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Membrane vesicles derived by osmotic lysis of spheroplasts of Paracoccus denitrificans (ATCC 13543) grown aerobically in continuous culture under conditions of carbon limitation with succinate as sole carbon and energy source accumulate radioactivity in an uncoupler-sensitive respiration-dependent manner when incubated in the presence of [14C]succinate or L-[14C]malate. Membranes prepared from cells grown under conditions of sulphate limitation with succinate as the sole carbon source do not accumulate L-[14C]malate during ascorbate + N,N,N′,N′-tetramethyl-p-phenylene diamine (TMPD) oxidation. The apparent Km for succinate and L-malate uptake is 6.7 and 10 μM respectively with a Vmax for uptake of either substrate being 8.3 nmol/min per milligram of membrane protein. Intravesicular radioactivity was largely confined to C4 dicarboxylic acids, succinate, fumarate, and malate and was freely exchangeable with external unlabeiled C4 dicarboxylic acids but not aspartate. Both ubiquinol1 and ascorbate (+ TMPD) oxidation supported accumulative uptake of succinate or L-malate but NADH did not. Since energization of the dicarboxylate transport system is accomplished by the oxidation of ascorbate + TMPD in the presence of antimycin A, it is concluded that heterotrophically grown P. denitrificans containing cytochrome a3 possess a functional energy-coupling site 3 (terminal energy-transducing region of the respiratory chain) despite claims to the contrary.

The steady-state levels of zinc in Candida utilis yeast grown in continuous culture under conditions of zinc limitation are 2+/mg dry weight of cells. Unlike carbon-limited cells, zinc-depleted cells from a zinc-limited chemostat possess the capacity to accumulate and store zinc at levels far in excess of the steady-state level of 4 nmol/mg dry biomass observed in carbon-limited chemostat cultures. Zinc uptake is energy-dependent and apparently unidirectional since accumulated 65Zn neither exits from preloaded cells nor exchanges with cold Zn2+. The transport system exhibits a high affinity for Zn2+ (Km = 0.36 μM) with a Vmax of 2.2 nmol per minute per milligram dry weight of cells. Growth during the period of the uptake assay is responsible for the apparent plateau level of 35 nmol Zn2+/mg dry weight of cells achieved after 20–30 min in the presence of 65Zn at pH 4.5 and 30 °C. Inhibition of growth during the uptake assay by cycloheximide results in a biphasic linear pattern of zinc accumulation where the cellular zinc is about 60 nmol/mg dry weight after 1 h. The enhanced level of accumulated zinc is not inhibitory to growth. Zinc-depleted C. utilis contains elevated amounts of polyphosphate and this anionic polymer must be considered a possible zinc-sequestering and storage agent. Present experimental evidence does not allow discrimination between possible regulation of zinc homeostasis either by inhibition of zinc efflux through control of the membrane carrier or by control of the synthesis of a cytoplasmic zinc-sequestering macromolecule.

A small-scale chemostat has been used to assess the basic nutritional requirements of curdlan producing strains of Alcaligenes faecalis. A lean, chemically-defined mineral salts medium has been formulated compatible with operation of a continuous culture at dilution rates equivalent to μxax observed in batch-culture. Molar growth yields for carbon, nitrogen, phosphorus and oxygen have been determined. The optimized minimal salts medium with 2.5% glucose and 90mM NH4Cl supported a steady-state nitrogen-limited biomass of 8.9g dry biomass/L at a max. dilution rate of 0.23hr−1. By virtue of its ability to increase μmax, the addition of yeast extract to the chemostat nutrient feed resulted in a significant increase in the max. biomass productivity of the continuous fermenter. Under these conditions curdlan is not synthesized in chemostat culture. Operation of a nitrogen-limited chemostat at Dmax resulted in the complete replacement of the original uracil-requiring mutant (ATCC 21680) by a non uracil-requiring variant strain of A. faecalis. Both the parent auxotroph and apparent revertant strain display similar physiological properties including the capacity to synthesize an extracellular, water-insoluble, neutral β-1,3-glucan polymer (curdlan) under appropriate non-growing conditions.

The biosynthesis of a thermogelable, extracellular homo-β-(1 → 3)-glucan called "curdlan," has been studied in batch and continuous cultures of Alcaligenes faecalis var. myxogenes. Curdlan production is associated with the poststationary phase of a nitrogen-depleted, aerobic batch culture. Exopolymer is not detected in single-stage, carbon-limited continuous cultures but curdlan can be isolated from the effluent of a nitrogen-limited chemostat operating at a dilution rate (D) of −1. A spontaneous variant of strain ATCC 21680 was isolated and found to be compatible with long-term, nitrogen-limited chemostat culture. The specific rate of curdlan production is approximately four times higher in poststationary batch cultures than in single-stage continuous fermentations. The product yield (Yp/s) associated with batch processing (nongrowing cultures) is approximately 0.5 g curdlan/g glucose, with CO2 being the only detectable by-product.

Candida utilis NRRL Y-900 was grown in aerobic continuous culture in a minimal salts medium with sucrose (1% w/v) as the carbon source. Increasing the concentration of zinc in the medium from 2.3 μM to about 30 μM results in an increase in the apparent critical dilution rate from 0.3 to 0.47 h−1, and in the maximum biomass productivity from 1.5 g dry weight per litre per hour (at D = 0.33 h−1) to 2.56 g per litre per hour (at D = 0.45 h−1). The maximum steady-state level of cell-associated zinc (at D = 0.4 h−1) is 4 nmol Zn2+/mg dry weight, during carbon-limited growth, and about 9 nmol Zn2+/mg dry weight when FeCl3 is omitted from the medium. At input zinc concentrations 2+ the culture becomes zinc-limited. Zinc-limitation results in a decrease in the growth yield Ysucrose from 0.54 to 0.38 g cells/g sucrose consumed, and Y0 from 22 to 13 g cells/g-atom O2, suggesting an altered efficiency of energy metabolism. The composition of the culture biomass with respect to protein and RNA content is not affected by zinc limitation.