Limiting Substrate Concentration Research Articles

Seasonal and spatial variations in pelagic community respiration on the southeastern U.S. continental shelf

Pelagic microbial community respiratory rates on the southeastern continental shelf show strong seasonal changes, with summer maxima and winter minima. In nearshore waters <20 m, community respiratory rates of 0.1–3 μM O 2 l −1 h −1 are similar to recent estimates of primary production. In outer shelf water >40 m, strongly influenced by the Gulf Stream, community respiration is 0.1–2.7 μM O 2 l −1 h −1, exceeding primary production in summer. The changes in microbial community respiration are partially explained by changes in bacterial numbers. A related potential cause is the inverse relationship between limiting substrate concentration and temperature, affecting rates of growth and respiration, that has been found to exist for aerobic heterotrophy bacterial isolates in the laboratory. These processes result in shifts between net autotrophy in winter and net heterotrophy in summer for the community as a whole, with little excess organic production remaining for export to the ocean's interior.

Kinetic evaluation of an anaerobic fluidised-bed reactor treating slaughterhouse wastewater

An anaerobic fluidised-bed reactor for purification of slaughterhouse wastewater was modelled as a continuous-flow, completely-mixed homogeneous microbial system, with the feed COD as the limiting-substrate concentration. The average microbial residence time in the reactor was defined in terms of conventional sludge-retention-time. The experimental data obtained indicated that the Michaelis-Menten expression was applicable to a description of substrate utilisation (i.e. COD removal) in the anaerobic fluidised-bed system. The maximum substrate utilisation rate, k, and the Michaelis constant, K s, were determined to be 1·2/day and 0·039 g/l. The observed biomass yield in the reactor decreased with increasing sludge-retention-time. The specific methane production rate observed was a linear function of the specific substrate-utilisation rate.

Kinetics of batch production of single-cell protein from cheese whey

A kinetic model for single-cell protein batch fermentation was developed using the numerical simultaneous integration approach of the fourth-order Runge-Kutta method. The model takes into account the effect of substrate inhibtion, maintenance energy, and cell death on the cell growth and substrate utilization during the fermentation process. The theoretical results obtained from the model compared well with the experimental data. The model was used to study the effect of the initial substrate concentration on the lag period, fermentation time, specific growth rate, population size, and cell productivity of batch fermentation. Increasing the initial substrate concentration increased the lag period and fermentation time and decreased the specific growth rate and cell yield. The growth limiting substrate concentration was 2.9 g/L, whereas the growth inhibiting substrate concentration was 69.0 g/L. Increasing the initial substrate concentration above 150 g/L significantly decreased the yeast population size.

In vitro growth and microcycle conidiation of Idriella bolleyi, a biocontrol agent of cereal pathogens

The minimum nutrient requirements for growth of Idriella (= Microdochium ) bolleyi in shaken liquid culture were mineral salts, nitrate nitrogen, glucose and thiamine. Typical batch culture kinetics were displayed in this medium and were assessed by culture turbidity when sodium alginate was used to induce growth by dispersed mycelia. Conidia were produced during exponential growth and reached maximum numbers ( ca 6 × 10 7 ml −1 ) by the deceleration or early stationary phase. Applying the Monod equation to culture parameters at different glucose concentrations, the minimum doubling time in glucose-nitrate-thiamine medium at 25 °C was calculated as between 5·25 and 6·1 h, and K s (limiting substrate concentration at half maximum specific growth rate) was between 0·049 and 0·077% glucose. Conidial germination was oxygen-dependent but did not require nutrients. On water agar or 0·1% glucose agar most spores germinated by germ-tubes, but up to 15% of spores swelled and produced further conidia (2–5) from one or both poles of the parent spore. This microcycle conidiation was suppressed at higher glucose levels. The findings are discussed in relation to production of biocontrol inocula and efficiency of I. bolleyi in biocontrol on roots.

Exponentially fed-batch cultures as an alternative to chemostats: The case of penicillin acylase production by recombinant E. coli

Exponentially fed-batch cultures (EFBCs), fed with medium containing a highly concentrated carbon source, are commonly employed for attainment of high cell densities. However, large variations in environmental conditions occur, and quasi-steady-state is usually achieved only for the limiting substrate concentration, restricting the use of such cultures in kinetic characterization studies. In this work we report the production of recombinant penicillin acylase (PA) in EFBC of an E. coli JM101 transformed with the pPA102 plasmid, which includes the PA gene under regulation of the lacZ gene promoter and using isopropyl- β-thio-galactopyranoside (IPTG) as inducer. The culture was fed with nonconcentrated complete medium, resulting in the attainment of quasi-steady-state conditions not only in substrate concentration, but also in cell concentration, and in the specific rates of growth, product production, and substrate consumption. Similar transient behavior was observed between EFBC and chemostat results. At quasi-steady-state, the dilution rate in the EFBC equaled the growth rate. Specific PA production rate during the fed-batch phase remained relatively constant at each dilution rate and followed typical Luedeking-Piret kinetics, with growth-associated and non-growth-associated constants of 142 U g dcw −1 and 7.2 U g dcw −1 h −1, respectively. Specific glucose consumption rate linearly increased from 0.025 to 0.6 g g dcw −1 h −1 as the dilution rate increased from 0.01 to 0.35 h −1. The maximum specific PA activity increased with decreasing dilution rate, reaching its highest value of 2.0 U mg −1 at a dilution rate of 0.01 h −1, the lowest dilution tested. Such an activity was 7-fold higher than in batch culture and, to our knowledge, represents the highest value reported in the literature.

Kinetic analysis of the michaelis-menten mechanism in which the substrate and the product are unstable

1. 1. A kinetic analysis of the Michaelis-Menten mechanism for the case in which both the substrate and the product are unstable, either spontaneously or as the result of the addition of a reagent, has been made. 2. 2. The explicit time course equations of the immediate product and the species into which it subsequently is transformed have been derived under the conditions of rapid equilibrium and limiting substrate concentration. 3. 3. The validity of these equations has been checked using numerical simulations. 4. 4. The kinetic data analysis which we suggest is based on the time progress curves of the product or, in the case in which the product accumulation cannot be monitored experimentally, on the time progress curve of the species into which the immediate product is transformed. 5. 5. This analysis allows the determination of the rate and the equilibrium constants if adequate experimental results are available. 6. 6. We have chosen a numerical example, with which we illustrate the procedure of the kinetic data analysis by simulating some curves with assumed experimental errors.

PH-mediated control methods for continuous ethanol fermentation using Zymomonas mobilis

Automated substrate supply by three types of pH-mediated control methods for ethanol fermentation using Zymomonas mobilis was investigated. Method 1 involved continuous substrate feeding with pH control using alkali. The system was stabilized by the growth rate of the microorganisms depending on a limiting substrate concentration; however, the growth rate could not be maintained at the maximum specific growth rate (μ max) value. Method 2 involved pH self-stabilization without alkali. When the system was operated under this method, a stopped-flow situation occurred and the growth rate became substrate-limited due to the extremely low glucose concentration. In Method 3, pH-stat culture with separate inflows of alkali and feed medium was carried out. The rate of medium input in this system was determined by the growth rate of the culture in response to a change of pH and it was possible to culture the microorganism at the maximum growth rate without the risk of washout. In this method, the dilution rate and biomass concentration were influenced by the pH set-points, the control interval time and the ratio of inflow of feed medium to alkali. A small difference between the higher pH set-point limit and the culture pH as well as a short control interval time for a computer-controlled digital set point resulted in a large specific growth rate (μ) and higher ethanol productivity. A setup involving a digital set-point control (DSC) system demonstrated advantages over a setup involving a feedback control loop using an analog signal. Under optimum operation conditions of DSC, high ethanol productivity of over of 11g l −1 h −1 was obtained.

The Rise and Decay of Cell Age Synchrony in the Model of Continuous Culture with McKendrick - Von Foerster Equation

A system including the equation for cell age distribution function, as well as the equation for growth limiting substrate concentration, is considered. Solutions for steady states are found analytically, and those for transient states are obtained by numerical integration. Regularities of cell age synchrony decay, as well as conditions of single-time synchronization of culture, were found.

Estimation of Growth Yield and Maintenance Coefficient of Plant Cell Suspensions

Methodology is presented for the determination of growth yield (Y(g)) and maintenance coefficient (m) for carbon utilization of plant cells grown in suspension culture. Estimation of Y(g) and m requires measurements of specific growth rate (micro) and specific rate of substrate uptake (q) at different growth limiting substrate concentrations. Batch culture of tobacco cells did not permit evaluation of Y(g) and m because micro is constant and maximal during most of the growth cycle. In batch culture, the period of declining specific growth rate is extremely brief because of the rapid transition from logarithmic growth to stationary phase. This occurs because the K(m) for growth is relatively small compared to the initial sucrose concentration. Thus, when the substrate level reaches the K(m), the large mass of cells rapidly depletes the remaining substrate. In contrast, semicontinuous culture facilitates the determination of Y(g) and m because various steady-state growth rates can be achieved. Mathematical expressions were developed to determine the effective values of micro and q over the semicontinuous replacement interval. The validity of this approach was verified by conducting simulations using experimentally determined parameters.

Inactivation of Highly Activated Spinach Leaf Sucrose-Phosphate Synthase by Dephosphorylation

Spinach (Spinacia oleracea L.) leaf sucrose-phosphate synthase (SPS) can be phosphorylated and inactivated in vitro with [gamma-(32)P]ATP (JLA Huber, SC Huber, TH Nielsen [1989] Arch Biochem Biophys 270: 681-690). Thus, it was surprising to find that SPS, extracted from leaves fed mannose in the light to highly activate the enzyme, could be inactivated in an ATP-independent manner when desalted crude extracts were preincubated at 25 degrees C before assay. The "spontaneous" inactivation involved a loss in activity measured with limiting substrate concentrations in the presence of the inhibitor, Pi, without affecting maximum catalytic activity. The spontaneous inactivation was unaffected by exogenous carrier proteins and protease inhibitors, but was inhibited by inorganic phosphate, fluoride, and molybdate, suggesting that a phosphatase may be involved. Okadaic acid, a potent inhibitor of mammalian type 1 and 2A protein phosphatases, had no effect up to 5 micromolar. Inactivation was stimulated about twofold by exogenous Mg(2+) and was relatively insensitive to Ca(2+) and to pH over the range pH 6.5 to 8.5. Radioactive phosphate incorporated into SPS during labeling of excised leaves with [(32)P]Pi (initially in the dark and then in the light with mannose) was lost with time when desalted crude extracts were incubated at 25 degrees C, and the loss in radiolabel was substantially reduced by fluoride. These results provide direct evidence for action of an endogenous phosphatase(s) using SPS as substrate. We postulate that highly activated SPS contains phosphorylated residue(s) that increase activation state, and that spontaneous inactivation occurs by removal of these phosphate group(s). Inactivation of SPS in vivo caused by feeding uncouplers to darkened leaf tissue that had previously been fed mannose in the dark, may occur by this mechanism. However, there is no evidence that this mechanism is involved in light-dark regulation of SPS in vivo.

Role of Sucrose Phosphate Synthase in Sucrose Biosynthesis in Ripening Bananas and Its Relationship to the Respiratory Climacteric

During ripening of bananas (Musa spp. [AAA group, Cavendish subgroup]), there is a massive conversion of starch to sucrose. Also during ripening there is a rise in respiration known as the respiratory climacteric. In this study changes in carbohydrate content, activities of starch and sucrose metabolizing enzymes, and respiration were measured to assess their potential interrelationships. Sucrose phosphate synthase activity increased dramatically during the first 4 days after initiation of ripening by ethylene treatment. Starch concentration decreased and sucrose concentration increased during this time period. Developmental changes in sucrose phosphate synthase activity were measured with limiting substrate (plus Pi) and saturating substrate concentrations. Activities were not parallel under the two assay conditions, providing tentative evidence that kinetically different forms of the enzyme may exist at different stages of ripening. Sucrose accumulation rate was most highly correlated with sucrose phosphate synthase activity assayed with limiting substrate concentrations (plus Pi). The cumulative amount of CO(2) respired during ripening was positively correlated with sugar accumulation (R(2) = 0.97). From this linear regression it was calculated that a constant 0.605 millimoles of CO(2) was evolved per mole of sucrose formed throughout ripening. Using this quantity, the percentage of the total respiratory ATP produced which was required for the conversion of starch to sucrose was calculated assuming different models for carbon export from the amyloplast. The results suggest that sucrose biosynthesis during ripening constitutes a significant sink for respiratory ATP.

Characterization of glucose uptake by Trichuris globulosa (Nematoda) in vitro

14C-glucose uptake by adult Trichuris globulosa is found to be a non-linear function of time and limiting substrate concentration. The uptake is a two component process, an initial rapid burst is followed by a lower steady state, implying a mediated process. The uptake is dependent on Na+ ions which cannot be replaced by K+, Li+ or choline. The uptake is also dependent on pH, being maximal at pH 7.4. 14C-glucose absorption is markedly inhibited by glucose, phlorizin, ouabain and to a smaller extent by a number of monosaccharides and other sugar-phosphates, nucleosides and metabolic inhibitors like p-nitrophenyl phosphate and iodoacetate. The inhibition constant for glucose, phlorizin and ouabain has been found to be 8 mM, 5 mM and 7 mM, respectively. A modified Dixon-plot shows that glucose is a completely competitive inhibitor for 14C-glucose uptake while the nature of competitive inhibition of phlorizin and ouabain are found to be partial.

QUINONE SENSITIZED ELECTRON TRANSFER PHOTOOXIDATION OF NUCLEIC ACIDS: CHEMISTRY OF THYMINE AND THYMIDINE RADICAL CATIONS IN AQUEOUS SOLUTION*

The 2-methyl-1,4-naphthoquinone (MQ) sensitized photooxidation of nucleic acid derivatives has been studied by laser flash photolysis and steady state methods. Thymine and thymidine, as well as other DNA model compounds, quench triplet MQ by electron transfer to give MQ radical anions and pyrimidine or purine radical cations. Although the pyrimidine radical cations cannot be directly observed by flash photolysis, the addition of N,N,N',N'-tetramethyl-1,4-phenylenediamine (TMPD) results in the formation of the TMPD radical cation via scavenging of the pyrimidine radical cation. The photooxidation products for thymine and thymidine are shown to result from subsequent chemical reactions of the radical cations in oxygenated aqueous solution. The quantum yield for substrate loss at limiting substrate concentrations is 0.38 for thymine and 0.66 for thymidine. The chemistry of the radical cations involves hydration by water leading to C(6)-OH adduct radicals of the pyrimidine and deprotonation from the N(1) position in thymine and the C(5) methyl group for thymidine. Superoxide ions produced via quenching of the quinone radical anion with oxygen appear to be involved in the formation of thymine and thymidine hydroperoxides and in the reaction with N(1)-thyminyl radicals to regenerate thymine. The effects of pH were examined in the range pH 5-8 in both the presence and absence of superoxide dismutase. Initial C(6)-OH thymine adducts are suggested to dehydrate to give N(1)-thyminyl radicals.

Effect of thiabendazole and fenbendazole on glucose uptake and carbohydrate metabolism in trichuris globulosa

14C-glucose uptake by adult Trichuris globulosa was found to be a non-linear function of time and limiting substrate concentration. The uptake was a two component process, an initial rapid burst, followed by a lower steady state. Linear transformation of the substrate saturation kinetics by Eadie plot gave a K t value of 5×10 3 μM and J max of 5 μmol mg −1 dry weight min −1. Presence of the benzimidazole drugs, thiabendazole and fenbendazole, markedly inhibited the uptake process at concentrations in which the motility of the parasites was not affected. T. globulosa was found to possess the full complement of enzyme systems of glycolysis, Krebs cycle, phosphatases, transaminases and other NAD- and NADP-dependent enzymes. Thiabendazole and fenbendazole lowered the V max of most of these enzymes and affected the substrate-affinity constant (K m) in some of them. The mode of action of these drugs was discussed in the light of the data obtained.

Structured segregated models and analysis of self-oscillating yeast continuous cultures

A structured segregated model of budding yeast (Saccharomyces cerevisiae) populations is analysed in order to verify its ability to predict the spontaneous oscillations arising in continuous cultures. To obtain tractable and useful information about the relationships among the metabolic modifications during the cell cycle, the control over division and the occurrence of oscillations, very simple assumptions are considered and added to the model. The cell metabolism has been taken into account by assuming a diversification in the yield coefficient during the cell cycle. Moreover, in the oscillatory range, the cell mass is assumed to be constant at budding and to depend on the limiting substrate concentration at division. For a suitable range of parameter values, sustained oscillations are obtained, which can be compared to the experimental ones.

Model for the feedback control system of bacterial growth II. Growth in continuous culture

A mathematical model is developed that describes substrate limited bacterial growth in a continuous culture and that is based upon the conceptual framework elaborated in a previous paper for describing the feedback control system of cell growth [S. Bleecken, (1988). J. theor. Biol. 133, 37.] Central to the theory are the ideas that the limiting substrate is converted into low molecular weight building blocks of macromolecular synthesis which again are converted into biomass (RNA and protein) and that the rates of RNA and protein synthesis are controlled by the intracellular concentration of building blocks. It is shown that a continuous culture can be simulated by two interconnected feedback control systems the actuating signals of which are limiting substrate concentration and the intracellular concentration of building blocks, respectively. Three types of steady-states are found to appear in a continuous culture, besides the well-known stable steady-state of the whole culture there exist two batchlike steady-states of the biotic part of the culture which are metastable. The model is used to analyse the steady-states and their stability properties as well as the dynamic responses of biomass, RNA, protein, building block and substrate concentrations to changes in environmental conditions. Especially the inoculation of a continuous culture and the effects of step changes in dilution rate, inlet substrate concentration and growth temperature are studied in detail. Relations between the growth behaviour of a single cell and that of a continuous culture are derived. The RNA to protein ratio is introduced as a rough measure of the physiological state of cells and it is shown that a cell reacts to environmental changes with a simple pattern of basic responses in growth rate and physiological state. There are reasons to assume that the model presented is the minimal version of a structured model of bacterial growth and represents an optimum compromise between biological relevance and mathematical practicability.

Optimization of fed‐batch penicillin fermentation: A case of singular optimal control with state constraints

The determination of the optimal feeding profile of a fed-batch fermentation requires the solution of a singular optimal control problem that is often complicated by constraints imposed on the state variables. Such constraints can be a maximum allowable biomass concentration beyond which oxygen transfer is severely limited or maximum substrate concentration to avoid side reactions. Due to these state constraints and the singular nature of the problem, complete solutions are especially hard to obtain and almost nonexistent in the literature. In this work the feed concentration is employed as the control variable in a solution approach that first determines the optimal reactor substrate concentration and subsequently solves for the feed concentration profile that results in the optimal reactor profile. Calculations for the fed-batch penicillin fermentation indicate sharp increases in productivity over frequently employed strategies.

Effect of limiting substrate concentration, growth rate and aeration on alginate composition and production by Azotobacter vinelandii in continuous culture

The extracellular production of the acetylated alginate of Azotobacter vinelandii strain E was studied in PO 4 2− limited continuous cultures. The uronate composition and fine structure of the isolated alginates was determined using 1H-n.m.r. Cultures maintained at low phosphate concentrations (0.24 mM) produced less biomass and polysaccharide than those maintained at higher PO 4 2− concentrations (2.0 mM). The ratio of polysaccharide produced:dry cell wt was not influenced by the degree of PO 4 2− limitation. Alginate production rates were not influenced greatly by changes in culture dilution rates but at very low (<0.08/h) and very high (>0.25/h) dilution rates, reduced production was observed. At low (100 ml/min) and high (800 ml/min) aeration rates alginate production was less than at intermediate aeration rates (300–500 ml/min). Alginates with a great diversity of uronate composition and fine structure were isolated from the cultures although no relationship between composition and growth rate or aeration rate was observed. From cultures with 2.0 mM PO 4 2−, alginates with high mannuronate levels (60–70%, with an essentially polymannuronate and heteropolymeric structure) were isolated whilst those from cultures with 0.24 mM PO 4 2− had high guluronate levels (70%, with essentially polyguluronate and heteropolymeric structure). Increasing the dilution rate of cultures (0.05–0.3/h) resulted in the isolation of alginates with increasing degrees of acetylation.

Unsteady-state behaviour of enzyme membrane reactors with substrate rejection

Substrate mass transfer in an immobilized-enzyme membrane reactor was studied assuming Michaelis—Menten reaction kinetics and taking into account a possible partial rejection of the substrate by the membrane. Time and axial coordinate dependence of the solution is fully taken into account in an approximate analytical model relying on techniques already successfully applied to membrane problems. The analysis shows how after some time from the introduction of the substrate in the reactor (start-up time) a steady state is reached in which the substrate concentration at the wall, and with it the reaction rate, varies with the axial coordinate. It either increases or decreases, depending on rejection, over a definite length scale (inlet length), eventually reaching a limiting value. The dependence of the limiting substrate concentration, start-up time and inlet length on the dimensionless parameters governing the system is illustrated in several plots.

Modelling oscillatory behavior in batch fermentations

AbstractA batch fermentation is modelled with constant and variable yeild terms. It is shown that the model cannot exhibit oscillations if a constant yield term is used. Oscillations in the cell concentration, but not in the limiting substrate concentration, can be simulated if a variable yield function of the limiting substrate is used. Conditions, for which the variable yield needs to satisfy in order to generate oscillations in the cell concentration, are discussed. The primary condition is that the yield must become zero at the same time that the specific growth rate does. Experimental results exhibiting oscillatory behavior are presented and compared with the proposed model.