Week 4 Lecture Kinetics and Monitoring Growth Flashcards
What is Kinetics of growth
Orderly increase in cellular components, resulting in cell enlargement and eventually leading to cell division
-Ideally growth kinetics is a homogeneous unicellular suspension culture
-We can use differential equations in continuum model
-Divisions produce identical daughter cells
-Generation vs. generation time
Why do we need good mixing in bioreactor for kinetics of growth?
-Biomass and number of cells will increase
-We need good mixing in bioreactor because we want to have an even heat transfer, even concentration of substrate, want it to be homogenous
-If there is no synchronous growth everything will be upside down, certain microbes in stationary phase and growth phase when they’re not supposed to be
-In stationary phase strictly control concentration of substrates so we don’t go to exponential phase too soon
What are the issues of Kinetics?
-Increase of biomass vs. increased number of cells due to division
-Asynchronous growth vs. synchronous
-Fermentation under different operating conditions (batch, fed-batch, continuous)
What happens if there is no synchronous growth?
-If there is no synchronized growth and homogenous conditions, microbes will all be in different phases, no correlation between biomass growth and increasing number of cells in bioreactor
-Growth rate in stationary phase slower than growth rate in exponential phase
What are the different growth phases?
-Lag
-Acceleration
-Exponential
-Deceleration
-Stationary
-Death phase
What is Lag phase?
-Intense metabolic activity
-Length of lag time depends on the age, -phase of growth microorganisms were taken from
-Chemical composition of fermentation media influences the length of lag time
-Physiological stress (inoculum moved from low osmotic to high osmotic pressure)
What is acceleration phase?
Cell division with increased rate until max growth rate umax is reached
What is the exponential phase?
-Growth begins cell number/biomass increase at a constant rate (x biomass, N number of cells)
-When a cell divides we say generation and time it takes is called mean generation time
-Mean generation time (doubling the number) td=t/n
-Division rate constant n/t
–Once substrates are consumed growth rate of microbes is going into deceleration phase
What is exponential growth?
Exponential growth begins when all nutrients are supplied in excess, then there is a relationship between # of cells and biomass then we have balanced growth
What is the Deceleration phase?
-Level of substrate decreases, becoming limiting and can not sustain max growth rate
-Limiting substrate is metabolized then growth is no longer sustainable
What is the Stationary phase?
-No net change in cell number; rate of divisions=rate of death of cells; but microorganisms are still metabolically active using intracellular storage compounds
What is Death phase?
-Cells die or can form spores to survive depending
What is Batch fermentation?
-Not steady state because: rate constant (u) changes, substrate concentration decreases, product-biomass concentration increases, pH changes, temperature might change (if we do not control the heat), and there is an accumulation of byproducts
-operations involve; charging water and substrates, sterilization, inoculation, production of products, harvesting, cleaning
Fed-batch (continuous process)?
-CSTR experiment is an example
What is the Kinetics growth equation?
xt=x0 * e^ut or ln xt= u*t + lnx0
x= grams of biomass
t=time
u= rate constant
Kinetics doubling time equation?
t= ln2/u
What are the Direct Methods of Monitoring Growth?
-Dry weight determination
-Cell counting by microscopy
-Plate counting methods
What are the Indirect Methods of Monitoring Growth?
-Turbiditmetry
-Spectrophotometry (optical density)
-Estimation of cell compounds (protein, DNA, ATP)
-On-line monitoring of CO2, O2
What are the issues in Monitoring of Growth?
-Issues include: duration, sensitivity, properties of material to be tested, costs
Direct Methods
-Petrof-Hauser or Neubauer type counting chambers
-Counting # of cells within proportion of grid
-Rapid but can not distinguish between dead and living cells
-Samples must contain higher cell count minimum is 5*10e^6 cells/ml
What are Electronic counters?
-Coulter counter- measurement of change in electrical resistance produced by non conducting particles suspended in an electrolytes
-For toner particles , how many microbial particles are forming and what shape they are
-The more particles you have the more resistance from the original reference
What is Plate counting?
-Detection of viable cells able to form colonies
-Spread plating (aerobic)
-Pour plating (anaerobic)
-Calculating of the colonies by taking into consideration dilution and volume of the substrate
-CFU colony forming units
-Time 1-2 days
What is turbidity measurement?
-Light scattering by suspension of cells
-It is proportional to cell concentration
-Particles in sample, light scatters because of this, will measure the amount of light that is scattered from your particles, light that is reflected at 90 degrees
What is dry weight estimation?
-Living and dead cells
-Liquid culture samples
-Filtration (0.2-0.45um membrane) under vacuum required
-Drying at 105*C to a constant weight
-Milligrams of cell per ml of culture
-High volume of samples required
What is ATP bioluminometry?
-To determine concentration of viable cells (living)
-Uses enzyme-substrate complex luciferase-luciferin extracted from the firefly which generates photon of light per one molecule of ATP
-This complex is added to the sample and resulting signal (light intensity due to photons attached to each molecule of ATP) is measured by bioluminometer
-Luciferin interacts with ATP and produces photon
What is On line monitoring?
-Optical density, capacitance based probes
-Measurement of CO2 generated or O2 consumed
-Bug-lab
-CO2 detector, O2 detector, Ethanol detector
