Cell batch culture phases

It may also be economical to remove the inhibitory product directly from the ongoing fermentation by extraction, membranes, or sorption. The use of sorption with simultaneous fermentation and separation for succinic acid has not been investigated. Separation has been used to enhance other organic acid fermentations through in situ separation or separation from a recycled side stream. Solid sorbents have been added directly to batch fermentations (18,19). Seevarantnam et al. (20) tested a sorbent in the solvent phase to enhance recovery of lactic acid from free cell batch culture. A sorption column was also used to remove lactate from a recycled side stream in a free-cell continuously stirred tank reactor (21). Continuous sorption for in situ separation in a biparticle fermentor was successful in enhancing the production of lactic acid (16,22). Recovery in this system was tested with hot water (16). 

Figure 2.5 Possible technological solutions to bioprocess problems a) Fed-batch culture b) Continuous product removal (eg dialysis, vacuum fermentation, solvent extraction, ion exchange etc) c) Two-phase system combined with extractive fermentation (liquid-impelled loop reactor) d) Continuous culture, internal multi-stage reactor e) Continuous culture, dual-stream multi-stage reactor f) Continuous culture with biomass feedback (cell recycling). (See text for further details).

The rate of product formation, rfi, depends upon the state of the cell population, environmental condition, temperature, pH, media composition and morphology with cell age distribution of the microorganism.2 3 A similar balance can be formulated for microbial biomass and cell concentration. The exponential phase of the microbial growth in a batch culture is defined by ... 

The effect of substrate concentration on specific growth rate (/i) in a batch culture is related to the time and p,max the relation is known as the Monod rate equation. The cell density (pcell) increases linearly in the exponential phase. When substrate (S) is depleted, the specific growth rate (/a) decreases. The Monod equation is described in the following equation ... 

Models for batch culture can be constructed by assuming mechanisms for each phase of the cycle. These mechanisms must be reasonably comph-cated to account for a lag phase and for a prolonged stationary phase. Unstructured models treat the cells as a chemical entity that reacts with its environment. Structured models include some representation of the internal cell chemistry. Metabolic models focus on the energy-producing mechanisms within the cells. 

Typically, the process is set to control temperature to a value that results in high cell growth or production rate. Sometimes, however, a different temperature may be selected, which could result in a better quality product or a respiration rate compatible with the oxygen transfer system (Chuppa et al., 1996). For some processes, a temperature shift batch culture may be desirable in which there are two controlled temperature values, one for the growth phase and another for the production phase, in which the cell division is interrupted due to the low temperature (Fox et al., 2004). 

Cells in primary culture Phase I and II present, whole metabolic expensive, batch variability, quality control, com-... 

Cell lysis generally becomes important when viability is low. Examples include continuous culture (with or without cell retention) at low dilution rates and the late stationary and death phases of batch culture. Cell lysis is also important in stirred reactors with very high agitation rates. In order to quantify cell growth parameters under these conditions, the lysed cells must be accounted for. One way to do this is to measure the amount of the cytosolic enzyme lactate dehydrogenase (LDH) released into the medium (see Chapter 2, section 2.5). A procedure for measuring LDH activity is described in Chapter 4, section 4.7. 

Cells can be inoculated at a normal inoculum level, e.g. 1-2 x 10 cells mL, and grown as a batch culture until the mid-exponential phase of growth. The supply of medium from the feed reservoir can then be started at a flow rate that gives the required dilution rate. In order that the cells are not washed out of the fermenter, the initial dilution rate should be below the maximum specific growth rate of the cells, although too low a dilution rate may result in low cell viability... 

The model was able to predict the culture dynamics for batch, fed-batch, and cell growth arrested cultures, especially up to the exponential growth phase, after which certain variable predictions deviated from the experimental results in fed-batch cultures, e.g. the viable cell concentration in the optimised fed-batch culture tended to be overestimated, and the simulated glucose uptake rate near the end of the fed-batch cultures was higher than observed. The model closely predicted the monoclonal antibody concentration in the optimised fed-batch culture despite an underestimation of the viable cell concentration. The model developed was able to direct experimental efforts to a more focused area in this case study. The monoclonal antibody yield in the optimised fed-batch culture was 3.5xl0 mg L" which was about 40% higher than the initial fed-batch culture. Further improvement of the model structure may be necessary to enhance its predictive capability. 

FIGURE 2-27 Cell density versus time in a batch culture. During the lag phase, the cells become acclimated. During the exponential phase, the number of cells increases exponentially as described by Monod growth kinetics, see Eq. [2-73]. During the stationary phase, cell density is constant for some time before the culture declines due to substrate depletion, waste accumulation, and/or excessive cell density. 

A new bioreactor, consisting of a liquid-gas two-phase system, was devised for utilization with berberine-secreting T. minus cells immobilized in calcium alginate beads. The cells were alternately soaked in medium, and then exposed to air, and the maximum yield of berberine production was 875 mg/L. The berberine productivity of immobilized cells was as high as that of freely suspended cells under such conditions of batch culture. In addition, the rate of production of berberine by the immobilized cells remained constant at a high value (50 mg/L/day) for 60 days of semicontinuous culture, achieved by the renewal of medium at 10 day intervals [154]. 

Unstructured distributed models such as Monod s equation satisfactorily predict the growth behavior in many situations. However, they cannot account for lag phases, sequential uptake of substrates, or changes in mean cell size during the growth cycle of a batch culture. Structured models recognize the multiplicity of cell components and their interactions. Many different models have been proposed based on the assumptions made for cell components and their interactions. 

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