**2.1 Batch fermentation**

In microbial batch, fermentation cultivation is done in a fixed volume of medium in a fermenter. The standard inoculum in the fermenter is 50–200 mL of shake flask volume in 2–5 L of fermentation media. The batch fermentation

*Strategies for Enhancing Product Yield: Design of Experiments (DOE) for* Escherichia coli*… DOI: http://dx.doi.org/10.5772/intechopen.99288*

typically OD600 is 20–40 in 8–12 h time. The microbial growth depletes the nutrients resulting in the accumulation of by-products; there is a continuous change in the culture environment. After completion of the batch, media and cells are harvested. The advantages are batch fermentation, ease of operation, low risk of contamination, high yield of protein to biomass in less time of fermentation, and majorly for soluble or excreted proteins. Typical disadvantages are relatively long downtime between batches due to vessel setup and sterilisation, low cell/biomass densities, due to cleaning. DOE is needed to optimise the required nutrients and minimise product accumulation during fermentation. Typical batch fermentation media constitutes Yeast extract, Bactotyptone (or Soybean meal) 10 g �<sup>l</sup> to 24 g �<sup>l</sup> , respectively. Buffers of Sodium and potassium phosphate in combination to reach pH 6.8. to 7.0, 100 X amino acid solution, Trace elements (400x) is CoCl2. 6H2O (1 mg/ml�<sup>1</sup> ), MnCl2.4H2O (6 mg ml�<sup>1</sup> ), CuCl2.2H2O (0.6 mg ml�<sup>1</sup> ), H3BO3 (1.2 mg ml�<sup>1</sup> ), Na2MoO4.2H2O(1 mg ml�<sup>1</sup> ), Zn(II)acetate.2H2O (5.2 mg ml�<sup>1</sup> ) and Fe(III)citrate (40 mg ml�<sup>1</sup> ) [54]. The typical medium components are listed in the (**Table 2**) for batch fermentation as a base design to start optimisation.

#### **2.2 Fed-batch fermentation**

Fed-batch fermentation is a standard mode of fermentation in the bioprocess industry. Typically, fed-batch fermentation starts at the end of batch fermentation. *E. coli* is adapted and cultivated in defined media. In Fed-batch fermentation, cells are inoculated and grown in batch mode for 10–15 h. Once all the nutrients are depleted, evident by analysing the amount of glucose in the medium, dissolved oxygen levels are increased to 60–80%. The Fed-batch fermentation is initiated by starting to feed of Glucose, Vitamins, amino acids, and trace elements. The feed is added to the medium to allow the volumetric cell to increase the mass concentration exponentially. The growth rate of is changed to 0.12–0.22 μ h�<sup>1</sup> during fed batch stage. These equations determine the growth rate in the medium.

$$ms(i) = (set + mYX/S)VSo,\tag{1}$$

$$ms(t) = ms(i)eset\*(t - ti)\tag{2}$$

The first equation, ms (i), is the value of feed rate at the initiation of the fedbatch phase at time ti. μset is the specific growth rate, m is specific maintenance coefficient, YX/S is yield coefficient, V is the bioreactor volume, and So is the initial glucose concentration. In the second equation, ms(t) is the rate of addition of substrate (g <sup>h</sup>�<sup>1</sup> ). After induction of protein expression, the specific growth rate of *E. coli* is typically reduced to 0.1 μ <sup>h</sup>�<sup>1</sup> . The cells are harvested after completion of the run. Suppose the growth rate is not specified during fermentation, constant accumulation of several toxic metabolites produced during the fed-batch process acetate, formate, succinate, and lactate, resulting in oxygen limitation fed-batch. Therefore, it is recommended to wash cells with Tris-EDTA buffers after washing *E. coli* cells are stored or lysed for downstream processing. The distinct advantage of resuspending *E. coli* after completion of batch reduces protein degradation due to metalloproteases [46].

Start of feed is determined by measuring the concentration of substrate in the fermentation broth typically after 10–12 hrs of batch. The feeding strategy should be designed so that the growth rate is maintained to limit the production of toxic formate, acetate and other metabolic compounds, enhancing bacterial growth. The growth of bacteria and conversion of feed to biomass is maximum when the exponential growth phase is maintained. The utility of fed-batch and importance is obtaining high cell density and biomass, leading to increased production of the high amount of product yield. The fed-batch is applicable to increase product yield by limiting growth rate and controlled substrate utilisation (**Table 2**). The media for batch and fed-batch fermentation is listed (**Table 2**).
