Cultivation of sediment microorganisms

Transcription

1 Cultivation of sediment microorganisms Martin Könneke Cultivation of microbes What s so important about cultivation Essentials of cultivation Essentials of isolation How to apply cultivation Cultivation of anaerobes Martin Könneke 1

2 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 2

3 Quelle: Brock Biology of Microorganisms Quelle: Brock Biology of Microorganisms 3

4 Quelle: Brock Biology of Microorganisms Quelle: Brock Biology of Microorganisms 4

5 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 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 5

6 Use in old and modern biotechnology Food production Identification of infective agents and diseases Production of pharmaceuticals Precursor for chemical products Martin Könneke 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 6

7 Nature Reviews Microbiology Vol. 5, Oct Martin Könneke 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 7

8 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 8

9 What do I need for successful cultivation Organism source Media Culture vessel Incubator Detection system Creativity 9

10 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)

11 Trace elements of prokaryotic cell Trace element Cellular function (example) Cobalt (Co) Copper (Cu) Molybdenum (Mo) Nickel (Ni) Selenium (Se) Tungsten (W) Vanadium (V) Zinc Iron (Fe) Vitamin B12 respiration, photosynthesis nitrogenase, nitrate reductase hydrogenase Hydrogenase, formate dehydrogenase Formate dehydrogenase Vanadium nitrogenase Alcohol dehydrogenase, RNA and DNA polymerases, DNA-binding protein 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) 11

12 Chemically defined versus undefined (complex) media Defined medium for E. coli Undefined medium for E. coli K 2 HPO 4 7 g KH 2 PO 4 2 g (NH 4 )SO 4 1 g MgSO g CaCl g Glucose 5-10 g Trace element solution Destilled water 1000 ml Glucose 15 g Yest extract 5 g Peptone 5 g KH 2 PO 4 2 g 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! 12

13 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 validly described species versus about species in 1L sea water about species in 1g soil total of 10 millions (estimations) 13

14 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 14

15 Quelle: Brock Biology of Microorganisms Continuous culture- culture in steady state Quelle: Brock Biology of Microorganisms 15

16 Selective enrichment and isolation of an relevant physiological group Example: Cultivation of sulfate-reducing bacteria from the German Wadden Sea (Antje Gittel) 16

17 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 17

18 Cultivation Liquid dilution series in anoxic media SO 4 2- Lactate Acetate H 2 /CO 2 Growth of sulfate-reducers Production of sulfide Identification Molecular analysis of the highest sulfide-positive dilutions 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 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. H 2 /CO cm 250 cm 400 cm Desulfosarcina spp. Acetate Lactate Desulfotalea spp. H 2 /CO 2 Acetate Lactate A. Gittel, Paleomicrobiology, ICBM 18

19 Selective enrichment and isolation of an abundant phylotype Example: The abundant marine, mesophilic Crenarchaeota The domain Archaea 19

20 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 (~10 28 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 20

21 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) 21

22 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 NH O 2 NO H 2 O + H + ( G 0 = kj mol -1 ) The first nitrifyer within the domain Archaea 22

23 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 23

24 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 24

25 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