Cultivation of microorganisms from sediments

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

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

Quelle: Brock Biology of Microorganisms

Quelle:

Brock Biology of Microorganisms

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

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.

What do I need for successful cultivation

• Organism source

• Media

• Culture vessel

• Incubator

• Detection system

• Creativity

Chemical composition of a prokaryotic cell

1 Inorganic ions

1 Nucleotides and precursors

2 Sugars and precursors

1 Amino acids and precursors

19 RNA

3 DNA

4 Lipopolysaccharide

9 Lipid

5 Polysaccharide

55 Protein

Percent of dry weight Molecule

Macro elements of a prokaryotic cell

0.2 Iron (Fe)

0.5 Calcium (Ca)

0.5 Magnesium (Mg)

1 Potassium (K)

1 Sulfur (S)

3 Phosphorus (P)

14 Nitrogen (N)

20 Oxygen (O)

8 Hydrogen (H)

50 Carbon (C)

Percent of dry weight Macro element

Trace elements of prokaryotic cell

Cytochromes, catalases, oxygenases Iron (Fe)

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

Vanadium nitrogenase Vanadium (V)

Formate dehydrogenase Tungsten (W)

Hydrogenase, formate dehydrogenase Selenium (Se)

hydrogenase Nickel (Ni)

nitrogenase, nitrate reductase Molybdenum (Mo)

respiration, photosynthesis Copper (Cu)

Vitamin B12 Cobalt (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)

Chemically defined versus undefined (complex) media

Destilled water 1000 ml Trace element solution Glucose 5-10 g

Destilled water 1000 ml CaCl₂ 0.002 g

KH₂PO₄ 2 g MgSO₄ 0.1 g

Peptone 5 g (NH₄)SO₄ 1 g

Yest extract 5 g KH₂PO₄ 2 g

Glucose 15 g K₂HPO₄ 7 g

Undefined medium for E. coli Defined 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!

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.

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

Quelle: Brock Biology of Microorganisms

Continuous culture- culture in

steady state

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

Wadden Sea (Antje Gittel)

pSRR /nmol*cm^-3 *d^-1

0 10 20 30 40 50

Sediment depth /cm

0

100

200

300

400

500

SO₄^2- concentration /mM

0 5 10 15 20 25 30

pSRR sulfate 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

Cultivation

Liquid dilution series in anoxic media

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

SO₄^2- 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

Desulfotaleaspp.

Desulfosarcinaspp.

Desulfobaculaspp. H₂/CO₂

Acetate Lactate

H₂/CO₂ Acetate Lactate Who is there?

Selective enrichment and isolation of the abundant marine, mesophilic Crenarchaeota

The domain Archaea

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(~10²⁸cells) [Karneret al., 2001]

• Detected in marine and terrestrial habitats

• Isotopic analyses and tracer experiments suggest possible autotrophy [Pearsonet al., 2001; Wuchter et al. 2003]

• No cultivated representatives

• Physiology has remained uncertain

• May play important roles in global geochemical cycles

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

bFISH

Scale: 1 µm

cTEM

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

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.

¾

Remove oxygen

¾

Keep it away

• Low redox potential

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

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