D. W. Tempest

Metabolic and energetic aspects of the growth of Bacillus stearothermophilus in glucose-limited and glucose-sufficient chemostat culture

AbstractWith a glucose-limited chemostat culture of Bacillus stearothermophilus, increasing the incubation temperature progressively from 45°C to 63°C led to a progressive marked increase in the maintenance rates of glucose and oxygen consumption. Hence, at a fixed low dilution rate the yield values with respect to glucose and oxygen decreased substantially with increased temperature. However, the apparent Yglucosemaxand

Metabolic fluxes during the growth of thermotolerant methylotrophic Bacillus strains in methanol-sufficient chemostat cultures

Y_{{\text{O}}_2 }^{\max }

The influence of the culture pH value on the direct glucose oxidative pathway in Klebsiella pneumoniae NCTC 418.

values did not decrease but actually increased with temperature, being highest at 63°C (i.e., close to the maximum growth temperature). With glucose-sufficient cultures growing at a fixed low dilution rate (0.2 h−1) and at their optimum temperature (55°C), glucose and oxygen consumption rates invariably were higher than that of a corresponding glucose-limited culture. Cation (K+ or Mg2+)-limited cultures expressed the highest metabolic rates and with the K+ limited culture this rate was found to be very markedly temperature dependent. As the temperature was increased from 45°C to 63°C the rate of glucose consumption increased 1.8-fold, and that of oxygen consumption by 3.7-fold. The culture pH value also exerted a noticeable effect on the metabolic rate of a glucose-limited culture, particularly at the extremes of pH tolerance (5.5 and 8.5, respectively). A K+-limited culture was less affected with respect to metabolic rate by the culture pH value though the steady state bacterial concentration, and thus the cellular K+ content, changed substantially. These results are discussed in relation to previous findings of the behaviour of this organism in batch culture, and to the behaviour of other thermophilic Bacillus species in chemostat culture.

Fermentation shifts and metabolic reactivity during anaerobic carbon-limited growth of Klebsiella aerogenes NCTC 418 on fructose, gluconate, mannitol and pyruvate

Recently we have isolated a number of thermotolerant, spore-forming methylotrophic bacilli in pure culture. With a methanol-limited chemostat culture of strain Ts1, incremental increases in the incubation temperature from 45°C to 62.5°C revealed an optimum with respect to growth yield of 52.5°C, and a maximum of 62.5°C. Similar investigations revealed a pH optimum of 7.5 and a broad growth rate optimum with respect to growth yield. The organism displayed a low maintenance energy requirement and high growth yield (attained simultaneosly with high growth rates) during growth on methanol. Under all conditions of methanol limitation, substrate was oxidized solely to biomass and CO2 and carbon recoveries greater than 90% were manifest. Our data suggested that this resulted from an ability of the organism to precisely adjust its catabolic and anabolic pathways to suit prevailing growth conditions. These results are discussed in relation to previously reported data on thermophiles in both batch and chemostat culture.

The separate roles of PQQ and apo-enzyme syntheses in the regulation of glucose dehydrogenase activity in Klebsiella pneumoniae NCTC 418.

Growth of the thermotolerant methylotrophic Bacillus strain TS1 in methanol-limited chemostat culture showed that the substrate was oxidized solely to biomass and CO2. When a pulse of methanol was added to the growth vessel anabolism could be shown to be dissociated from catabolism for a transient period of time. Present data shows that when the organism was grown with a limitation other than carbon, some of the substrate was channelled into metabolite over-production. When the organism was grown under N-limitation 2-oxoglutarate accumulated in the culture medium in small amounts whilst acetate accumulated under all carbon excess conditions. Although the average carbon recovery was 92%, analysis of the culture filtrates for other metabolites failed to show significant amounts of any individual product above those detected in carbon-limited growth comditions. The results are discussed in relation to published data.

Overflow metabolism during anaerobic growth of Klebsiella aerogenes NCTC 418 on glycerol and dihydroxyacetone in chemostat culture

The regulation of the PQQ-linked glucose dehydrogenase in different organisms is reviewed. It is concluded that this enzyme functions as an auxiliary energy-generating mechanism, because it is maximally synthesized under conditions of energy stress. It is now definitively established that the oxidation of glucose to gluconate generates metabolically useful energy. The magnitude of the contribution of the oxidation of glucose to gluconate via this enzyme to the growth yield of organisms such asAcinetobacter calcoaceticus is not yet clear.

Growth yield and energy distribution

Klebsiella pneumoniae NCTC 418 was cultured aerobically in chemostat cultures (D=0.3 h-1; 35°C) under respectively carbon-, phosphate-, potassium-, sulphate-, and ammonia-limited conditions with glucose as the sole carbon and energy source. The effect of the external pH value on glucose metabolism and on the enzymes of the direct glucose oxidative pathway was examined. The pH value of the medium had a profound influence on both the activity and the synthesis of the glucose dehydrogenase and the gluconate dehydrogenase. At pH values ranging from pH 5.5 to pH 6.0 maximal activity and synthesis of these enzymes resulted in a more than 80% conversion of the glucose consumed into gluconate and 2-ketogluconate under potassium-or phosphate-limited conditions. On the other hand, no gluconate and/or 2-ketogluconate production could be detected when K. pneumoniae was cultured at pH 8.0. Whereas the synthesis of gluconate dehydrogenase seemingly was completely repressed, still some glucose dehydrogenase was present. The lack of glucose dehydrogenase activity at pH 8.0 was shown not to be due to the dissociation of the cofactor PQQ from the enzyme.

Replacement of potassium ions by ammonium ions in different micro-organisms grown in potassium-limited chemostat culture

Klebsiella aerogenes NCTC 418 was grown in chemostat culture under carbon limitation, with fructose, mannitol, gluconate or pyruvate as the growth-limiting substrates, respectively. It was found that under these conditions the carbon sources were fermented with maximal stoichiometry of ATP generation. The YATP values (g cells per mol ATP generated net) were similar for mannitol- and fructose-limited cultures, but gluconate-limited cultures expressed a value that was 20% lower. From these data it was concluded that gluconate-limited cells invest 0.5 ATP in the uptake of 1 gluconate.If the carbon limitation was instantaneously relieved by addition of a saturating amount of the growth-limiting substrate it was found that the response depended on the nature of the carbon substrate, and in particular on the ability to shift the fermentation pattern towards new products. In fructose- and gluconate-limited cultures the specific uptake rate of the carbon source increased substantially, without a concomitant increase in growth rate, and D-lactate appeared as a new fermentation product, in the case of gluconate accompanied by pyruvate. In contrast, with mannitol- and pyruvate-limited cultures the uptake rate of the carbon source and the fermentation pattern did not change. These results are discussed in connection with the functioning of the methylglyoxal by-pass and its role in sustaining metabolic reactivity.