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