Cell Growth Flashcards
________ is autocatalytic
Growth
Characterized by specific growth rate, μ
Cell Growth
Cell Growth
cells + substrate → extracellular products + new cells + original cells
Cell concentration can be measured directly and/or indirectly.
Measuring Cell Concentration
__________: mass or cell number basis.
Direct
Cell number counting: ______________
hemocytometer, plate counts and particle counts
Advantage: accurate, typically low noise in measurement.
Counting Cells: Hemocytometer.
Disadvantage: time consuming, carcinogenic, mutagenic stains.
Counting Cells: Hemocytometer.
petri dish or dilution plate counts: count colonies formed by individual cells (dilute sample)
Counting Cells: Plate Counts
CFUs = ___________
colony forming units
Advantages: counts viable cells, fairly accurate.
Counting Cells: Plate Counts
Disadvantages: noisy, takes days.
Counting Cells: Plate Counts
Particle counters (Coulter counter): measure particle size distributions.
Counting Cells: Particle Counters
Advantages: very quick, obtain a size distribution in addition to a count.
Counting Cells: Particle Counters
Disadvantages: solutions must be particle free for accurate count, hardware, expensive, complicated.
Counting Cells: Particle Counters
Most common units to report biomass.
Mass Concentration
Obtained by centrifuging sample, drying and weighing.
Mass Concentration
Advantages: concentration Mass is typical variable in models, simple, low-tech method.
Mass Concentration
Disadvantages: Presence of solids makes inaccurate, difficult to measure low biomass concentrations.
Mass Concentration
Turbidimeter or spectrophotometer (most common).
Indirect Concentration Measurements
Substrate uptake or product evolution.
Indirect Concentration Measurements
Luciferin/ATP fluorescence
Indirect Concentration Measurements
Protein or DNA/RNA concentration measurements.
Indirect Concentration Measurements
Is a measure of the amount of light that passes through a turbid sample.
Optical density
Reported with the wavelength of the light used in the measurement. For example OD600 = optical density at 600 nm.
Optical density
________ is often measured in OD and converted to mass per volume with a standard curve.
Biomass
Fixed amount of substrate (growth medium) present at beginning.
Batch Cultures
_______ is seeded with an inoculum (small amount of live cells to start growth).
Batch
5 phases of growth:___________
lag, exponential, deceleration, stationary, death.
Adaptation as inoculum cells adjust enzyme systems to new environments (repression of unneeded systems, induction of useful enzyme systems).
Lag Phase
Growth is suppressed, duration 1-10 hours
Lag Phase
Extended by low temperature, small inoculum, radical substrate/temperature changes, low nutrient levels, inoculum “age”.
Lag Phase
Inoculum should be 5 volumes % and from exponential phase culture.
Lag Phase
___________ can exist with multiple growth substrates (diauxic growth).
Multiple lag phases
Growth is balanced (intracellular concentrations remain constant).
Exponential Growth Phase
No dependence on substrate concentration (growing at intrinsic maximum growth rate).
Exponential Growth Phase
Primary metabolites (growth associated) produced.
Exponential Growth Phase
Growth rate is 1st order with respect to cell concentration, Oth order with respect to substrate concentration.
Exponential Growth Phase
End of exponential phase.
Deceleration Phase
Caused by either build-up of toxic products or depletion of substrate.
Deceleration Phase
Cell physiology changes to favor survival overgrowth.
Deceleration Phase
Net growth rate is zero.
Stationary phase
Cells produce secondary metabolites (not growth associated).
Stationary phase
Many products important to the biotechnology industry are produced during this phase.
Stationary phase
Cells begin to lose ability to reproduce.
Stationary phase
Cells begin to lyse, cryptic growth occurs.
Stationary phase
Cells catabolize energy reserves (eg; PHB) in endogenous metabolism
Stationary phase
Although growth slows or ceases, maintenance requirements still exist.
Stationary phase
Death is relative to the population, death always occurs.
Death Phase
Commonly modeled as a 1st order process with respect to biomass.
Death Phase
Some portion of cells remain viable for a long time, but are altered
Death Phase
Growth yield microorganisms = produced per unit substrate utilized
Growth Yield, Yield Coefficient
_______________ is similarly affected relative to diffusion.
Biological reaction rate
pH optima: _______________
bacteria 3-8, yeast 3-6 molds 3-7 plant cells 5-6, animal cells 6.5-7.5
pH varies significantly during fermentation if system is not buffered or controlled for pH.
Effects of pH
CO2 evolution and ammonium as nitrogen source both lower pH.
Effects of pH
Nitrate utilization raises pH.
Effects of pH
________ can become a limiting substrate.
DO
At________ concentration, growth is independent of [O2].
high DO
O₂ solubility in water ~____________
7 ppm (25°C, 1atm)
Bacteria require ~___________
10% of saturation for [O2] independent growth, yeast 10-50%.
_____________ is usually limited by stagnant liquid around bubbles.
Rate of O₂ transfer
When O₂ transfer is limiting ____________ , so ….
OTR=OUR
Very high maybe toxic to some organisms
Dissolved Carbon Dioxide
Cells require certain DCO2 for proper metabolic functions
Dissolved Carbon Dioxide
Controlled by changing CO2 conc. In air supply and agitation speed Ionic Strength
Dissolved Carbon Dioxide
Affect the transport of certain nutrients in and out of the cell
Dissolved Carbon Dioxide
Metabolic functions of cells
Dissolved Carbon Dioxide
Solubility of certain nutrients
Dissolved Carbon Dioxide
Structured versus unstructured (cell composition does not change with time).
Models for Growth Kinetics
Segregated versus unsegregated (all cells are identical).
Models for Growth Kinetics
_________ is an unstructured, unsegregated model.
Monod equation
Single substrate controls growth. (low population density)
Monod equation
Analogous to Michaelis-Menten enzyme kinetics and the Langmuir isotherm.
Monod equation
Mechanistic if one enzyme system controlled growth
Monod equation
Maintenance: _________________
energy expenditures for repair, transport, motility
Endogenous metabolism: ____________
consumption of storage polymers for energy
There are other nonsegregated, unstructured models for cell growth but the difference is not worth the work.
Other Models for Cell Growth
___________ are not typically mechanistic, but are selected to fit data.
Inhibitory kinetic expressions
__________ are analogous to inhibited enzyme kinetics.
Expressions
If a substrate is inhibiting cell growth of a batch culture, the substrate should be added in a fed- batch mode.
Growth Inhibition by Substrate
Inhibitory kinetic expressions are not typically mechanistic, but are selected to fit data.
Growth Inhibition by Product
If a product inhibits cell growth of a batch culture, the product recovery will be expensive.
Growth Inhibition by Other Compounds
Instrumentation
pH probe and controller
DO probe and controller
Antifoam probe and controller
Level probe and controller
Nutrient addition pump
Agitation rate controller
Shuler uses ____ to represent changes in cell mass due to endogenous respiration and ka’ for changes in cell mass due to cell death and lysis.
Kd
__________: catabolism of cellular reserves for continued maintenance and energy.
Endogenous respiration
WWWebster’s Dictionary: 2.b) the quantitative relationship between two or more substances especially in processes involving physical or chemical change
Stoichiometry
If cells have a characteristic molecular composition (i.e.; CH₂ON) then yield coefficients can be determined through stoichiometry. In practice, these estimates.
Stoichiometry
The system is determinate and we can form the yield coefficients
Yield Coefficients
from the stoichiometric coefficients and formula weights.
Yield Coefficients
