Research questions:
- Which variable is important to study in which ecosystem?
o E.g. O2 and hydrogen sulphide (H2S) in marine sediments and stratified water
column
- Study anaerobic methane oxidation:
o Methane and nitrite/ammonium in N-rich Dutch peat
- Study Fe oxidisers/reducers
o Fe2+/Fe3+
Steps to methods:
What are the ecosystem characteristics?
1. In situ (in the field)
a. Field probes and microsensors to measure
i. PH Redox
ii. O2 O2
iii. Temperature H2S
iv. Redox H2
v. Depth N2O
vi. Light NO
vii. Salinity Chlorophyll-a
b. Measuring activity: adding labelled substrates to an enclosed system in the field,
and/or construct concentration profiles and perform flux measurements (for
example greenhouse gas fluxes)
i. Example: anaerobic methanotrophs: add CH4 and nitrite in the absence of
O2, measure removal of CH4 and appearance of N2. Use 15N label to detect
30N2 by GC-MS
2. Or take samples for the lab
a. Bottles, pore water sampler or core sampler for sediments and perform chemical
analysis in the lab
i. Dissolved NO3-, NO2-, NH4+, SO42-, HS-, Fe2+/Fe3+ via
spectrophotometric/colorimetric assay
ii. Dissolved gasses CH4, N2O, CO2, H2, HS- via gas chromatography (GC)
iii. Labelled gases 13CH4, 13CO2, 15N2O via gas chromatography – mass
spectrometry (GC/MS)
Identifying relevant microbial processes and measuring activity:
Lab incubation, measure disappearing substrates, appearing products. Stable/ radioactive isotopes.
SIP.
Enrichment of target microorganisms
To obtain pure culture in order to study physiology, metabolic pathway and activity of the
microorganisms.
3. Enrichment
a. Create the favourable and selective conditions to stimulate growth of specific
microorganisms by adding energy source and selective e-donor, e-acceptor, C-
source, N-source and other nutrients. Also light, O2, temperature, acidity, osmolarity
, 4. Isolation
a. To get pure cultures by
i. Dilution and growth in liquid or on solid media
ii. Single cell techniques: flow cytometry. Density gradient centrifugation to
separate species in an enrichment
5. Great plate count anomaly
a. Only a small proportion of cells is culturable, the rest is the microbial dark matter
b. Unculturability: slow growth rates, very specific nutrient requirements, requirement
of cross feeding or signalling
i. Improved by diluting nutrient media, reducing the complexity of the
community, adding helper strains or mimicking the natural environment
Who’s there? Identifying microbes and exploring their diversity
6. Sequencing DNA/RNA of microbes:
a. Amplification-dependent methods
i. Original DNA to be replicated -> denaturation (2 strands) -> annealing
(adding probe) -> elongation (probe is making second strand, duplication)
ii. Amplicon sequencing (one gene) can be used for marker genes: 16S rRNA
genes (for bacterial or archaeal diversity) and functional genes (nirS/nirK for
denitrification, pmoA for methane oxidation)
1. Sequencing the 16S rRNA gene (transcriptional machinery of all
microbes) -> identify microbial species
iii. qPCR is used to quantify the number of target genes in your sample and
therefore quantify the composition of your community
b. Sequencing of PCR products
i. Done via clone library (cloning the PCR products in plasmids and sequencing
the inserts of the isolated plasmids) -> representation of the PCR products
ii. Or via amplicon sequencing using next generation sequencing (NGS)
methods -> sequencing all PCR products = more complex.
7. Sequencing DNA/RNA of microbes directly after extraction: without targeted PCR step
a. If you sequence the whole genome, you know the entire genetic content of an
organism.
b. Metagenomics gives all genomic information present in the sample.
i. Difficult to extract individual genomes from the metagenome, you need
powerful bioinformatics
- Which variable is important to study in which ecosystem?
o E.g. O2 and hydrogen sulphide (H2S) in marine sediments and stratified water
column
- Study anaerobic methane oxidation:
o Methane and nitrite/ammonium in N-rich Dutch peat
- Study Fe oxidisers/reducers
o Fe2+/Fe3+
Steps to methods:
What are the ecosystem characteristics?
1. In situ (in the field)
a. Field probes and microsensors to measure
i. PH Redox
ii. O2 O2
iii. Temperature H2S
iv. Redox H2
v. Depth N2O
vi. Light NO
vii. Salinity Chlorophyll-a
b. Measuring activity: adding labelled substrates to an enclosed system in the field,
and/or construct concentration profiles and perform flux measurements (for
example greenhouse gas fluxes)
i. Example: anaerobic methanotrophs: add CH4 and nitrite in the absence of
O2, measure removal of CH4 and appearance of N2. Use 15N label to detect
30N2 by GC-MS
2. Or take samples for the lab
a. Bottles, pore water sampler or core sampler for sediments and perform chemical
analysis in the lab
i. Dissolved NO3-, NO2-, NH4+, SO42-, HS-, Fe2+/Fe3+ via
spectrophotometric/colorimetric assay
ii. Dissolved gasses CH4, N2O, CO2, H2, HS- via gas chromatography (GC)
iii. Labelled gases 13CH4, 13CO2, 15N2O via gas chromatography – mass
spectrometry (GC/MS)
Identifying relevant microbial processes and measuring activity:
Lab incubation, measure disappearing substrates, appearing products. Stable/ radioactive isotopes.
SIP.
Enrichment of target microorganisms
To obtain pure culture in order to study physiology, metabolic pathway and activity of the
microorganisms.
3. Enrichment
a. Create the favourable and selective conditions to stimulate growth of specific
microorganisms by adding energy source and selective e-donor, e-acceptor, C-
source, N-source and other nutrients. Also light, O2, temperature, acidity, osmolarity
, 4. Isolation
a. To get pure cultures by
i. Dilution and growth in liquid or on solid media
ii. Single cell techniques: flow cytometry. Density gradient centrifugation to
separate species in an enrichment
5. Great plate count anomaly
a. Only a small proportion of cells is culturable, the rest is the microbial dark matter
b. Unculturability: slow growth rates, very specific nutrient requirements, requirement
of cross feeding or signalling
i. Improved by diluting nutrient media, reducing the complexity of the
community, adding helper strains or mimicking the natural environment
Who’s there? Identifying microbes and exploring their diversity
6. Sequencing DNA/RNA of microbes:
a. Amplification-dependent methods
i. Original DNA to be replicated -> denaturation (2 strands) -> annealing
(adding probe) -> elongation (probe is making second strand, duplication)
ii. Amplicon sequencing (one gene) can be used for marker genes: 16S rRNA
genes (for bacterial or archaeal diversity) and functional genes (nirS/nirK for
denitrification, pmoA for methane oxidation)
1. Sequencing the 16S rRNA gene (transcriptional machinery of all
microbes) -> identify microbial species
iii. qPCR is used to quantify the number of target genes in your sample and
therefore quantify the composition of your community
b. Sequencing of PCR products
i. Done via clone library (cloning the PCR products in plasmids and sequencing
the inserts of the isolated plasmids) -> representation of the PCR products
ii. Or via amplicon sequencing using next generation sequencing (NGS)
methods -> sequencing all PCR products = more complex.
7. Sequencing DNA/RNA of microbes directly after extraction: without targeted PCR step
a. If you sequence the whole genome, you know the entire genetic content of an
organism.
b. Metagenomics gives all genomic information present in the sample.
i. Difficult to extract individual genomes from the metagenome, you need
powerful bioinformatics
