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Cultivation of microorganisms from

sediments

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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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)

Quelle: Brock Biology of Microorganisms

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Quelle: Brock Biology of Microorganisms

Quelle: Brock Biology of Microorganisms

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Quelle:Brock Biology of Microorganisms

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 usuallynecessary to culture (grow) them.

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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

1Inorganic ions

1Nucleotides and precursors2Sugars and precursors1Amino acids and precursors

19RNA3DNA4Lipopolysaccharide9Lipid5Polysaccharide

55Protein

Percent of dry weight

Molecule

Macro elements of a prokaryotic cell

0.2Iron (Fe)0.5Calcium (Ca)0.5Magnesium (Mg)1Potassium (K)

1Sulfur (S)3Phosphorus (P)14Nitrogen (N)20Oxygen (O)8Hydrogen (H)

50Carbon (C)

Percent of dry weightMacro element

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Trace elements of prokaryotic cell

Cytochromes, catalases, oxygenasesIron (Fe)

Alcohol dehydrogenase, RNA and DNA polymerases, DNA-binding protein

ZincVanadium nitrogenaseVanadium (V)Formate dehydrogenaseTungsten (W)Hydrogenase, formate dehydrogenaseSelenium (Se)hydrogenaseNickel (Ni)nitrogenase, nitrate reductaseMolybdenum (Mo)respiration, photosynthesisCopper (Cu)Vitamin B12Cobalt (Co)

Cellular function (example)Trace element

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

Destilled water 1000 mlTrace element solutionGlucose 5-10 g

Destilled water 1000 mlCaCl2 0.002 gKH2PO4 2 gMgSO4 0.1 gPeptone 5 g(NH4)SO4 1 gYest extract 5 gKH2PO4 2 gGlucose 15 gK2HPO4 7 g

Undefined medium for E. coliDefined medium for E. coli

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.

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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 Biologyof Microorganisms

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Selective enrichment and isolation of sulfate-reducing bacteria from the German

Wadden Sea (Antje Gittel)

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pSRR /nmol*cm-3 *d-1

0 10 20 30 40 50

Sed

imen

t dep

th /c

m

0

100

200

300

400

500

SO42- concentration /mM

0 5 10 15 20 25 30

pSRRsulfate

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 sulfideBuffer: Carbonate/Carbon dioxideRedox indicator: ResazurinCarbon source: Lactate, acetate, or carbon dioxideElectron donor: Lactate, acetate, or hydrogenElectron 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

AcetateH2/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 marinesulfate-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

AcetateLactate

H2/CO2

AcetateLactate

Who is there?

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Selective enrichment and isolation of 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 culture1) 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

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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 culture1) 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 oxygenKeep it away

• Low redox potentialAddition 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