19.01.

2009

Cultivation of sediment
microorganisms

Martin Könneke www.icbm.de

Cultivation of microbes

•  What’s so important about cultivation

•  Essentials of cultivation
•  Essentials of isolation
•  How to apply cultivation
•  Cultivation of anaerobes

Martin Könneke www.icbm.de

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Recommended literature

Accessing uncultured Microorganisms

(K. Zengler 2008)

Principles of enrichment, isolation, cultivation

and preservation of Prokaryotes
(J. Overmann 2006)

Early milestones in microbiology

•  Louis Pasteur - Settled spontaneous generation

controversy (1864)

•  Robert Koch - Methods for study bacteria in pure

culture (1881)

Martin Könneke www.icbm.de

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Quelle: Brock Biology of Microorganisms

Quelle: Brock Biology of Microorganisms

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Quelle: Brock Biology of Microorganisms

Quelle: Brock Biology of Microorganisms

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Koch’s Postulates
•  The microorganism should be constantly present in
animals suffering from disease, but should not be
present in healthy individuals

•  Microorganism must be cultivated in pure culture

outside the diseased animal

•  Healthy animals infected with these pure cultures

must display the characteristic disease symptomes

•  Microorganism should be reisolated from the

experimental animals and shown to be the same
Martin Könneke www.icbm.de

Early milestones in microbiology

•  Louis Pasteur - Settled spontaneous generation

controversy (1864)

•  Robert Koch - Methods for study bacteria in pure

culture (1881)

•  Sergey Winogradsky - Concept of lithoautotrophy

(1889)

•  Martinus Beijerinck - Selective cultures (1901)

Martin Könneke www.icbm.de

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Use in old and modern

biotechnology
•  Food production
•  Identification of infective agents and
diseases
•  Production of pharmaceuticals
•  Precursor for chemical products

Martin Könneke www.icbm.de

Scientific use of cultivation

based methods
•  Physiology
•  Biochemistry
•  Identification
•  Quantification

To study microorganisms in the lab, it is usually

necessary to culture (grow) them.

Martin Könneke www.icbm.de

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Nature Reviews Microbiology Vol. 5, Oct. 2007

Martin Könneke www.icbm.de

•  Pure cultures provides whole genomes

essential to evaluate metagenomes

•  Proof of hypothesis constructed from

metagenomes

•  Complete reconstruction of whole genomes

is still not possible

•  But, metagenomes can provide hints to

physiological properties

Martin Könneke www.icbm.de

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Giovannoni and Stingl 2007

Essentials of successful
cultivation
•  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 successful

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

•  Creativity

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Chemical composition of a prokaryotic cell

Molecule Percent of dry

weight

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

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 DNA
polymerases, DNA-binding protein
Iron (Fe) Cytochromes, catalases, oxygenases

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 versus

undefined (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

Isolation of microorganisms into pure

cultures

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

The majority of microbes

present in nature have no
counterpart among previously
cultured organism.

4700 validly described species

versus
about 20000 species in 1L sea water
about 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 of
members belonging to one physiological
group
•  Culturing an abundant phylotype

•  Cultivation of all microorganisms from a

marine environment

Estimation of bacteria numbers

by tenfold dilution series
“MPN - most probable number”
•  Estimation of viable microorganisms
•  Obtained by the statistical method of
maximum likelihood
•  Many variations in cultivation conditions
possible (complex - defined medium)

•  Detection of growth essential

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Quelle: Brock
Biology of Microorganisms

Continuous culture- culture in

steady state

Quelle: Brock Biology

of Microorganisms

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Selective enrichment and

isolation of
an relevant physiological group

Example: Cultivation of sulfate-reducing

bacteria from the German Wadden Sea
(Antje Gittel)

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SRR at the study site Janssand, September 2005

A. Gittel, Paleomicrobiology, ICBM

Selective enrichment and isolation of

sulfate-reducing bacteria from the German
Wadden Sea (Antje Gittel)

Chemically defined medium (Widdel& Bak, modified)

Basic medium (salt concentration adapted to sea water)

Reducing agent: Sodium sulfide
Buffer: Carbonate/Carbon dioxide
Redox indicator: Resazurin
Carbon source: Lactate, acetate, or carbon dioxide
Electron donor: Lactate, acetate, or hydrogen
Electron acceptor: Sulfate

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Cultivation

Liquid dilution series in anoxic media

SO42- Lactate
Acetate
H2/CO2
Growth was stimulated in liquid dilution
Growth of sulfate-reducers cultures from each depth and with
each substrate
Production of sulfide
Variety of partial 16S rRNA genes,
Identification most of them related to known marine
sulfate-reducing bacteria
Molecular analysis of the highest
sulfide-positive dilutions

Pure cultures

Repeated application of the liquid and

deep agar dilution method (in progress)
A. Gittel, Paleomicrobiology, ICBM

Who is there?

50 cm Desulfobacula spp. H2/CO2

100 cm

H2/CO2
250 cm Desulfotalea spp. Acetate
Lactate
Acetate
Desulfosarcina spp.
Lactate
400 cm

A. Gittel, Paleomicrobiology, ICBM

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Selective enrichment and

isolation of
an abundant phylotype
Example: The abundant marine, mesophilic
Crenarchaeota

The domain Archaea

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Abundance of marine Crenarchaea

What did we know about marine Crenarchaea

•  Discovered in 1992 by Furhman et al. and DeLong

•  Account for nearly 20% of all oceanic bacterioplankton (~1028 cells)

[Karner et al., 2001]

•  Detected in marine and terrestrial habitats

•  Isotopic analyses and tracer experiments suggest possible

autotrophy [Pearson et al., 2001; Wuchter et al. 2003]

•  No cultivated representatives

•  Physiology has remained uncertain

•  May play important roles in global geochemical cycles

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Starting point

•  Detection in a tropical fish tank > Organism source

•  Molecular techniques (quantitative PCR) > screening tool

•  Some hints to autotrophy and ammonium oxidation

Steps to the pure culture

1)  Enrichment in filtered aquarium water + ammonium > increase of
phylotype and nitrite production

2)  Isolation by liquid dilution in chemically defined medium,

facilitated by filtration (size) and addition of antibiotics (archaea)

Strain SCM1
a DAPI

b FISH

Scale: 1 µm

c TEM

b SEM

Scale: 0.1 µm

Koenneke et al. Nature 2005)

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Starting point

•  Detection in a tropical fish tank > Organism source

•  Molecular techniques (quantitative PCR) > sreening tool

•  Some hints to autotrophy and ammonium oxidation

Steps to the pure culture

1)  Enrichment in filtered aquarium water + ammonium > increase of
phylotype and nitrite production

2)  Isolation by liquid dilution in chemically defined medium,

facilitated by filtration (size) and addition of antibiotics (archaea)

3)  Prove of its physiology by monitoring growth, ammonium

consumption and nitrite formation

Growth of Strain SCM1 at 28 ˚C

NH3 + 1.5 O2 → NO2- + H2O + H+ (∆G0’ = - 235 kJ mol-1)

The first nitrifyer within the domain Archaea

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Cultivating the uncultured (K. Zengler)

How many microbes can we stimulate to grow?

Simulate the environmental condition as good as possible!

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 or
chambers
•  Cultivation without gaseous headspace
•  Co-culture with oxygen consuming bacteria

The Widdel-flask

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Take home messages!

•  There is no microbiology without cultivation

•  We have no universal media nor technique
to culture all microbes with
•  We need more pure cultures
•  Be creative

Giovannoni and Stingl 2007

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