Potential for Methanosarcina to Contribute to Uranium Reduction during Acetate-Promoted Groundwater Bioremediation
- 2.1k Downloads
Previous studies of acetate-promoted bioremediation of uranium-contaminated aquifers focused on Geobacter because no other microorganisms that can couple the oxidation of acetate with U(VI) reduction had been detected in situ. Monitoring the levels of methyl CoM reductase subunit A (mcrA) transcripts during an acetate-injection field experiment demonstrated that acetoclastic methanogens from the genus Methanosarcina were enriched after 40 days of acetate amendment. The increased abundance of Methanosarcina corresponded with an accumulation of methane in the groundwater. In order to determine whether Methanosarcina species could be participating in U(VI) reduction in the subsurface, cell suspensions of Methanosarcina barkeri were incubated in the presence of U(VI) with acetate provided as the electron donor. U(VI) was reduced by metabolically active M. barkeri cells; however, no U(VI) reduction was observed in inactive controls. These results demonstrate that Methanosarcina species could play an important role in the long-term bioremediation of uranium-contaminated aquifers after depletion of Fe(III) oxides limits the growth of Geobacter species. The results also suggest that Methanosarcina have the potential to influence uranium geochemistry in a diversity of anaerobic sedimentary environments.
KeywordsUranium bioremediation Methanogenesis U(VI) reduction Acetate amendment Methanosarcina
Injection of acetate into the groundwater of uranium-contaminated aquifers has been shown to be an effective way to stimulate microbially mediated reductive precipitation of soluble U(VI) to poorly soluble U(IV) [1, 2, 3]. A wide diversity of microorganisms are capable of U(VI) reduction [4, 5, 6, 7, 8, 9] but only Geobacter species have been shown to reduce U(VI) with acetate as an electron donor. Although growth with acetate as the electron donor and U(VI) as the electron acceptor is possible , the low concentrations of U(VI), even in heavily contaminated subsurface environments requires that microbes use other forms of respiration as their primary means of energy conservation . Geobacter species grow rapidly in the initial phases of subsurface uranium bioremediation with added acetate because Fe(III) oxides are typically abundant in subsurface environments [1, 11, 12, 13, 14] and Geobacter species outcompete other Fe(III) reducers under conditions of high acetate availability [15, 16]. However, the potential for other microorganisms to contribute to acetate oxidation coupled to U(VI) reduction, especially after the Fe(III) oxides that support Geobacter growth are depleted, has not been intensively investigated. Sulfate reducers that can reduce U(VI) have been identified, but none of these are known to use acetate as an electron donor [5, 7, 9, 17]. Furthermore, relying on sulfate reducers to reduce U(VI) may not be a good long-term strategy because acetate additions can rapidly deplete sulfate from groundwater [18, 19, 20].
Unlike Fe(III)- and sulfate-reducers, methanogens can thrive for long periods of time in organic-rich environments without external inputs of electron acceptors because they can conserve energy either from acetate dismutation or from the reduction of carbon dioxide, an electron acceptor generated by fermentation in their environment. If methanogens were capable of U(VI) reduction then this would make long-term in situ bioremediation of U(VI) a more attractive practice. To our knowledge, U(VI) reduction by methanogens has not been previously described. Previous studies have shown that methanogens can transfer electrons to various Fe (III) forms [21, 22, 23, 24, 25, 26], as well as vanadate , molecular sulfur  and quinones [22, 29]. However, acetate has not been shown to serve as an electron donor for these processes.
Evidence for methane production in response to acetate amendments during in situ uranium bioremediation  led us to investigate the potential for methanogens to further contribute to uranium bioremediation. The results suggest that Methanosarcina species that can couple the oxidation of acetate to the reduction of U(VI) might aid in the bioremediation process.
Materials and Methods
Description of Sampling Site
The Rifle 24-acre experimental site is located close to the Colorado River, on the premises of an earlier uranium ore processing facility. Uranium concentrations in the water table of the Rifle aquifer are 2–8 times higher than the drinking water contamination limit (0.126 μM) established by the uranium mill tailings remedial action (UMTRA). A detailed review of geochemical characteristics of the site has already been published  and in situ bioremediation of U(VI) has been intensely studied at this site [1, 2, 3]. Similar to previous years, acetate was injected into the subsurface at a concentration of ~ 15 mM between August and October, 2011 and monitored from six different wells . Groundwater and sediments for this study were collected from well CD-01 (a down gradient well) and a background well (CU-01) that never received any acetate additions.
Nucleic Acid Extraction and cDNA Preparation
For nucleic acid extraction, it was first necessary to concentrate 50 L of groundwater by impact filtration on 293 mm diameter Supor membrane disc filters with pore sizes of 1.2 and 0.2 μm (Pall Life Sciences). All filters were placed into whirl-pack bags, flash frozen in a dry ice/ethanol bath, and shipped on dry ice back to the laboratory where they were stored at – 80 °C. RNA was extracted from the filters using a modified phenol–chloroform method, as previously described . DNA was extracted from the filters with the FastDNA SPIN Kit for Soil (MP Biomedicals, Santa Ana, CA) according to the manufacturer’s instructions.
Extracted RNA and DNA were quantified with a NanoDrop spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and stored at – 80 °C until further analyses. A DuraScript enhanced avian RT single-strand synthesis kit (Sigma, Sigma-Aldrich, St Louis, MO, USA) was used to generate cDNA from RNA, as previously described . In order to ensure that RNA samples were not contaminated with DNA, PCR with primers targeting the 16S rRNA gene was conducted on RNA samples that had not undergone reverse transcription.
PCR Amplification Parameters and Microbial Community Analysis
For clone library construction, fragments from the mcrA gene which codes for the large subunit of methyl CoM reductase and the 16S rRNA gene were amplified from cDNA with mcrAf/mcrAr primers  and with 344f/915r  (ESM 1: Supplementary Table S1). Amplicons were ligated into the pCR-TOPO2.1 TA cloning vector according to the manufacturer’s instructions (Invitrogen, the Netherlands). Inserts from the recombinant clones were directly amplified by PCR with M13 primers, purified and sequenced at the University of Massachusetts sequencing facility.
Quantification of Methanosarcina mcrA Transcript Abundance
The quantitative PCR primer set (msa_mcrA173f/271r) targeted mcrA genes from Methanosarcina species found in the Rifle subsurface and was designed according to the manufacturer’s specifications (Applied Biosystems) (ESM 1: Supplementary Table S1). Quantitative PCR amplification and detection was performed with the 7500 Real Time System (Applied Biosystems) using cDNA made by reverse transcription from total RNA extracted from groundwater collected during the bioremediation experiment. Each reaction mixture consisted of a total volume of 25 μl and contained 1.5 μl of the appropriate primers (stock concentration 1.5 μM), 5 ng cDNA, and 12.5 μl Power SYBR Green PCR Master Mix (Applied Biosystems). All qPCR experiments followed MIQE guidelines  and qPCR efficiencies were 98%. Optimal thermal cycling parameters consisted of an activation step at 50 °C for 2 min, an initial 10 min denaturation step at 95 °C, and 50 cycles of 95 °C for 15 s and 60 °C for 1 min. A dissociation curve generated by increasing the temperature from 58 to 95 °C at a ramp rate of 2% showed that the PCR amplification process yielded a single predominant peak, further supporting the specificity of the qPCR primer pair.
mcrA gene sequences were compared to Genbank nucleotide and protein databases with the BLASTn and BLASTx algorithms [36, 37]. Alignments were generated with MAFFT  and PRANK  algorithms. The phylogenetic tree was inferred with the Maximum Likelihood method using MEGA7 software . The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (100 replicates) is shown next to the branches . All positions with less than 95% coverage were eliminated and a total of 117 positions were considered in the final dataset.
Nucleotide sequences of mcrA genes used for phylogenetic analyses have been deposited in the Genbank database under accession numbers MF616623-MF616647.
U(VI) Reduction Studies
Methanosarcina barkeri (DSM 800) was selected for U(VI) reduction studies because sequences most similar to this strain were significant members of the Methanosarcina community (37% of the sequences). Although M. barkeri was isolated from an anaerobic sewage digester , it grows in freshwater medium and can utilize acetate as a substrate for methanogenesis, similar to methanogens likely to be enriched from the acetate-amended Rifle aquifer. In addition, the majority of studies examining reduction of extracellular electron acceptors by Methanosarcina have focused on M. barkeri [22, 23, 24, 25].
Batch cultures of 500-mL M. barkeri were grown under strictly anaerobic conditions  on modified DSMZ medium 120  with acetate (40 mM) as substrate, and incubated at 37 °C for ~ 3 weeks. Cultures were harvested when they reached an optical density at 600 nm of 0.19. All cell suspension preparations were performed in an anaerobic chamber to minimize oxygen exposure. Cells were pelleted by centrifugation for 10 min at 4000 × g in a Sorval RC 5B Plus centrifuge. These pellets were then washed twice in anoxic phosphate depleted buffer (PDB), which consisted of the following salts 0.2 g/L MgSO4 × 7H2O, 0.025 g/L CaCl2 × 2H2O, 1 g/L NaCl, and 2 g/L NaHCO3. Cell pellets were then resuspended in 10-mL anoxic PDB to a cell density of ~ 0.4–0.5 at 600 nm. To generate heat-killed cells, 3 mL of this suspension was autoclaved at 122 °C for 30 min. Six replicates were prepared by diluting 1 mL of the cell-suspension in 9-mL PDB buffer. For the heat-killed incubation, 1-mL autoclaved cell suspension was diluted in 9-mL PDB buffer. Sulfide (0.5 mM) was added to all inoculated tubes to ensure anoxic conditions. Acetate (40 mM) was also added to the tubes to fuel methanogenic activity. Triplicate live cell suspensions (active cells) and triplicate heat-killed controls (heat-killed cells) were incubated at 37 °C. The other three live cell suspensions were incubated at 4 °C (inactive cells). All cell suspensions were incubated with 0.2 mM U6+ prepared from a stock of uranyl-acetate (5 mM). Cell densities were determined with a bench top spectrophotometer, by absorbance measurements at 600 nm with mili-Q water as a blank.
The ability of Methanosarcina barkeri to reduce U(VI) was verified with U(VI) depletion measurements carried out on different cell suspensions over the course of 24 h. Samples (0.1 mL) were retrieved anaerobically and diluted in 14.9 mL anoxic bicarbonate (100 mM) and 14.9 mL Uraplex solution. Concentrations of U(VI) were then measured with a kinetic phosphorescence analyzer, as previously described .
Groundwater samples for geochemical analyses were collected after purging 12 L of groundwater from the wells with a peristaltic pump. The phenanthroline method  was used to determine ferrous iron concentrations. Sulfate and thiosulfate concentrations were measured with an ion chromatograph (ICS-2100, Dionex, CA) equipped with an AS18 column under isocratic elution with 32 mM KOH as the eluent. Acetate concentrations were determined with a high performance liquid chromatograph equipped with an ion exclusion HPX-87H column (Biorad, Hercules, CA) using 8 mM sulfuric acid as eluent. In situ methane production was monitored as previously described . Methane in the headspace of sediment/groundwater incubations was measured as previously described  using a gas chromatograph with a flame ionization detector (Shimadzu, GC-8A).
Results and Discussion
Evidence for Acetoclastic Methanogenic Activity During Acetate Amendments
The increase in Methanosarcinales coincided with an increase in free sulfide in the groundwater, producing highly reducing conditions that favor the growth of methanogens. Another consideration is the slow growth rate of Methanosarcinales, which might have limited their growth after acetate injections even under the most favorable conditions. The lack of sufficient reducing conditions coupled with the slow growth rate of Methanosarcinales may explain the finding that although acetate concentrations were high during the Fe(III) reducing phase of the experiment (days 0–33) (Fig. 1c), the number of Methanosarcinales sequences stayed low until sulfate reduction became the primary subsurface metabolism (Fig. 1a, d). The increase in abundance of Methanosarcinales was later followed by a decline, which coincided with acetate limitation associated with the halt in acetate injections on day 68.
Measurements of methane concentrations in the groundwater were not initiated until day 79 (Fig. 1e). The high concentration of methane at this time demonstrated that methanogens had been active in the preceding days. Methane concentrations steeply declined over time coincident with the steep decline in acetate availability.
Phylogenetic Analysis of the In Situ Methanosarcina Community
U(VI) Reduction by Metabolically Active Methanosarcina Cells
Our findings that acetate additions during in situ uranium bioremediation promotes the growth of Methanosarcina and that a Methanosarcina can reduce U(VI) has important implications for the design of long-term in situ uranium bioremediation strategies. Previous interpretations of U(VI) reduction during acetate-amendment at the Rifle, Colorado study site have focused on the U(VI) reduction capacity of Geobacter species because of their prevalence at the site [1, 2, 3, 51, 52, 53, 54] and because the sulfate-reducers that are enriched with acetate amendments [18, 19, 55, 56] are not likely to be effective U(VI) reducers. In fact, there has yet to be a description of an acetate-utilizing sulfate-reducing microorganism capable of U(VI) reduction. The results presented here suggest that Methanosarcina may also contribute to U(VI) reduction in the field experiments. Unlike Geobacter species, Methanosarcina do not require an external electron acceptor for acetate metabolism. Therefore, in long-term in situ uranium bioremediation, Methanosarcina may emerge as an important microbial catalyst for uranium removal.
Furthermore, microbial reduction of U(VI) may play an important role in the uranium geochemistry of a diversity of sedimentary environments . Thus, the potential contribution of Methanosarcina to U(VI) reduction in anaerobic environments should be considered.
This work is a contribution to grant by the Office of Science (BER), US Department of Energy, Awards no. DE-SC0004080 and DE-SC0004814 and Cooperative Agreement no. DE-FC02-02ER63446. AER has been supported by grants from the Novo Nordisk Foundation, Innovationsfonden (grant no. 410600017) and Danish Research Council (418100203).
- 1.Anderson RT, Vrionis HA, Ortiz-Bernad I, Resch CT, Long PE, Dayvault R, Karp K, Marutzky S, Metzler DR, Peacock A, White DC, Lowe M, Lovley DR (2003) Stimulating the in situ activity of Geobacter species to remove uranium from the groundwater of a uranium-contaminated aquifer. Appl. Environ. Microbiol. 69:5884–5891. https://doi.org/10.1128/aem.69.10.5884-5891.2003 CrossRefPubMedPubMedCentralGoogle Scholar
- 2.Williams KH, Long PE, Davis JA, Wilkins MJ, N'Guessan AL, Steefel CI, Yang L, Newcomer D, Spane FA, Kerkhof LJ, McGuinness L, Dayvault R, Lovley DR (2011) Acetate availability and its influence on sustainable bioremediation of uranium-contaminated groundwater. Geomicrobiol J. 28:519–539. https://doi.org/10.1080/01490451.2010.520074 CrossRefGoogle Scholar
- 7.Tebo BM, Obraztsova AY (1998) Sulfate-reducing bacterium grows with Cr(VI), U(VI), Mn(IV), and Fe(III) as electron acceptors. FEMS Microbiol. Lett. 162:193–198. https://doi.org/10.1111/j.1574-6968.1998.tb12998.x CrossRefGoogle Scholar
- 9.Wall JD, Krumholz LR (2006) Uranium reduction. Annu. Rev. Microbiol. 60:149–166. https://doi.org/10.1146/annurev.micro.59.030804.121357 CrossRefPubMedGoogle Scholar
- 11.Holmes DE, Finneran KT, O'Neil RA, Lovley DR (2002) Enrichment of members of the family Geobacteraceae associated with stimulation of dissimilatory metal reduction in uranium-contaminated aquifer sediments. Appl. Environ. Microbiol. 68:2300–2306. https://doi.org/10.1128/Aem.68.5.2300-2306.2002 CrossRefPubMedPubMedCentralGoogle Scholar
- 12.Holmes DE, Nevin KP, O'Neil RA, Ward JE, Adams LA, Woodard TL, Vrionis HA, Lovley DR (2005) Potential for quantifying expression of the Geobacteraceae citrate synthase gene to assess the activity of Geobacteraceae in the subsurface and on current-harvesting electrodes. Appl. Environ. Microbiol. 71:6870–6877. https://doi.org/10.1128/aem.71.11.6870-6877.2005 CrossRefPubMedPubMedCentralGoogle Scholar
- 13.Holmes DE, O’Neil RA, Vrionis HA, N’Guessan LA, Ortiz-Bernad I, Larrahondo MJ, Adams LA, Ward JA, Nicoll JS, Nevin KP, Chavan MA, Johnson JP, Long PE, Lovley DR (2007) Subsurface clade of Geobacteraceae that predominates in a diversity of Fe(III)-reducing subsurface environments. ISME J 1:663–677. https://doi.org/10.1038/ismej.2007.85 CrossRefPubMedGoogle Scholar
- 14.Holmes DE, Giloteaux L, Barlett M, Chavan MA, Smith JA, Williams KH, Wilkins M, Long P, Lovley DR (2013) Molecular analysis of the in situ growth rates of subsurface Geobacter species. Appl. Environ. Microbiol. 79:1646–1653. https://doi.org/10.1128/AEM.03263-12 CrossRefPubMedPubMedCentralGoogle Scholar
- 16.Lovley DR, Ueki T, Zhang T, Malvankar NS, Shrestha PM, Flanagan KA, Aklujkar M, Butler JE, Giloteaux L, Rotaru AE, Holmes DE, Franks AE, Orellana R, Risso C, Nevin KP (2011) Geobacter: the microbe Electric’s physiology, ecology, and practical applications. In: Poole, RK (ed.) Adv microb Phys, pp. 1–100Google Scholar
- 19.Vrionis HA, Anderson RT, Ortiz-Bernad I, O'Neill KR, Resch CT, Peacock AD, Dayvault R, White DC, Long PE, Lovley DR (2005) Microbiological and geochemical heterogeneity in an in situ uranium bioremediation field site. Appl. Environ. Microbiol. 71:6308–6318. https://doi.org/10.1128/aem.71.10.6308-6318.2005 CrossRefPubMedPubMedCentralGoogle Scholar
- 30.Holmes DE, Giloteaux L, Orellana R, Williams KH, Robbins MJ, Lovley DR (2014) Methane production from protozoan endosymbionts following stimulation of microbial metabolism within subsurface sediments. Front. Microbiol. 5. doi: Artn 366 Doi https://doi.org/10.3389/Fmicb.2014.00366
- 31.Zachara JM, Long PE, Bargar J, Davis JA, Fox P, Fredrickson JK, Freshley MD, Konopka AE, Liu CX, McKinley JP, Rockhold ML, Williams KH, Yabusaki SB (2013) Persistence of uranium groundwater plumes: contrasting mechanisms at two DOE sites in the groundwater-river interaction zone. J. Contam. Hydrol. 147:45–72. https://doi.org/10.1016/j.jconhyd.2013.02.001 CrossRefPubMedGoogle Scholar
- 32.Giloteaux L, Holmes DE, Williams KH, Wrighton KC, Wilkins MJ, Montgomery AP, Smith JA, Orellana R, Thompson CA, Roper TJ, Long PE, Lovley DR (2013) Characterization and transcription of arsenic respiration and resistance genes during in situ uranium bioremediation. ISME J 7:370–383. https://doi.org/10.1038/ismej.2012.109 CrossRefPubMedGoogle Scholar
- 34.Casamayor EO, Massana R, Benlloch S, Ovreas L, Diez B, Goddard VJ, Gasol JM, Joint I, Rodriguez-Valera F, Pedros-Alio C (2002) Changes in archaeal, bacterial and eukaryal assemblages along a salinity gradient by comparison of genetic fingerprinting methods in a multipond solar saltern. Environ. Microbiol. 4:338–348. https://doi.org/10.1046/J.1462-2920.2002.00297.X CrossRefPubMedGoogle Scholar
- 35.Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin. Chem. 55:611–622. https://doi.org/10.1373/Clinchem.2008.112797 CrossRefPubMedGoogle Scholar
- 44.Orellana R, Leavitt JJ, Comolli LR, Csencsits R, Janot N, Flanagan KA, Gray AS, Leang C, Izallalen M, Mester T, Lovley DR (2013) U(VI) reduction by diverse outer surface c-type cytochromes of Geobacter sulfurreducens. Appl Environ Microb 79:6369–6374. https://doi.org/10.1128/Aem.02551-13 CrossRefGoogle Scholar
- 46.Kendall M, Boone D (2006) The order Methanosarcinales. In: Dworkin M, Falkow S, Rosenberg E, Schleifer K, Stackebrandt E (eds) The prokaryotes: Vol 3: archaea bacteria: Firmicutes, Actinomycetes. Springer Science+Business Media, LLC, New York, pp 244–256Google Scholar
- 51.Wilkins MJ, VerBerkmoes NC, Williams KH, Callister SJ, Mouser PJ, Elifantz H, N’Guessan AL, Thomas BC, Nicora CD, Shah MB, Abraham P, Lipton MS, Lovley DR, Hettich RL, Long PE, Banfield JF (2009) Proteogenomic monitoring of Geobacter physiology during stimulated uranium bioremediation. Appl. Environ. Microbiol. 75:6591–6599. https://doi.org/10.1128/aem.01064-09 CrossRefPubMedPubMedCentralGoogle Scholar
- 52.Wilkins MJ, Callister SJ, Miletto M, Williams KH, Nicora CD, Lovley DR, Long PE, Lipton MS (2011) Development of a biomarker for Geobacter activity and strain composition; Proteogenomic analysis of the citrate synthase protein during bioremediation of U(VI). Microb Biotech 4:55–63. https://doi.org/10.1111/j.1751-7915.2010.00194.x CrossRefGoogle Scholar
- 53.Wilkins MJ, Wrighton KC, Nicora CD, Williams KH, McCue LA, Handley KM, Miller CS, Giloteaux L, Montgomery AP, Lovley DR, Banfield JF, Long PE, Lipton MS (2013) Fluctuations in species-level protein expression occur during element and nutrient cycling in the subsurface. PLoS One 8:e57819. https://doi.org/10.1371/journal.pone.0057819 CrossRefPubMedPubMedCentralGoogle Scholar
- 54.Yun J, Ueki T, Miletto M, Lovley DR (2011) Monitoring the metabolic status of Geobacter species in contaminated groundwater by quantifying key metabolic proteins with Geobacter-specific antibodies. Appl. Environ. Microbiol. 77:4597–4602. https://doi.org/10.1128/aem.00114-11 CrossRefPubMedPubMedCentralGoogle Scholar
- 55.Holmes DE, Giloteaux L, Williams KH, Wrighton KC, Wilkins MJ, Thompson CA, Roper TJ, Long PE, Lovley DR (2013) Enrichment of specific protozoan populations during in situ bioremediation of uranium-contaminated groundwater. ISME J 7:1286–1298. https://doi.org/10.1038/ismej.2013.20 CrossRefPubMedPubMedCentralGoogle Scholar
Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.