MOLECULAR BIOLOGY TOOLS FOR ENVIRONMENTAL

MANAGEMENT

PRESENTED BY,

POORANACHITHRA M

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

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

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.

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

Molecular Biological Toolbox

MICROARRAY SIP

CENSUS(qPCR) DGGE

CSIA

PCR MBTs

FISH

TRFLP

SEQUENCING PLFA

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

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

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

Reductive Dechlorination

PCE- TetraChloro Ethane TCE- TriChloro Ethane

CisDCE-cis Dichloro Ethane VC-vinyl Chloride

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

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.

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

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

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)

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

FISH (Fluorescent in situ hybridization)

a cytogenetic technique which can be used todetect and localize the presence or absence ofspecific DNA sequences on chromosomes.

It uses fluorescent probes binding parts of thechromosome to show a high degree of sequence similarity.

Fluorescence microscopy can be used to findout where the fluorescent probe bound to the chromosome.

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

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.

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

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").

Thank you