Traditional culturebased methods are generally considered to be insufficient to describe microbial c

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• 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.

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Two 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.
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DGGE 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).

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Can analyze multiple products on same gel.
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Once 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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Microbial 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.

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DGGE 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.
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The 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.
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Thermophilic 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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Experiment

• 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).

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Result 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
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DGGE 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

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t (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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