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Title:
Introduction
Text: There is growing interest in the development and use of molecular biological tools (MBTs) for environmental restoration applications. MBTs are laboratory tests that can measure the presence and activity of microbes at a site. They can be used to assess the potential for and performance of monitored natural attenuation and bioremediation strategies for remediation of environmental contaminants. A few MBTs are already used to support field-scale projects and newer technologies continue to emerge.
A good example of this would be routine real-time polymerase chain reaction or other analysis to measure Dehalococcoides sp. presence to assess whether the indigenous microbial population includes dechlorinating microorganisms. This could be used to determine if bioaugmentation is necessary at a chlorinated-solvent contaminated site.
This Web Tool provides an introduction to MBTs applied through gene-based, protein-based, lipid-based and isotope-based approaches. It also highlights Navy sites that have used these new techniques.
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Visual Description: Graphic of laboratory technician looking at microbes through a microscope.
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Title:
Background
Text: The information obtained from MBTs may help to improve the design, application and/or optimization of remediation processes by:
Allowing for qualitative and quantitative assessment of the types of microorganisms present and/or their levels of metabolic activity.
Providing evidence that contaminant removal is due to biodegradation.
Assessing microbial population shifts in response to remedial approaches (e.g., substrate addition, physical and/or chemical treatment).
Aiding in performance assessments to delineate the treatment area impacted by substrate addition and the distribution of microbes in the subsurface.
Addressing performance and/or engineering issues such as the causes of incomplete biodegradation or biofouling problems. The graphic shows how the evaluation of the Dehalococcides reductase genes can provide information regarding the efficiency of different steps in the dechlorination pathway. For example, it shows that the presence of only the tceA enzyme may indicate inefficiency in the vinyl chloride to ethene path and therefore result in accumulation of vinyl choloride in the field, which is highly undesirable.
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Visual Description: Contaminant degradation pathway of tceA, vcrA and bvcA enzymes from PCE, to TCE, to cis-DCE, to vinyl chloride, and to ethene.
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Title:
Microbial Cell (1 of 3)
Text: To understand how MBTs work, it is important to understand the components of microorganisms that are used as targets for MBTs. Microorganisms are generally single cell organisms that can be classified as prokaryotes (e.g., bacteria) or eukaryotes (e.g., fungi). These microscopic organisms are comprised of four main macromolecules called proteins, nucleic acids, lipids, and polysaccharides.
In general, proteins, nucleic acids and lipids are the most important in the context of MBTs.
A wide variety of microbes can reductively dechlorinate. For example, Dehaloccoides reduces TCE and PCE into ethene, Dehalobacterium converts the toxic dichloromethane into the fatty acid acetate plus formate. MBTs are used to access the capabilities of the microoganisms.
The figure shows one generalized organism. Organisms in the subsurface may or may not take that shape, they can be circular, rod-shaped, etc.
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Visual Description: Graphic illustration of a microbial cell including proteins (cytoplasm, membrane, and enzyme), nucleic acids (ribosomes) and lipids (membrane and cell wall).
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Title:
Proteins
Text: Proteins are large organic compounds made of amino acids arranged in a linear chain and joined together by peptide bonds. Many proteins are enzymes or have structural functions. Enzymes are catalysts for the chemical reactions that occur inside or outside cells including biodegradation of contaminants. Structural proteins are used to build the cell wall, cytoplasm, and other components.
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Title:
Nucleic Acids
Text: Each microbial cell contains genetic instructions for how to reproduce, grow, and function. These instructions are coded in nucleic acids that are found in two forms.
Deoxyribonucleic acid (DNA) is a double-stranded nucleic acid and it carries the genetic material of the cell. DNA is contained in each microbial cell. It is present within either a nucleoid in prokaryotes (i.e., bacteria) or in a nucleus in eukaryotes (i.e., fungi).
Ribonucleic acid (RNA) is a single-stranded nucleic acid and it acts to convert genetic information into proteins.
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Title:
Lipids
Text: Lipids are fatty compounds that will not readily dissolve in water. They play an important role in the cell wall and other structural layers that protect the bacteria cell from the outside environment.
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Title:
Polysaccharide
Text: Polysaccharides are long chain polymers of sugars. They are mainly located in the cell wall.
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Title:
DNA Structure (2 of 3)
Text: Each microbial cell's DNA contains genes that specify the structure and metabolic capabilities of that type of microbe. Specific genes within the DNA strand can be used to “fingerprint” or identify types of microorganisms.
As shown here, the DNA molecule has a spiral structure. It is made up of a sugar-phosphate backbone and four types of organic bases, adenine (A), thymine (T), guanine (G), and cytosine (C).
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Visual Description: Graphic illustration of DNA structure with sugar-phosphate backbone, A-T base pair, and C-G base pair.
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Title:
A-T Bases
Text: Adenine (A) bonds with thymine (T) in DNA.
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Title:
C-G Bases
Text: Cytosine (C) and guanine (G) are organic bases found in both DNA and RNA molecules.
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Title:
Protein Synthesis (3 of 3)
Text: Every cellular function is carried out by proteins. The presence of proteins and/or precursor compounds can be used to gauge microbial activity. Therefore, it is very important to understand the processes of transcription and translation which allow genetic information in the microbial chromosome to be converted into proteins. Click on the figure for more information.
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Visual Description: Graphic illustration of protein synthesis. DNA first transferred to a messenger RNA (mRNA) strand in a process called transcription. mRNA undergoes a process called translation to help in the production of proteins.
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Title:
Transcription
Text: DNA does not participate directly in protein synthesis. The genetic code for a specific protein is first transferred to a messenger RNA (mRNA) strand in a process called transcription.
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Title:
Translation
Text: Before the mRNA can help in the production of a protein, it must undergo a process called translation. In translation, mRNA along with transfer RNA (tRNA), and ribosomes, work together to produce proteins.
Translation occurs in the cytoplasm where the ribosomes are located. In translation, genetic information that the messenger RNA carries is decoded and the sequence is used as a template to guide the synthesis of a chain of amino acids that form a protein.
The ribosome is made of ribosomal RNA and proteins and is where amino acids are assembled into proteins. tRNAs are small RNA chains that have a site for amino acid attachment and transport amino acids to the ribosome. The amino acids that the tRNAs carry are then used to assemble a protein.
The lifespan of the mRNA is usually short, as it is quickly broken down after translation.
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Visual Description: Graphic animation of the translation process in which messenger RNA (mRNA) along with transfer RNA (tRNA) and ribosomes work together to produce proteins.
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Title:
Molecular Biological Tools
Text: Once the components of a microbial cell are understood, a number of molecular-based techniques can be used to investigate the presence and metabolic activity of microbes.
MBTs have been categorized as gene-based, protein-based, lipid-based, and isotope-based tools. Each type of MBT can help to answer several useful questions at environmental restoration sites.
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Visual Description: Molecular biological tools include gene based tools, protein based tools, lipid based tools and isotopic tools.
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Title:
Gene-Based Tools (1 of 9)
Text: Gene-based MBTs use DNA or RNA to help to "fingerprint" microbes at a given site. Gene-based MBTs can identify microbes that degrade chlorinated solvents, total petroleum hydrocarbons, benzene, toluene, ethylbenzene, and toluene (BTEX), and other compounds.
As shown in the table, gene-based tools can be divided into two categories: biodegradation potential and biodegradation activity. Specifically, tests can be run to evaluate whether the potential for microbes to contribute to a system exists or whether microbial biodegradation activity within a system is occurring.
The potential only indicates that specific microbes are present in the system. However, the test does not reveal whether specific microbial populations are active.
To determine activity, a different set of gene-based tests must be conducted. These tests test for expression of specific genes (RNA or enzymes) known to contribute to biodegradation.
As RNA and enzymes are short lived in a cell, the detection of expression indicates that the microbes are actively contributing to the system.
As all the gene-based techniques in the table rely on Polymerase Chain Reaction (PCR), the next slide provides a quick overview on the PCR technique.
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Visual Description: Table entitled, "MBTs for Biodegradation Potential and Activity", lists biodegradation potential and biodegradation activity.
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Title:
16S rRNA Gene
Text: 16S rRNA is the name of a molecule located on the microbial ribosome. The gene that codes for the 16S rRNA molecule is present in all microbes because it is required in protein synthesis.
Evolution has resulted in differences in the genetic sequence of this gene between microbial species. Microbes can be identified by selecting the 16S rRNA gene for analysis. 16S rRNA gene sequences contain hypervariable regions that can provide species-specific signature sequences useful for microbial identification.
Even unknown bacteria can be placed within a framework of known microorganisms. Closely related microbes will have more similar genetic sequences than distantly related ones.
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Title:
PCR (2 of 9)
Text: PCR is a technique for amplifying a specific region of DNA or RNA. As shown here, each PCR cycle results in a doubling of DNA or RNA. PCR derives its name from one of its key components, a DNA polymerase used to amplify a piece of DNA.
As PCR progresses, the DNA generated is used as a template for replication. This sets in motion a chain reaction in which the DNA template is exponentially amplified. With PCR it is possible to amplify a single or few copies of a piece of DNA across several orders of magnitude, generating millions or more copies of the DNA piece. PCR can be extensively modified to perform a wide array of genetic manipulations.
Almost all PCR applications employ a heat-stable DNA polymerase, which assembles a new DNA strand from DNA building blocks, by using single-stranded DNA as a template and DNA primers, which are required for initiation of DNA synthesis. Many PCR methods alternately heat and cool the PCR sample to selectively amplify the target DNA.
PCR helps to identify if a target microbe is present in a sample. A technique called real-time or quantitative PCR can also be used to quantitatively estimate the number of microbial cells in a sample. Reverse transcription PCR allows the RNA to be converted to DNA.
PCR can be performed by specialized biological laboratories and/or universities.
Click here to learn more about the use of real-time PCR in the field.
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Visual Description: Graphic animation of the Polymerase Chain Reaction (PCR). Each PCR cycle consists of heat, DNA polymerase and primer, and primer extension, resulting in a doubling of DNA.
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Title:
Real-Time PCR or Quantitative PCR
Text: Real-time PCR (RTm PCR), also called quantitative PCR (qPCR) is a method that allows direct measurement of the number of microbes in a sample. This technique is based on the PCR, which is used to amplify and simultaneously quantify a targeted DNA molecule. It enables both detection and quantification of a specific sequence in a DNA sample.
RTm PCR uses a fluorescent dye which binds to double stranded DNA, or a fluorescent marker which is tagged to each gene copy. With each cycle of PCR, more copies of the DNA or RNA target are produced, which in turn increases the amount of the fluorescence dye or marker within the sample in a predictable fashion. The rate of increase in the fluorescence measured throughout the amplification process is directly related to the number of target microbes through the use of a standard curve similar to chemical analytical techniques.
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Title:
Reverse Transcription PCR
Text: Reverse transcription polymerase chain reaction, abbreviated as RT-PCR, is a laboratory technique for amplifying a defined piece of a ribonucleic acid (RNA) molecule. The RNA strand is first reverse transcribed into its DNA complement or complementary DNA, followed by amplification of the resulting DNA using PCR.
RT-PCR can be used to quantify mRNA levels from much smaller samples. RT-PCR is the most sensitive technique of mRNA detection and quantitation currently available, this technique is sensitive enough to enable quantitation of RNA from a single cell.
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Title:
Potential: qPCR (3 of 9)
Text: Quantitative Polymerase Chain Reaction (qPCR) is currently one of the most widely available technologies to identify contaminant degraders and can be used to indicate the presence of the desired organism within environmental samples and/or to track relative changes in concentration of time and/or space.
qPCR is a semi-quantitative method for estimating the concentration of target microbe (i.e., Dehalococcoides DNA) with high specificity, sensitivity and reproducibility. qPCR is based on PCR, in which a target DNA sequence (i.e., the 16S rRNA gene within Dehalococcoides sp.) is amplified exponentially by performing multiple cycles of DNA replication. It enables both detection and quantification of a specific sequence in a DNA sample.
One of the common methods of quantification are the use of fluorescent dyes, as shown in the figure. It is commonly carried out with an RNA-based probe with a fluorescent reporter at one end and a quencher of fluorescence at the opposite end of the probe.
The PCR reaction is prepared and the reporter probe is added.
As the reaction commences, during the annealing stage of the PCR both probe and primers anneal to the DNA target.
Polymerization of a new DNA strand is initiated from the primers, and once the polymerase reaches the probe, it degrades the probe, physically separating the fluorescent reporter from the quencher, resulting in an increase in fluorescence.
Fluorescence is detected and measured.
Many university and commercial laboratories offer this technology as a service. The technique can be used before a remedial strategy to assess the potential use of the micrbes in the strategy. It can be used during and after to assess treatment progress and success.
Such applications can be seen in the graphs.
The first set of graphical results (second graphic) shows qPCR results from groundwater collected from a chlorinated solvent source area undergoing thermal remediation using electrical resistance heating. Results show changes in Dehalococcoides sp. 16S rRNA and reductase genes tceA, bvcA and vcrA with temperature.
The second graph displays qPCR results showing changes in Dehalococcoides sp. 16S rRNA and reductase genes tceA, bvcA and vcrA following thermal treatment.
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Visual Description: Graphic illustration of (1) denature, (2) primer annealing/probe hybridization, and (3) extension.
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Title:
Potential: T-RFLP (4 of 9)
Text: After PCR amplification terminal restriction fragment length polymorphism (T-RFLP) can be used to evaluate different populations within a sample. The method generates a chromatogram that represents the different populations on the X axis and their relative abundance on the Y axis. This provides a quick and effective way to track the diversity of predominant microbial populations over time and/or space. For example, it is often used to evaluate population shifts before, during, and after remediation treatment.
The figure shows the T-RFLP procedure. The gene of interest is amplified using PCR (as seen in earlier slides) with a fluorescently labeled primer, yielding a mixture of DNA fragments with the fluorescent label on one end. The mixture is digested with a restriction enzyme and the fragments are separated by different size. Separation occurs through gel or capillary electrophoresis and a laser reader detects the labeled fragments to generate a profile based on fragment lengths. These data can be correlated with site properties to provide information on the ongoing microbial processes to assess the potential for biodegradation.
The first graph shows a T-RFLP chromatogram of the bacterial groundwater community undergoing bioremediation for chlorinated solvents. Dehalococcoides, a chlorinated solvent-degrader, is identified. This chromatogram was prior to thermal treatment at ambient temperature (11°C).
The second graph shows the effect of electrical resistance heating with increased groundwater temperatures (77°C) on the chlorinated-solvent degrading microbial community. Dehalococcoides was absent from the community profile.
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Visual Description: Graphic illustration of the T-RFLP procedure which includes (1) extract DNA from the community, (2) PCR with a fluorescently labeled 16S rRNA forward primer, (3) restriction digest of PCR product, (4) fragment separation in sequencing gel, and (5) recognition of labeled fragments.
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Title:
Potential: Clone Library (5 of 9)
Text: After PCR amplification, clone libraries can be constructed to isolate target genes to allow for DNA sequencing and identification of microbial populations. This information can then be used to assess which microbes are present with the potential capability for contaminant degradation.
Clone libraries generate a series of DNA fragments that correspond to different populations and provide an estimate of relative abundance of populations represented. This method is very effective for identification of unknown populations in environmental samples.
In general, the process is laborious and relatively expensive. Clone library construction is available through commercial and/or university laboratories.
The first figure shows a clone library constructed from groundwater contaminated with chlorinated solvents. It identifies major bacterial populations and their relative proportion in the microbial community (including chlorinated-solvent degrading Dehalococcoides).
The second figure is a clone library constructed from the same groundwater following thermal treatment using electrical resistance heating at elevated temperature. The pie chart illustrates shifts in predominant populations including the notable absence of Dehalococcoides observed prior to thermal treatment.
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Visual Description: Pie chart of microbial populations. Firmicutes, proteobacteria, actinobacteria, bacteroidetes, dehalococcoides, OD1, OP11, and spirochaetes are shown.
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Title:
Potential: DGGE (6 of 9)
Text: After PCR amplification, denaturing gradient gel electrophoresis (DGGE) can be used to identify the types of microbial genes in a sample and is used to assess microbial communities. The profiles for each type of microorganism are visible as bands in the gel shown here.
DGGE can be used to profile and assess dominant members of the microbial community to correlate data with the conditions of the site to assess various remedial strategies.
The ability of DGGE to separate individual species within a sample also enables one to follow the progression of communities over a period of time. This application is useful for remediation studies where sites require sampling over extended periods of time to follow the degradation of contaminants and the organisms degrading them.
DGGE analysis is used less often than real-time PCR in environmental applications.
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Visual Description: Graphic illustration showing DGGE gel profile of sample A and sample B. Prominent gel bands are excised and sequenced, and phylogenetic analysis identifies organisms.
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Title:
Activity: qPCR for RNA (7 of 9)
Text: RNA is an immediate precursor to the production of protein; therefore, RNA is a more direct measurement of activity within a cell and cellular components such as enzymes capable of biodegradation (halogenase, oxygenase, etc.). Therefore, detection and quantification of RNA can be useful for evaluation of bioremediation-based technologies.
qPCR for rRNA or mRNA targets requires a reverse transcription step to convert the RNA into complimentary DNA. Once in DNA form, qPCR techniques are used to quantify the target microorganism.
qPCR for RNA is also widely available through commercial and/or university laboratories.
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Visual Description: Graphic shows 4 steps: (1) incubate with reverse transcriptase to synthesize cDNA strand, (2) when cDNA strand is completed, hydrolyze RNA strand, (3) incubate with DNA polymerase to synthesize second DNA strand, (4) cDNA ready for qPCR analysis.
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Title:
Activity: FISH (8 of 9)
Text: Fluorescence in situ hybridization (FISH) is a whole cell technique that targets either DNA or RNA using labeled probes that fluoresce when bound to the targeted microorganisms or genes.
FISH involves the preparation of short sequences of single-stranded DNA, called probes. The probe is labeled with fluorescent dye and is then applied to the chromosome DNA. Hybridization occurs and the unhybridized or partially-hybridized probes are washed away. The probes hybridize, or bind, to the complementary DNA and enable researchers to see the location of those sequences of DNA because they were labeled with the fluorescent tags. The results are then visualized and quantified using a microscope that is capable of exciting the dye and recording images.
FISH is widely used to identify microorganisms and multiple probes can be used on one sample to visualize the presence and/or activity of various populations within that sample.
In general, the cost-per-test and the technical complexity of current FISH protocols has limited its widespread use. FISH is available through commercial and/or university laboratories.
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Visual Description: Graphic illustration of Fluoresence In Situ Hybridization (FISH).
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Title:
Activity: DNA Microarray (9 of 9)
Text: A DNA microarray is a powerful tool used to detect and quantify the presence of targeted genes and is most often used to evaluate their expression.
DNA microarrays are small, solid supports onto which the sequences from thousands of different genes are immobilized, or attached, at fixed locations. Each DNA fragment representing a gene is assigned a specific location on the array, usually a glass slide, and then microscopically spotted to the location. The spots are single stranded DNA fragments that are strongly attached to the slide, allowing cellular DNA or RNA to be fluorescently labeled and laid overtop of the array. DNA or RNA in the overlaid sample will stick (through a process called hybridization) to a complementary spot on the array. By exposing the microarray to a fluorescently labeled sample, the DNA that hybridizes will be identifiable as glowing spots on the array, while the spots that have nothing hybridized to them will not be visible.
By using an array containing many DNA samples, scientists can determine the expression levels of hundreds or thousands of genes within a cell by measuring the amount of mRNA bound to each site on the array. The amount of mRNA bound to the spots on the microarray is measured, generating a profile of gene expression in the cell.
Microarrays are significant because they may contain a very large number of genes and because of their small size.
Two main types of commercial microarrays are oligonucleotide arrays and cDNA arrays.
Oligonucleotide arrays use small 25 base pair gene fragments (called probes) as the DNA to be spotted onto an array. As shown in the graphic, only one sample is hybridized to a single array during these experiments. Such samples are prepared by extracting mRNA from a cell and turning it back into DNA through a process that involves the reverse transcription of the mRNA into what is referred to as a cDNA. The cDNA is then transcribed to cRNA while incorporating a label (e.g., biotin). Once labeled, the sample of cRNAs can be hybridized to the array and bound by the various oligonucleotide probes. Lastly, a staining reaction is performed in order to visualize the amount of hybridization.
A cDNA array uses the same principle; the probes are larger pieces of DNA that are complementary to the genes one is interested in studying.
DNA microarrays are an emerging technology and research is underway to develop remediation-specific microarrays. Currently, there is limited availability of global microorganism arrays at universities and research labs.
The GeoChip is a a comprehensive microarray for investigating biogeochemical, ecological and environmental processes. It was successfully used for tracking
the dynamics of metal-reducing bacteria and associated communities for an in situ bioremediation study.
The GeoChip is the first comprehensive microarray currently available for studying biogeochemical processes and functional activities of microbial communities important to human health, agriculture, energy, climate change, ecosystem management, and environmental restoration.
The PhyloChip is a microarray for rapid profiling of microbial populations. Much of the bacteria in a sample cannot survive in a culture and the PhyloChip provides an accurate means for sample testing without any culturing required. It has the ability to identify multiple bacterial and archaeal organisms from complex microbial samples.
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Visual Description: Graphic illustration of DNA microarray making, hybridization and results delivery.
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Title:
DNA microarray
Text: A DNA microarray is a high-throughput technology consisting of a series of up to hundreds of thousands of spots on a microscope slide; each spot is composed of targeted DNA oligonucleotides probes (genes, organisms via 16S, etc.) with fluorescent markers. These probes are used to hybridize cDNA or cRNA targets within a sample (i.e., environmental DNA extraction).
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Title:
Protein-Based Tools (1 of 2)
Text: Microbes readily adapt and make only the proteins or enzymes that they need to survive in their environment. Although certain microbes may be present at a site, they may or may not be actively producing the enzymes needed to biodegrade a given contaminant. Protein-based tools help to determine whether or not active enzymes associated with biodegradation are present within an environmental sample.
There are several types of protein-based tools including enzyme probes and various mass spectrometry techniques used for the quantitative analysis of large biomolecules.
Protein-based MBTs are currently experimental in nature. Only a few enzyme probes have been developed targeting a limited number of contaminants. Proteomic analyses of specific degradation genes within contaminant plumes are limited to a few sites. Thus, validation of these types of tools is required before widespread application is recommended. Protein-based MBTs do, however, provide the most direct measurement of microbial activity.
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Visual Description: Flowchart asks three questions: (1) Are bacteria present with capability for contaminant degradation? (DNA probe, QPCR), (2) Are those bacteria active? (FISH, rRNA, probe & QPCR), (3) Are the degrading enzymes active? (enzyme activity probe, stable isotopes, mRNA probe & QPCR). TCE degradation is possible only with answering yes to all three questions. No TCE or on-going TCE degradation is possible with a no answer.
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Title:
Proteomics
Text: Proteomics involves the identification of proteins expressed by a microbial cell and the determination of their role in the physiology of that cell. While still relatively novel, these analyses can provide direct evidence of any one targeted protein or all proteins, such as biodegrading enzymes, being expressed by microbial populations.
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Title:
Enzyme Probe (2 of 2)
Text: Enzyme activity probes (EAPs) have been developed to assess the presence and activity of specific microorganisms in contaminated subsurface environments. These probes are innovative research tools that have the potential to provide direct evidence that the mechanisms for degradation are occurring.
EAPs are substrates that can bind to specific enzymes of interest, and are subsequently transformed by those enzymes into fluorescent products. If the appropriate enzyme is not present, or it is present but not active in a given sample, then the probes will not be transformed and no fluorescence will be detected. Therefore, the technology directly measures both the presence and activity of the degradative enzyme.
Currently, EAPs have been developed to evaluate aerobic cometabolic oxidation of chlorinated solvents and include a suite of aromatic compounds (toluene, phenol, benzene), and the soluble methane monooxygenases (sMMO). These techniques have been used to verify natural attenuation mechanisms at chlorinated solvent-contaminated sites.
EAPs are available through one commercial laboratory in addition to research laboratories.
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Visual Description: Graphic animation of enzyme activity probes (EAPs) showing positive and negative results.
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Title:
Examples of Known Enzymes
Text: Only a few enzyme probes have been developed to date. The target enzyme must be known and more research is needed to identify the range of enzymes produced by bacteria during biodegradation.
A few examples of enzymes produced by known degraders of chlorinated solvents are listed below:
D. ethenogenes uses a trichloroethene (TCE)-reductive dehalogenase (TceA) for vinyl chloride (VC) reduction
Bacteria that cometabolize TCE produce four separate toluene oxygenases and soluble methane monooxygenase (sMMO)
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Title:
Lipid-Based Tools (1 of 2)
Text: Lipid-based tools like phospholipid fatty acid (PLFA) analysis can gauge three key aspects of the microbial community including viable biomass, community composition, and metabolic status. PLFA is a substance found in the cytoplasmic membrane of the microbial cell.
The use of PLFA analysis for environmental applications is gradually being replaced by more specific gene-based tools.
PLFA analysis is based on the extraction and separation of lipid classes followed by quantitative analysis using gas chromatography and mass spectrometry (GC/MS). The classes of lipids are defined in this table.
The individual fatty acids differ in their chemical composition depending on the organisms present and the environmental conditions. Therefore, PLFA analysis can help to determine how much biomass is in a given sample and what general types of microorganisms are present.
While PFLA can provide indications regarding general biomass and community structure, it is generally non-specific and does not provide information on populations of interest (i.e. contaminant-degrading populations). With the increasing number of activity-based tests currently available and gene-based tests providing information on specific communities and microbes of interest, PFLA is slowly being replaced.
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Visual Description: Table entitled, "Description of PLFA Structural Groups". PLFA structural groups and general classifications are listed.
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Title:
PLFA (2 of 2)
Text: PLFA analysis results are shown here from a site where bioventing was used to promote aerobic biodegradation.
The first figure shows the increase in biomass after the site was biovented. The biomass content in the sample is calculated by the total amount of PLFA extracted from a given sample. The units are presented as cells/gram of sample.
The second figure shows the types of microorganisms present based on the various chemical structures of the PLFA found in the sample. Their relative abundance is displayed as a percentage of the total biomass. The classes of PLFA are defined in the table on the previous slide. After bioventing, the second figure shows an increase in proteobacteria, which are associated with aerobic biodegradation.
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Visual Description: Barchart showing the biomass before bioventing and after bioventing. A second chart shows the community structure before and after bioventing.
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Title:
Isotope-Based Tools (1 of 2)
Text: Compound specific isotope analysis (CSIA) can be used to study trends in biodegradation and contaminant source identification.
Many contaminants contain elements that are present in two stable isotope forms. The ratio of the isotopes can change over time due to various physical, chemical, and biological processes.
Isotope analyses can be performed by the United States Geological Survey (USGS), specialized commercial laboratories, and universities.
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Visual Description: Graphic illustration of a compound specific isotope analysis (CSIA) is shown with gas inlet, ion source, ion beam, magnet, lighter and heavier ions, and collector array. Photograph of lab equipment is shown. Isotopes Hydrogen, Oxygen, Carbon, Chlorine, Nitrogen, and Sulfur are listed.
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Title:
Isotopes
Text: Isotopes are atoms of the same element, but with different atomic masses due to a difference in the number of neutrons in the nucleus. Stable isotopes of hydrogen, oxygen, carbon, chlorine, and others are common in nature.
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Title:
Isotope-Based Tools (2 of 2)
Text: During biodegradation, the lighter isotopic species, such as carbon 12 compared to carbon 13, tend to be preferentially metabolized by the microorganism, resulting in an increase in the isotope ratios of the residual substrate. This means that the substrate will become heavier and the product will become lighter over time. This trend can be used to distinguish between biodegradation and mass loss due to physical processes such as sorption, dispersion, and volatilization.
Two examples are shown here of the change in isotope ratios from biodegradation processes. Isotope ratios are displayed as "delta," which is a measure of the difference in the isotopic composition of a compound relative to an internationally-accepted standard. Roll over the figures for more information.
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Visual Description: Graphic plot entitled, "Chlorine Isotope Fractionation during Biodegradation of Perchlorate".
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Title:
Isotope Fractionation with Perchlorate
Text: The first figure shows the increase in the delta value of the chlorine element as perchlorate is biodegraded. The heavier 37Cl is left behind because the microbes favor use of the lighter 35Cl when perchlorate is used as an electron acceptor during biodegradation.
This means that the perchlorate that is left behind is "heavier" and the sample has a higher delta value.
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Title:
Isotope Fractionation with cis-DCE
Text: The second figure shows that there is no change over time in the delta value of the carbon element for cis-dichloroethene (DCE) present in groundwater samples. The samples shown were taken during a time period from April 2003 to August 2004 at several different monitoring well locations (X axis). These data suggest that after 1.3 years there is no significant change in the isotope fractionation of carbon at each monitoring location, suggesting cis-DCE stall.
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Title:
Applicability
Text: This Web Tool describes MBTs that have already been applied at environmental field sites including real-time PCR (or qPCR), DGGE, enzyme probes, PLFA analysis, and CSIA. Real-time PCR and PLFA analysis are MBTs that are currently available for widespread use for field-scale projects. DGGE, enzyme probes, and CSIA applications are less common, but practical information can be obtained from these tools for bioremediation projects.
The use of MBTs for environmental restoration projects is rapidly evolving and this table summarizes additional MBTs that may prove useful in the future.
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Visual Description: Table of molecular biological tool type, current relative frequency of use, degradation potential, specific organism detection, organism activity, process rates and completeness, environmental limitations, operational improvements, and continuous monitoring/process control for gene based tools, lipid based tools, protein-based tools, and isotope-based tools.
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Title:
Case Study: Naval Weapons Station Seal Beach
Text: Naval Weapons Station (NAVWPNSTA) Seal Beach is a 5,000-acre facility located in California. NAVWPNSTA Seal Beach provides deployment-ready ordnance to ships.
Installation Restoration (IR) Site 40 includes a concrete pit located near a Locomotive Shop (Building 240) and a gravel area north of and adjacent to the building. Spent oil and solvents were sent to the concrete pit during historic locomotive maintenance activities. Materials that collected in the pit were discharged into the gravel area through a drain pipe until the pipe was removed in 1978.
Currently, the gravel area is paved over on the northeast side of Building 240 and remains unpaved to the northwest.
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Visual Description: Map showing the location of Naval Weapons Station at Seal Beach, California.
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Title:
Conceptual Site Model
Text: Click on the figure for more site information.
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Visual Description: Cross sectional 3-D graphic showing the PCE concentration in groundwater under building 240 and building 239.
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Title:
Geology and Hydrogeology
Text: Groundwater at IR site 40 is encountered at a depth of 8 to 9 feet below ground surface (bgs). The hydraulic gradient undergoes changes in the magnitude and direction due to tidal influences. Flow direction is to the southeast with a gradient ranging from approximately 0.0004 to 0.002 ft/ft for groundwater at depths less than 30 feet bgs. Seasonal variations in water level at IR Site 40 appear to be minimal. The geologic units observed at IR Site 40 are as follows:
Surficial soils: silty sands and clayey sands with considerable lateral variation
First sand unit: sands to silty sands within a few feet of the water table extending to 7.5 to 10 feet bgs
Second sand unit: saturated sands to silty sands at 9 to 21 feet bgs, extending to 28 to 41 feet bgs
Third sand unit: saturated sands to silty sands at 38 to 52 feet bgs, depending on the location
Lower permeability intervals: clay, silty clay, and silt separate the coarser grained units noted above.
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Title:
Contaminant Distribution
Text: The lateral extent of the perchloroethene (PCE) and TCE plume at IR site 40 is several hundred feet east-southeast downgradient of the source area(s) and the depth is limited to approximately 66 feet bgs. Previous site characterization data also indicated the presence of cis-1,2-DCE, trans-1,2-DCE, and chloroform in the groundwater plume.
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Title:
Contaminant Fate and Transport
Text: An assessment of site conditions indicated that:
The potential for continued leaching of constituents of concern (COCs) from vadose zone soil to groundwater was low to negligible.
The potential for transport of soil COCs through runoff was low to negligible.
Downward migration of chlorinated volatile organic compounds (CVOCs) was limited to approximately 66 feet bgs. The absence of higher CVOC levels below the second interbedded unit indicated that the slight downward gradient had not caused a significant impact at lower intervals.
The potential for the COC plume to reach the site boundary at concentrations exceeding acceptable levels was low because of lithologic controls on groundwater flow and the apparent CVOC degradation taking place.
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Title:
Risk Assessment
Text: A human-health risk screening for IR Site 40 groundwater estimated a total cancer risk of 0.0041 and a hazard index (HI) of 85, resulting primarily from PCE and TCE. Approximately 88% of the total cancer risk is from PCE, and approximately 76% and 9% percent of the total HI is from PCE and TCE, respectively. All other COCs contributed 5% or less to the total cancer risk or total HI.
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Title:
Bioremediation
Text: PCE and TCE can be completely biodegraded to ethene by members of the bacterial genus Dehalococcoides. Bioaugmentation with this microbe was selected as a remedial approach for IR Site 40 and a pilot test was conducted in two phases.
In Phase I, the addition of sodium lactate as a carbon source was used to stimulate native microorganisms. Monitoring well MW-40-28 was used to inject sodium lactate, but the injection resulted in incomplete dechlorination resulting in a build up of DCE and VC. In Phase II, a Dehalococcoides culture was added to further stimulate the biodegradation of PCE and TCE to ethene. The microbial culture was injected into monitoring wells MW-40-25 and MW-40-22.
MBTs were used to evaluate the microbial strains present in groundwater during Phase I and to evaluate the distribution of augmented microbes during Phase II. The bioaugmentation led to the reduction of the DCE to VC and further to ethene, indicating that the use of bioaugmentation was beneficial at this site.
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Visual Description: Sulfate, Methane and COD concentrations over time.
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Title:
MBT Results
Text: The figure shows the results of the PLFA analyses. PLFA analyses showed an increase in the biomass due to biostimulation in Phase I, which indicated that sufficient substrate was injected to promote biomass growth, but that the microorganisms capable of complete TCE reduction to ethene were not active. In addition, Dehalococcoides was not detected during Phase 1 although conditions were conducive for the Dehalococcoides to grow.
The addition of the Dehalococcoides culture in Phase II made the qPCR analysis especially useful. It enabled tracking of the survival and transport of the microbe, along with its effect on dechlorination. The results of the qPCR analyses are shown in Figure 2 and the Table. The qPCR analyses confirmed the presence of the bioaugmented microbes at locations 8 ft upgradient and 17 ft downgradient of the injection location after a period of four months.
Therefore, the qPCR analysis demonstrated that Dehalococcoides not only survived, but was transported within 4 months throughout the entire Phase II treatment area. Figure 3 illustrates the contaminant degradation response during Phases I and II in response to biostimulation and bioaugmentation.
At all locations where qPCR indicated Dehalococcoides was present in high numbers, only VC and ethene were detected.
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Visual Description: PCE, TCE, cDCE, VC, and ethene concentrations over time.
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Title:
Case Study: Norfolk Naval Base
Text: At Norfolk Naval Base in Virginia, the U.S. Naval Research Laboratory used isotopic fractionation of carbon dioxide and methane to investigate the biodegradation of petroleum hydrocarbons at an IR site.
In the presence of sufficient oxygen, microorganisms will biodegrade petroleum hydrocarbons to form carbon dioxide. Therefore, carbon dioxide levels in soil vapor are generally higher at fuel-impacted sites compared to clean sites.
However, as oxygen levels are depleted over time, methanogenic conditions can develop in the presence of petroleum hydrocarbons. Under methanogenic conditions, carbon dioxide is used as an electron acceptor and methane is produced by the microorganisms.
The fractionation of carbon isotopes in carbon dioxide and methane can be used to evaluate the biodegradation of petroleum hydrocarbons in soil vapor at impacted sites. Under methanogenic conditions, the creation of methane from carbon dioxide enriches 13C in carbon dioxide which makes it "heavier" and methane "lighter."
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Visual Description: Map of Norfolk Naval Base site in Virginia.
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Title:
Case Study: Isotope Results
Text: At the Norfolk site, soil-gas samples were analyzed for stable isotopes of carbon dioxide (delta 13CO2) and methane (delta 13CH4) in terms of percent modern carbon (pMC). The results of the isotope analysis indicated that active biodegradation was occurring at this site as follows:
In monitoring points near the plume, carbon dioxide and methane levels were elevated in soil-gas samples which indicated ongoing petroleum hydrocarbon biodegradation under methanogenic conditions.
Under methanogenic conditions, the "left over" carbon dioxide was heavier, while the methane was lighter based on isotope analyses shown here.
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Visual Description: Plots of percent modern carbon for carbon dioxide and methane.
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Title:
Summary
Text: The use of MBTs in environmental restoration applications is rapidly evolving. RPMs can draw upon the expertise of specialized biological laboratories, universities, and others to perform these state-of-the-art tests. Techniques such as real-time PCR, PLFA analysis, and others are currently available for field-scale applications.
However, because MBTs are relatively new techniques there are no standardized analytical methods or protocols established among service providers and laboratories. RPMs must carefully weigh the potential costs and benefits to their projects on a site-specific basis.
Further research and development are planned to standardize methods and improve the ability of MBTs to support the design, implementation, and optimization of remedial technologies. Click here for more information.
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Title:
Delete
Text: Delete
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Title:
Delete
Text: Delete
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