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Gene to Function - prokaryotes > Lecture 12 - Continuous culture of microbes > Flashcards

Lecture 12 - Continuous culture of microbes Flashcards

1
Q

What are the features of the stirred tank reactor?

A
  • 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
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2
Q

What is the vessel constructuion of a stirred tank reactor?

A
  • stainless/mild steel lined with glass/plastic

- smooth and capable of withstanding cleaning

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3
Q

How can stirred tank reactors be sterilised?

A
  • 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

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4
Q

How is temp and pH regulated in a stirred tank bioreactor?

A
  • 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

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5
Q

What are the features of stirring and aeration of a stirred tank bioreactor?

A
  • 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
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6
Q

What does the combination of stirring and aeration result in in a stirred tank bioreactor?

A
  • 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
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7
Q

How is foam control achieved in a stirred tank bioreactor?

A

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
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8
Q

What control is most necessary in a STR?

A

-pH, temperature, aeration, mixing and foam control

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9
Q

What alternative fermenter can be used instead of a stirred tank bioreactor, and why might this be advantageous?

A

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

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10
Q

What are the features of an Air lift fermenter?

A
  • 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
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11
Q

What are the three fermenter operation modes?

A
  • Batch fermentation
  • Fed Batch fermentation
  • Continous culture
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12
Q

What are the features of batch fermentation?

A
  • closed system in which a fixed volume of medium is inoculated
  • culture grows and media is altered as a result of growth
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13
Q

What are the features of Fed batch fermentation?

A
  • 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
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14
Q

What are the features of a continuous culture?

A
  • 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
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15
Q

What is a big advantage of continuous culture?

A
  • 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)
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16
Q

Through what equation can growth pattern be represented by and how was this formulae derived?

A

Monod equation

-derived empirically by observing growth of microbial cultures

17
Q

What is the monod equation?

A

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)

18
Q

What are the features of the Monod equation?

A
  • 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
19
Q

How is the growth rate set in a continuous system?

A

-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)

20
Q

How can the net biomass balance be described under steady state conditions?

A

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

21
Q

How can chemostats be used to measure growth rates?

A

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
22
Q

What can the growth rate graph show when using a chemostat to measure growth rates, and what action of the microorganisms does this reflect ?

A

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
23
Q

What is a problem with studying the physiological funtion of my E.coli genes?
How can this be overcome?

A

-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

24
Q

Give an example of the use of a competition experiement in E.coli

A
  • 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

25
Q

What is the formula for Quantataive growth?

A

Nt = N0X2^n

Gene to Function - prokaryotes (12 decks)

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