Growth and Kinetics IV batch culture Flashcards
1
Q
batch culture cultivation
A
- most commonly for kinetic studies in batch culture, we grow the organism in 50 mL volumes of a defined medium in a 250-mL glass Erlenmeyer flask – this is the so-called ‘Pirt
ratio’ of liquid:vessel volumes for optimal gas exchange. - we can inoculate them by addition of a small volume of cells in suspension or a small amount of biomass from an agar plate (ideally the former) and then time how long it takes for a culture to grow and then stop growing.
- we shake the flask very vigorously, either reciprocal shaking (side-to-side) or orbital shaking (in circles about a point) – the latter is usually better for gas exchange. Always report the revolutions/reciprocations per minute (rpm), the model of shaker and the throw or amplitude or radius of the shaking (in cm). The reason for these measurement is to calculate the gas exchange from them.
- batch culture is a closed culture – finite amount of nutrients are added, nothing is added or removed during growth, which stops when nutrients run out.
growing an autotroph better to use a wider neck flask as more CO2, more dense, more in flask by diffusion. Shake a lot as dissolve CO2 into medium.
If it’s growing at low pH then grow as static as CO2 solubility changes.
2
Q
quantifying growth
A
- we could count the cells by hand using a microscope or using an instrument such as CellFACTS or other flow cytometer, but this is folly. Consider how a culture grows – before division cells enlarge –this is more biomass but same cell number. As such, cell number is not very useful.
- we usually quantify dry weight of biomass (dry wt) in g dry biomass. Rather than drying samples from cultures, we normally calibrate the organism so that we know what the relationship is between the optical density (kind of the same as turbidity but measured differently (measuring how much light goes through however turbidity is how much light back scatters) at several wavelengths and the concentration of dry biomass (g dry biomass/L).
- if we know the above, we can just measure the optical density and derive the dry weight of biomass.
- shorter wavelengths are generally more sensitive but you need to check several as not all are linear and some are only useful in a small range.
- we will often know that e.g. OD440 = 0.1 at 28 mg dry biomass/L [e.g.for example on the right].
- therefore if we have an OD440 of 0.85, that would be 238 mg/L, so in a 50 mL culture, 12 mg dry biomass.
- why do we write “OD440 = 0.1 at 28…” and not “…1.0 = 280…”?
Because often calibrations aren’t linear that far and more importantly, no spectrophotometer can measure OD for any wavelength > 0.9 accurately. If we get a reading of 0.95, we do a 1/10 dilution of the culture in buffer and re-measure and will get e.g. 0.35, which means 3.5 in the culture before dilution.
3
Q
basis for growth curves
A
- the amount of biomass formed (χ) is what we have
just discussed – remember, amount, not concentration –
OD440 (etc) convert to concentration, and you then need
to use culture volume to get amount - if we measure χ at time intervals and plot against time,
we have the basis of a growth curve, but as growth
itself is logarithmic, we have to use a log axis for the y-axis. - growth really obeys ln (natural logarithm) but it’s hard
to plot, so we use log10 (common logarithm) and
convert during equations. - the part of the growth curve in which growth is logarithmic is the log phase or exponential phase – the
curves on the right only show this phase, but can you
see how the top curve misleads?
4
Q
real growth curves
A
- in reality, cells often need time to adapt to changes in
conditions e.g. if the inoculum was previously grown at 30
°C and you have just put it into medium at 45 °C, it might
need a time to produce heat-tolerance proteins. - if the inoculum were grown on succinate (an intermediate of Krebs’ cycle) but you are now growing it on D-(+)-glucose, it might need time to express enough of each enzyme of the
glycolytic pathways it uses. - if the inoculum was grown on a defined medium with lactose and now you have moved it to the same medium with the addition of penicillin G, it might require time to express penicillin G-resistance proteins.
- this initial phase for acclimatising/expressing enzymes etc is the lag phase.
- in the lag phase (adaptation), the carbon and/or energy source may well
be being consumed, but you won’t see a change in the
amount of biomass. - not always present e.g. if inoculum was grown under
identical conditions
more realistic growth curve, showing lag phase, log phase (exponential phase),
stationary phase and death phase.
5
Q
log phase (exponential phase)
A
- in the log phase growth follows a true exponential pattern and we can use kinetic models to describe this.
- cells in late-exponential phase are particularly good quality in terms of being relatively consistent in properties and are often harvested for downstream work.
- more cells are being produced than are dying, the
population is growing.
6
Q
Stationary phase
A
- in stationary phase the population is no longer
growing and has reached a static population in which
the number of dividing cells are equal to the number of
dying cells. - normally the C or E (electron donor) source doesn’t run out first – N source or one of the metals (Cu or Zn) (zn mostly) usually seems be the thing that actually runs out in most media. Could be something is built up in medium that is inhibition growth such as cells are producing acid then too many protons.
7
Q
Death phase
A
The population as a whole is dying, and many more cells are dying than are dividing to make new cells.
8
Q
kinetic parameters
(maths only on exponential phase)
A
- from the curve in exponential phase we can determine
the doubling time (tD) and the specific growth rate (µ). Both measure form a variation of hours - the former is easy to determine visually.
1) find any value on y-axis.
2) double it.
3) read off the values on the x-axis.
4) the difference between them is tD
.
RED lines on right are original y value (50) and doubled
(100) – on upper and lower edges, respectively.
GREEN are reading down to the x-axis – the interval is 1
h (doesn’t look like it but read from inner edges of green
lines!). - then, we take tD, we can determine µ:
µ = (ln 2)/tD = 0.693/tD
so from the 1 h doubling time we just obtained:
ln 2/1 = 0.693 h^-1
. - We conventionally report µ in h^-1
(for reasons that will become clear in Kinetics V next week), so if you obtained tD in minutes, convert it first!
9
Q
the growth equation
χ this is actually called kai and has one s-shape line and a straight line looking like an X
(In my coursework i will get a question that asks calculate the maximum amount of biomass formed in the experiment which is simply most biomass formed at any time point minus the starting amount of biomass. This does NOT involve the question (χ = χo^× eµt)
A
- allows you to determine the amount of biomass (χ) during
exponential phase based on the initial amount (χo, at start of exponential phase) if you know the specific growth rate (µ) and how long has passed since the start of exponential phase(t):
χ = χo^× eµt - not actually that useful!
- if we know how much substrate was consumed (ΔS)
during growth (meaning we measured it!) we could also
determine the specific molar growth yield (Y)
Y = χ/ΔS
Y will be in “[mass] dry biomass/[amount] of [whatever S
is]” e.g. if 120 mg dry biomass formed during growth in which 37 mmol acetate (CH3COO-) was consumed:
Y = 3.24 mg dry biomass/mmol acetate consumed OR
Y = 3.24 g dry biomass/mol acetate consumed OR
Y = 1.62 g dry biomass/mol acetate-carbon consumed etc (2 C so we divide value by 2)
10
Q
batch culture is flawed
A
- products of metabolism build up in the culture – e.g. causing pH to fall – over time.
- you can only change one parameter at a time.
- not very realistic – very seldom in Nature is an organism growing in a high concentration of substrate and then sitting in its waste (changes pH as there will be protons)
- all of this ultimately lead to the formation of the chemostat as an improvement – see next lecture!
