Citrate efflux in glucose-limited and glucose-sufficient chemostat culture of Penicillium simplicissium

Citrate excretion by Penicillium simplicissimum was investigated in a chemostat. Carbon-limited grown P. simplicissimum did not excrete no citrate. Citrate was excreted, however, when growth was nitrogen-limited. Further effects of nitrogen-limitation were a slightly increased rate of glucose and oxygen consumption. This behaviour is typical for a so-called `overflow metabolism', i.e. the uncoupling of anabolism from catabolism under conditions of carbon excess. Still more citrate was excreted by nitrogen-limited P. simplicissimum when (i) the extracellular osmolarity was increased from 0.2 to 1.5 osm kg^–1 or (ii) when the pH was increased from 4 to 7; or (iii) when the extracellular potassium concentration was lowered from 6 to 0.5 mM. These results were interpreted in terms of a higher energy-consumption under these conditions.     

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.     

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.     

A continuous culture study of the bioenergetic aspects of growth and production of exocellular protease in Bacillus licheniformis

Summary Bacillus licheniformis S 1684 is able to produce an alkaline serine protease exocellularly. In glucose-limited chemostat cultures the specific rate of protease production was maximal at a -value of 0.22. Above this growth rate protease production was repressed. Dependent on 10–20% of the glucose input was used for exocellular product formation. The degree of reduction of exocellular products was 4.1.Maximum molar growth yields were high and indicate a high efficiency of growth. The values of Y _glu ^max and YO ₂ ^max were 83.8 and 53.3, respectively. When Y _glu ^max was corrected for the amount of glucose used for product formation a value of 100.3 was obtained. These high maximum molar growth yields are most probably caused by a high Y _ATP ^max . Anaerobic batch experiments showed a Y _ATP of 14.6.Sometimes the used strain was instable in cell morphology and protease production. Non-protease producing cells most probably develop from producing cells by mutation in the rel-gene. Producing cells most probably are relaxed (rel ^-) and non-producing cells stringent (rel ⁺).Glossary specific growth rate (h^-1) - Y _sub growth yield permol substrate (g biomass/mol) - Y ^max maximum molar growth yield, corrected for maintenance requirements (g biomass/mol) - Y ^max(corr) Y ^max corrected for product formation (g biomass/mol) - m _sub maintenance requirements (mol/g biomass·h) - m _sub(corr) maintenance requirements corrected for product formation (mol/g biomass·h) - Y _c fraction of organic substrate converted in biomass - z fraction of organic substrate converted in exocellular products - d fraction of organic substrate converted in CO₂ (g mol/g atom C) - Crec% carbon recovery % - average degree of reduction of exocellular products - P/O amount of ATP produced during electron-transport of 2 electrons to oxygen     

Bioenergetic aspects of aerobic growth of Klebsiella aerogenes NCTC 418 in carbon-limited and carbon-sufficient chemostat culture

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. Thus we propose an alternative theory which allows for this possibility, and present evidence to show that it may be applicable to both carbon-limited and carbon-sufficient chemostat cultures. Finally we offer an explanation of the high maintenance rate of oxygen consumption found with carbon-sufficient cultures, and consider this phenomenon in relation to the loose coupling between respiration and growth extant in most microbial cultures.     

Plasmid instability in an industrial strain ofBacillus subtilis grown in chemostat culture

Summary A pUB110-derived plasmid/Bacillus subtilis host combination was segregationally unstable when grown in chemostat culture with complex or minimal medium and under starch, glucose or magnesium limitation. The kinetics of plasmid loss were described in terms of the difference in growth rates between plasmid-containing and plasmid-free cells (d) and the rate at which plasmid-free cells were generated from plasmid-containing cells (R). Loss of plasmid-containing cells from the population was d dominated. Changes in medium composition and the nature of growth limitation caused variations in both d and R. The plasmid was most stable in glucose-limited chemostat cultures with minimal medium and least stable under starch limitation with complex complex medium. R and d were smaller for cultures in complex media than those in minimal media. Limitation by starch induced expression of the plasmid-encoded HT amylase gene and was associated with increased values of R and d. Magnesium limitation in minimal medium caused a significant increase in d and a decrease in R.Abbreviations Cm chloramphenicol - Kan kanamycin - Cm^r cells resistant to chloramphenicol (5 mg L^–1) - Kan^r cells resistant to kanamycin (5 mg L^–1) - Cm^sKan^s cells sensitive to chloramphenicol and kanamycin     

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

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

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.When steady state glycerol-limited cultures were instantaneously relieved of the growth limitation a vastly enhanced glycerol uptake rate was observed, accompanied by a shift in the fermentation pattern towards 1,3-propanediol and acetate. This observation was consistent with the extremely high glycerol dehydrogenase activity that was measured in vitro. Some mechanisms that could be responsible for the energy dissipation during this response are discussed.     

An analysis of the growth of Gluconobacter oxydans in chemostat cultures

Gluconobacter oxydans was grown successively in glucose and nitrogen-limited chemostat cultures. Construction of mass balances of organisms growing at increasing dilution rates in glucose-limited cultures, at pH 5.5, revealed a major shift from extensive glucose metabolism via the pentose phosphate pathway to the direct pathway of glucose oxidation yielding gluconic acid. Thus, whereas carbon dioxide production from glucose accounted for 49.4% of the carbon input at a dilution rate (D)=0.05 h^-1, it accounted for only 1.3% at D=0.26 h^-1. This decline in pentose phosphate pathway activity resulted in decreasing molar growth yields on glucose. At dilution rates of 0.05 h^-1 and 0.26 h^-1 molar growth yields of 19.5 g/mol and 3.2 g/mol, respectively, were obtained. Increase of the steady state glucose concentration in nitrogen-limited chemostat cultures maintained at a constant dilution rate also resulted in a decreased flow of carbon through the pentose phosphate pathway. Above a threshold value of 15–20 mM glucose in the culture, pentose phosphate pathway activity almost completely inhibited. In G. oxydans the coupling between energy generation and growth was very inefficient; yield values obtained at various dilution rates varied between 0.8–3.4 g/cells synthesized per 0.5 mol of oxygen consumed.     

Localized insertion of new S-layer during growth of Bacillus stearothermophilus strains

Bacillus stearothermophilus strains PV 72 and ATCC 12980 carry a crystalline surface layer (S-layer) with hexagonal (p6) and oblique (p2) symmetry, respectively. Sites of insertions of new subunits into the regular lattice during cell growth have been determined by the indirect fluorescent antibody technique and the protein A/colloidal gold technique.During S-layer growth on both bacillus strains the following common features were noted: 1. shedding of intact S-layer or turnover of individual subunits was not seen; 2. new S-layer was deposited in helically-arranged bands over the cylindrical surface of the cell at a pitch angle related to the orientation of the lattice vectors of the crystalline array; 3. little or no S-layer was inserted into pre-existing S-layer at the poles, and 4. septal regions and, subsequently, newly formed cell poles were covered with new S-layer protein.     

Growth and physiology of potassium-limited chemostat cultures of Paracoccus denitrificans

In potassium-limited chemostat cultures of Paracoccus denitrificans the maximum specific growth rate (µ_max) was found to depend on the input potassium concentration: At 0.21mM µ_max was 0.10–0.11 h^-1; at 0.44 mM 0.15–0.16 h^-1 and at 0.66 mM 0.20–0.21 h^-1. The plots of the specific rates of oxygen-, succinate-and potassium consumption against gave straight lines. The intracellular potassium concentration was a linear function of and varied from 1% (0.13 M) at a value of 0.034 h^-1 to 2.2% (0.29 M) at =0.26 h^-1; the potassium concentration gradient and the potassium concentration in the culture fluid in the steady state were dependent on the input potassium concentration. The potassium concentration gradient varied from 8,900-1,200. At all values 20–25% of the total energy production was used for potassium transport. 350,100 and 30 ATP molecules were calculated to be required to maintain one potassium ion intracellular during 1 h at values of 0.034, 0.197 and 0.257 h^-1 respectively. It is concluded that the amount of circulation of potassium is dependent on the potassium concentration gradient or on the potassium concentration in the culture in the steady state. The dependency of µ_max on the input potassium concentration was explained by the assumption that at low input potassium concentrations the net uptake of potassium (influx-efflux) is not rapidly enough to maintain the high potassium gradient in the existing cells and to establish it in the newly formed cells. At high values and at high input potassium concentrations µ_max is limited by the specific rate of oxygen consumption, which was found to be 11–12 mmol O₂ g dry weight^-1 h^-1 at µ_max for potassium-, succinate-and sulphate-limited chemostat cultures.     

Calcium uptake and survival of Bacillus stearothermophilus

The calcium transport in resting vegetative cells of Bacillus stearothermophilus was studied by determining the retention of ⁴⁵Ca in a membrane filter assay. The kinetics of death by vegetative cells, when suspended in buffer at 55°C, was also investigated. The calcium influx required the presence of an energy source, e.g. glucose-1-phosphate and the system exhibited saturation kinetics. The requirements for survival of the thermophilic cells reflected those of the calcium transport system. Thus, cells treated with nitrogen gas showed an increased thermal stability and a decreased efflux of calcium. The initial velocity of calcium influx correlated linearly with the survival of the cells after 1 min heating at 55° C. Lanthanum inhibited calcium influx and reduced survival. Magnesium did not inhibit calcium influx but could replace calcium as a stabilizing agent. The results suggest that the thermophilic cells are not intrinsically heat stable but survive due to a high cellular concentration of divalent ions.Abbreviations CFU colony forming units - CPM counts per min - NCA National canners association - CCCP carbonyl cyanide m-chlorophenylhydrazone - PMS phenazine methosulfate     

Glucose catabolism by Thiobacillus A2 grown in chemostat culture under carbon or nitrogen limitation

Thiobacillus A2 was grown in glucose- or ammonium-limited chemostats and relative contributions of the Embden-Meyerhof (EM), Entner-Doudoroff (ED) and pentose phosphate (PP) pathways to glucose catabolism estimated by ¹⁴C-glucose radiorespirometry. In fast growing strain GFI, the EM pathway predominated (41–79%) under all growth conditions with the PP pathway contributing 18–30%. The ED pathway was apparently absent under some conditions of glucose limitation. In contrast, wild type Thiobacillus A2 exhibited predominance of the EM pathway (43–48%) under ammonium-limitation but apparent predominance of the PP pathway (43–55%) under glucose-limitation, although all three pathways were calculated to operate. Under some conditions of glucose limitation the EM pathway was possibly considerably depressed. No clear pattern of response of the three pathways to altered environmental conditions could be deduced, although marked change in pathway activities were obviously induced. Growth yield was apparently unaffected by variation in pathways. The problems of interpreting such complex radiorespirometric data are discussed.Abbreviations EM Embden-Meyerhof - ED Entner-Doudoroff - KDPG 2-keto-3-deoxy-6-phosphogluconate - 6-PG 6-phosphogluconate - PK phosphoketolase - PP pentose phosphate     

Autotrophic growth and inorganic sulphur compound oxidation by Sulfolobus sp. in chemostat culture

Sulfolobus strain LM was grown in tetrathionate and thiosulphate-limited continuous culture. CO₂ limitation resulted in a decrease of the steady-state biomass and an increase in the specific rate of thiosulphate oxidation so that substrate did not accumulate in the medium. The initial step in thiosulphate utilization appeared to be its conversion to tetrathionate. The affinity for tetrathionate oxidation appeared to increase with prolonged continuous culture giving an apparent K _m of about 6 M tetrathionate, a higher affinity than for thiosulphate oxidation and in the same range as values observed with acidophilic, sulphur-oxidizing eubacteria.     

Determination of adenine and pyridine nucleotides in glucose-limited chemostat cultures of Penicillium simplicissimum by one-step ethanol extraction and ion-pairing liquid chromatography

Under specific conditions Penicillium simplicissimum excretes large amounts of organic acids, mainly citrate. As the energetic status of the hyphae might play a role in that respect, we developed a method for the determination of adenine (adenosine triphosphate, adenosine diphosphate, and adenosine monophosphate) and pyridine (nicotinamide adenine dinucleotide and reduced nicotinamide adenine dinucleotide (NADH)) nucleotides in hyphae of P. simplicissimum. An optimum separation of the five compounds in less than 15 min was possible on a C-8 column, utilizing 50 mM aqueous triethylamine-buffer (pH 6.5) and acetonitrile as mobile phase; detection was performed at 254 nm. With the exception of NADH, which could not be determined accurately due to stability problems, the method was sensitive (LOD < or = 0.7 ng on-column), repeatable (sigma(rel) < or = 4.4%), accurate (recovery rates between 97.9 and 104.9%), and precise (intraday variation < or = 9.4%, interday variation < or = 6.2 %). For an optimum extraction of the nucleotides the chemostat samples were directly placed into hot (90 degrees C) 50% ethanol, and shaken for 10 min, followed by evaporation of the solvent and a solid phase extraction cleanup of the redissolved aqueous samples. With this method the nucleotide concentrations in hyphae from a glucose-limited chemostat culture and the respective energy charge were determined. Additionally, the effect of the time lag between sampling and extraction and the effect of a glucose pulse on nucleotide concentrations were determined.     

Regulation of glucose catabolism in Thiobacillus A2 grown in the chemostat under dual limitation by succinate and glucose

In contrast to its diauxic behaviour in batch culture, Thiobacillus A2 grew in chemostat culture using glucose and succinate as dual limiting substrates. Biomass production under dual limitations was the sum of that on single substrates with each substrate being oxidized and assimilated to similar extents in single and dual substrate-limited cultures. In glucose and glucose + succinate-limited cultures glucose was oxidized largely by the Entner-Doudoroff and pentose phosphate pathways, but other mechanisms also contributed and the ratios of pathways depended on substrate ratios and the previous substrate-history of the culture. Variations in specific activities of enzymes of carbohydrate metabolism following switches from single to mixed substrates were considerable, ranging from fourfold for fructose diphosphate aldolase to more than 200-fold for hexokinase, fructose diphosphatase, glucose 6-phosphate and 6-phosphogluconate dehydrogenases. Changes in specific activities occurred only over prolonged time periods in the chemostat, probably reflecting low concentrations of free substrates in carbon-limited cultures and consequent low levels of catabolite repression.     

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

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 acid tolerance response of Bacillus cereus ATCC14579 is dependent on culture pH, growth rate and intracellular pH

The food pathogen Bacillus cereus is likely to encounter acidic environments (i) in food when organic acids are added for preservation purposes, and (ii) during the stomachal transit of aliments. In order to characterise the acid stress response of B. cereus ATCC14579, cells were grown in chemostat at different pH values (pH_o from 9.0 to 5.5) and different growth rates (μ from 0.1 to 0.8 h⁻¹), and were submitted to acid shock at pH 4.0. Cells grown at low pH_o were adapted to acid media and induced a significant acid tolerance response (ATR). The ATR induced was modulated by both pH_o and μ, and the μ effect was more marked at pH_o 5.5. Intracellular pH (pH_i) was affected by both pH_o and μ. At a pH_o above 6, the pH_i decreased with the decrease of pH_o and the increase of μ. At pH_o 5.5, pH_i was higher compared to pH_o 6.0, suggesting that mechanisms of pH_i homeostasis were induced. The acid survival of B. cereus required protein neo-synthesis and the capacity of cells to maintain their pH_i and ΔpH (pH_i - pH_o). Haemolysin BL and non-haemolytic enterotoxin production were both influenced by pH_o and μ.     

Anaerobiosis and low specific growth rates enhance hemolysin BL production by <Emphasis Type="Italic">Bacillus cereus</Emphasis> F4430/73

Bacillus cereus F4430/73 produced the highest levels of hemolysin BL (HBL) when grown under anaerobiosis in MOD medium. Anaerobic cells grown in a chemostat at low specific growth rate (0.1–0.2 h^–1) expressed up to sevenfold more HBL than did cells held at a faster growth rate. At 0.2 h^–1, the presence of 90 mM glucose resulted in inhibition of HBL production. Glucose was found to repress HBL induction at the mRNA level, indicating the potential involvement of catabolite repression in the regulation of HBL. Based on these data, it is suggested that growth rate could be an effector of catabolite regulation of HBL.     

Growth and physiology of Candida utilis NCYC 321 in potassium-limited chemostat culture

When grown in a defined simple salts medium, plus vitamins, Candida utilis displayed an absolute requirement for potassium. But the potassium content of this yeast was exceedingly variable and, with aerobic chemostat cultures (grown at a dilution rate of 0.1 h^-1; 30° C; pH 5.5), was low (< 0.2%, w/w) when they were potassium-limited and high (> 2%, w/2) when glucose-limited. With potassium-limited cultures, the cell-bound potassium content also varied markedly with growth rate, though hardly at all with glucose-limited cultures; magnesium- and phosphate-limited cultures gave intermediate responses.Changes in cell-bound potassium content correlated only weakly with changes in the cellular contents of magnesium, phosphate and RNA, but strongly with changes in both the Y _glucose and Y _O values, indicating an involvement of potassium in the generation of energy by oxidative phosphorylation reactions and/or the utilization of this energy for growth processes.Studies with isolated mitochondria revealed that potassium-limited organisms had a changed content of cytochrome b relative to cytochrome a, and lacked coupling at either site 2 or site 3 of the respiratory chain.These results are discussed in relation to the reported functions of potassium in the growth of micro-organisms, and the organizational differences between prokaryotic and eukaryotic cells.