Cultivation of microorganisms from sediments4 Quelle: Brock Biology of Microorganisms Martin...
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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