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Growth of microorganisms in culture: example of the phytoplancton - - PowerPoint PPT Presentation
Growth of microorganisms in culture: example of the phytoplancton - - PowerPoint PPT Presentation
Growth of microorganisms in culture: example of the phytoplancton Christophe Six UE Evolution of marine phytoplancton UE Evolution of marine phytoplancton and biogeochemistry Cultures of marine cyanobacteria Master 2 nd year What is
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How to measure the growth of a phytoplankton culture ?
Monitor the number of cells in the culture function of time
- Counting slides (Malassez, Thoma, etc…)
250 µm 250 µm 50 µm 200 µm
= 0,01 mm3 = 0,01 µL = 10-5 mL
200 µm
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- Cell counter (type Wallace H. Coulter)
Detection of conductance
- f the electrolyte
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- Flow cytometry
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Monitoring a parameter that varies proportionally to the cell density
- Photosynthetic pigments
- Concentration of elements (C, N)
- Absorption of non-pigmented material (OD 750 nm)
But be careful to acclimation !
How to measure the growth of a phytoplankton culture ?
0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1 400 450 500 550 600 650 700 750 800 Longueur d'onde (nm) Absorbance (UA)
Non pigmented material Chl a
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Growth in continuous culture: Turbidostat
Sampling
Culture medium
Peristaltic pump
The culture is constantly diluted Culture Trash
- Constant volume of culture
- Constant cell density
- Constant growth rate
- No limitation
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Growth in closed medium ‘batch cultures)
Cell density
Growth is limited by the capacities of the culture medium
Time
Latence phase Exponentielle phase Transition phase Stationnary phase Decreasing phase
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Latence phase
- Abrupt medium change = stress
- ajustment of the physiology to the new medium
- All transferred cells are not viables
- The duration and dynamics of the latence phase is often dependent on the mother culture
Temps Cellules
Culture mère Cultures filles
Temps Cellules
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The exponential phase of growth 2 1 4
t 1 t 2 t 3 Binary division
Temps Cellules
8
Cells Time t 4
Time
1 2 4 8
cells
1 2 3 4
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N = N0 . ekT
N0 K : growth constant (= growth rate µ
µ µ µ)
Time Cell density
The exponential phase of growth
ln(N2) – ln (N1) K = t2 – t1 N1 N2 t2 t1
Time Cell density
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. Generation / doubling time tg or td : The time necessary for a cell to divide tg = ln(2) / k
Generation 0 1 2 3
The exponential phase of growth
. Number of divisions per day n :
Generation time tg = 1 day n = 1 division per day Growth rate K = 0,69 jour-1
!
n = 1/ tg => n = k / ln(2)
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The stationnary and decreasing phases
- Change of the physiology of the cells in response to the limitation
Resistance
Time Cells
- Dynamics of these phases mal connue
Time Cells Time Cells
k
k
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Measuring a growth rate experimentally
. Estimation from two time points ln(N2) – ln (N1) K =
30 000 000 35 000 000
ion
Time (day) 1 2 3 4 5 6 7 8 9 10 11 12 cell/mL 1 849 596 960 232 1 235 454 2 001 565 2 945 654 5 621 245 10 236 458 20 804 561 26126876 29463189 30109473 30533249 28112286
K = t2 – t1 ln(26126876) – ln (5621245) K = 8 – 5
5 000 000 10 000 000 15 000 000 20 000 000 25 000 000
5 10 15
Time
Cell concentratio N2 N1 t1 t2
K = 0.5121
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Time (day) 1 2 3 4 5 6 7 8 9 10 11 12 cell/mL 1 849 596 960 232 1 235 454 2 001 565 2 945 654 5 621 245 10 236 458 20 804 561 26126876 29463189 30109473 30533249 28112286 Ln (Cell/mL) 14,4305 13,7749 14,02695 14,50944 14,89584 15,54206 16,14147 16,85068 17,07848 17,19865 17,22035 17,23433 17,151717
. Estimation from a natural logarithm regression ln(N) – ln (N0) K = t ln(N) = kt + lnN0
=>
Slope of the regression
Measuring a growth rate experimentally
Ln (Cell/mL) 14,4305 13,7749 14,02695 14,50944 14,89584 15,54206 16,14147 16,85068 17,07848 17,19865 17,22035 17,23433 17,151717
5 000 000 10 000 000 15 000 000 20 000 000 25 000 000 30 000 000 35 000 000
5 10 15
Time
Cell concentration 10 12 14 16 18 20 5 10 15 Ln (Cell concentration)
y = 0,5617x + 12,8 R
2 = 0,9904
10 12 14 16 18 20 2 4 6 8 Ln (Cell concentration)
Time (day)
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Exercise: The influence of light on the growth of a cyanobacterium
Synechococcus sp. WH8102 15 25 50 85 200 700
µmol photons . m-2 . s-1
Strain isolated from oligotrophic waters
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What is the impact of light irradiance on the growth of Synechococcus sp. WH8102 ? Experimental protocole :
- The cultures were transferred at low cell density
- The number of cells was determined by flowr cytometry for several days