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Basics of Cell Culture

Higher category : 



1. Classification of incubation

⑴ Batch culture

① Grows cells by limiting the amount of culture medium.

○ Growth-limiting substrate : Substrates with limited amounts that affects cell growth

② 1st. Lag phase

○ In order for microbes to recognize and adapt to new molecules, the number of cells does not increase and the level of internal change is taken.

○ Shortening strategy : Adapting to incubation environment before cell culture, Metabolic improvement substance(e.g., Mg), Cell factor concentration optimization

③ 2nd. Exponential phase

○ Balanced growth : A pattern of growth in which all cells multiply at a certain rate

④ 3rd. Deceleration phase

○ Unbalanced growth starts.

⑤ 4th. Stationary phase

○ Useful secondary metabolites such as antibiotics begin to be produced, i.e. non-replicating metabolism begins.

○ As the stage where oxygen and nutrients are insufficient, sufficient supply of oxygen and nutrients can delay the stationary phase.

⑥ 5th. Death phase or decline phase

○ Errors are severe because dead cells are counted during absorbance analysis (using a spectrophotometer).

⑵ Fed-batch culture

① Cultivation method that increases productivity of products by adding organic nutrition ingredients only during incubation but not extracting the culture fluid.

○ The volume of culture fluid continuously increases.

○ The continuously supplied nutrient is called ‘feed’.

② Advantages : Overcomes substrates inhibition problem.

○ If E. coli grows at maximum speed using glucose, it will produce organic acids, a by-product, which will inhibit growth. In the fed-batch culture, it is possible to reduce the production of by-products and cultivate highly concentrated E. coli by maintaining the concentration of substrate and maintaining the growth of E. coli properly.

⑶ Chemostat

① An open-system culture method that maintains environmental conditions while continuously providing nutrients and removing wastes.

② CSTR(continuous stirred-tank reactor model) is generally used for incubator.

③ Parameter

○ Cell density(X) : g cells / L

○ Dilution rate : F(feed) / V(incubator volume)

○ Productivity of biomass : D × X

○ Space time : The duration of the reactant’s stay in the reactor, V(incubator volume) / F(volume flow)

○ Space velocity : Inverse number of space time, Relative velocity concept

⑷ Basic equation

① Parameter

○ Biomass yield : Indicates how much more cells can multiply per substrate of unit mass.

○ Unit : g cells / g substrates

○ Generally marked as Yx/s 

○ Product yield : Indicates how much product per substrate of unit mass can be produced.

○ Unit : g products / g substrates

○ Generally marked as Yp/s 

○ Production speed : The rate of formation of acellular products, and the rate of formation of products per cell of unit mass

○ Unit : g products / g cells · h

○ Generally marked as qp

② Biomass’s rate of change equation


drawing

③ Substrates’ rate of change equation


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④ Products’ rate of change equation


drawing

⑤ Monod equation : Well applied when cell concentration is low.

○ Similar to Michaelis-Menten formula.


drawing

○ Residual substrate concentration(S) is independent of the incoming substrate concentration(S0).

⑥ Contois equation

○ Developed from Monod equation.


drawing

○ Residual substrate concentration(S) is independent of the incoming substrate concentration(S0).

⑸ Cell recycle : If there is cell recycle in a chemostat, dilution rate can be greater than the specific growth rate.

① General phenomenon : μ = D

② μm < D : Generally cells continue to escape.

③ Proof


drawing


drawing

⑹ Equation abount amount of expression recombinant proteins from recombinant microorganisms in fed-batch culture.


drawing

: concentration of cloned-gene protein (mg proteins / mg cells)

: time(h)

ke : maximum rate of protein synthesis (mg proteins / me cells per hour)

: intracellular plasmid concentration (mg plasmids / mg cells)

Kt : transcription rate saturation constant (mg / mg cells)

k-p : cloned-gene protein denaturation rate constant (1st-order reaction) (h-1)

μ : specific growth rate (h-1)



2. Biofuel process

⑴ Conducted in the order of pre-treatment, saccharification, and fermentation.

① Example of pre-treatment : Cellulase and hemicellulase production.

② Example of saccharification : Hydrolysis that converts cellulose and hemicellulose into monomer sugars.

③ Example of fermentation : Fermentation of hexose and pentose.

⑵ SHF(separate hydrolysis and fermentation)

① Traditional process consisting of 1st stage(pre-treatment, saccharification) and 2nd stage(fermentation).

② Advantage : Since each stage is executed under optimal conditions, it is executed without the cleaning pre-treated biomass, eliminating poison and supplying nutrients. 

⑶ SSF(simultaneous saccharification and fermentation)

① Process in which the preparation phase occurs first and the fermentation takes place simultaneously with saccharification.

Advantage 1. Biomass hydrolysis rate is high due to removal of final product inhibition.

Advantage 2. Cleaning pre-treated materials improves biomass conversion rate due to removal of inhibitory mixtures.

Advantage 3. Use of heat-resistant yeast is permitted.

⑷ CBP(consolidated bio-processing)

① The pre-treatment, saccharification and fermentation are treated as a single process.

② Advantage : Provides low cost, high efficiency potential for biomass decomposition enzyme production.



3. Conservation of strains

⑴ Method for long-term preservation of strains : Cryopreservation

① Note. -70 ℃ deep freezer, liquid nitrogen (-196 ℃)

⑵ Method for short-term preservation of strains : Incubation in agar plate, agar slant, etc. → Refrigerate them(0-5 ℃)

① Agar molecules function as contact-dependent growth inhibition.



Input : 2019.03.14 08:59

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