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Microbial Growth in Batch Fermentation,As the cells in a batch. fermentation grow they,follow a growth curve,similar to the one shown. here The growth curve,contains four distinct,regions known as. phases They are as,1 Lag Phase,2 Exponential Phase. 3 Stationary Phase,4 Death Phase,Growth curve is from Shuler p 161.
Microbial Growth in Batch Fermentation, The first major phase of microbial growth in a batch fermentation. A period of adaptation of the cells to their new environment. Minimal increase in cell density,May be absent in some fermentations. Shuler p 161 162,Microbial Growth in Batch Fermentation. Exponential Phase, The second major phase of microbial growth in a batch fermentation. Also known as the logarithmic growth phase,Cells have adjusted to their new environment.
The cells are dividing at a constant rate resulting in an exponential. increase in the number of cells present This is known as the specific. growth rate and is represented mathematically by first order kinetics. as the following, where X is the cell concentration is the cell growth rate and kd. is the cell death rate The term kd can be referred to as net. The cell death rate is sometimes neglected if it is considerably. smaller than the cell growth rate,Shuler p 162 163. Microbial Growth in Batch Fermentation,Exponential Phase continued. Cell growth rate is often substrate,limited as depicted in the figure to. The growth curve is well,represented by Monod batch.
kinetics which is mathematically,depicted on the following slide. Shuler p 163,Microbial Growth in Batch Fermentation. Exponential Phase continued, Monod batch kinetics is represented mathematically in the. following equation, where is the specific growth rate max is the maximum specific. growth rate S is the growth limiting substrate concentration and. KS is the saturation constant which is equal to the substrate. concentration that produces a specific growth rate equal to half. the maximum specific growth rate All specific growth rates. account for the term kd and should be considered to be net. Shuler p 176,Microbial Growth in Batch Fermentation.
Exponential Phase continued, There are other models used to determine cell growth rate that. depend upon inhibition,Substrate Inhibition,Product Inhibition. Toxic Compounds Inhibition, The type of inhibition causes mathematical changes in the. previously presented Monod equation for batch kinetics. Shuler p 178 180,Microbial Growth in Batch Fermentation. Exponential Phase continued,Substrate Inhibition, In batch fermentation this can occur during the initial growth.
phases while substrate concentrations are high, If this is a major problem continuous or fed batch. fermentation methods should be considered,Product Inhibition. In batch fermentation this can occur after induction of the. recombinant gene,Shuler p 178 180,Microbial Growth in Batch Fermentation. Stationary Phase, The third major phase of microbial growth in a batch fermentation. Occurs when the number of cells dividing and dying is in. equilibrium and can be the result of the following. Depletion of one or more essential growth nutrients. Accumulation of toxic growth associated by products. Stress associated with the induction of a recombinant gene. Primary metabolite or growth associated production stops. Secondary metabolite or non growth associated production may. Shuler p 163 164,Microbial Growth in Batch Fermentation.
Death Phase, The fourth major phase of microbial growth in a batch. fermentation process,Also known as the decline phase. The rate of cells dying is greater than the rate of cells dividing. Similar to Exponential phase it is represented mathematically by. first order kinetics as the following,Shuler p 164 165. Microbial Growth in Batch Fermentation, There are a two main methods primarily used to establish a growth. curve Both of which are represented on the previously shown. growth curve,Viable Cell Count, Initially lower curve representing the number of cells that are.
actually viable,Determined by plating a sample from the culture. Optical Density, Initially higher curve representing the number of cells that. are both viable and non viable, Determined by taking an optical measurement using a. spectrophotometer,Shuler p 161,Microbial Growth in Batch Fermentation. Measuring the optical density with a,spectrophotometer is a quick and.
easy way to to develop a growth,curve One takes a sample of the. fermentation broth and measures the,absorbance at a particular. wavelength in the,spectrophotometer For E coli cells. in a typically LB medium the,wavelength used in 600 nm The. measured value can be compared to,previous measurements made in.
conjunction with cell plating or cell,counting The negative side of using. the optical density is that both viable Spectrophotometer pictured above is. and non viable cells absorb this a copyright of Perkin Elmer. wavelength As a result the values,taken are not representative of only. viable cells,Batch Fermentation, Now that you understand how microbial cells grow in a batch. process it is time to see how a general biotechnology fermentation. process works An example of a fermentation process is. represented in the block flow diagram shown below The different. blocks depicted are described in detail in the following slides. Shake Flask,1st Seed 2nd Seed Production,Media Prep Purification. Fermentor Fermentor Fermentor,Batch Fermentation,First a frozen vial containing a few.
milliliters of one recombinant E coli,strain is taken out of a freezer and. thawed This vial is sometimes referred,to as an inoculum vial and its contents. is known as an inoculum,After thawing the inoculum is. transferred in a sterile manner to a,shake flask containing growth media. This process is known as inoculation,For E coli the initial pH of the media is.
typically around 7 and is controlled by,using a buffering agent in the media A. picture of a shake flask is depicted to, the right The volume of media in the Shake flask photo above is a copyright. shake flask is usually on the order of of Kimax Kimble USA. magnitude of hundreds of milliliters,Batch Fermentation. After inoculation the shake flask is,placed in an incubator shaker so the. cells can grow and reproduce The,shaker is operated at a constant.
temperature which is around 37 C for,E coli The shake flask holders in the. shaker are attached to an orbital plate,that rotates horizontally at a. programmable rate This shaking,motion has two purposes. Keep the cells and the nutrients,in the growth media homogeneous. Increases the rate of oxygen uptake,by the media for the aerobic E coli.
cells The cells are grown to a,particular density near the end of. their exponential phase and used to Incubator shaker photo above is a copyright. of New Brunswick Scientific,inoculate a small fermentor known as. Introduction to Fermentation Genetically modified Escherichia coli have been chosen as the host organism for each of the co proteins to be produced Each strain of E coli will contain a different gene that is responsible for producing the desired co protein The modified E coli cells will be separately grown through the process of batch fermentation This tutorial w ill introduce