Environmental Remediation can take the form of several basic strategies. Molecular Biological Tools (MBTs) can help whether your strategy is primarily Biological or whether microbes are helpful boosts to other strategies.
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When the remediation of petroleum-impacted sites by engineered methods are not feasible (e.g., large dilute plumes or far downgradient areas), MNA may be the most promising strategy to achieve remedial objectives. Natural attenuation results from physical processes (dispersion, volatilization, and dilution), abiotic degradation, and biological degradation (biodegradation). The monitoring of natural attenuation involves the characterization of chemical (abiotic) and biological (biodegradation, by microbes) processes using molecular biological tools (MBTs) as described below.
MBTs in MNA of Petroleum hydrocarbon–impacted sites. Biodegradation can be critically important for carrying out crucial degradative steps. MBTs are now routinely employed, such as CENSUS® qPCR and QuantArray®-Petro, to quantify concentrations of bacteria and their genes (functional genes) responsible for degrading the petroleum hydrocarbons (including BTEX, MTBE) — to assess a site’s biodegradative potential for supporting MNA (see Top Tips for MNA). Complementing these MBTs, Stable Isotope Probing (SIP) is used to conclusively demonstrate the biodegradation of specific contaminants (e.g., benzene). The method uses a contaminant of interest synthesized with 13C-label (e.g. synthesized 13C‑benzene) that is loaded into the matrix of a vessel (Bio-Trap®) that is placed into a site-monitoring well for approximately 30-45 days. After retrieval of the Bio-Trap, the presence of 13C-labeled biodegradative end products — biomass and CO2 — provides conclusive evidence that the contaminant is being biodegraded, providing support to MNA as a remediation strategy.
MBTs in MNA of Chlorinated solvent–impacted sites. Chlorinated compounds can be aerobically cometabolized (i.e., co-oxidized along with the growth-supporting nutrient but without providing energy). Chlorinated compounds can also be abiotically degraded via catalysis by common iron-bearing minerals. Although aerobic cometabolism and abiotic degradation can be important and even predominant components of the MNA of chlorinated solvent sites, these degradative mechanisms are a challenge to quantify because they do not produce measurable daughter products (unlike anaerobic reductive dechlorination). Therefore, to assess these mechanisms’ contribution in MNA, other lines of evidence complementary to traditional chemical and geochemical monitoring are needed. As described in the white paper, “MNA of Chlorinated Solvents: Aerobic Cometabolism & Abiotic Degradation”, a chlorinated-solvent site’s Aerobic cometabolism can be assessed by quantifying bacteria and their genes responsible for co-oxidizing chlorinated hydrocarbons (using CENSUS® qPCR or QuantArray®-Chlor). One method available for assessing the potential for abiotic degradation is Magnetic Susceptibility. A variety of chlorinated solvents (including PCE, TCE, cis-DCE, and vinyl chloride) react with the mixed-valence iron mineral magnetite (Fe3O4), which is the most abundant magnetic mineral in natural sediments. Thus, Magnetic Susceptibility provides an inexpensive but valuable estimate of the potential for abiotic reactivity of magnetite with chlorinated solvents in environmental samples.
Electron-donor addition stimulates the growth of halorespiring bacteria (e.g., Dehalococcoides, Dehalogenimonas, Dehalobacter) and is established as a treatment for remediating sites contaminated with chlorinated solvents. However, the effectiveness of electron-donor addition can be limited by competing electron-accepting processes, inhibitory co-contaminants, and inadequate mass or distribution of the electron donor.
Therefore, such potential confounders at a site should be characterized regarding the existing halorespiring bacterial concentrations, the electron donor quantity needed to overcome terminal electron-accepting processes (TEAPs), and whether the treatment is likely to change the groundwater pH so it can be corrected into a favorable range.
Quantification of the contaminant-degrading microorganisms (e.g., Dehalococcoides) and their responsible genes (e.g., vinyl chloride reductase) is accomplished using CENSUS qPCR or QuantArray‐Chlor. An In Situ Microcosm study can also be used to test different amendments and evaluate donors in situ.
The biodegradation of petroleum hydrocarbons can be limited by low concentrations of electron acceptors such as dissolved oxygen, nitrate, and sulfate. Therefore, an electron acceptor is often added or injected — air (bioventing/biosparging), oxygen (oxygen infusion), or a product (e.g. ORC®, PermeOx®, EAS®) — to stimulate the growth of petroleum hydrocarbon–degrading bacteria, to enhance bioremediation.
For planning for the addition of electron-acceptor, the site should be characterized regarding the existing bacteria and their genes capable of degrading the petroleum hydrocarbons — BTEX degraders (aerobic and anaerobic), MTBE and TBA degraders (aerobic), and naphthalene and other PAH degraders (aerobic and anaerobic). This information is provided by CENSUS® qPCR or QuantArray®‐Petro, e.g., and can be part of an in-situ microcosm study.
This understanding of the community of petroleum hydrocarbon–degrading bacteria informs whether an oxygen-releasing product should be used to stimulate aerobic bioremediation, or a sulfate product should be used to stimulate anaerobic bioremediation — and how much of the electron acceptor should be added.
Before the electron-acceptor addition, QuantArray®‐Petro provides critical information on the baseline microbiological community and for monitoring the effectiveness of the treatment to stimulate the petroleum hydrocarbon–degrading microbes.
NSZD refers to the loss of light non-aqueous phase liquid (LNAPL) petroleum hydrocarbons from the subsurface due to the naturally occurring processes of dissolution, volatilization, and biodegradation. NSZD can be used at LNAPL-contaminated sites after active remediation and where removal of LNAPL is limiting. The dominant process for NSZD is methanogenesis. Recently, a study of the microbial communities at an LNAPL-contaminated site undergoing NSZD found a large population of Archaea, particularly Methanomicrobia and Methanobacteria, and an indication of complex syntrophic relationships among methanogens, methanotrophs, and bacteria. During environmental cleanup, hydrophobic contaminants can become bound to soil particles making them difficult to remediate. To increase contaminant desorption, natural surfactants can be utilized in combination with soil and water remediation technologies. Using CENSUS® qPCR to quantify the microorganisms with the capability of producing biosurfactants can be particularly useful at sites undergoing NSZD.
QuantArray®-NSZD quantifies a broad spectrum of microorganisms and functional genes involved in biodegrading LNAPL in the source zone. Additionally, QuantArray®‐Petro can be used to assess the genetic potential for both anaerobic and aerobic biodegradation of petroleum hydrocarbons.
Next Generation Sequencing (NGS) indicates the microorganisms present down to the genus and even the species, as well as their relative proportions, which can provide insight into the types of microbial processes occurring.
In Situ Chemical Oxidation (ISCO). ISCO is used to remediate a wide range of volatile and semi-volatile contaminants, including dense nonaqueous phase liquids (DNAPL) source zones and their dissolved-phase plumes. The oxidizing agents most commonly used are hydrogen peroxide, catalyzed hydrogen peroxide, potassium permanganate, sodium permanganate, sodium persulfate, and ozone. Advantages of ISCO include the rapid degradation of contaminants, with measurable reductions in weeks or months, without producing significant wastes. Additionally, ISCO can potentially enhance biodegradation of residual hydrocarbons in subsequent bioremediation or monitored natural attenuation. Thus, CENSUS® qPCR or QuantArray®‐Chlor can be used to understand the capacity of the site for these final steps in remediation.
In Situ Chemical Reduction (ISCR). ISCR is most often used to clean up chromium and the industrial solvent trichloroethene (TCE). For remediating chromium and other metals/metalloids (e.g., arsenic and uranium), energetics, and halogenated ethenes and ethanes, the most commonly used reductant is the engineered reductant, zero valent iron (ZVI). For treating chlorinated solvents, ZVI can be coupled with bioremediation. The reducing conditions resulting from ZVI treatment are favorable for biological reductive dechlorination, including compounds having lower reactivity with ZVI, such as cDCE, VC, or 1,2-DCA.
ISCR reducing conditions can also be produced by biotic reactions. Anaerobic biostimulation can induce iron-reducing conditions (ferric to ferrous), with ferrous iron reducing hexavalent chromium to insoluble trivalent chromium. Additionally, microbial reduction of sulfate to sulfide can result in insoluble precipitates of divalent metal sulfides.
Commercial products for ISCR include composites containing ZVI-organic carbon, biodegradable carriers, electron acceptors, and nutrients — to promote both abiotic and biotic degradation.
Thus, CENSUS® qPCR or QuantArray®‐Chlor can assess the potential of the microbial community for biological reduction-dechlorination and for anaerobic biostimulation to induce reductants.
Min-Trap™ samplers are applicable to combined biotic/abiotic degradation of chlorinated solvents as well as in situ oxidation and in situ chemical reduction. Min-Traps™ can assess the formation of reactive iron minerals for biotic/abiotic degradation of chlorinated solvents, the co-precipitation of metals like arsenic with iron oxides during in situ chemical oxidation, the formation of metal sulfides during in situ chemical reduction at Cr(VI) sites, and whether pH neutralization will increase solid phase minerals. These samplers are useful in estimating the long-term stability and/or reactivity of minerals, implications for transition from active to passive treatment, and to confirm ongoing effectiveness of active in-situ remediation, passive treatment, and long-term monitoring programs.
CSIA conclusively determines whether contaminant degradation (biotic or abiotic) has occurred and can provide the baseline to assess the effectiveness of remediation options including biostimulation, bioaugmentation, and combined bioremediation/ISCR.
In-situ thermal remediation (ISTR) uses thermal conductive heating (TCH) elements to heat the ground to temperatures above 100°C to accelerate the dissolution, desorption, volatilization/removal, and abiotic degradation of contaminants. Additionally, lower temperature heating can stimulate biodegradation (and desorption) of contaminants. ISTR is typically one of multiple methods used at a site. Along with it, other methods are used, for example, to treat the downgradient plume, which include heat-stimulated bioremediation (30 to 40°C), and, after ISTR shutdown, to treat the source zone, which include monitored natural attenuation (MNA) or bio-polishing whereby the bacteria use the residual heat energy and the newly dissolved organic matter to “polish off” the residual contaminants. These methods can be used with amendments such as electron donors, electron acceptors, and bacterial cultures to promote post-ISTR biodegradation.
When assessing treatment strategies such as ISTR, multiple lines of evidence — site chemistry (concentrations of contaminant, daughter products, etc.), geochemistry (redox status, electron acceptors, electron donors) and microbiology (CENSUS® qPCR and QuantArray®-Chlor or QuantArray®-Petro) — provide the most complete picture.
At chlorinated-solvent contaminated sites, CENSUS® qPCR and QuantArray®-Chlor are used to quantify Dehalococcoides and vinyl chloride reductase genes as an indispensable component of the performance monitoring of anaerobic-bioremediation (reductive dechlorination). Similarly, at sites contaminated with petroleum hydrocarbons, CENSUS® qPCR and QuantArray®-Petro are routinely used to quantify the genes for aerobic and anaerobic biodegradation of BTEX, naphthalene, and other petroleum hydrocarbons.
Phytoremediation uses plants and associated soil microbes to reduce the quantity and toxicity of contaminants at a site by: (1) retaining them in the roots or rhizosphere to prevent their dispersal (phytostabilization), (2) converting them to less harmful substances (phytodegradation), (3) converting them to gases released via evapotranspiration into the atmosphere (phytovolatilization), and (4) accumulating them so they can be harvested (phytoextraction). Phytoremediation can be a cost-effective alternative to more soil-destructive engineering methods.
The soil microorganisms, in symbiotic relationships with plant roots, feed off the rhizosphere and can make nutrients available to the plant, secrete growth-promoting phytohormones, and protect the plant against abiotic and biotic stressors — and thereby impact the uptake, sequestration, and detoxification of contaminants. For example, root exudates can promote the growth of oil-degrading bacteria; thus, in bioremediating petroleum, plants and bacteria can be more effective together than either organism alone. Based on this knowledge, phytoremediation can be further enhanced by adding bacteria and symbiont fungi (bioaugmentation) or growth-supporting nutrients (biostimulation).
During the development of the site conceptualization model, MBTs are used to quantify the microbes existing at the site that can biodegrade the contaminant as well as their distribution across the site’s existing soil types (e.g., CENSUS® qPCR or QuantArrays®). Should a phytoremediation strategy be selected, pilot testing can be lengthy, running for 1−3 years; therefore, during this time, it is important to evaluate the factors in the effectiveness of the phytoremediation strategy, such as can be obtained using MBTs. For example, MBTs can quantify the root soil for contaminant-degrading bacteria or microorganisms able to enhance the uptake of the contaminant into the plant as wells as the potential effectiveness of biostimulation (e.g., air sparging of individual trees) or bioaugmentation (CENSUS® qPCR or QuantArrays®). Additionally, Next Generation Sequencing (NGS) can identify the microorganisms present down to the genus and even the species, as well as their relative proportions, to provide insight into the microbial relationships and processes, including their potential to promote the growth and health of the plants.