation of vaccines and other immunogenic products, antibiotics, legume inoculants, yeast, and other fermentation products. The fact that the apparatus has been operated with no infections of personnel or serious accidents, in growing an organism as virulent and infective as Brucella suis, is considered to be of significance. The utilization of a closed system for culture and the decreased amount of manual attention required have contributed materially to this factor of safety. Thus, the possible applications to experimental preparation of virulent or toxic biologicals are felt to be of particular importance. There are several factors limiting such applications: the organism must have stable strain characteristics; there can be no accumulation of limiting toxic by-products of the organism; and the method Downloaded from on January, 0 by guest

10 PHILIPP GERHRDT [VOL. probably could not be adapted to the production of sporulating bacteria. With continued study and modification, however, it is hoped that the concept of continuous culture of bacteria may find further practical applications. SUMMARY Preliminary experiments were conducted to determine the time required for regeneration of Brucelia suis cultures, using different levels of inocula taken from various periods in the growth phase. These results were applied to the construction and operation of so-called "cyclic" continuous culture systems, in which the addition of fresh medium and the withdrawal of culture were made at periodic intervals, a portion of the product being retained as starter for the ensuing cycle. As a consequence of these initial studies, a laboratory scale apparatus was developed in which the addition of fresh medium and the withdrawal of product were accomplished continuously and simultaneously. This apparatus was operated safely and without contamination for prolonged periods of time with little manual attention, and produced a Brucella suis suspension of very high concentration, uniformity, and purity. ACKNOWLEDGMENT The author wishes to express his appreciation to st Lt. Lynn L. Gee, Army Air Force, for his close co-operation and advice, and to PhM/c Elizabeth McIntyre, WAVE, for her excellent technical assistance. The study was carried out under the supervision of Dr. J. L. Roberts and Major S. S. Elberg, Sanitary Corps. REFERENCES BILFORD, H. R., SCALF, R. E., STARK, W. H., AND KoLAcHov, P. J. Alcoholio fermentation of molasses. Rapid continuous fermentation process. Ind. Eng. Chem., Ind. Ed.,, 0-0. CLzARY, J. P., BEARD, P. J., AND CLI7rON, C. E. Studies of certain factors influencing the size of bacterial populations. J. Bact.,, 0-. Gzz, LYNN L., AND GERHARDT, PHILIPP Brucella sui in aerated broth culture. II. Aeration studies. J. Bact.,, 7-. HADDON, E. C. Apparatus for obtaining a continuous bacterial growth. Trans. Roy. Soc. Trop. Med. Hyg.,, -00. MCCULLOUGH, W. G., et al. Unpublished. MOYER, H. V. A continuous method of culturing bacteria for chemical study. J. Bact.,, -7. ROGERS, L. A., AND WHITTIER, E The growth of bacteria in a continuous flow of broth. J. Bact., 0, 7-. UNGER, E. D., STARK, W. H., SCALF, R. E., AND KOLACHOV, P. J.. Continuous aerobic process for distillers yeast. Engineering and design factors. Ind. Eng. Chem., Ind. Ed.,, 0-0. Downloaded from on January, 0 by guest