Title: Introduction Text: There is growing interest in the development and use of molecular biological tools (MBTs) for environmental restoration applications. MBTs are field and laboratory tests that can measure the presence and activity of microbes at a site. They can be used to assess the performance of monitored natural attenuation and bioremediation remedies. A few MBTs are already used to support field-scale projects and newer technologies continue to emerge. This Web Tool provides an introduction to MBTs applied through gene-based, lipid-based, protein-based, and isotope-based approaches. It also highlights Navy sites that have used these new techniques.

Title: Background Text: The information obtained from MBTs may help to improve remediation processes by: Allowing for quantitative assessment of the types of microorganisms present and their levels of metabolic activity. Providing supporting 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 issues such as the causes of incomplete biodegradation pathways or biofouling problems.

Title: Bacterial Cell (1 of 3) Text: To understand how MBTs work, it is important to understand the components of a bacterial cell. Bacteria are single cell organisms and do not have a membrane-bound nucleus like human cells. Their genes are contained in a single loop called the nucleoid. All bacterial cells include four groups of macromolecules called proteins, nucleic acids, lipids, and polysaccharides.

Title: Proteins Text: There are two types of proteins including enzymes and structural proteins. Enzymes are catalysts for the chemical reactions that occur in cells including biodegradation of contaminants. Structural proteins are used to build the cell wall, cytoplasm, and other components.

Title: Nucleic Acids Text: Each bacterial 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. Ribonucleic acid (RNA) is a single-stranded nucleic acid and it acts to convert genetic information into proteins.

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.

Title: Polysaccharide Text: Polysaccharides are long chain polymers of sugars. They are mainly located in the cell wall.

Title: DNA Structure (2 of 3) Text: Each bacterial cell has a single chromosome or strand of DNA that contains all of its genes. Specific genes on the DNA strand can be used to "fingerprint" or identify types of bacteria. 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).

Title: A-T Bases Text: Adenine (A) bonds with thymine (T) in DNA. Cytosine (C) bonds with guanine (G) in DNA.

Title: C-G Bases Text: Cytosine (C) and guanine (G) are organic bases found in both DNA and RNA molecules.

Title: Protein Synthesis (3 of 3) Text: Every cellular function is carried out by proteins. The presence of proteins and/or intermediate 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 bacterial chromosome to be converted into proteins. Click on the figure for more information.

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.

Title: Translation Text: Next, the messenger RNA (mRNA) strand carries genetic information from the bacterial chromosome to the ribosome, where protein synthesis occurs. This genetic material is used to code for a specific sequence of amino acids to form a protein as shown here. This process is called translation.

Title: Molecular Biological Tools Text: Once you understand the components of a bacterial cell, a number of molecular-based techniques can be developed to investigate the presence and metabolic activity of bacteria. 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.

Title: Gene-Based Tools (1 of 3) 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. A few examples are shown here of bacteria that are known to biodegrade chlorinated solvents. They were identified based on variances in their 16S rRNA gene. The two most common gene-based techniques are Polymerase Chain Reaction (PCR) and Denaturing Gradient Gel Electrophoresis (DGGE).

Title: 16S rRNA Gene Text: 16S rRNA is the name of a molecule located on the bacterial ribosome. The gene that codes for the 16S rRNA molecule is present in all bacteria 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. Even unknown bacteria can be placed within a framework of known microorganisms. Closely related bacteria will have more similar genetic sequences than distantly related ones.

Title: PCR (2 of 3) Text: Polymerase Chain Reaction (PCR) is a technique for amplifying a specific region of DNA. As shown here, each PCR cycle results in a doubling of DNA through synthesis of new strands. PCR helps to identify if a target bacteria is present in a sample. A technique called real-time PCR can also be used to determine the number of microbial cells in a sample. PCR can be performed by specialized biological laboratories and/or universities. Typical costs are $150 to $500 per sample depending on the number of gene targets and number of samples. Click here to learn more about the use of real-time PCR in the field.

Title: Real-Time PCR or Quantitative PCR Text: Real-time PCR (RTm PCR) or quantitative PCR (qPCR) is a method that allows direct measurement of the number of bacteria in a sample. RTm PCR uses a fluorescent marker, which is tagged to each gene copy. The amount of fluorescence measured throughout the amplification process is used to determine the number of targeted genes in the sample. This information is then correlated to the cell count of the target microorganism.

Title: DGGE (3 of 3) Text: After amplification, Denaturing Gradient Gel Electrophoresis (DGGE) can be used to identify the types of bacterial genes in a sample. The profiles for each type of bacteria are visible as bands in the gel shown here. DGGE analysis is expected to be used less often than real-time PCR in environmental applications. DGGE profiles can be performed by specialized biological laboratories and/or universities. Typical costs are $275 to $350 for a DGGE profile.

Title: Protein-Based Tools Text: Bacteria readily adapt and make only the proteins or enzymes that they need to survive in their environment. Although a certain bacteria may be present at a site, it may or may not be actively producing the enzymes needed to biodegrade a given contaminant. Protein-based tools help to determine whether or not the microbes are actively producing enzymes known to lead to biodegradation. 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 to detect enzymes necessary for biodegradation. Future development may lead to more widespread application.

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)

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 bacterial cell. 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. The use of PLFA analysis for environmental applications is gradually being replaced by more specific gene-based tools.

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.

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. The costs can range from $80 to over $1,000 per sample depending on the type and number of isotopes to be studied.

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.

Title: Isotope-Based Tools (2 of 2) Text: During biodegradation, the lighter isotopic species tend to be preferentially metabolized by bacteria, 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.

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.

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. This data suggests that after 1.3 years there is no significant change in the isotope fractionation of carbon at each monitoring location suggesting cis-DCE stall.

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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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.

Title: Conceptual Site Model Text: Click on the figure for more site information.

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.

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.

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.

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.

Title: Bioremediation Text: PCE and TCE can be completely biodegraded to ethene by the microbe, Dehalococcoides ethenogenes (D. ethenogenes). 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 D. ethenogenes 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 bacterial 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.

Title: MBT Results Text: Figure 1 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. The addition of the D. ethenogenes culture in Phase II made the qPCR analysis especially useful. It enabled tracking of the survival and transport of the bacteria, 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 D. ethenogenes not only survived, but was transported within 4 months throughout the entire Phase II treatment area.

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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."

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). 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.

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 is 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.

Title: References Text: Clark, I. and P. Fritz. 2000. Environmental Isotopes in Hydrogeology, Lewis Publ., Boca Raton, FL. Environmental Security Technology Certification Program (ESTCP). 2005. SERDP and ESTCP Panel Workshop on Research and Development Needs for the Environmental Remediation Applications of Molecular Biological Tools. October. Madigan, M. T., J. M. Martkinko, and J. Parker. 2002. Brock Biology of Microorganisms. 10th Ed. Prentice Hall. Upper Saddle River, NJ.

Title: Contact Text: For more information about MBTs, please contact: NFESC POC (805) 982-1656 PRTH_NFESCT2@navy.mil