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Jennifer J. Mosher, Robert H. Findlay, and Carl G. Johnston ... Ohio, USA, has led to the accumulation of a wide variety of pollutants within its sediments.
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Physical and chemical factors affecting microbial biomass and activity in contaminated subsurface riverine sediments Jennifer J. Mosher, Robert H. Findlay, and Carl G. Johnston

Abstract: Over 80 years of direct discharge of industrial effluents into the Mahoning River, located in northeastern Ohio, USA, has led to the accumulation of a wide variety of pollutants within its sediments. This study examined the physical and chemical parameters, including lipophilic pollutants, affecting microbial activity and biomass in subsurface (10–40 cm horizon) sediments. Microbial biomass was higher in anthropogenically contaminated sediments, and stepwise linear regression showed that approximately 82% of the variation in microbial biomass could be explained by total hexane extractable hydrocarbons, sediment particle size, and water content. There was no correlation between microbial activity and biomass. Independent variables influencing anaerobic activity were temperature and water holding capacity. The results of this study indicate that freshwater, sedimentary anaerobic microbial communities respond to a range of environmental parameters, many of which influence subsurface river sediments, and that lipophilic pollutants, when present, can cause increases in total microbial biomass. Key words: microbial activity, microbial biomass, anaerobic sediments, PAH, metals. Résumé : Plus de 80 ans de déversement d’effluents industriels dans la rivière Mahoning, situé dans le nord-est de l’Ohio, États-Unis, a mené à l’accumulation d’une grande variété de polluants à l’intérieur de ses sédiments. Cette étude s’est intéressée aux paramètres physiques et chimiques, incluant les polluants lipophiles, affectant l’activité et la biomasse microbienne dans les sédiments de subsurface (horizon de 10–40 cm). La biomasse microbienne était plus élevée dans les sédiments à contamination anthropogène et une régression linéaire séquentielle a démontré qu’environ 82 % de la variation de la biomasse microbienne pourraient être expliqués par les hydrocarbures totaux extractibles à l’hexane, la taille des particules sédimentaires et le contenu en eau. Il n’y eut aucune corrélation entre l’activité microbienne et la biomasse. Les variables indépendantes influençant l’activité anaérobie étaient la température et la capacité de rétention de l’eau. Les résultats de cette étude indiquent que les communautés microbiennes anaérobies sédimentaires d’eau douce répondent à un éventail de paramètres environnementaux dont plusieurs influencent la subsurface des sédiments des rivières et que les polluants lipophiles, lorsque présents, peuvent causer une augmentation dans la biomasse microbienne totale. Mots clés : activité microbienne, biomasse microbienne, sédiment anaérobie, HPA, métaux. [Traduit par la Rédaction]

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Introduction Microbial communities in riverine sediments may be influenced by many abiotic factors, including various types of organic and inorganic nutrients, sediment physical properties, and environmental conditions, and in many systems may be negatively impacted by human activity. An understanding of Received 30 June 2005. Revision received 10 November 2005. Accepted 14 November 2005. Published on the NRC Research Press Web site at http://cjm.nrc.ca on 20 April 2006. J.J. Mosher1,2 and C.G. Johnston. Department of Biological Sciences, Youngstown State University, Youngstown, OH 44555, USA. R.H. Findlay. Department of Biological Sciences, The University of Alabama, Tuscaloosa, AL 35487, USA. 1 2

Corresponding author (e-mail: [email protected]). Present address: Department of Biological Sciences, The University of Alabama, Box 807206, Tuscaloosa, AL 35487, USA.

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relationships among microbial biomass, microbial activity, and these variables may be important to researchers interested in remediation of riverine systems suffering from historical degradation due to industrial contamination (Barkey and Pritchard 1988; Langworthy et al. 1998). In stream sediments, Bott and Kaplan (1985) demonstrated correlations among microbial biomass and activity and sedimentary physical properties with higher microbial biomass associated with smaller grain sizes. Also, positive correlations were observed between biomass and dissolved organic carbon, dissolved organic nitrogen, selected anions and cations, and organic matter content in the sediments. Bacterial activities showed positive correlations with dissolved organic carbon, temperature, anions, and sedimentary organic matter. In shallow subsurface sediments, researchers found little or no differences in microbial community structure and biomass, but microbial metabolic potential increased with levels of moisture and Fe (III) availability, whereas it decreased with increasing sediment particle size (Musslewhite et al. 2003). In surface sediments of a regulated stream, flow

doi:10.1139/W05-144

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dynamics and temperatures associated with seasonal changes were correlated with shifts in microbial community dynamics (Sutton and Findlay 2003). Other external factors found to be important in structuring sedimentary microbial communities in streams include water temperature, organic carbon, anthropogenic pollution, sediment water content, and pH (Dale 1974; Schallenberg and Kalff 1993; Pusch and Schwoerbel 1994; Franken et al. 2001; Battin et al. 2003; Feris et al. 2003). In sediments contaminated with organic pollutants, the microorganisms present have either adapted to the contaminant and (or) utilized it as a food source (Barkey and Pritchard 1988; Grosser et al. 1991). Recent studies have shown a variety of changes in microbial biomass and activity of sediments contaminated with polycyclic aromatic hydrocarbons (PAHs). For example, Langworthy et al. (2002) demonstrated higher levels of microbial biomass in sediments with intermediate levels of PAH concentration and decreased biomass levels in sediments with high levels of PAH concentration. Meso- and micro-cosm experiments reported microorganisms thriving with intermediate concentrations of introduced PAH mixtures (Carman et al. 1995; Verrhiest et al. 2002). Our objective was to explore the physical and chemical parameters, including anthropogenic pollutants, affecting microbial activity and biomass in subsurface-polluted (10– 40 cm horizon) riverine sediments. We expected to see a negative impact on microbial biomass and activity due to the high concentrations of anthropogenic pollutants present in the sediments.

Materials and methods Experimental design We conducted a correlative study in the Lower Branch of the Mahoning River, Ohio, USA, a fourth-order stream, to determine the impact of environmental factors and multiple pollutants on the distribution and activity of sedimentary microbes. The Upper Branch of the Mahoning River headwaters in Columbiana County, Ohio, and flows northward through a primarily agricultural watershed. At approximately river mile (RM) 50, the river turns southeastward becoming the Lower Mahoning River. The Lower Mahoning River flows through several towns and cities, including Leavittsburg (RM 46.3; little to no industrialization), Warren (RM 27.0; heavily industrialized), Youngstown (RM 20.5; heavily industrialized), and Lowellville (RM 13.3; moderate industrialization). The river continues southeastward into Pennsylvania forming a confluence with the Shenango River (RM 0). Site 1 (RM 46.3) served as the ambient station, as it is located upstream from heavily industrialized areas. Site 2 (RM 27.0) and Site 3 (RM 20.5) were stations chosen in the midst of the industrial regions of the Mahoning Valley. Site 4 (RM 13.3) is downstream of the heavily industrialized region but is the site of the first coke plant in the United States at the turn of the 20th century (US Army Corps of Engineers (USACE) 1999). Subsurface sediments, collected from run (actively flowing) areas of the river, were analyzed for a suite of biological, chemical, and physical parameters. These included microbial activity, microbial biomass, sediment toxicity, PAH concentration, total hexane-extractable hydrocarbon concentration (THEH), pH, ash-free dry mass (AFDM), water holding capacity (WHC), sediment particle

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size, temperature, and water content. Total sediment metal concentrations were obtained from previously published research conducted at these sites (USACE 1999). Step-wise linear regression was used to investigate the relationships between measures of microbial activity and biomass and chemical and physical parameters. Sediment collection and sample processing Sediments were collected, in triplicate, using a Wildco corer (Buffalo, New York). The corer was fitted with a Plexiglas™ tube measuring 48 cm in length and 5.25 cm in diameter. Upon recovery of the core, the Plexiglas™ tube was removed, capped on both ends, placed on ice, and transported in an upright position. In the laboratory, the samples were immediately placed in an anaerobic glove bag, the overlying water and top 10 cm of sediments were discarded, and the 10–40 cm horizon collected. Previous studies on the Mahoning River have shown the sediments at these depths to be anaerobic (Martin 2000). The sediments were placed in a clean Mason jar and homogenized. The sediment used for anaerobic microbial activities remained in sealed containers, while the rest of the sediments were exposed to the atmosphere. The sediments were immediately used for microbial activity and microbial biomass measurements, as timing and handling were crucial for these assays. Physical parameters The United States Environmental Protection Agency (USEPA) method 9045C was used for pH measurements (USEPA 1995). For AFDM, oven-dried sediments were weighed into aluminum boats and combusted at 550 °C for 24 h and then gravimetrically analyzed (Tiessen and Moir 1993). WHC was measured by saturation experiments with dried sediments (Livingston 1993). Settling time was measured with a hydrometer (Fisher Scientific, Suwanee, Georgia) and analyzed to determine particle size of the sediment (Sheldrick and Wang 1993). Water content was determined gravimetrically after drying in a Fisher Scientific Isotemp 500 oven at 105 °C for 24 h. Chemical parameters PAHs were extracted from sediments using the USEPA sonication extraction method 3550 (USEPA 1996a). The samples were quantified on a Hewlett Packard 5890 Gas Chromatograph/5970B Mass Spectrometer (GC/MS, Palo Alto, California) in accordance with USEPA method 8270C (USEPA 1996b). Total hydrocarbons were extracted from sediments by sonication with hexanes (Grosser et al. 1991; USEPA 1998). Total sediment metal concentrations were obtained from USACE (1999). Biological parameters Microbial activity was analyzed by dehydrogenase enzyme activity via the reduction of the compound 2-(p-iodophenyl)3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT) to a red-colored formazan (INTF) and analyzed colorimetrically in a Shidmadzu spectrophotometer (Columbia, Maryland) (Mosher et al. 2003). Microbial biomass was estimated by extracting phospholipid phosphate with organic solvents, oxidation to ortho-phosphate, and colorimetrical analysis with malachite green M-9636 (Sigma-Aldrich Co., St. Louis, Missouri) © 2006 NRC Canada

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Table 1. Physical and chemical characteristics of subsurface sediments in the Lower Mahoning River. Assay

Site 1 (RM 46.3) –1

PAH (µg g ) THEH (mg g–1) pH AFDM (%) WHC (%) Sediment particle size Sand Clay Silt Temperature (°C) Water content (%) Metals* (g kg–1)

2.0±0.7 0.01±0.02 6.1±0.9 5.4±2.2 48±12 (%) 18±5 7±3 75±8 21±1 34±8 33.5

Site 2 (RM 27.0)

Site 3 (RM 20.5)

Site 4 (RM 13.3)

23.0±1.0 1.40±0.30 7.0±0.5 9.9±6.8 40±11

32.0±7.5 1.70±1.00 6.8±0.3 9.7±2.9 55±7

230.0±96.3 8.70±3.30 7.5±0.1 14.6±6.4 47±8

12±8 6±4 82±12 24±1 34±6 389.7

18±8 6±6 76±13 25±3 54±1 203.7

29±22 7±3 64±13 22±2 48±4 190.2

Note: PAH, polycyclic aromatic hydrocarbons; THEH, total hexane-extractable hydrocarbon concentration; AFDM, ash-free dry mass; WHC, water holding capacity; RM, river mile. *Data from USACE (1999).

Table 2. Biological characteristics of subsurface sediments in the Lower Mahoning River. Assay

Site 1 (RM 46.3)

Site 2 (RM 27.0)

Site 3 (RM 20.5)

Site 4 (RM 13.3)

Microbial activity (µmol g–1 h–1) Biomass (nmol P g–1) Activity (per unit biomass) Toxicity (ln EC50)

1.1±0.9 39.5±20.6 35.4±33.3 7.0±0.8

3.1±0.3 93.3±18.8 33.8±7.9 4.5±1.3

2.2±0.4 147.8±66.4 16.7±6.4 4.9±0.8

2.6±0.4 100.5±42.2 22.2±16.7 6.8±1.2

Note: RM, river mile.

(Findlay et al. 1989). Transformation of INT to INTF per unit biomass was calculated to determine the rate of activity per unit of microbial biomass. Sediment toxicity was determined by using Vibrio fisheri, a freeze-dried bioluminescent marine bacterium, on the MicrotoxOmni® M500 (AZUR Environmental Inc., Newark, Deleware) (Guzzella 1998). The method was a modification provided from the manufacturer in which 14 serial dilutions were used instead of the usual nine dilutions because of high toxicity of the sediments. Statistics One-way ANOVA with Tukey’s Post Hoc test was used to determine differences of individual parameters between sampling stations (SPSS 12.0). Before analyzing data with step-wise linear regression to identify factors determining microbial biomass, microbial activity, and toxicity, all independent variables were screened for auto-correlations using Pearson’s correlation coefficient. When significant correlation between a pair of variables was found, only one of the pair was used for further statistical analysis. The Durbin– Watson d statistic was used to determine any further autocorrelation of independent variables within the step-wise linear regression. Mean metal concentrations were correlated to biological parameters by linear regression of site means.

Results Physical parameters The following physical parameters of the study showed no differences between sites (Table 1); pH (p = 0.081; range: 6.1–7.5); WHC (p = 0.378; range: 40%–55%); sediment

particle size (p = 0.541; range: sand 12%–29%, clay 6%–7%, silt 64%–82%); and temperature (p = 0.108; range: 21–25 °C). AFDM ranged from 5%–15%, with Site 1 significantly lower than Sites 3 and 4, whereas Site 2 was higher than Site 3 (p = 0.003, Tukey’s honestly significant difference (HSD) α = 0.05). The water content (range: 34%–54%) of sediments at Sites 3 and 4 were significantly higher than the other sites (p < 0.001; Tukey’s HSD α = 0.05). Chemical parameters PAH concentrations were significantly higher for Site 4, whereas Site 1 was lower than all other sites (p = 0.003; Tukey’s HSD α = 0.05) with a range of 2–230 µg g–1 (Table 1). THEH concentrations (0.01–9.00 mg g–1) were significantly higher at Site 4 than all other sites (p < 0.001; Tukey’s HSD α = 0.05; Table 1). Total sediment metal concentrations ranged from 33.5 to 389.7 g kg–1 (Table 1). Biological parameters Microbial activity ranged from 1.1 to 3.1 µmol g–1 h–1 INTF produced throughout the sites (Table 2). One-way ANOVA indicated microbial activity was significantly lower for Site 1 than the contaminated sites, and Site 2 was significantly greater than Site 3 (p < 0.001; Tukey’s HSD α = 0.05). Microbial biomass values varied between 40 and 150 nmol g–1 and were higher in anthropogenically contaminated sediments, with Sites 3 and 4 significantly higher than Site 1 (p = 0.001; Tukey’s HSD α = 0.05; Table 2). Transformation of INT per unit biomass values ranged from 17 to 35 and had no differences between sites (p = 0.400; Table 2). Values for sediment toxicity ranged from 4.5 to 7.0 ln EC50, © 2006 NRC Canada

400 Fig. 1. Correlation of microbial activity with temperature and water holding capacity as determined by step-wise linear regression in subsurface sediments of the Lower Mahoning River.

Can. J. Microbiol. Vol. 52, 2006 Fig. 2. Correlation of microbial biomass with total hydrocarbons, sediment particle size, and water content as determined by stepwise linear regression for microbial biomass in subsurface sediments of the Lower Mahoning River.

with no significant differences between sites (p = 0.171; Table 2). Correlations The Pearson’s correlation coefficient found auto-correlations between THEH and PAH concentrations (R2 = 0.903) and between water content and WHC (R2 = 0.642). Therefore, only one parameter of each pair was used in each subsequent statistical analysis. Step-wise linear regression showed that approximately 65% of the variation in microbial activity was due to temperature and WHC (R2 = 0.6517; Tukey’s HSD α = 0.043; Durbin–Watson d Statistic = 1.1219; Fig. 1). Independent variables influencing microbial biomass were THEH, inverse sediment particle size, and water content (R2 = 0.8258; Tukey’s HSD α = 0.045; Durbin–Watson d Statistic = 1.989; Fig. 2). There was no correlation between microbial activity and biomass, indicating that activity was not driven by sampling disturbance and (or) limited by microbial biomass. None of the physical and chemical variables measured in this study correlated with toxicity (p = 0.171).

Discussion This study showed that a subsurface riverine microbial community responded to a complex mixture of environmental and anthropogenic factors. Microbial biomass was correlated to THEH concentration, sediment particle size, and water content, while microbial activity was positively correlated with temperature and WHC. These patterns contain aspects that have been found in previous studies, as well as others that have not been previously reported (Dobbs and Findlay 1993; Sutton and Findlay 2003; Ben-David et al. 2004). In a summary of previous reports, Dobbs and Findlay (1993) found that microbial biomass of freshwater surface sediments, measured as phospholipid phosphate, ranged from 0.40 to 2.17 mg C (g sediment dry mass)–1 (for the ease of comparison, all biomass and abundance values were converted to C using the conversion factors provided in Balkwill et al. (1988)). Two recent reports from Ohio streams found

that microbial biomass of noncontaminated surface sediments ranged from 0.02 to 1.34 mg C (g sediment dry mass)–1 (Sutton and Findlay 2003), whereas PAH-contaminated sediments ranged from 1.11 to 6.02 C (g sediment dry mass)–1 (Langworthy et al. 2002). Ben-David et al. (2004) found total microbial biomass ranged from 0.02 to 0.12 mg C (g sediment dry mass)–1 for an acid drainage impacted stream in South Australia. Bacterial biomass of metalcontaminated subsurface riverine sediments in Western Montana ranged from 0.43 to 3.17 mg C (g sediment dry mass)–1 (Feris et al. 2003). We report here that total microbial biomass in anaerobic Mahoning River sediments ranged from 0.40 to 1.48 mg C (g sediment dry mass)–1. These cross-system comparisons indicated that stream-bed microbial biomass had a range of approximately two and one-half orders of magnitude. In addition, both natural (surface vs. subsurface) and anthropogenic (ambient vs. metal contaminated and (or) hydrocarbon contaminated) variations in biomass were within this range. We found that total microbial biomass was positively correlated with total hydrocarbon concentration and water content, while inversely correlated with sediment particle size. The most often reported trend associated with microbial communities in sediments is the inverse correlation between sediment particle size and microbial biomass (Dale 1974; Bott and Kaplan 1985; Peters et al. 1989; Albrechtsen and Winding 1992; Phelps et al. 1994; Jeng and Chen 1995; Kieft et al. 1995; Chafiq et al. 1999; Fleituch et al. 2001; Musslewhite et al. 2003). Our finding that biomass was correlated with sediment particle size further extends this trend to anaerobic stream-bed sediments. Hydrocarbon pollution has the potential to either stimulate total microbial biomass if the community responds to the pollutants as a food resource or to decrease total microbial biomass if the pollutants are toxic or inhibitory (Carman et al. 1995; Langworthy et al. 1998). An earlier study found similar correlations in mangrove sediments, where microbial biomass was significantly higher in contaminated © 2006 NRC Canada

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vs. noncontaminated sites (El-Tarabily 2002). In contrast, Langworthy et al. (2002) demonstrated both responses in stream-bed sediments with sites having high PAH levels (>50 µg g–1) containing lower biomass than sites with intermediate levels (5–50 µg g–1) of PAH concentrations. The differences in our findings compared with those of Langworthy et al. (2002) may have resulted from changes in sediment grain size among our sites. Site 4, the site with PAH concentrations predicted by Langworthy et al. (2002) to induce lower total microbial biomass, had greater grain size (a factor also known to decrease total microbial biomass) than Sites 1–3. Although Site 4 showed lower total microbial biomass than the sites with intermediate PAH contamination, the increase in sediment grain size likely obscured the changes in biomass attributable to inhibition by PAH. Our finding of a positive relationship between microbial biomass and water content is similar to previous studies in vadose zone sediments, sandy aquifer sediments, stream sediments, and desert soils (Colwell 1989; Johnson and Wood 1992; Kieft et al. 1993; Byappanahalli et al. 2003). Schallenberg and Kalff (1993) also found a correlation between microbial biomass and water content in a crosssystem comparison in sediment ecosystems. They argued for the use of sediment wet mass vs. dry mass for normalizing microbial biomass, citing the dilution effect of water content on biomass in sediments with greater than 50% water content. Schmidt et al. (1998), working with marine sediments, found a decrease in variation when microbial biomass was scaled with respect to the volume of pore water. Water content of subsurface Mahoning River sediments ranged from 34% to 54%. The use of both wet and dry mass for normalizing biomass was analyzed, and no significant difference with either method was observed. Smoot and Findlay (2001) also found no difference in variation when normalizing microbial biomass with either wet or dry sediment mass. Microbial activity, measured as INTF produced under anaerobic conditions in Mahoning River sediment, was similar to that of chemoheterotrophic bacteria isolated from anoxic activated sludge in a wastewater treatment pilot plant (Maurines-Carboneill et al. 1998). Most studies utilizing INT reduction in sediments were performed aerobically and (or) in noncontaminated systems. For example, previous reports of microbial activity in noncontaminated stream sediments were similar to those of ambient Mahoning River sediments, in spite of methodological differences (Trevors et al. 1982; von Mersi and Schinner 1991). However, similar comparisons between contaminated sediments indicated that Mahoning River microbial communities showed INT reduction rates that were 2- to 30-fold greater than previous measurements conducted under aerobic conditions (Mathew and Obbard 2001). The proximate cause of these differences is not known. We found correlations between microbial activity in Mahoning River sediments and temperature and WHC. This result is similar to those of a wide range of studies using stream-bed, hyporheic, and alluvial aquifer sediment that have found correlations between either temperature or moisture (and a suite of other parameters, including dissolved organic carbon, nutrients, oxygen and other electron acceptor concentrations, and sediment grain size) and microbial activity (Bott and Kaplan 1985; Albrechtsen and Windling 1992;

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Phelps et al. 1994; Pusch and Schwoerbel 1994; Mauclaire and Gibert 2001; Musslewhite et al. 2003). Concentrations of metals, previously reported for these sediments by USACE (1999), were not correlated with microbial biomass and microbial activity. Other studies have reported a similar lack of impact of high metals concentration on total microbial biomass for hyporheic sediments and soils (Pennanen et al. 1996; Feris et al. 2003). In contrast, other studies have demonstrated decreased microbial biomass in the presence of high metals concentration in river streambed sediments, river embankment sediments, and marine coastal and harbor sediments (Togna et al. 2001; Zarcinas and Rogers 2002; Dell’Anno et al. 2003). The proximate cause for the observed differences among systems is not known, although bioavailability of metals has been shown to vary among systems and to strongly influence microbial responses to metal contamination (Deaver and Rodgers 1996; Dell’Anno et al. 2003; Hsieh et al. 2004). Our data suggest that microbial communities in sediments contaminated with petroleum and metals are influenced by multiple environmental factors in ways similar to that observed in other streams that are relatively free of pollution (Dale 1974; Kaplan and Bott 1985; Osgood and Boylen 1990; Feris et al. 2003). The presence of metals did not inhibit microbial responses, in terms of both microbial biomass and activity, to chemical and physical factors. These results suggest that the microorganisms present in this historically contaminated river were tolerant of metals; or possibly the age of the contamination limited their bioavailability. Total microbial biomass increased with sediment hydrocarbon contamination. Other studies in contaminated sediments found that the abundance of microorganisms was influenced by pollutants and sedimentary environmental conditions. Langworthy et al. (2002) showed a complex response of total microbial biomass to PAH concentrations; however, periods of high flow and cold temperature obscured these differences. Williams and Fulthorpe (2003) studied invertebrate and microbial communities in chlorobenzenecontaminated sediments and reported that the major variances in both communities were attributed to down-welling zones, redox potential, and ammonium concentrations rather than to hydrocarbon contamination concentration. In summary, the natural and anthropogenic factors affecting microbial biomass and activity in this environment were sediment particle size, hydrocarbon concentration, water content, temperature, and percentage of moisture. This is the first study (known to us) to examine the effects of both hydrocarbon and metal pollution on microbial biomass and microbial activity in anaerobic freshwater sediments. Shi et al. (2002), working in soils contaminated with metals and hydrocarbons, found similar responses where the microbial communities were predominantly affected by hydrocarbons, but not metals. The 80-plus year legacy of pollution of this river may account for the adaptation of the microbial community to the presence of hydrocarbon pollution and the tolerance to, or decreased bioavailability of, metal pollution. These findings contribute to the overall knowledge of external factors that shape sedimentary microbial communities, while also addressing tolerances and adaptations of the indigenous microorganisms and their possible benefit in remediation of © 2006 NRC Canada

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anthropogenically-impacted ecosystems. In particular, the findings highlighted that anaerobic stream sediments responded in much the same way as aerobic stream sediment to natural and anthropogenic determinants. The high levels of anaerobic metabolic activity documented in this study for subsurface stream sediments suggest that these systems may be equally as important to ecosystem function as the often-studied surface sediments.

Acknowledgements This project was funded by The Department of Biological Sciences at Youngstown State University. Sarah Ellis, Josh Kollat, and Neil Hoffman provided valuable sampling and technical assistance. David Lineman provided analytical assistance and the use of a GC/MS. Bruce Levison, Scott Martin, and Lauren Schroeder provided valuable technical advice.

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