Microbial growth
Steve Zinder MBL June 11, 2013
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
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)
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
Growth in batch culture
dN/dt = µN N = number of cells present
µ = specific growth rate (a.k.a. “k”) = 0.69/Td
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
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
Measurement by qPCR
Measurement of Dhc in PCB utilizing mixed culture AEM 73:2513, 2007
Assumptions?
Life in the slow lane: Pelagibacter growth in seawater
10x increase (3.3 doublings) in 9 days ≈ 2.7 day Td
Colonies from soil dilute medium
From AEM 68:2391 (2002)
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
Growth ain’t just binary fission: Epulopiscium
Metabacterium From: Angert, Nature Rev. Microbiol 5:214, 2005
Multiple cells and budding
Rhodomicrobium vannielii
4 buds/hyphum
Actinobacterial filamentous growth
Growth curve revisited
Problems?
Lag phase
Inoculum with 10% viability
From: Neidhardt et al, Physiology of the Bacterial Cell, Sinauer, 1990
Diauxic (biphasic) growth of E. coli on glucose and lactate
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)
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
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
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
Growth physiology
Neidhardt book
tRNA/total RNA
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)
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
“Wall growth” = Biofilms
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)
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