Growth and Kinetics V: continuous culture: the chemostat

Question 1

batch culture problems

Answer 1

• cells are at unnatural levels of substrate at the start e.g. using 0.5 mM acetate would be environmental relevant to soil organisms as that is the standing concentration in soil but you’d get v little growth. In situ there is a continuous feed of acetate so the flux (thing build up and then leaving) of acetate through soil is very high. You could keep adding more acetate each time it is consumed, this is a fed batch culture, but we normally use 10 mM acetate instead in batch culture for speed!
• cells are in unnatural levels of waste products e.g. H3O+
  ions, fatty acids etc during growth that get washed away in situ.
• if you change any parameter, you automatically change the growth rate (µ). If you wanted to look at expression of a given gene at 15 °C versus 37 °C
  for Escherichia coli (optimal growth 37 °C), the growth rate would be slower at the lower temperature, so has gene expression changed as a result of temperature or growth rate or both?

Question 2

continuous culture types
and important chapter form book

Answer 2

• there are lots – we are only concerned with the chemostat in this module.
• you may also see auxostats (feed themselves), pHauxostats, cytostats (mini ones) like ones set), turbidostats (grow organism on light) etc when you are reading around the subject

Boden R and Hutt LP (2018) Determination of kinetic parameters and metabolic modes using the chemostat. In: Steffan R (ed) Consequences of microbial interactions with hydrocarbons, oils and lipids: biodegradation and bioremediation. Handbook of hydrocarbon and lipid microbiology. pp. 1-42.

Question 3

bioreactors
(bioreactors are not chemostats)

Answer 3

• stirred reactor.
• oxygenated (or sparged with e.g. sterile argon to remove oxygen) usually with sterile compressed air.
• dO2 measured and can be set.
• pH measured and can be set and auto-titrated with sterile acid and base.
• temperature is set and maintained by heating elements/jackets and cold-fingers.
• outflowing gas runs through a condenser to keep volume stable.
• posh ones measure pO2 and pCO2 (analyser) in the outflowing gas.
• can be run as batch cultures or continuous cultures.

media reservoir
pump
every drop of liquid going in goes out so volume remains the same. The liquid forced out goes into waste vessel due to aeration
air filter so that air going in is sterile

if what’s inside unstable you don’t want air so you use argon as its more stable

Question 4

the chemostat

what does V mean

Answer 4

• keeps the culture chemically stable with respect to one defined limiting substrate. This could be the carbon or energy source, the nitrogen source, the iron source, the respiratory electron acceptor source – anything.
• at any point in time, the limiting substrate has a concentration of 0.000 M at any time. This is by definition, so it’s not e.g. very low, it is actually zero. We intentionally keep it that way. This is quite handy for toxic substrates as they are never at high concentrations.
• culture is grown in a bioreactor that holds a culture of (V mL) in volume.
  This is the culture volume not the volume of the container as a whole but people do say “vessel volume” colloquially!
• a defined medium is used in which the limiting substrate, S, is held quite low so that it runs out before anything else.
• medium flows through the bioreactor via a pump (in) and usually a weir overflow (out). The medium flows at a rate of F – normally used in mL/min but we must use mL/h for calculations!
• the hydrodynamic dilution rate D is determined from F/V
  e.g. if F = 76 mL/h and culture is 400 mL then D = 0.190 h-1
  e.g. if F = 76 mL/h and culture is 10,000 mL then D = 0.008 h-1
• D of e.g. 0.5 h-1 means that half (0.5) of the culture volume of medium is replaced every hour.
• reactor is grown in batch culture until late exponential phase when [S] is very low, then pumps are turned on.
• as [S] is effectively 0 mM when pumps are turned on, the first droplet containing x mmol S lands and that x mmol is degraded near instantly, returning the system to [S] = 0 mM. This repeats with each droplet.
  the system will self-settle over time until [S] is truly zero and a steady state is achieved.
  can’t measure acetate in the vessel, it will always disappear

Question 5

steady state

Answer 5

• if the specific growth rate µ > D then the amount of biomass χ will keep increasing even though the pumps are on, until µ = D.
• if the specific growth rate µ < D then the amount of biomass χ will decrease
  (‘washing out’) until µ = D.
• if µ = D then χ becomes constant: a physiological steady state has been reached.
• normally we would determine χ lots of times (around 5 culture volumes) at this point and ensure it is really stable and we would measure [S] too to ensure it really is not present in the vessel. It is not a steady state until 5V of medium has passed through the reactor, minimum – so this is very time-consuming and requires small reactors! 500 mL is typical for kinetic work, 10,000 mL for growing biomass to use in experiments.
• SO as you can see, as D = µ at steady state, we can define µ and fix it at a set value whilst we change e.g. temperature etc etc.
• there is a limit on D – all organisms have a maximum value called Dcrit (= µMAX) above which the culture can no longer grow as D is above their maximum growth rate. You may see this, and its determination using wash-out kinetics

Question 6

specific molar growth yield

Answer 6

• the amount of biomass formed (χ) is, alone, not that useful – compare:
• 1 g dry biomass formed during growth on 10 mM acetate
• 1 g dry biomass formed during growth on 100 mM acetate
• same amount of biomass but much more substrate was present at the start. If we measure substrate depletion during an experiment, we could do something more useful
  e.g. if we know how much acetate was consumed:
• 1 g dry biomass formed when 10 mmol acetate consumed.
• 1 g dry biomass formed when 90 mmol acetate consumed.
• we could divide through and normalise χ to substrate, and this gives us the specific molar growth yield (Y):
• 0.1 g dry biomass formed per mmol acetate consumed.
• better written as 100 g dry biomass per mole acetate consumed!
• 11.1 mg dry biomass formed per mmol acetate consumed.
• again, better as 11.1 g dry biomass per mole acetate consumed!
• units can be very variable – pay close attention to them! e.g.
• Y = 100 g dry biomass/mol acetate consumed is the same as:
• YC = 50 g dry biomass/mol C [as acetate = C2]
• “YC” is specific molar growth yield per mole of C.
• myriad others, particularly in more complex kinetics.

Question 7

More on specific molar growth yield

Answer 7

• Y and so on allow us to compare organisms properly – if you have an organism growing on D-(-)-fructose (C6) and one growing on acetate (C2) and one growing on succinate (C4), you can make more useful comparisons by working
  per mole of C or whatever.
• because the chemostat is always at [S] = 0 at steady state, we can thus measure [S]i in the inflowing medium and determine the amount of substrate consumed
  (ΔS) given ΔS = [S]i at steady state for a 1 L culture, so you can just adjust to any size culture by using ΔS = [S]i × V (if V in L) e.g. if a 400 mL culture, if [S]i = 30 mM then ΔS = 30 × 0.4 = 12 mmol. Remember, mmol, not mM or mmol/L!!!
• SO, we can really easily convert χ to Y in a chemostat. In the days before we realised substrates in the medium reservoir degrade and it needs to be measured 2-3 times a day, this meant Y was often assumed based on how much S was added to the reservoir, of course. Bit dodgy but not that inaccurate, actually.

Question 8

Y versus D and the Pirt kinetic

Answer 8

• if we grow chemostats at e.g. 10 values of D, each to
  steady-state and then determine Y, we can plot Y versus D,
  and we get a Pirt plot, which is very similar to a Michaelis
  plot
• as in BIOL131Z you learned that Michaelis plots are an
  annoying shape, so we USED TO USE Lineweaver-Burk
  plots (1/v versus 1/S) to linnearise the data and make
  determination of VMAX easier, we USED TO USE the same
  method for determining YMAX.
  [I say “used to use” because in real life, no one uses L-B
  plots anymore, they’re too inaccurate but they’re a useful
  tool for understanding the data!]
• if we do this, YMAX = the y-intercept just as VMAX would be for an enzyme.
• YMAX is the maximum specific growth yield coefficient
  and shares units with the Y used to determine it. It’s not
  real, it’s a coefficient determined to allow us to compare
  different organisms that grow only at v different rates by
  normalising the data to kind of see “Y an infinite D”.
  Michaelis plot would be this shape with v on y-axis and S
  on x-axis. The Pirt plot would have Y on the y-axis and D on the x-axis.
  There is also a Monod plot of µ on the y-axis and [S] on
  the x-axis which we don’t care about in this module,
  I’m just telling you so you don’t confuse the plots!
  Ŷ is the same as YMAX - may see it in Pirt’s work.

Question 9

maintenance

Answer 9

• this is a measure of cost-of-living. There are many versions of it, but I use Pirt’s version, or Pirtian maintenance, mS. (small s) change s to whatever the limiting substrate is
• mS is the maintenance coefficient with respect to substrate (it could be with respect to nitrogen source or electron donor or whatever, instead).
• mS is a measure of how much substrate is needed to maintain 1 g dry biomass per hour so usually has ‘funny units’ like e.g. 32 mg thiosulfate per g dry biomass per hour or 76 µmol acetate per g dry biomass per hour.
• if you grew Escherichia coli (optimal growth pH 6-8) at pH 7 and pH 5, the mS at pH 5 would be much, much higher and Y would be much lower. Think about what it is spending its C/E source on
• the gradient of a 1/Y versus 1/D plot is the specific maintenance rate (a) which is useless on its own, but we can use it as mS = a/YMAX.
• because gradient of double-reciprocal plots are really dodgy, even by the 1950s, we stopped doing this, and we used an analogue of the Hanes-Woolf plot (S/v versus S plots), except that as D/Y = q, we call it a ‘q versus D’ plot and q is the metabolic quotient aka the specific rate of substrate uptake.
• in reality, we determine mS and YMAX by hyperbolic fitting which I will be uploading a video on shortly and you can practice it at your own pace ready for our assessment next month.