Formulation of the CBC-Model for Modelling the Contaminants and Footprints in Natural Attenuation of BTEX

The Refuse Hideaway Landfill (23-acre) received municipal, commercial, and industrial waste between 1974 and 1988. It was designed as a "natural attenuation" landfill and no provision was made to collect and treat contaminated water. Natural biological degradation through sequential reductive dechlorination had been an important mechanism for natural attenuation at the site. We used the concentration of hydrogen to forecast whether reductive dechlorination would continue over time at particular locations in the plume. Based on published literature, reductive dechlorination and natural attenuation of PCE, TCE, and cis-DCE can be expected in the aquifer if the concentration of molecular hydrogen in monitoring wells are adequate (> 1 nanomolar). Reductive dechlorination can be expected to continue as the ground water moves down gradient. Natural attenuation through reductive dechlorination is not expected in flow paths that originate at down gradient monitoring wells with low concentrations of molecular hydrogen (< 1 nanomolar). In three monitoring wells at the margin of the landfill and in five monitoring wells down gradient of the landfill, ground water maintained a molecular hydrogen concentration, ranging from 1.30 to 9.17 nanomolar, that is adequate for reductive dechlorination. In three of the monitoring wells far down gradient of the landfill, the concentration of molecular hydrogen (0.33 to 0.83 nanomolar) was not adequate to support reductive dechlorination. In wells with adequate concentrations of hydrogen, the concentrations of chlorinated volatile organic compounds were attenuated over time, or concentrations of chlorinated volatile organics were below the detection limit. In wells with inadequate concentrations of hydrogen, the concentrations of chlorinated organic compounds attenuated at a slower rate over time. In wells with adequate hydrogen the first order rate of attenuation of PCE, TCE, cis-DCE and total chlorinated volatile organic compounds varies from 0.38 to 0.18 per year. In wells without adequate hydrogen the rate varies from 0.015 to 0.006 per year.     

The Effect of Groundwater Aeration on PCE Natural Attenuation Patterns

Attempts to remediate ground water contaminated with tetrachloroethylene at a Superfund site in Minnesota included the installation of a vacuum vaporizer well. Prior to the remedial system installation, the contaminant source half-life was approximately 0.3 years. Aquifer aeration by the vacuum vaporizer well unintentionally disrupted the ambient natural attenuation rate. Although the overall plume size did not increase, concentrations of the anaerobic breakdown products of tetrachloroethylene—trichloroethylene, cis-dichloroethylene, and vinyl chloride—all increased in downgradient monitoring wells after startup of the vacuum vaporizer well. At a well 360 feet downgradient of the source, trichloroethylene increased from concentrations below 10?µg/L to over 35?µg/L, while cis-dichloroethylene concentrations increased from 70?µg/L to 370?µg/L. Vinyl chloride, which was below detection limits at this location prior to operation of the vacuum vaporizor well, increased in concentration to 83?µg/L. Concentrations of these contaminants returned to pre-sparging levels after deactivation of the system, indicating that existing anaerobic natural attenuation processes play an important role in the remediation of ground water at this site. Investigations should routinely assess the role of natural attenuation in remediation before implementing engineered remedies that may disrupt existing beneficial attenuation processes.     

Challenges in Monitoring the Natural Attenuation of Spatially Variable Plumes

Monitored natural attenuation may be applied as a risk-based remediation strategy if it can be established that contaminants are or will be reduced to some acceptable level at or before a compliance point. Contaminant attenuation is often attributed to intrinsic biodegradation, which in some circumstances may occur only at the plume fringes where electron acceptors from the surrounding uncontaminated zones mix by dispersion and diffusion with the plume. However, due to the common spatial and temporal variability exhibited by many plumes, the centreline monitoring approaches advocated in many natural attenuation protocols may be unable to detect natural attenuation occurring primarily by fringe processes. Snapshot data from a multilevel sampling well transect across an MTBE plume at Vandenberg Air Force Base, CA, USA, illustrate the difficulty of centreline monitoring and the challenge of providing sufficient detail to detect attenuation processes that may be occurring primarily at plume fringes. In a study of a phenols plume in Wolverhampton, UK, high-resolution multilevel wells demonstrated that the key biodegradation processes were restricted spatially to the upper fringe of the plume and were rate-limited by transverse dispersion and diffusion of electron acceptors into the plume. Thus the overall extent of biodegradation was considerably less than suggested by a plume-scale analysis of total electron acceptor and contaminant budgets. These examples indicate that more robust and cost-effective MNA assessments can be obtained using monitoring strategies that focus on the location of key biodegradation processes.     

Evaluation of Natural Attenuation of Benzene and Dichloroethanes at the KL Landfill

Natural attenuation of benzene and dichloroethanes in groundwater contaminated by leachate from the West KL Avenue landfill in Kalamazoo, Michigan, was evaluated in three phases. Existing data from the previous site investigations were used to locate a series of high-resolution vertical profile samples. By analyzing data from the discrete vertical profile samples, the rates of attenuation of benzene and dichloroethanes in the plume were forecasted. Permanent monitoring wells were installed over the depth intervals associated with high concentrations in the vertical profile sampling. These wells were monitored over time to extract independent estimates of the rates of degradation of benzene and dichloroethanes. Estimates of first-order attenuation rate constants were obtained using two methods: a method due to Buscheck and Alcantar (1995), which is based on a one-dimensional steady-state analytical solution, and the tracer correction method of Wiedemeier et al. (1996). The rates of attenuation predicted from the vertical profile sampling were found to be in good agreement with the rates obtained from the permanent monitoring well data, indicating that the long-term behavior of the contaminant plumes is consistent with the initial forecast. The results also indicated that the natural attenuation of benzene, 1,1-dichloroethane (DCA), and 1,2-DCA was statistically significant (at the 0.05 level).     

Monitored Natural Attenuation of Explosives

Explosives are subject to several attenuation processes that potentially reduce concentrations in groundwater over time. Some of these processes are well defined, while others are poorly understood. The objective of the project was to optimize data collection and processing procedures for evaluation and implementation of monitored natural attenuation of explosives. After conducting experiments to optimize data quality, a protocol was established for quarterly monitoring of thirty wells over a 2-year period at a former waste disposal site. Microbial biomarkers and stable isotopes of nitrogen and carbon were explored as additional approaches to tracking attenuation processes. The project included a cone penetrometry sampling event to characterize site lithology and to obtain sample material for biomarker studies. A three-dimensional groundwater model was applied to conceptualize and predict future behavior of the contaminant plume. The groundwater monitoring data demonstrated declining concentrations of explosives over the 2 years. Biomarker data showed the potential for microbial degradation and provided an estimate of the degradation rate. Measuring stable isotopic fractions of nitrogen in TNT was a promising method of monitoring TNT attenuation. Overall, results of the demonstration suggest that monitored natural attenuation is a viable option that should be among the options considered for remediation of explosives-contaminated sites.     

Methyl-tertiary Butyl Ether Natural Attenuation Case Studies

The occurrence of methyl-tertiary butyl ether (MtBE), a gasoline additive in ground water and surface water, is causing regulatory agencies, owner/operators, environmental professionals, and researchers to reevaluate remediation strategies at sites where gasoline containing this additive has been released. Over the last 5 to 10 years, monitored natural attenuation has been applied at petroleum hydrocarbon-impacted sites with increasing frequency. The efficacy of this remediation method for releases containing MtBE is now coming under increased scrutiny. The question of natural attenuation efficacy stems from uncertainty about MtBE biodegradability and behavior in the subsurface. Researchers and applied environmental scientists have completed and are continuing studies concerning MtBE biodegradability and behavior. This article briefly summarizes the history of MtBE, its physicochemical properties, its behavior in the environmental, and the applicability of monitored natural attenuation as a remediation tool. Case studies representing past and current research are then presented and followed by a brief discussion. Results from the documented research reviewed show that MtBE does biodegrade in the laboratory and at actual release sites. “Plumathon” studies document MtBE concentration and mass reduction and/or plume stabilization over time. MtBE concentrations in monitoring wells may show declining concentrations indicating natural attenuation. The data reviewed from past and current research suggest that natural attenuation may be the appropriate remediation strategy at some release sites. Yet, the data also indicate that care must be exercised in determining the efficacy of applying monitored natural attenuation at sites impacted with MtBE.     

Implementation of natural attenuation at a JP-4 jet fuel release after active remediation

After eighteen months of active remediation at a JP-4 jet-fuel spill, aresidual of unremediated hydrocarbon remained. Further site characterizationwas conducted to evaluate the contribution of natural attenuation to controlexposure to hazards associated with the residual contamination in thesubsurface. Activities included the detailed characterization ofground-water flow through the spill; the distribution of fuel contaminantsin groundwater; and the analysis of soluble electron acceptors moving intothe spill from upgradient. These activities allowed a rigorous evaluation ofthe transport of contaminants from the spill to the receptor of groundwater,the Pasquotank River. The transport of dissolved contaminants of concern,that is benzene, toluene, ethyl benzene, xylene isomers (BTEX) andmethyl-tertiary-butyl ether (MTBE), into the river from the source area wascontrolled by equilibrium dissolution from the fuel spill to the adjacentgroundwater, diffusion in groundwater from the spill to permeable layers inthe aquifer, and advective transport in the permeable layers. The estimatedyearly loading of BTEX compounds and MTBE into the receptor was trivial evenwithout considering biological degradation. The biodegradation ofhydrocarbon dissolved in groundwater through aerobic respiration,denitrification, sulfate reduction, and iron reduction was estimated fromchanges in ground-water chemistry along the flow path. The concentrations oftarget components in permanent monitoring wells continue to decline overtime. Long term monitoring will ensure that the plume is under control, andno further active remediation is required.     

Determination of major pollutant and biogeochemical processes in an oil-contaminated aquifer using human health risk assessment and multivariate statistical analysis

Techniques for monitored natural attenuation usually produce large complex datasets that are difficult to interpret. Here, human health risk assessments and multivariate statistical analyses are combined to extract and analyze useful information from large monitoring datasets to identify the main pollutants in a petroleum-contaminated aquifer in northeast China and the main biogeochemical processes affecting the pollutants. The data included organic and inorganic geochemical species concentrations, physicochemical indicators, C and S stable isotope data collected for four years of more than 10 monitoring. The health risk assessment indicated that benzene was a representative pollutant. Cluster analysis classified the groundwater samples into two groups and indicated strong biodegradation occurred near the core and upgradient of the petroleum hydrocarbon plume. The factors explaining most variability were extracted by principal component analysis, which correlated with biodegradation and mineral dissolution processes. The factor scores and spatial distributions of hydrogeochemical and isotope indicators confirmed that biodegradation effects weakened and mineral dissolution strengthened upgradient to downgradient of the contaminated plume. The analysis method could be useful for rapidly studying pollution characteristics and identifying biodegradation processes in contaminated aquifersfrom large complex datasets. The results will provide a basis for developing an enhanced bioremediation scheme for the study site.     

Diversity of Planktonic and Attached Bacterial Communities in a Phenol-Contaminated Sandstone Aquifer

Polluted aquifers contain indigenous microbial communities with the potential for in situ bioremediation. However, the effect of hydrogeochemical gradients on in situ microbial communities (especially at the plume fringe, where natural attenuation is higher) is still not clear. In this study, we used culture-independent techniques to investigate the diversity of in situ planktonic and attached bacterial communities in a phenol-contaminated sandstone aquifer. Within the upper and lower plume fringes, denaturing gradient gel electrophoresis profiles indicated that planktonic community structure was influenced by the steep hydrogeochemical gradient of the plume rather than the spatial location in the aquifer. Under the same hydrogeochemical conditions (in the lower plume fringe, 30 m below ground level), 16S rRNA gene cloning and sequencing showed that planktonic and attached bacterial communities differed markedly and that the attached community was more diverse. The 16S rRNA gene phylogeny also suggested that a phylogenetically diverse bacterial community operated at this depth (30 mbgl), with biodegradation of phenolic compounds by nitrate-reducing Azoarcus and Acidovorax strains potentially being an important process. The presence of acetogenic and sulphate-reducing bacteria only in the planktonic clone library indicates that some natural attenuation processes may occur preferentially in one of the two growth phases (attached or planktonic). Therefore, this study has provided a better understanding of the microbial ecology of this phenol-contaminated aquifer, and it highlights the need for investigating both planktonic and attached microbial communities when assessing the potential for natural attenuation in contaminated aquifers.     

Selecting Remediation Goals by Assessing the Natural Attenuation Capacity of Groundwater Systems

Remediation goals for the source areas of a chlorinated ethene-contaminated groundwater plume were identified by assessing the natural attenuation capacity of the aquifer system. The redox chemistry of the site indicates that sulfate-reducing (H₂ ∼ 2 nanomoles [nM]) per liter conditions near the contaminant source grade to Fe(III)-reducing conditions (H₂ ∼ 0.5 nM) downgradient of the source. Sulfate-reducing conditions facilitate the initial reduction of perchloroethene (PCE) to trichloroethene (TCE), cis-dichloroethene (cis-DCE), and vinyl chloride (VC). Subsequently, the Fe(III)-reducing conditions drive the oxidation of cis-DCE and VC to carbon dioxide and chloride. This sequence gives the aquifer a substantial capacity for biodegrading chlorinated ethenes. Natural attenuation capacity (the slope of the steady-state contaminant concentration profile along a groundwater flowpath) is a function of biodegradation rates, aquifer dispersive characteristics, and groundwater flow velocity. The natural attenuation capacity at the Kings Bay, Georgia site was assessed by estimating groundwater flowrates (∼0.23±0.12 m/d) and aquifer dispersivity (∼1 m) from hydrologic and scale considerations. Apparent biodegradation rate constants (PCE and TCE ∼0.01 d^-1; cis-DCE and VC ∼0.025 d^-1) were estimated from observed contaminant concentration changes along aquifer flowpaths. A boundary-value problem approach was used to estimate levels to which contaminant concentrations in the source areas must be lowered (by engineered removal), or groundwater flow velocities lowered (by pumping) for the natural attenuation capacity to achieve maximum concentration limits (MCLs) prior to reaching a predetermined regulatory point of compliance.     

Spatial Electron Acceptor Variability: Implications for Assessing Bioremediation Potential

An extensive network of multilevel samplers was established in a hydrocarbon-contaminated wetland aquifer. Results of groundwater sampling for benzene, toluene, ethylbenzene, and xylenes (BTEX), and electron acceptors show that both pristine and contaminated groundwater have spatially variable chemical signatures, owing primarily to microbially mediated oxidation-reduction reactions. Due to these spatial variations, estimates of the efficiency of intrinsic bioremediation can vary significantly depending on how geochemical data are collected. Use of data collected from monitoring wells with screens longer than the vertical extent of the plume will generally underestimate the potential for intrinsic bioremediation for the most chemically active horizon of the plume. A comparison of pristine and contaminated redox patterns demonstrates that, although BTEX exerts the highest demand for electron acceptors, oxidation of natural organic matter also contributes to electron acceptor utilization. If natural and other non-BTEX losses of electron acceptors are ignored, the assimilative capacity, defined as the amount of a contaminant that can potentially be degraded with known amounts of electron acceptors, will be overestimated. Many numerical and analytical models designed to simulate biodegradation are directly or indirectly based on assimilative capacity estimates. Proper estimation of assimilative capacity is crucial if models are to accurately quantify solute concentrations over time and space.     

Stereoscopic virtual reality models for planning tumor resection in the sellar region

Background

It is difficult for neurosurgeons to perceive the complex three-dimensional anatomical relationships in the sellar region.

Methods

To investigate the value of using a virtual reality system for planning resection of sellar region tumors. The study included 60 patients with sellar tumors. All patients underwent computed tomography angiography, MRI-T1W1, and contrast enhanced MRI-T1W1 image sequence scanning. The CT and MRI scanning data were collected and then imported into a Dextroscope imaging workstation, a virtual reality system that allows structures to be viewed stereoscopically. During preoperative assessment, typical images for each patient were chosen and printed out for use by the surgeons as references during surgery.

Results

All sellar tumor models clearly displayed bone, the internal carotid artery, circle of Willis and its branches, the optic nerve and chiasm, ventricular system, tumor, brain, soft tissue and adjacent structures. Depending on the location of the tumors, we simulated the transmononasal sphenoid sinus approach, transpterional approach, and other approaches. Eleven surgeons who used virtual reality models completed a survey questionnaire. Nine of the participants said that the virtual reality images were superior to other images but that other images needed to be used in combination with the virtual reality images.

Conclusions

The three-dimensional virtual reality models were helpful for individualized planning of surgery in the sellar region. Virtual reality appears to be promising as a valuable tool for sellar region surgery in the future.

     

Changes in Prokaryote and Eukaryote Assemblages Along a Gradient of Hydrocarbon Contamination in Groundwater

Groundwater biota are particularly sensitive to environmental perturbations such as groundwater contamination. The diversity of prokaryotic and eukaryotic biota has been examined along a gradient of chlorinated hydrocarbon (CHC) contamination in the Botany Sands, an urban coastal sand-bed aquifer (Sydney, Australia). Molecular techniques were used to analyze the richness and composition of prokaryote and eukaryote assemblages using 16S and 18S rDNA, respectively. Taxon richness did not change significantly along the gradient for either prokaryotes or eukaryotes; however, significant shifts in assemblage composition were evident for both groups. Assemblage changes were most strongly correlated with concentrations of the major CHC, cis-1,2-dichloroethene, but the concentrations of a number of the contaminants were also correlated, making it difficult to infer if effects were due to any particular contaminant. The presence of cis-1,2-dichloroethene and other secondary ethenes suggests in situ breakdown of the primary CHCs via natural attenuation. The current focus of management of the Botany aquifer is to stop the contaminant plume reaching the adjoining estuary. This approach is clearly justified given the changes evident in the microbial assemblages in the groundwater, which are a likely consequence of the contamination.     

Cone penetrometer testing,hydropunch®, and borehole geophysics applications for environmental investigations

An effective groundwater monitoring system can be implemented by the combined utilization of cone penetrometer (CPT), HydroPunch^® sampling, and borehole geophysical methods. The combined techniques provide a cost‐effective method for the design of a groundwater monitoring system for geologists or hydrogeologists assessing a site. With the relatively high costs associated with determining groundwater quality for site assessments, coupled with regulatory agency compliance, these combined methods can provide an effective edge in an increasingly competitive environmental industry. CPT combined with HydroPunch sampling can delineate the horizontal and vertical extent and concentration of a contaminant plume, define the extent and thickness of a free product plume, define soil and aquifer characteristics, and aid in the proper selection of well location and screen placement. The use of borehole geophysics further enhances the interpretation provided from the CPT. The interpretation of borehole geophysics provides additional information about the deposition regime of the area of investigation and a more detailed investigation of the stratigraphy. The CPT and HydroPunch can be used in unconsolidated sediments, and HydroPunch sampling can be combined with a hollow‐stem auger system. Borehole geophysics can be run in almost any environment. CPT and borehole geophysics provide information on specific lithologic characteristics necessary to obtain a groundwater sample from vertically separated aquifers. The HydroPunch can obtain a discrete, chemically representative groundwater sample from the targeted aquifer. CPT and borehole geophysics can also be used to determine lithology and for correlation of equivalent stratas from one borehole or well to the next. Borehole geophysical interpretation also provides a means of determining not only the stratigraphy and lithology but also the aquifer parameters and the type of fluids in the aquifer. Hydrogeologic and geologic data obtained from using these three methods can be employed to maximize the cost‐effectiveness and design efficiency of a groundwater monitoring system. Proper location of wells and screened interval placements are determined by a coherent design process rather than by random chance. Two studies demonstrating the combined applications of CPT, HydroPunch, and borehole geophysics for the design and placement of groundwater monitoring wells are presented in the following discussion.     

Field-Scale Modeling of Benzene,Toluene, Ethylbenzene,and Xylenes (BTEX) Released from Multiple Source Zones

A field-scale three-dimensional numerical flow and transport model was applied to simulate the fate and transport of benzene, toluene, ethylbenzene, and xylenes (BTEX) from six source zones of light non-aqueous-phase liquids (LNAPLs). A number of scenarios were defined to evaluate the effect of natural attenuation and enhanced remediation methods on the fate of BTEX. Two strategies were defined for cleaning up the site: (1) reduction of BTEX over the entire site down to the maximum concentration limit (MCL), and (2) remediation of the site to a level that reduces BTEX below MCLs only at the nearest discharge wells. The results show that under natural attenuation alone, the remediation time for benzene (time needed to decrease concentrations to a specified level) is more than 60 years, whereas that of TEX is longer, but only benzene will reach the receptors. LNAPL removal and enhanced bioremediation techniques could decrease this time to about 30 years for benzene. A cost-effective two-stage enhanced remediation operation should be applied. In the first stage, the entire dissolved BTEX plume should be treated, whereas in the second stage, after the remediation of benzene, the operation area should be limited to the remaining TEX plume areas. In the second strategy, air injection will be sufficient to prevent the BTEX plume from reaching the receptors. Addition of oxygen and hydrogen peroxide is likely too expensive.     

Assessing the correlation between anaerobic toluene degradation activity and <Emphasis Type="Italic">bssA</Emphasis> concentrations in hydrocarbon-contaminated aquifer material

The assessment of biodegradation activity in contaminated aquifers is critical to demonstrate the performance of bioremediation and natural attenuation and to parameterize models of contaminant plume dynamics. Real time quantitative PCR (qPCR) was used to target the catabolic bssA gene (coding for benzylsuccinate synthase) and a 16S rDNA phylogenetic gene (for total Bacteria) as potential biomarkers to infer on anaerobic toluene degradation rates. A significant correlation (P = 0.0003) was found over a wide range of initial toluene concentrations (1–100 mg/l) between toluene degradation rates and bssA concentrations in anaerobic microcosms prepared with aquifer material from a hydrocarbon contaminated site. In contrast, the correlation between toluene degradation activity and total Bacteria concentrations was not significant (P = 0.1125). This suggests that qPCR targeting of functional genes might offer a simple approach to estimate in situ biodegradation activity, which would enhance site investigation and modeling of natural attenuation at hydrocarbon-contaminated sites.     

Modeling the Impact of Oxygen Reaeration on Natural Attenuation

Based on studies of leaking petroleum storage tank (LPST) sites in Texas and California, the average plume of benzene, toluene, ethylene, and xylenes (BTEX) is between 61 and 132 m (200 and 400 ft) long. Standard modeling of BTEX plumes produces plumes well in excess of observed plume lengths. The amount of oxygen carried into the plume zone with clean upgradient water often is insufficient to account for the levels of biodegradation observed in these studies. Traditional recharge of oxygen-containing water into an aquifer adds insufficient oxygen to the system and cannot account for the observed plume lengths. Research has shown that anaerobic processes can contribute to biodegradation in certain cases; however, anaerobic pathways are not included in this work. Reaeration of oxygen-depleted aquifers by diffusive transport of oxygen through the vadose zone has generally been neglected as a way to introduce oxygen into surficial aquifers. The observed plume lengths and preliminary laboratory results indicate that this source of oxygen should be accounted for in any natural attenuation model of BTEX contamination. This approach to modeling reaeration has been incorporated into the finite-element groundwater flow and contaminant transport code, FLOTRAN. Adding diffusion-driven reaeration to the modeling process produces BTEX plumes consistent with observed plume lengths.     

Rate of Natural Attenuation of Tert-Butyl Alcohol at a Chemical Plant

Tert-butyl alcohol (TBA) may be present in groundwater as an original component of leaked gasoline, or as a degradation product of methyl tert-butyl ether (MTBE). Evidence for natural attenuation of TBA in groundwater is presented from a chemical plant in Pasadena, Texas. Shallow groundwater in several areas of the plant has been affected by historic leaks and spills of TBA. A decade of regular groundwater monitoring of one groundwater plume, consisting primarily of TBA, shows generally declining concentrations and a plume area that is shrinking. Natural attenuation mechanisms are limiting the advective transport of TBA. The principal mechanism of attenuation in this case is probably biodegradation as the other physical components of natural attenuation (dilution, dispersion, diffusion, adsorption, chemical reactions, and volatilization) cannot explain the behavior of the plume over time. Biodegradation was also indicated by the enrichment of stable carbon isotope composition (¹³C/¹²C) of TBA along the flow path. Preliminary dissolved gas and electron acceptor analyses indicate the groundwater is at least under sulfate reducing condition in the core of the plume and the process responsible for biodegradation of TBA may include fermentation under aerobic (plume fringes) and possible anaerobic conditions. This case history demonstrates that natural attenuation of TBA is important, and can be used as a groundwater management tool at this site.     

Microbiological Comparison of Core and Groundwater Samples Collected from a Fractured Basalt Aquifer with that of Dialysis Chambers Incubated In Situ

Microorganisms associated with basalt core were compared to those suspended in groundwater pumped from the same well in the eastern Snake River Plain Aquifer (Idaho, USA). Two wells located at different distances from the source of a mixed-waste plume in the fractured basalt aquifer were examined. In the well more distal from the plume source, an array of dialysis chambers filled with either deionized water or crushed basalt was equilibrated to compare the microorganisms collected in this fashion with those from core and groundwater samples collected in a traditional manner from the same well. The samples were characterized to determine the total amount of biomass, presence of specific populations or physiological groups, and potential community functions. Microorganisms and their activities were nearly undetectable in core and groundwater collected from the well farthest from the plume source and substantially enriched in both core and groundwater from the well closest to the plume source. In both wells, differences (statistically significant for some measures) were found between bacteria associated with the cores and those suspended in the groundwater. Significantly higher populations were found in the basalt- and water-filled dialysis chambers incubated in the open well compared with core and groundwater samples, respectively. For a given parameter, the variation among dialysis chambers incubated at different depths was much less than the high variation observed among core samples. Analyses on selected basalt- and water-filled dialysis chamber samples suggested that these two communities were compositionally similar but exhibited different potential functions. Documented knowledge of cell physiological changes associated with attachment and potential differences between attached and unattached communities in aquifers indicate that careful consideration should be given to the type of sample media (i.e., core, groundwater, substrata incubated in a well) used to represent a subsurface environment.     

Proteomic and targeted qPCR analyses of subsurface microbial communities for presence of methane monooxygenase

The Test Area North (TAN) site at the Idaho National Laboratory near Idaho Falls, ID, USA, sits over a trichloroethylene (TCE) contaminant plume in the Snake River Plain fractured basalt aquifer. Past observations have provided evidence that TCE at TAN is being transformed by biological natural attenuation that may be primarily due to co-metabolism in aerobic portions of the plume by methanotrophs. TCE co-metabolism by methanotrophs is the result of the broad substrate specificity of microbial methane monooxygenase which permits non-specific oxidation of TCE in addition to the primary substrate, methane. Arrays of experimental approaches have been utilized to understand the biogeochemical processes driving intrinsic TCE co-metabolism at TAN. In this study, aerobic methanotrophs were enumerated by qPCR using primers targeting conserved regions of the genes pmoA and mmoX encoding subunits of the particulate MMO (pMMO) and soluble MMO (sMMO) enzymes, respectively, as well as the gene mxa encoding the downstream enzyme methanol dehydrogenase. Identification of proteins in planktonic and biofilm samples from TAN was determined using reverse phase ultra-performance liquid chromatography (UPLC) coupled with a quadrupole-time-of-flight (QToF) mass spectrometer to separate and sequence peptides from trypsin digests of the protein extracts. Detection of MMO in unenriched water samples from TAN provides direct evidence of intrinsic methane oxidation and TCE co-metabolic potential of the indigenous microbial population. Mass spectrometry is also well suited for distinguishing which form of MMO is expressed in situ either soluble or particulate. Using this method, pMMO proteins were found to be abundant in samples collected from wells within and adjacent to the TCE plume at TAN.