Aggregate-formation by Clostridium butyricum

Summary Clostridium butyricum was grown in a glucose-limited chemostat culture at a dilution rate of 0.1 h^–1 at pH 6.0. With 0.9% w/v input glucose in the medium the cells were found to grow in suspension and glucose was fermented completely to acetate and butyrate. An increase in the input concentration of glucose resulted in increased concentrations of end-products, but not all extra glucose was consumed. It could be demonstrated that this was due to a lowering of the maximal growth rate by elevated levels of butyric acid. However, prolonged growth in the presence of high glucose concentrations led to an increase in biomass. This was caused by the selection of a variant that was less sensitive to butyrate. This variant was able to form aggregates in an anaerobic gas-lift reactor at high dilution rates. Inoculation of these aggregates in a conventional chemostat culture with high glucose input resulted in an aggregated culture that remained stable for at least 6 months, and in which all glucose was consumed. Whether the organisms grew in suspension or in aggregates was found to be determined by the concentration of butyrate. The isolation of aggregate-forming variants from chemostat cultures leads to a very simple and new type of immobilization technique.Offprint requests to: G. R. Zoutberg     

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

The biomass concentration extant in potassiumlimited cultures of either Klebsiella pneumoniae or Bacillus stearothermophilus (when growing at a fixed temperature and dilution rate in a glucose/ammonium salts medium) increased progressively as the medium pH value was raised step-wise from 7.0 to 8.5. Because the macromolecular composition of the organisms did not vary significantly, this increase in biomass could not be attributed to an accumulation of storage-type polymers but appeared to reflect a pH-dependent decrease in the cells' minimum K⁺ requirement. Significantly, this effect of pH was not eviden with cultures in which no ammonium salts were present and in which either glutamate or nitrate was added as the sole nitrogen source; however, it was again manifest when various concentrations of NH₄Cl were added to the glutamate-containing medium. This suggested a functional replacement of K⁺ by NH ₄ ⁺ , a proposition consistent with the close similarity of the ionic radii of the potassium ion (1.33 Å) and the ammonium ion (1.43 Å). At pH 8.0, and with a medium containing both glutamate (30 mM) and NH₄Cl (100 mM), cultures of B. stearothermophilus would grow without added potassium at a maximum rate of 0.7 h^-1. Under these conditions the cells contained maximally 0.1% (w/w) potassium (derived from contaminating amounts of this element in the medium constituents), a value which should be compared with one of 1.4% (w/w) for cells growing in a potassiumlimited medium containing initially 0.5 mM K⁺. Qualitatively similar findings were made with cultures of K. pneumoniae; and whereas one may not conclude that NH ₄ ⁺ can totally replace K⁺ in the growth of these bacteria, it can clearly do so very extensively.     

Metabolic and energetic aspects of the growth of Clostridium butyricum on glucose in chemostat culture

The influence of a number of environmental parameters on the fermentation of glucose, and on the energetics of growth of Clostridium butyricum in chemostat culture, have been studied. With cultures that were continuously sparged with nitrogen gas, glucose was fermented primarily to acetate and butyrate with a fixed stoichiometry. Thus, irrespective of the growth rate, input glucose concentration specific nutrient limitation and, within limits, the culture pH value, the acetate/butyrate molar ratio in the culture extracellular fluids was uniformly 0.74±0.07. Thus, the efficiency with which ATP was generated from glucose catabolism also was constant at 3.27±0.02 mol ATP/mol glucose fermented. However, the rate of glucose fermentation at a fixed growth rate, and hence the rate of ATP generation, varied markedly under some conditions leading to changes in the Y _glucose and Y _ATP values. In general, glucose-sufficient cultures expressed lower yield values than a correponding glucose-limited culture, and this was particularly marked with a potassium-limited culture. However, with a glucose-limited culture increasing the input glucose concentration above 40g glucose·l^-1 also led to a significant decrease in the yield values that could be partially reversed by increasing the sparging rate of the nitrogen gas. Finally glucose-limited cultures immediately expressed an increased rate of glucose fermentation when relieved of their growth limitation. Since the rate of cell synthesis did not increase instantaneously, again the yield values with respect to glucose consumed and ATP generated transiently decreased.Two conditions were found to effect a change in the fermentation pattern with a lowering of the acetate/butyrate molar ratio. First, a significant decrease in this ratio was observed when a glucose-limited culture was not sparged with nitrogen gas; and second, a substantial (and progressive) decrease was observed to follow addition of increasing amounts of mannitol to a glucose-limited culture. In both cases, however, there was no apparent change in the Y _ATP value.These results are discussed with respect to two imponder-ables, namely the mechanism(s) by which C. butyricum might partially or totally dissociate catabolism from anabolism, and how it might dispose of the excess reductant [as NAD(P)H] that attends both the formation of acetate from glucose and the fermentation of mannitol. With regards to the latter, evidence is presented that supports the conclusion that the ferredoxin-mediated oxidation of NAD(P)H, generating H₂, is neither coupled to, nor driven by, an energy-yielding reaction.     

Metabolic shift analysis at high cell densities

Abstract: In high cell density cultures it is virtually inevitable that the environment to which the cells are exposed is heterogeneous. Thus, with suspended cultures, individual cells are subject to temporal changes in their environment whereas with aggregated or immobilized cells, the culture can be considered as being formed by a number of subpopulations, each with its own environmental characteristics. In addition, in a high cell density environment, high concentrations of end products may negatively influence the growth rate. This may result in the selection of organisms with an altered metabolic behaviour or with a decreased sensitivity to the adverse effects of the product. We discuss the consequences of this heterogeneity with regard to carbon source metabolism in view of the ability of many bacterial species to adapt to environmental conditions. Selection of variant organisms was found to occur with Clostridium butyricum when grown for a prolonged time in a medium containing approx. I-50 mM glucose. In contrast to the original strain, these variants could sustain a high maximal growth rate in the presence of butyric acid. In addition, they had acquired the capacity to spontaneously form aggregates and were able to carry out a completely solventogenic fermentation. Heterogeneous metabolic activity in aggregated cells is demonstrated with cultures of Lactobacillus laevolacticus , an aggregateforming lactic acid bacterium that converts glucose completely to o-lactate. By using microelectrodes, we show that the fraction of metabolically active cells decreases with increasing aggregate size: in larger aggregates steep pH gradients occur with the effect that only the outer layer of the aggregate is metabolically active, i.e. contributes to lactic acid formation, whereas with smaller aggregates all cells remain active. As a result, the net specific lactic acid production rate of the population as a whole is not invariably increased with increased aggregate size.     

Aerobic 2-ketogluconate metabolism of Klebsiella pneumoniae NCTC 418 grown in chemostat culture.

Klebsiella pneumoniae NCTC 418 is able to convert 2-ketogluconate intracellularly to 6-phosphogluconate by the combined action of an NADPH-dependent 2-ketogluconate reductase and gluconate kinase. Synthesis of the former enzyme was maximal under 2-ketogluconate-limited growth conditions. An instantaneous transition to a 2-ketogluconate-excess condition resulted in an acceleration of catabolism of this carbon source, accompanied by complete inhibition of biosynthesis. It is suggested that the cause of this inhibition resides in depletion of the NADPH pool due to the high rate at which NADPH is oxidized by 2-ketogluconate reductase.     

Quantitative aspects of glucose metabolism by Escherichia coli B/r,grown in the presence of pyrroloquinoline quinone

Escherichia coli B/r was grown in chemostat cultures under various limitations with glucose as carbon source. Since E. coli only synthesized the glucose dehydrogenase (GDH) apo-enzyme and not the appropriate cofactor, pyrroloquinoline quinone (PQQ), no gluconate production could be observed. However, when cell-saturating amounts of PQQ (nmol to mol range) were pulsed into steady state glucose-excess cultures of E. coli, the organisms responded with an instantaneous formation of gluconate and an increased oxygen consumption rate. This showed that reconstitution of GDH in situ was possible.Hence, in order to examine the influence on glucose metabolism of an active GDH, E. coli was grown aerobically in chemostat cultures under various limitations in the presence of PQQ. It was found that the presence of PQQ indeed had a sizable effect: at pH 5.5 under phosphate- or sulphate- limited conditions more than 60% of the glucose consumed was converted to gluconate, which resulted in steady state gluconate concentrations up to 80 mmol/l. The specific rate of gluconate production (0.3–7.6 mmol·h^-1·(g dry wt cells)^-1) was dependent on the growth rate and the nature of the limitation. The production rate of other overflow metabolites such as acetate, pyruvate, and 2-oxoglutarate, was only slightly altered in the presence of PQQ. The fact that the cells were now able to use an active GDH apparently did not affect apo-enzyme synthesis.Abbreviations HEPES N-2-hydroxy-ethylpiperazine-N-2-ethane sulphonic acid - MES 2-morpholinoethane sulphonic acid - PQQ pyrroloquinoline quinone (systematic name: 2,7,9-tricarboxy-1H-pyrrolo-(2,3-f)-quinoline-4,5-dione) - WB Wurster's Blue (systematic name: 1,4-bis-(dimethylamino)-benzene perchlorate     

Influence of growth environment on the phosphoenolpyruvate: Glucose phosphotransferase activities of Escherichia coli and Klebsiella aerogenes: A comparative study

A consistent difference was found between glucose-limited cultures of Escherichia coli and Klebsiella aerogenes strains in the manner which their apparent cellular content of glucose: phosphoenolpyruvate phosphotransferase (glucose-PTS) varied with growth rate. With the former strains, activity increased as a function of growth rate; in the latter it decreased. However, under glucose-sufficient conditions (potassium-or ammonia-limitation) both species behaved similarly; the glucose-PTS activity was lower and bore no obvious relationship to the rate of glucose consumption expressed by the growing culture. These results are discussed in relation to the role of glucose as a regulator of glucose-PTS synthesis, and to the likely contribution which the glucose-PTS makes to the overall rate of glucose uptake, particularly by cells growing in glucose-sufficient environments.Abbreviation Glucose-PTS phosphoenolpyruvate phosphotransferase From May to November 1978 on study leave in the University of Amsterdam     

The role of energy-spilling reactions in the growth of Klebsiella aerogenes NCTC 418 in aerobic chemostat culture.

When cell-saturating amounts of glucose and phosphate were added to steady state cultures of Klebsiella aerogenes that were, respectively, glucose- and phosphate-limited, the organisms responded immediately with an increased oxygen consumption rate. This suggested that in neither case was glucose transport the rate-limiting process, and also that organisms must possess effective mechanisms for spilling the excess energy initially generated when a growth-limitation is temporarily relieved. Steady state cultures of mannitol- or glucose-limited organisms also seemingly generated energy at a greater rate than was required for cell synthesis since gluconate-limited cultures consumed oxygen at a lower rate, at each corresponding growth rate, than did mannitol- or glucose-limited cultures, and therefore expressed a higher YO value. Thus, mannitol- and glucose-limitations must be essentially carbon (and not energy) limitations. The excess energy generated by glucose metabolism is one component of "maintenance" and could be used at lower growth rates to maintain an increased solute gradient across the cell membrane, imposed by the addition of 2%, w/v, NaCl to the growth environment. The maintenance rates of oxygen consumption of K. aerogenes also could be caused to increase by adding glucose discontinuously (drop-wise) to a glucose-limited chemostat culture, or by exchanging nitrate for ammonia as the sole utilizable nitrogen source. The significance of these findings to an assessment of the physiological factors circumscribing energy-spilling reactions in aerobic cultures of K. aerogenes is discussed.     

Production of gluconic acid and 2-ketogluconic acid by Klebsiella aerogenes NCTC 418

2-Ketogluconic acid and, to a lesser extent, gluconic acid were found to be major products of glucose catabolism by phosphate-limited cultures of Klebsiella aerogenes NCTC 418, and together accounted for up to 46% of the glucose carbon that was metabolized.Although the concentrations of both acids increased sub-stantially at low growth rates, their specific rates of synthesis decreased markedly, as did the proportion of glucose converted into these products.Determination of the affinity constant, for glucose, of phosphate-limited organisms showed it to be not significantly different from that of glucose-limited organisms (K _s 50 M), indicative of the phosphotransferase uptake system. And since these organisms possessed an active glucose 6-phosphate dehydrogenase, and had no detectable glucose dehydrogenase activity, it was concluded that gluconic acid and 2-ketogluconic acid arose from their corresponding phosphorylated metabolites, and not directly from glucose.