Scale-down model to simulate spatial pH variations in large-scale bioreactors.

For the first time a laboratory-scale two-compartment system was used to investigate the effects of pH fluctuations consequent to large scales of operation on microorganisms. pH fluctuations can develop in production-scale fermenters as a consequence of the combined effects of poor mixing and adding concentrated reagents at the liquid surface for control of the bulk pH. Bacillus subtilis was used as a model culture since in addition to its sensitivity to dissolved oxygen levels, the production of the metabolites, acetoin and 2,3-butanediol, is sensitive to pH values between 6.5 and 7.2. The scale-down model consisted of a stirred tank reactor (STR) and a recycle loop containing a plug flow reactor (PFR), with the pH in the stirred tank being maintained at 6.5 by addition of alkali in the loop. Different residence times in the loop simulated the exposure time of fluid elements to high values of pH in the vicinity of the addition point in large bioreactors and tracer experiments were performed to characterise the residence time distribution in it. Since the culture was sensitive to dissolved oxygen, for each experiment with pH control by adding base into the PFR, equivalent experiments were conducted with pH control by addition of base into the STR, thus ensuring that any dissolved oxygen effects were common to both types of experiments. The present study indicates that although biomass concentration remained unaffected by pH variations, product formation was influenced by residence times in the PFR of 60 sec or longer. These changes in metabolism are thought to be linked to both the sensitivity of the acetoin and 2,3-butanediol-forming enzymes to pH and to the inducing effects of dissociated acetate on the acetolactate synthase enzyme.

Using the Microcyte flow cytometer to monitor cell number, viability, and apoptosis in mammalian cell culture.

The Microcyte is a novel, portable flow cytometer based on diode laser technology whose use has been established for yeast and bacterial analysis. We present data that demonstrate its suitability for routine mammalian cell counting and viability determination. To extend its range of applications in the field of animal cell culture biotechnology, a test to determine the number of apoptotic cells present has been developed for use with the instrument. Apoptosis was induced in hybridoma cell cultures by treatment with camptothecin. Apoptotic cells were labeled with biotinylated Annexin V and then visualized using a streptavidin-allophycocyanin conjugate. Their numbers were counted, and the cell size of the apoptotic cell population was determined using the Microcyte.

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.

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 m(3)) and bench (5 x 10(-3) m(3)). 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.

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.

Multi-parameter flow cytometric techniques have been developed for the 'at-line' study of bacterial cultivations. Using a mixture of specific fluorescent stains it is possible to resolve an individual cells physiological state beyond culturability, based on the presence or absence of an intact polarised cytoplasmic membrane, enabling assessment of population heterogeneity. It has been shown that during the latter stages of small-scale (5 l), well mixed fed-batch cultivations there is a considerable drop in cell viability, about 17%, as characterised by cytoplasmic membrane depolarisation and permeability. These phenomena are thought to be due to the severe and steadily increasing stress associated with glucose limitation at high cell densities, during the fed-batch process. Such effects were not found in either batch or continuous culture cultivations. The possibility of using these findings for improved process control using 'on-line' flow cytometry are discussed.

Growth and enumeration of the meat spoilage bacterium Brochothrix thermosphacta.

Brochothrix thermosphacta is a common meat spoilage bacterium. The morphology of this bacterium changes from coccobacilli and short rods to chains during growth, which may give a false estimation in numbers using some enumeration techniques. Methods for the quantification of this bacterium have been compared. Turbidimetric readings showed good agreement with cell dry weight indicating that the former provides a good measure of the change in cell mass during growth. The turbidimetric method also correlated well with bacterial numbers determined by plate counts, flow cytometry and manual counts (by microscope) over a limited range of 10(7)-10(9) cells/ml. Flow cytometry and manual counts gave a linear relationship over a wider range of 10(5)-10(9) cells/ml. The sensitivity of analysis, growth rates and lag time attained using these methods were also compared. As a consequence of changes in bacterial cell size during growth, turbidimetry over-estimated the growth rate. The plate count method proved unable to detect the difference between bacteria existing as chains or single cells. The sensitivity of analysis and the calculated growth related parameters were similar for flow cytometry and manual counts. This suggests that flow cytometry is capable of counting individual cells in a chain. Further investigation showed that passage of B. thermosphacta cells through the flow cytometer resulted in the breakage of chains into single cells. The reliability, low error and rapidity of this technique make it attractive for bacterial enumeration, something which has been demonstrated using B. thermosphacta, a bacterium which exhibits complex morphologies.

Visualization and modelling of the thermal inactivation of bacteria in a model food.

A large number of incidents of food poisoning have been linked to undercooked meat products. The use of mathematical modelling to describe heat transfer within foods, combined with data describing bacterial thermal inactivation, may prove useful in developing safer food products while minimizing thermal overprocessing. To examine this approach, cylindrical agar blocks containing immobilized bacteria (Salmonella typhimurium and Brochothrix thermosphacta) were used as a model system in this study. The agar cylinders were subjected to external conduction heating by immersion in a water bath. They were then incubated, sliced open, and examined by image analysis techniques for regions of no bacterial growth. A finite-difference scheme was used to model thermal conduction and the consequent bacterial inactivation. Bacterial inactivation rates were modelled with values for the time required to reduce bacterial number by 90% (D) and the temperature increase required to reduce D by 90% taken from the literature. Model simulation results agreed well with experimental results for both bacteria, demonstrating the utility of the technique.

Use of multi-staining flow cytometry to characterise the physiological state of Escherichia coli W3110 in high cell density fed-batch cultures.

High cell density fed-batch fermentations of Escherichia coli W3110 have been carried out at specific growth rates of less than 0.3 h-1, to investigate the effect of glucose limitation on the physiological state of individual cells. After an initial exponential batch phase, the feed rate was held constant and a final dry cell weight of approximately 50 g per litre was achieved. The fermentations were monitored by mass spectrometry whilst measurements of pH, DOC, CFU/mL, TCN, OD500nm and residual glucose concentrations were made. Satisfactory and reproducible results were obtained. Flow cytometric analysis of cells in broth samples, based on either of two multi-staining protocols, revealed a progressive change in cell physiological state throughout the course of the fermentations. From these measurements it was concluded that the loss in reproductive viability towards the end of the fed-batch process is due to cell death and not due to the formation of a "viable but nonculturable state" as had previously been reported. Since the presence of a high proportion of dead or dying cells at any time during a fermentation has a detrimental effect on the synthesis of any desired product it is proposed that an on-line flow cytometric analysis and control strategy could be used as a means of increasing overall process efficiency.

The use of flow cytometry to study the impact of fluid mechanical stress on Escherichia coli W3110 during continuous cultivation in an agitated bioreactor.

Continuous culture fermentations of Escherichia coli W3110 have been carried out at controlled dissolved oxygen levels of 40% and 10% of saturation. Satisfactory and reproducible results were obtained. Agitation speeds of 400 and 1200 rpm at an aeration rate of 1 vvm have been used as well as an aeration rate of 3 vvm at 400 rpm. The upper levels of these variables represent much higher agitation and aeration intensities than those normally used in practical fermentations. The fermentations were monitored by mass spectrometry and optical density, and cell samples were studied by flow cytometry, SEM, and TEM. Protocols were developed so the state of both cell membranes and cell size could be measured by flow cytometry. Under all the conditions of agitation and aeration, flow cytometric analysis indicated that both cell membranes were intact and that a cytoplasmic membrane potential existed; also the cell size did not change, results confirmed by SEM and TEM. There were no detectable changes in off-gas analysis or optical density during the continuous fermentation nor in the cell structure as revealed by SEM or TEM, except at the highest agitation intensity. Under the latter conditions, after 7 h, the outer polysaccharide layer on the cell was stripped away. It is concluded that any changes in biological performance of this E. coli cell line due to variations in agitation or aeration intensity or scale of operation cannot be attributed to fluid dynamic stresses associated with the turbulence generated by impellers or with bursting bubbles.