Automatic Assessment of Respiration During Growth in Stirred Fermentors

APPLIED MICROBIOLOGY, Sept. 1969, p. 438-443 Copyright 1969 American Society for Microbiology Vol. 18, No. 3 Printed in U.S.A. Automatic Assessment of Respiration During Growth in Stirred Fermentors DOUGLAS W. RIBBONS' Milstead Laboratory of Chemical Enzymology, "Shell" Research, Ltd., Sittingbourne, Kent, England Received for publication 12 February 1969 An apparatus for the continuous and automatic measurement of respiration during growth of micro-organisms in stirred aerated culture is described. The effluent atmosphere from the culture vessels is passed through commercially available oxygen and carbon dioxide gas analyzers, and their electrical output is fed to a multipoint recorder. The apparatus has been used to measure the respiration of Escherichia coli, Pseudomonas aeruginosa, Bacillus thuringiensis, and Methylococcus capsulatus during growth on complex and defined media. In addition, ph values and dissolved oxygen concentrations were measured. Growth of P. aeruginosa in glucoselimited media showed unexpected interruptions in the oxygen consumption curves which resembled diauxic growth; growth of B. thuringiensis in complex media showed similar discontinuities of respiration. These results are explained as the sequential utilization of preferred substrates which, in the case of P. aeruginosa were provided as transient intermediates of glucose. Most of the available data on the respiration of microorganisms have been obtained with nonproliferating suspensions (and very likely suspensions of low viability) in manometric apparatus. On some occasions, bacteria have been cultivated in Warburg apparatus, but respiration has usually been assessed in the absence of carbon dioxide (19; I. MacKechnie, Ph.D. Thesis, Univ. of Hull, 1965). Gaseous exchange during growth has also been measured by sampling techniques; analyses of the culture atmosphere have been made by selective absorption of the component gases (12) or gas chromatography (2) or by continuously recording the changes in pressure in closed culture vessels with manometers or pneumatic transducers (6, 18). Occasionally, indirect inference of gas consumption has been obtained by providing growth yield-limiting quantities of substrate vapors to the cultures (18). All of these methods depend upon an atmosphere with limited volume, a situation rarely encountered with aerated, stirred fermentors. A continuous-flow recording respirometer has also been described by O'Brien and Clark (9) in which a dissolved oxygen probe senses the oxygen demand of the culture and oxygen is supplied by constant-current electrolysis of water and measured by recording the operating time of the power supply. 1 Present address: Department of Biochemistry, Medicine, University of Miami, Miami, Fla. 33152. School of 438 It has been known for more than a decade that physical measurements with infrared analyzers (for carbon dioxide) and paramagnetic analyzers (for oxygen) of the atmospheres emanating from cultures are entirely suitable for assessing respiration of cultures grown with a constantly replenished atmosphere (3, 4, 5, 11, 14, 16). The measurements made are often illuminating and they provide good parameters by which to assess the state or stage of the culture. However, documentation of the results of oxygen and carbon dioxide measurements during batch growth have been few. This paper describes some observations made during the growth of Escherichia coli, Pseudomonas aeruginosa, Bacillus thuringiensis, and Methylococcus capsulatus in stirred, aerated fermentors. The apparatus was assembled primarily to investigate the gaseous exchange that occurs during bacterial growth on methane as the sole source of carbon. It consists of three analyzers for methane, oxygen, and carbon dioxide, but it is easily used to measure only the oxygen consumption and carbon dioxide evolution during the growth of other microorganisms. The results reported here with E. coli, P. aeruginosa, and B. thuringiensis show unexpected variations in respiration during the socalled exponential-growth phase. The use of the apparatus to describe the gaseous exchange during bacterial growth on methane is also shown.

VOL. 18, 1969 GROWTH IN STIRRED FERMENTORS 439 MATERIALS AND METHODS Organisms. E. coli W (NCTC 8666), P. aeruginosa T1 (10, 13), B. thuringiensis Mattes and B. thuringiensis Tolworth (8), and M. capsulatus (1) were used. E. coli, P. aeruginosa, and B. thuringiensis were maintained on Lab-Lemco (Oxoid) agar, and M. capsulatus was maintained in the liquid culture of Leadbetter and Foster (7) and on slopes of the same medium solidified with Ionagar (Oxoid; 10 g/liter). Cultivation. Culture media were contained in 10-liter (working volume) stirred fermentors (Type F-14 New Brunswick Scientific Co., N.J.) for E. coli, P. aeruginosa, and B. thuringiensis and in 10-liter (working volume) stirred fermentors provided with magnetic couplings to the stirrer paddles (Biotec, Stockholm) for M. capsulatus. Inocula (5 to 10% by volume) of the respective organisms, grown into stationary phase on the same medium, were used. Media. The measurements of gaseous exchange of the bacteria were made during growth in media of the following compositions. Medium C1 for P. aeruginosa and E. coil (g/liter): NaH2PO4-2H20, 0.78; (NH4)2 HPO4, 5.95; K2SO4, 1.75; and (mg/liter) CaCO3, 5; ZnO, 1.02; FeCl3-6H20, 13.5; MnCl2*4H20, 2.47; CuCl2*2H20, 0.425; CoCl2-2H20, 0.595; H3BO3, 0.155; MgC12-6H20, 254; Na2MoO4-2H20, 1. Carbon source was added as required and indicated in individual legends (15). Medium C2 for P. aeruginosa (g/liter): glucose, 20; NaH2PO4-2H20, 0.39; K2HPO4, 3.92; K2SO4, 0.875; and (mg/liter) CaCO3, 5; ZnO, 1.02; FeCl3-6H20, 13.5; MnCl2-2H20, 2.47; CuC12-2H20, 0.425; CoCl2-2H20, 0.595; H3BO3, 0.155; MgCl2 6H20, 254; Na2MoO4-21H20, 1. Nitrogen source was added as required and indicated in individual legends (15). Medium Norris for B. thuringiensis (g/liter): KH2PO4, 5.1; KOH, 0.242; KNO3, 7.5; Lab Lemco Broth (Oxoid CM15), 3.2; bacteriological peptone (Oxoid, L37), 2.4; and (mg/liter) Na2SO4, 111; MgSO4-7H20, 93; CaCl2-6H20, 164; MnSO4-4H20, 1.7; Fe2(SO4)3, 15; ZnSO4 7H20, 11. Medium M5 for M. capsulatus (g/liter): Na2HPO4, 1.2; KH2PO4, 0.8; MgSO4-7H20, 0.2; NH4Cl, 0.4; and (mg/liter) FeCl3 * 6H20, 16.7; CaCl2* 2H20, 0.66; ZnSO4-6H20, 0.18; CuSO4-5H20, 0.16; MnSO4. 4H20, 0.15; CoCl3-6H20, 0.18; yeast extract (Difco), 100 mg. Measurement of gaseous exchange during growth. The analytical system is depicted in Fig. 1. Air (or methane and air) was metered to the bottom of the fermentor via constant-pressure regulators usually at 10 psi, constant differential-flow controllers (model 62 BU, Associated Electrical Industries, Ltd., Harlow, England), flow meters (G. A. Platon, Ltd., Croydon, England), and sterile cotton-wool filters. The effluent atmosphere was passed through a heated cotton-wool filter (60 to 70 C) and a water-cooled condenser (4 ft; Mines Safety Appliances Co., Pittsburgh, Pa.), which was sufficient to remove the water vapor that would otherwise affect the analyzers. The analyzers were arranged in series so that the gas stream passed sequentially through two infrared gas analyzers (model SRB2, Sir Howard Grubb Parsons, Newcastle-upon-Tyne, England) for methane and carbon dioxide analyses and a paramagnetic resonance analyzer (type OA137 Industrial oxygen analyzer, Servomex Controls, Ltd., Crowborough, England). The signals from each of the three analyzers were continuously fed to a multipoint single-channel potentiometric recorder (mark III, 50 mv; George Kent, Ltd., Luton, England). The outputs received from a ph meter (model 23A or 91B, Electronic Instruments Ltd., Richmond, England) and a dissolved oxygen electrode (model 15A, Electronic Instruments, Ltd.), were also displayed on the recorder. At aeration rates of 2.5 liters/min, the response of the analyzers is 95% of the true compositions of the effluent gas after 6 min flow-through time. Oxygen analysis. The OA 137 analyzer is capable of range changes by simple switching on the front panel. However, backing off is not possible; therefore, to measure oxygen concentration in the appropriate range (16 to 21% 02 in air) with a full-scale deflection to the recorder in that range, an electronic variable backing-off device (Fig. 2) was incorporated into the analyzer circuitry. For most applications, the signal to the recorder was linear with oxygen concentration. Carbon dioxide analysis. The selected analyzer had sample tubes constructed for nominal ranges 0 to 0.5, 0 to 5, and 0 to 50% of CO2 in the atmosphere. For most applications, the 0 to 5% range was used. The calibration curve relating the partial pressure of CO2 to recorder (or meter) reading was not linear, and the calibrations provided by the manufacturers were satisfactory. Methane analysis. The infrared analyzer used had three available ranges for methane analysis, 0-1, 0-10, and 0 to 100% of methane in the atmosphere. The middle range was used entirely with gain settings on the instrument of 2 or 3. This effectively expands FIG. 1. Flow diagram of apparatus used to measure gaseous exchange during growth in fermentors. FIG. 2. Circuit diagram of the backing-off device used for the Servomex OA137 oxygen analyzer.

440 RIBBONS APPL. MICROBIOL. the scale by a little more than two- or fourfold. When the initial methane partial pressures were about 16% of the atmosphere, the mechanical zero control was used to back-off the output to the recorder. Consequently, it was possible to measure methane partial pressures with full-scale deflection of approximately 5 and 2.5% of the atmosphere in the region of 10 or 20% methane in the atmosphere. Changes in partial pressure of methane are not represented linearly, and the manufacturers calibrations relating methane concentration to meter readings are unsatisfactory for this application. For each individual experiment, the scale was calibrated with mixtures of methane and air prepared by flowing methane and air into the analyzer at predetermined rates. Bacterial dry weight. Absorbance values of cell suspensions at 270 nm were determined in a Unicam SP 600 and were related to dry weight by calibration curves. RESULTS AND DISCUSSION Figures 3-8 show the gaseous exchange that occurs during the growth of E. coli, P. aeruginosa, B. thuringiensis, and M. capsulatus. Legends to the figures provide complete details of the conditions in individual experiments. The respiratory activities of E. coli during growth on defined salts media, in which the quantity of glucose supplied limits the final population attained, are as expected (Fig. 3). Oxygen consumption and carbon dioxide evolution follow approximately exponential patterns, as does the increase in cell mass. During the last doubling of the population, oxygen consumption and carbon dioxide evolution abruptly change and fall to values approaching those of the input air. The decline of oxygen consumption does not coincide with the apparent evolution of carbon dioxide, and the delay shown by the carbon dioxide curve probably reflected the release of carbon dioxide from bicarbonate as the concentration of carbon dioxide in equilibrium with the culture decreased. Buffering in this medium was sufficient to maintain the ph value during growth under these conditions. When P. aeruginosa is grown in similar media Downloaded from http://aem.asm.org/ on April 22, 2018 by guest Fig 3 FIG. 3. Growth and respiration of E. coli in glucoselimited media. Growth medium: Cl (10 liters) with glucose as carbon source (30 g). Air flow rate: 3.8 liters/min. Stirrer speed: 590 rev/min. Temperature, 24 C. Lines for oxygen, carbon dioxide, and ph are the actual recorder traces. Approximate full-scale deflections on recorder: oxygen, 16 to 21%; carbon dioxide, 0 to 5%; ph, 3.5 to 8.5. Hours FIG. 4. Growth and respiration of P. aeruginosa in ammonium-limited media. Growth medium: C2 (10 liters), but with twice the phosphate concentration, with ammonium sulfate as nitrogen source (8.8 g). Air flow rate: 4 liters/min. Stirrer speed: 570 rev/min. Temperature, 25 C. Lines for oxygen, carbon dioxide, and ph are the actual recorder traces. Approximate full-scale deflections on recorder: oxygen, 15 to 21%; carbon dioxide, 0 to 5%; ph, 3.5 to 8.5. Final cell density, 2.12 mg (dry weight) ml.

VOL. 18, 1969 GROWTH IN STIRRED FERMENTORS 441 but the ammonium ions are the growth yield- 16-0 limiting nutrient, the curves for oxygen consumption, carbon dioxide evolution and the increase in bacterial dry weight are again exponential. (Fig. 4). An interruption in the carbon dioxide curve is usually seen. Deviations in the oxygen 075 consumption pattern at this point are barely 17 detectable. The ph value of the cultures at this C point also changes slightly, which is only seen dioxbde with a continuous trace, and then recovers (Fig. 4). However, when the nitrogen source has been exhausted and growth ceases, oxidation of 18 15 55 12 the remaining glucose continues, the acidity falls to approximately ph 4.0, and respiration ceases. 2 The results obtained with P. aeruginosa culti- e. vated on glucose-limited media were rather un- expected (Fig. 5, 6). The curve for oxygen con- - 19 235 _ 6.5 0352 sumption always shows two periods during X which respiration has been interrupted. This is not so pronounced with the carbon dioxide evolution curve which, in any case, would be complicated by the ph-dependent equilibrium of / 3 carbon dioxide with the medium components. 2 Oxygen When the increase in bacterial mass is plotted, there appears to be no break in the exponential curve (Fig. 6). This may reflect the insensitivity Dry of the estimation and low frequency of sampling relative to the measurements of gaseous ex- 0 2 4 6 - Hours FIG. 6. Growth and respiration of P. aeruginosa on l-rglucose-limited media. Growth medium: Cl (10 liters) with glucose as carbon source (45 g). Air flow rate, 4.75 liters/min. Stirrer speed, 600 rev/min. Tempera- -I0 ture, 30 C. Lines for oxygen, carbon dioxide, and ph 4.5 aw are the actual recorder traces. 17 0 7 Carbon \ 5 - / Approximate full-scale deflections on recorder: oxygen, 16 to 21%; carbon dioxide, 0 to 5%; ph, 3.5 to 8.5. _183> 1.-t\5 5 e c change. The changes in oxygen consumption are reflected also by the level of dissolved oxygen in ph X the i medium _ S (Fig. 5). Thus, the routine use of a -!tzo23 < ffi [- ~ 65 Am dissolved oxygen electrode in fermentors easiy provides information on events such as these. The ph value of these cultures falls during the 20 3 45 iss~l~ed r 7.5 period preceding the first break in oxygen consumption, and continues until the rate of oxygen uptake has recovered for the second time. The L0 _^last phases of rapid oxidation in these cultures is 21L SC 2i 6 1la 2a characterized ce lo by an increase in ph value; the final ph value of the spent medium assumes a FIG. 5. Growth and respiration of P. aeruginosa on value a little more acidic than the initial medium. glucose-limited media. Growth medium: Cl (10 liters) The extent of these ph changes is also a function with glucose as carbon source (30 g). Air flow rate, of the initial concentration of the growth-limiting 2.3 liters/min. Stirrer speed, 580 rev/min. Tempera- glucose. The events with glucose-limiting media ture, 30 C. Lines for oxygen, carbon dioxide, ph, anda dissolved oxygen are the actual recorder traces. Ap- are characteristic of the diauxic growth of microproximate full-scale deflections on recorder: oxygen, organisms on mixed substrates. It seems very 16 to 21%; carbon dioxide, 0 to 5%; ph, 3.5 to 8.5. probable that acids such as gluconate, 2-keto- Final cell density, 1.87 mg/ml. gluconate, a-ketoglutarate, or pyruvate may

442 RIBBONS APPL. MICROBIOL. transiently accumulate in the culture medium, and a short periodof adjustment of the respiratory activities of the cells is required before they are completely oxidized. Tigerstrom and Campbell (17) have indeed demonstrated that washed suspensions of P. aeruginosa accumulate a-ketoglutarate from glucose and that large amounts of pyruvate are transiently formed during growth in glucose-limited cultures. Pyruvate has also been identified as a transient metabolite in glucoselimited cultures of our strain of P. aeruginosa. It is possible that new enzymes are elaborated during these lag periods, and a true diauxie is being observed by oxygen and carbon dioxide measurements that is not detected by estimations of bacterial dry weight. Growth of microorganisms in complex media or media containing more than one oxidizable carbon source, then, is likely to provide patterns of oxygen consumption and carbon dioxide evolution that are not exponential. This was clearly seen when the rates of growth and respiration were measured for two strains of B. thuringiensis grown on identical complex media. The growth curve is not exponential after the mass has increased to 20 to 30% of the final value. Oxygen uptake and carbon dioxide output curves are almost linear and show shoulders with the Mattes strain and distinct troughs with the Tolworth strain (Fig. 7). The absorbance (X 570 nm, but expressed as dry weight) of the Mattes culture increased linearly for 20 hr after the highest respiratory rates had been observed, during which time the ph of the culture rose from 5.95 to 7.2. It was clear that many oxidizable compounds remained in the culture media after the maximal rates of oxygen consumption had been attained, as oxygen was consumed by and carbon dioxide was evolved from the culture at a slowly decreasing rate (for several hours). This was quite characteristic for the complex media; with the minimal defined media (Fig. 3-6), respiratory rates declined abruptly after growth had ceased, even in N-limited cultures that had no ph control. Growth of the Tolworth strain ceased abruptly about 2-hr after the maximal oxygen consumption rate was achieved. The troughs in oxygen consumption parallel the record of dissolved oxygen concentration (Fig. 7). When the carbon sources provided in the Norris medium are increased fourfold, exponential growth of B. thuringiensis occurs for a longer period; this is also reflected by the oxygen and carbon dioxide traces. However, these cultures do not sporulate and the ph can rise as high as 8.8. The growth of the obligate methane-utilizer M. capsulatus is shown in Fig. 8, and methane consumption, in addition to oxygen and carbon dioxide measurements, is seen. Exponential Downloaded from http://aem.asm.org/ on April 22, 2018 by guest FH-UFS FIG. 8. Growth, respiration, and methane utilization FIG. 7. Growth and respiration of B. thuringiensis by M. capsulatus. Growth medium M5 (10 liters). Gas (Tolworth) on Norris media (10 liters). Air flow rate, flow rates: air, 460 ml/min.; methane, 80 ml/min. 2 liters/min. Stirrer speed, 570 rev/min. Temperature, Stirrer speed, 700 rev/min. Temperature, 38 C. Lines 30 C. Lines for oxygen, carbon dioxide, ph, and dis- for oxygen, carbon dioxide, and methane are the actual solved oxygen are actual recorder traces. Approximate recorder traces. Approximate full-scale deflections on full-scale deflections on recorder: oxygen, 16 to 21%; recorder: oxygen, 14 to 19%; carbon dioxide, 0 to 5%; carbon dioxide, 0 to 5%G; ph, 3.5 to 8.5. methane, 11 to 16%.

VOL. 18, 1969 GROWTH IN STIRRED FERMENTORS 443 growth of this thermotolerant bacterium at 38 C ceases when the ph falls to about 6.2, after which time the rates of oxygen and methane consumption and carbon dioxide evolution decay rather slowly over the next 8 to 10 hr. The recorder traces for methane partial pressures were usually very noisy when the instrument was backed off to the extent shown. The continuous measurement of respiration during microbial growth in batch culture, and also in continuous culture (16), is a valuable aid to analyzing and understanding growth processes. The data presented emphasize the transient and unexpectedly fluctuating nature of the environment in a batch culture during the often quoted "steady state" phase of the growth cycle. Although we have not utilized any of the data presented for quantitative relationships, several computations are possible. Thus, Qo,, Qco,, and RQ values may be determined at any stage of the growth curve (provided the ph value is known). The total oxygen consumption, or carbon dioxide evolution, may be assessed at any stage; thus, carbon or oxygen balances may be made at desired periods during the fermentation. Respiratory measurements such as those shown here assume some importance in industrial fermentations in which the oxygen demand alters, possibly unpredictably, and in which mixtures of oxidizable nutrients are nearly always provided. ACKNOWLEDGMENTS I am grateful to W. H. Kingham and K. Sage for experimental assistance. I greatly appreciate the help of A. J. W. Hewett, F. A. Smith, and G. Joy in the choice and modification of instruments. LITERATURE CITED 1. Foster, J. W., and R. H. Davis. 1966. A methane-dependent coccus, with notes on classification and nomenclature of obligate, methane-utilizing bacteria. J. Bacteriol. 91: 1924-1931. 2. Hamer, G. 1967. Methane as a carbon substrate for the production of microbial cells. Biotechnol. Bioeng. 9:499-514. 3. Herbert, D. 1958. Some principles of continuous culture, p. 381-396. In G. Tunevall (ed.), Recent progress in microbiology. Almquist & Wiksell, Stockholm. 4. Herbert, D. 1965. Microbial respiration and oxygen tension. J. Gen. Microbiol. 41:viii-ix. 5. Jensen, A. L., and J. S. Schultz. 1966. Apparatus for monitoring the oxygen uptake and carbon dioxide production of fermentations. Biotechnol. Bioeng. 8:539-548. 6. Jensen, A. L., J. S. Schultz, and P. Shu. 1966. Scale up of antibiotic fermentations by control of oxygen utilization. Biotechnol. Bioeng. 8:525-537. 7. Leadbetter, E. R., and J. W. Foster. 1958. Studies on some methane-utilizing bacteria. Arch. Mikrobiol. 30:91-100. 8. Norris, J. R. 1964. The classification of Bacillus thuringiensis. J. Appl. Bacteriol. 27:439-447. 9. O'Brien, N. J., and J. W. Clark. 1967. A continuous-flow recording respirometer. Process Biochem. 2:21-23. 10. Pankhurst, E. 1959. The biological oxidation of spent gas liquor. J. Appl. Bacteriol. 22:202-215. 11. Phillips, K. L. 1963. Determination of oxygen and carbon dioxide using a paramagnetic oxygen analyzer. Biotechnol. Bioeng. 5:9-15. 12. Pirt, S. J. 1957. The oxygen requirement of growing cultures of an Aerobacter species determined by means of the continuous culture technique. J. Gen. Microbiol. 16:59-75. 13. Ribbons, D. W. 1966. Metabolism of o-cresol by Pseudo. monas aeruginosa strain Ti. J. Gen. Microbiol. 44:221-231 14. Telling, R. C., R. Elsworth, and D. N. East. 1958. A continuous infrared analyzer for measurement of CO2 in effluent air from bacterial cultures. J. Appl. Bacteriol. 21: 26-44. 15. Tempest, D. W. 1965. An 8-liter continuous flow culture apparatus for laboratory production of micro-organisms. Biotechnol. Bioeng. 7:367-386. 16. Tempest, D. W., and D. Herbert. 1965. Effect of dilution rate and growth-limiting substrate on the metabolic activity of Torula utilis cultures. J. Gen. Microbiol. 41:143-150. 17. Tigerstrom, M. von, and J. J. R. Campbell. 1966. The accumulation of a-ketoglutarate by suspensions of Pseudomonas aeruginosa. 12:1005-1013. 18. Vary, P. S., and M. J. Johnson. 1967. Cell yields of bacteria grown on methane. Appl. Microbiol. 15:1473-1478. 19. Whitaker, A. M., and S. R. Elsden. 1963. The relation between growth and oxygen consumption in micro-organisms. J. Gen. Microbiol. 31:xxii.