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Keywords:

  • iron oxidation;
  • ferrous iron;
  • iron cycle;
  • gradient culture

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

A species of Dechlorospirillum was isolated from an Fe(II)-oxidizing, opposing-gradient-culture enrichment using an inoculum from a circumneutral, freshwater creek that showed copious amounts of Fe(III) (hydr)oxide precipitation. In gradient cultures amended with a redox indicator to visualize the depth of oxygen penetration, Dechlorospirillum sp. strain M1 showed Fe(II)-dependent growth at the oxic–anoxic interface and was unable to utilize sulfide as an alternate electron donor. The bacterium also grew with acetate as an electron donor under both microaerophilic and nitrate-reducing conditions, but was incapable of organotrophic Fe(III) reduction or nitrate-dependent Fe(II) oxidation. Although members of the genus Dechlorospirillum are primarily known as perchlorate and nitrate reducers, our results suggest that some species are members of the microbial communities involved in iron redox cycling at the oxic–anoxic transition zones in freshwater sediments.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Redox cycling of iron in aquatic systems can be closely tied to biogeochemical transformations of C, N, and other elements, in addition to being involved in pollutant transformation and mobility (Lovley, 2000; Picardal & Cooper, 2005; Roden & Emerson, 2007). Because of the rapid, abiotic oxidation of Fe2+ by oxygen (O2) in aqueous systems (Stumm & Lee, 1961), Fe(II)-oxidizing bacteria (FeOB) at a circumneutral pH typically are found in greatest numbers in environments where dissolved O2 concentrations are sufficiently low, for example, <5% of air-saturated values, to minimize abiotic reaction rates relative to the rates of biological catalysis (Emerson et al., 1999; Emerson & Moyer, 2002; Neubauer et al., 2002; Emerson & Weiss, 2004).

Although the increased use of reduced-O2 cultivation techniques with opposing gradients of Fe2+ and O2 (Kucera & Wolfe, 1957) has stimulated further research with FeOB over the last 15 years (Emerson & Moyer, 1997; Sobolev & Roden, 2001; Emerson & Weiss, 2004), the isolation of FeOB nevertheless still presents unique challenges that have limited the number of available pure cultures demonstrated to benefit from Fe(II) oxidation (Emerson et al., 2010). To date, Fe(II)-dependent or -enhanced growth has been shown only for a handful of freshwater isolates including species from the genera Gallionella and Sideroxydans (Hallbeck & Pederson, 1991; Emerson & Moyer, 1997; Weiss et al., 2007). Since the known FeOB are phylogenetically and physiologically diverse and the functional genes unique to Fe(II) oxidation are unknown, the use of nonculture-based, molecular methods to study FeOB ecology and distribution can be problematic. It therefore remains critical to further our knowledge of FeOB using enrichment and isolation techniques.

The genus Dechlorospirillum has been primarily described in the literature as a perchlorate and nitrate reducer (Coates, 1999; Bender et al., 2004; Bardiya & Bae, 2008), and Fe(II)-oxidation-dependent growth of this genus has not been demonstrated previously. The objective of our studies was to determine whether a Dechlorospirillum sp. isolated from an Fe(II)-oxidizing, microbial mat is involved in and benefits from microaerophilic Fe(II) oxidation in gradient cultures.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Inoculum source

The inoculum consisted of sediment and microbial mat samples collected in June 2007 from a portion of Jackson Creek (Bloomington, IN) fed by an iron-rich groundwater spring. In addition to irregular mats several centimeter thick on the creek bed, the creek also contained orange, bulbous, and filamentous formations of up to 20 cm diameter. Under microscopic examination, we found that these formations primarily consist of both iron (oxy)hydroxide precipitates and mostly empty, Leptothrix-like sheaths. In addition to the sheaths, large numbers of other bacteria were observed including occasional spiral stalks characteristic of Gallionella. The pH of the site water was 6.6 on the day of inoculum collection and typically ranges from 5.8 to 6.8. During the period that samples were obtained, the spring water typically contained 0.36–1.8 mM Fe2+, 0.02–0.18 mM NO3, and approximately 2 mg L−1 dissolved organic carbon. Samples of the flocculent mat and sediment were collected in sterile bottles, returned to the laboratory, and used to inoculate gradient-culture bottles on the day of collection.

Enrichment and isolation

Opposing-gradient-culture systems, inoculation procedures, and enrichment transfers were similar to those described elsewhere (Emerson & Moyer, 1997; Sobolev & Roden, 2001). Initially, we used 250- or 40-mL screw-cap bottles containing a lower layer of 50 mM FeCl2, stabilized by 2% Difco noble agar (Becton, Dickinson and Company, MD) and buffered at pH 7 by 20 mM 1,4-piperazinediethanesulfonic acid (PIPES). The upper layer consisted of 0.5% noble agar, 30 mM NaHCO3, 10 mM NH4Cl, 1 mM KH2PO4, 5 mL L−1 vitamin solution (Strąpoćet al., 2008), and 2.5 mL L−1 trace mineral solution (Strąpoćet al., 2008). Because of concerns that heterotrophic growth might be occurring on trace organic contaminants in the noble agar or PIPES buffer, we subsequently substituted purified agarose (Agarose Molecular Grade, Bioline, Boston, MA, or peqGOLD Universal Agarose, PEQLAB Biotechnologie GmbH, Erlangen, Germany) for the noble agar and 20 mM NaHCO3 for the PIPES in later enrichments and pure culture studies. We increased the agarose concentrations to 0.75±0.5% in the upper layer to provide the necessary layer stability.

The enriched gradient culture was streaked onto plates of MG medium that were incubated under reduced-O2 (approximately 5–10% of saturation) conditions in anaerobic culture jars (GasPak System, BBL) containing a Campy Pak microaerophilic pouch (BBL CampyPak Plus, Becton, Dickinson and Company). MG medium was a modified medium based on that described for the isolation of Magnetospirillum by Blakemore et al. (1979), consisting of 18 g L−1 Bacto agar, 1.2 mM NaNO3, 5 mM KH2PO4, 5 mM NaHCO3, 2 mM sodium acetate, 3.7 mM sodium succinate, 7.2 μM FeCl3, 1.0 mL L−1 vitamin solution (Strąpoćet al., 2008), and 1.0 mL L−1 SL-10 trace minerals solution (Atlas, 2004). A single colony of spirilla (strain M1) was restreaked to obtain a pure culture and maintained on plates of MG medium under reduced-O2 conditions or in gradient cultures.

When air was used in the headspace, the Fe2+ in gradient cultures was abiotically oxidized relatively quickly, for example, within approximately 2 weeks. In later experiments, we therefore reduced the initial O2 headspace concentrations by partially purging the vial headspace with sterile 80% N2 : 20% CO2 before tightening vial caps. Reduced initial O2 and the subsequent slow entry of O2 into the vials was sufficient to allow Fe(II) oxidation. Using this method, we were able to maintain viable cultures for over 30 days before complete oxidation and culture transfer.

The capacity for the growth of a pure culture under various physiological conditions was evaluated in a liquid medium using an anoxically prepared basic medium containing 0.6 mM CaCl2, 0.2 mM KCl, 0.5 mM MgCl2, 1.0 mM NH4Cl, 0.1 mM KH2PO4, 2.5 mL L−1 SL-10 trace mineral solution, 5.0 mL L−1 vitamin solution, and 50 mg L−1 Difco yeast extract buffered with 10 mM PIPES at pH 6.9–7.1. To determine whether the bacterium was capable of nitrate-dependent Fe(II) oxidation, the basic medium was amended with 5 mM FeCl2 and 5 mM NaNO3 in the presence and absence of 0.5 mM sodium acetate. Fe(III) reduction ability coupled to either 20 mM lactate or 5 mM acetate oxidation was determined by adding the carbon source and either 50 mM Fe(III) citrate or 10 mM Fe(III)–nitrilotriacetic acid (NTA) to the basic medium. Nitrate reduction ability was evaluated in the basic medium amended with 5 mM acetate and 5 mM sodium nitrate. Where indicated, acetate consumption was measured via HPLC. In all cases, inoculated tubes were incubated without shaking at room temperature in sealed anaerobic tubes containing an N2 headspace.

16S rRNA gene sequencing

PCR amplification of the 16S rRNA gene was performed using the primers 63F (5′-CAG GCC TAA CAC ATG CAA GTC-3′) and 1387R (5′-GGG CGG WGT GTA CAA GGC-3′). Three colonies from an M1 pure culture plate were initially vortexed with 50 μL lysing solution (0.05% sodium dodecyl sulfate; 30 mM NaOH). Following incubation for 15 min at 95 °C and brief centrifugation, the solution was diluted with 450 μL H2O and centrifuged for 15 min. In addition to 2.0 μL alkaline lysis supernatant as the template, the 50-μL PCR mixture contained 10 mM Tris-HCl (pH 9.0), 50 mM KCl, 0.1% Triton X-100, 1.2 mM MgCl2, each dNTP (0.1 mM each), 0.2 μM of each primer, and 0.625 U of Perpetual OptiTaq DNA polymerase (Roboklon, Berlin, Germany). Amplification was performed using the following conditions: 95 °C for 2 min, followed by 33 cycles of 95 °C for 20 s, 55 °C for 30 s, and 72 °C for 1.5 min, with a final step of 10 min at 72 °C. The PCR product was sequenced by SMB (Berlin, Germany). Chromatogram sequences were trimmed using Chromas Lite (Technelysium Pty Ltd, Tewantin, Qld, Australia), alignments were performed using bioedit (Hall, 1999), and the phylogenetic tree was constructed using the neighbor-joining method in the arb program (Ludwig et al., 2004).

Iron-oxidation experiments

Fe(II) oxidation experiments were performed using the bicarbonate-buffered, gradient-culture medium described above. To rule out the possibility that the growth of strain M1 was occurring on organic compounds in the agarose gel, we designed an experiment with three treatments (three replicates each). The first treatment utilized gradient vials with 50 mM FeCl2 in the lower layer. The second treatment excluded FeCl2 from the lower layer and the third treatment substituted 5 mM Na2S for FeCl2. The sulfide was used to establish a redox and O2 gradient in the vials in case the growth of M1 required microoxic conditions.

In all cases, resazurin (0.0001%) was included in the 15-mL, upper layer to allow visualization of the depth of O2 penetration. Inoculum was prepared by resuspending colonies from plates in 2 mL sterile upper layer. Two 50-μL aliquots of the resultant suspension were used to inoculate gradient systems at a depth of about 1 cm below the upper-layer surface. Two additional vials containing FeCl2 in the lower layer were inoculated with only sterile upper layer and used as abiotic controls. All gradient vials were purged with N2 : CO2 for 10 s before closing the screw-caps to partially remove O2. Vials were incubated statically in the dark at room temperature.

At the conclusion of the 8-day experiment, cells were counted by epifluorescence microscopic examination after staining cells with 4′,6-diamidino-2-phenylindole (DAPI) after fixation with 3.4% formaldehyde (Kepner & Pratt, 1994). Where necessary, iron oxide precipitates were removed before staining using an oxalate dissolution method as described elsewhere (Roden & Zachara, 1996). To determine the vertical distribution of cells in an iron-oxidizing, gradient-culture system, aliquots of an upper-layer suspension for DAPI counting were withdrawn at 5-mm depth intervals using a sterile syringe. For statistical evaluation, at least 10 microscopic fields (125 × 125 μm) were chosen randomly and a minimum of 1000 cells were counted. Data are presented as the mean cell number±the SD.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Isolation and phylogenetic characterization of Dechlorospirillum sp. strain M1

After several transfers, we developed a stable, iron-oxidizing enrichment that showed the presence of relatively long, morphologically distinctive spirilla. Repeated efforts to obtain a pure, iron-oxidizing culture by serial dilution to extinction in either gradient cultures or liquid culture (Emerson & Floyd, 2005) over a period of 1 year were unsuccessful. Using gradient-culture enrichments, a preliminary 16S rRNA gene clone library to identify predominant organisms (data not shown) revealed that the closest relatives for many of the clones were either Magnetospirillum or Dechlorospirillum sp. Because the latter organism had been described primarily as a perchlorate reducer (Achenbach et al., 2001; Bardiya & Bae, 2008), we initially thought that the enriched spirillum could be physiologically related to Magnetospirillum, a genus known to be active in iron metabolism (Taoka et al., 2009).

After streaking of a gradient-culture enrichment onto plates of modified MG medium used for the growth of Magnetospirillum and incubation under reduced-O2 conditions, we obtained a pure culture of a spirillum that, when transferred to gradient systems, appeared identical to the morphologically distinct spirilla observed in enrichment cultures (Fig. 1). Phylogenetic analysis placed strain M1 in a clade with other Dechlorospirillum isolates within the Alphaproteobacteria (Fig. 2). The 1045-bp, partial 16S rRNA gene sequence showed 99% sequence similarity to perchlorate-reducing Dechlorospirillum sp. WD (Coates, 1999), Dechlorospirillum sp. VDY (Thrash et al., 2007), and Dechlorospirillum sp. DB (Bender et al., 2004). We have therefore tentatively classified the isolate as Dechlorospirillum sp. strain M1 (GenBank accession number GQ262802).

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Figure 1.  Phase-contrast photomicrograph showing the typical morphology of Dechlorospirillum sp. strain M1 grown in an Fe(II)-oxidizing, gradient culture. Scale bar=5 μm.

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Figure 2.  Phylogenetic tree showing Dechlorospirillum sp. strain M1 and related isolates in the Alphaproteobacteria. Bootstrap values (1000 replicates) are indicated at the nodes. The Fe(III)-reducing bacterium, Shewanella putrefaciens AC-1, was isolated from the same sediment that yielded strain M1. Geobacter metallireducens was used as an outgroup, but pruned from the tree. Bar indicates 10% sequence difference.

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Microaerophilic iron oxidation by strain M1

Preliminary experiments with strain M1 in opposing Fe(II)-O2 gradient cultures showed that cell numbers reached 108–109 mL−1 near or within the lower boundary of precipitated Fe(III) oxide. To determine whether Fe(II) oxidation was responsible for cell growth and to rule out the possibility that cells were instead growing heterotrophically on either the agarose or the trace organics in the agarose, we conducted an experiment using gradient cultures with a lower layer of varying composition. One set of vials lacked Fe(II) or other reductants in the lower layer to allow aerobic conditions throughout the vial. In these vials, the resazurin remained pink (oxidized) throughout the experiment (see Supporting Information, Fig. S1). The lower layer in another set of vials contained 5 mM Na2S, which resulted in the formation of a reduced, colorless layer overlaid by an oxidized pink layer in two of the three vials. The sulfide was used to establish a redox and O2 gradient in the vials in case the growth of M1 required microoxic conditions.

This redox gradient was also observed in the third set of vials that contained 50 mM FeCl2 in the lower layer. In the latter vials, the resazurin was decolorized to a point just below the zone of Fe(III) oxide precipitation. Because both resazurin and Fe2+ are rapidly oxidized by O2 at neutral pH and Fe3+ quickly precipitates in the absence of a chelator, the point of resazurin decolorization and Fe(III) oxide precipitation roughly corresponds to the depth of O2 penetration. The resazurin in the third, Na2S-containing vial (vial 2C) never became decolorized, suggesting that the incorrect amount of sulfide was inadvertently added to this vial (see Fig. S1).

Figure 3 shows the results of cell enumerations in the upper 10 mL of the gradient cultures for each of the three treatments after 8 days of incubation. No cells were observed in the lower 5 mL of the upper layer. With the exception of vial 2C, in which resazurin did not become decolorized, there was no significant difference in cell numbers in fully oxic vials lacking a reductant or in gradient vials containing sulfide in the lower layer. All of these vials contained between 1.8 × 108 and 2.3 × 108 cells. Since 3.7 × 107 cells were added in the inoculum, cells underwent two to three doublings following addition to the vials. The relatively slight increase in cell numbers (equivalent to two to three doublings) likely resulted from the consumption of trace organics in the agarose, metabolism of intracellular storage products, or cells in the inoculum that were in the process of division. In all vials that contained Fe(II) in the lower layer, however, cell numbers were approximately one order of magnitude greater and ranged from 1.2 × 109 to 1.6 × 109 in the upper 10 mL of medium. Microscopic observations showed that these cells were highly concentrated in a thin layer at or just below the lower layer of oxide precipitation.

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Figure 3.  Fe(II)-dependent growth and cell numbers of strain M1 after an 8-day incubation in gradient-culture vials with varying lower layer composition. Although cell growth was localized to a narrow band, the entire upper 10 mL of culture was removed to insure that all cells were counted. Individual vials are shown to depict the variations between replicate treatments. Vials 1A, 1B, and 1C lacked any reductant in the lower layer. Vials 2A, 2B, and 2C contained 5 mM Na2S and vials 3A, 3B, and 3C contained 50 mM FeCl2 in the lower layer to establish a redox gradient. Error bars represent the SD.

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To explore the vertical distribution of cells in the redox gradient, cells were also enumerated in vertically sampled aliquots of the upper layer in an additional iron-oxidizing, gradient-culture replicate. As shown in Fig. 4, cell numbers were the highest (∼5 × 108 mL−1) at a depth of 5 mm below the surface. This depth approximately corresponded to the lower border of the oxide precipitation layer immediately above the decolorized resazurin. At samples collected below this depth, the cell numbers decreased by approximately one order of magnitude with each 5-mm depth interval.

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Figure 4.  Vertical distribution of Dechlorospirillum sp. strain M1 cell numbers after an 8-day incubation in a gradient-culture vial containing 50 mM FeCl2 in the lower layer. Error bars represent the SD.

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Growth of strain M1 under different culture conditions

Strain M1 was able to grow organotrophically on 5 mM acetate using either O2 or NO3 as an electron acceptor. On solid MG medium, colonies arose more rapidly and were larger when plates were incubated under reduced-O2 conditions than when incubated at ambient O2 concentrations. M1 was unable to couple the oxidation of lactate or acetate to the reduction of Fe(III) citrate or Fe(III)–NTA. Cultures grown under organotrophic NO3-reducing conditions or in Fe(II)-oxidizing gradient cultures did not exhibit magnetotaxis.

To determine whether Dechlorospirillum M1 was capable of NO3-dependent Fe(II) oxidation, cultures were used to inoculate an anoxic medium containing 5 mM FeCl2 and 5 mM NO3. After 2 weeks, no growth was observed, no oxide precipitation was noted, and no motile cells were observed under the microscope, regardless of whether or not 0.5 mM acetate was provided as a cosubstrate. Pure cultures previously grown organotrophically with acetate and nitrate were also incapable of anaerobic Fe(II) oxidation, lost motility, and did not consume acetate when incubated in a medium containing Fe(II), NO3, and low concentrations of acetate.

We also attempted to culture strain M1 in a liquid culture as described by Emerson & Floyd (2005). Using a medium identical to that in the upper layer in gradient cultures, but lacking agarose, inoculated media under a 1% headspace were fed daily with small amounts of O2 and Fe2+. In two separate experiments, we observed very little growth (zero to three doublings) when Fe2+ was present vs. controls lacking Fe2+. In all cases, any growth observed was not sustainable in liquid culture and microscopic examination showed that most cells had become nonmotile by the end of the 10–16-day experiment.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Although the genus Dechlorospirillum is most associated with perchlorate reduction (Coates, 1999; Bender et al., 2004; Bardiya & Bae, 2008), we have demonstrated that Fe(II) oxidation by strain M1 was clearly linked to an increase in cell numbers. Other recent reports, however, suggest that members of this genus may also be sometimes enriched at the redox interface found in gradient-culture systems. Wang et al. (2009) recently described gradient-culture enrichment of FeOB using various wetland sediments. Although their use of FeS-based gradient cultures yielded Gallionella-related enrichment cultures, community analysis of bacteria in the zone of Fe(II) oxidation was also performed using denaturing gradient gel electrophoresis (DGGE). After sequencing of bands excised from DGGE gels and a blast search of the NCBI database, Wang et al. (2009) showed that the closest relatives to two of the sequences, B17 (FJ391522) and B16 (FJ391521), were Magnetospirillum sp. When we compared these sequences (provided by J. Wang) with that of Dechlorospirillum sp. strain M1, we found a 97% sequence similarity. In addition, bacteria morphologically identical to strain M1 as depicted in Fig. 1 were commonly observed by J. Wang in gradient-culture enrichments (J. Wang, pers. commun.).

Geelhoed et al. (2009) reported the isolation of three spirilla from FeS-gradient-culture microcosms inoculated with freshwater sediment. Strains L70 and LD2 were subsequently isolated using an anaerobic dilution series with lactate as an electron donor and Fe(III) hydroxide as an electron acceptor. Based on 16S rRNA gene sequence similarity, strain L70 was found to be 99.2–99.4% related to other Dechlorospirillum isolates and LD2 equally related (97.6–97.8%) to Dechlorospirillum and Magnetospirillum. Although these isolates appear similar to strain M1 and were also initially enriched from gradient-culture systems, L70 and LD2 were isolated in Fe(III)-reducing, dilution series, whereas strain M1 was unable to reduce Fe(III) in the presence of either lactate or acetate. In addition, Geelhoed et al. (2009) reported that L70 and LD2 did not oxidize Fe(II) and suggest that these bacteria grew in Fe(II) gradient systems using Fe(III) hydroxide as a terminal electron acceptor. Regardless of slight differences in phylogeny and physiology, these reports support our contention that Dechlorospirillum sp., in addition to its more commonly known role as a perchlorate and nitrate reducer, can be enriched in Fe(II)-oxidizing, gradient cultures and may be an important member of microbial communities involved in iron redox cycling at oxic–anoxic transition zones in sediments.

It would be premature to suggest that this bacterium is capable of chemolithoautotrophic growth, however, because we have no evidence that strain M1 can fix CO2 or can harness the energy from Fe(II) oxidation for growth. One can speculate about other mechanisms that could provide explanations for the observed Fe(II)-oxidation-dependent growth in gradient cultures. One such possibility involves the formation of reactive species, for example, OH, O2, or H2O2, during the chemical oxidation of Fe(II) by O2 (King et al., 1995). Such reactive species might lead to a partial breakdown of complex organic matter, for example agarose or dissolved organic matter, into smaller molecules that can be degraded heterotrophically or utilized mixotrophically. If such a mechanism was operative, propagation of cells at zones of abiotic Fe(II) oxidation would also be expected. Although Fe(II)-oxidation-dependent growth of strain M1 was clear in our studies, further work is therefore necessary to determine whether the increase in the growth yield at the Fe(II)/Fe(III) interface was linked to microbial energy conservation from Fe(II) oxidation or resulted from other mechanisms.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

This research was supported by National Science Foundation Biogeosciences Program Grant 0525069 to F.W.P. and E. Roden and by grant EXB04-0017-0111 from the National Aeronautics and Space Administration to J.S. The authors would like to thank David Emerson and Eric Roden for useful suggestions during the initial stages of the research and Burga Braun for her assistance in rDNA sequencing and phylogenetic characterization.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Fig. S1. Replicate gradient-culture vials for three different treatments after 8 days of incubation.

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