As a result of World War II and the subsequent Cold War, a large nuclear complex was developed in the United States, including large land tracts in Nevada, Idaho, and Washington state. Over a 40-year period, approximately 104 metric tons of plutonium was extracted from irradiated uranium at various sites within this complex. The result of the fuel chemical reprocessing at the Hanford Site, near Richland, Washington, and the Savannah River Site, near Aiken, South Carolina, was an accumulation of approximately 90 million gallons of high-level radioactive waste (HLW). Most of the waste was stored in tanks of various sizes and designs at Hanford and Savannah River, with lesser amounts at other sites across the United States.
At Hanford alone, approximately 107,000 tons of nuclear fuel was irradiated in nine reactors. Pu was extracted from the irradiated fuel by three different reprocessing schemes: reduction-oxidation process, bismuth-phosphate, and plutonium-uranium extraction process (
27). Much of the waste from irradiated fuel processing was stored in 177 single-shell and double-shell underground storage tanks that now contain approximately 55 million gallons of poorly characterized but highly radioactive waste. The tanks are below ground and are covered with approximately 3 m of soil and gravel. The earliest tanks, used since 1944, had a design life of 10 to 20 years; leaks were first suspected in 1956 and were confirmed in 1959. The amount and distribution of waste leakage from the Hanford tanks is unknown, but present estimates range from 0.6 to 1.5 million gallons. This waste contains approximately 1 million Ci of radiation, primarily from
137Cs, but the HLW soon after reprocessing contained high levels of short-lived radionuclides, including
106Ru,
144Ce,
147Pm, and others (
28). The wastes leaked from these tanks have been in contact with surrounding soils and vadose sediments for decades and have undergone significant geochemical and radiological transformations. Wastes also contained an estimated 870 tons of chemicals.
Microorganisms in terrestrial subsurface environments play a major role in the cycling of elements as well as weathering of rocks and sediments and can affect the geochemical properties of groundwater (
25) by modifying the fate and transport of organic and inorganic contaminants. While the vadose region of the subsurface generally does not support robust microbial populations, particularly in arid regions, there have been numerous reports of viable microorganisms associated with unsaturated zone soils and sediments (
15,
21,
31,
33), including at the Hanford Site (
9,
24,
30). Water potentials in the vadose zone generally do not directly restrict microbial activity, because many microorganisms are relatively tolerant to the matric water potentials typical of vadose sediments (
30). Rather, it is relatively thin, discontinuous water films that retard the diffusion of solutes, including nutrients and metabolic waste products that restrict microbial metabolism (
41).
During the summer of 2000, a slant borehole was drilled beneath tank SX-108 at Hanford's S-SX tank farm that intercepted a vadose zone contaminant plume of high-level nuclear waste. The purpose of this sampling effort was to assess the distribution of contaminants and to obtain scientific information regarding processes that may influence the fate and transport of the contaminants. The plume was characterized by high concentrations of radionuclide and chemical contaminants, elevated temperature, and low moisture content. Some samples exhibited the highest levels of radioactivity (>50 μCi g−1) of any soils or sediments yet collected at Hanford. As part of this effort, core samples were analyzed for viable microbial populations, and DNA from the isolates and sediments was subjected to phylogenetic analysis to identify the microorganisms. The main objectives of this research were to analyze the microbiological properties of SX-108 sediment samples in relation to sediment properties and contaminant distributions and to assess potential biogeochemical effects on contaminant fate and transport.
RESULTS
Vadose sediment physical and chemical properties.
The chemical and physical properties of the cored sediments (Table
1) reflect the complex effects of waste leakage from Hanford tank SX-108, subsequent migration of the tank liquor through the vadose zone, and geochemical reaction with vadose sediments. The slant borehole successfully traversed and allowed sampling of sediments beneath tank 108 that were contaminated with
137Cs and other chemical and radiological contaminants. Leaked wastes were very hot due to radioactive decay of short-lived isotopes during waste storage in the 1950s and 1960s and high concentrations of
137Cs associated with the HLW. Heating of the vadose sediments altered water seepage patterns in the subsurface and resulted in large-scale moisture redistributions. Thermal modeling of the SX tank farm and the SX-108 subsurface (
43,
48) indicated that the temperature may have exceeded 100°C as deep as 24 m beneath the tanks at the time of the SX-108 leak (ca. 1962). At the time the samples were collected (2000), the temperatures had cooled from the estimated maximum (100°C) and ranged from near ambient (∼37°C) to 75°C (Fig.
1). The maximum subsurface temperature occurred near the lower depth of
137Cs penetration (e.g., ∼19 m). The effects of the thermal load were evident in the moisture contents of the various sediment samples as sediments were desiccated to depths of >20 m beneath the tanks (Table
1 and Fig.
1).
The pH of the sediments varied from 7.2 near the base of the borehole to >9 for several of the sediment samples collected from the upper region of the profile (Table
1). The moderately alkaline pH indicated that significant waste-sediment reaction had occurred that neutralized the high pH (>14) of the original waste from the reduction-oxidation process. The samples that were higher in the profile also contained the greatest concentrations of
137Cs, with sample 6a exceeding 50 μCi g
−1 (Table
1 and Fig.
1 and
2). These high
137Cs concentrations resulted from the sorptive concentration of Cs
+ by the abundant micaceous fraction of the sediment. These samples represent some of the most highly radioactive sediment samples yet collected at the Hanford Site. The highest concentrations of water-extractable Cr and nitrate are coincident and generally occur deeper in the profile than Cs, except in the cases of samples 6a to 8a. These differences result from the relative mobility of Cs
+ and the negatively charged chromate and nitrate ions (for examples see references
36 and
49). The nitrate concentration in many of the samples was strikingly high, exceeding 10 g liter
−1 in 1:1 water extracts in 50% of the samples. Computed pore water concentrations of NO
3− based on the measured water contents of the sediments ranged between 5 and 15 mol liter
−1 in the core of the plume (e.g., 24.4 to 29.5 m and 34.5 to 39.5 m; Fig.
2). Nitrite concentrations were substantially lower than those of nitrate but nonetheless exceeded 30 mg liter
−1 in 1:1 water extracts in 6 out of 16 samples.
Technetium-99, the other major radiologic contaminant in the SX-108 vadose zone plume, existed deeper in the profile than
137Cs (Fig.
2).
99Tc is a long-lived mobile radionuclide (
t1/2 = 2.13 × 10
5 years) that decays by beta emission in the form of the pertechnetate anion [Tc(VII)O
4−]. The distribution of
99Tc was nearly identical to that of NO
3− and defined the extent of the HLW vadose zone plume. The sorption status of
137Cs and
99Tc was distinct.
137Cs was strongly adsorbed as a high-affinity exchange complex on micaceous minerals that resist desorption except in saline electrolytes (
35). In contrast,
99Tc was not adsorbed and existed as a solute in pore waters and as salt in air-filled pores.
Viable microbial populations.
In general, the populations of aerobic heterotrophic bacteria as determined by dilution plate counts were low, ranging from below detection to >10
4 CFU g
−1 in the deepest sediment collected (17a) (Table
2). Of the three different agar media used in this study, PTYG yielded the highest populations of aerobic heterotrophic bacteria while R2A yielded fewer or no colonies for three samples; however, it provided for growth of a few colonies on two samples (10a and 15a) where PTYG agar did not.
Based on previous investigations, we anticipated relatively low population densities of aerobic heterotrophic bacteria in the contaminated vadose sediments. Therefore, liquid enrichments were included in the microbiological analyses. For a number of sediment samples, including highly radioactive sediments 3a, 5a, 6a, and 8a, positive broth enrichments were obtained where populations were below detection by dilution plate count techniques. Although most transfer attempts from the enrichments into fresh broth medium were unsuccessful, a number of isolates from the original enrichments were obtained by streak plate purification on agar medium, including several from the highly radioactive sediments. Many of the sediments that yielded successful enrichments at pH 7 also exhibited growth in the same medium where the pH was initially adjusted to 10. It is not possible from these analyses to establish whether the organisms that grew in the pH 10 enrichments were similar or distinct from those that grew at pH 7. Regardless, these results indicate the presence of organisms in the contaminated vadose sediments that were able to grow at alkaline pH values.
Because we anticipated elevated temperatures of the sediments beneath SX-108, replicate PTYG and R2A broth enrichments were also incubated at 50°C. Similar to the pH 10 enrichments, growth was common in many of the original enrichments but only a few of the cultures were successfully transferred (Table
2). Interestingly, the cultures that successfully transferred originated from some of the same samples for which the 21
°C enrichment cultures also were successfully transferred; these included samples 1a, 9a, and 12a. A temperature of 50°C was selected for incubation of enrichment cultures, because it was estimated (e.g., Fig.
1) that this would approximate the in situ temperature for most of the sampled depths, although for some of the samples the temperatures were found to be higher (Fig.
1).
Because NO3− was a common tank waste constituent and the concentrations were remarkably high in a majority of the sediments examined, we initiated enrichments for denitrifying bacteria. Cores 12a and 17a were the only samples where the presence of viable denitrifying bacteria was confirmed. No sulfate-reducing or fermentative bacteria were cultured from any of the samples that were analyzed.
Uncontaminated vadose sediment microbial populations.
Two vadose samples were obtained from uncontaminated sediments from a borehole adjacent to the SX-108 slant borehole for comparison. These samples, designated RG1 and RG4, were from the same depths as the SX-108 samples that had the highest concentrations of
137Cs and, therefore, were stratigraphically similar. The population of viable aerobic heterotrophic bacteria in sample RG1 (Fig.
3) was low (2.4 log CFU g
−1) but was comparable to the population size associated with sample 7a (Table
2) from the SX-108 borehole obtained from approximately the same depth. In contrast, the population from untreated RG4 sediment was relatively high at 5.5 log CFU g
−1. This result is in considerable contrast to that for the sediment from SX-108 collected at approximately the same depth (8a), which exhibited no growth on PTYG agar even at the lowest dilution.
In order to assess the potential effects of drying and ionizing radiation on the population of viable vadose zone bacteria, uncontaminated sediments were subjected to desiccation or exposure to gamma radiation. The results from these experiments revealed that desiccation decreased the population sizes of aerobic heterotrophic bacteria in RG1 and RG4 by 2.5- and 10-fold, respectively (Fig.
3). Exposure to ionizing radiation had a much greater effect on the population size of viable aerobic bacteria, eliminating growth from the RG1 sample at both doses and decreasing the population size in RG4 by 3 and 4 orders of magnitude for acute exposures of 5 and 10 kGy, respectively.
Phylogeny and radiation resistance of isolates.
More than 110 cultures of aerobic heterotrophic bacteria were isolated and purified from the various enrichments and dilution plates (Table
3). To obtain insights into the genetic diversity and phylogeny of the isolates, the cultures were subjected to 16S rDNA gene sequencing.
The genera represented among the isolates from the SX-108 samples included gram-positive bacteria high in G+C content that are typical inhabitants of soil and vadose sediments. Isolates whose closest match was a member of the genus
Arthrobacter were the most common for cultures from both SX-108 and 299-W22-48 boreholes (Table
3). Other gram-positive genera commonly represented among the isolates included
Staphylococcus and
Nocardia in addition to relatives to several unclassified bacteria high in G+C content. Gram-negative genera were less common, but representatives included
Pseudomonas and
Sphingomonas as well as close relatives to a number of unclassified α-, β-, and γ-Proteobacteria. Interestingly, several isolates from sample 7a, one of the most radioactive samples collected, were closely related to
Deinococcus radiodurans, a bacterium that can withstand acute doses of ionizing radiation to 15 kGy without lethality (
17).
There are several interesting observations regarding the phylogenetic distributions of the isolates in Table
3. Only gram-positive and/or organisms high in G+C content were cultured from the most highly radioactive sediments, 1a to 7a (Table
1). In contrast, below sample 7a organisms related to gram-negative bacteria were relatively common, representing ∼45% of the isolates. Many of the same genera in the SX-108 vadose sediments were also present in the uncontaminated vadose sediments from borehole 299-W22-48.
Nineteen of the 20 radiation-resistant isolates were gram-positive bacteria high in G+C content, 13 of which were phylogenetically related to members of
Arthrobacter and its close relative
Micrococcus. Only one (10c-1) of the 13 isolates related to gram-negative bacteria exhibited any resistance to 2.5 kGy of gamma radiation. Three of the four isolates with some resistance to 5 kGy were most closely related to an uncultured
Micrococcus luteus-like bacterium identified in a clone library obtained from a sludge sample from a recirculating two-stage bioreactor (
14). The two isolates (7b-1 and 7c-1) that exhibited the highest levels of radiation resistance, with >0.2% of the population of 7b-1 cells surviving exposure to 20 kGy, were most closely related to
D. radiodurans, one of the most radiation-resistant organisms known. The source of these strains was sample 7a, which had the second highest concentration of
137Cs at 21.4 μCi g
−1.
Community 16S rDNA analysis.
The direct extraction of nucleic acids from vadose sediments followed by PCR amplification, cloning, and sequencing allowed for a cultivation-independent analysis of microbial phylogeny to complement the characterization of sediment isolates. With the bacterial primers, the 1:5-diluted DNA template produced the strongest bands on agarose gels for samples 12a and 17a, with very weak bands present with template at full strength and no bands present at 1:50 and 1:150 dilutions. For samples 3a, 5a, 6a, and 8a, no PCR products were observed on gels regardless of template level. Use of a seminested PCR produced visible products in these samples, with the exception of 8a. The archaeal primers failed to produce a PCR product in any of the sample extracts, regardless of template concentration. Competitive PCR containing 1:5 dilutions of indigenous template spiked with various amounts of Escherichia coli genomic DNA showed (with the exception of sample 8) between 300 and 900 copies of indigenous 16S target in the reaction, equivalent to 150,000 to 450,000 copies on a per-gram-of-sediment basis (data not shown). The extent to which PCR was able to sample these low-biomass communities was poor because the detection level, determined to be 80,000 copies by spiking the 1:5 dilutions of indigenous template with known amounts of nonindigenous 16S target into PCRs, was only two- to sixfold lower than the indigenous template concentrations (data not shown). Nevertheless, blastN analysis of sequences revealed between 2 and 11 genera per sample and 22 genera across all samples.
There was relatively good agreement, at the genus level, between the bacterial phylogenies obtained by the cultivation-independent cloning and sequencing approach and the samples from which isolates were obtained and characterized. Gram-positive bacteria high in G+C content, including members of
Arthrobacter,
Bacillus,
Streptomyces, and
Nocardioides, were among the most common genera represented among the cloned sequences (Table
4) and were also represented among the isolates (Table
3), especially
Arthrobacter. Among the gram-negative genera represented in the clone libraries,
Sphingomonas and
Pseudomonas were also present, including a sequence closely related to
Pseudomonas stutzeri from sample 12a (Table
4), the same sample from which an isolate closely related to
P. stutzeri was obtained (Table
3). A
P. stutzeri-like sequence was also obtained from the 17a clone library that was phylogenetically similar to three of the nine isolates from this sample.
DISCUSSION
In spite of harsh chemical and physical conditions imposed on vadose sediments by wastes leaked from tank SX-108 (Tables
1 and
2), viable aerobic heterotrophic bacteria were recovered from 11 of the 16 sediment samples. Due to low population densities it is difficult to discern trends in either population size or presence of aerobic heterotrophic bacteria in relation to sediment properties such as pH, water content, and contaminant concentration (Table
1). Several sediment samples, 1a, 4a, and 7a, with relatively low water contents and high radioactivity also contained moderate populations of heterotrophic bacteria. The highest viable populations were associated with samples that had not been subjected to heating and drying or severe contaminant exposure: 17a (>4.3 log CFU g
−1) from SX-108 and RG4 (5.5 log CFU g
−1) from the 299-W22-48 uncontaminated borehole. RG4 was from an uncontaminated region of the vadose zone, and 17a was among the least contaminated samples from SX-108. Because no attempts were made to measure total microbial biomass in these samples, it was not possible to draw any conclusions regarding relationships between total microbial biomass and sediment properties.
One of the caveats that must be recognized with a study of this type is the limitation associated with using cultivation-based methods exclusively for microbiological characterization. In some environments, the population size of the cultured prokaryotic community can be as much as 2 to 4 orders of magnitude below the population size determined by direct microscopic counting (
2). In spite of their limitations, cultivation methods have previously been successfully applied to characterizing subsurface microbial populations in saturated (
4,
23) and unsaturated (
9,
24) nonradioactive subsurface sediments. The use of cultivation-based methods over sequence-based methods has the advantage that cultures can be used for physiologic and metabolic analyses (
1). In this study, we applied both methods to investigate the phylogenetic composition of the microbial populations associated with contaminated subsurface sediments from the Hanford Site. We found the results (Tables
3 and
4) of both methods to be in reasonably good agreement, and they were consistent with previous findings (
24,
30), supporting the idea that viable populations in Hanford vadose sediments are sparse but are typically higher in regions where the moisture contents are elevated.
Isolates related to members of the gram-positive bacteria high in G+C content dominated the cultures obtained from both the contaminated and uncontaminated vadose sediments, and they exclusively represented organisms isolated from either highly radioactive SX-108 samples or irradiated uncontaminated sediments (Table
3). The same group also dominated the phylogeny of cloned sequences obtained from sediment DNA extracts (Table
4). In contrast to the highly radioactive and gamma-irradiated samples, nearly half of the isolates from sediment samples 17a and RG4 that had little or no contamination and relatively high water contents were gram-negative Proteobacteria. Although the results are not quantitative, the phylogenetic diversity and the dominance of gram-positive bacteria high in G+C content was greater in the sequenced sediment DNA clones from samples 12a and 17a (Table
4) than was represented among the isolates from these same samples (Table
3). Desiccation alone did not eliminate the isolation of gram-negative bacteria from RG1 or RG4, as did gamma irradiation (Table
3), suggesting that ionizing radiation, perhaps in combination with other contaminants, may have had a significant effect on the phylogenetic composition of the vadose microbial population.
Previous studies have indicated that, in general, gram-positive bacteria such as
Arthrobacter spp. are more drought tolerant than gram-negative organisms like
Pseudomonas spp. (
13,
32,
44). In fact,
Arthrobacter members appear to be well adapted to life in arid soils (
12), and some members are adept at surviving for extended periods of desiccation (
8). Members of the genus
Arthrobacter also appear to be well adapted to vadose sediments of the Hanford Site, as approximately one-third of the total isolates and a significant number of cloned sequences (11 out of 48) from this study were related to members of this genus. This is about the same proportion of total viable aerobic chemoheterotrophic bacteria as was isolated from pristine Ringold Formation sediments obtained from another location on the Hanford Site (
6).
Arthrobacter spp. were also common isolates in a third study of vadose zone sediments at the Hanford Site (
10). The phylogeny of the Ringold Formation
Arthrobacter strains has been investigated in detail, and many of the isolates appear to represent novel species within the genus (
16). Additional genera represented among the vadose zone cultures and sediment DNA-cloned sequences from this study that were also found in previous analyses of uncontaminated subsurface sediments from the Hanford Site (
6,
10) include
Rhodococcus,
Staphylococcus,
Streptomyces,
Nocardioides,
Bacillus, and
Sphingomonas.
One of the more intriguing results from this study was the isolation of two cultures from core 7a (25.6 m) that were resistant to extreme (20 kGy) laboratory doses of gamma radiation. This sample was obtained from the highest
137Cs concentration region of the plume. Both of these isolates were closely related to
D. radiodurans, a bacterium that is well recognized for its remarkable ability to withstand high levels of ionizing radiation. To our knowledge, this is the first time that
D. radiodurans-like strains have been isolated from a radionuclide-contaminated environment. It is possible that
Deinococcus is indigenous to Hanford soils and vadose zone sediments and that the harsh environment of the SX-108 contaminant plume led to conditions that selected for this highly stress-resistant organism. The ecological habitat of deinococci is poorly defined, but they do appear to be widely distributed in soils (
11,
39). Additional studies are presently under way to determine if
Deinococcus is a cosmopolitan inhabitant of Hanford Site soils. Mattimore and Battista (
37) have shown that in
D. radiodurans some genes that are necessary to survive irradiation are also necessary for desiccation resistance. However, a recent report (
7) has shown the existence of genes in
D. radiodurans that affect desiccation resistance but not radiation resistance, indicating that resistance to these conditions may involve different mechanisms.
Although all the factors influencing the microbiological characteristics of the SX-108 vadose sediments are unclear at this time, finding viable aerobic heterotrophic bacteria in radioactive sediments beneath the SX-108 tank may have important implications for the fate and transport of waste-associated contaminants. Microorganisms, in general, have the capacity for a wide range of biogeochemical transformations, including various reactions with waste constituents. For example, microorganisms are capable of degrading a wide range of organic compounds, oxidizing and reducing multivalent metals and radionuclides, such as Cr, U, and Tc, oxidizing ammonium to nitrite and nitrate, reducing nitrate or nitrite to ammonium or N
2, and for sorption and/or assimilation of a range of cations, including Cs and Sr. An important consideration for microbial-driven biogeochemical processes in the vadose sediments, including interactions with contaminants, is the availability of water. Assuming that the water contents measured on the core sediment samples accurately reflect in situ water distributions, it is clear that microbial processes in the upper 31 m are presently of little consequence to contaminant fate and transport because diffusion of solutes would be extremely limited and microbial cells are sparse and will likely be inactive or dormant. However, any future increases in moisture content due to either episodic natural or artificial (
24) recharge or alteration in regional climate patterns could result in significant increases in the size and activity of microbial populations in vadose sediments. Indeed, moisture calculations for the S-SX tank farm indicate that subsurface water contents are increasing as the system slowly re-equilibrates from the extreme thermal loads imposed through HLW waste boiling. This high thermal load decreased in the early 1970s as the decay of short-lived radionuclides declined.
We have confirmed the presence of viable bacteria in vadose zone sediments contaminated with high-level radioactive waste beneath waste tank SX-108 on DOE's Hanford Site. The site has experienced extreme geochemical, thermal, and radiological conditions in the past and still represents a harsh chemical and radiological environment. The culturable microbiota was comprised predominantly of aerobic chemoheterotrophic bacteria, mainly gram-positive organisms, including several highly radiation-resistant isolates related to D. radiodurans. Although these organisms are likely inactive or dormant under present environmental conditions, the ability of these organisms to survive under extreme conditions for extended periods in vadose sediments indicates that they could influence contaminant fate and transport should moisture regimes be altered in the future.