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A striking property of many prokaryotes is their enormous metabolic flexibility with respect not only to catabolic and anabolic substrates but also with respect to the continuously changing availability of nutrients. The phenotypic responses to low-nutrient growth conditions involve structural changes in the cellular make-up, changes in the specific capacity of the enzyme system(s) involved in uptake and/or assimilation of the limiting nutrient and changes in the affinity of these enzymes. Here the responses of some members of the Enterobacteriaceae to potassium-, ammonium- and energy source-limited conditions will be reviewed. The focus will be on the energetic consequences of these adaptations as reflected by the growth yield value for the energy source (Y energy source). It will be illustrated that Y energy source values can be dramatically lowered as a result of incomplete oxidation of the energy source (overflow metabolism), bypassing potential sites of energy conservation (uncoupling) or catabolic cycles that have no other apparent effect than the hydrolysis of ATP (futile cycles). Thus, it is concluded that adaptation to low nutrient conditions aims at maintaining high metabolic fluxes at low nutrient concentrations at the cost of a loss in the energetic efficiency of the overall metabolism.

Streptomyces coelicolor was grown in variously limited chemostat cultures and the specific rate of extracellular actinorhodin production (q(actinorhodin)) was measured. The highest q(actinorhodin) values were observed in glucose- or ammonia-limited cultures, whereas almost no actinorhodin was produced in sulfate-, phosphate-, potassium-, or magnesium-limited cultures. The effect of the dilution rate on actinorhodin production was studied in glucose-limited cultures. It was found that q(actinorhodin) was highest at D = 0.06h(-1), which was well below the maximal D value tested (0.14 h(-1)). This explains why, in batch cultures, actinorhodin production starts at the onset of the stationary phase. It was also found that the use of nitrilotriacetate instead of citrate as a chelating agent had a negative effect on actinorhodin production. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 577-582, 1997.

The presence of the pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase in several strains of different Rhizobium species and of Bradyrhizobium japonicum was investigated. It was found that most strains were only able to synthesize the apo-enzyme, whereas in three strains, Rhizobium meliloti 102F34 and Bradyrhizobium japonicum strains USDA110 and USDA138 holo-enzyme activity could be observed. In washed suspensions, however, only Rhizobium meliloti 102F34 produced gluconate from glucose in the absence of added PQQ. All strains showed positive chemotaxis towards PQQ. This chemotaxis could already be observed with nanomolar concentrations of PQQ.

Enterococcus faecalis was grown in chemostat culture on various energy sources at dilution rates ranging from 0.05 h-1 to 0.5 h-1, under both aerobic and anaerobic conditions. NADH/NAD ratios and total nicotinamide adenine dinucleotide pool size (NAD(H)) were determined. It was found that the NADH/NAD ratio was controlled by the steady state product concentrations rather than by the degree of reduction of the energy source. Highest ratios were observed when NADH was reoxidized via ethanol formation, whereas in aerobic cultures, in which predominantly acetate was produced and oxidation of NADH occurred via the NADH oxidase, ratios were lowest. Addition of ethanol to the medium resulted in an increase of the NADH/NAD ratio, both aerobically and anaerobically. The total amount of NAD(H) was found to be influenced by the culture conditions. Under anaerobic conditions, the NADH oxidation (NAD reduction) rate appeared to correlate with the total amount of nicotinamide nucleotides. In contrast, no effect of the culture conditions on the total amount of NAD(H) was observed in aerobically grown cells.

Size distributions and glucose and pH profiles of aggregates of the d -(-)-lactic acid-producing organism Bacillus laevolacticus were measured. The organisms were grown in continuous culture with a medium glucose concentration of either 280 or 110 mM. A maximal aggregate diameter of 2.2 mm, with a Sauter mean of 1.46 mm, was determined for the former culture condition, whereas aggregates from a culture with 110 mM glucose input had a maximal diameter of 1.9 mm (Sauter mean of 1.07 mm). A pH gradient of approximately 2 U was observed for large aggregates (above 1.5 mm). In smaller aggregates (0.75 mm), the pH value in the interior part was approximately 0.4 U lower than that in the culture fluid. It could be concluded that, in cultures with the high glucose input, lactic acid accumulated within the aggregates to such an extent that metabolism in the central region of the larger aggregates could not proceed further. In these cultures, approximately 90% of the total biomass was active. In aggregates from cultures with a low glucose input, glucose only partly penetrated the larger-sized aggregates, and the activity of this culture was reduced to approximately 70% of the biomass. These aggregates were found to decrease in size after prolonged periods of cultivation. It is suggested that this is caused by glucose depletion in the interior of the aggregates. It is concluded that the availability of glucose is an important factor in determining the size of aggregates of B. laevolacticus.

The stability of several pBR322-derived recombinant plasmids, carrying thethr operon fromEscherichia coli, was investigated in sulfur-limited chemostat cultures ofE. coli GT123. A marked increase in the segregational stability of one of these plasmids was observed. It is concluded that the increased stability was due to the spontaneous insertion of Tn1000 from the chromosome of the host into the plasmid.

Potassium-limited cultures of Candida utilis grown with ammonium chloride as the sole nitrogen source attained a higher dry weight than similar cultures grown with sodium nitrate as the sole nitrogen source. This increase depended on the extracellular ammonia concentration and was not due to accumulation of storage polymers. We conclude that in this yeast ammonium ions can fulfill to some extent the physiological role of potassium ions and that the intracellular concentration of ammonium ions is predominantly determined by the ammonia concentration in the culture medium.

Under anaerobic 2-ketogluconate-limited growth conditions (D = 0.1 h-1), Klebsiella pneumoniae NCTC 418 was found to convert this carbon source to biomass, acetate, formate, CO2, ethanol and succinate. The observed fermentation pattern is in agreement with the simultaneous functioning of the pentose phosphate pathway and the Entner-Doudoroff pathway in 2-ketogluconate catabolism. When cultured at pH 8.0 apparent YATP values were lower than those found at culture pH 6.5. This difference can be explained by assuming that at high culture pH values approximately 0.5 mol ATP was invested in the uptake of 1 mol 2-ketogluconate. Sudden relief of 2-ketogluconate-limited conditions led to lowering of the intracellular NADPH/NADP ratio and (possibly as a result of this) to inhibition of biosynthesis. Whereas production of ethanol stopped, lactate was produced at high rate. This product was formed, at least partly, via the methylglyoxal bypass.

Enterococcus faecalis was grown under anaerobic conditions in chemostat cultures on energy sources with different degress of reduction (i.e. mannitol, glucose, pyruvate) at various culture pH values. Intracellular NADH/NAD ratios were measured and were found to be influenced both by the nature of the energy source and by the culture pH value. Highest ratios were found with mannitol as energy source and with high culture pH values. A role for the redox potential of the NADH/NAD couple as a regulatory effector is suggested by a correlation of the redox potential with the in vivo distribution of the carbon flux between pyruvate formate lyase and the pyruvate dehydrogenase complex.

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 mumol 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.

A new method of d(−)lactic acid production based on the aggregated form of growth of Bacillus laevolacticus in continuous culture in an anaerobic gaslift reactor is presented. With glucose as the substrate a bacterial dry weight of 25 g·1−1 and a lactic acid production rate of 13 g·1−1·h−1 was attained. In conventional glucose-limited chemostat cultures elevated levels of lactic acid stimulated specific lactic acid production while the formation rates of other end-products remained unaffected. In glucose-limited aggregated cultures lactic acid positively influenced the aggregation of cells, improving the volumetric production rate. It is concluded that lactic acid itself is a positive effector in the optimisation of lactic acid production with aggregated cultures of B. laevolacticus.

In this contribution we describe the occurrence of futile cycles in growing bacteria. These cycles are thought to be active when organisms contain two uptake systems for a particular nutrient (one with a high, the other with a low affinity for its substrate). The high-affinity system is responsible for uptake of the nutrient, some of which is subsequently lost to the medium again via leakage through the low-affinity-system. A special futile cycle is caused under some growth conditions by the extremely rapid diffusion of ammonia through bacterial membranes. When the ammonium ion is taken up via active transport, the couple NH 3 NH + 4 will act as an uncoupler. This is aggravated by the chemical similarity of the potassium and the ammonium ion, which leads to ammonium ion transport via the Kdp potassium transport system when the potassium concentration in the medium is low. Other examples of futile cycles, such as those caused by the production of fatty acids by fermentation, are briefly discussed.

Escherichia coli is attracted by pyrroloquinoline quinone (PQQ), and chemotaxis toward glucose is enhanced by the presence of PQQ. A ptsI mutant showed no chemotactic response to either glucose or PQQ alone but did show a chemotactic response to a mixture of glucose and PQQ. A strain lacking the methylated chemotaxis receptor protein Tar showed no response to PQQ.

Summary The growth yield of microbial cultures can be used to estimate the efficiency of energy generation during a fermentation or respiration, in the past, the assessment of this efficiency in organisms carrying out a respiration has been the subject of many heated debates. This has partly been caused by the complexity of microbial respiratory chains. Strains of Escherichia coli specifically modified in their respiratory chain have been used recently to re-evaluate the energetic efficiency of the bacterial respiration using chemostat cultures. The different strains indeed show different growth efficiencies. The physiological significance of energetically less-efficient branches of the respiratory chain is discussed.

The effect of NADH on the activity of the purified pyruvate dehydrogenase complexes (PDHc) of Enterococcus (Ec.) faecalis, Lactococcus lactis, Azotobacter vinelandii and Escherichia coli was determined in vitro. It was found that the PDHc of E. coli and L. lactis was active only at relatively low NADH/NAD ratios, whereas the PDHc of Ec. faecalis was inhibited only at high NADH/NAD ratios. The PDHc of Azotobacter vinelandii showed an intermediate sensitivity. The organisms were grown in chemostat culture under conditions that led to different intracellular NADH/NAD ratios and the PDHc activities in vivo could be calculated from the specific rates of product formation. Under anaerobic growth conditions, only Ec. faecelis expressed PDHc activity in vivo. The activities in vivo of the complexes of the different organisms were in good agreement with their properties determined in vitro. The physiological consequences of these results are discussed.

Summary: The role of the pyruvate dehydrogenase complex (PDC) in the formation of different fermentation products by Enterococcus faecalis was studied. This organism was grown on a semi-defined medium under various conditions in the presence or absence of lipoic acid, an essential cofactor of the enzyme complex. When grown on a medium without added lipoic acid, a very low activity, both in vivo and in vitro, of the PDC was observed. When pyruvate served as the energy source, lipoic acid was found to be essential for growth under anaerobic conditions at low culture pH values. The presence of lipoic acid in the culture medium had a marked effect on the production of acetoin: in the presence of lipoic acid, acetoin was produced only when the intracellular pyruvate concentration was relatively high, whereas in the absence of lipoic acid, acetoin was a common product. Under potassium-limited conditions, lactate was the main product and culture pH significantly affected the bacterial dry weight. After instantaneous addition of lipoic acid to a glucose + pyruvate-limited chemostat culture, an immediate activation of the PDC took place as deduced from the change in fermentation pattern. Reconstitution of the PDC by the addition of lipoic acid was also possible in cell-free extracts, although pre-incubation with ATP and lipoic acid for 90 min was necessary for maximal activation. The effects of an active PDC on product formation and the physiological role of the complex under anaerobic growth conditions are discussed.

When grown in carbon source-limited chemostat cultures with lactate or glucose as the carbon and energy source and xylose as an additional source of reducing equivalents. Pseudomonas putida NCTC 10936 oxidized xylose to xylonolactone and xylonate. No other products were formed from this pentose, nor was it incorporated into biomass. The presence of xylose in these cultures resulted in higher Yglucose and Ylactate values as compared to cultures without xylose indicating that biologically useful energy was conserved during the periplasmic oxidation of xylose. As the Y0 values for growth on glucose or on lactate alone were equal to the Y0 values for growth with xylose as co-substrate, it is concluded that for glucose- or lactate-limited growth energy conservation by PQQH2 oxidation is as efficient as by NADH2 oxidation.

NADH/NAD+ ratios and internal pyruvate concentrations were determined during switches between aerobic and anaerobic steady-state conditions of glucose-limited chemostat cultures of Enterococcus faecalis. During the switch experiments, changes in catabolic fluxes were observed: transition from anaerobic to aerobic conditions resulted in a complete and instantaneous conversion of glucose into acetate and CO2 via the pyruvate dehydrogenase complex, while during a switch from aerobic to anaerobic conditions the culture became homolactic. A similar switch to a homolactic fermentation was observed upon release of the limitation by addition of a glucose pulse to the culture. In sharp contrast to this, a pyruvate pulse resulted in an increase of both pyruvate formate-lyase and pyruvate dehydrogenase complex activity. Furthermore, acetoin was formed during a pyruvate pulse, probably due to a dramatic increase in internal pyruvate concentration. Regulation of the catabolic fluxes over the various pyruvate-catabolizing enzymes is discussed in view of the observed changes in internal pyruvate concentrations and NADH/NAD+ ratios.

In this contribution the isolation and some of the structural and kinetic properties of the pyruvate dehydrogenase complex (PDC) of anaerobically grown Enterococcus faecalis are described. The complex closely resembles the PDC of other Gram-positive bacteria and eukaryotes. It consists of four polypeptide chains with apparent molecular masses on SDS/PAGE of 97, 55, 42 and 36 kDa, and these polypeptides could be assigned to dihydrolipoyl transacetylase (E2), lipoamide dehydrogenase (E3) and the two subunits of pyruvate dehydrogenase (E1 alpha and E1 beta), respectively. The E2 core has an icosahedral symmetry. The apparent molecular mass on SDS/PAGE of 97 kDa of the E2 chain is extremely high in comparison with other Gram-positive organisms (and eukaryotes) and probably due to several lipoyl domains associated with the E2 chain. NADH inhibition is mediated via E3. The mechanism of inhibition is discussed in view of the high PDC activities in vivo that are found in E. faecalis, grown under anaerobic conditions.

Escherichia coli Frag1 was grown under various nutrient limitations in chemostat culture at a fixed temperature, dilution rate and pH both in the presence and the absence of a high concentration of ammonium ions by using either ammonium chloride or DL-alanine as the sole nitrogen source. The presence of high concentrations of ammonium ions in the extracellular fluids of potassium-limited cultures of E. coli Frag1 caused an increase of the specific rate of oxygen consumption of these cultures. In contrast, under phosphate-, sulphate- or magnesium-limited growth conditions no such increase could be observed. The presence of high concentrations of ammonium ions in potassium-limited cultures of E. coli Frag5, a mutant strain of E. coli Frag1 which lacks the high affinity potassium uptake system (Kdp), did not increase the specific rate of oxygen consumption. These results indicate that ammonium ions, very similar to potassium ions both in charge and size, are transported via the Kdp leading to a futile cycle of ammonium ions and ammonia molecules (plus protons) across the cytoplasmic membrane. Both the uptake of ammonium ions and the extrusion of protons would increase the energy requirement of the cells and therefore increase their specific rate of oxygen consumption. The involvement of a (methyl)ammonium transport system in this futile cycle could be excluded.

Enterococcus faecalis NCTC 775 was grown anaerobically in chemostat culture with pyruvate as the energy source. At low culture pH values, high in vivo and in vitro activities were found for both pyruvate dehydrogenase and lactate dehydrogenase. At high culture pH values the carbon flux was shifted towards pyruvate formate lyase. Some mechanisms possibly involved in this metabolic switch are discussed. In particular attention is paid to the NADH/NAD ratio (redox potential) and the fructose-1,6-bisphosphate-dependent lactate dehydrogenase activity as possible regulatory factors.

Magnesium-limited chemostat cultures of Klebsiella pneumoniae NCTC 418 with 20 μM CaCl2 in the medium showed a low rate of gluconate plus 2-ketogluconate production relative to potassium- or phosphate-limited cultures. However, when the medium concentration of CaCl2 was increased to 1 mM, the glucose dehydrogenase (GDH) activities also increased and became similar to those observed in potassium- or phosphate limited cultures. It is concluded that this is due to Mg2+ and Ca2+ ions being involved in the binding of pyrroloquinoline quinone (PQQ) to the GDH apoenzyme. There seems to be an absolute requirement of divalent cations for proper enzyme functioning and in this respect Ca2+ ions could replace Mg2+ ions. The high GDH activity which has been found in cells grown under Mg2−-limited conditions in the presence of higher concentrations of Ca2+ ions, is compatible with the earlier proposal that GDH functions as an auxiliary energy generating system involved in the maintenance of high transmembrane ion gradients., Centro de Investigación y Desarrollo en Fermentaciones Industriales

Cells of Klebsiella pneumoniae NCTC 418 grown at low culture pH values (4.5–5) in a glucose-limited chemostat culture contained elevated levels of glutamate synthase (EC 2.6.1.53). This can be taken as an indication that these cells show the physiology of nitrogen-limited cells, in spite of the fact that high concentrations (about 80 mM) of ammonium ions were present in the culture extracellular fluids. This phenomenon can be explained by the rapid diffusion of ammonia (NH3) through the cell membrane, leading to very low cytoplasmic ammonium (NH4+) and NH3 levels in cells that possess an almost neutral cytoplasmic pH value, but are growing at low culture pH values.

When grown on glucose in K+-limited chemostat culture, or in batch culture with or without 2,4-dinitrophenol, several strains of Escherichia coli (including the type strain) were found to synthesize a quinoprotein glucose dehydrogenase apoenzyme. The pyridine nucleotides, NAD+ and NADP+, would not serve as cofactor, but activity could be demonstrated upon addition of 2,7,9-tricarboxy-1 H-pyrrolo(2,3-f)quinoline-4,5-dione (PQQ). Thus, in the presence of PQQ, but not in its absence, glucose was oxidized to gluconic acid. A mutant of E. coli PC 1000 was isolated that lacked Enzyme I of the phosphoenolpyruvate phosphotransferase system (PTS) but still synthesized the glucose dehydrogenase apoenzyme. Whereas this mutant would not grow on glucose in the absence of PQQ, it would do so in the presence of low concentrations (1 μM) of this cofactor. On the basis of these observations, it is concluded that the protein (apoenzyme) formed is a genuine glucose dehydrogenase, but that it is not functional in growing cells due to their inability to synthesize the appropriate cofactor (PQQ), at least under these conditions.

When cell-saturating amounts of glucose and phosphate were added to steady state cultures ofKlebsiella 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 posses 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 there-fore expressed a higherY o 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 ofK. 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 ofK. aerogenes is discussed.

The biomass concentration extant in potassium-limited 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 evident 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 NH4+, a proposition consistent with the close similarity of the ionic radii of the potassium ion (1.33 A) and the ammonium ion (1.43 A). 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 potassium-limited medium containing initially 0.5 mM K+.(ABSTRACT TRUNCATED AT 250 WORDS)

Klebsiella aerogenes NCTC 418 was grown anaerobically in chemostat culture with glycerol as source of carbon and energy. Glycerol-limited cultures did not ferment the carbon source with maximal efficiency but produced considerable amounts of 1,3-propanediol. The fraction of glycerol converted to this product depended on the growth rate and on the limitation: faster growing cells produced relatively more of this compound. Under glycerol excess conditions the energetic efficiency of fermentation was decreased due to the high 1,3-propanediol excretion rate. Evidence is presented that 1,3-propanediol accumulation exerts a profound effect on the cells' metabolic behaviour.

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.

We report a new pathway for galactose metabolism in Escherichia coli and Salmonella typhimurium. Growth of gal mutants on galactose is restored by the addition of pyrrolo-quinoline quinone (PQQ) to the medium. In such strains galactose is oxidized to galactonate by a PQQ-dependent, membrane-bound dehydrogenase. A pathway for galactonate metabolism in these organisms has already been described.

Like other members of the Enterobacteriaceae, Klebsiel la aerogenes transports glucose into the cell by means of a PEP-dependent phosphotransferase system (PTS; for review see [1]). This system has a high affinity for glucose and, not surprisingly, was found to be exceedingly active in cells of a slowly growing glucose-limited chemostat culture (apparent specific activity > 250 nmo l /min . (mg dry wt cells)-1 at D = 0 .1 /h [2]). Interestingly, the apparent activity of this system declined sharply as the growth rate was increased such that, at growth rates greater than 0.7/h, its activity was lower than that necessary to account for the actual rate of glucose uptake by the growing cells. More significantly, glucose-sufficient cultures (that were K +-, phosphateor ammonia-limited) consumed glucose at substantially higher rates than the corresponding glucose-limited culture, yet seemingly possessed uniformly low glucose-PTS activities. These discrepancies suggested that, at the higher growth rates a n d / o r with glucose-sufficient conditions, ancillary systems might contribute substantially to the overall rate of glucose flux, though such a supposition rested heavily on the assumption that the apparent glucose-PTS activity measured in vitro approximated closely to the actual activity of the undisturbed cells. However, the further observation that some glucose-sufficient cultures excreted gluconic and 2-ketoghiconic acid in substantial amounts [3] again pointed to the possibility of there being present in K. aerogenes processes of glucose catabolism that might not directly involve the glucose-PTS. Hence we undertook a search for this putative alternative glucosemetabolizing system which resulted in the finding of a quinoprotein glucose dehydrogenase (EC 1.1.99.17). To the best of our knowledge, this enzyme has not previously been reported to be present in K. aerogenes, or in related species.

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 substantially at low growth rates, their specific rates of synthesis decreased markedly, ad did the proportion of glucose converted into these products. Determination of the affinity constant, for glucose, of phosphate-limited organisms showed it ot be not significantly different from that of glucose-limited organisms (KS less than or equal to 50 muM), 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-keto-gluconic acid arose from their corresponding phosphorylated metabolites, and not directly from glucose.

Klebsiella pneumoniae NCTC 418 was cultured aerobically in chemostat cultures (D = 0.3 h-1; 35 degrees 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.

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.

Klebsiella aerogenes NCTC 418 was grown in chemostat cultures (D = 0.17 hr-1; pH 6.8;35 degrees C) that were, successively, carbon-, sulphate-, ammonia-, and phosphate-limited, and which contained as the sole carbon-substrate first glucose, then glycerol, mannitol and lactate. Quantitative analyses of carbon-substrate used and products formed allowed carbon balances to be constructed and direct comparisons to be made of the efficiency of substrate utilzation. With all sixteen cultures, carbon recoveries of better than 90% were obtained. Optimum utilization of the carbon substrate was invariably found with the carbon-limited cultures, the sole products being organisms and carbon dioxide. But the extent to which excess substrate was over-utilized varied markedly with both the nature of the growth-limitation and the identity of the carbon-substrate. In general, sulphate-, ammonia-, and phosphate-limited cultures utilized glycerol more efficiently than mannitol, mannitol better than lactate, and glucose least efficiently. Glucose-containing cultures also synthesized some extracellular polysaccharide. When the carbon source was in excess, a range of acidic compounds generally were excreted. Sulphate-limited cultures, growing on glucose, excreted much pyruvate and acetate, whereas similarly-limited cultures growing on glycerol, mannitol or lactate produced only acetate. Ammonia-limited cultures invariably excreted 2-oxoglutarate and acetate, whereas phosphate-limited cultures produced gluconic acid, 2-ketogluconic acid and acetate, when growing on glucose, but only acetate when growing on mannitol or lactate. From the rates of substrate and oxygen consumption, and the rates of cell synthesis, yield values for both substrate and oxygen were calculated. These showed different trends, but were similar in being highest under carbon-limitation and substantially lower under all other limitations. The physiological significance of these findings, and the probable nature of the regulatory mechanisms underlying "overflow metabolism" are discussed.

Department of Microbiology, University of Groningen, Kerklaan 30, 9751 NN Harem ~ Department of Microbiology, University of Amsterdam, Nieuwe Achtergracht 127, 1018 WS Amsterdam," b Department of Microbiology, Free University, De Boelelaan 1087, 1007 MC Amsterdam, and c Department of Biochemistry, University of Amsterdam, Plantage Muidergracht 12, 1018 TV Amsterdam, The Netherlands Received 8 December 1981 Accepted 20 April 1982

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.

The mechanism of granule formation by Selenomonas ruminantium was investigated. A basic protein has been isolated from the lysate of S. ruminantium which triggers cluster formation (small aggregates of 20–100 cells) of suspended cells. Evidence is presented that these basic proteins were of ribosomal origin. It is suggested that ribosomes are released into the culture broth by lysis and that the associated basic proteins are subsequently dissociated by high monovalent cation concentrations. It was found that these positively charged basic proteins interact with the negatively charged lipopolysaccharide of the organism to form the clusters. Adding lysate to suspended cells, followed by lowering of the pH from 5.8 to 4.5 also induced clustering. At dilution rates exceeding the maximum growth rate clusters were retained in anaerobic gas-lift reactors and grew into granules (1–3 mm). It is postulated that granules evolve from clusters. Within the clusters, lysis and a low pH are induced due to diffusion limitations. As a consequence dividing cells are entrapped within the clusters, resulting in growth.

Publisher Summary This chapter discusses the regulation of carbon substrate metabolism in bacteria growing in chemostat culture. Many of the regulatory processes of carbon substrate catabolism best might be rationalized in terms of optimization of rate of ATP generation required to fuel biosynthesis and to promote growth at the maximum permissible rate in any particular environment. To grow, microorganisms require an adequate supply of other essential nutrients such as a utilizable source of nitrogen, phosphorus, sulfur, magnesium, and potassium, each of which frequently might be present only in low sub-saturating concentration in many natural ecosystems. The chapter highlights the processes of microbial adaptation to steady-state nutrient-limited conditions and discusses the response of microbial cells to transient changes in the concentration of limiting nutrient. It also describes the special case of a carbon substrate-limited culture.

SUMMARY: Glucose-limited cultures of Klebsiella aerogenes NCTC 418 (and the supposedly identical strain NCIB 418) possessed a glucose phosphoenolpyruvate (PEP) phosphotransferase activity that varied markedly and progressively with growth rate, from more than 250 nmol min-1 (mg dry wt cells)-1 at D = 0.1 h-1 to less than 100 nmol min-1 (mg dry wt cells)-1 at D = 0.8 h-1. When relieved of the glucose limitation, substrate was used at a rate that bore no precise relationship to the cells’ phosphotransferase activity. Similarly, glucose-sufficient (phosphate- or potassium-limited) cultures metabolized glucose at high rates, whereas the cells possessed only moderate glucose PEP phosphotransferase activities. These results are compared with those reported for glucose-limited cultures of Escherichia coli and for variously limited cultures of K. aerogenes. Glucose-sufficient cultures, as well as glucose-limited cultures that had been temporarily relieved of glucose limitation, excreted partially oxidized products of glucose catabolism in considerable amounts. The relevance of this ‘overflow’ metabolism to studies of glucose transport using [U-14C]glucose is emphasized.

The principal advantages of chemostat culture over batch culture, in studies of microbial physiology, reside in (i) being able to vary environmental parameters independent of each other without invoking concomitant changes in growth rate, (ii) being able to impose upon organisms a range of unique (nutrient-limited) environments, and (iii) being able to establish and maintain steady state conditions, thereby facilitating quantitative and meaningful analyses of cell structure and functioning. Selected examples are presented that illustrate the ways in which each of these properties of the chemostat culture have been exploited.

Carbon-limited chemostat cultures of Klebsiella aerogenes NCTC 418 consumed more oxygen per unit of cell synthesis when growing on mannitol or glycerol than when growing on glucose; and since the "maintenance" requirements were similar, this suggested that the extra reducing equivalents present in these compounds were oxidized wastefully. By comparison with carbon-limited cultures, carbon-sufficient cultures that were ammonia-, sulphate- or phosphate-limited generally consumed considerably more oxygen per unit of cell synthesis, particularly at low growth rates. Thus, according to the theory of Pirt, these carbon-sufficient cultures had a greatly increased "maintenance energy" requirement but nevertheless used the remaining energy with a much increased efficiency compared with carbon-limited cultures. This, we suggest, is a false conclusion which stems from the basic assumption that the maintenance requirement does not change with growth rate.

Progressively increasing the input concentration of growth-limiting nutrient (glucose, ammonia, K+) to anaerobic chemostat cultures of Klebsiella aerogenes (D = 0.38 h-1; 35 degrees C; pH 6.8) led to a non-linear increase in bacterial cell concentration. At modest population densities, residual growth-limiting substrate levels increased substantially, with increasing input concentration, and the culture bacterial dry weight tended to a constant value. With the glucose-limited culture, increasing the glucose input concentration above 20 g X 1(-1) led to accumulation of unused glucose and a change in the fermentation pattern. There was a concomitant lowering of the yield value with respect to glucose consumption, and the calculated YATP value similarly declined. Addition of extra essential (non-limiting) nutrients to the culture was without effect. Similarly, addition of individual fermentation products (acetate, ethanol, D-lactate, 2,3-butanediol, succinate) to the feed medium, in varying concentrations and in different combinations, failed to influence the fermentation pattern or the energetics of cell synthesis. However, a clear correlation was observed between the yield values (of both glucose- and K+-limited cultures) and the steady state concentration of CO2 in the effluent gas. Increasing the concentration CO2 either by increasing the population density or lowering the sparging rate of nitrogen gas through the culture, effected a lowering of the yield values. It is suggested that dissolved CO2 exerts an effect on both metabolism and the energetics of cell synthesis. A possible mechanism of energy dissipation (i.e., a futile cycle) involving carboxylation and decarboxylation reactions is proposed.

A study was undertaken of the microbial composition of aggregates from an acidifying anaerobic gas-lift reactor. For this purpose a simple 100 ml anaerobic gas lift reactor was developed. It was found that the predominant organism in the aggregates was Selenomonas ruminantium. Both, microscopical observations and a newly developed enumeration technique led to the conclusion that the mixed granules consisted mainly of this organism. Grown in pure culture, S. ruminantium was capable of forming aggregates. These aggregates resembled the mixed aggregates both macro- as well as microscopically. Furthermore the fermentation pattern of this pure aggregated culture was similar to that of a mixed aggregated culture.