Download DNA Fingerprinting – Your Bioremediation “Taq”ometer

DNA Fingerprinting – Your Bioremediation
“Taq”ometer
Annemarie L. Douglas1, Michael J. Borda2, and
J. Stephen Goudey1
1HydroQual
Laboratories Ltd. and 2Golder Associates Inc.
Outline
¾ Who I am and who I work for
¾ Explanation of DNA profiling
¾ Examples of how it can be used
¾ Other possible uses
¾ New tests that are being developed
HydroQual Laboratories Ltd
¾ HydroQual Laboratories Ltd. (HydroQual)
does applied biology for environmental
management.
¾ We are a wholly-owned subsidiary of Golder
Associates Ltd.
¾ Our talented team of professionals all have
degrees and expertise in the natural
sciences (microbiology, biochemistry,
toxicology, ecology, botany and zoology).
¾ We measure effects for assessing
environmental quality.
Groups
water
soil
air
water quality
applied
science
goldfish
DNA Profiling
¾ Sampling
¾ How it is performed
¾ Results
All living things have DNA
¾ DNA encodes for all cellular processes
¾ Proteins and enzymes
¾ Cellular machinery
¾ Cellular structure
Fingerprinting
¾ Identify individuals within a population
¾ CSI, genetic testing, genealogy
¾ Create a fingerprint of the population
¾ Monitor population changes
Conventional Approach
Sample
Extract bacteria
Grow in media
Identify
Disadvantages
¾ No detection of bacteria that can’t grow
¾ Lots of time - 3 to 21 days or longer
Fingerprint Approach
Sample
Extract DNA
Advantages
¾ No culturing = Fast
¾ Sensitive
¾ Small sample size
Fingerprint
How Do You Fingerprint DNA?
Sample
Isolate DNA
PCR
DGGE
¾ Break open the bacteria
¾ Specifically isolate DNA from everything
else
Polymerase Chain Reaction
Sample
Isolate DNA
PCR
DGGE
¾ Need just a small piece of DNA, but lots of it.
¾ What section of the chromosome?
¾ Ribosomal RNA
• Involved in protein synthesis
• Used for taxonomy
conserved
¾ Target population
divergent
conserved
PCR
¾ What’s needed
¾ Thermophilic enzyme = Taq
¾ DNA bases – AGCT
¾ Primers – DNA targets
Denaturing Gradient Gel Electrophoresis
Sample
Isolate DNA
PCR
DGGE
¾ To separate the PCR DNA
¾ Special gel
¾ Special chemicals to denature DNA
¾ Gradient of chemicals
DGGE
¾ A,T – separates at lower conc.
¾ G,C – separates at higher conc.
Case Studies
¾ 5 case studies
¾ Range of issues
¾ Demonstrate the capabilities of DNA profiling
Study 1
¾ Effects of nickel on plant growth
¾ 5 types of plants
¾ 11 concentrations of Ni2+
• 0 to 5 g/kg
¾ Soil samples were analyzed by fingerprinting
Ni2+ Effects on Plant Growth
¾ Large changes in bacterial population when plants can’t
grow
¾ Smaller changes when Ni2+ concentration is lower
Study 2
¾ Biofouling of Bag Filters
GW
Bag Filter
Zero-Valent Iron Wall
Air Stripper
¾ Question: Was bacterial growth in the bag filters
caused by the remediation system?
¾ Water samples were analyzed by Fingerprinting
Biofouling of Bag Filters
GW
6
Bag Filter
4, 5
1, 2, 8, 9
Zero-Valent Iron Wall
3
¾ Bacteria in the bag filters
¾ Don’t appear to be from the incoming GW
¾ Are not leaving the bag filters
Air Stripper
7
Study 3
¾ Contamination with vinyl acetate
¾ Decision: Dig & Dump or Bioremediate
¾ DNA profiling of soil
Vinyl Acetate in Soil
¾No bacteria are present in the soil.
¾Bioremediation is not possible.
Study 4
¾ Remediation of chlorinated hydrocarbons
with nZVI
¾ 6 wells
¾ 2 treatments with nanoscale zero-valent iron
(nZVI)
¾ Fingerprinting of water samples before
and after treatment
nZVI Remediation
¾ Treatment affected bacterial populations at each well
¾ Bacterial populations got less diverse with treatment
6 months later
¾ Populations are returning to their original state.
¾ Bacterial populations differ by depth.
Study 5
¾ Anaerobic Benzene Bioremediation
¾ Previous treatment with nZVI
¾ Biotreatability study
¾ Addition of N and P
¾ Nitrate – Dechloromonas, etc
¾ Sulfate – Desulfuromonas, etc
¾ Soy protein
¾ Analyze for chemicals and bacteria
¾ DNA profiling for population changes
¾ PCR for detection of specific bacteria
Biotreatability Study
¾ N&P alone causes big changes
¾ Soy protein has a short term effect on the bacterial populations
¾ nZVI abrogates some of the nutrient effects
Other Uses - DGGE
¾ Bacterial ID – sequencing of fingerprints
for definitive ID
¾ Monitoring of microbes, invertebrates,
plants, etc.
¾ Baseline studies of soil
¾ Indoor air quality
Other Uses - PCR
¾ Microbial ID
¾ Dehalococcoides and other hydrocarbon
degraders
¾ Legionella
¾ Cryptosporidium
¾ Biological Activity ID
¾ Culture Maintenance
¾ Plant ID
¾ Source Tracking
¾ Enterococcus
¾ Bacteriodes
¾ Animal Tracking
New Products
¾ Quantitative PCR
¾ Species ID and Quantitation
¾ Quantitative RT-PCR
¾ Gene activity measurements
¾ Isolation of RNA
TCE Bioremediation RT-PCR
cis-Dichloroethene
gene 1
TCE
Ethene
Vinyl Chloride
gene 2
¾ PCR
¾ Detects Dehalococcoides spp.
¾ Detects gene 1 & 2 in the genome
¾ PCR indicates if the bacteria with the correct genes are
present but not if the bacteria are alive and degrading
TCE
¾ RT-PCR
¾ Detects the expression of gene 1 & 2
¾ RT-PCR indicates that TCE is already being degraded
to ethene
Summary
¾ DGGE/PCR
¾ Used for monitoring soil and water
¾ Superior to culturing – speed and sensitivity
¾ More complete picture of in situ populations
Questions?
¾ Contact information
¾ Annemarie Douglas
¾ (403) 253-7121
¾ [email protected]