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Microbial growth
Steve Zinder MBL June 11, 2013
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The obvious goal of any bacterium is to become bacteria. In e-mail signature of: Lizzie Wilbanks – UC Davis – Course student ‘10, TA ’11 – Queen of the berries
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E. coli dividing • Plos Biol. 3:295, 2005 • YFC-labeled cells
grown in slide culture on Luria broth at 30 oC
• 505 cells (~9 doublings) in 315 min ~34 minute Td
• Cells with initial poles (old) grow slightly slower (1%/gen) • Old cells more likely to die than newer ones • More protein aggregation at poles? (EMBO 29:910. 2010)
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E. coli dividing • E. coli dividing optimally doubles every 20 minutes
• Makes up to 20,000 new ribosomes, • > 1,000,000 molecules of >1000 different
proteins • >22,000,000 lipid molecules • ≥ 2 simultaneous chromosome replications (each
takes 40 minutes) • If growth continued unimpeded, a culture’s weight
would be > than the Earth’s after 48 hours
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Growth in batch culture
dN/dt = µN N = number of cells present
µ = specific growth rate (a.k.a. “k”) = 0.69/Td
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Measuring culture biomass by turbidity Beer’s law: A(λ) = e(λ) l c
A = absorption at wavelength λ c = concentration of solute l = length of light path e(λ) = (molar) absorptivity constant
What are we measuring?
From: Neidhardt et al, Physiology of the Bacterial Cell, Sinauer, 1990
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Other ways to measure growth
Metabolic product: CH4 Thermophilic Methanosaeta Arch. Microbiol. 146:315, 1987
Cell protein and microscopic counts Metabolic product: vinyl chloride Dehalococcoides culture Science 276:1578, 1997
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Measurement by qPCR
Measurement of Dhc in PCB utilizing mixed culture AEM 73:2513, 2007
Assumptions?
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Life in the slow lane: Pelagibacter growth in seawater
10x increase (3.3 doublings) in 9 days ≈ 2.7 day Td
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Colonies from soil dilute medium
From AEM 68:2391 (2002)
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Organisms carrying out biogeochemical processes often grow on a geological time scale
Gas production from hexadecane by a methanogenic consortium. 4C16H34 + 45 H2O --> 49CH4 + 15HCO3
- +15 H+ ∆G’ = -1430 kJ/rxn or -29.2 kJ/CH4 From: Nature 401:266, 1999
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Growth ain’t just binary fission: Epulopiscium
Metabacterium From: Angert, Nature Rev. Microbiol 5:214, 2005
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Multiple cells and budding
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Rhodomicrobium vannielii
4 buds/hyphum
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Actinobacterial filamentous growth
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Growth curve revisited
Problems?
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Lag phase
Inoculum with 10% viability
From: Neidhardt et al, Physiology of the Bacterial Cell, Sinauer, 1990
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Diauxic (biphasic) growth of E. coli on glucose and lactate
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Effect of substrate concentration on growth
[substrate]
Reac
tion
rat
e
Km
Vmax
Michaelis Menten enzyme kinetics V = Vmax * [S] [S] + Km
[substrate]
Grow
th r
ate
Ks
µmax
Monod growth kinetics µ = µmax *[S] [S] + Ks
Liebig’s law of the minimum Only one thing limits growth at any one time. (bottom up control)
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The chemostat
D = flow/volume = 1/Retention time e.g. if volume is 10 L and flow is 1 L/h, D = 0.1 h-1 At steady state µ = D
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Uses: Constant growth rate - omic studies Feeding toxic substrates at low concentration Competition experiments
Chemostat kinetics
S = Ks at 0.5 µmax J. Gen Micro 14:601, 1956
Add Pirt plot
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Competition
Physiological basis of the selective advantage of a Spirillum sp. in a carbon-limited environment.
Matin A., Veldkamp H. (1978) J Gen Microbiol. 105:187
Pseudomonas: µmax = 0.64 h-1, Ks = 91 µm Spirillum: µmax = 0.35 h-1, Ks = 23 µm
D = 0.05 h-1 P/S
D = 0.24 h-1 P/S
D = 0.32 h-1 S/P
R vs K selection
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Growth physiology
Neidhardt book
tRNA/total RNA
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Growth physiology – maintenance energy Pirt double reciprocal plots: 1/yield vs 1/dilution rate
Butyrivibrio fibrisolvens Maint coeff = 0.049
Bacteroides ruminicola Maint coeff = 0.135
Shifts from acetate and propionate to lactate (less ATP) at high growth rates
Selenomonas ruminantium
Russell and Baldwin AEM 37:537 (1979)
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Other continuous feed methods
• Turbidistat – dilute when culture reaches certain turbidity
• pH auxostat: substrate neutralizes pH change by organism’s growth – Conversion of formic or acetic acid to CO2
• Gradient cultures – e.g. SOB
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“Wall growth” = Biofilms
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Growth on slides at 90o C
Suspending cover slips in Boulder Spring a boiling water spring extensively studied by Brock and coworkers
Microbial colonization of cover slips incubated in boiling water springs -
J. Bacteriol. 107:303 (1971)
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Growth on slides
EM pictures of some of the Boulder Spring "bacteria" which were clearly Archaea.
Uptake of radiolabeled lactate, acetate, or thymidine by Boulder Spring bacteria attached to slides