Biochar amendment as a remediation strategy for surface soils impacted by crude oil.

The impact of organic bulking agents on the biodegradation of petroleum hydrocarbons in crude oil impacted soils was evaluated in batch laboratory experiments. Crude oil impacted soils from three separate locations were amended with fertilizer and bulking agents consisting of biochars derived from walnut shells or ponderosa pine wood chips produced at 900 °C. The batch reactors were incubated at 25 °C and sampled at pre-determined intervals to measure changes in total petroleum hydrocarbons (TPH) over time. For the duration of the incubation, the soil moisture content was adjusted to 75% of the maximum water holding capacity (MWHC) and prior to each sampling event, the sample was manually stirred. Results show that the addition of fertilizer and bulking agents increased biodegradation rates of TPH. Soil samples amended with ponderosa pine wood biochar achieved the highest biodegradation rate, whereas the walnut shell biochar was inhibitory to TPH biodegradation. The beneficial impact of biochars on TPH biodegredation was more pronounced for a soil impacted with lighter hydrocarbons compared to a soil impacted with heavier hydrocarbons. This study demonstrates that some biochars, in combination with fertilizer, have the potential to be a low-technology and eco-friendly remediation strategy for crude oil impacted soils.

Using DNA-Stable Isotope Probing to Identify MTBE- and TBA-Degrading Microorganisms in Contaminated Groundwater.

Although the anaerobic biodegradation of methyl tert-butyl ether (MTBE) and tert-butyl alcohol (TBA) has been documented in the laboratory and the field, knowledge of the microorganisms and mechanisms involved is still lacking. In this study, DNA-stable isotope probing (SIP) was used to identify microorganisms involved in anaerobic fuel oxygenate biodegradation in a sulfate-reducing MTBE and TBA plume. Microorganisms were collected in the field using Bio-Sep® beads amended with 13C5-MTBE, 13C1-MTBE (only methoxy carbon labeled), or13C4-TBA. 13C-DNA and 12C-DNA extracted from the Bio-Sep beads were cloned and 16S rRNA gene sequences were used to identify the indigenous microorganisms involved in degrading the methoxy group of MTBE and the tert-butyl group of MTBE and TBA. Results indicated that microorganisms were actively degrading 13C-labeled MTBE and TBA in situ and the 13C was incorporated into their DNA. Several sequences related to known MTBE- and TBA-degraders in the Burkholderiales and the Sphingomonadales orders were detected in all three13C clone libraries and were likely to be primary degraders at the site. Sequences related to sulfate-reducing bacteria and iron-reducers, such as Geobacter and Geothrix, were only detected in the clone libraries where MTBE and TBA were fully labeled with 13C, suggesting that they were involved in processing carbon from the tert-butyl group. Sequences similar to the Pseudomonas genus predominated in the clone library where only the methoxy carbon of MTBE was labeled with 13C. It is likely that members of this genus were secondary degraders cross-feeding on 13C-labeled metabolites such as acetate.

Effect of different transport observations on inverse modeling results: case study of a long-term groundwater tracer test monitored at high resolution.

Conservative tracer experiments can provide information useful for characterizing various subsurface transport properties. This study examines the effectiveness of three different types of transport observations for sensitivity analysis and parameter estimation of a three-dimensional site-specific groundwater flow and transport model: conservative tracer breakthrough curves (BTCs), first temporal moments of BTCs (m1), and tracer cumulative mass discharge (Md) through control planes combined with hydraulic head observations (h). High-resolution data obtained from a 410-day controlled field experiment at Vandenberg Air Force Base, California (USA), have been used. In this experiment, bromide was injected to create two adjacent plumes monitored at six different transects (perpendicular to groundwater flow) with a total of 162 monitoring wells. A total of 133 different observations of transient hydraulic head, 1,158 of BTC concentration, 23 of first moment, and 36 of mass discharge were used for sensitivity analysis and parameter estimation of nine flow and transport parameters. The importance of each group of transport observations in estimating these parameters was evaluated using sensitivity analysis, and five out of nine parameters were calibrated against these data. Results showed the advantages of using temporal moment of conservative tracer BTCs and mass discharge as observations for inverse modeling.

Impacts of an ethanol-blended fuel release on groundwater and fate of produced methane: Simulation of field observations.

In a field experiment at Vandenberg Air Force Base (VAFB) designed to mimic the impact of a small-volume release of E10 (10% ethanol and 90% conventional gasoline), two plumes were created by injecting extracted groundwater spiked with benzene, toluene, and o-xylene, abbreviated BToX (No-Ethanol Lane) and BToX plus ethanol (With-Ethanol Lane) for 283 days. We developed a reactive transport model to understand processes controlling the fate of ethanol and BToX. The model was calibrated to the extensive field dataset and accounted for concentrations of sulfate, iron, acetate, and methane along with iron-reducing bacteria, sulfate-reducing bacteria, fermentative bacteria, and methanogenic archaea. The benzene plume was about 4.5 times longer in the With-Ethanol Lane than in the No-Ethanol Lane. Matching this different behavior in the two lanes required inhibiting benzene degradation in the presence of ethanol. Inclusion of iron reduction with negligible growth of iron-reducers was required to reproduce the observed constant degradation rate of benzene. Modeling suggested that vertical dispersion and diffusion of sulfate from an adjacent aquitard were important sources of sulfate in the aquifer. Matching of methane data required incorporating initial fermentation of ethanol to acetate, methane loss by outgassing, and methane oxidation coupled to sulfate and iron reduction. Simulation of microbial growth using dual Monod kinetics, and including inhibition by more favorable electron acceptors, generally resulted in reasonable yields for microbial growth of 0.01-0.05.

An ex situ evaluation of TBA- and MTBE-baited bio-traps.

Aquifer microbial communities can be investigated using Bio-traps(®) ("bio-traps"), passive samplers containing Bio-Sep(®) beads ("bio-beads") that are deployed in monitoring wells to be colonized by bacteria delivered via groundwater flow through the well. When bio-beads are "baited" with organic contaminants enriched in (13)C, stable isotope probing allows assessment of the composition and activity of the microbial community. This study used an ex situ system fed by groundwater continuously extracted from an adjacent monitoring well within an experimentally-created aerobic zone treating a tert-butyl alcohol (TBA) plume. The goal was to evaluate aspects of bio-trap performance that cannot be studied quantitatively in situ. The measured groundwater flow through a bio-trap housing suggests that such traps might typically "sample" about 1.8 L per month. The desorption of TBA or methyl tert-butyl ether (MTBE) bait from bio-traps during a typical deployment duration of 6 weeks was approximately 90% and 45%, respectively, of the total initial bait load, with initially high rate of mass loss that decreased markedly after a few days. The concentration of TBA in groundwater flowing by the TBA-baited bio-beads was estimated to be as high as 3400 mg/L during the first few days, which would be expected to inhibit growth of TBA-degrading microbes. Initial inhibition was also implied for the MTBE-baited bio-trap, but at lower concentrations and for a shorter time. After a few days, concentrations in groundwater flowing through the bio-traps dropped below inhibitory concentrations but remained 4-5 orders of magnitude higher than TBA or MTBE concentrations within the aquifer at the experimental site. Desorption from the bio-beads during ex situ deployment occurred at first as predicted by prior sorption analyses of bio-beads but with apparent hysteresis thereafter, possibly due to mass transfer limitations caused by colonizing microbes. These results suggest that TBA- or MTBE-baited bio-traps could be baited at lower initial total mass loading with no detriment to trapping ability. The bio-traps were able to collect detectable amounts of microbial DNA and thus allow some insight into the sparse microbial community present in the aquifer during remediation of the low concentration plume.

Mass discharge in a tracer plume: evaluation of the Theissen Polygon Method.

A tracer plume was created within a thin aquifer by injection for 299 d of two adjacent "sub-plumes" to represent one type of plume heterogeneity encountered in practice. The plume was monitored by snapshot sampling of transects of fully screened wells. The mass injection rate and total mass injected were known. Using all wells in each transect (0.77 m well spacing, 1.4 points/m(2) sampling density), the Theissen Polygon Method (TPM) yielded apparently accurate mass discharge (M(d) ) estimates at three transects for 12 snapshots. When applied to hypothetical sparser transects using subsets of the wells with average spacing and sampling density from 1.55 to 5.39 m and 0.70 to 0.20 points/m(2) , respectively, the TPM accuracy depended on well spacing and location of the wells in the hypothesized transect with respect to the sub-plumes. Potential error was relatively low when the well spacing was less than the widths of the sub-plumes (>0.35 points/m(2) ). Potential error increased for well spacing similar to or greater than the sub-plume widths, or when less than 1% of the plume area was sampled. For low density sampling of laterally heterogeneous plumes, small changes in groundwater flow direction can lead to wide fluctuations in M(d) estimates by the TPM. However, sampling conducted when flow is known or likely to be in a preferred direction can potentially allow more useful comparisons of M(d) over multiyear time frames, such as required for performance evaluation of natural attenuation or engineered remediation systems.

In Situ Biotreatment of TBA with Recirculation/Oxygenation.

The potential for in situ biodegradation of tert-butyl alcohol (TBA) by creation of aerobic conditions in the subsurface with recirculating well pairs was investigated in two field studies conducted at Vandenberg Air Force Base (VAFB). In the first experiment, a single recirculating well pair with bromide tracer and oxygen amendment successfully delivered oxygen to the subsurface for 42 days. TBA concentrations were reduced from approximately 500 µg/L to below the detection limit within the treatment zone and the treated water was detected in a monitoring transect several meters downgradient. In the second experiment, a site-calibrated model was used to design a double recirculating well pair with oxygen amendment, which successfully delivered oxygen to the subsurface for 291 days and also decreased TBA concentrations to below the detection limit. Methylibium petroleiphilum strain PM1, a known TBA-degrading bacterium, was detectable at the study site but addition of oxygen had little impact on the already low baseline population densities, suggesting that there was not enough carbon within the groundwater plume to support significant new growth in the PM1 population. Given favorable hydrogeologic and geochemical conditions, the use of recirculating well pairs to introduce dissolved oxygen into the subsurface is a viable method to stimulate in situ biodegradation of TBA or other aerobically-degradable aquifer contaminants.

Role of back diffusion and biodegradation reactions in sustaining an MTBE/TBA plume in alluvial media.

A methyl tert-butyl ether (MTBE) / tert-butyl alcohol (TBA) plume originating from a gasoline spill in late 1994 at Vandenberg Air Force Base (VAFB) persisted for over 15 years within 200 feet of the original spill source. The plume persisted until 2010 despite excavation of the tanks and piping within months after the spill and excavations of additional contaminated sediments from the source area in 2007 and 2008. The probable history of MTBE concentrations along the plume centerline at its source was estimated using a wide variety of available information, including published details about the original spill, excavations and monitoring by VAFB consultants, and our own research data. Two-dimensional reactive transport simulations of MTBE along the plume centerline were conducted for a 20-year period following the spill. These analyses suggest that MTBE diffused from the thin anaerobic aquifer into the adjacent anaerobic silts and transformed to TBA in both aquifer and silt layers. The model reproduces the observation that after 2004 TBA was the dominant solute, diffusing back out of the silts into the aquifer and sustaining plume concentrations much longer than would have been the case in the absence of such diffusive exchange. Simulations also suggest that aerobic degradation of MTBE or TBA at the water table in the overlying silt layer significantly affected concentrations of MTBE and TBA by limiting the chemical mass available for back diffusion to the aquifer.

Comparative assessments of benzene, toluene, and xylene natural attenuation by quantitative polymerase chain reaction analysis of a catabolic gene, signature metabolites, and compound-specific isotope analysis.

A controlled-release study conducted at Vandenberg Air Force Base involved the injection of anaerobic groundwater amended with benzene, toluene, and o-xylene (BToX; 1-3 mg/L each) in two parallel lanes: lane A injectate contained no ethanol, whereas lane B injectate contained approximately 500 mg/L ethanol. As reported previously by Mackay and co-workers, ethanol led to slower BToX disappearance in lane B. Here, we report on assessments of BToX natural attenuation by three independent and specific monitoring approaches: signature metabolites diagnostic of anaerobic TX metabolism (benzysuccinates), compound-specific isotope analysis (CSIA), and quantitative polymerase chain reaction (qPCR) analysis of a catabolic gene involved in anaerobic TX degradation (bssA). In combination, the three monitoring methods provided strong evidence of in situ TX biodegradation in both lanes A and B; however, no single method provided strong evidence for TX biodegradation in both lanes. Benzylsuccinates were detected almost exclusively in lane B, where slower TX degradation and higher residual TX concentrations led to higher metabolite concentrations. In contrast, CSIA provided evidence of TX biodegradation almost exclusively in lane A, as greater degradation rates led to more pronounced isotopic enrichment. qPCR analyses of bssA were more complex. Evidence of increases in bssA copy number (up to 200-fold) after the release started was stronger in lane A, but higher absolute bssA copy number (and bacterial abundance, based on 16S rRNA genes) was observed in lane B, where bacteria genetically capable of anaerobic TX degradation may have been growing primarily on ethanol or its metabolites rather than TX.

Quantifying MTBE biodegradation in the Vandenberg Air Force Base ethanol release study using stable carbon isotopes.

Compound-specific isotope analysis (CSIA) was used to assess biodegradation of MTBE and TBA during an ethanol release study at Vandenberg Air Force Base. Two continuous side-by-side field releases were conducted within a preexisting MTBE plume to form two lanes. The first involved the continuous injection of site groundwater amended with benzene, toluene and o-xylene ("No ethanol lane"), while the other involved the continuous injection of site groundwater amended with benzene, toluene and o-xylene and ethanol ("With ethanol lane"). The delta(13)C of MTBE for all wells in the "No ethanol lane" remained constant during the experiment with a mean value of -31.3 +/- 0.5 per thousand (n=40), suggesting the absence of any substantial MTBE biodegradation in this lane. In contrast, substantial enrichment in (13)C of MTBE by 40.6 per thousand, was measured in the "With ethanol lane", consistent with the effects of biodegradation. A substantial amount of TBA (up to 1200 microg/L) was produced by the biodegradation of MTBE in the "With ethanol lane". The mean value of delta(13)C for TBA in groundwater samples in the "With ethanol lane" was -26.0 +/- 1.0 per thousand (n=32). Uniform delta(13)C TBA values through space and time in this lane suggest that substantial anaerobic biodegradation of TBA did not occur during the experiment. Using the reported range in isotopic enrichment factors for MTBE of -9.2 per thousand to -15.6 per thousand, and values of delta(13)C of MTBE in groundwater samples, MTBE first-order biodegradation rates in the "With ethanol lane" were 12.0 to 20.3 year(-1) (n=18). The isotope-derived rate constants are in good agreement with the previously published rate constant of 16.8 year(-1) calculated using contaminant mass-discharge for the "With ethanol lane".

Impact of ethanol on the natural attenuation of benzene, toluene, and o-xylene in a normally sulfate-reducing aquifer.

Side-by-side experiments were conducted in a sulfate-reducing aquifer at a former fuel station to evaluate the effect of ethanol on biodegradation of other gasoline constituents. On one side, for approximately 9 months we injected groundwater amended with 1-3 mg/L benzene, toluene, and o-xylene (BToX). On the other side, we injected the same, adding approximately 500 mg/L ethanol. Initially the BToX plumes on both sides ("lanes") extended approximately the same distance. Thereafter, the plumes in the "No Ethanol Lane" retracted significantly, which we hypothesize to be due to an initial acclimation period followed by improvement in efficiency of biodegradation under sulfate-reducing conditions. In the "With Ethanol Lane", the BToX plumes also retracted, but more slowly and not as far. The preferential biodegradation of ethanol depleted dissolved sulfate, leading to methanogenic/acetogenic conditions. We hypothesize that BToX in the ethanol-impacted lane were biodegraded in part within the methanogenic/acetogenic zone and, in part, within sulfate-reducing zones developing along the plume fringes due to mixing with sulfate-containing groundwater surrounding the plumes due to dispersion and/or shifts in flow direction. Overall, this research confirms that ethanol may reduce rates of biodegradation of aromatic fuel components in the subsurface, in both transient and near steady-state conditions.

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.

Carbon and hydrogen isotopic fractionation during biodegradation of methyl tert-butyl ether.

Carbon and hydrogen isotopic fractionation during aerobic biodegradation of MTBE by a bacterial pure culture (PM1) and a mixed consortia from Vandenberg Air Force Base (VAFB) were studied in order to assess the relative merits of stable carbon versus hydrogen isotopic analysis as an indicator of biodegradation. Carbon isotopic enrichment in residual MTBE of up to 8.1/1000 was observed at 99.7% biodegradation. Carbon fractionation was reproducible in the PM1 and VAFB experiments, yielding similar enrichment factors (epsilon) of -2.0/1000 +/- 0.1/1000 to -2.4/1000 +/- 0.3/1000 for replicates in the PM1 experiment and -1.5/1000 +/- 0.1/1000 to -1.8/1000 +/- 0.1/1000 for replicates in the VAFB experiment. Hydrogen isotopic fractionation was highly reproducible for the PM1 pure cultures, with epsilon values of -33/1000 +/- 5/1000 to -37/1000 +/- 4/1000 for replicate samples. In the VAFB microcosms, there was considerably more variability in epsilon values, with values of -29/1000 +/- 4/1000 and -66/1000 +/- 3/1000 measured for duplicate sample bottles. Despite this variability, hydrogen isotopic fractionation always resulted in 2H enrichment of the residual MTBE of >80/1000 at 90% biodegradation. The reproducible carbon fractionation suggests that compound-specific carbon isotope analysis may be used to estimate the extent of biodegradation at contaminated sites. Conversely, the large hydrogen isotopic fractionation documented during biodegradation of MTBE suggests that compound-specific hydrogen isotope analysis offers the most conclusive means of identifying in-situ biodegradation at contaminated sites.

In situ MTBE biodegradation supported by diffusive oxygen release.

Microcosm studies with sediments from Vandenberg Air Force Base, CA, suggest that native aerobic methyl tert-butyl ether (MTBE)-degrading microorganisms can be stimulated to degrade MTBE. In a series of field experiments, dissolved oxygen has been released into the anaerobic MTBE plume by diffusion through the walls of oxygen-pressurized polymeric tubing placed in contact with the flowing groundwater. MTBE concentrations were decreased from several hundred to less than 10 microg/L during passage through the induced aerobic zone, due apparently to in situ biodegradation: abiotic MTBE loss mechanisms were insignificant. Lag time for initiation of degradation was less than 2 months, and the apparent pseudo-first-order degradation rate was 5.3 day(-1). Additional MTBE was added in steps to raise the influent concentration to a maximum of 2.1 mg/L. With each step, MTBE was degraded within the preestablished aerobic treatment zone at rates ranging from 4.4 to 8.6 day(-1). Excess dissolved oxygen suggested that even higher MTBE concentrations could have been treated. Continued flow through the treatment zone was repeatedly confirmed through tracer and other tests. These and others' results suggest that it is possible to create permeable in situ treatment zones solely by releasing oxygen to support native microbial degradation of MTBE.