The document discusses molecular biological tools (MBTs) used for environmental management, which include techniques like quantitative PCR, PLFA analysis, and microarray-based assessments, focusing on the quantification of microorganisms involved in biodegradation processes. It highlights advantages over traditional microbiology, such as improved representation of in-situ communities and the ability to track specific functional genes. The document also outlines various sampling methods and applications for evaluating and managing environmental contaminants.

1. MOLECULAR
BIOLOGY TOOLS FOR
ENVIRONMENTAL
MANAGEMENT
PRESENTED BY,
POORANACHITHRA M

2. What are Molecular Biological Tools?
Tools that target biomarkers to provide information about organisms
and processes relevant to the assessment and/or remediation of
contaminants in the environment.
MBTs target Cellular Components
Nucleic Acids
Phospholipids
Proteins
Quantify total biomass & assess the entire microbial population

3. What kinds of genes?
TAXONOMIC GENES - Counting 16SrRNA allows us to count
members of taxonomic groups
Sulfate reducers
what type? Methanotrophs
Dehalococcoides spp. (DHC)
Total Bacteria

4. FUNCTIONAL GENES
Genes that code for the production of enzymes involved in
biodegradation processes.
An increase in numbers of specific Functional genes can indicate that
the biodegradation process has been stimulated.
Progress of the process of management.

5. Why not Traditional Microbiology?
Results are not very representative of in-situ community
Highly selective based upon media conditions
1 to 10% of bacteria are culturable
Highly time consuming

6. Molecular Biological Toolbox
MICROARRAY SIP
CENSUS(qPCR) DGGE
CSIA
PCR MBTs
FISH
TRFLP
SEQUENCING PLFA

7. MBT: CENSUS
Molecular biological tool to analyze microbial communities in
environmental media.
It works by counting genes
#Genes are segments of DNA that code for the production
of an individual protein or enzyme
#rRNA

8. Sample Collection
Groundwater, soil, or Bio-
Trap samples collected and
shipped overnight on ice
(4c)
DNA extracted from
sample upon arrival
Quantitative Real-Time PCR is used to
detect and quantify targets of interest (i.e.
Dehalococcoides spp.)
Results are integrated with other site parameters to evaluate
site management decisions
DNA Extraction
Amplification
Assessment
Working of qPCR

9. Applications include
#Directly evaluate the feasibility of monitored natural attenuation (MNA)
#Evaluate the efficacy of enhanced bioremediation approaches
#Assess the need for bio augmentation
Site specific applications of CENSUS or qPCR
This method is used to identify and quantify the microbes that are responsible for
the reduction of
#Chlorinated hydrocarbons - Reductive Dechlorination, Co-metabolism
# Petroleum hydrocarbons – Aerobic, Anaerobic
# Fuel oxygenates
# Emerging contaminants

10. Reductive Dechlorination
PCE- TetraChloro Ethane TCE- TriChloro Ethane
CisDCE-cis Dichloro Ethane VC-vinyl Chloride

11. Reductive Dechlorination Bacteria
• Dehalococcoides - Capable of complete dechlorination of PCE/TCE
to ethene
• Desulfuromonas - Capable of dechlorination of PCE and TCE
to cis- DCE
• Desultiftobacterium - Capable of dechlorination of PCE and TCE
to cis-DCE
• Dehalogenimonas - Capable of dechlorination of Trichloropropane

12. Community Profiling (TRFLP and sequencing)
Terminal Restriction Fragment Length Polymorphisms (TRFLP)
DNA based technique that provides a profile of the microbial community
Used to identify microorganisms to the genus level.
sequencing
DNA based technique that provides an in depth profile of the microbial
community
Used to identify microorganisms to the genus level.

13. Sample Collection
Groundwater, soil, or Bio-
Trap samples collected and
shipped overnight on ice
(4c)
DNA extracted from
sample upon arrival
DNA
Extraction
Amplification with Fluorescent
Primer
PCR Amplification
Digestion
with
Restriction
Enzyme
Separation of
Products
Steps Involved In The TRFLP

14. MBT: PLFA (Phospholipid Fatty Acid Analysis )
Class of organic compounds that are fatty acids (primarily
phospholipids) or their derivatives and are insoluble in water but soluble
in organic solvents. They include many natural oils, waxes, and steroids.
Why are Phospholipids useful?
Found in living organisms, and associated fatty acids can be useful as
biomarkers
Phospholipids are major membrane lipids of microorganisms, and are
rapidly turned over to upon cell death

15. Sample Collection
Groundwater, soil, or Bio-
Trap samples collected and
shipped overnight on ice
(4c)
PLFA biomarkers
extracted from sample
upon arrival
PLFA Extraction Gas Chromatography
Assessment
Biomarkers are identified by gas
chromatography with flame
ionization detection (GC-FID)

16. Indicator PLFAs
PLFA Structural Group General Classification
Normal Saturated (Nsats) #Found in all organisms
Monoenoic (Monos) #Abundant in Gram negative bacteria, particularly Proteobacteria
which are typically fast growing, utilize many carbon sources, and
adapt quickly to variety of environments
Terminally Branched Saturated (TerBrSats) #Characteristic of Firmicutes (Low G+C Gram-positive bacteria), and
also found in Bacteriodes, and some Gram-negative bacteria
(especially anaerobes)
Branched Monoenoic (BrMonos) #Found in the cell membranes of micro-aerophiles and anaerobes, such
as sulfate or iron-reducing bacteria
Mid-Chain Branched Saturated (MidBrSats) Common in sulfate reducing bacteria and also Actinobacteria
(High G+C Gram-positive bacteria)
Polyenoic #Found in eukaryotes such as fungi, protozoa, algae, higher plants, and
animals

17. FISH(Fluorescent in situ hybridization)
a cytogenetic technique which can be used to
detect and localize the presence or absence of
specific DNA sequences on chromosomes.
 It uses fluorescent probes binding parts of the
chromosome to show a high degree of sequence
similarity.
 Fluorescence microscopy can be used to find
out where the fluorescent probe bound to the
chromosome.

18. CSIA (Compound-specific isotope analysis)
 an in situ monitoring tool that characterizes the natural abundance of
stable isotope signatures (C, N, H, and O) of individual dissolved
contaminants.
 Used to measure in-situ transformation processes of pollutants in
contaminated aquifers as well as to help determine the sources of
groundwater Contaminants

19. DGGE (denaturing gradient gel electrophoresis)
 Screening tool for presence/absence of indicator genes; sequencing
of amplicons for positive identification.
 DGGE also has been applied to evaluate microbial diversity because
it can distinguish closely related species, or even strains within a
species, and it could potentially be applied to RNA to detect
metabolic activity.

20. SIP(stable isotope probing)
Stable Isotope Probing involves the incorporation of stable-isotope-
labeled substrates (typically 13C) into process-specific biomarkers
(DNA, RNA, proteins, lipids).
SIP in combination with PLFA/nucleic acid/protein analysis can
identify which populations are active in a complex environment.
SIP/PLFA has been used with 13C carbon

21. DNA microarrays
microarrays may be subsequently developed and used in situ to
understand which genes are being expressed and how the key
metabolic activities are regulated under different environmental
conditions
consisting of rRNA-targeted oligonucleotide probes. Multiple
oligonucleotide probes are included that target 16S rRNA gene
sequences of organisms at different or the same phylogenetic levels
("multiple probe concept").

22. Thank you