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Cultivation of sediment microorganisms

Martin Könneke www.icbm.de

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

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Recommended literature

Accessing uncultured Microorganisms (K. Zengler 2008)

Principles of enrichment, isolation, cultivation and preservation of Prokaryotes

(J. Overmann 2006)

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)

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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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Martin Könneke www.icbm.de

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)

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Martin Könneke www.icbm.de

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.

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Martin Könneke www.icbm.de

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

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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

Repeated application of the liquid and deep agar dilution method (in progress)

SO42- Lactate

Acetate H2/CO2

Growth of sulfate-reducers

Production of sulfide

Identification

Molecular analysis of the highest sulfide-positive dilutions

Pure cultures

Growth was stimulated in liquid dilution cultures from each depth and with each substrate

Variety of partial 16S rRNA genes, most of them related to known marine sulfate-reducing bacteria

A. Gittel, Paleomicrobiology, ICBM

A. Gittel, Paleomicrobiology, ICBM

50 cm

100 cm

250 cm

400 cm

Desulfotalea spp.

Desulfosarcina spp.

Desulfobacula spp. H2/CO2

Acetate Lactate

H2/CO2

Acetate Lactate

Who is there?

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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