Stoichiometric and kinetic characterisation of Nitrosomonas sp. in mixed culture by decoupling the growth and energy generation processes

A novel method that relies on the decoupling of the energy production and biosynthesis processes was used to characterise the maintenance, cell lysis and growth processes of Nitrosomonas sp. A Nitrosomonas culture was enriched in a sequencing batch reactor (SBR) with ammonium as the sole energy source. Fluorescent in situ hybridization (FISH) showed that Nitrosomonas bound to the NEU probe constituted 82% of the bacterial population, while no other known ammonium or nitrite oxidizing bacteria were detected. Batch tests were carried out under conditions that both ammonium and CO2 were in excess, and in the absence of one of these two substrates. The oxygen uptake rate and nitrite production rate were measured during these batch tests. The results obtained from these batch tests, along with the SBR performance data, allowed the determination of the maintenance coefficient and the in situ cell lysis rate, as well as the maximum specific growth rate of the Nitrosomonas culture. It is shown that, during normal growth, the Nitrosomonas culture spends approximately 65% of the energy generated for maintenance. The maintenance coefficient was determined to be 0.14-0.16 mgN mgCOD(biomass)(-1)h(-1), and was shown to be independent of the specific growth rate. The in situ lysis rate and the maximum specific growth rate of the Nitrosomonas culture were determined to be 0.26 and 1.0 day(-1) (0.043 h(-1)), respectively, under aerobic conditions at 30 degrees C and pH 7.     

Stoichiometric and kinetic characterisation of Nitrobacter in mixed culture by decoupling the growth and energy generation processes

The growth, maintenance and lysis processes of Nitrobacter were characterised. A Nitrobacter culture was enriched in a sequencing batch reactor (SBR). Fluorescent in situ hybridisation showed that Nitrobacter constituted 73% of the bacterial population. Batch tests were carried out to measure the oxygen uptake rate and/or nitrite consumption rate when both nitrite and CO2 were in excess, and in the absence of either of these two substrates. The results obtained, along with the SBR performance data, allowed the determination of the maintenance coefficient and in situ cell lysis rate of Nitrobacter. Nitrobacter spends a significant amount of energy for maintenance, which varies considerably with the specific growth rate. At maximum growth, Nitrobacter consume nitrite at a rate of 0.042 mgN/mgCOD(biomass) . h for maintenance purposes, which increases more than threefold to 0.143 mgN/mgCOD(biomass) . h in the absence of growth. In the SBR, where Nitrobacter grew at 40% of its maximum growth rate, a maintenance coefficient of 0.113 mgN/mgCOD . h was found, resulting in 42% of the total amount of nitrite being consumed for maintenance. The above three maintenance coefficient values obtained at different growth rates appear to support the maintenance model proposed in Pirt (1982). The in situ lysis rate of Nitrobacter was determined to be 0.07/day under aerobic conditions at 22 degrees C and pH 7.3. Further, the maximum specific growth rate of Nitrobacter was estimated to be 0.02/h (0.48/day). The affinity constant of Nitrobacter with respect to nitrite was determined to be 1.50 mgNO2(-)-N/L, independent of the presence or absence of CO2.     

Respiratory energy costs for the maintenance of biomass, for growth and for ion uptake in roots of Carex diandra and Carex acudformis

van der Werf, A., Kooijman, A., Welschen, R. and Lambers, H. 1988. Respiratory energy costs for the maintenance of biomass, for growth and for ion uptake in roots of Carex diandra and Carex acutiformis. - Physiol. Plant. 72: 483–491. The respiratory characteristics of the roots of Carex diandra Schrank and Carex acutiformis Ehrh. were investigated. The aims were, firstly to determine the respiratory energy costs for the maintenance of root biomass, for root growth and for ion uptake, and secondly to explain the higher rate of root respiration and ATP production in C. diandra. The three respiratory energy components were derived from a multiple regression analysis, using the relative growth rate and the net rate of nitrate uptake as independent variables and the rate of ATP production as a dependent variable. Although the rate of root respiration and ATP production was significantly higher in C. diandra than in C. acutiformis, the two species showed no significant difference in their rate of ATP production for the maintenance of biomass, in the respiratory energy coefficient for growth (the amount of ATP production per unit of biomass produced) and the respiratory energy coefficient for ion uptake (amount of ATP production per unit of ions absorbed). It is concluded that the higher rate of root respiration of C. diandra is caused by a higher rate of nitrate uptake. At relatively high rates of growth and nitrate uptake, the contribution of the rate of ATP production for ion uptake to the total rate of ATP production amounted to 38 and 25% for C. diandra and C. acutiformis, respectively. At this growth rate, the respiratory energy production for growth contributed 37 and 50%, respectively, to the total rate of ATP production. The relative contribution of the rate of ATP production for the maintenance of biomass increased from 25 to 70% with increasing plant age for both species. The results suggest that ion uptake is one of the major sinks for respiratory energy in roots. These experimentally derived values for the rate of ATP production for the maintenance of biomass, the respiratory energy coefficient for growth and the respiratory energy coefficient for ion uptake are discussed in relation to other experimentally and theoretically derived values.     

A method for the determination of confidence limits for the P/2e − ratio for chosen values of Y ATP max from the results of continuous culture experiments

A model is described, which allows the determination of 95% confidence limits for the maintenance coefficient and the efficiency of oxidative phosphorylation for chosen values of the growth yield for ATP corrected for energy maintenance (Y _ATP ^max ). As experimental data the specific rates of substrate consumption, product formation and oxygen uptake in chemostat cultures at various growth rates are used.     

Determination of the energy maintenance coefficient ofZymomonas mobilis

Summary Continuous culture experiments withZymomonas mobilis Z-1-81, indicated the existence of linearity between specific substrate uptake rates and dilution rates. The value of the energy maintenance coefficient was estimated at 2,521 g/g.h. The percentage of carbon used for maintenance energy was shown to increase with the decrease in the dilution rate.     

Cellular cycling of substrate as a possible cryptic way for energy spilling in suspended cellular continuous cultures

Mass balances analysis of polyphasic dispersed systems showed that cellular cycling of the substrate involves (a) an increase in the specific metabolisation rate and (b) a decrease in the biomass yield coefficient (energy spilling). In biomass calculations, the substrate release rate can replace the maintenance coefficient concept. Thus, decreasing the substrate transfer rates could improve cells or microorganisms strains of industrial importance.     

Effect of external pH on yield,morphology and cell components ofCandida utilis growing in continuous culture

The physiological state ofCandida utilis growing in continuous culture was controlled by external pH and dilution rate. The pH of the medium influences the physiological mechanisms coupled with the maintenance of living functions. The consumption of nitrogen source for biomass formation is a linear function of growth rate, independent of external pH. The statistical distribution of cell volumes was identified as log-normal where the dispersion is indirectly proportional to the maintenance coefficient and the mean of the logarithm of cell volume is a linear combination of specific growth rate and maintenance coefficient. The content of RNA, protein, glucan and mannan in dry biomass may also be expressed as a simple function of specific growth rate and maintenance coefficient.     

On material balance models and maintenance coefficients in CSTRs with various extents of biomass recycle

This work considers the consumption of substrate as needed to maintain cell populations in systems that are closed or partially closed with respect to biomass. The maintenance coefficient is divided between mass and energy maintenance terms. Analysis of data on anaerobic degradation of glucose to lactic acid and ethanol shows the mass maintenance term to be negligibly small. The energy maintenance term is large and variable. It is speculated that the energy is consumed in a futile cycle and is not directly related to cell viability.     

Effect of nutrient limitation on product formation during continuous fermentation of xylose with <Emphasis Type="Italic">Thermoanaerobacter ethanolicus</Emphasis> JW200 Fe(7)

Thermoanaerobacter ethanolicus JW200 Fe(7) was grown in continuous culture, using xylose as the primary carbon source, with progressively lower concentrations of supplementary yeast extract. This enabled the comparison of metabolic flux to fermentation end-products under carbon-limited and carbon-sufficient (yeast extract-limited) conditions and the determination of process data under fully mass-balanced conditions. Under carbon-limitation, the specific ethanol-formation rate was described by q (p)=40.34 micro +3.74, the specific rate of substrate utilisation for maintenance was 0.31+/-0.02 g x g(-1) x h(-1) and the maximum cell yield on xylose, corrected for maintenance requirements, was 0.15+/-0.04 g x g(-1). Based on the product profiles, these corresponded to a maintenance coefficient of m(ATP)=4.1+/-0.5 mmol x g(-1) x h(-1) and a maximum cell yield of = 14.7+/-0.8 x g x mol(-1). Limitation by a component in yeast extract resulted in incomplete xylose utilisation, increased catabolic flux rates (primarily resulting in increased lactate production, due to limitations in the flux through the phosphoroclastic reaction), a reduction in cell yield = 10.0+/-1.0 g x mol(-1) and an increase in maintenance energy requirements of m(ATP)=7.95+/-0.7 mmol x g(-1). The latter was also reflected in a shift from ethanol to acetate production at lower growth rates. An analysis of ethanol and acetate tolerance indicated that any high-intensity process employing this strain would require a bioreactor design which incorporated continuous ethanol stripping.     

Fermentation products, amino acid utilization, maintenance energies and growth yields for the fibrillar Streptococcus salivarius HB and a non-fibrillar mutant HB-B grown in continuous culture under glucose limitation

The fibrillar strain Streptococcus salivarius HB and a non-fibrillar mutant, strain HB-B, were grown in a defined medium under glucose limitation in a chemostat. Fermentation balances were produced for both strains in batch culture and at growth rates between 0.1/h and 1.1/h. In batch culture both strains fermented glucose to lactate, but in continuous culture glucose was fermented to formate, acetate and ethanol with increasing amounts of lactate as the growth rate was increased. Lactate never became the major fermentation product even at the highest growth rate. Amino acid analysis showed that only lysine was more than 50% utilized, while proline and tyrosine showed net production. The non-fibrillar strain HB-B showed, in general, a reduced utilization of amino acids compared with the fibrillar strain HB. Calculated growth yields and maintenance energies for the two strains showed that there was a reduction in the true growth yield and the maintenance energy coefficient of the non-fibrillar strain HB-B when compared with the fibrillar strain HB. The increase in the maintenance energy of the fibrillar strain HB (1.382 mmol/g/h) when compared with the non-fibrillar strain HB-B (0.546 mmol/g/h) of 153% is proposed to be the energy required for the maintenance of the fibrillar surface of the cell.     

Fermentation products, amino acid utilization, maintenance energies and growth yields for the fibrillar Streptococcus salivarius HB and a non-fibrillar mutant HB-B grown in continuous culture under glucose limitation

The fibrillar strain Streptococcus salivarius HB and a non-fibrillar mutant, strain HB-B, were grown in a defined medium under glucose limitation in a chemostat. Fermentation balances were produced for both strains in batch culture and at growth rates between 0.1/h and 1.1/h. In batch culture both strains fermented glucose to lactate, but in continuous culture glucose was fermented to formate, acetate and ethanol with increasing amounts of lactate as the growth rate was increased. Lactate never became the major fermentation product even at the highest growth rate. Amino acid analysis showed that only lysine was more than 50% utilized, while proline and tyrosine showed net production. The non-fibrillar strain HB-B showed, in general, a reduced utilization of amino acids compared with the fibrillar strain HB. Calculated growth yields and maintenance energies for the two strains showed that there was a reduction in the true growth yield and the maintenance energy coefficient of the non-fibrillar strain HB-B when compared with the fibrillar strain HB. The increase in the maintenance energy of the fibrillar strain HB (1.382 mmol/g/h) when compared with the non-fibrillar strain HB-B (0.546 mmol/g/h) of 153% is proposed to be the energy required for the maintenance of the fibrillar surface of the cell.     

Relationship between substrate inhibition and maintenance energy ofChlamydomonas reinhardtii in heterotrophic culture

The relationship between substrate inhibition and maintenance energy ofChlamydomonas reinhardtii grown heterotrophically on acetate was investigated. At low acetate concentrations (<0.4 g l^–1), where no inhibition of cell growth was observed, the cell growth yield and specific growth rate could be represented by the Pirt model, 1/Y=1/Y _g +m/ with a constant value of maintenance energy coefficient m. However, at high acetate concentrations (>0.4 g l^–1), inhibition of cell growth occurred, in which m became variable and dependent on the acetate concentration. A simple mathematical model was proposed to predict the actual maintenance energy coefficient m in the inhibited cultures and experimentally validated.Author for correspondence     

Muscle contraction: energy rate equations in relation to efficiency and step-size distance

We derive the energy rate equation for muscle contraction. Our equation has only two parameters m, the maintenance heat rate and 1/S, the shortening heat coefficient. The impulsive model (previously described in earlier papers) provides a physical basis for parameter 1/S as well as for constants a and b in Hill’s force–velocity equation. We develop new theory and relate the efficiency and the step-size distance to our energy rate equation. Correlation between the efficiency and the step-size distance is established. The various numbers are listed in Table 1: we use data from five different muscles in the literature. In summary, our analysis strongly supports the impulsive model as the correct model of contraction.     

Energetics of growth of a defined mixed culture of Desulfovibrio vulgaris and Methanosarcina barkeri: maintenance energy coefficient of the sulfate-reducing organism in the absence and presence of its partner

The maintenance energy coefficient of Desulfovibrio vulgaris was studied by using a chemostat, with Methanosarcina barkeri or sulfate as the electron acceptor; lithium lactate or sodium pyruvate served as the electron donor. The experiments showed that the growth energetics of D. vulgaris or M. barkeri were greatly affected by maintenance energy coefficients. When D. vulgaris grew on lactate or pyruvate medium with sulfate, these coefficients reached 4.40 and 2.80 mM g-1 h-1, respectively; on lactate medium in the presence of M. barkeri the same coefficient reached a value of 2.90 mM g-1 h-1. Results also showed that the increase of the value of the maintenance energy coefficient corresponded to a decrease of the biomass produced. D. vulgaris maximal growth yield values calculated by use of the Pirt equation were slightly higher with M. barkeri (maximal growth yield, 10 g/mol) than with sulfate (maximal growth yield, 7.5 g/mol). This finding could be interpreted by reference to the ATP-generating reactions involved in D. vulgaris growth in the presence of sulfate or M. barkeri.     

Relationship Between Energy Substrate Utilization and Specific Growth Rate in Aspergillus nidulans

The maintenance coefficient of glucose-limited Aspergillus nidulans chemostat cultures at 30 C was 0.018 g per g (dry weight) per hr for glucose and 0.55 mmoles per g (dry weight) per hr for oxygen. These values can only be approximate because melanin was produced by the mold at low growth rates and because it is unlikely that this polymer contributed to the maintenance energy requirement although it contributed to the dry weight. Biomass (defined here as dry weight minus melanin) was used to calculate a more meaningful maintenance coefficient for glucose (0.029 g of glucose per g of biomass per hr). At the highest growth rates examined, a nonlinear relationship between growth rate and glucose utilization rate was obtained, suggesting a qualitative change in the metabolic activities of the mold at high growth rates. The oxidative capacity of the mold was highest at the highest growth rates. This observation indicates that the increased substrate utilization rate observed at the higher growth rates is a reflection of enhanced enzyme synthesis. This hypothesis was verified by assaying the specific activities of several enzymes at different growth rates. However, in contrast to all the other enzymes assayed, the activities of reduced nicotinamide adenine dinucleotide phosphate: (acceptor) oxido-reductases were highest at the lowest growth rates.     

Cultivation of Saccharomyces cerevisiae in continuous culture

Summary Glucose limited growth of a respiratory deficient mutant of Saccharomyces cerevisiae was studied in continuous culture under steady state conditions. The maximal growth rate, the Michaelis constant, the cell yield, the maintenance coefficient and the ethanol yield of the growing cell population were determined. The steady state concentrations of cells, glucose and ethanol were measured as functions of the dilution rate and compared with theoretical predictions. A far-reaching agreement between theory and experiment was observed. The decrease of the cell yield in the range of low dilution rates is well explained by introducing the concept of maintenance energy in the general theory of continuous cultures. A deviation of the cell yield from the predicted values, which has been found in the range of high dilution rates, is discussed.     

Effect of malic acid on the growth kinetics of<Emphasis Type="Italic"> Lactobacillus plantarum</Emphasis>

The fermentation kinetics of Lactobacillus plantarum were studied in a specially designed broth formulated from commercially available, dehydrated components (yeast extract, trypticase, ammonium sulfate) in batch and continuous culture. During batch growth in the absence of malic acid, the specific growth rate was 0.20 h^–1. Malic acid in the medium, at 2 mM or 10 mM, increased the specific growth rate of L. plantarum to 0.34 h^–1. An increase in the maximum cell yield due to malic acid also was observed. Malic acid in the medium (12 mM) reduced the non-growth-associated (maintenance energy) coefficient and increased the biomass yield in continuous culture, based on calculations from the Luedeking and Piret model. The biomass yield coefficient was estimated as 27.4 mg or 34.3 mg cells mmol^–1 hexose in the absence or presence of malic acid, respectively. The maintenance coefficient was estimated as 3.5 mmol or 1.5 mmol hexose mg^–1 cell h^–1 in the absence or presence of malic acid. These results clearly demonstrate the energy-sparing effect of malic acid on the growth- and non-growth-associated energy requirements for L. plantarum. The quantitative energy-sparing effect of malic acid on L. plantarum has heretofore not been reported, to our knowledge.     

The effects of reproductive state on digestive efficiency in three sympatric bat species of the same guild

The functional link between food as an energy source and metabolizable energy is the digestive tract. The digestive organs may change in size, structure, or retention time in response to energetic demands of the animal. Very efficient digestive tracts may be better at processing food but require higher energetic investments for maintenance even when post-absorptive. These costs influence the resting metabolic rate (RMR) that is defined as the energy necessary to fuel vital metabolic functions in a resting animal. In bats a trade-off between the necessity for a highly efficient digestive tract and moderate energetic maintenance costs may be particularly important. We hypothesized that low RMR coincides with low digestive efficiency (defined as apparent metabolizable energy coefficient (MEC)) and that phases of increased energetic demand are compensated for by increased digestive efficiency. We measured RMR and apparent MEC in the bats species Myotis nattereri, M. bechsteinii, and Plecotus auritus. In support of our hypothesis, M. nattereri has the lowest mass-specific RMR of the three species and the lowest apparent MEC. However, apparent MEC did not change during phases with differing energetic demands in any of the bat species, probably because bats operate at the limit of their sustainable energy demand.     

Assessment of macroenergetic parameters for an anaerobic upflow biomass bed and filter (UBF) reactor

This article reports the steady-state performance of two hybrid anaerobic digesters treating soluble synthetic sugar wastes of 1 and 0.5% strength and the assessment of the associated macroenergetic parameters (growth yield, so-called maintenance coefficient). A theoretical development shows a "nongrowth" parameter concept to be more appropriate than maintenance or decay. Combined energy and mass balances are used to develop a model for growth rate which compares well with experimental data. The COD removal efficiency had no significant effect on growth yield and the maintenance parameter, although a dual combined balance indicated the possibility of such an effect. Macroenergetic parameters did not vary significantly with the specific feeding rate of the system. We thus conclude that a single model may be used over a broad range of feeding and performance conditions.     

Growth kinetics of Apple plant cell cultures

Summary Aerobic batch cultures of Apple fruit cells were carried out using sucrose as the sole carbon source. The determination of the growth parameters was used in order to estimate the maintenance coefficient m and the maximum growth yield Y_G; their values at 25°C are respectively 0.32. 10^-2 g sugar/g cell dry weight.h and 0.57 g cell dry weight/g sugar. Then, a mathematical expression of the cell growth is discussed, with respect to the specific growth rate evolution during cultivation.