Determination of Microbial Carbon Sources and Cycling during

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Environ. Sci. Technol. 2010, 44, 2322–2327

Determination of Microbial Carbon Sources and Cycling during Remediation of Petroleum Hydrocarbon Impacted Soil Using Natural Abundance 14C Analysis of PLFA BENJAMIN R. COWIE,† BRUCE M. GREENBERG,‡ AND G R E G O R Y F . S L A T E R * ,† School of Geography and Earth Sciences, McMaster University, 1280 Main Street West, Hamilton, ON L8S 4K1 Canada, and Department of Biology, University of Waterloo, Waterloo, ON N2L 3G1 Canada

Received September 29, 2009. Revised manuscript received January 26, 2010. Accepted February 2, 2010.

Inapetroleumimpactedland-farmsoilinSarnia,Ontario,compoundspecific natural abundance radiocarbon analysis identified biodegradation by the soil microbial community as a major pathway for hydrocarbon removal in a novel remediation system. During remediation of contaminated soils by a plant growth promoting rhizobacteria enhanced phytoremediation system (PEPS), the measured ∆14C of phospholipid fatty acid (PLFA) biomarkers ranged from -793‰ to -897‰, directly demonstrating microbial uptake and utilization of petroleum hydrocarbons (∆14CPHC ) -1000‰). Isotopic mass balance indicated that more than 80% of microbial PLFA carbon was derived from petroleum hydrocarbons (PHC) and a maximum of 20% was obtained from metabolism of more modern carbon sources. These PLFA from the contaminated soils were the most 14C-depleted biomarkers ever measured for an in situ environmental system, and this study demonstrated that the microbial community in this soil was subsisting primarily on petroleum hydrocarbons. In contrast, the microbial community in a nearby uncontaminated control soil maintained a more modern ∆14C signature than total organic carbon (∆14CPLFA ) +36‰ to -147‰, ∆14CTOC ) -148‰), indicating preferential consumption of the most modern plant-derived fraction of soil organiccarbon.Measurementsofδ13Cand∆14CofsoilCO2 additionally demonstrated that mineralization of PHC contributed to soil CO2 at the contaminated site. The CO2 in the uncontaminated control soil exhibited substantially more modern ∆14C values, and lower soil CO2 concentrationsthanthecontaminatedsoils,suggestingincreased rates of soil respiration in the contaminated soils. In combination, these results demonstrated that biodegradation in the soil microbial community was a primary pathway of petroleum hydrocarbon removal in the PEPS system. This study highlights thepowerofnaturalabundanceradiocarbonfordeterminingmicrobial carbon sources and identifying biodegradation pathways in complex remediation systems. * Corresponding author e-mail: [email protected]; phone: 905525-9140x-26388; fax: 905-546-0463. † McMaster University. ‡ University of Waterloo. 2322

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Introduction Directly demonstrating biodegradation in contaminated environments is integral for the validation of natural attenuation and engineered environmental remediation systems. However, multiple degradation pathways and mechanisms may be involved in contaminant removal, particularly when more than one remediation approach is applied (1-4). Therefore, effective tools for monitoring and verifying contaminant removal processes in bioremediation systems must be employed to delineate between biodegradation and abiotic mass-removal processes. Increasingly, compound-specific isotope analysis is being applied to demonstrate biodegradation. In addition to isotopic assessment of contaminants themselves, compound specific carbon isotope analysis of phospholipid fatty acid (PLFA) biomarkers has emerged as an effective indicator of organic contaminant biodegradation (5-9). Stable isotope analysis of PLFA has been applied to elucidate microbial processes and carbon cycling pathways in many environments (7-15). However, for both contaminant and PLFA based approaches, the limited range of δ13C in petroleum hydrocarbons (δ13CPHC ) -18 to -35‰) relative to natural organic matter (δ13CNOM ) -24 to -34‰ for C3 plants) can preclude the identification of biodegradation using 13C (16). Natural abundance radiocarbon (14C) profiling of PLFA is a promising new approach to elucidate microbial carbon sources, and therefore constrain major degradation pathways of petroleum biodegradation (8, 9, 17-19). Petroleum hydrocarbons (PHC) contain no detectable 14C due to radioactive decay over millions of years (∆14CPHC ) -1000‰). In contrast, natural organic carbon in soil is a complex mixture that contains components with variable radiocarbon ages, and carbon recently fixed from the atmosphere has modern levels of 14C (∆14Catm ≈ +55‰) (20, 21). The distinction in 14C between petroleum and natural organic carbon in soil provides a label that can be exploited to overcome the limitations of indistinguishable δ13C signatures between natural soil organic matter and petroleum hydrocarbons. A negative shift in ∆14C of microbial PLFA can therefore directly demonstrate uptake and metabolism of petroleum by microbial communities in complex remediation systems (8, 14). Phytoremediation of organic contaminants utilizes synergies between plant roots and soil microbes to accelerate the removal of contaminants from surface soils (3). While phytoremediation has been more extensively studied for metal remediation, it is now developing into a useful tool for removal of organic contaminants (22). Recently, a plant growth promoting rhizobacteria enhanced phytoremediation system (PEPS) was developed to remove recalcitrant PHC from soil (23-27). The PEPS was demonstrated to be an effective method for removal of 30% of PHC per growing season, however, the role and activity of soil microorganisms versus the role of plants for petroleum hydrocarbon degradation remains a central question within PEPS and phytoremediation systems in general (3, 22, 24, 27-29). Processes that occur in the rhizosphere, the fraction of the soil most closely associated with plant roots, are potentially responsible for driving remediation in these systems, however, differentiating the relative roles of soil microbes and plants is often impossible using conventional approaches. For example, aqueous dilution, transport, or volatilization can mobilize and/or diminish the concentration of PHC in soil without any biodegradation occurring. Biodegradation can be inferred by using genetic profiling and microbial 10.1021/es9029717

 2010 American Chemical Society

Published on Web 03/02/2010

culturing studies to identify the presence of microbes or enzymes capable of degrading a contaminant (30); however, these approaches do not directly demonstrate that degradation processes are actively occurring in situ. The objective of this study was to use natural abundance compoundspecific radiocarbon analysis of PLFA biomarkers to assess whether microbial biodegradation of PHC plays a major role when PEPS is employed to remediate impacted soils. It should be noted that this study did not compare the efficiency of the PEPS remediation system against other natural attenuation or engineered approaches for remediation of PHC in soils.

Experimental Section Site Description. An experimental PEPS phytoremediation plot and an uncontaminated control site at a land farm in Sarnia, ON were selected for this study. Over the past 20 y, the land farm site has been sprayed with a recalcitrant high molecular weight petroleum sludge and the soil has been tilled over between the months of May and October (27). The land farming process has produced fairly homogeneous soils with consistent properties (Supporting Information, Table S1) (27). The PEPS plot (“BRF+”) was planted with barley (Hordeum vulgare), annual ryegrass (Lolium multiflorum), and tall fescue (Festuca arundinacea) seeds that were treated with Plant Growth Promoting Rhizobacteria (PGPR, Pseudomonas sp., Pseudomonas putida) as previously described (27). The “uncontaminated control” site was a nearby grassy knoll that had not been exposed to PEPS treatments or petroleum contamination. In addition to the two primary experimental sites, soil CO2 was also obtained from an “untreated control”, and an alternate PEPS experimental plot, “RF-” that was planted with annual ryegrass and tall fescue, but not treated with PGPR. The untreated control was a bare petroleum-contaminated soil on the land farm that had not received PEPS treatment or planting. These experiments occurred in 2006 as part of a three-year PEPS field study conducted by Gurska et al. (27). Soil Gas Collection and Analysis. Soil gas was collected by drilling holes into a piece of plastic tubing, and installing the tubing into the soil at 10 cm depth, creating an ∼80 cm3 void space. The tube was attached to a length of Tygon tubing with a silicone seal, and the tubing was clamped at the soil surface to prevent atmospheric mixing with subsurface gases. Soil gas was sampled after a 1-month equilibration period and analyzed by GC-TCD (see Supporting Information). Stable carbon isotope ratios of CO2 were determined using an Agilent 6890 GC equipped with a GS/Q column, isothermal at 120 °C, coupled to a Thermo Delta Plus XP isotope ratio mass spectrometer (IRMS) via a Conflo III interface. Reproducibility of CO2 standards was e0.5‰, and the standard deviation for triplicate analyses was e0.2‰. CO2 for radiocarbon analysis was cryogenically sealed in quartz tubes on a vacuum line and transported to the National Ocean Sciences Accelerator Mass Spectrometer (NOSAMS) facility at Woods Hole Oceanographic Institution for analysis. Stable isotope data is reported in delta notation (δ13C) relative to the Vienna PeeDee Belemnite standard reference material. Radiocarbon values are expressed in ∆14C notation, in per mille (‰) deviation from the 14C Standard Reference Material 4990B, oxalic acid (31). Data were normalized to δ13C of -25‰, and were age corrected for the year of collection (2006). Soil δ13CCO2 values were independently verified at NOSAMS and were within 0.5‰ of the reported values in this study. Soil Sampling and Bulk Organic Carbon Isotope Analysis. Bulk soil samples were obtained from each PEPS treated site and the two control sites in August 2006 from a soil depth of 5-10 cm. Soils were sampled directly into precombusted 500 mL glass jars with Teflon faced lids, and frozen at -20 °C until analysis. Prior to extraction, roots were

removed and soils were homogenized using a mortar and pestle. Homogenized soils were acidified 3 times with excess 1 N HCl to remove carbonates, rinsed 3 times with distilled, deionized water, and dried at 50 °C for 24 h. Triplicate samples were combusted and δ13C was analyzed by Elemental Analyzer/IRMS, with standards IAEA 600, IAEA CH-7, NBS 21, USGS 24, and USGS 40. Standard deviation for triplicate analysis of standards was e0.2‰. BRF+ and uncontaminated control soils were analyzed for bulk organic 13C and 14C at NOSAMS by standard methods (32). The δ13C values measured at NOSAMS were within 0.5‰ of the values measured at McMaster. PLFA Extraction and Preparation. Approximately 150 g of homogenized soil samples was extracted 3 times using the modified Bligh and Dyer process (8, 9, 33), and subsequently fractionated by silica gel chromatography to separate the polar lipids including phospholipids. The high concentration of PHC in the soils required use of large volumes of silica gel and solvents, including a secondary silica gel purification step, to separate phospholipids from other organic compounds in these samples. The details of these separations are described in the Supporting Information. After separation, the polar lipid fraction was evaporated to dryness under a stream of nitrogen gas, and reacted to fatty acid methyl esters (FAME) as previously described (33). FAME were further purified using a third silica gel chromatography step, on 0.5 g of fully activated silica gel and eluted with 5 mL of hexane (f1), 5 mL of dichloromethane (f2 FAME), and 5 mL of methanol (f3). Approximately 100 mL of of UW4 PGPR bacterial culture (24) was filtered using a 0.2 µm Whatman filter and extracted as described above. The first (100 g) silica column was omitted from the pure culture procedure. Identification and Quantification of FAME. An Agilent 6890 gas chromatograph (30 m × 0.25 mm DB-5 MS column, 0.25 µm film thickness) coupled to an Agilent 5973 quadrupole mass spectrometer was used for identification and quantification of FAME. The temperature program was 40 °C hold 1 min, ramp to 130 at 20 °C min-1, to 160 at 4 °C min-1, and finally to 300 at 8 °C min-1. FAME were quantified using external calibration standards methyl tetradecanoate and methyl eicosanoate (Supelco Inc.). FAME were identified using several bacterial reference standards (Bacterial Acid Methyl Esters CP Mix, Matreya Inc., Fatty-Acid Methyl Ester Mix, Supelco Inc.), mass-fragmentation patterns, and retention times. Double bond positions were determined by analysis of dimethyl-disulfide adducts (34). GC-C-IRMS Analysis of FAME. Stable carbon isotope ratios of individual FAME were determined using an Agilent 6890 GC (30 m × 0.25 mm DB-5 MS column, 0.25 µm film thickness) coupled to a Thermo Delta Plus XP isotope ratio mass spectrometer via a Conflo III interface. The GC program was 50 °C for 1 min, 10 °C min-1 to 150 °C, 1.5 °C min-1 to 180 °C for 20 min, 10 °C min-1 to 280 °C, and 15 °C min-1 to 320 °C for 15 min. Reproducibility for isotopically characterized hexadecane, octacosane, and m-terphenyl laboratory standards was better than 0.3‰ (1σ) and accuracy was always within 2σ of expected values. FAME δ13C and ∆14C values were corrected during data processing for the addition of methanol carbon during derivatization. Precision for analysis of microbial FAME was typically 0.5‰ for minimum of three analyses, and all analyses were less than 1.5‰ (1σ). Compound Specific Radiocarbon Analysis. Concentrated FAME extracts were collected cryogenically on a preparative capillary gas chromatograph (PCGC) to obtain sufficient material for 14C analysis (35). FAME were separated on an Agilent gas chromatograph (60 m × 0.5 mm DB-5 column, 0.25 µm film thickness) interfaced with a Gerstel preparative fraction collection (PFC) system (for details, see Supporting VOL. 44, NO. 7, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 1. Concentration, δ13C and ∆14C Measurements of Soil CO2, Total Organic Carbon, and PLFAa treatment type

%CO2 ( 0.10%

δ13CCO2 ( 0.5‰

∆14CCO2 ( 40‰

δ13CTOC ( 0.5‰

∆14CTOC ( 20‰

∆14CPLFA ( 40‰

BRF+PGPR RFuntreated control uncontaminated control

0.42 0.55 0.44 0.11

-22.4 -22.9 -23.4 -16.9

-621 -712 -664 -182

-28.5 -29.1 -28.9 -27.8

-941 n.a. n.a. -148

-793 to -898 n.a. n.a. +36 to -147

a

n.a. indicates not analyzed.

Information). Individual FAME were submitted for 14C analysis by AMS at the NOSAMS via previously described methods (32), and data is reported in ∆14C notation, as described above. Submitted sample sizes for individual FAME ranged from 4.3 to 14.2 µmolC.

Results Bulk Organic Carbon. The δ13C and ∆14C values for total organic carbon (TOC) are presented in Table 1. The BRF+ soil was significantly depleted in ∆14CTOC (∆14CTOC ) -941‰; δ13CTOC ) -28.5‰), indicating a large contribution of petroleum hydrocarbons to soil organic matter at the contaminated sites. In contrast, the measured isotopic values of the uncontaminated control soil (∆14CTOC ) -148‰, 1230 ( 50 years; δ13CTOC ) -27.8‰,) were typical of C3 planted uncontaminated soils (36, 37). The uncontaminated control TOC values were assumed to be representative of natural organic matter in the contaminated soils prior to petroleum contamination. To constrain the relative contribution of petroleum (∆14CPHC ) -1000‰) relative to modern organic carbon in the contaminated soils, a mass balance was derived using the uncontaminated control (∆14CTOC ) -148‰) as the nonPHC soil organic matter, including contributions from plant litter. ∆14CBRF+TOC ) ∆14CPHC × (f ) + ∆14CTOC × (1 - f )

(1)

where f ) fraction of petroleum in soil organic matter. Using eq 1, petroleum hydrocarbons (f ) were calculated to represent 93% of soil organic carbon in the BRF+ treated soil. Nearest to the time of sampling, 74 g kg-1 petroleum hydrocarbons were measured in the BRF+ soil (Table S1) (27), and this calculation estimated an additional 6 g kg-1 natural organic carbon. PLFA. PLFA distributions at both uncontaminated and contaminated sites were typical for surface soils (Table S2) (38). However, the increased abundance of unsaturated PLFA in the uncontaminated control soil contrasted with the greater proportion of saturated and branched PLFA observed in the contaminated soils (Table S3). The greater abundance of unsaturated PLFA in the uncontaminated control may have indicated a community composed mainly of Gram negative bacteria (39-41). In contrast, the contaminated sites were dominated by branched and saturated PLFA, which indicated a community composed mainly of Gram positive bacteria (39-41). The PLFA distribution at the PGPR treated site was not different from that at the untreated control site. Furthermore, analysis of pure cultures of PGPR bacteria did not produce unique biomarker lipids (Table S4). Radiocarbon analysis of PLFA (Figure 1) revealed a 14Cdepleted microbial community in the BRF+ soil (∆14CPLFA ) -793‰ to -897‰), and a relatively modern community at the uncontaminated control (∆14CPLFA ) +36‰ to -147‰). In both the uncontaminated control and BRF+ treated soils, the microbial communities were enriched in ∆14CPLFA relative to ∆14CTOC. Using an isotopic mass balance (eq 1) between PLFA, TOC, and PHC, it was determined that PLFA in the contaminated soils were derived from 80-90% petroleum 2324

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FIGURE 1. ∆14C results for PLFA from the BRF+ treated soil (circles) and the uncontaminated control soil (triangles). Error bars represent 2 times the standard radiocarbon age error ((40‰) for ∆14C. PLFA in this figure represent >50% of all detected PLFA by mole percent. Dashed line represents atmospheric ∆14C, solid line represents uncontaminated control ∆14CTOC, and dotted line represents BRF+ ∆14CTOC. hydrocarbons, compared to the nearly modern ∆14CPLFA signature in the uncontaminated control soil (Figure 1). In contrast, δ13CPLFA in the BRF+ soil and the uncontaminated control were virtually indistinguishable from each other (Figure 2). In both soils, δ13CPLFA were depleted by less than 5‰ relative to δ13CTOC, consistent with the expected variability in a natural system (42). Soil Gases. Soil gas CO2 concentrations are presented in Table 1 for all soils. CO2 comprised 0.4-0.6% of the total gas composition in the contaminated soils and 0.1% in the uncontaminated control. In addition to CO2, a substantial O2 chromatographic peak was observed in all samples. O2 concentrations were not precisely quantified due to an interfering N2 peak; however, the presence of O2 in the gas samples indicated that predominantly aerobic conditions prevail in these shallow, loamy soils. Stable isotope analysis of the soil CO2 revealed isotopic depletions in δ13CCO2 in the contaminated soils relative to the uncontaminated control (Table 1). Soil ∆14CCO2, also presented in Table 1, indicated that CO2 was substantially 14C-depleted in the contaminated soils (∆14C ) -621‰ to -712‰) relative to the uncontaminated control (∆14C ) -182‰). The BRF+, RF-, and untreated control soils did not vary in δ13CCO2 or ∆14CCO2 beyond instrument reproducibility.

Discussion Petroleum Hydrocarbon Metabolism by the Soil Microbial Community. The extremely depleted ∆14CPLFA values observed at the PEPS treated site directly demonstrated petroleum uptake and metabolism in the microbial community of the BRF+ treated soil. This observation was

FIGURE 2. δ13CPLFA at the uncontaminated control (triangles) and BRF+ (circles) soils. Error bars represent standard deviations of triplicate analysis, with a 0.5‰ minimum error. Dashed line denotes δ13CTOC for the uncontaminated control, and dotted line denotes δ13CTOC for the BRF+ soil. concurrent with previously observed 10-100 fold increases in microbial populations, and 30% per year reduction in PHC content in the land farm soils treated with PEPS (27). The observation of petroleum carbon uptake and utilization by the soil microbial community in conjunction with increased microbial biomass and observed PHC mass loss confirms that petroleum mineralization is occurring (27). Further, the observation of highly depleted microbial PLFA in the treated soil indicates that metabolism and utilization of petroleum hydrocarbons by the soil microbial community is the primary mechanism for remediation in PEPS. The large difference between the uncontaminated control and BRF+ treated PLFA indicated that the majority (>50% of PLFA by mol %) of the microbes in the contaminated community are actively consuming petroleum hydrocarbons and converting this carbon to biomass. The extensively depleted 14C signature of these bacteria further indicated that they are obtaining almost all of their carbon (>80%) from petroleum sources. This level of 14C depletion in these PLFA is comparable only with one previous laboratory study, in which microbial enrichment cultures were grown with ancient shale-derived kerogen as an exclusive carbon source and produced very depleted PLFA (∆14CPLFA ) -711.3 to -921.6‰) (14). The signature of petroleum metabolism observed in the present study is far more pronounced than in previously characterized environmental systems where petroleum biodegradation was suspected (8, 17). Petroleum Hydrocarbon Contribution to Soil Gas. Complementary to the ∆14CPLFA approach, δ13C and ∆14C of soil gas CO2 has been used previously as evidence for in situ petroleum mineralization (43, 44). By measuring ∆14CPLFA in conjunction with soil gases, we have overcome the ambiguity of the soil gas observations, and directly related microbial carbon uptake and utilization to petroleum hydrocarbons, and therefore verified biodegradation. In the contaminated soils, 13C-depleted CO2 (δ13CCO2 ) -22.4‰ to -23.4‰) indicated that the elevated CO2 concentrations relative to the uncontaminated control were derived from the degradation of organic carbon. However, because there is no difference in stable isotope signatures between petroleum and natural organic matter at this site, biodegradation cannot

be conclusively demonstrated using a 13C approach. All of the PHC contaminated samples, including the untreated control, exhibited 14C-depletion in CO2, up to -712‰, consistent with PHC mineralization in all soils. Within analytical error, neither planted treatment was isotopically distinct from the untreated control (∆14CCO2 ) -664‰). Therefore in this case it was not possible to distinguish the contribution of plants to soil remediation using isotopic analysis of soil gas. While this is indicative of petroleum respiration in the untreated control, Gurska et al. demonstrated that there was substantially more petroleum remediation in the PEPS treated soils relative to the untreated control (27). Soil gas ∆14CCO2 was less depleted than the ∆14CPLFA at the BRF+ site, suggesting an input of more modern CO2 to the soil gas than what was produced by microbial respiration. Such an input is likely contributed by plant root respiration. However, at the untreated control soil, with no root respiration, CO2 was equally 14C-depleted within error when compared to two planted soils that do have contributions from root respiration (∆14CCO2 ) -620‰ and -712‰). While this may suggest an input from atmospheric CO2 to the contaminated soils, soil CO2 concentrations were elevated more than an order of magnitude above atmospheric levels, implying a diffusive gradient out of the soil gas rather than into it. In this case, the soil gas carbon isotope data could not unambiguously resolve petroleum mineralization from inputs of root respiration, diffusion, or other subsurface processes. The source of soil gas CO2 at the uncontaminated control site is likewise complicated. Here the CO2 was more 14Cdepleted (∆14CCO2 ) -182‰) than the root respiration (∆14Cplant ) +55‰, (15)), and the microbial community (∆14CPLFA ) +36‰ to -147‰) but was within analytical error of the natural organic matter at the site (∆14CTOC ) -148‰). The major contributors to soil CO2 are respiration from microbes and plants, both of which are substantially 14Cenriched compared to the soil CO2. Therefore, another 14Cdepleted source of CO2 must contribute to soil gas at the uncontaminated control. Upward diffusion of CO2 from microbial respiration of older, 14C-depleted TOC in deeper soils is a possible explanation (21). Other potential explanations include lateral gas flux from the contaminated soil nearby, or dissolution of older soil carbonates (45-48). Stable Isotopes of PLFA. Compound specific radiocarbon analysis also overcame the limitations of stable isotope analysis of microbial PLFA that were incapable of resolving differences in carbon source between the uncontaminated control and PEPS treated microbial communities due to the similarity in δ13C of the two potential sources. PLFA were depleted in δ13C between 0‰ and 5‰ relative to δ13CTOC in both the BRF+ treated soil and uncontaminated control (Figure 2). This range of depletion from carbon source to PLFA has previously been identified as a signature of aerobic, heterotrophic microbes, however this signature alone is not a unique indicator of this type of community (42, 49). As expected, petroleum metabolism by soil bacteria could not be identified using stable isotope analysis because the δ13C of natural organic matter was indistinguishable from that of the petroleum hydrocarbons. Isotopic composition of PLFA was consistent with aerobic heterotrophic microbial communities in both the BRF+ soil and the uncontaminated control with the exception of cycC19:0 and cyc-C17:0. These PLFA were depleted in δ13C by more than 3‰ relative to δ13CTOC, which is uncharacteristic of aerobic heterotrophs. It was hypothesized that this depletion was related to a biosynthetic isotope effect observed during the synthesis of the cyclopropyl group in the fatty acid chain. During this process, a very 13C-depleted methyl group from the amino acid methionine is added to an unsaturated fatty VOL. 44, NO. 7, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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acid chain, resulting in a slightly more depleted δ13C value for the cyclopropyl fatty acid in cell membranes (50, 51). This biosynthetic effect could explain the observed depletions for the cyclopropyl fatty acids observed in both the uncontaminated and contaminated soils. However, it is important to consider that another study found cyclopropyl PLFA that were enriched relative to the precursor unsaturated PLFA, therefore this explanation remains tentative (52). It is possible that the cyclopropyl PLFA represent a community of microbes consuming an older, more 13C-depleted carbon source such as methane. However, methane was not detected in any uncontaminated control soil gas samples. Preferential Degradation of Modern Organic Carbon: Implications for Natural Systems. The PLFA in the uncontaminated control and contaminated soils are enriched in ∆14C relative to their respective bulk ∆14CTOC values (Figure 1). This indicates that the microbial community is preferentially degrading recent, more labile organic carbon in the upper soil, as opposed to the older, more recalcitrant component of soil organic carbon. This finding correlates with previous work on soil formation and microbial processes in surface soils (21, 36). Similar results were obtained previously using a radiocarbon approach, demonstrating that preferential microbial metabolism of younger natural organic carbon sources does occur (9, 21, 36). Identification of a nearly modern microbial community also partially explains the radiocarbon age of the uncontaminated control soil. As the most labile carbon is consumed, the most recalcitrant organic matter would remain, and over time the average radiocarbon age for total organic matter in the soil would increase if no new carbon were added to the system (37). Therefore compound specific radiocarbon analysis of PLFA has the potential to contribute valuable information about microbial carbon cycling in natural soil systems, by directly measuring the isotopic composition of the organic carbon affected by microbial degradation in soils. Implications to Assessing and Monitoring Remediation. The use of compound specific radiocarbon to identify biodegradation is a sensitive approach to trace the fate of petroleum hydrocarbons in contaminated systems. In this study, 14C depleted PLFA biomarkers directly indicated uptake and metabolism of petroleum in the soil microbial community of an engineered phytoremediation system, and determined that the primary carbon source to the microbial community was indeed petroleum hydrocarbons. Radiocarbon analysis demonstrated that observed PHC mass losses were related to biodegradation and that metabolism by the soil microbial community was the primary mechanism for petroleum removal in PEPS. Isotopic signals of soil CO2 supported the hypothesis of increased organic carbon respiration in PEPS treated soils relative to the uncontaminated control, however contributions from multiple source mixing in this system illustrated the complexity of using the isotopic signature of CO2 to monitor biodegradation. Therefore compound specific radiocarbon analysis of biomarkers conveys a substantial advantage, when compared to gas phase radiocarbon or stable isotope biomarker approaches, for establishing microbial carbon sources and degradation mechanisms in environmental systems contaminated with petroleum-derived organic compounds.

Acknowledgments We gratefully acknowledge funding provided by an NSERC Collaborative Research and Development Grant to B.M.G. and by NSERC funding to G.F.S. Special thanks are owed to Jennie Kirby and Martin Knyf (McMaster University) and Georges Lacrampe-Couloume (University of Toronto) for analytical assistance and technical expertise. Additional thanks to Jola Gurska, Wenxi Wang, Xiao-Ming Yu, XiaoDong Huang (University of Waterloo), Ayan Chakraborty and 2326

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Larry Lawlor (Imperial Oil). Thank you to Jason M.E. Ahad (Geological Survey of Canada) and to three anonymous reviewers who greatly improved this manuscript by providing helpful suggestions and discussion.

Supporting Information Available Additional information including detailed method descriptions, soil properties (Table S1) and PLFA distributions for soils and PGPR cultures (Tables S2-S4). This material is available free of charge via the Internet at http:// pubs.acs.org.

Literature Cited (1) Atlas, R. M. Microbial degradation of petroleum hydrocarbons: an environmental perspective. Microbiol. Rev. 1981, 45 (1), 180– 209. (2) Bamforth, S. M.; Singleton, I. Bioremediation of polycyclic aromatic hydrocarbons: current knowledge and future directions. J. Chem. Technol. Biotechnol. 2005, 80, 723–736. (3) Cohen, M.; Yamasaki, H.; Mazzola, M. Bioremediation of soils by plant-microbe systems. Int. J. Green Energy 2004, 1 (3), 301– 312. (4) Vidali, M. Bioremediation. Pure Appl. Chem. 2001, 73 (7), 1163– 1172. (5) Schmidt, T. C.; Zwank, L.; Elsner, M.; Berg, M.; Meckenstock, R. U.; Haderlein, S. B. Compound-specific stable isotope analysis of organic contaminants in natural environments: a critical review of the state of the art, prospects, and future challenges. Anal. Bioanal. Chem. 2004, 378, 283–300. (6) Slater, G. F. Stable isotope forensics: when isotopes work. Environ. Forensics 2003, 4, 13–23. (7) Boschker, H.; Nold, S.; Wellsbury, P.; Bos, D.; de Graaf, W.; Pel, R.; Parkes, R.; Cappenberg, T. Direct linking of microbial populations to specific biogeochemical processes by 13Clabelling of biomarkers. Nature 1998, 392, 801–805. (8) Slater, G. F.; Nelson, R. K.; Kile, B. M.; Reddy, C. M. Intrinsic bacterial biodegradation of petroleum contamination demonstrated in situ using natural abundance, molecular-level 14C analysis. Org. Geochem. 2006, 37, 981–989. (9) Slater, G. F.; White, H. K.; Eglinton, T. I.; Reddy, C. M. Determination of microbial carbon sources in petroleum contaminated sediments using molecular 14C analysis. Environ. Sci. Technol. 2005, 39 (8), 2552–2558. (10) Fang, J.; Lovanh, N.; Alvarez, P. J. The use of isotopic and lipid analysis techniques linking toluene degradation to specific microorganisms: applications and limitations. Water Res. 2004, 38 (10), 2529–2536. (11) Hanson, J. R.; Macalady, J. L.; Harris, D.; Scow, K. M. Linking toluene degradation with specific microbial populations in soil. Appl. Environ. Microb. 1999, 65 (12), 5403–5408. (12) Kindler, R.; Miltner, A.; Thullner, M.; Richnow, H.; Kaestner, M. Fate of bacterial biomass derived fatty acids in soil and their contribution to soil organic matter. Org. Geochem. 2009, 40 (1), 29–37. (13) Moore-Kucera, J.; Dick, R. P. Application of C-13-labeled litter and root materials for in situ decomposition studies using phospholipid fatty acids. Soil Biol. Biochem. 2008, 40 (10), 2485– 2493. (14) Petsch, S. T.; Eglington, T. I.; Edwards, K. J. 14C-dead living biomass: evidence for microbial assimilation of ancient organic carbon during shale weathering. Science 2001, 292 (5519), 1127– 1131. (15) Rubino, M.; Lubritto, C.; D’Onofrio, A.; Terrasi, F.; Kramer, C.; Gleixner, G.; Cotrufo, M. F. Isotopic evidences for microbiologically mediated and direct C input to soil compounds from three different leaf litters during their decomposition. Environ. Chem. Lett. 2009, 7 (1), 85–95. (16) Faure, G. Principles of Isotope Geology; Wiley: New York, 1986. (17) Wakeham, S. G.; McNichol, A. P.; Kostka, J. E.; Pease, T. K. Natural-abundance radiocarbon as a tracer of assimilation of petroleum carbon by bacteria in salt marsh sediments. Geochim. Cosmochim. Acta 2006, 70, 1761–1771. (18) Mollenhauer, G.; Rethemeyer, J. Compound-specific radiocarbon analysis - analytical challenges and applications. IOP Conf. Ser.: Earth Environ. Sci. 2009, 5, 012006. (19) Ingalls, A. E.; Shah, S. R.; Hansman, R. L.; Aluwihare, L. I.; Santos, G. M.; Druffel, E. R. M.; Pearson, A. Quantifying archaeal community autotrophy in the mesopelagic ocean using natural radiocarbon. Proc. Natl. Acad. Sci. 2006, 103, 6442–6447.

(20) Turnbull, J.; Lehman, S.; Miller, J.; Sparks, R.; Southon, J. R.; Tans, P. A new high precision 14CO2 time series for North American continental air. J. Geophys. Res 2007, 112, D11310. (21) Trumbore, S. Radiocarbon and Soil Dynamics. Annu. Rev. Earth Planet. Sci. 2009, 37, 47–66. (22) Pilon-Smits, E. Phytoremediation. Annu. Rev. Plant Biol. 2005, 56, 15–39. (23) Greenberg, B. M.; Huang, X. D.; Dixon, D.; Glick, B. R. An integrated multi-process phytoremediation system for removal of persistent contaminants from soils. In Proceedings of the Remediation Technologies Symposium, Environmental Services Association of Alberta, 2004. http://www.esaa-events.com/ remtech/2004/pdf/16Greenberg.pdf. (24) Huang, X. D.; El-Alawi, Y.; Gurska, J.; Glick, B. R.; Greenberg, B. M. A multi-process phytoremediation system for removal of persistent total petroleum hydrocarbons (TPHs) from soils. Microchem. J. 2005, 81, 139–147. (25) Huang, X. D.; El-Alawi, Y.; Penrose, D. M.; Glick, B. R.; Greenberg, B. M. Responses of three grass species to creosote during phytoremediation. Environ. Pollut. 2004, 130 (3), 453–463. (26) Huang, X. D.; El-Alawi, Y.; Penrose, D. M.; Glick, B. R.; Greenberg, B. M. A multi-process phytoremediation system for removal of polycyclic aromatic hydrocarbons from contaminated soils. Environ. Pollut. 2004, 130 (3), 465–476. (27) Gurska, J.; Wang, W.; Gernhardt, K. E.; Khalid, A. M.; Isherwood, D. M.; Huang, X. D.; Glick, B. R.; Greenberg, B. M. Three year field test of a plant growth promoting rhizobacteria enhanced remediation system at a land farm for treatment of hydrocarbon waste. Environ. Sci. Technol. 2009, 43, 4472–4479. (28) Wang, W. Toxicity Assessment of PAHs and Metals to Bacteria and the Roles of Soil Bacteria in Phytoremediation of Petroleum Hydrocarbons. Ph.D. Dissertation, University of Waterloo, Waterloo, Ontario, Canada, 2008. (29) Gao, Y.; Zhu, L. Phytoremediation for phenanthrene and pyrene contaminated soils. J. Environ. Sci.-China 2005, 17 (1), 14–18. (30) Cappello, S.; Caruso, G.; Zampino, D.; Monticelli, L. S.; Maimone, G.; Denaro, R.; Tripodo, B.; Troussellier, M.; Yakimov, M.; Giuliano, L. Microbial community dynamics during assays of harbour oil spill bioremediation: a microscale simulation study. J. Appl. Microbiol. 2007, 102 (1), 184–194. (31) Stuiver, M.; Polach, H. A. Discussion: Reporting of 14C Data. Radiocarbon 1977, 19 (3), 355–363. (32) Pearson, A.; McNichol, A. P.; Schneider, R. J.; von Reden, K. F. Microscale AMS 14C Measurement at NOSAMS. Radiocarbon 1998, 40 (1), 6175.29. (33) White, D. C.; Davis, W.; Nickels, J.; King, J.; Bobbie, R. Determination of the sedimentary microbial biomass by extractible lipid phosphate. Oecologia 1979, 40, 51–62. (34) Nichols, P. D.; Guckert, J. B.; White, D. C. Determination of monounsaturated fatty acid double-bond position and geometry for microbial monocultures and complex consortia by capillary GC-MS of their dimethyl disulphide adducts. J. Microbiol. Meth. 1986, 5, 49–55. (35) Eglinton, T. I.; Aluwihare, L. I.; Bauer, J. E.; Druffel, E. R. M.; McNichol, A. P. Gas chromatographic isolation of individual compounds from complex matrices for radiocarbon dating. Anal. Chem. 1996, 68, 904–912. (36) Rethemeyer, J.; Grootes, P. M.; Bruhn, F.; Andersen, N.; Nadeau, M. J.; Kramer, C.; Gleixner, G. Age heterogeneity of soil organic matter. Nucl. Instrum. Meth. B 2004, 223, 521–527 .

(37) Rethemeyer, J.; Kramer, C.; Gleixner, G.; Wiesenberg, G. L. B.; Schwark, L.; Andersen, N.; Nadeau, M. J.; Grootes, P. M. Complexity of soil organic matter: AMS C-14 analysis of soil lipid fractions and individual compounds. Radiocarbon 2004, 46, 465–473. (38) Zelles, L. Fatty acid patterns of phospholipids and lipopolysaccharides in the characterisation of microbial communities in soil: a review. Biol. Fertil. Soils 1999, 29, 111–129. (39) Green, C. T.; Scow, K. M. Analysis of phospholipid fatty acids (PLFA) to characterize microbial communities in aquifers. Hydrogeol. J. 2000, 8, 126–141. (40) Kaur, A.; Chaudhary, A.; Choudhary, R.; Kaushik, R. Phospholipid fatty acid - A bioindicator of environment monitoring and assessment in soil ecosystem. Curr. Sci. 2005, 89 (7), 1103– 1112. (41) Ratledge, C.; Wilkinson, S. G. Microbial Lipids; Academic Press Limited: San Diego, CA, 1988; Vol. 1. (42) Hayes, J. M. , Fractionation of the Isotopes of Carbon and Hydrogen in Biosynthetic Processes. In Stable Isotope Geochemistry - Mineral Society of America; Valley, J., Cole, D., Eds.; Washington, D.C., 2001; Vol. 43. (43) Aelion, C. M.; Kirtland, B. C.; Stone, P. A. Radiocarbon assessment of aerobic petroleum bioremediation in the vadose zone and groundwater at an AS/SVE site. Environ. Sci. Technol. 1997, 31, 3363–3370. (44) Kirtland, B. C.; Aelion, C. M.; Stone, P. A. Assessing in situ mineralization of recalcitrant organic compounds in vadose zone sediments using δ13C and ∆14C measurements. J. Contam. Hydrol 2005, 76 (1-2), 1–18. (45) Cerling, T. E.; Solomon, D. K.; Quade, J.; Bowman, J. R. On the isotopic composition of carbon in soil carbon-dioxide. Geochim. Cosmochim. Acta 1991, 55 (11), 3403–3405. (46) Davidson, G. R. The stable isotopic composition and measurement of carbon in soil CO2. Geochim. Cosmochim. Acta 1995, 59 (12), 2485–2489. (47) Kuzyakov, Y.; Shevtzova, E.; Pustovoytov, K. Carbonate recrystallization in soil revealed by 14C labeling: Experiment, model and significance for paleo-environmental reconstructions. Geoderma 2006, 131, 45–58. (48) Poulsen, T.; Christophersen, M.; Moldrup, P.; Kjeldsen, P. Modeling lateral gas transport in soil adjacent to old landfill. J. Environ. Eng.-ASCE 2001, 127 (2), 145153.39. (49) Blair, N.; Leu, A.; Mun ˜ oz, E.; Olsen, J.; Kwong, E.; Des Marais, D. Carbon isotopic fractionation in heterotrophic microbial metabolism. Appl. Environ. Microb. 1985, 50 (4), 996–1001. (50) Grogan, D. W.; Cronan, J. E. Cyclopropane ring formation in membrane lipids of bacteria. Microbiol. Mol. Biol. R. 1997, 61 (4), 429–441. (51) Sacks, G. L.; Brenna, J. T. High-precision position-specific isotope analysis of 13C/12C in leucine and methionine analogues. Anal. Chem. 2003, 75 (20), 5495–5503. (52) Jahnke, L. L.; Eder, W.; Huber, R.; Hope, J. M.; Hinrichs, K. U.; Hayes, J. M.; Des Marais, D. J.; Cady, S. L.; Summons, R. E. Signature lipids and stable carbon isotope analyses of Octopus Spring hyperthermophilic communities compared with those of Aquificales representatives. Appl. Environ. Microb. 2001, 67 (11), 5179–5189.

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