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
③ Substrates’ rate of change equation
④ Products’ rate of change equation
⑤ Monod equation : Well applied when cell concentration is low.
○ Similar to Michaelis-Menten formula.
○ Residual substrate concentration(S) is independent of the incoming substrate concentration(S0).
⑥ Contois equation
○ Developed from Monod equation.
○ 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
⑹ Equation abount amount of expression recombinant proteins from recombinant microorganisms in fed-batch culture.
P : concentration of cloned-gene protein (mg proteins / mg cells)
t : time(h)
ke : maximum rate of protein synthesis (mg proteins / me cells per hour)
p : 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.
② Advantage 1. Biomass hydrolysis rate is high due to removal of final product inhibition.
③ Advantage 2. Cleaning pre-treated materials improves biomass conversion rate due to removal of inhibitory mixtures.
④ 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