Unique Organic Matter and Microbial Properties in the Rhizosphere of

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Unique Organic Matter and Microbial Properties in the Rhizosphere of a Wetland Soil Daniel I. Kaplan,*,† Chen Xu,‡ Shan Huang,§ Youmin Lin,‡ Nikola Tolić,∥ Kristyn M. Roscioli-Johnson,⊥ Peter H. Santschi,‡ and Peter R. Jaffé§ †

Savannah River National Laboratory, Aiken, South Carolina 29808, United States Texas A&M University, Galveston, Texas 77553, United States § Princeton University, Princeton, New Jersey 08540, United States ∥ Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, Washington 99352, United States ⊥ Idaho National Laboratory, Idaho Falls, Idaho 83415, United States ‡

ABSTRACT: Wetlands attenuate the migration of many contaminants through a wide range of biogeochemical reactions. Recent research has shown that the rhizosphere, the zone near plant roots, in wetlands is especially effective at promoting contaminant attenuation. The objective of this study was to compare the soil organic matter (OM) composition and microbial communities of a rhizosphere soil (primarily an oxidized environment) to that of the bulk wetland soil (primarily a reduced environment). The rhizosphere had elevated C, N, Mn, and Fe concentrations and total bacteria, including Anaeromyxobacter, counts (as identified by qPCR). Furthermore, the rhizosphere contained several organic molecules that were not identified in the nonrhizosphere soil (54% of the >2200 ESI-FTICR-MS identified compounds). The rhizosphere OM molecules generally had (1) greater overall molecular weights, (2) less aromaticity, (3) more carboxylate and N-containing COO functional groups, and (4) a greater hydrophilic character. These latter two OM properties typically promote metal binding. This study showed for the first time that not only the amount but also the molecular characteristics of OM in the rhizosphere may in part be responsible for the enhanced immobilization of contaminants in wetlands. These finding have implications on the stewardship and long-term management of contaminated wetlands.

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conducive to contaminant immobilization.14,15 Carbon and nitrogen flow in the rhizosphere is dependent on plant and environmental conditions and can vary spatially and temporally along the root.16 The amount and type of C released, whether it is CO2, mucilage, exudates, sloughed-off root cells, or root dieback, is dependent on the plant type, plant age, and habitat.17,18 Furthermore, C flow in the rhizosphere is bidirectional, where plants release organic and inorganic C, while taking up CO2(g) and varying forms of dissolved inorganic C and low molecular weight organic C, such as organic acids, sugars, and amino acids.19,20 The influence of plant roots on soil contaminant-binding properties is especially pronounced in wetlands where plants can also create steep dissolved O2 gradients. Releasing O2 into the rhizosphere is a physiological adaptation of wetland plants to enable them to grow in water-logged, reducing conditions.21−23 The elevated organic C concentrations and more oxidized redox conditions in the rhizosphere are favorable for

INTRODUCTION Subsurface contaminants move with groundwater flow and often pass through wetlands before entering surface aquatic environments. While many contaminants can move readily through aquifers, the movement of most contaminants is strongly attenuated in wetlands because of the presence of geochemical, microbiological, and hydrological conditions that can promote contaminant binding.1−4 Furthermore, these ecosystems can accumulate high concentrations of soil organic matter (OM) as a result of slow carbon turnover. The resulting soil OM has several direct and indirect properties that enhance the capacity of wetland soils to bind contaminants, including increasing microbial activity, lowering the oxidation state, and creating more surface binding sites.5−7 These conditions often lead to groundwater metals8,9 and radionuclides10−12 becoming immobilized in the wetlands. The wetter conditions near the soil surface of wetlands can create an environment conducive for plant growth. In turn, the release of C and N from the roots stimulate soil microbial activity. Together these conditions commonly result in wetlands acting as C sinks, where C accumulation exceed release.13 Roots and their rhizosphere, the soil region influenced hydraulically, chemically, and/or microbially by roots, have been shown to promote environmental conditions © XXXX American Chemical Society

Received: October 21, 2015 Revised: March 28, 2016 Accepted: April 4, 2016

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DOI: 10.1021/acs.est.5b05165 Environ. Sci. Technol. XXXX, XXX, XXX−XXX

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Environmental Science & Technology microbial activity, which together may influence inorganic contaminant mobility.24,25 Uranium migration has been significantly attenuated in the Tims Branch wetland on the Savannah River Site (SRS) in South Carolina, a nuclear processing facility.26,27 Of the 44 T of depleted U introduced between 1954 and 1985 into the Tims Branch system, 70% remains in the wetland. A series of laboratory, greenhouse mesocosm, spectroscopy, and field studies have been conducted to understand better the biogeochemical processes responsible for this attenuation.28−30 Among the key findings, was that U concentration could be 15 times greater in the rhizosphere than that in the bulk soil, the U existed almost exclusive in the +6 oxidation state, and the U(VI) formed bidentate bonds to carboxylate sites on the OM. The objective of this study was to build upon these results and to determine how rhizosphere and nonrhizosphere soils differ in terms of microbiology and organic C composition. Our hypothesis was that the rhizosphere soil, compared to the nonrhizosphere soil, not only contains greater concentrations of OM but that the OM had a different composition because of its proximity to the roots and its age in the soil. The general approach was to collect a soil sample from just upstream from a U-contaminated wetland along Tims Branch. Rhizosphere and nonrhizosphere subsamples were then separated and analyzed for OM composition by electrospray ionization−Fourier transform ion cyclotron resonance−mass spectrometry (ESIFTICR-MS) and microbial structure by quantitative polymerase chain reaction (qPCR).

Figure 1. Schematic of Tims Branch study site, locating the former MArea nuclear materials processing facility and the minimally contaminated sample site upstream of the highly contaminated Steeds Pond.

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MATERIALS AND METHODS Sampling and General Soil Characterization. The study site was a wetland along Tims Branch on the SRS, described in the Introduction (Figure 1). This stream received waste from a former target and fuel processing facility for nuclear materials.27 A 15 cm deep soil sample was collected in April 2014 from a portion of the wetland located just upstream of the Ucontaminated region. The soil sample was collected from a nonradioactive portion of the wetland to permit analysis with instruments that were not housed in laboratories that can accept radiological material. The ∼400 g sample contained a largely undisturbed root and soil mass from a soft rush plant (Juncus eff uses). It was placed in a zip locked bag and stored in an ice chest. The season that the sample was collected is expected to be one of many influences on the rhizosphere composition.31 While the impact of seasonality on rhizosphere composition is recognized, clarifying its impact is outside the scope of the present project. Once transported to the Savannah River National Laboratory, the soil samples were placed in tripled zip locked bags; the space between each bag was filled with N2 gas, forming a double-N2 gas envelope around the samples. The refrigerated samples were shipped overnight on blue ice to the Environmental Molecular Sciences Laboratory (EMSL, Richland, WA), where they were stored in a moist state at 4 °C. There was no evidence that the N2 gas envelope had deflated prior to subsampling at EMSL. In a N2 glovebag, the soil sample was divided into two subsamples: rhizosphere and nonrhizosphere. The rhizosphere subsample was operationally defined as soils near the root (generally within 3−10 mm of the root) that had a distinctive reddish color. This moist subsample was manually separated from the roots and bulk soil with a spatula. The nonrhizosphere subsample was composed of the remaining soil without roots.

Less than 2 g of a field-moist rhizosphere sample was collected. This limited amount of rhizosphere sample hindered general soil characterization but permitted duplicate CHNO and pH analyses, triplicate qPCR and XRF analyses, and a single solvent extraction for the ESI-FTICR-MS analyses. The XRF analysis was conducted on a PANalytical Epsilon 5 instrument on different regions of a single pellet. CHNO analysis was conducted on a LECO CNS-2000 analyzer. XRD analysis was conducted on the clay-size fraction of the nonrhizosphere subsample using a Scintag XRD unit and the scans were processed using a JADE XRD pattern processing software. Measurements were made on Mg- and K-saturated samples at room temperature and 550 °C. The pH values of 1:4 solid:liquid suspensions were measured after a 24 h equilibration period. qPCR. DNA was extracted from 500 mg of triplicate soil samples using the FastDNA spin kit for soil (MP Biomedicals, U.S.A.) as described by the manufacturer. The concentrations of DNA were measured using a Nanodrop 2000 spectrophotometer (Thermo Scientific, U.S.A.). DNA concentrations were around 89−102 ng/μL. Total bacterial abundance was estimated using qPCR with the BACT1369F-PROK1492R primers and the TaqMan 1389F probe to enumerate copies of 16S rRNA genes.32 Potential sulfate-reducing and iron-reducing bacteria of the δ-Proteobacteria class were enumerated using the 361F-685 primer set and the TaqMan 1839F probe.33 Geobacter was enumerated by using 561F-825R primer set and the TaqMan Gbc2 probe.34 Anaeromyxobacter was enumerated by using the 60F-461R primer set.35 Gallionellalike Fe-oxidizing bacteria were enumerated by using the 628F998R primer set.36 PCR products of each primer set from the soil samples were cloned into the pGEM-T Easy Vector System B


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Environmental Science & Technology Table 1. General Soil Properties of Rhizosphere and Nonrhizosphere Subsamples pH total C (wt %) total N (wt %) total P (wt %) total S (wt %) total Mn (wt %) total Fe (wt %) sand/silt/clay (wt %) mineralogy of goeth > HIV > hem (no qtz)b

a

NA: not available because of insufficient amount of sample. bRanking of the XRD peaks: kao = kaolinite, HIV = hydroxyl-interlayered vermiculite, qtz = quartz, goeth = goethite, gib = gibbsite, and hem = hematite.

Calc version 1.0 NHMFL, 1998) generated empirical formula matches within 1.0 ppm using formula criteria of C(5−50)H(5−100)O(0−30)N(0−8)S(0−2)P(0−2). A mass error window of 1.0 ppm was used to compute the possible molecular formulas. All formulas obtained from the formula calculator were screened to eliminate those that are unlikely to occur in natural OM, according to a list of selection criteria, which were applied in previous studies.42−44 Peaks with large mass ratios (m/z values >500 Da) often have multiple possible formulas. These peaks were assigned formulas through the detection of homologous series (CH2, CH2O, COO, O, H2O, H2, etc.).45 Specifically, when the m/z of a homologous series group and the m/z of an already confidently assigned compound were summed to an m/z that was observed >500 Da, the formula assigned to the smaller compound was then appended by the atoms of CH2, and the new formula was assigned to the larger compound. The double bond equivalent (DBE) was calculated as DBE = 1 + 0.5(2C−H+N+P). Aliphatic compounds were assigned to formulas with DBE:C < 0.3 and H:C = 1.0−3.0. Aromatic compounds and condensed aromatic compounds were assigned to formulas with aromaticity indices (AI, calculated by (1+C−O−S−0.5H)/(C−O−S−N−P)) greater than 0.5 and 0.67, respectively. Carboxylic-rich alicyclic molecules (CRAM) were assigned to formulas that had very certain, yet restricted, compositions of DBE:C 0.30−0.68, DBE:H 0.20−0.95, and DBE:O 0.77−1.75. Because some formulas assignable as CRAM can also be assigned as lignin, there is a small region within the van Krevelen diagrams where CRAM and lignin overlap.37,46 The carboxyl-containing aliphatic (number of COO-R functionality ≥1) and/or alicyclic molecules (CCAM) were assigned to formulas with H:C 0.85− 2.00 and O:C 0−0.40.47

(Promega, France) according to the instructions of the manufacturer. Approximately 10 clones from each primer set were then randomly chosen for sequencing (Genewiz.Inc.). All qPCR measurements were carried out using a StepOnePlus Real-Time PCR System (Life Technologies, U.S.A.). For DNA quantification, each 20 μL qPCR mixture was composed of 10 μL of SYBR Premix Ex Taq II (Takara, Japan), 0.8 μL of 10 μM of each primer, and ∼10 ng og DNA template. Each assay contained a standard produced by serial dilution of plasmids containing specific target sequences. Independent triplicate templates for each soil sample and triplicate no template controls (NTC) were carried through the analyses. ESI-FTICR-MS. To obtain compositional differences between the soil OM recovered from the rhizosphere and nonrhizosphere samples, the samples were extracted with a methanol/water solvent for a brief period (in the order of minutes, to avoid methylation of the soil OM). The method of soil OM extraction impacts the type of organic compounds extracted and therefore the characterization results.37,38 The methanol/water solvent was selected to provide a mild extraction, yet still extract organic polar components from the soils. By comparison, alkaline extractants (e.g., NaOH or NH4OH) typically have higher soil OM recovery (as high as 66% to 74%) but can increase the salt contents in the OM, which in turn may greatly suppress the signals of organic molecules in the negative ion mode, especially if an extra saltremoval step is not conducted in the extraction procedure.37,38 Extraction with volatile alkaline solvent, such as NH4OH, increases the incorporation of N into the organic molecules.39,40 Therefore, none of the commonly used soil OM extractants recover 100% of the OM from soil, and some create sampling artifacts. The methanol/water solvent extractant used in this study permitted evaluation of the relative compositional differences between the two soil types of a readily extractable fraction, which is potentially more mobile in the environment. The extractants were infused directly at a flow rate of 2 μL/ min into a standard ESI ion source connected to a Bruker Daltonics 12 T Apex Qe FTICR-MS instrument. Analyses were conducted in negative and positive ion modes. Most of the discussion and data presented were in the negative ion mode because it is more representative of the acidic functional groups that dominate humic materials, including phenolic and carboxylic acids.41 The instrument was externally calibrated prior to sample analysis. Mass spectra were internally calibrated with fatty acids, dicarboxylic acids, and other naturally present CH2 homologous series within the sample itself. All m/z lists, with a signal-to-noise (S/N) ≥ 7 were exported for further data analysis. A molecular formula calculator (Molecular Formula

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RESULTS General Soil Properties. Carbon and nitrogen concentrations were about 4 times greater in the rhizosphere than in the nonrhizosphere subsamples (Table 1). Elevated C and N concentrations can be attributed to, as noted earlier, direct plant releases of exudates, mucilage, sloughed-off cells, and root die-back and to indirect root influences on stimulating microbial populations.48 Rhizosphere soil also had greater Fe and Mn concentrations than the nonrhizosphere soil (Table 1), which has also been reported previously.49 Again, this can be attributed to various rhizosphere biogeochemical processes that are conducive to Fe- and Mn-(oxyhydr)oxide precipitation. Among the more important processes is that roots pump oxygen into the rhizosphere, promoting the oxidation of Fe(II) and Mn(II) to the oxide-forming Fe(III) and Mn(IV) species. C


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Environmental Science & Technology

Table 2. qPCR Microbial Counts (Average Copy Number of Genes/G Dry Wt Soil ± Standard Deviation of Three Replicated Soil Extractions)a rhizosphere nonrhizosphere

16S (total bacteria)

δ-Proteobacteriab

Geobacter

Anaeromyxobacter

(6.50 ± 0.17) × 10 (5.93 ± 1.35) × 107

(6.58 ± 2.88) × 104 (1.90 ± 0.32) × 105c

(6.10 ± 2.52) × 104 (1.09 ± 0.12) × 105c

(1.77 ± 0.24) × 105c (6.87 ± 0.89) × 104

7c

a

Rhizosphere sediment and nonrhizosphere sediment contained 67.3% and 29.8% moisture, respectively. The probes used for the 16S (total bacteria) was BACT1369F-PROK1492R with the TaqMan probe 1389F; δ-proteobactria was 361F-685R with TaqMan probe 1839F; Geobacter was 561F-825R with TaqMan probe Gbc2; Anaeromyxobacter was 60F-461R. bThe δ-Proteobacteria detected with this primer include only the potential sulfate-reducing and iron-reducing bacteria. cIdentifies significantly (p ≤ 0.05; n = 3, 1-tail, type 1 t test) greater means of microbial counts between rhizosphere and nonrhizosphere samples.

Table 3. ESI-FTICR-MS Negative Mode Formula Assignments for Rhizosphere and Nonrhizosphere OM rhizosphere

nonrhizosphere % formulas

total formulas shared formulas different formulas CHO CHNO CHOS CHOP CHNOS CHNOP CHOSP CHNOSP nonoxygen-containing formulas

% formulas

number of formulas

by number

by intensity

number of formulas

by number

by intensity

2265 1040 1225a 643 550 155 56 397 212 31 75 146

100 46 54 28 24 7 2 18 9 1 3 6

100

2396 1040 1356b 912 604 175 27 281 215 28 38 116

100 47 53 38 26 8 1 12 9 1 1 5

100

16 22 9 3 22 13 2 6 8

38 23 13 2 9 9 1 1 5

a

Formulas identified in the rhizosphere sample but not identified in the nonrhizosphere sample. bFormulas identified in the nonrhizosphere sample but not found in the rhizosphere sample.

For example, δ-Proteobacteria (e.g., Pseudomonadaceae or Burkholderiaceae families) have been shown to be fast growing in the rhizosphere because of their ability to utilize a broad range of root-originating C substrates.53 It is possible that while refrigeration, inert gas chambers, and sterile sample handling were used in this study to minimize sampling artifacts, additional precautions may be warranted in future studies. Soil Organic Matter Composition. The rhizosphere OM had 2265 identified formulas, of which 1040 were similar to those identified in the nonrhizosphere OM; the remaining 1225 formulas were unique to the rhizosphere OM (Table 3). Similarly, the nonrhizosphere OM had a total of 2396 identified formulas, of which 1356 formulas were unique to the nonrhizosphere OM. While ESI-FTICR-MS is not a quantitative method, the number of peaks and peak intensities can provide some information about relative concentrations within a given sample and to a lesser extent between samples.54 There were fewer CHO formulas in the rhizosphere OM (28% by peak number and 16% by peak intensity) than in the nonrhizosphere OM (38% by both peak number and peak intensity) (Table 3). The two samples had high heteroatom (N, S, O, or P) contents, with over 50% of identified peaks consisting of heteroatom-containing compounds. Previous measurements of dissolved OM recovered from environments with strong redox gradients also had elevated heteroatom contents.55,56 Nitrogen containing formulas accounted for 54% of the rhizosphere and 47% of the nonrhizosphere OM formulas, consistent with the trend observed with total N contents in these two samples (Table 1). The intensity-averaged molecular weight was greater by ∼20% in the rhizosphere OM (653 Da) than in the

Phosphorus and sulfur concentrations were greater in the nonrhizosphere than in the rhizosphere subsample. One potential explanation for this observation may be that the roots took up P and S as nutrients from the rhizosphere to a greater extent than they did from the more distal nonrhizosphere soil. Root exudates, such as citrate and to a lesser extent malate, are highly effective at extracting P from soils.50 The pH of the rhizosphere soil was slightly more acidic than the nonrhizosphere soil. There are several plant-mediated processes that can influence soil pH, and these processes vary depending on the soil conditions, plant conditions, plant age, and distance from the apical growth area.51 Some of the key processes controlling pH include the root compensation for unbalanced cation−anion uptake, exudation, respiration (contributing to CO2 and carbonic acid concentration), and redox reactions.51 Microbial Populations. The soils contained a wide variety of bacteria, including iron and sulfate reducing bacteria from the δ-Proteobacteria class, Geobacter, and general metal-reducing bacteria, Anaeromyxobacter (Table 2). It is not surprising that various redox actives microbes were in these samples because the sampling site periodically underwent flooding. Slightly greater concentrations of total bacteria (as estimated by copies of 16S rRNA genes) and Anaeromyxobacter were detected in the rhizosphere than in the nonrhizosphere soil sample. Conversely, slightly greater δ-Proteobacteria and Geobacter were detected in the nonrhizosphere than in the rhizosphere soil sample. Given the relatively large differences in C, Fe, and Mn concentrations between the two samples, it is not clear why such small differences were observed in the populations of the probed microorganism. Soil microorganisms have been shown to feed on and be attracted to the organic C released by roots.52 D


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Table 4. Molecular Information for Rhizosphere and Nonrhizosphere Soil OM as Revealed by ESI-FTICR-MS in Negative Mode rhizosphere molecular classa

number of peaks

all identified formulas aliphaticsd aromatics (AI > 0.5)e condensed aromatics (AI ≫ 0.67)f carboxylic-rich alicyclic molecules (CRAM)g carboxyl-containing aliphatic molecules (CCAM)h N-containing CCAM

2265 1428 100 44 282 1666 955

nonrhizosphere

% peak intensitya ave MWb (Da) 100 74 2.45 0.80 5.79 83 57

653/527c 680 578 481 513 663 690

number of peaks 2396 1582 110 61 329 1696 849

% peak intensitya ave MWb (Da) 100 81 5.64 3.04 5.13 78 32

551/495c 564 428 417 552 561 673

a

% relative intensity was the percentage of the summed intensity of one category divided by that of the whole sample. bMW is intensity-normalized molecular weight. c653 is the intensity-averaged MW, and 527 is the number-averaged MW for the rhizosphere OM; 551 is the intensity-averaged MW, and 496 is the number-averaged MW for nonrhizosphere OM. dAliphatics were estimated by assigning double bond equivalent (DBE) DBE = 1 + 0.5x(2C−H+N+P), where C < 0.3 and H:C 1.0−3.0). eAromatics are molecules with an aromatic index (AI) larger than 0.5; AI = aromatic index = (1+C−O−S−0.5H)/(C−O−S−N−P). Aromatics include condensed aromatics. fCondensed aromatics have an AI > 0.67 (AI defined in footnote e). gCRAM = carboxylic-rich alicyclic molecules was recently reported as one of the major refractory nonliving components of freshwater DOM and likely derived from terpenoids and are defined as having double bond equivalents (DBE)/C = 0.30−0.68, DBE/H = 0.20−0.9, DBE/O = 0.77−1.75, DBE = 1 + 0.5 × (2C−H+N+P). hCCAM = carboxyl-containing aliphatic molecules (CCAM) = O:C 0−0.40; H: C= 0.85- 2.00.

immobilization. However, when environmental conditions result in the dispersion of OM, such as may occur if there is a sharp increase in pH or a sharp reduction in ionic strength, a reduction in metal immobilization may occur.65 N-containing CCAM were more enriched in rhizosphere extracts than the nonrhizosphere extract (Table 4). Recently Xu et al.66 reported the presence of a strong metal-binding ligand, hydroxamate siderophores (HO−NH−COOH), in the particulate and colloidal OM of soil aggregates. The occurrence of these N- and COO-containing molecules was correlated to soil Pu concentrations. Hydroxamate siderophores constituted only a very minor component of total organic C ( lipid-like > protein-like. Furthermore, the CHO molecules extracted from the rhizosphere as compared to the nonrhizosphere tended to have more aromaticity: the nonrhizosphere aromatic index (AI = (1+C−O−S−0.5H)/(C−O−S−N−P) was −0.39, and the rhizosphere aromatic index was 0.13 (also see molecule distributions in the aromatic and condensed aromatic regions in Figure 2A). A ranking of the abundance of CHON formulas identified only in the rhizosphere OM (i.e., the different formulas) is lignin-like ≈ lipid-like > protein-like (Figure 2B). A ranking of the abundance of formulas identified only in the nonrhizosphere is lipid-like > lignin-like > proteinlike. In contrast, the CHON molecules were more aromatic in the nonrhizosphere than in the rhizosphere OM (AI = −0.05 for the rhizosphere and 0.37 for the nonrhizosphere); this trend is consistent with that noted for all the identified molecules (Table 4). One explanation of these contrasting results is that the rhizosphere CHO compounds consisted mostly of plant exudates with freshly produced proteinaceous and lignin

nonrhizosphere OM (551 Da), and the number-averaged molecular weight was greater by ∼6% in the rhizosphere OM (527 Da) than in the nonrhizosphere OM (496 Da) (Table 4). While there is debate about the use of ESI-FTICR-MS to estimate molecular weights of natural OM molecules,57 these results are consistent with those of Takeda et al.,58 who used size exclusion chromatography and ultrafiltration to demonstrate that dissolved OM in the rhizosphere soil contained higher molecular weight OM molecules than the dissolved OM recovered from nonrhizosphere soil. To highlight the compositional differences of these two soil OM samples, the ESI-FTICR-MS data were organized further into different formula categories: aliphatics, aromatics, condensed aromatics, carboxylic-rich alicyclic molecules (CRAM), carboxyl-containing aliphatic molecules (CCAM), and Ncontaining CCAM molecules (Table 4).46,59 These operationally defined constructs help to organize the assigned formulas into pools that have functional chemical significance. These formula categories are not mutually exclusive, and therefore, their sums do not equal 100%. The aliphatic compounds accounted for a vast majority of the identified formulas: 74% of the rhizosphere and 81% of the nonrhizosphere OM fractions (Table 4). Among the larger differences between the two OM samples were that the nonrhizosphere OM contained greater number and intensity of aromatics and condensed aromatic compounds. Such compounds tend to be more recalcitrant to abiotic and biotic degradation (however, molecular structure alone does not control soil OM stability).60 Carboxylate concentrations were slightly greater in the rhizosphere than in the nonrhizosphere samples, as indicated by the CRAM and CCAM percent peak intensities (Table 4). Carboxylate groups on OM can form strong complexes with many metals,61 including radionuclide (U and Pu).62,63 Organic acids high in carboxylate groups could increase the acidity of rhizosphere soil and enhance the aqueous activity of ambient trace metals, resulting in an increased mobility of some metals (e.g., Al, Fe, Cu, Y, La, and U).58,64 The rhizosphere pH was 0.36 units lower than the nonrhizosphere soil (Table 1). While most soil OM is bound to soil, a smaller fraction exists in the mobile aqueous phase. As such, the presence of more carboxylate functions groups in the rhizosphere would be expected to generally enhance metal E


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were defined as having a NSOP:C ≤ 0.1 and a MW > 850; moderately hydrophobic molecules were defined as having a NSOP:C ratio between the range of 0.1 to 0.49 and a MW < 850; hydrophilic molecules were defined as having a NSOP:C ≥ 0.49 and a MW < 850 (Figure 3).68 On the basis of both

Figure 3. Number of formulas in rhizosphere and nonrhizosphere OM classified as hydrophobic (NSOP:C ≤ 0.1 or molecular mass (MW) ≥ 850 Da), moderately hydrophobic (0.1 < NSOP:C < 0.49 and MW < 850 Da), and hydrophilic (NSOP:C ≥ 0.49 and MW < 850 Da).

peak number (Figure 3) and peak intensity (not shown), rhizosphere OM tended to have more hydrophilic molecules than the nonrhizosphere OM, a property that may promote metal binding.

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DISCUSSION Wetlands can concentrate contaminants that are introduced via groundwater and surface runoff. They are unusually complex systems because their hydrology and biology are constantly in a flux. Furthermore, these systems have steep biogeochemical gradients, and the hydraulic, biological, mineralogical, and geochemical processes have strong interactions, confounding simple interpretations. Understanding the processes responsible for contaminant retention becomes especially important when predicting long-term stewardship of these systems or attempting to evaluate remediation options, including monitored natural attenuation. Our previous work has measured significant U concentrations in the rhizosphere compared to the bulk wetland soils. The implications of this observation are that plants not only promote contaminant sequestration by creating OM, but they also actively create a biogeochemical environment in the rhizosphere that is especially well suited to contaminant sequestration. Rhizosphere organic molecules can either increase or decrease metal mobility, depending primarily on whether the OM is bound to soil. If the metal binds to an organic molecule that is strongly bound to soil particles, then the OM−metal complex will be immobilized. Conversely, if the metal binds to a mobile organic molecule, then the OM−metal complex will be mobile. Because a vast majority of OM is strongly bound to soil surfaces, the net effect is that the OM immobilizes the vast majority of the metal, while potentially enhancing the mobility of a much smaller (but potentially quite important) fraction. The release of root exudates, especially N-containing siderophores, are a well-known plant mechanism to promote the solubilization of nutrients, most notably Fe(III) and Zn, from the rhizosphere.69−71 Conversely, Li et al.65 discovered that both uranium oxidation states (IV) and (VI) were coexistent in the rhizosphere and bonded mostly to carboxylic groups

Figure 2. van Krevelen diagrams of compound pools of unique molecules in the rhizosphere or nonrhizosphere of (A) CHO molecules consisting of atomic composition as C5−66H6−120O1−26 and (B) CHON molecules consisting of atomic composition as C8−67H7−125O1−17N1−8. (AI = aromatic index = (1+C−O−S−0.5H)/ (C−O−S−N−P); CCAM = carboxyl-containing aliphatic molecules; CRAM = carboxylic-rich alicyclic molecules; the “Aromatic line” identifies where the AI = 0.5; the “Condensed-aromatic line” identifies where the AI = 0.67).

material that contains abundant aromatic structures (Figure 2). The nonrhizosphere OM may have undergone greater degradation as a result of being in contact with the soil longer, which may have promoted an enrichment of aliphatic and oxygenated molecules and/or the formation of more recalcitrant N-containing aromatics (Figure 2). With regard to this latter process, Schmidt-Rohr et al.67 made measurements of OM derived from rice paddies and reported that anilides (i.e., CO−NH groups bonded to aromatic carbon compounds) could be formed through a reaction of lignin-derived-phenolic compounds with ammonia, COOH or NCO groups of amino acids, or peptides. However, interpretation of these findings is limited by the fact that ESI-FTICR-MS provides molecular formula information but not molecular structures. Most of these large formulas could have multiple structural isomers. The ratios of the number of heteroatoms to carbon (NSOP:C) and the molecular weights (MW) can be used as an index of hydrophobicity.68 Following the classification method used by Liu and Kujawinski,68 hydrophobic molecules F


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provided by root exudates or microbial activity. Even the oxidized U(VI), which was expected to exhibit limited absorption to sediment minerals under acidic conditions, was immobilized due to the formation of U(VI)−OM complexes, where the OM was also strongly bound to the wetland soil.65 This study showed that the rhizospheres of wetland soils may have unique microbial and OM attributes with respect to the bulk soil. It did not attempt to identify active OM structures or microbes responsible for contaminant sequestration. The rhizosphere had elevated C, N, Mn, and Fe concentrations and elevated total bacteria and Anaeromyxobacter counts. Furthermore, the OM was different in the rhizosphere soil compared to the nonrhizosphere soil; rhizosphere organic molecules contained (1) greater overall molecular weights, (2) less aromaticity, (3) more carboxylate and N-containing COO functional groups (which likely affects local pH and provide extra binding sites for trace metals and radionuclide contaminants), and (4) more heteroatoms (N, S and P), thus more hydrophilic that may promote metal binding. While this study provided for the first time a detailed description of many rhizosphere properties, the study was limited to a single site, single plant, and single set of environmental conditions. As such, additional work is needed to evaluate how widespread and robust these findings are.

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AUTHOR INFORMATION

Corresponding Author

*Phone +1-803-725-2363. Fax: +1-803-725-2363. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

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REFERENCES

This work was supported by the Subsurface Biogeochemistry Research Program within the Climate and Environmental Sciences Division in the Office of Biological and Environmental Research, Office of Science, U.S. Department of Energy (DOE), Grants DR-FG02-08ER64567 and ER652221038426-0017532. The ESI-FTICR-MS analyses were conducted at EMSL, a national scientific user facility sponsored by DOE’s Office of Biological and Environmental Research program. EMSL is located at the PNNL in Richland, WA, USA.

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Unique Organic Matter and Microbial Properties in the Rhizosphere of

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