Fed Batch Culture

In fed batch culture, growth rate is controlled by the rate of feeding of the limiting nutrient. The feed rate can therefore be used to set qp and the desired productivity of protein production. 

At a steady state concentration of substrate, Eqs. (7-9) result in Eq. (18) Equation (18) and Fig. 4 can be taken together to determine three regimes of 

In many enzyme fermentations, the limiting component, usually the C-source, has to be added semi-continuously to keep its concentration at a predetermined, usually low, value. This measure makes it possible either to influence selectivity between different pathways or to uncouple predominantly cell growth during the first phase of the fermentation from predominantly product (i.e., enzyme) formation in the later stages of the fermentation cycle. Often, protein formation is induced by adding an inducer (see Chapter 4). During the fed-batch phase, the broth volume increases. Either the broth is harvested when the maximum volume is reached, or broth is withdrawn from time to time. The product is present in high concentrations. 

When analyzing a fed-batch fermentation, we cannot assume constant volume as in the batch fermenter, so the balances for cell density, substrate, product, and volume read according to Eqs. (8.10)—(8.13). 

Sf is the substrate feed rate, and FSf consequently is the molar flow into the fermenter (there is no flow out of the fermenter). Yx/s and YpyS are the two empirical yield coefficients of cell X or product P on substrate S [g (g substrate)-1] and qp is the specific production formation rate [g product (g cells)-1 h-1] (qp-X = rp, the product formation rate [g product h 11). 

Combining cell and substrate mass balance [Eqs. (8.14) and (8.15) respectively] and integrating yields Eq. (8.17), which, with the feed concentration satisfying the condition stipulated by Eq. (8.18), transforms into Eq. (8.19). 

The most important special case of fed-batch fermentation increases F exponentially so as to keep F/V constant. Then, Eq. (8.13) can be rewritten as Eq. (8.20), or integrated to give Eqs. (8.21) and (8.22).  

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Fed-batch culture A cell cultivation technique in which one or more nutrients are supplied to the bioreactor in a given sequence during the growth or bioconversion process while the products remain in the vessel until the end of the run. 

As you might have already gathered, the majority of industrial fermentations are batch processes. In closed batch systems, the growth medium is inoculated with cells and growth and product formation is allowed to proceed until the required amount of conversion has taken place. After harvesting the culture the vessel is cleaned, sterilised and filled with fresh medium prior to inoculation. For some processes, addition of all the feedstock prior to inoculation, as is done in closed batch fermentations, is undesirable and it is preferable to incrementally add the carbon source as the fermentation proceeds. Such a process is known as fed-batch culture and the approach is often used to extend the lifetime of batch cultures and thus product yields fed-batch cultures are considered further in Section 2.7.4. 

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).

When excess substrate interferes with growth and/or product formation. One example is the production of baker s yeast. It is known that relatively low concentrations of certain sugars repress respiration and this will make the yeast cells switch to fermentative metabolism, even under aerobic conditions. This, of course, has a negative effect on biomass yield. When maximum biomass production is aimed at, fed batch cultures are the best choice, since the concentration of limiting sugar remains low enough to avoid repression of respiration. 

Mars AE, J Houwing, J Dfolfing, DB Janssen (1996) Degradation of toluene and trichloroethylene by Burk-holderia cepacia G4 in growth-limited fed-batch culture. Appl Environ Microbiol 62 886-891. 

In this case, there is a continuous supply of nutrients and a continuous withdrawal of the culture broth including the submerged free cells. The governing equations for continuous cultures are the same as the ones for fed-batch cultures (Equations 7.20-7.22). The only difference is that feed flowrate is normally equal to the effluent flowrate (Fm=Fout=F) and hence the volume. V, stays constant throughout the culture. 

In this case we assume that we know the dilution rate (D-F/V) precisely as a function of time. In a chemostat D is often constant since the feed flowrate and the volume are kept constant. In a fed-batch culture the volume is continuously increasing. The dilution rate generally varies with respect to time although it could also be kept constant if the operator provides an exponentially varying feeding rate. 

To perform fed-batch experiments with P. putida a method had to be developed to prevent carbon limitation and to prevent a buildup of the concentration of the fatty acids to inhibitory levels. HPLC methods to measure the concentration of aliphatic substrates and octanoic acid have been reported, but these are not suitable for the detection of long chain fatty acids in a watery phase due to their low solubility. Instead Huijberts et al. [55, 56] developed a method in which discrete pulses of fatty acids were added to fed-batch cultures. Substrate exhaustion was detected by a sudden increase in dissolved oxygen tension and this signal was used to trigger the injection of another fatty acid pulse into the... 

Fig. 2. Time profiles of cell dry weight, P(3HB) concentration and the content during the fed-batch culture of recombinant E. coli XLl-Blue(pJC4) (reproduced from [48] with permission)...

Fig. 4 A, B. Time profiles of A cell dry weight and PHA concentration B PHA content (wt %) and 3HV fraction in PHA (mol%) during the fed-batch culture of XLl-Blue(pJC4) with oleic acid supplementation after acetic acid induction. The feeding solution was added to increase the concentrations of glucose and propionic acid to 20 g/1 and to 5 mmol/1, respectively, after each feeding (reproduced from [62] with permission)... 

Wang, P., and Krawiec, S., Kinetic analyses of desulfurization of dibenzothiophene by Rhodococcus erythropolis in batch and fed-batch cultures. Applied and Environmental Microbiology, 1996. 62(5) pp. 1670-1675. 

Konishi, M. Kishimoto, M. Omasa, T., et al., Effect of Sulfur Sources on Specific Desulfurization Activity of Rhodococcus Erythropolis KA2-5-1 in Exponential Fed-Batch Culture. J. Bioscience and Biotechnology, 2005. 99(3) p. 259. 

For investigations of microorganisms three regimes of cultivation with some modifications or combinations are used batch cultures, fed-batch cultures, and continuous cultures. 

Fed-batch cultures differ from batch cultures by the possibility of additional input of the main substrate. Potentially, fed-batch cultures are very promising since in these cultures the possibility to prolong a hydrogen production phase with approximately constant rate exists. Unfortunately publications reporting the application of this cultivation regime for hydrogen production systems are not known to us. 

T. Suizuki, H. Mori, T. Yamane, S. Shimizu (1985) Automatic supplementation of minerals in fed-batch culture to high cell mass concentration Biotechnol. Bioeng., 27 192-201. 

Suspension systems can be operated in different modes batch, fed-batch, chemostat, and perfusion (Fig. 1). These operation modes differ basically in the way nutrient supply and metabolite removal are accomplished, which in turn determines cell concentration, product titer and volumetric productivity that can be achieved [8]. The intrinsic limitation of batch processes, where cells are exposed to a constantly changing environment, limits full expression of growth and metabolic potentials. This aspect is partially overcome in fed-batch cultures, where a special feeding strategy prolonges the culture and allows an increase in cell concentration to be achieved. In perfusion and chemostat processes nutrients are continuously fed to the bioreactor, while the same amount of spent medium is withdrawn. However, in perfusion cultures the cells are retained within the bioreactor, as opposed to continuous-flow culture (chemostat), which washes cells out with the withdrawn medium [9]. 

Fed-Batch Cultures of Escherichia coli Cells with Oxygen-Dependent nar Promoter Systems... 

Several excellent review articles have addressed this issue from molecular biology to high density cell cultures of Escherichia coli cells [1-4]. We will briefly review E. coli promoter system with emphasis on oxygen-dependent VHb and nar promoters and discuss fed-batch cultures of the nar promoter system requiring nitrates or no nitrates. These new systems will be compared with other existing promoter systems. 

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