Glutathione-dependent synthesis of deoxyribonucleotides. Purification and characterization of glutaredoxin from Escherichia coli.

Multiparameter flow cytometric techniques developed in our laboratories have been used for the “at-line” study of fed-batch bacterial fermentations. These fermentations were done at two scales, production (20 m3) and bench (5 × 10−3 m3). In addition, at the bench scale, experiments were undertaken where the difficulty of achieving good mixing (broth homogeneity), similar to that found at the production scale, was simulated by using a two-compartment model. Flow cytometric analysis of cells in broth samples, based on a dual-staining protocol, has revealed, for the first time, that a progressive change in cell physiological state generally occurs throughout the course of such fermentations. The technique has demonstrated that a changing microenvironment with respect to substrate concentration (glucose and dissolved oxygen tension [DOT]) has a profound effect on cell physiology and hence on viable biomass yield. The relatively poorly mixed conditions in the large-scale fermentor were found to lead to a low biomass yield, but, surprisingly, were associated with a high cell viability (with respect to cytoplasmic membrane permeability) throughout the fermentation. The small-scale fermentation that most clearly mimicked the large-scale heterogeneity (i.e., a region of high glucose concentration and low DOT analogous to a feed zone) gave similar results. On the other hand, the small-scale well-mixed fermentation gave the highest biomass yield, but again, surprisingly, the lowest cell viability. The scaled-down simulations with high DOT throughout and locally low or high glucose gave biomass and viabilities between. Reasons for these results are examined in terms of environmental stress associated with an ever-increasing glucose limitation in the well-mixed case. On the other hand, at the large scale, and to differing degrees in scale-down simulations, cells periodically encounter regions of relatively higher glucose concentration. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 70: 381–390, 2000.

Studies related to the scale-up of high-cell-density E. coli fed-batch fermentations using multiparameter flow cytometry: effect of a changing microenvironment with respect to glucose and dissolved oxygen concentration.

Publisher Summary The scale-up of single-celled aerobic microbial fermentation processes is complicated that can lead to unpredictable process performance. However, this is not due to the introduction of fluid dynamic generated stresses (or so-called “shear damage”), whether arising from agitator generated turbulence or bursting bubbles, rather it is because the large-scale fed-batch bioreactor provides a very dynamic environment with large spatial and temporal heterogeneities. Such environmental heterogeneities can induce multiple physiological responses in cells. These responses consume energy and resources such that biomass concentration and product yields can be reduced. These phenomena are not observed in well-mixed homogeneous laboratory-scale reactors where much process development is done and their effects are difficult to model mathematically. Therefore, the ability to obtain data on how a recombinant laboratory process may perform at the large scale, dependent on feeding regime employed or controlling action taken is invaluable for any detailed and informed development program.

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The Scale‐Up of Microbial Batch and Fed‐Batch Fermentation Processes

Oxygen supply is one of the main factors which influences aerobic cell growth in a fermentor. Maximal rates at which E. coli can grow on glucose as carbon source under various limiting oxygen-supply conditions were determined in a bench-scale fermentor. Culture conditions are described which gave yields of about 38 g dry cells per liter medium.

Maximal exponential growth rate and yield of E. coli obtainable in a bench-scale fermentor.

The use of multi-parameter flow cytometry to compare the physiological response of Escherichia coli W3110 to glucose limitation during batch, fed-batch and continuous culture cultivations

In further experiments on the adsorption of n-alkyltrimethylammonium corrosion inhibitors at a dropping mercury electrode in N-potassium chloride solution, the potential applied to the dropping mercury electrode was taken as far as -1.9 volts S.C.E. The electrocapillary and differential capacitance curves for n-dodecyl-and n-hexadecyltrimethylammonium bromides in N-potassium chloride were generally similar to those in N-sulphuric acid, and the calculated surface concentrations of the adsorbed quaternary ammonium ions were almost the same.



The wider range of potential in the present experiments enabled the desorption peaks in the differential capacitance curves for the more dilute solutions to be demonstrated. The stronger solutions gave subsidiary peaks, or steps, which may be associated with the formation of double or multiple layers.



The results of these and previous experiments are explained on the basis that the adsorbed layer of n-alkyltrimethylammonium ions does not completely exclude solvated ions, but hinders their approach to the metal surface.

R. J. Meakins

Papers

The properties of the electrical double layer at a dropping mercury electrode in n-potassium chloride containing n-alkyltrimethylammonium ions