Environ. Sci. Technol. 2010, 44, 7652–7658
Evaluating the Potential of Native Ureolytic Microbes To Remediate a 90 Sr Contaminated Environment Y O S H I K O F U J I T A , * ,† J O A N N A L . T A Y L O R , ‡ LYNN M. WENDT,† DAVID W. REED,† AND ROBERT W. SMITH‡ Idaho National Laboratory, Idaho Falls, Idaho 83415, and University of Idaho-Idaho Falls, Idaho Falls, Idaho 83402
Received May 26, 2010. Revised manuscript received July 23, 2010. Accepted August 6, 2010.
This study was a preliminary evaluation of ureolytically driven calcite precipitation and strontium coprecipitation for remediating 90Sr contamination at the Hanford 100-N Area in Washington; in particular the approach is suitable for treating sorbed 90Sr that could otherwise be a long-term source for groundwater contamination. Geochemical conditions at the site are compatible with long-term calcite stability, and therefore groundwater and sediment samples were examined to assess the ureolytic capabilities of the native microbiota. Quantitative assays detected up to 2 × 104 putative ureC gene copies mL-1 in water and up to 9 × 105 copies g-1 in sediment. The ureC assays and laboratory-based estimates of ureolytic activity indicated that the distribution of in situ ureolytic potential was very heterogeneous with depth and also that the ureolytic activity was predominantly associated with attached organisms. A mixed kinetic-equilibrium model was developed for the 100-N site to simulate urea treatment and predict strontium removal. Together, the microbial characterization data and modeling suggest that the site has the requisite biogeochemical characteristics for application of the calcite precipitation remediation approach for 90Sr.
Introduction At the 100-N Area on the U.S. Department of Energy’s Hanford Reservation in Washington, past waste disposal practices have resulted in extensive vadose zone and groundwater contamination by radionuclides such as 90Sr, 137Cs, 60Co, 241 Am, 63Ni, 238Pu, and 234, 235, 238U (1). Currently 90Sr is the primary groundwater contaminant of concern, with some measured 2008 groundwater concentrations exceeding 1000 pCi/L (2). The U.S. Environmental Protection Agency maximum contaminant level for 90Sr is 8 pCi/L. Groundwater at 100-N discharges into the Columbia River through shoreline springs, and 0.14 to 0.19 Ci of 90Sr have been estimated to reach the river annually (3). A pump and treat operation initiated at Hanford 100-N in 1995 was discontinued ten years later, as the quantity of 90 Sr removed by the system was only one-tenth of that removed by natural radioactive decay (4). The major drawback of the pump and treat scheme at Hanford 100-N, and the case for most such systems, is that the majority of * Corresponding author phone: (208)526-1242: fax: (208)526-0870; e-mail:
[email protected]. † Idaho National Laboratory. ‡ University of Idaho-Idaho Falls. 7652
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contaminant is associated with the solid phase rather than the extracted water (5). Mass balance calculations using sitespecific distribution coefficients for Hanford (6) suggest that >99% of the 90Sr is sorbed. However, the bulk is expected to be readily susceptible to displacement by cations (e.g., Na+, K+, Ca2+, Mg2+, nonradiogenic Sr2+) incoming with the natural water flux. Consequently this sorbed fraction is a potential source for continuing groundwater contamination. An apatite permeable reactive barrier is now being deployed at Hanford 100-N to intercept dissolved 90Sr before discharge into the river. The apatite is emplaced using a novel biogeochemical approach, where calcium citrate and sodium phosphate are injected into the subsurface and microbial activity is responsible for liberating the calcium necessary for apatite formation (7, 8). We report here an evaluation of another potential biogeochemical approach for in situ remediation which could be particularly useful for treatment of sorbed 90Sr in a contamination source zone: coprecipitation in calcite driven by urea hydrolysis. Strontium partitioning into calcite is well-known (9), and microbial ureolysis has been demonstrated to drive calcite precipitation and coprecipitation of Sr within calcite (10-12). Urea hydrolysis produces bicarbonate and ammonium. Bicarbonate participates directly in calcite precipitation, and ammonium can exchange for sorbed strontium, calcium, and other metals, resulting in their enhanced susceptibility to recapture via a more robust sequestration mechanism, namely carbonate mineral formation. The reliance on in situ microbes to generate alkalinity facilitates a more extensive treatment zone than could be achieved by direct introduction of basic solutions (e.g., bicarbonate or hydroxide). The latter would likely result in immediate mineral formation at the well. A conceptual diagram of the approach is shown in Figure 1. In environments where calcite is stable, newly precipitated calcite and coprecipitated strontium should remain after active remediation ceases and “background” conditions return. Given 90Sr’s short half-life (29 yrs), within 300 years more than 99.9% of the immobilized 90Sr will have decayed. Such long-term compatibility of the treatment with the environment is an important factor in the success of in situ remediation approaches for elemental contaminants (13). Previous work demonstrated that ureolytic organisms are common in the Eastern Snake River Plain Aquifer (ESRPA) in Idaho and that these organisms can induce calcite precipitation (14). Using similar as well as newer methods developed in subsequent investigations (15, 16), in this study we evaluated the potential for ureolytically driven calcite precipitation in the Hanford 100-N subsurface. Well water and sediment samples from the site were characterized for ureolytic activity using culture-dependent and -independent methods. In addition, a mixed equilibrium-kinetic biogeochemical model was constructed to simulate the impacts of urea treatment on strontium mobility at Hanford 100-N. The analytical and simulation results indicated that substantial microbial ureolysis activity can occur at Hanford 100-N, and engineered calcite precipitation has potential for treating 90Sr contamination at this site.
Experimental Section Site Description, Sample Collection, and Handling. Groundwater was collected from three wells (199-N-119, 199-N-120, and 199-N-121; hereafter referred to as N-119, N-120, and N-121) adjacent to the Columbia River at the Hanford 100-N area, near the pilot-scale study of the apatite remediation approach (Figure 2; a map showing the location within the Hanford Reservation is provided in the Supporting Informa10.1021/es101752p
2010 American Chemical Society
Published on Web 09/03/2010
FIGURE 1. Conceptual diagram of ureolytically driven calcite precipitation approach for remediation of 90Sr contaminated geomedia. A. Microbially catalyzed hydrolysis of urea. B. Cation exchange and calcite precipitation. C. Continued precipitation of calcite. tion). The 10 cm internal diameter (stainless steel casing) wells are located ∼3 m apart and span the Hanford and Ringold formations. The younger (10,000-13,000 year old) highly heterogeneous gravel-dominated facies of the Hanford Formation overlie the much older (5-8 million year old) Ringold Formation (Unit E), which consists of relatively uniform, bimodal, clast-supported sandy gravel to silty sandy gravel (17). The boundary between the two formations at this location occurs approximately 4.6 m below land surface (mbls). The wells are screened at different depths, within the Ringold formation (depths shown in Table 1). The deepest well completion is bottomed in a lower permeability silt and clay-rich unit that is locally the base of the 100-N upper aquifer (17). Water levels fluctuate at this location due to changes in the river stage. The drilling log for N-121 indicates that at the time of sediment sample collection the water table was at 5 mbls; at the time of water sampling for this study the water table was at approximately 3 mbls. Representative water chemistry data for 100-N area wells are provided in the Supporting Information. Following purging of 3 borehole volumes and/or until specific conductance, pH, temperature, and turbidity stabilized, triplicate water samples (10 L each) were collected in June 2004 from each well and shipped on ice to the Idaho National Laboratory (INL). The N-120 water arrived the next day, with intact ice. Due to weather-related shipping delays, N-119 and N-121 samples were not received until 3 days after collection; the samples were cool to the touch, but the ice was melted. Immediately upon receipt, portions (40 mL) were preserved with 2% formaldehyde for microscopic counts and stored at 4 °C until processing. Also immediately after sample receipt, microbial cells were collected on 47 mm Supor-200 filters (0.2 µm pore size; Pall Corporation, East Hills, NY) from each of the triplicate samples and frozen (-80 °C) for later DNA extraction, and aliquots were used for MPN enumerations and ureolysis rate estimations as described below. Core samples were collected aseptically during the installation of well N-121 in March 2004 and stored at 4 °C. Eleven subsamples (∼50 g each) were shipped on ice overnight to the INL in June 2004. The subsamples were composites over 0.2 m intervals, from depths between 3.0 to 12.6 mbls, except for the shallowest sample which was composited over 1.5 to 3.0 mbls. Immediately upon receipt, 1 g portions were preserved for cell counts by formaldehyde addition and stored at 4 °C. Other portions reserved for DNA extraction were stored also at 4 °C until extraction, within 5 days of receipt. Solids for the ureolytic rate estimation were stored at 4 °C at the University of Idaho-Idaho Falls (UI-IF) for 2.5 months after receipt and prior to analysis; the extended storage time was due to procedural constraints associated with the sample origin. Direct Cell Enumeration. Total microbial cells were enumerated using acridine orange (AO) staining and epif-
luorescence microscopy. Cells in the sediment samples were prepared as follows: 4.5 mL of TRIS buffer (25 mM, pH 8.0) and 0.5 mL of 37% formaldehyde were added to the sediment aliquots preserved with formaldehyde. Samples were vortexed and then centrifuged at 400 x g for 1 min to separate cells from sediment. Three to 5 mL of fixed supernatant from the core samples and 10 mL of the fixed groundwater samples were then stained with AO and counted using standard protocols (18). Triplicate samples were counted for the water samples; only single samples were counted for the sediments due to burdens (because of sample origin) associated with waste disposal from sample processing. Most Probable Number Method for Estimation of Ureolytic Cell Numbers. The numbers of ureolytic organisms in the groundwater samples were estimated using the Most Probable Number (MPN) method. Urea R Broth (19) was used as the indicator medium. For each of the triplicate samples, 10-fold serial dilutions (from 10-1 to 10-8) were prepared in sterile phosphate buffered saline, and 5 replicates for each of the 8 dilutions were assayed. The MPN tubes were incubated at room temperature for 12 weeks before being scored as positive or negative for urea hydrolysis. MPN values were calculated as described by Koch (1994) (20). DNA Extraction. DNA was extracted from samples using MoBio kits (UltraClean Soil and UltraClean Water; MoBio, Solano Beach, CA) and the manufacturer’s protocols. The filters used to concentrate cells from the groundwater were cut into 0.5 cm strips prior to insertion into the bead beating tubes. DNA was extracted from positive (Sporosarcina pasteurii; ATCC 11859) and negative (Escherichia coli str. 25922; ATCC 25955) control strains for urease using the UltraClean Microbial DNA Isolation Kit. UreC Gene Quantification. Bacterial ureC genes were quantified using a quantitative polymerase chain reaction (qPCR) assay with degenerate PCR primers targeting a 389bp amplicon of the ureC gene. The ureC gene sequence codes for the catalytic subunit of urease and is strongly conserved among the bacteria (21). Primers L2F (5′-ATHGGYAARGCNGGNAAYCC-3′) and 733R (5′-GTBGHDCCCCARTCYTCRT3′) were used (22). The detailed qPCR protocols are provided in the Supporting Information. Ureolysis Rate Estimations. To estimate ureolysis rates, trace amounts of 14C-labeled urea were added to the samples, and the evolution of 14CO2 was measured over time, as described previously (15, 16). For the groundwater samples (triplicates), 9 mL of groundwater was amended with 59 nmol of [14C] urea (7.7 mCi/mmol, Sigma, St. Louis, MO) for a total volume of 10 mL. For the solid samples (ten of the eleven depths; the deepest sample was not assayed), approximately 2 g of sediment in 9 mL of sterile synthetic groundwater (10) were amended with the 14C-urea. The microcosms (triplicate for each sample and time-point) were incubated at 21 °C (similar to in situ temperatures at 100-N; see Table SI-1 in the Supporting Information) for periods between 0 and 73 h. VOL. 44, NO. 19, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 2. Map showing location of wells N-119, N-120, and N-121 at the Hanford 100-N site, just northeast of the apatite injection barrier. Figure from ref 2.
TABLE 1. Microbial Characterization Data, Well Screen Depth, and Selected Geochemical Parameters for Well Waters N-119 -1
direct counts (cells mL ) ureolytic MPN (cells mL-1) ureC copies (mL-1) ureolysis rates (nmol L-1 h-1) screen depth (mbls) pH dissolved O2 (mg L-1) strontium-90 (pCi L-1) SICalcite
N-120
2.0 (0.15) × 10
