Nutrient-limited continuous culture in the phauxostat.

Stable steady-state growth of Escherichia coli B limited by succinate, phosphate, or sulfate ion over the range of specific growth rates of 0.025-0.51 h(-1) was achieved using pH-controlled auxostasis in the phauxostat. The concentration of the growth-limiting substrate in the growth vessel could be varied at will in the region of the Monod half-maximal saturation constants by adjusting the concentration of that substrate in the reservior (at constant buffering capacity) or by varying the population density of the culture through changing the buffering capacity of the medium in the reservoir.

Oxygen-limited continuous culture and respiratory energy conservation in Escherichia coli.

Escherichia coli B was cultured continuously in succinate-minimal medium under conditions of oxygen limitation in the phauxostat. With decreasing oxygenation and consequent decreasing growth rates, the complement of terminal cytochrome oxidases changed as follows: high growth rates, cytochrome o; intermediate growth rates, cytochromes o and d; lowest growth rates, cytochromes o, d, and a1. Respiratory kinetics exhibited by nongrowing cell suspensions obtained from continuous cultures indicated that terminal oxidase activity was exhibited by cytochrome o (Km for O2 = 0.2 micron; Vmax = 1.1 to 1.5 mumol of O2 per nmol of cytochrome o per min) and cytochrome d (Km for O2 = 0.024 micron; Vmax = 0.7 mumol of O2 per nmol of cytochrome d per min). During oxygen-limited growth, the molar growth yield referred to respiration, and corrected for maintenance respiration [Yo(max)], was 12.6 g (dry weight) per g-atom of oxygen, not significantly different from the succinate-limited value of 12.0 g (dry weight) per g-atom of oxygen. The rate of maintenance respiration of the oxygen-limited culture was only 3.4 mg-atoms of O per g (dry weight) per h, some threefold less than that of the succinate-limited culture. Respiration-driven proton extrusion did not vary with the growth rate or with the complement of terminal oxidases (H+/O = 3.7; standard deviation, 0.07). We conclude that the content of terminal oxidases is without effect on the efficiency of respiratory energy conservation.

Effects of growth temperature on yield and maintenance during glucose-limited continuous culture of Escherichia coli.

The effects of growth temperature on the aerobic growth yield with respect to oxygen consumption (Y0-grams [dry weight] per gram-atom of O) and the rate of maintenance respiration (m0-milligram-atoms of O/gram [dry weight] per hour) are reported for Escherichia coli B cultivated continuously in the presence of oxygen with limiting glucose. During anaerobic continuous culture, YATP(max) (grams [dry weight] per mole of ATP corrected for maintenance) increases from 10.3 to 12.7 as the growth temperature is lowered from 37 to 25 C. Over this same range, Y0(max) (Y0 corrected for maintenance respiration) rises from 12.5 to 28.8 and remains at the higher value down to 17.5 C. From 37 to 32 C, m0 increases from 0.9 to 4.4 but then falls to 1.5 as the temperature is lowered to 17.5 C. The value of m0 sharply rises some 13-fold as the temperature is raised to 42 C without a significant change in the value of Y0(max). Changes of Y0(max) are consistent with a temperature-sensitive doubling of the efficiency of oxidative phosphorylation, but the reasons for the changes of the rate of maintenance respiration are not known.

A method for the regulation of microbial population density during continuous culture at high growth rates.

A method for the continuous culture of microorganisms is described which employs growth-dependent pH changes to control the rate of addition of fresh medium to a culture vessel. The apparatus (the "phauxostat") supports, at constant pH, long-term continuous culture at rates near or at the maximum of which the organisms are capable. The buffering capacity of the inflowing medium determines the steady-state population density of the culture, but the rate of growth is independent of the buffering capacity. The fundamental theory of operation is tested and some basic parameters of growth are estimated using Escherichia coli B growing continuously in media containing glucose, glycerol or DL-lactate.

Effects of varying the carbon source limiting growth on yield and maintenance characteristics of Escherichia coli in continuous culture.

The magnitudes of Yo (grams [dry weight] formed per gram of atom O) and mo, the maintenance respiration (milligram-atoms of O per gram [dry weight] per hour), of Escherichia coli B have been determined by growing the organism in aerobic continuous culture limited by a number of different substrates. The value found were as follows: glucose--tyo = 12.5, mo = 0.9; glucose plus 2.7 mM cyclic adenosine 3',5'-monophosphate (cAMP)--Yo = 31.2, mo = 9.3; galactose--Yo = 13.2, mo = 1.8; mannitol--Yo = 20.1, mo = 6.1; L-glutamate--Yo = 25.5, mo = 17.7; glycerol--Yo = 14.9, mo = 10.0; succinate--Yo = 11.2, mo = 12.1; and acetate--Yo = 14.7, mo = 25.4. During growth in anaerobic continuous culture with limiting glucose YATP was found to be 10.3 g (dry weight)/mol of adenosine 5'-triphosphate (ATP) and m ATP was 18.9 mmol of ATP/g (dry weight) per h. The aerobic growth yields of cells growing on glucose, glucose plus cAMP, mannitol, and glutamate were consistent with the hypothesis that carbohydrates partially repress oxidative phosphorylation, but the yields of cells growing on glycerol, succinate, acetate, and galactose were all lower than expected. We conclude that, like the efficiency of oxidative phosphorylation, both the maintenance respiration and the amount of ATP necessary to serve maintenance processes are determined by the identity of the growth substrates. Yields smaller than expected may be explained by the absence of respiratory control exerted by phosphate acceptors.

Thiosulfate- and sulfide-dependent pyridine nucleotide reduction and gluconeogenesis in intact Thiobacillus neapolitanus.

Nicotinamide adenine dinucleotide phosphate (reduced form) is formed more rapidly after the addition of thiosulfate to suspensions of intact Thiobacillus neapolitanus in the absence of CO(2) than nicotinamide adenine dinucleotide (reduced form). Measurement of acid-stable metabolites shows this phenomenon to be the result of rapid reoxidation of nicotinamide adenine dinucleotide (reduced form) by 3-phosphoglyceric acid and other oxidized intermediates, which are converted to triose and hexose phosphates, and that, in reality, the rate of nicotinamide adenine dinucleotide (oxidized form) reduction exceeds that of nicotinamide adenine dinucleotide phosphate (oxidized form) by approximately 4.5-fold. The overall rate of pyridine nucleotide reduction by thiosulfate (264 nmol per min per mg of protein) is in excess of that rate needed to sustain growth. Pyridine nucleotide reduction, adenosine triphosphate synthesis, and carbohydrate synthesis are prevented by the uncoupler m-Cl-Carbonylcyanide phenylhydrazone. Sodium amytal inhibits pyridine nucleotide reduction and carbohydrate synthesis are prevented by the uncoupler m-Cl-carbonylcyanide observations are reproduced when sulfide serves as the substrate. The rate of pyridine nucleotide anaerobic reduction with endogenous substrates or thiosulfate is less than 1% of the aerobic rate with thiosulfate. We conclude that the principal, if not the only, pathway of pyridine nucleotide reduction proceeds through an energy-dependent and amytal-sensitive step when either thiosulfate or sulfide is used as the substrate.

The 503-nm pigment of Escherichia coli B: characterization and nutritional conditions affecting its accumulation.

Escherichia coli B was shown to contain a pigment with a single symmetrical absorption band at 503 nm, which cannot be attributed to a cytochrome. The absorption of tetrahydroporphyrin in vitro closely resembled that of P-503 in intact cells. The compounds which rendered P-503 colorless, such as cyanide, azide, hydrazine, thiocyanate, hydroxylamine, dithionite, sulfite, and methylcyanide, also rendered tetrahydroporphyrin colorless. The pigment was present when the cells were grown aerobically or anaerobically in glucose minimal medium, or aerobically in either lactate or succinate minimal medium, but the pigment was not found in cells grown in complex media or in minimal media supplemented with methionine. A model is presented to suggest the involvement of methionine in the conversion of coproporphyrinogen to protoporphyrin. A variety of evidence suggesting that the 503-nm chromophore is in kinetic equilibrium with flavoprotein is discussed. However, it is not a participant in main line respiration, as its rate of reduction upon exhaustion of oxygen was too slow, and the rate of respiration in resting-cell suspensions was independent of P-503 concentration.

Yield coefficients of Thiobacillus neapolitanus in continuous culture.

Thiobacillus neapolitanus, when grown in continuous culture with thiosulfate limiting growth, possessed an apparent maximal molar growth yield of 8.0 g (dry weight) per mole of thiosulfate. The substrate requirement for energy of maintenance was the highest yet reported, amounting to 21.8 mmoles of thiosulfate per g per hr. The molar growth yield, corrected for this maintenance energy requirement, was 13.9 g (dry weight) per mole of thiosulfate. It was concluded that substrate-level phosphorylation during sulfite oxidation accounted for about 45% of the adenosine triphosphate (ATP) requirement for CO(2) assimilation and maintenance during growth on limiting thiosulfate, that three sites of energy conservation exist in the electron-transport chain terminating in oxygen, and that 7.8 moles of ATP are required to fix and assimilate 1 mole of CO(2) into cell material.