Low-Temperature Microbial Aerobic Degradation of Polychlorinated

William A. Williams*, and Ralph J. May. GE Research and .... Use in Biotechnology. David Allen , Adrienne Huston , Lloyd Wells , Jody Deming. 2003, ...
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Environ. Sci. Technol. 1997, 31, 3491-3496

Low-Temperature Microbial Aerobic Degradation of Polychlorinated Biphenyls in Sediment WILLIAM A. WILLIAMS* AND RALPH J. MAY GE Research and Development Center, P.O. Box 8, Schenectady, New York 12301-0008

To test for low-temperature microbial aerobic PCB degradation, water-saturated samples of PCB-contaminated upper Hudson River sediment spiked with Aroclor 1242 were incubated at 4 °C for several months. Principal components analysis of the PCBs, quantified by GCECD and GC-MS, indicated the loss of specific congeners within the top 15 mm of sediment. In contrast, no loss of PCBs was observed in the deeper region (>15 mm from the surface) of the sediment or in any region of similarly treated sediment samples that were autoclaved prior to Aroclor 1242 addition. Loss of specific di- and trichlorobiphenyls typical of microbial aerobic degradation was first indicated at 1.4 months, and >50% loss had occurred by 5 months. Initially, the sediment was dark brown. However, by 2 months a lighter, rust-colored layer developed at the top 6 mm that increased to ∼10 mm by 5 months and to ∼15 mm by 10 months. The color change, due at least in part to formation of hydroxylamine-reducible ferric oxide, indicated the depth to which oxygen was available and the depth to which PCB degradation occurred. In addition, chlorobenzoic acid metabolites of microbial aerobic PCB degradation were detected in the rust-colored layer. These observations demonstrate that low-temperature microbial aerobic PCB degradation can occur in PCB-contaminated sediment.

Introduction Polychlorinated biphenyls (PCBs) are a group of compounds that are banned from large-scale production in the United States. These compounds were manufactured as mixtures based on the extent of biphenyl chlorination; the mixtures were given trade names such as Aroclor. Because of their widespread use and recalcitrant nature, PCBs have accumulated in soils, sediments, and biota. Aerobic PCB degradation by bacteria in PCB-contaminated upper Hudson River sediment is well characterized. The PCBdegrading bacterium Alcaligenes eutrophus H850 was isolated by Bedard and co-workers (1-3) from PCB-contaminated dredge spoils of the upper Hudson River. This bacterium has a particularly broad PCB congener specificity as compared to many other PCB-degrading bacterial isolates from the upper Hudson River and other sites (1-4). In addition to PCB degradation by bacterial isolates, in situ microbial aerobic PCB degradation was demonstrated at a PCB-contaminated site in the upper Hudson River (5). Fish and Principe (6, 7) also described microbial aerobic PCB degradation and * Corresponding author present address: Department of Biology, SUNY at Albany, 1400 Washington Avenue, Albany, NY 12222; e-mail address: [email protected].

S0013-936X(97)00241-1 CCC: $14.00

 1997 American Chemical Society

anaerobic dechlorination of Aroclor 1242 in test tube microcosms of PCB-contaminated upper Hudson River sediment. Each of the studies mentioned above focused on microbial aerobic PCB degradation in a mesophilic temperature range of 18-35 °C. However, many PCB-contaminated soils and sediments are located at global regions where the temperature is below 15 °C for several months of the year. Microbial life at low temperatures (i.e., psychrotrophic and psychrophilic microorganisms) has been studied for several decades (8). While it is known that some xenobiotic compounds such as toluene (9, 10), naphthalene (9), phenol (11), and chlorophenols (8) undergo low-temperature microbial degradation, there are no reports of similar degradation of PCBs. This paper describes an experiment where specific di- and trichlorobiphenyls were depleted in a surface layer of PCBcontaminated upper Hudson River sediment incubated at 4 °C. The loss of specific PCB congeners from the sediment is indicative of microbial aerobic PCB degradation and demonstrates that PCB degradation can occur in sediment samples at low temperatures.

Experimental Section Sediment Collection Site. Hudson River sediment was collected at river mile 188.8 just upstream of the Thompson Island Dam and stored at 4 °C. The sediment contained dechlorinated Aroclor 1242 (∼10 µg/g dry sediment); approximately 40% of the PCBs were a mixture of 2-chlorobiphenyl (2-CB); 2-2-; 2,6-; and 2,6-2-CB based on our analysis. Sediment and Sample Preparation. Wet sediment was passed through a 2-mm sieve and split into two portions. One portion was autoclaved at 120 °C for 3.5 h, and the other was untreated (i.e., the live sediment sample). Distilled water (one-tenth volume of wet sediment) was mixed with each portion of sediment using an overhead impeller; the suspension was kept in an ice water bath. During a 2-h period, a methanol solution containing Aroclor 1242 and 2,4,6-2,4,6hexachlorobiphenyl (2,4,6-2,4,6-CB) was added to the suspension (while mixing) using a syringe pump. After PCB addition, the suspension was mixed for 30 min before sample preparation. The concentrations of Aroclor 1242 and 2,4,62,4,6-CB in each suspension were ∼100 and ∼3 ppm by the volume of wet sediment, respectively (70 and 2.2 ppm by dry weight basis). The concentration of methanol was 0.1% of the total volume of the suspension. The samples of sediment with the added PCBs were prepared in 25 mm × 200 mm glass test tubes. During preparation, the samples were set in an ice water bath. While the sediment was stirring, ∼14-mL aliquots were removed and added to the test tubes, filling the bottom 30-34 mm of the tubes. Each tube was then capped with a Teflon-backed, plastic screw-cap and placed in a cold box set at 4 °C. The temperature in the cold box remained at 2-5 °C throughout the experiment. After 1 week, the sediment had settled in each tube, and a water layer ∼5 mm deep had formed above the sediment surface. The water layer was removed to e1 mm from the sediment surface in each sample, and the screwcap was replaced with a foam plug. Most of the samples were placed in a covered container in the cold box; the container had a small layer of water on the bottom to maintain constant humidity. Four live samples were placed in a similar container and incubated at room temperature (22-25 °C). This was the start of the experiment (time zero). Sampling and PCB Quantitation. Several samples (test tubes) of the live and the autoclaved sediment were removed at time zero and stored at -20 °C until PCB analysis 2 weeks later. Three tubes of live and two tubes of autoclaved

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sediment were removed from 4 °C for fractionation and PCB analysis at 14, 43, 67, 98, 123, 149, 188, 228, and 300 days from the start of the experiment. One tube of live sediment incubated at 25 °C was removed at 14, 43, 67, and 98 days from the start of the experiment. The sediment in each tube was removed by cutting off the bottom of the glass tube using a diamond wheel and allowing the sediment to slide out as an undisturbed mass. Two regions of the sediment mass were fractionated using a metal spatula: a surface (0-5 mm from the surface) and a subsurface layer (g16 mm from the surface). Starting at day 123, a third region (6-10 mm from the surface, increasing to 6-15 mm at 300 days) was fractionated from live sediment samples incubated at 4°C. For each fraction, ∼1.5 g wet sediment was placed in a 8-mL, screw-cap glass vial for ether extraction. Sediment samples were extracted twice with 4 mL of anhydrous ethyl ether by reciprocal shaking overnight at ∼24°C. The vials were capped with Teflon-backed, plastic screw-caps. After extraction, each sediment sample was dried in an oven at 90 °C for 3 days before it was weighed. The two ether extracts of each sample were combined, evaporated to ∼0.5 mL, and applied to a 1.5-mL LC-CN solid-phase extraction column (Supelco, Inc.; Bellefonte, PA) that was equilibrated with hexane; the PCBs were eluted from the column in 6.0 mL of hexane. Vacuum-dried, acid-washed copper powder (∼1 g/sample) was added to the PCB solutions to precipitate the elemental sulfur extracted with the PCBs. PCB solutions were analyzed in two separate runs by gas chromatography using a 30 m × 0.25 mm (i.d.) SPB-octyl fused silica capillary column (Supelco, Inc.; Bellefonte, PA) connected to an electron capture detector (GC-ECD) or a mass selective detector set up for selected ion monitoring of PCBs (GC-MS). The concentrations of the PCB congeners were determined from quadratic, six-point calibration curves of standard solutions containing Aroclor 1242 (1-15 ppm) and 2,4,6-2,4,6-CB (7-105 ppb) in hexane. The peak identifications and mole percent compositions of the PCB congeners in Aroclor 1242 were determined from the data of Frame (13, 14). The operating conditions for the SPB-octyl column were the following: an injection temperature of 260 °C and a temperature program of 70 °C for 2 min, 160 °C at 15 °C/min, 270 °C at 3.0 °C/min and 270 °C for 5 min. The electron capture detector was set at 300 °C. Selected ion monitoring GC-MS was used to quantify certain PCB congeners; PCB quantification ions were m/z 188, 222, 256, 292, 326, and 360 with a dwell time of 300 ms for each. Aroclor 1242 and 2,4,6-2,4,6-CB (>99% purity) were purchased from Accustandard, Inc. (New Haven, CT). The concentrations of PCB congeners in each sample were calculated as micromoles per gram dry sediment and normalized to the average concentration of 2,4,6-2,4,6-CB in the time zero samples of the sediment; normalization of PCB congener data was described by Harkness et al. (5). The congener 2,4,6-2,4,6-CB is recalcitrant to aerobic degradation by microorganisms in upper Hudson River sediment (J. Lobos, personal communications). GC-MS was used to quantitate PCB homologs that co-migrate on the SPB-octyl capillary column (e.g., 2,3-2-CB and 4-4-CB) or to quantitate PCB congeners in Aroclor 1242 that give a signal (peak height) beyond the linear range of the ECD. Hence, only the following PCB congeners were quantitated using GC-MS: 2-; 2-4-; 2,52-; 2,4-2-; 2,3-2-; 4-4-; 2,5-4-; 2,4-4-/2,3-3-; 2,3,6-2-/2,4-2,6-; 3,4-2-; 2,3-4-; 2,4-2,4-/2,3-2,5-; 3,4-4-; and 2,4,6-2,4,6-CB. The combined GC-ECD and GC-MS analyses provided quantitative data on each mono-, di-, and trichlorobiphenyl in Aroclor 1242. Principal Components Analysis (PCA). PCA was carried out on a matrix of the PCB compositional data of all the samples. Each sample was mean centered and scaled to unit variance before PCA; the mean PCB congener concentration was subtracted from each PCB congener concentration, and

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the resulting value was divided by the standard deviation of the sample’s PCB congener concentrations. The numerical computation was done using Matlab for Microsoft Windows software (The Math Works Inc.; Natick, MA) and PCA analysis routines from the PLS Toolbox for use with Matlab Version 1.5 (Eigenvector Technologies; Manson, WA). The first two principal components (eigenvalues) captured 91% and 6% of the total variance between the samples. Iron Analyses. The amounts of Fe(II) and Fe(III) in the sediment samples were measured using the methods described by Lovley and Phillips (15, 16). Three tubes of live sediment were taken from 4 °C incubation at 320 days. The sediment was removed from each tube as described above and fractionated into two regions: 0-15 mm from the surface and g16 mm from the sediment surface. Each region was split into two parts of ∼1 g each and placed in 8-mL glass vials. The vials were placed in an anaerobic chamber (Coy Laboratory Products; Ann Arbor, MI) containing an atmosphere of g98% N2 and e2% H2. To each vial was added either 5 mL of 0.25 M hydroxylamine in 0.25 M HCl or 5 mL of 0.5 M HCl. Each vial was capped with Teflon-backed, screwcaps; vortexed for 20 s; and left in the chamber for 70 min. After 70 min, the vials were removed from the chamber and centrifuged to separate the extract from the sediment. After extraction, each sediment sample was dried in an oven at 90 °C for 3 days before it was weighed. A 0.1-mL sample of the extract was added to 4.9 mL of ferrozine (1 g/L) in 50 mM HEPES [N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid] buffer at pH 7.0. After a 10-s vortexing, the solution was passed through a nylon filter (pore diameter, 0.2 µm). The amount of Fe(II) in the solution was determined from the absorbance at 562 nm. The Fe(II) concentrations in the extracts were determined from a linear, six-point calibration curve of standard solutions prepared with ferrous ammonium sulfate (1-125 ppm) in 0.25 mM HCl. The Fe(II) and Fe(III) concentrations in each extract were calculated as milligram Fe(II) or Fe(III) per gram dry sediment. Under acidic conditions, hydroxylamine reduces Fe(III) to Fe(II). Therefore, the amount of hydroxylamine-reducible Fe(III) was calculated as the difference between the Fe(II) measured in the hydroxylamine and HCl extractions (16). PCB Metabolite Analysis. Chlorobenzoic acids (CBAs) in the sediment samples were extracted and analyzed using a modified method of Flanagan and May (17). Three tubes of live sediment and one tube of autoclaved sediment were taken from 4 °C incubation at 385 days. The sediment was removed from each tube as described above and fractionated into two regions: 0-15 mm and 16-26 mm from the sediment surface. The PCBs in the sediment were extracted with a mixture of a sodium metasilicate solution and a hexane/acetone (90:10) solvent. The sediment was acidified using HCl, and the CBAs were extracted with anhydrous ethyl ether. The organic phase of the extraction that contained the CBAs was removed and derivatized with pentafluorobenzyl bromide (Pierce). After extraction, each sediment sample was dried in an oven at 90 °C for 3 days before it was weighed. Derivatized CBAs were separated and detected by congener-specific GC-MS (17); a Hewlett-Packard Model 5890 Series II gas chromatograph equipped with a 30 m × 0.25 mm (i.d.) column of 0.25 mm DB-1 phase was connected to a Hewlett-Packard Model 5971A quadrapole mass selective detector operated at an electron energy of 70 eV. Selected ion monitoring was used to detect each CBA congener. CBA detection ions were m/z 139, 181, and 336 for monochlorinated CBAs, and m/z 173, 181, and 370 for dichlorinated CBAs, with a dwell time of 300 ms for each. A calibration standard of 2-; 4-; 2,6-; 2,5-; 2,4-; 2,3-; 3,4-; and 3,5-CBA was analyzed with the samples to determine the relative retention times in the system. The relative amounts of the CBAs in the samples were calculated by comparing the area counts of the monochlorinated fragment ion (m/z 139) or the dichlorinated

FIGURE 1. Principal component plot of the surface layer fraction of all sediment samples. fragment ion (m/z 173) after adjustment to amount sediment extracted (g dry sediment).

Results Single Sample Observations. Low-temperature microbial aerobic PCB degradation was first indicated in a single sample of PCB-contaminated upper Hudson River sediment. This sample was contained in a sealed glass jar that was stored at 8 °C for 9 months, during which time a lighter-colored layer appeared at the sediment surface. Congener-specific analysis of PCBs in the top 5 mm of the sediment showed loss of specific di- and trichlorobiphenyls as compared to those in the subsurface sediment. Greater than 50% of the dichlorobiphenyls 2-2-; 2,5-; 2,4-; 2-3-; and 2-4-CB and the trichlorobiphenyls 2,5-4- and 3,4-2-CB were depleted in the surface layer relative to the subsurface sediment. However, not all the di- and trichlorobiphenyls were depleted from the surface layer. For example, the trichlorobiphenyls 2,6-2-; 2,3,6-; and 2,4-4-CB were not significantly depleted. Furthermore, two PCB congeners, 2,6- and 2,6-3-CB, that contain di-orthochlorines on one ring showed only limited depletion (e15%). This apparent PCB congener selectivity in the surface sediment layer was similar to the PCB congener specificity of PCB-degrading upper Hudson River bacteria (2, 4, 5). Immediately after sampling, the surface layer in the jar was mixed with the subsurface sediment, and the sample was returned to the refrigerator. When the sample was examined 6 months later, the lighter-colored surface layer had reappeared. PCB analysis of the two regions as before indicated the same loss of specific PCB congeners from the surface layer. Since these observations were made on a single sample, an experiment that included autoclaved sediment samples as the killed, control samples was conducted. Analysis of the Multi-Sample Experiment. Compilation of the PCB congener data of all the samples resulted in a matrix of over 6500 values. Significant errors were observed when simple statistical analyses (i.e., mean and standard deviation) were used to identify changes in PCB congener concentrations. Such error (e.g., 15-35% differences between replicates) is typical in analysis of environmental samples because nonhomogeneous PCB concentrations in the sediment and microbiological effects, possibly due to low populations, contribute to the overall error. This problem often obscures identification of significant trends that might develop during the experiment. A multivariate analysis method was needed to succinctly correlate the data.

PCA of the PCB compositional data was used to distinguish trends between the samples. The analysis showed that changes in PCB composition only occurred in the surface layer fraction (0-5 mm from the surface) of the live samples (i.e., samples that were not autoclaved). A comparison of the scores of the first two principal components (PC1 and PC2) revealed that the live samples were systematically distributed away from a cluster of data representing the autoclaved and time zero samples (Figure 1). The data representing subsurface fractions (g16 mm from the surface) of both the live and the autoclaved samples were also clustered with the autoclaved surface layer fraction and the time zero samples but were not included in Figure 1. Further examination of the data for the surface layer of sediment samples at 4 °C showed a systematic correlation with incubation time. A time course of the PC1 scores of these samples (Figure 2) showed a decrease in total PCB concentration. Since there was a linear correlation between the PC1 scores and the total PCB concentration of the unscaled data (data not shown), the PC1 scores represented the total PCB concentration in the samples. Hence, a change in the PC1 scores represents a change in the total PCB concentration between the samples. The change was the result of congenerspecific PCB degradation in the samples. Congener-Specific PCB Degradation. Several mono-, di-, and trichlorobiphenyls were depleted from the surface layer of the live sediment samples. This conclusion was made after reconstruction of data matrix after PCA using the scores and loadings of the first two principal components. This procedure provided an enhanced signal to noise ratio for comparing the samples by significantly diminishing the variations in the data that typically obscure identification of trends in chemical analyses of environmental samples. Loss of specific di- and trichlorobiphenyls (2-2-; 2-4-; 2,3-2-; and 3,4-2-CB) was observed from the surface layer of the live sediment samples at 4 °C (Figure 3). Degradation of these congeners was first indicated at 43 days and continued through the last sampling time at 300 days. The largest change in the relative concentration of each PCB congener that was degraded occurred between the 67 and the 98 day sampling periods, suggesting that the largest degradation rate occurred during the period between those two sampling times. The specificity of PCB congener degradation was identical in the surface layer fractions of the live sediment samples incubated at 4 and 25 °C. A comparison of the mono-, di-, and trichlorobiphenyls that were degraded or not degraded

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FIGURE 2. Time course of PC1 scores for the surface layer fraction of the live sediment samples at 4 °C. The scores reflect the relative difference in the total PCB concentration between the samples and the average sample of the data set. Each point is the mean of three replicates, and the error bars represent one standard deviation.

FIGURE 3. PCB congener degradation in the surface layer fraction of the live sediment samples at 4 °C. The data are ratios of the PCB congener concentration at each time point relative to its starting concentration. Each point is the mean of three replicates, and the error bars represent one standard deviation. in these samples is shown in Table 1. PCB congeners containing di-ortho-chlorines on one ring (e.g., 2,6- or 2,63-CB) or di-para-chlorines (e.g., 4-4- or 2,4-4-CB) were not degraded in samples incubated at either temperature. The overall rate of PCB degradation was estimated to be at least 3-4-fold lower in the 4 °C samples as compared to the 25 °C samples since the extent of degradation was roughly equal between samples incubated for 149 days at 4 °C and for 43 days at 25 °C. Ferric Oxide Formation in the Sediment. Initially, the sediment samples were uniformly dark brown. At 43 days, a lighter, rust-colored layer was observed in the top 4 mm of

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the live sediment samples at 4 and 25 °C. This rust-colored layer steadily increased from the surface to 15 mm deep by 300 days (Figure 4) in the samples incubated at 4 °C. In contrast, the rust-colored layer did not deepen in samples incubated at 25 °C, but gas pockets that were presumed to be methane (19-21) occurred in regions of the sediment deeper than 4 mm from the surface. No color change was observed in autoclaved sediment samples. Some of live sediment samples at 4 °C were assayed at 320 days for hydroxylamine-reducible ferric iron and acid-soluble ferrous iron in the rust-colored layer (0-15 mm from the surface) and the subsurface region (g16 mm from the surface). The

FIGURE 4. Appearance of the rust-colored layer in the live sediment samples at 4 °C (measured as depth from the surface). Each datum point varies by approximately 1 mm.

TABLE 1. Degradation of PCB Congeners in Sediment Samples at 4 and 25 °C PCB homolog

congeners degradeda

monochlorobiphenyl dichlorobiphenyl

trichlorobiphenyl

congeners not degraded 2-CB

2,3-CB 2,4-CB 2,5-CB 2-2-CB 2-3-CB 2-4-CB 2,3-2-CB 2,3-3-CB 2,4-2-CB 2,4-3-CB 2,5-2-CB 2,5-3-CB 2,5-4-CB 3,4-2-CB

2,6-CB 4-4-CB 2,3,6-CB 2,3-4-CB 2,4-4-CB 2,6-2-CB 2,6-3-CB 2,6-4-CB 3,4-4-CB

a Congeners degraded at g20% in live samples that were incubated at 4 °C for 149 days or at 25 °C for 43 days.

rust-colored layer contained 2.7 mg of hydroxylaminereducible ferric iron/g dry sediment as compared to 0.0 mg/g dry sediment in the subsurface region. The amounts of acidsoluble ferrous iron were nearly reversed between the two regions: the rust-colored layer contained 0.2 mg/g dry sediment as compared to 2.5 mg/g dry sediment in the subsurface region. PCB congener degradation was also observed in the deeper, rust-colored region of the live samples at 4 °C (6-15 mm from the surface). Initially, the PCB composition was measured in two regions of each sediment sample (0-5 mm and g16 mm from the surface). However, since the rustcolored layer had deepened in the samples, a third region was fractionated, and the PCB composition was assayed starting at 123 days. The third fraction was of the rust-colored layer deeper than 5 mm (e.g., 6-10 mm at 123 days, increasing to 6-15 mm at 300 days). The same PCB congener degradation specificity was observed in the deeper fraction of the rust-colored layer as in the surface layer fraction (Table 1). PCB dechlorination was not observed in the subsurface sediment (g16 mm below the surface) of the live samples incubated at 4 °C. In contrast, extensive microbial PCB dechlorination was observed in the gaseous, subsurface

sediment of the live samples incubated at 25 °C. The PCB dechlorination patterns observed in this region were the same as those reported in the literature (18-21). Detection of Aerobic PCB Degradation Metabolites. Surface and subsurface fractions of live and autoclaved sediment samples were assayed for mono- and dichlorobenzoic acid metabolites of microbial aerobic PCB degradation. Low levels of chlorobenzoic acid metabolites were found in all fractions except the rust-colored fraction of the live samples, which contained significantly larger amounts of 4-chloro-; 2,3-dichloro-; 2,4-dichloro-; and 2,5-dichlorobenzoic acid. These metabolites were found at 5-11-fold higher concentrations in the rust-colored fraction of the live samples at 4 °C as compared to the autoclaved sediment sample.

Discussion The data clearly indicate that microbial aerobic degradation of mono-, di-, and trichlorobiphenyls occurred in sediment samples at 4 °C. This conclusion is supported by several lines of evidence. First, there was a systematic change, identified as depletion, in the PCB composition from the surface layer fractions of the live sediment samples but no change in the subsurface (g16 mm from the surface) or autoclaved sediment samples. Second, not all but certain di- and trichlorobiphenyls were degraded in the surface sediment layer. Any abiotic depletion of PCBs (i.e., vaporization) would likely result in approximately equal losses of the congeners in each di- and trichlorobiphenyl homolog group. Third, the PCB congener specificity in the surface layer of the live sediment was similar to the PCB congener specificities of Alcaligenes eutrophus H850 (1-3) and other bacteria isolated from upper Hudson River sediment (4, 5). Lastly, increased levels of specific chlorobenzoic acid metabolites of microbial aerobic PCB degradation were identified in the surface, rust-colored layer of live sediment incubated at 4 °C. Our study of low-temperature PCB degradation was initiated by our discovery of a surface rust-colored layer in PCB-contaminated upper Hudson River sediment stored in the refrigerator. When sediments are collected and placed in cold storage, they are uniformly dark brown. However, we noticed that a surface rust-colored layer formed in Hudson River sediment samples, except an autoclaved sediment sample within 2 months of storage, suggesting a microbial role in the color change. Lovley and Phillips (15) demonstrated that the amount of hydroxylamine-reducible ferric iron in sediment is correlated with the amount of ferric iron

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available for microbial reduction. Therefore, formation of hydroxylamine-reducible ferric iron in the rust-colored layer probably resulted from microbial oxidation of ferrous iron. Furthermore, Fish and Principe reported formation of a similar surface rust-colored layer in their Hudson River test tube microcosms incubated at 24 °C (6, 7); they also reported that microbial aerobic PCB degradation occurred in this layer. As mentioned above, the PCB congener selectivity in the surface sediment layer at both 4 and 25 °C was the same as the PCB congener specificity of many PCB-degrading bacterial isolates, particularly those isolated from upper Hudson River sediment (1-5). Bedard et al. (1-3) noted that in addition to their diminished ability to degrade di-para-chlorinecontaining congeners such as 2,4-4-CB, PCB-degrading upper Hudson River bacteria have a reduced ability to degrade the mono-ring-substituted, di-ortho-chlorine-containing congeners such as 2,6-; 2,6-2-; 2,6-3-; and 2,6-4-CB. In a recent report, several PCB-degrading bacteria were classified into two groups based on their congener specificity and their 16S rRNA gene sequences (4). One group, identified as Grampositive, Rhodococcus species had strong abilities to degrade the di-para-chlorine-containing congeners. In contrast, the other group was identified as members of the β subdivision of the Proteobacteria. This group of all upper Hudson River bacterial isolates, including A. eutrophus H850, had strong abilities to degrade a broad range of PCBs but not the dipara-chlorine-containing congeners (4). Consequently, lowtemperature aerobic PCB degradation could be caused by these Gram-negative species. Potential Low-Temperature PCB Degradation in Situ. The data presented in this paper suggest that microbial PCB degradation potentially could occur in Hudson River sediment during the winter and accordingly throughout the year. This conclusion is supported by several lines of evidence. First, PCB degradation at 4 °C occurred within 6 weeks, and extensive degradation of most of the dichlorobiphenyls, particularly 2-2-CB and 2-4-CB, occurred by 5 months. Winter temperatures of