Lecture 12 - Continuous culture of microbes Flashcards
What are the features of the stirred tank reactor?
- can keep the parameters constant for a long period of time
- allows high control
- smooth inside so no cell accumuation and no differences in growth
- allows bacteria to grow in a biofilm
- must be able to be autoclaved/sterilise with steam
- cooling jacket
- pH control - respiration is acidic
- O2 transfer by blowing air in under pressure
- stirred
- addition of an antifoaming agent (issue at high biomass)
- can be done to study physiology or recombinant protein production
- uses liquid media
What is the vessel constructuion of a stirred tank reactor?
- stainless/mild steel lined with glass/plastic
- smooth and capable of withstanding cleaning
How can stirred tank reactors be sterilised?
- by pressured steam at around 120*c for 15 mins
- fully charged vessed injected with steam and the latent heat of vapourisation kills contaminating microbes
How is temp and pH regulated in a stirred tank bioreactor?
- microbes generate heat so must have a cooling system
- small - can use cooling jackets
- large have cooling coils
pH control is by pH electrodes immersed in the medium
-adds alkali under automatic control to neutalise the metabolic acids produced
What are the features of stirring and aeration of a stirred tank bioreactor?
- acheived by stirring gear
- vertical paddles create circular movement
- baffles prevent formation of vortices and increase turbulence
- most fermentations are aerobic and require large volumes of air supplied through a sparger
What does the combination of stirring and aeration result in in a stirred tank bioreactor?
- increases the rate of oxygen transfer from the bubbles to the medium
- keeps bubbles small and increases the time bubbles are in the liquid
- prevents formation of clumps of cells
How is foam control achieved in a stirred tank bioreactor?
Proteinaceous compound act as surfectants
- under vigorous agitation and aeration generate lots of foam
- antifoaming agents are added that prevent the production of stable foams
What control is most necessary in a STR?
-pH, temperature, aeration, mixing and foam control
What alternative fermenter can be used instead of a stirred tank bioreactor, and why might this be advantageous?
Air-lift fermenter
-most stirred systems have the disadvantages of high energy requirement for agitation and they also damage cells because of the shear effect
What are the features of an Air lift fermenter?
- for microbes that cannot survive the stress of stirring
- medium moved by injection of air at the bottom of the riser column
- air is trapped in the downward flow of liquid and dissolution is enhanced by compression
- often used in large industrial fermentation as well as in the lab
- sterilized automatically
What are the three fermenter operation modes?
- Batch fermentation
- Fed Batch fermentation
- Continous culture
What are the features of batch fermentation?
- closed system in which a fixed volume of medium is inoculated
- culture grows and media is altered as a result of growth
What are the features of Fed batch fermentation?
- media is gradually added to the vessel as the process proceeds
- fermentaiton is stopped when the vessel is full
- can extend any growth phase you want by ensuring those condition and nutirent levels are present
- NOT a closed system
What are the features of a continuous culture?
- an open system where fresh medium is added at the same rate as spent media and cells leave the fermentor
- volume is kept constant throughout
- cells essentially are kept growing in the exponential phase
- similar to fed batch, however cells need to be diluted
- hard in the lab as open and can be easily contaminated
What is a big advantage of continuous culture?
- can limit growth by the amount of a particular nutrient
- altering the concentration of an essential nutrient gives a hyperbolic curve
- at saturating concentration, growth is fast, however when nutrient concentration is limiting, growth slows
- 1/2μmax = Ks (half saturation constant)
Through what equation can growth pattern be represented by and how was this formulae derived?
Monod equation
-derived empirically by observing growth of microbial cultures
What is the monod equation?
μ = μ(max)Xs/ ((K(s)+s)
Where:
μ = specific growth rate (h^-1)
μmax = maximal growth rate obtained in excess nutrients
s= concentration of growth limiting substrate (g/l)
k(s) = half maximal saturation constant (g/l)
What are the features of the Monod equation?
- allows to represent the growth pattern in continuous culture
- analogous to the Michaelis-menton equation for the rate of enzyme reation
- Ks values for most growth substrates is low, reflecting the high affinities of the systems for substrate uptake
- possible to calculate the yield of biomass from a defined amount of substrate -> how efficiently a particular nutrient is being used
How is the growth rate set in a continuous system?
-growth rate is set by the dilution rate
-D=F/V
D=dilution rate (h^-1)
F=flow rate (l/h)
V=reactor volume (I)
mean retention time = V/F (reciprocal)
How can the net biomass balance be described under steady state conditions?
dx/dt = rate of growth in the reactor - rate of loss from the reactor
aka
dx/dt = μx - Dx
-At steady state the rate of growth = the rate of loss, aka dx/dt = 0, therefore…
μx=Dx and μ=D
How can chemostats be used to measure growth rates?
Experimentally detemining growth rates
- grow cells with a limiting concentration of a nutrient and start with a low dilution rate (D)
- let biomass reach steady state
- then increase the D stepwise and follow the biomass at each steady state
- this eventually drops as dilution rate gets close to maximal growth rate (μmax) and unused substrate will accumulate
- cells will eventually not be able to maintain the growth rate required at the set dilution rate. Will not reach steady state and will be washed out
- in these conditions the highest dilution rate at which the steady state can be reached (Dcrit) approximates to the μmax
What can the growth rate graph show when using a chemostat to measure growth rates, and what action of the microorganisms does this reflect ?
Relationship between biomass and growth rate at a particular limiting concentration of a nutrient
- as it is the nutient concentration that stays the same and the growth rate (D=dilution rate) that is varied
- As D is increased (tends to be stepwise), cells are forced to grow faster and faster
- can do initially but struggle to continue dividing and will reach a point where the addition of fresh medium is faster than their ability to divide and they are washed out => μmax
- if working around or below Ks then increasing S will increase yield
What is a problem with studying the physiological funtion of my E.coli genes?
How can this be overcome?
-have no obvious phenotypes in batch culture when studied using reverse genetics
-can use continuous systems to investigate gene function
e.g.
E.coli has a set of genes each for a second potential nitrate reductase (required for anaerobic respiriation)
-genetic deletion of the second system (P) resulted in no obvious phenotype for anaerobic growth with nitrate whilst the first system (G) was present, while deletic of G gave a strong phenotype in batch culture
-must then construt strains of E.coli expressing only one nitrate reductase e.g. P+G- or P-G+
-then examine growth in conditions where growth is limited by nitrate conc. show different phenotypes:
P+G- strain has a Ks for nitrate of 15μM
P-G+ strain has a Ks for nitrate of 50μm
As batch culture nitrate is added at 20mM, P is not even expressed in these conditions and the cell exclusively used G
-test that P is more useful that G at low conc can use a competition experiment
Give an example of the use of a competition experiement in E.coli
- in a continuous system make a 50:50 mixture of P-G+ and P+G- (having first constructed the strains so that they have different Ab resistance gene so can be quantified in viable counts after treatment with Ab)
- set system so that nitate is the limiting factor for growth
- P outcompetes G (P+G- more successful than P-G+) when nitrate is limiting
- when nitrate is in excess G outcompetes P
Conc. - having both P and G alllows cells to grow efficiently over a wide range of concentration of nitrate and the role of P is to allow the cell to use v low conc of nitrate, could not do with G alone
