Kinetics substrate uptake

A weakness of the model discussed above is its reliance on detailed information about C N ratios of bacterial substrates that is currently unavailable. A later study modeled bacterial growth by optimizing electron flow, substrate uptake kinetics, and bacterial C N ratio (VaUino et al., 1996). This study found that the C Ns ratio is not always an accurate predictor of whether bacteria wiU remineralize NH4. ... 

Fatty Acid Transporters. Figure 2 Quencher-based real-time fatty acid uptake assay with a fluorescently labeled FFA analogue (C1-Bodipy-C12). Predominantly protein-mediated fatty acid uptake by 3T3-L1 adipocytes (diamonds) was compared with diffusion-driven uptake by fibroblasts (squares) using the QBT Fatty Acid Uptake reagent (Molecular Devices Corp., CA, USA), which contains C1-Bodipy-C12 as substrate in conjunction with a cell impermeable quencher. Uptake kinetics was recorded using a Gemini fluorescence plate reader. Error bars indicate the standard deviations from 12 independent wells. RFU relative fluorescence units. 

In vitro and ex vivo studies have shown that FATPs transport LCFAs and very long-chain fatty acids (VLCFAs) but no medium-chain fatty acids, fatty acid esters, or lipid-soluble vitamins [4]. LCFA transport is inhibited by prior protease treatment. Synthetic substrates for FATPs include 14C-labeled fatty acids and the fluorescently labeled fatty acid analogue C1 -BODEP Y-Cl 2. Using the latter substrate, differences in fatty acid uptake kinetics between FATP expressing 3T3 LI adipocytes and 3T3 LI fibroblasts, which are devoid of FATPs, can be readily appreciated (Fig. 2). 

Figure 3. Schematic view of the substrate uptake rate versus concentration relationship as described by the whole-cell Michaelis-Menten kinetics. Q is the substrate uptake rate, <2max the biologically determined maximum uptake rate per biomass, c the substrate concentration, and Kj the whole-cell Michaelis constant, i.e. the concentration resulting in 2max/2 (mass of substrate per volume). At c

As shown in Fig. 6B, a two-phase pattern occurred for the substrate uptake. It can be observed that during the exponential growth phase, sucrose assimilation by the bacteria was small, corresponding to about 20% of the initial amount introduced into the medium. However, after a 40-h process corresponding to the end of the growth phase, there was a rise in the substrate uptake, suggesting that the carbon source was directed to biosurfactant production, for the conditions tested. It should be emphasized that the fermentative process, when the medium was supplemented with microsalts and EDTA (Fig. 6A), generated a different substrate kinetics in comparison with that obtained for the nonsupplemented medium (Fig. 6B). 

Generally, the expressions proposed for the kinetics of glucose or glutamine uptake do not differ significantly from each other. Table 8.5 (for formulations) and Table 8.6 (parameters) sum up the kinetic models for the description of substrate uptake rates. 

Laboratory studies have suggested that there are three modes of transport for silicic acid (reviewed by Martin-Jezequel et al., 2000) first, silicic acid may be rapidly transported across the cell membrane, following surge uptake kinetics. This occurs primarily in Si-starved cells with cell quotas (Droop, 1968, 1973) near minimal values. Second, sdicic acid uptake can be controlled internally, presumably due to regulation ofsihcaprecipitation and deposition (e.g., Hildebrand et al., 1997). Third, silicic acid uptake may be controlled externally due to substrate hmitation. 

An interesting alternative, however, is batch culture using cellulose as the feedstock. Because cellulose will only be consumed gradually, the effective value of S is much lower than the actual cellulose concentration, which decreases the rate of cell growth u and, according to Fig. 4, increases. In this system, the substrate uptake rate will depend on the amount of cellulase present. Because of its complexity, the kinetics of fermentation of cellulose will not be developed further here. However, batch culture has been used for cellulase production and is an option to consider in a fermentation design, as the feedstock is inexpensive. Handling and sterilization of the insoluble solid is a concern. 

I hus, with a typical diffusivity of 10 5 cm2s 1, a 3- im cell obtains at the most I 10 20 mol cell 1 s 1 from a substrate at 10-9 M. This rate increases pro-l-i a tionally with (. and R, but the need for substrate increases with R3, of course, i This discussion of enzyme kinetics is applicable in toto to uptake kinetics, mi ilher mechanism by which cells iniluence the chemistry of their surroundings.)... 

Figure 7-3 shows that glucose uptake by erythrocytes and liver cells exhibits kinetics characteristic of a simple enzyme-catalyzed reaction involving a single substrate. The kinetics of transport reactions mediated by other types of proteins are more complicated than for uniporters. Nonetheless, all protein-assisted transport reactions occur faster than allowed by passive diffusion, are substrate-specific as reflected in lower Kjn values for some substrates than others, and exhibit a maximal rate (Vjjjax)-... 

Assuming that oxygen supply is sufficient to avoid local oxygen limitations, the kinetic model required for the simulation includes only the material balance equation for the substrate. As suggested in earfier simulations based on recirculation models (micro-macromixer) by Bajpai and Reuss [60], the uptake kinetics are only considered in the vicinity of the so-called critical sugar concentration. Thus, a rather simple unstructured empirical model is chosen for the purpose of this study. It involves a Monod type of kinetics for substrate uptake... 

The situation is different when simulating the dynamics of the uptake of the carbon and energy source. Here, there is a high risk of failure if the dynamic behavior is predicted with Monod kinetics verified at different snapshot steady states in continuous or fed batch cultures. Application of these kinetics is questionable, because the steady state data of substrate uptake at different dilution rates may be corrupted by induction of different transporter systems depending on the steady state substrate concentrations. In addition to the variability of the affinity of the various transporter systems as clearly demonstrated for the yeast Saccharomyces cerevisiae, we do expect pronounced differences between permeases and phospho-transferase systems because of the clear distinctions in the influence of intracellular metabolites upon the uptake dynamics. 

EMA is a useful metabolic pathway analysis tool for rational strain design and metabolic pathway evolntion of designed strains. EMA is entirely based on the strnctnral analysis of invariant metabolic networks withont reqniring kinetics parameters or experimental flnx data, snch as substrate uptake or secretion rates, which enables the prediction of genetic modifications (e.g. gene deletion and over- or down-expression) to reprogram microbial metabolic pathways and achieve desirable phenotypes. 

Andreasen K, Nielsen PH (1997) Application of microautoradiography to the study of substrate uptake by filamentous microorganisms in activated sludge. Appl Environ Microbiol 63(9) 3662—3668 Arnold FH, Blanch HW, Wilke CR (1985) Analysis of affinity separations II the characterization of affinity columns by pulse techniques. Chem Eng J 30 25-36 Axelrod D, Koppel DE, Schlesinger J, Elson E, Webb WW (1976) Mobility measurement by analysis of fluorescence photobleaching recovery kinetics. Biophys J 16 1055-1069... 

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