Determination of Microbial Carbon Sources in Petroleum

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Environ. Sci. Technol. 2005, 39, 2552-2558

Determination of Microbial Carbon Sources in Petroleum Contaminated Sediments Using Molecular 14C Analysis G R E G O R Y F . S L A T E R , * ,† HELEN K. WHITE,‡ TIMOTHY I. EGLINTON,‡ AND CHRISTOPHER M. REDDY‡ School of Geography and Geology, McMaster University, Hamilton, Ontario, Canada, L8S 4K1, and Department of Marine Chemistry and Geochemistry, Woods Hole Oceanographic Institution, Woods Hole, Massachusetts 02543

Understanding microbial carbon sources is fundamental to elucidating the role of microbial communities in carbon cycling and in the biodegradation of organic contaminants. Because the majority of anthropogenic contaminants are either directly or indirectly derived from fossil fuels that are devoid of 14C, radiocarbon can be used as a natural inverse tracer of contaminant carbon in the contemporary environment. Here, 14C analysis of individual microbial phospholipid fatty acids (PLFA) was used to characterize the carbon sources utilized by the active microbial community in salt marsh sediments contaminated by the Florida oil spill of 1969 in Wild Harbor, West Falmouth, MA. A specific goal was to determine whether this community is actively degrading petroleum residues that persist in these sediments. The ∆14C values of microbial PLFA in all sediment horizons (contaminated and noncontaminated) matched the ∆14C of the total sedimentary organic carbon after petroleum removal, indicating that no measurable metabolism of petroleum residues was occurring. This result agrees with ancillary data such as the δ13C content and distribution of PLFA, and the residual hydrocarbon composition determined by comprehensive two-dimensional gas chromatography (GC×GC) analysis. We hypothesize that microbes have chosen to respire the natural organic matter rather than the residual petroleum hydrocarbons because the former is more labile. Future efforts directed at determining indices of microbial degradation of petroleum hydrocarbons should consider competition with natural organic matter.

Introduction Elucidating the processes by which microbes utilize carbon in the environment is fundamental to our understanding of biogeochemical cycles both past and present. This information is also vital for elucidation of controls on the occurrence and extent of microbial degradation of organic contaminants in both natural and engineered systems. At many contaminated environmental sites, heterogeneity in matrix composi* Corresponding author phone: 905-525-9140, ext 26388; fax: 905546-0463; e-mail: [email protected]. † McMaster University. ‡ Woods Hole Oceanographic Institution. 2552

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tion, contaminant distribution, and geochemical parameters renders definitive demonstration of in situ microbial degradation of contaminants extremely difficult. Existing techniques for assessment of degradation are often limited because they are based on indirect parameters or carry uncertainty as to whether current or past microbial activity is responsible. Measurement of concentration changes in products or reactants are further complicated by concurrent degradative and nondegradative processes of mass loss (i.e., sorption). Although culturing of organisms can provide insights into the identity and metabolic capabilities of organisms at a site, differences in conditions between laboratory cultures and environmental systems introduce large uncertainties concerning the relevance of this information to the field. Addition of tracers can identify in situ processes occurring at a site but often require long times for resolvable signal and in some cases have been shown to behave differently to native contaminants. For example, Steinberg et al. (1) observed that 14C-labeled ethylene dibromide (EDB) added to EDB-contaminated soils was metabolized preferentially over the native contaminant. Recent work using compound-specific stable carbon isotopic analysis has allowed in situ measurements and identification of reactants and products of some reactions (2, 3), however, this approach can be limited by similarities in isotopic composition between contaminants and naturally occurring compounds. Approaches utilizing compound-specific 14C measurements have the potential to provide answers to such challenges, particularly those concerning the environmental processing of anthropogenic contaminants. Since the majority of anthropogenic contaminants are fossil fuel based and are 14C-free due to their geologic age, 14C analysis can thus be used as a naturally occurring inverse tracer of contaminant carbon in the environment. Microbial uptake of petroleum carbon will result in a reduction of the 14C content of microbial cellular components relative to those utilizing natural organic matter since the latter generally contains modern levels of 14C. Microbial phospholipid fatty acids (PLFA) are integral components of cell membranes that are labile and hydrolyze within weeks after cell death (4, 5). Consequently, they are ideal for study of carbon sources of active microbial populations. The isotopic composition of in situ microbial PLFA can thus reveal a near-instantaneous snapshot of the carbon source of the active microbial community at a site. Petsch et al. (6) first used this approach to show that a laboratory enrichment culture of aerobic microbes was capable of respiring the 14C-free kerogen in organic-rich sedimentary rocks. In the present study, we have taken this approach out of the laboratory and applied it directly to environmental samples. We exploit the large disparity in the 14C content of petroleum (14C-free) and of natural organic matter in salt marsh sediments (modern or near-modern 14C content) to constrain the carbon source of microbes currently active at a petroleum-contaminated site. Specific goals of this work were to establish compoundspecific 14C analysis as a tool to determine carbon sources of microbial communities and to apply this approach to examine whether the active microbial community in salt marsh sediments from Wild Harbor, MA, are actively respiring the petroleum contaminants that have persisted at depth since the Florida spill of 1969. Studies have shown the presence of oil residues in these sediments seven years after the spill (7) and at some sites 20 (8) and 30 (9) years later, demonstrating that petroleum hydrocarbons can, in certain cases, persist in the environment for many years. Recent work by Reddy et al. (9) and Frysinger et al. (10) also provided 10.1021/es048669j CCC: $30.25

 2005 American Chemical Society Published on Web 03/05/2005

a refined inventory of existing petroleum hydrocarbons in Wild Harbor based on analysis of hydrocarbon residues by comprehensive two-dimensional gas chromatography (GC × GC). This novel technology revealed that only the n-alkanes were completely degraded and that all other compound classes typical of No. 2 fuel oil remained. Reddy et al. (9) also hypothesized that degradation of branched alkanes, such as pristane and phytane, ceased in the mid 1970s. These results are noteworthy as studies have shown that microbes are capable of degrading petroleum hydrocarbons such as n-alkanes (11-13), naphthalene, methyl and ethyl naphthalenes and other polycyclic aromatic hydrocarbons (1318), acyclic hydrocarbons (ethylcyclopentane) (19), and alkylbenzenes (20), under the anaerobic conditions that have likely existed in these sediments for the last three decades. This disparity between the aforementioned studies and the apparent recalcitrance of petroleum hydrocarbons in Wild Harbor sediments is difficult to reconcile. One possibility is that microbial metabolism of the petroleum hydrocarbons is occurring but at rates too slow to be resolved by concentration-based approaches such as those used by Reddy et al. (9). Alternatively, metabolism of these petroleum hydrocarbons may not be occurring at all at this site. To address this question, a marsh sediment core was sectioned into discrete depth intervals and solvent-extracted. Microbial phospholipids were isolated by silica gel chromatography and high-performance liquid chromatography (HPLC) and then converted to their respective fatty acid methyl esters (FAMEs). Individual or groups of PLFA were isolated by preparative capillary gas chromatography (PCGC) and then analyzed for 14C by accelerator mass spectrometry (AMS). Compound-specific 14C analysis of PLFA was used to assess whether microbial metabolism of petroleum based contaminants was occurring.

Methods Sampling. In November 2001, a 30-cm × 30-cm box core was obtained from the intertidal marsh sediment at station M-1 in Wild Harbor, West Falmouth, MA (9, 21). The core was returned immediately to the laboratory and sectioned at 2-cm intervals from 0 to 20 cm. The outer 1 cm of each section was discarded to avoid cross contamination. Each sediment horizon (approximately 500 g of wet sediment) was placed in precombusted glass jars with Teflon-lined caps and frozen (-40 °C) until analysis. Extraction. Each horizon was spiked with R-cholestane and extracted three times with a modified Bligh and Dyer solution (22) using a 2:1 mixture of methanol/chloroform. Because Laarkamp (23) showed that the efficiency of PLFA extraction was not dependent on using a phosphate buffer, none was used. The resulting total lipid extracts (TLEs) were combined and back-extracted with MilliQ water to remove any remaining water-soluble components. An aliquot of each of the TLEs was derivatized with bis-(trimethylsilyl)-trifluoroacetamide (BSTFA) and pyridine to improve chromatography by silylating active hydrogens present in the samples. These derivatives were analyzed by gas chromatography with flame ionization detection (GC-FID) to determine the concentration and distribution of total petroleum hydrocarbons (TPHs) present as an unresolved complex mixture (UCM) similar to the approach of Reddy et al. (9). The majority of each TLE was charged onto 60 g of fully activated silica gel (45-350 mesh) and eluted with 100 mL of chloroform (f1), 100 mL of acetone (f2), and 300 mL of methanol (f3). The polar f3 fraction was evaporated under N2 to a small volume and then separated by preparative HPLC on a LiChrospher DIOL 100 5µm-column to further purify the phospholipids from other polar organic components in these samples, which can otherwise interfere with the determination of PLFA distribution (24, 25). Once the PLFA had been purified, they

were subjected to mild alkaline methanolysis (22) to yield the corresponding fatty acid methyl esters (FAMEs). The methanol used for methanolysis was characterized for its 13C and 14C content before the reaction so that corrections for the isotopic composition of the methanol-derived methyl groups to the FAMEs could be made. Quantification of TPH. TPH present as UCM in the TLE were quantified by methods similar to Reddy et al. (3). Analysis was carried out on an HP-5890 GC with a Gerstel CIS (PTV) injector and a Flame Ionization Detector (FID). The CIS program was initially held at 40 °C (0.3 min) then ramped to 350 °C at 12 °C s-1. The column used was a CP Sil 5 CB (60 m; 0.25-mm i.d.; 0.25-µm film) the oven temperature was initially held at 40 °C (1 min), then ramped at 30 °C min-1 to 120 °C and then ramped to 320 °C at 6 °C min-1. Identification and Quantification of Microbial PLFA. Microbial PLFA were identified and quantified on an Agilent 6890 gas chromatograph equipped with a J&W DB-XLB column (60 m; 0.25-mm i.d.; 0.25-µm film) and coupled to an Agilent 5973 MSD operating under electron ionization (EI, 70 eV) in the full scan mode. The oven temperature was initially held at 40 °C (1 min.), then ramped at 20 °C min-1 to 130 °C, at 4 °C min-1 to 160 °C, and finally at 8 °C min-1 to 300 °C. Identification was based on mass fragmentation patterns and retention time as compared with a bacterial reference standard (Bacterial Acid Methyl Esters CP Mix, Matreya Inc.). Compounds not present in this standard mixture were tentatively identified based on mass fragmentation patterns and relative retention times. FAMEs generated from PLFA are designated according to Cn:m, where n is the number of carbon atoms in the chain and m is the number of double bonds. Double bond positions were not determined definitively. Methyl group position is indicated by i ) iso for pentultimate methyl groups, a ) anteiso for ante-penultimate methyl groups, or xMeCn:m where x ) methyl group position. Cyclopropyl PLFA are indicated by cycCn:m. The cyclohexyl PLFA observed is designated by ωC11cyclohexyl. Compound-Specific Stable Carbon Isotopic Analysis. Compound-specific stable carbon isotope analysis of the PLFA was performed in triplicate on a gas chromatographyisotope ratio mass spectrometry (GC-IRMS) system consisting of a Hewlett-Packard 6890 gas chromatograph interfaced to a Finnigan-MAT DeltaPlus via a GC Combustion III interface. The GC was equipped with a CP-Sil 5CB column (50 m; 0.25-mm i.d.; 0.25-µm film). Samples were injected into a Gerstel CIS (PTV) injector initially at 10 °C (0.25 min hold) followed by a ramp at 12 °C s-1 to 350 °C (3 min hold); GC temperature program was initially held at 60 °C (3 min), then ramped at 20 °C min-1 to 130 °C, at 3 °C min-1 up to 260 °C, and finally at 10 °C min-1 up to 340 °C and held for 2 min. Compound-Specific Radiocarbon Analysis. Individual or groups of PLFA were isolated by PCGC (see Eglinton et al. (26) for a more detailed discussion on this method). Briefly, concentrated solutions of PLFA were repeatedly injected ∼100 times onto a Hewlett-Packard 5890 gas chromatograph coupled to a Gerstel preparative fraction collector. The PLFA were separated on a J&W DB-XLB column (60 m; 0.53-mm i.d.; 0.5-µm film) with hydrogen carrier gas and an oven temperature program starting at 50 °C (1 min hold) then ramped at 10 °C min-1 to 150 °C, then ramped at 1.5 °C min-1 to 180 °C and held for 10 min, and finally ramped at 10 °C min-1 to 320 °C. Individual PLFA were trapped in cooled (0 °C), glass u-tubes. Purified individual or groups of compounds were recovered by dissolution in DCM and transferred to precombusted quartz tubes (12 mm × 20 cm). The solvent was evaporated under a stream of nitrogen and then ∼100 mg of copper oxide was added to each tube. The tubes were evacuated on a vacuum line, sealed, and combusted at 850° C for 5 h, yielding carbon dioxide, water, VOL. 39, NO. 8, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 1. Distributions of PLFA Above, Within, and Below the Contaminated Zone Expressed as Mole Percentagesa depth (cm)

FIGURE 1. Profile of TPH concentrations (solid line) versus depth observed in this study and the relative total PLFA concentration (dashed line) normalized to the upper sample.

and other combustion gases. The tubes were reattached to the vacuum line and the carbon dioxide was isolated and purified through a series of cold traps and quantified by manometry. About 10% of the carbon dioxide was reserved for δ13C analysis by isotope ratio mass spectrometry. The remaining 90% was reduced to graphite (27). Targets of the graphite were pressed and mounted on target wheels for 14C analysis by AMS at the National Ocean Sciences Accelerator Mass Spectrometry (NOSAMS) facility at Woods Hole Oceanographic Institution, Woods Hole, MA (27). In this study, all 14C measurements are normalized to δ13C values of -25‰ and expressed as ∆14C values. The latter term is the per mille (‰) deviation from the international standard for 14C dating, Standard Reference Material 4990B “Oxalic Acid” (24). In this context, petroleum has a ∆14C of -1000‰ while recently photosynthesized materials are more enriched (0 to 200‰) depending on when and where the organism grew.

Results Concentration of TPH and Microbial PLFA. The concentration of TPH present as an UCM determined for this core is shown in Figure 1. The peak concentration of 4.5 mg g-1 (dry weight) occurred at 14-16 cm depth. This is consistent with Reddy et al. (9) and Peacock et al. (28) who have observed variation in TPH concentration between ∼4 and ∼8 mg g-1 at and around the M-1 site. Such variability is not unexpected in a highly heterogeneous system such as a salt marsh. Figure 1 also shows the relative concentration of PLFA, normalized to the 0-2 horizon, to illustrate the overall trend of decreasing viable cell density over the depth of the core. There is nearly an order of magnitude decrease in PLFA content over the upper 4 cm of the core. The total PLFA concentration in the uppermost sample was 1.9 × 104 ng g-1 (dry weight), decreased to 8.1 × 103 ng g-1 at 4-6 cm, and further decreased to 2.1 × 103 ng g-1 through the contaminated zone (12-16 cm). Using generic conversion factors of 2 × 104 cells pmol-1 of PLFA and 6 × 104 cells pmol-1 (29) and converting PLFA concentrations to pmoles, this corresponds to cell densities of 1.4-4.0 × 109 cells g-1 in the uppermost sediments decreasing to 1.5-4.5 × 108 cells g-1 in the deeper sediments. These estimates are consistent with cell densities that might be expected for an active sedimentary microbial community (30). There is no resolvable variation in cell density associated with the contaminated zone. PLFA Distribution. Table 1 shows the distribution, as mole percentage, of microbial PLFA in these sediments for 2554

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FAME

0-2

2-4

6-8

10-12

14-16

18-20

isoC14:0 C14:0 isoC15:0 anteisoC15:0 C15:0 isoC16:0 C16:1 C16:1 C16:0 10 MeC16:0 isoC17:0 anetisoC17:0 C17:1 CycC17:0 C17:1 C17:0 phytanate C18:2 C18:1 C18:1 ωC11 cyclohexyl C18:0 CycC19:0

1.52 2.20 3.46 7.84 0.81 1.22 16.14 2.24 17.6 2.07 0.68 0.75 0.60 1.56 0.21 0.63 0.74 4.62 3.63 28.6 nd 1.66 1.17

3.40 3.47 7.30 20.7 1.30 4.22 6.07 1.43 18.1 3.49 1.35 2.30 0.54 1.58 1.10 0.99 0.92 3.48 3.27 6.57 5.27 2.05 1.08

2.50 2.88 5.51 19.7 1.07 3.62 5.12 1.33 21.0 2.45 0.95 2.10 0.79 1.06 0.79 0.96 0.99 5.60 4.42 9.86 4.39 2.04 0.93

3.87 4.73 8.45 25.0 1.27 6.38 2.57 1.06 16.6 2.26 1.07 3.67 na 0.90 1.65 0.56 1.18 1.79 1.45 3.63 9.71 1.47 0.71

2.44 2.60 6.68 18.3 1.03 6.02 3.27 1.33 19.8 2.65 1.37 3.00 0.48 1.08 1.62 0.66 1.41 5.90 5.10 5.31 7.81 1.89 0.23

3.41 4.96 7.34 26.4 1.18 5.53 2.68 1.04 16.6 2.88 1.04 4.03 0.19 0.81 1.28 0.63 1.23 0.91 1.24 3.83 10.4 1.50 0.82

a

Values in bold indicate FAMEs analyzed for 14C. na ) not available.

representative depths above (0-2, 2-4, and 6-8 cm), within (10-12 and 14-16 cm), and below (18-20 cm) the contaminated zone. Mole percentage is used as a more reliable descriptor of microbial community distribution as it is not affected by variations in recovery and response that might occur between samples. The distribution of PLFA observed in sediments from the M-1 site is consistent with that observed by Boschker et al (31) for PLFA recovered from surficial salt marsh sediments in the United States and The Netherlands. There is no significant observable relationship between PLFA distribution and TPH concentration, indicating that the presence of TPH is not resulting in a resolvable effect on the microbial community. Such an effect has been observed in other research (32-35). The most significant trends in microbial PLFA distribution observed in this core are a rapid decrease in the relative abundance of unsaturated C16 and C18 PLFA between the O-2 cm and 2-4 cm sections, from 18.3 to 7.5% and from 36.9 to 13.3%, respectively. Concurrent with this decrease is an increase in the relative abundance of iso and anteiso C15:0 PLFA from 3.5 to 7% and from 7.8 to 20.7%, respectively. This change in PLFA distribution between the shallower and deeper sediment intervals is consistent with a transition to anaerobic conditions (36). The iso and anteiso C15:0 PLFA are produced in large proportions by sulfate reducing bacteria (37-39) that are generally abundant in salt marsh sediments (40) and capable of degrading petroleum hydrocarbons (11, 17, 18, 41). One interesting observation is the increasing relative abundance of a fatty acid tentatively identified as a ωC11cyclohexyl fatty acid. This compound is not present in the upper sample, but increases from 5.3% in the 2-4 cm sample to 10.4% in the deepest sample. This fatty acid, which has been ruled out as a sampling artifact based on blank and duplicate analyses, is known to occur in hotspring bacteria (42-44), however, the reasons for its presence at this site is unclear and the subject of continuing investigation. In the present study, this compound was included in radiocarbon analyses of the C18 unsaturated PLFA due to their partial coelution during PCGC. The ubiquitous C16:0 is consistently a large proportion of the total PLFA at 16.6-21.0% throughout the core.

FIGURE 2. ∆14C PLFA compared to the in situ ∆14C of EXT-RES and TOC from the core of White et al. (21) (not corrected for decay since deposition) versus depth at the site. Error bars on PLFA represent 20‰ accuracy and reproducibility. Error bars on TOC represent 10‰ accuracy and reproducibility. Compound-Specific Radiocarbon Measurements. PLFA chosen for 14C analysis were those that made up a majority of the microbial PLFA and were thus present in sufficient quantity for collection and analysis. Three sets of PLFA were collected over the complete depth profile; C16:0, combined iso and anteiso C15:0, and combined C18 unsaturated (including the FAME tentatively identified as ωC11cyclohexyl). These three samples represented between 65 and >70% of the total PLFA at each depth. Iso and anteiso C15:0 were chosen as markers of sulfate reducing bacteria. Unsaturated C18 PLFA and ωC11cyclohexyl PLFA are associated with bacteria, and ωC11cyclohexyl PLFA have been observed in sulfate reducing bacteria (42-44). C16:0 is a ubiquitous fatty acid is representative of the overall community 14C composition. In the upper sections, sufficient mass was present to collect a combined cycC17:0 and cycC19:0 PLFA that are indicative of microaerophillic gram negative bacteria (45). C14:0 and iso C16:0 are bacterial PLFA whose mole percentage increased with depth, allowing these PLFA to be collected deeper in the profile. Radiocarbon analysis of microbial PLFA was performed on the 0-2, 2-4, and 4-6 cm horizons, below which decreasing PLFA concentrations necessitated combining of sections and analysis was only possible for the 8-12, 12-16, and 16-20 cm intervals. The PLFA collected for 14C analysis represented >75% of the PLFA mass for all samples and >80% of all but one of the samples. The results from compound-specific radiocarbon analysis of the microbial PLFA are compared with the in situ (uncorrected, see below) ∆14C of the total organic carbon (TOC) and solvent extracted sediment residues (EXT-RES) from White et al. (21) in Figure 2. EXT-RES reflects the 14C content of the residual organic carbon remaining in the sediments after removal of petroleum hydrocarbons since White et al. (21) determined that the TPH were completely removed during solvent extraction. We therefore interpret EXT-RES ∆14C values as indicative of the naturally occurring organic matter at this site. In contrast, TOC values represent the ∆14C of both the natural organic carbon and petroleum hydrocarbons. It should be noted that data of White et al. (21) are from a core from the same site taken 1.25 years before the present study, however the cores were from the same site and the depth distribution of TPH from the two cores was consistent. Further, the 14C data published in White et al. (21) were corrected for radioactive decay since deposition using the 210Pb -210 ages determined for the sediments; no correction was applied to the 14C data reported here as the goal is a comparison of in situ ∆14C contents. Both the TOC and EXT-RES show an increasing trend of ∆14C with depth, increasing to a peak dated at ∼1967 (21) and then a subsequent decrease in ∆14C. This trend is a record

FIGURE 3. δ13C PLFA as compared to the δ13C of EXT-RES and TOC from the core of White et al. (21) versus depth at this site. Error bars represent 0.5‰ accuracy and reproducibility. of the changes in the 14C inventory of atmospheric CO2 associated with above-ground thermonuclear weapons testing in the 1950s and 1960s, the so-called “bomb spike” (21). In the contaminated zone, there is an offset between the extracted and unextracted samples due to the contribution of the radiocarbon-free TPH to the organic carbon in the latter samples (21). If the microbial community is utilizing the petroleum as a carbon source (∆14C ) -1000 ‰), a depletion in 14C should be observed for the microbial PLFA. ∆14C values for the microbial PLFA are similar for all samples at each depth interval, indicating a consistent carbon source for the members of the microbial community. Moreover, PLFA ∆14C values parallel the trend in ∆14C of the EXT-RES (i.e., that reflects naturally occurring organic matter), including the increase and subsequent decrease in ∆14C content associated with the bomb spike. In the upper samples, within the precision of this analysis, the ∆14C PLFA (( 20 ‰) and the EXT-RES (( 10 ‰) of all samples are the same. Within the contaminated zone (8-12 and 12-16 cm), the ∆14C values of the PLFA also agree well with the EXTRES. In the 12-16 cm and 16-20 cm intervals there is a slight offset to more positive PLFA ∆14C values than those of EXT-RES. This may be due in part to the integration of signal over a 4-cm interval that may incorporate a greater proportion of bomb-derived 14C, or it may be due to slight differences between the two cores. Most importantly, however, both of these contaminated zone samples show no evidence of incorporation of petroleum carbon that would otherwise result in a decrease in 14C content. Instead, the similarity between PLFA and EXT-RES ∆14C values implies that the microbial community is using natural organic matter as a carbon source. Stable Carbon Isotopes. Figure 3 shows δ13C values of representative PLFA compared with the δ13C values of the EXT-RES and TOC from White et al (21). As for the 14C data, TOC δ13C values show the impact of petroleum carbon on isotopic values within the contaminated zone. The EXT-RES δ13C values are consistent with those observed by other researchers in salt marsh systems and trend to a value of ∼-18 ‰, characteristic of the recalcitrant components of Spartina cordgrass, the dominant macrophyte in these systems (46). The δ13C values of the C14:0, i-C15:0, a-C15:0, and C16:0 PLFA are shown versus depth in Figure 3. These PLFA were chosen as they are well resolved during GC-IRMS analysis and include bacterial indicators such as C14:0, iso and anteiso C15: 14 13 0, as well as the ubiquitous C16:0. As for ∆ C, the δ C values of all of these PLFA are highly coherent for any given section. The PLFA δ13C values are, in general 4-6 ‰ offset from δ13C values of the EXT-RES (naturally occurring organic carbon), consistent with the observations of Boschker et al. (31). There is some greater variation in δ13C values in the upper 4 cm VOL. 39, NO. 8, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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of the core, which may be related to a greater range of potential carbon sources including dissolved organic carbon from terrestrial and algal inputs (31, 46). The consistency of the δ13C values of these PLFA agrees with the 14C data and indicates a common carbon source for the microbial community.

Discussion What is the Microbial Carbon Source in Wild Harbor Sediments? All lines of evidence consistently indicate that microbial communities at the Wild Harbor site are not significantly metabolizing petroleum hydrocarbon residues in these sediments but instead are using the naturally occurring organic matter as a carbon source. The agreement between ∆14C values of the individual PLFA and the EXTRES (Figure 2) is compelling evidence that this is the case. To test the limits of this analytical approach, we estimated the extent of incorporation of fossil carbon that would be necessary to produce a resolvable shift in PLFA ∆14C values. This model employs the precision of isotopic measurements and the range in isotopic compositions of the end-members. The results of this model are plotted in Figure 4a, which shows that for the isotopic difference between fossil carbon (∆14C ≈ 1000 ‰) and a modern carbon source (∆14C ) 100 ‰) microbes would only need to incorporate 3% of petroleum-derived carbon into their PLFA to produce a resolvable signal. This resolution is significantly greater than that for δ13C (Figure 4b), which would require a 10% incorporation at this site assuming the isotopic difference between petroleum hydrocarbons and naturally occurring organic matter is ∼10‰. In the many terrestrial environments where the OC is predominantly derived from C3 vegetation with δ13C values (∼ -25 ‰) in the same range as petroleum compounds, δ13C would be unable to provide any resolution of microbial carbon sources. The use of δ13C values is further complicated by uncertain and potentially variable isotopic fractionations associated with biosynthetic production of PLFA from carbon substrates (31, 47, 48) (Figure 4b). Instead of showing any evidence for incorporation of fossil carbon, the ∆14C values of the PLFA and the solvent-extracted residues (EXT-RES) are in close agreement over the length of the core. In contrast, Petsch et al. (6) observed extremely depleted values of ∆14C PLFA (-990‰ to -770‰) for samples of a microbial enrichment culture growing on black shale organic matter as its sole carbon source (∆14C ) -1000‰). Since microbial utilization of more than 3% of fossil carbon would have resulted in a resolvable negative offset between the PLFA and EXT-RES, we conclude that metabolism of petroleum in Wild Harbor sediments is insignificant (Figure 2). The δ13C results are consistent with this interpretation. The high level of coherence in ∆14C and δ13C values of PLFA at a given depth indicates that not only is the microbial carbon source the local naturally occurring organic carbon, but also that the entire microbial community (to the extent that PLFA reflect community structure) at a given depth is using a similar carbon source. Since the PLFA ∆14C values closely track the natural OC ∆14C values across the bomb spike it suggests that the carbon source of the microbial community is the autochthonous natural organic matter. This is also supported by the 4-6‰ offset between the δ13C PLFA and the EXT-RES. The small positive ∆14C offset observed in the deeper samples could be argued to indicate the occurrence of some downward transport of “bomb” carbon. However, if this was a significant process over the entire core, then the overall agreement between isotopic compositions of the PLFA and EXT-RES would be less strong. It is more likely that this observation is due to the use of broader PLFA sampling intervals deeper in the core or to slight variations between the two cores. 2556

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FIGURE 4. Percentage incorporation of a contaminant carbon source of a given isotopic difference required to generate a resolvable isotopic signal that will change the isotopic composition outside the precision of the measurement. For 14C, an accuracy and reproducibility of 20‰ is used for small sample AMS analysis, and 10‰ for standard AMS analysis, resulting in a requirement of a difference of 30‰ for isotopic resolution. (a) The solid line shows that the required 30‰ isotopic difference in the ∆14C of the naturally occurring organic matter is achieved at 3% incorporation for the ∼1000‰ isotopic difference expected between petroleum based contaminants and modern organic matter. For 13C, an accuracy and reproducibility of 0.5‰ is used resulting in a required difference of 1‰ for isotopic resolution. (b) The solid line shows that the required isotopic difference occurs at 10% incorporation of a carbon source with an isotopic difference of 10‰, as might be expected in a salt marsh system. However, this resolution is reduced if variation in isotopic fractionation during biosynthesis is considered. A range of biosynthetic fractionation of 2 ‰ increases the required incorporation to 30% (dashed line). And for many systems the δ13C of naturally occurring organic matter and petroleum will be similar, making resolution impossible. What is the Form of the Microbial Carbon Source? It is likely that the microbial incorporation of carbon occurs via initial metabolism of organic compounds to either dissolved inorganic carbon or acetate (49, 50). Harvey et al (50) used isotopically labeled tracers to determine that the primary precursor for lipid synthesis by the microbial community in salt marsh sediments was DIC and argued that all carbon utilized is first metabolized to CO2 and then converted to biomass. Hines et al (49) have also shown acetate utilization in this type of sediments. In either case, the isotopic compositions of the DIC or acetate should reflect the organic matter from which it came, in this case the natural sedimentary organic matter. The observation of a systematic 4-6‰ offset between Spartina litter and microbial PLFA by

Boschker et al (31) and in this study implies that the net isotopic fractionation during uptake and utilization of this carbon source is 4-6‰. However, other researchers have observed greater variations in isotopic fractionation during synthesis of PLFA in pure cultures. In particular, Londry et al (51) cultured four species of sulfate reducing bacteria on acetate and lactate and observed a range of isotopic depletions between the δ13C of PLFA and their carbon sources of from 2.0 up to 37‰. The reason for the disparity between the findings of Londry et al (51) and those of Boschker et al (31) and the present study, both sampling an environment where significant sulfate reduction is expected, is unclear. However, it underlines the caution that is required in using δ13C to determine microbial carbon sources in the environment. In contrast, ∆14C analysis of microbial PLFA can provide more accurate and precise data on carbon sources because any effects of fractionation of 13C are removed in the calculation of ∆14C. This approach, however, also depends on the relative isotopic compositions of the potential carbon sources; thus for two modern carbon sources, 14C analysis will not provide source resolution. Reasons for Persistence of Petroleum Residues. The absence of fossil carbon in PLFA raises the question as to why the resident microbial community is not actively metabolizing the residual petroleum in Wild Harbor sediments. One explanation might lie in the availability and energetic favorability of degrading petroleum hydrocarbon residues versus the naturally occurring organic matter at the site. The petroleum residues, TPH, which make up a maximum of ∼9% of the TOC at the site (21), are less polar than most of the natural organic matter, rendering them more strongly sorbed to matrix material and potentially less bioavailable for the microbial community. The abundance of a more easily accessible carbon source may remove any pressure for microbes to utilize a niche carbon source such as petroleum contaminants. Future limitation of carbon supply at this site may therefore result in microbial metabolism of the petroleum hydrocarbons. This balance between abundance and bioavailability of naturally occurring organic matter and introduced petroleum hydrocarbons may be a critical determinant in the fate of these contaminants in the environment. These results imply that the presence of microbes capable of carrying out metabolism of a contaminant may not be sufficient to ensure that degradation takes place. Limitation or competition for carbon sources may be necessary for utilization of this less available carbon source. The presence of naturally occurring organic carbon may result in differences in the order and rates of degradation of components of a petroleum mixture as has been defined in laboratory studies (52, 53). In cases where there is more than one potential carbon source, the pattern of degradation of an oil spill may differ from situations where only petroleum carbon is present. Therefore, interpreting the extent of degradation of petroleum may require consideration of the other organic material present. It is also possible that the lack of degradation of the contaminants at this site is due to a limitation of electron acceptors, such as sulfate, or to reduction in microbial activity due to seasonal variations in temperature. However, the presence of PLFA indicative of sulfate reducers throughout the core, and the observation by others of the persistence of sulfate with depth in salt marsh systems (54), makes the first explanation unlikely. And while there is no information on the seasonal variation of the microbial population in salt marsh sediments, the relatively mild temperatures at the time of sampling, the observation of bacterial cell counts consistent with surficial sediments, and the observation of PLFA indicative of sulfate reducers make this explanation unlikely as well. Irrespective of the cause, the results described above imply that the naturally occurring organic carbon is the carbon

source for the active microbial community. Compoundspecific 14C analysis of microbial PLFA has unequivocally shown that no significant metabolism of the residual petroleum hydrocarbons is presently occurring in Wild Harbor salt marsh sediments. In the absence of future changes in the local geochemical conditions at the site, we speculate that hydrocarbon contamination at this site will not be subject to significant further degradation, leading to its persistence in the sediments indefinitely.

Acknowledgments We acknowledge D. Montlucon, L. Xu, B. Nelson, C. Johnson, and J. Hayes for their field, laboratory, and analytical assistance. This work was supported by funds from the National Science Foundation (CHE-0089172), an NSERC PDF award to G.F.S. and funding from the Postdoctoral Scholar Program at the Woods Hole Oceanographic Institution, the J. Seward Johnson Fund and from WHOI Coastal Ocean Institute. This is WHOI Contribution No. 11318.

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Received for review August 25, 2004. Revised manuscript received January 12, 2005. Accepted January 28, 2005. ES048669J