Title: Traditional culturebased methods are generally considered to be insufficient to describe microbial c
1- Traditional culture-based methods are generally
considered to be insufficient to describe
microbial communities. - Not all microbes can be readily cultured
- Not all culturable microbes have selectable media
(can be hard to ID all microbes). - Time-consuming
- Alternative molecule-based methods exist.
2Two DNA strands melt when heat or chemical
denaturant is applied. This temp is influenced
by 1) H-bonds (GC metls at higher temp than
AT) 2) Attraction between neighboring bases of
the same strand DGGE exploits that DNA molecules
differing by only 1 NT within a low melting
domain will have diff melting temps, and that the
mobility of these molecules is retarded when DNA
strand dissociate.
3DGGE Preliminary Preparation
- DGGE usually performed on PCR products, hence
primers must be carefully chosen so the region to
be screened has one or at most two melting
domains. - A GC clamp (40 NT long GC sequence added to one
PCR primer) is usually positioned adjacent to the
highest melting domain. Thus need full sequence
data. - To determine concentration of denaturant to use,
can do a gradient analysis (next slide).
4Can analyze multiple products on same gel.
5Once you establish denaturant concentration, can
do gels containing just that amount of
denaturant. Allows analysis of different samples
on different gel lanes.
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7Microbial ecology of oil spill bioremediation
- Removal of hydrocarbons from oil spills doesnt
occur as rapidly as desired because crude oil is
high in hydrocarbons but low in nitrogen and
phosphorous, which are required for
microorganisms to convert hydrocarbons into
biomass (which is rich in N and P). - Hydrocarbon degradation by indigenous microbes
can be stimulated by adding N and P. - There is some evidence that controlling the level
of nutrients may result in different patterns of
hydrocarbon degradation, most likely, due to
selection of different microbes. - This hypothesis can be tested.
8DGGE was used to follow broad scale changes in
the bacterial communities selected during HC
degradation. DGGE analysis of bacterial 16S
rRNA gene fragments from oil contaminated beach
sediments undergoing bioremediation, shows
succession of bacterial communities in response
to nutrient addition These gel bands can be
cloned for further analysis.
9The cloned gel fragments from DGGE can be further
analyzed. One method of analysis is ARDRA
(amplifieid rDNA restriction analysis. Amplify
16S rRNA-gene ? restriction digestions ? run
products on gel).
Results indicated Alcanivorax spp. May be
important contributors to hydrocarbon-degradation
in oil-impacted marine ecosystems. What do you
do with this information? Develop strategies to
select for this bacteria in the field.
10Thermophilic treatment of high-strength
wastewaters
- One potential alternative to treating wastewater
via the activated sludge process or anaerobic
treatment is autothermal thermophilic aerobic
biological treatment which uses the exogenous
energy released during microbial pollutant
metabolism to heat engineered reactor systems to
temps ranging from 45-65oC. - Benefits to this system include
- Rapid biodegradation rates ? smaller reactor
sizes - Low growth yields ? reduced residual biomass
disposal costs - Current status is analysis of microbial
communities supported by these reactors using
DGGE of 16S rRNA
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12Experiment
- Compare mixed microbial communities from
thermophilic aerobic biological treatment
reactors with analogous biological aeration
basins operated at a lower temperature. - Step 1 Extract total genomic DNA using a
commercially available kit (BIO 101) - Step 2 Amplify partial 16S rRNA sequences
specific for the domain Bacteria using the
polymerase chain reaction (PCR) - Step 3 Separate amplified sequences on the
basis of GC content using a polyacrylamide gel
containing gradually varying levels of denaturant
(urea).
13Result Have thermophilic cultures (left)
distinct from mesophilic cultures
(right) treating the same waste. Note each band
is cosidered likely to represent a
different organism. Conclusion Thermophilic
aerobic biological treatment reactors support
completely different cultures than similar
mesophilic reactors
14DGGE pros and cons
- Advantages
- High detection rate and sensitivity
- Simple methodology and non-radioactive
- PCR fragments can be gel isolated
- Disadvantages
- Preliminary expts required
- Specialized equipment
- More expensive primers ( GC clamp)
- Not good for high GC genes
15t (terminal) - RFLP
- In tRFLP the target gene is amplified from the
community using standard PCR techniques with a 5'
fluorescently tagged primer. The amplification
products are digested with a restriction
endonuclease and run on an automated sequencer.
Because only the restriction fragment proximal to
the labeled primer carries the flourescine, only
this fragment is detected by the automated
system. In general, each population of the
community contributes a terminal fragment of one
size.
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