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Growth of microbialcultures

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The dream of a bacterium...

• Microorganisms proliferate mostly by binary division.

• Hereby they are potentially immortal.

• By rapid and exponential growth ressources are consumed and starvation arises.

... is becoming two bacteria.

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What is your metabolism at thismoment?

! Growth rate = 0

! Maintenance metabolism

! Catabolism 100 % (net)

! Main substrates metabolised: Glucose + O2

(the only substrates used by the brain!)

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Cell division

• Biomass and length increase

• Replication of the chromosom, segregation of the daughter molecules

• Formation of new membranes and cell walls

Primitivescheme!

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Cell division

FtsZ: Tubulin-like protein

MreB: Actin-like protein

Forms helical structures alongthe membrane

filamenting temperature-sensitive mutant Z

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oCell numbers in a culture

Z = Z0 * 2g

with

Z0 = initial cell number g = number of generations

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To keep in mind:

210 = 1024 # 1000

220 # 1 million

230 # 1 billion (dt. Milliarde)

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Growth phases

A: Lag phase

B: Exponential or log phase

C: Stationary phase

D: Death phase

logarithmic

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Some terms

Generation time: time required for a bacterial cell todouble (h)

Division rate:1/generation time (v, h-1)

Growth rate: Increase per time per amount present (!,h-1)

Doubling time (td): Time required for a growthparameter as dry mass, protein and even cell numbersto double (h)

Maximum growth rate !max: Growth rate during theexponential phase (h-1)

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Growth kinetics

Exponential growth: 20 ! 21 ! 22 ! 23 ! 2n

N = N0 • 2n => lgN = lgN0 + n • lg2

n = lgN - lgN0 / lg2

Division rate (v): v = n/t [ h-1 ]

v = lgN - lgN0 / lg2 (t - t0)

Generation time (g): g = t/n [ h ]

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Growth kinetics

Exponential growth: 20 ! 21 ! 22 ! 23 ! 2n

Kinetic follows 1st order reaction:

!x = dx / dt (= change of biomass to every time point)

Growth rate ! is constant

x = x0 • e! • t

For doubling of x0 to 2 x0

2 x0 = x0 • e! • td => ln2 = ! • td

! = ln2 / td ( = 0.693 / td)

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Growth kinetics

Exponential growth: 20 ! 21 ! 22 ! 23 ! 2n

Kinetic follows 1st order reaction:

!x = dx / dt (= change of biomass to every time point)

Growth rate ! is constant

x = x0 • e! • t

For doubling of x0 to 2 x0

2 x0 = x0 • e! • td => ln2 = ! • td

! = ln2 / td ( = 0.693 / td)

V = 1/ td

If td = g

! = ln2 • v

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Direct conting

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Turbidity measurements of microbial growth

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The growth yield depends on

- Catabolic pathway or other possibilities of energy conservation

- Type of available carbon sources

- Energy demand for maintenance

The growth yield is better predictable thanthe growth rate. Often the specific growthyield (e.g. per mol of glucose consumed) isused.

Growth yield (Y)

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Essentials of successfulcultivation

• Scientific question/ hypothesis• Medium choice• Carbon and energy source• Other media components• Gelling agent• Inoculum and interaction• Growth conditions, temperature, pH, atmosphere• Incubation time

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What do I need for successfulcultivation

• Organism source• Media• Culture vessel• Incubator• Detection system

• Creativity

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Chemical composition of a prokaryotic cell

Molecule Percent of dryweight

Protein 55

Polysaccharide 5

Lipid 9

Lipopolysaccharide 4

DNA 3

RNA 19

Amino acids and precursors 1

Sugars and precursors 2

Nucleotides and precursors 1

Inorganic ions 1

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Macro elements of a prokaryotic cell

Macro element Percent of dry weight

Carbon (C) 50

Hydrogen (H) 8

Oxygen (O) 20

Nitrogen (N) 14

Phosphorus (P) 3

Sulfur (S) 1

Potassium (K) 1

Magnesium (Mg) 0.5

Calcium (Ca) 0.5

Iron (Fe) 0.2

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Trace elements of prokaryotic cell

Trace element Cellular function (example)

Cobalt (Co) Vitamin B12

Copper (Cu) respiration, photosynthesis

Molybdenum (Mo) nitrogenase, nitrate reductase

Nickel (Ni) hydrogenase

Selenium (Se) Hydrogenase, formate dehydrogenase

Tungsten (W) Formate dehydrogenase

Vanadium (V) Vanadium nitrogenase

Zinc Alcohol dehydrogenase, RNA and DNApolymerases, DNA-binding protein

Iron (Fe) Cytochromes, catalases, oxygenases

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General requirements in microbiological media

• Energy source• Source of macro elements (including carbon

and nitrogen)• Source of trace elements• Buffer• Growth factors (including Vitamins or

amino acids)

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Chemically defined versusundefined (complex) media

Defined medium for E. coli Undefined medium for E. coli

K2HPO4 7 g Glucose 15 g

KH2PO4 2 g Yest extract 5 g

(NH4)SO4 1 g Peptone 5 g

MgSO4 0.1 g KH2PO4 2 g

CaCl2 0.002 g Destilled water 1000 ml

Glucose 5-10 g

Trace element solution

Destilled water 1000 ml

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Isolation of microorganisms into purecultures

A culture containing only a single kind of microorganism, originate from a single cell (monoclonal).

Most common is the isolation of microbes by the use of solid media. Alternatives: serial agar dilution, serial liquid dilution

Highest priority: Avoid contaminants!

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Why do we need pure cultures?

• Precise physiology

• Biochemistry and structure

• Taxonomy

• Genetics

• Reproducibility of experiments

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The majority of microbespresent in nature have no

counterpart among previouslycultured organism.

4700 validly described speciesversus

about 20000 species in 1L sea waterabout 40000 species in 1g soil

total of 10 millions (estimations)

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How to apply cultivation?

• Estimation of bacterial numbers using MPN

• Selective enrichment and isolation ofmembers belonging to one physiologicalgroup

• Culturing an abundant phylotype

• Cultivation of all microorganisms from amarine environment

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Estimation of bacteria numbersby tenfold dilution series

“MPN - most probable number”

• Estimation of viable microorganisms

• Obtained by the statistical method ofmaximum likelihood

• Many variations in cultivation conditionspossible (complex - defined medium)

• Detection of growth essential

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Quelle: Brock Biology of Microorganisms

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Chemostat

In a chemostat cellscan be cultivatedunder contantconditions in anexponential phasewith ! < !max

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Essentials of successfulcultivation

• Scientific question/ hypothesis• Medium choice• Carbon and energy source• Other media components• Gelling agent• Inoculum and interaction• Growth conditions, temperature, pH, atmosphere• Incubation time

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Culturing anaerobes

• Oxygen free media.

!Remove oxygen

!Keep it away

• Low redox potential

!Addition of reducing agents

• Optional: oxygen (redox) indicator

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Culturing anaerobes

• Flush headspace (Hungate-technique)

• Cultivation in sealed anaerobic jars orchambers

• Cultivation without gaseous headspace

• Co-culture with oxygen consuming bacteria

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The Widdel-flask