International Biodeterioration & Biodegradation 70 (2012) 104e110

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International Biodeterioration & Biodegradation

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Correlation of soil microbial community responses to contamination with crude oil with and without chromium and copper
Eder da C. dos Santos a, *, Isis S. Silva b, Tiago H.N. Simes c, Karen C.M. Simioni c, Valria M. Oliveira c, Matthew J. Grossman b, Lucia R. Durrant b
a b c

Department of Soil science, University of So Paulo, 13418-900 Piracicaba, SP, Brazil Laboratory of Systematic and Microbial Physiology, State University of Campinas, CP 6171, 13081-970 Campinas, SP, Brazil Division of Microbial Resources, Research Center for Chemistry, Biology and Agriculture (CPQBA), State University of Campinas, CP 6171, 13081-970 Campinas, SP, Brazil

a r t i c l e i n f o
Article history: Received 8 August 2011 Received in revised form 31 January 2012 Accepted 31 January 2012 Available online Keywords: Soil contamination Crude oil Chromium Copper Microbial communities Enzyme activities

a b s t r a c t
Soil microcosms contaminated with crude oil with or without chromium and copper were monitored over a period of 90 days for microbial respiration, biomass, and for dehydrogenase, lipase, acid phosphatase, and arylsulfatase activities. In addition, the community structure was followed by enumerating the total heterotrophic and oil-degrading viable bacteria and by performing a denaturing gradient gel electrophoresis (DGGE) of the PCR amplied 16S rDNA. A signicant difference was observed for biochemical activities and microbial community structures between the microcosms comprised of uncontaminated soil, soil contaminated with crude oil and soil contaminated with crude oil and heavy metals. The easily measured soil enzyme activities correlated well with microbial population levels, community structures and rates of respiration (CO2 production). The estimation of microbial responses to soil contamination provides a more thorough understanding of the microbial community function in contaminated soil, in situations where technical and nancial resources are limited and may be useful in addressing bioremediation treatability and effectiveness. 2012 Published by Elsevier Ltd.

1. Introduction Soil is a vital natural resource that is largely non-renewable on a reasonable time scale (Trasar-Cepeda et al., 2000a). The physical, chemical and biological conditions of soils inuence not only food production, but also the efciency of environmental processes including water purication, carbon cycling, nitrogen cycling and the degradation of natural and man made pollutants (TrasarCepeda et al., 2000b). Pollution of soils is widespread and an increasingly serious problem contributing to the deterioration of soils on a global scale (van Beelen and Fleuren-Kemil, 1997). Among the most common pollutants are crude oil and petroleum fuels and chemicals and metals, specically chromium and copper, due to their widespread usage for a broad range of purposes and as result it is not uncommon to nd polluted environments containing a mixture of these pollutants (Saison et al., 2004; Mielke et al., 2005). Hexavalent chromium is widely used in many industrial processes including electroplating and wood preservation and the chromium manufacturing industry produces a large quantity of

* Corresponding author. Tel.: 55 1934172160. E-mail address: eder.c.santos@hotmail.com (E.daC. dos Santos). 0964-8305/$ e see front matter 2012 Published by Elsevier Ltd. doi:10.1016/j.ibiod.2012.01.010

solid and liquid waste containing Cr(VI) as potassium chromate and potassium dichromate (Jeyasingh and Philip, 2005). Copper is discharged in industrial wastes mostly in the form of Cu(II) ion, and in copper cleaning and plating and metal processing Cu(II) ion concentrations are in the range of 100 mg l1, well above the common allowable levels (w1.0 mg l1) (Chatterjee and Lalitagauri, 2008). Soil microorganisms play a dominant role in the biodegradation of organic compounds, such as crude oil and its products (Gianfreda and Rao, 2008). They also play a role in reducing the toxic charge of heavy metals through conversion of toxic Cr(VI) to relatively nontoxic Cr(III), which precipitates at pH higher than 5.5 or through biosorption of Cu(II) (Chen and Hao 1998; Nannipieri et al., 2002; Chatterjee and Lalitagauri, 2008). However, the chemical, physical and biological quality of the soil must be maintained for these and other benecial processes to operate efciently (Trasar-Cepeda et al., 2000a). Given the importance of active microbial communities in remediating pollutants, it is necessary to assess their pollutant removal abilities and to determine if bioremediation efforts are further stimulating these abilities. Moreover, the fact that polluted sites may be remote and/or sophisticated laboratory instruments and analytical techniques unavailable, it is of considerable value to

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be able to monitor microbial activities with simple test methods (Prince et al., 2003) The microbial community structure of soils and corresponding microbial activities can be used as indicators of environmental changes caused by anthropogenic activities. It has also been suggested that the biological and biochemical properties that are most useful for detecting the deterioration of soil quality are those that are most closely related to nutrient cycles, including microbial biomass, soil respiration and the activities of soil enzymes (Visser and Parkinson, 1992). Soil microbial biomass (Cmic) is both a labile nutrient pool and an agent of transformation and cycling of organic matter in soils. Several studies have indicated that Cmic responds more rapidly to changes resulting from forest management activities than other soil organic matter and consequently may be an early and sensitive indicator of changes in soil quality (Sparling 1997). In addition, the proportion of Cmic relative to soil organic C (Cmic/Corg) has been used as an indicator for C availability and can provide an effective early indicator of the deterioration of soil quality (Xue et al. 2006). Soil basal respiration (CO2 evolution) mainly comes from microbial respiration. The microbial respiration rate is the sum of all metabolic processes that produce CO2 (Trasar-Cepeda et al., 2000b), is a good indicator of the degradation of organic compounds, and is used as an indicator of total soil microbial activity (Xue et al. 2006). The metabolic quotient (qCO2), the ratio of basal respiration to microbial biomass, denes a dynamic relationship between microbial biomass and metabolic activities, and several studies have demonstrated higher values of qCO2 in soils containing pollutants in comparison to uncontaminated soils (Gianfreda and Rao, 2008). Assessing the changes in cell counts of the heterotrophic and hydrocarbon-degrading bacteria is also a useful approach for evaluating soil microbial communities contaminated with hydrocarbons. A more complete picture of the diversity of microbial communities can be made using 16S rDNA sequence analysis and denaturing gradient gel electrophoresis (DGGE) of PCR amplied 16S rDNA has been shown to be an effective means to determine spatial and temporal changes of soil communities within and between locations under different environmental conditions (Grossman et al., 2000; Renella et al., 2005; Nakatsu, 2007). Although the above approaches are very valuable for evaluating microbial community structure and activity, most are either fairly sophisticated and/or labor intensive techniques. Soil enzyme activities have been suggested to be suitable indicators of soil quality because: (i) they are measures of the soil microbial activity and therefore they are related to nutrient cycles and transformations; (ii) they can rapidly change in response to changes in soil caused by both natural and anthropogenic factors; and (iii) they are fairly easy to measure (Nannipieri et al., 2002; Glser and Erdogan, 2008). As a result of these advantages, it has been suggested that soil enzyme activities are useful as early and sensitive indicators of soil alteration in both natural ecosystems and ecosystems altered by man, and are well suited to measure the impact of pollution on soil quality (Trasar-Cepeda et al., 2000b). In this study we address the problem of rapidly evaluating microbial communities in soils which have been contaminated with crude oil, and in soils contaminated with crude oil and chromium and copper. This was performed by comparative analysis of Cmic/ Corg ratios, respiration rates (as determined by CO2 evolution), metabolic quotients (qCO2), DGGE of PCR amplied 16S rDNA analyses, and cell counts of heterotrophic and hydrocarbon-degrading bacteria, and correlating these results to the activities of soil enzyme that react quickly to environment change. In this work we monitored enzymes involved in the assimilation of carbon (lipase), sulfur (arylsulfatase), phosphorus (phosphatase), and oxidative metabolism (dehydrogenase) (Bergstrom et al., 1998).

2. Materials and methods 2.1. Soil sample The soil used for the microcosm experiments was collected from an experimental farm located at the College of Agricultural Engineering (22 480 5700 S, 47 030 3300 W, 640 m average altitude), State University of Campinas, So Paulo, Brazil. No heavy metals and hydrocarbons contamination had been previously reported in this area. Samples were collected in plastic bags, thoroughly mixed, sieved through 2 mm pore size mesh, and stored at room temperature. The basic soil properties were determined using standard methods recommended by the Brazilian Society of Soil Science. The soil had the following properties: pH 5.3, Ca 5.2 centimols of charge (cmolc) kg1, Mg 1.9 cmolc kg1, organic matter 4.4%, cation exchange capacity (CEC) 8.28 cmolc kg1, total P 3.9 mg kg1, total S 2.8 mg kg1, base saturation 68%, 351 g sand kg1, 93 g silt kg1 and 559 g clay kg1, classied as Typic Haplorthox. 2.2. Microcosms 400 g of soil was placed in each 1.5 l glass jar, and the soil moisture content was adjusted to 50e60% of the water holding capacity. Chromium and copper were added to soil as K2Cr2O7 and CuCl2 salt solutions, respectively, at a concentration of 300 mg kg1. Crude oil was added to soil in concentrations of 2000 or 10,000 mg kg1. All soil treatments in microcosms were run in triplicate: (1) Cdcontrol without any articial contamination; (2) T1dsoil contaminated with 2000 mg kg1 crude oil; (3) T2dsoil contaminated with 10,000 mg kg1 crude oil; (4) T3dsoil contaminated with 2000 mg kg1 crude oil and chromium; (5) T4dsoil contaminated with 10,000 mg kg1 crude oil and chromium; (6) T5dsoil contaminated with 2000 mg kg1 crude oil and chromium and copper; (7) T6dsoil contaminated with 10,000 mg kg1 crude oil and chromium and copper. 2.3. Microbial biomass and respiration rates Soil microbial biomass was estimated by the fumigationextraction method (Vance et al., 1987). Soil (2 g dry weight) was fumigated with ethanol-free chloroform for 24 h and immediately extracted with 0.5 M K2SO4 in 250 ml plastic bottles, and then ltered using Whatman 42 lter paper. Non-fumigated soils were extracted in the same way, and extracts were frozen prior to analysis. Carbon content of each extract was determined by the dichromate oxidation method and the microbial C-biomass was calculated according to Vance et al. (1987). Soil respiration was determined by a CO2-trapping system using 20 ml of 0.25 M NaOH in small plastic cups held by an aluminum support in each microcosm. BaCl2 was added to precipitate CO2, and NaOH excess was titrated with 0.5 M HCl. 2.4. Soil enzymes activities All enzymes analyzed in this work were selected to represent a range of processes involved in decomposition and nutrient cycling, reecting microbial activities. Dehydrogenase was determined as described by Alef (1995), with the following modication: 1 g soil mixed with 0.01 g of CaCO3 was incubated with 1 ml 3% 2,3,5 tri-phenyl tetrazolium chloride (TTC) and 3 ml water at 30  C in darkness. After 24 h, 8 ml acetone was added, and the suspension homogenized, then ltered and washed with methanol until the reddish color disappeared from the soil (indicating reduced triphenyl formazan). The optical density of the extract at 546 nm was compared with tri-phenyl formazan standards. Acid phosphatase

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(EC 3.1.3.2) and arylsulfatase (EC 3.1.6.1) activities were determined as described by Alef (1995), and lipase (EC 3.1.1.3) was determined as described by Margesin (2005), where the activities are reported as mg r-nitrophenol (rNP) g1 h1. 2.5. Microbial counting

2.9. Statistical data treatment Analysis of variance by the Tukey test was used to assess the signicance of differences (p < 0.05) of the means (n 3) using SYSTAT v.12 software. 3. Results and discussion

Quantications of soil microbial microorganisms were determined by the plate count method for viable cells. Soil suspensions were prepared by shaking 1 g (dw) of soil with 9 ml of sterile sodium pyrophosphate (0.2%) for 30 min at 20  C at 150 revolutions min1. A volume of 0.1 ml of appropriate dilutions of soil suspensions, prepared in 0.9% NaCl, was spread onto nutrient agar plates to determine heterotrophic microorganisms. Hydrocarbondegrading microorganisms were quantied using oil agar plates containing sterile crude oil as the sole carbon source (Margesin and Schinner, 1998). Colony-forming units (CFU) were counted after incubation for 48 h at 30  C. 2.6. Community DNA extraction The extraction of DNA from soil samples was carried out using the method described by van Elsas and Smalla (1995) with some modications: (i) the volumes were adapted to extraction of DNA from 0.5 g of sediment (from 5 to 9 replicates), (ii) lysozyme incubation for 2 h, (iii) SDS addition before the bead-beating step and (iv) three bead-beating cycles at 3800 rpm for 90 s. DNA extracts were pooled and puried using the Wizard DNA clean-up system (Promega), according to the manufacturers recommendations. 2.7. PCR amplication of 16S rRNA genes The primers used for amplication were 968f (attached to a 40nucleotide GC-rich sequence) and 1401r (Heuer et al., 1997), which are homologous to conserved bacterial 16S rDNA regions. PCR amplications were performed in 50 ml-reactions containing 5 ml of 10 TriseHCl reaction buffer, 1.5 mM MgCl2, 0.4 mM of each primer, 0.2 mM dNTP mix, 2 U Taq DNA Polymerase (Invitrogen, Grand Island, N.Y., USA) and approximately 100 ng of total genomic DNA extracted from the soil samples. The amplication program was optimized for the bacterial domain-specic 16S rRNA gene as follows: an initial denaturation step of 5 min at 94  C, 10 cycles of 1 min at 94  C, 30 s at 58  C, decreasing 0.5  C each cycle, and 2 min at 72  C, followed by another 25 cycles of 1 min at 94  C, 30 s at 53  C and 2 min at 72  C. The amplicons were rst checked on 1.2% agarose gels prior to DGGE analyses. 2.8. DGGE analyses DGGE analysis of PCR products was carried out using the D-Code Universal Mutation Detection System (Bio-Rad, USA) with a linear denaturing gradient of urea and formamide ranging from 45 to 65% (100% denaturant corresponding to 7 M urea and 40% (v/v) deionized formamide). Gels (6% polyacrylamide) containing about 250 ng of PCR products from each sample, in triplicate, were run at 50 V and 60  C for 14 h in 1 TAE buffer. The band patterns were visualized under UV light after staining with SYBR Green (Molecular Probes) diluted 1:4000 in 0.5 TAE buffer for 1 h in the dark. DGGE patterns were analyzed using GelCompar v. 4.1 (Applied Maths, Kortrijk, Belgium) and UPGMA-based dendrograms constructed from Pearson (product-moment) correlation coefcient matrices.

3.1. Soil samples The main criteria used for selecting soil samples was the absence of hydrocarbons and heavy metals contamination, which would suggest that the autochthonous microbial community was not acclimated to the presence of these compounds. Soil samples were collected from an experimental eld of the Agriculture Engineering Faculty, State University of Campinas, So Paulo, Brazil. This area is used for physical experiments on soils, has well established vegetation during most of the year, and the physical and chemical characteristics are typical of native elds in this region of Brazil. The soil samples had low values of macronutrients (P, S, Ca, Mg, K) essential to microbial metabolism, a pH of 5.3, and high levels of clay and organic matter (data not shown). 3.2. Microbial respiration CO2 production was low for the rst ve days in all microcosms (Fig. 1a). From the 6th day on there was a rapid increase in CO2 production in all microcosms except the control (without articial contamination), which also increased but to a lesser extent than the contaminated microcosms (all of which received crude oil). The contaminated soil microcosms showed higher respiration rates than the control soil throughout the experimental period (90 days). The highest value for C-CO2 was observed for treatment T6 (soil 10,000 mg kg1 crude oil chromium copper), which showed cumulative CO2 production 69.8% higher than the control. As a group, treatments T4eT6, all receiving crude oil and at least one heavy metal, had the highest CO2 production and there was no statistically signicant difference in CO2 production between these treatments (T4eT6, Tukey, p < 0.01). These results demonstrate that the addition of crude oil plus chromium (or chromium and copper) to soil can have a stimulating effect on the respiration rate above that of soils containing crude oil alone. These results are also consistent with studies carried out by Shi et al. (2002), on soil contaminated with lead, chromium, and hydrocarbons, who suggested the increased production of CO2 in the presence of contaminants is due to increased metabolic activity in response to metabolic stress. 3.3. Microbial biomass and metabolic quotient (qCO2) Microbial biomass and the ratio of basal respiration to microbial biomass (qCO2) were assessed to determine if a correlation could be made with the type of microcosm treatment. Fig. 1b shows the change in microbial biomass over time for the various microcosm treatments. In contrast to the increase in CO2 observed with the addition of crude oil and further with the addition of both crude oil and metals, the presence of these contaminants caused a reduction of microbial biomass relative to the control throughout the experimental period. Treatments T6 and T5 (oil Cr Cu) and T3 and T4 (oil Cr) showed the greatest relative decrease in biomass, with T6 having the greatest measured decrease (51%) by day 90. Both microcosms contaminated only with hydrocarbons (T1 and T2) had a reduction in biomass of 26.5%. These results show that all treatments negatively affected total biomass.

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production and qCO2 concomitant with an overall reduction in total biomass indicates that the overall population declined while selected members of the population increased in number and activity. The additional increase in these parameters in the presence of metals is consistent with their causing more stress and energy consumption for cellular maintenance (Al-Saleh and Obuekwe, 2005; Renella et al., 2007). 3.4. Dehydrogenase activity Biological oxidation processes of organic compounds are frequently related to the activities of dehydrogenases involved in respiratory metabolism and soil dehydrogenase activity is recognized as an indicator of the overall microbial metabolism (Riffaldi et al., 2006). The soils contaminated with both crude oil and metals, T3eT6, had dehydrogenase activities signicantly lower than the control (Fig. 2). In contrast, treatments T1 and T2 containing only crude oil demonstrated signicantly higher dehydrogenase levels than the control (Fig. 2a), consistent with results reported by Riffaldi et al. (2006). Vivas et al. (2008) evaluated enzyme activities related to microbial activity (dehydrogenase activity) and the soil carbon cycle (total and extracellular b-glucosidase activities) in soils historically contaminated with hydrocarbons and the heavy metals Cu, Pb, Ni, Zn, Cd, and Cr. They found the complexity of the microbial community as well as the dehydrogenase soil activity negatively correlated with contamination levels, while the bglucosidase activities positively correlated with TPH levels. 3.5. Arylsulfatase activity Arylsulfatase catalyzes the hydrolysis of organic sulfate esters (Nannipieri et al., 2002) and hence has a correlation with the biological demand for sulfur were inorganic sulfur is limiting. The expression prole of arylsulfatase, although similar to that of dehydrogenase and acid phosphatase, differed slightly in that the control values were statistically similar to that of treatments T1 and T2 throughout the experimental period, with the exception of day 60 (Fig. 2c). Treatments T3eT6 were signicantly lower than that of treatments T1 and T2 from day 15 onward. The soil used in these experiments contained low levels of inorganic sulfur, and would be expected to induce the microorganisms to mineralize organic sulfates. From the 45th day on T1 (2000 mg kg1 oil) and from 60th day on T2 (10,000 mg kg1 oil) had decreases in arylsulfatase activity, which may have represented the exhaustion of the readily degradable fractions of the crude oil. Prez de Mora et al. (2005) found a positive correlation between increased activities of arylsulfatase, b-glucosidase and dehydrogenase and glucose mineralization and a decrease in soluble heavy metals in heavy metal contaminated soil after series of organic amendments, which is consistent with our results in that the presence of metals decreased both the dehydrogenase and arylsulfatase levels. 3.6. Acid phosphatase activity
Fig. 1. Cumulative respiration rates (CO2 production) (A), microbial biomass carbon (Cmic) (B), metabolic quotient (qCO2) (C). Error bars are standard errors of the mean (n 3).

All the soils treated with crude oil or crude oil plus heavy metals showed increased qCO2 values over the 90 day period (Fig. 1c). The pattern of qCO2 increase was similar to that observed for cumulative CO2 production per g soil. Treatments T3eT6 (crude oil metals) showed the greatest increase in qCO2, with T6 producing the highest values with a 4-fold relative increase in CO2 production per unit biomass in comparison to the control. The increase in CO2

Soil acid phosphatases are ubiquitous in soils and are produced by microorganisms when inorganic P levels are low. These enzymes catalyze the hydrolysis of organic phosphate esters to orthophosphate, thereby converting biologically unavailable P to bioavailable P (Alvarenga et al., 2008). Acid phosphatase activities were similar to that observed for dehydrogenase activities with treatments T1 and T2 (crude oil only) showing a rapid increase in activity after day 15. In contrast, the control had a relatively consistent moderate level of activity, and treatments T3eT6 (crude oil and metals) a consistently low level of activity. Treatments T1 and T2 reached

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Fig. 2. Dehydrogenase (A), acid phosphatase (B), arylsulfatase (C) and lipase (D) activities of the soil microcosms. Error bars are standard errors of the mean (n 3).

levels 37% and 30% above the control on days 75 and 60 respectively (Fig. 2b). These results were similar to studies carried out by Wang et al. (2007) and Shen et al. (2005) on soil contaminated with herbicides or polynuclear aromatics (PAHs), with added heavy metals. 3.7. Lipase activity Many soil microorganisms produce lipase to hydrolyze lipids to fatty acids and glycerol for subsequent use as a food source. In the rst 30 days, the microbial lipase activity was low and approximately equivalent across all microcosms (Fig. 2d). From the 45th day on lipase activity increased dramatically relative to the control for treatments T1 and T2, which received 2000 and 10,000 mg kg1 crude oil respectively, with T2 demonstrating signicantly higher levels than T1 on days 60 and 75. A considerably lower increase in lipase activity was observed for treatments T3eT6 with soils contaminated with hydrocarbons and heavy metals. The reason for the increases in lipase activity is not clear, given the lack of lipids in crude oil, but may indicate microbial degradation of lipids released by dead microorganisms, which could account for the observed time delay. Lee et al. (2008) conducted studies that demonstrated a negative correlation between lipase activity and hydrocarbons present in soils, while Allard and Neilson (1997) reported no effect. 3.8. Heterotrophic microorganisms, total and hydrocarbon degraders Successive plate counts were performed on nutrient agar plates to evaluate cultivable heterotrophic bacterial populations. Relative to the uncontaminated control microcosm, during the rst 15 days heterotroph populations were greatly reduced in all contaminated microcosms. Treatments T1 and T2 showed a decrease from 106 to 104 cells/g soil and treatments T3eT6 showed a decrease to below 103 cells g1 soil (Fig. 3a). Treatments T1 and T2 leveled off at approximately 104 cells g1 soil by 45th day. The greatest reduction

was observed in soil treatments with crude oil plus heavy metals (a decrease from 106 to 102 cells g1 of soil). In contrast, all of the soils impacted with crude oil and oil plus heavy metals showed a considerable increase in hydrocarbon degraders after week two (Fig. 3b). Treatments T1 and T2 reached 106 cells g1 soil, an increase from an initial level of 102 cells g1 of soil, while treatments T3eT6 showed an increase to between 103 and 104 cells g1 of soil. Nakatsu et al. (2005) observed similar behavior in soil contaminated with PAHs and Cr and Pb. The oil degrader counts exceed that of total heterotroph counts for the soils contaminated with oil alone, indicating a signicant change in the microbial community structure with respect to population density and metabolic characteristics. 3.9. Analysis of bacterial diversity changes using DGGE In molecular genetics terms, biodiversity is the number of different DNA sequences in the environment. DNA sequences, notably 16S rDNA, can be used to determine phylogenetic relationships, and characterize specic populations. In this study, bacterial diversity was evaluated using the 16S rRNA gene (16S rDNA) as a molecular marker in prokaryotic cells. Amplicons generated with a universal bacterial primer set were separated on denaturing gradient polyacrylamide gels (DGGE), yielding relatively complex ngerprints for all soil samples analyzed (Fig. 4). Visual inspection of the proles revealed a high consistency of the proles between the replicate samples. Control samples produced multiple bands of low abundance, evenly distributed, whereas treatment samples presented multiple bands of low abundance interspersed with intense individual bands. In addition, there were substantial similarities in band richness among treatments, although particular bands were favored according to the treatment (examples given by arrows, Fig. 4). This is reected in the dendrogram obtained using GelCompar software. As expected, proles obtained by replicate samples from the same treatment clustered most closely together (similarity levels of

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Fig. 4. Cluster analysis of the DGGE proles generated from soil microcosms with primer set 968f-GC and 1401r, using the Pearson correlation coefcient and UPGMA method. Soil treatments are specied in the text.

indicate that all contaminated soils went through major diversity changes in relation to the control. 4. Conclusion
Fig. 3. Total heterotrophic microorganisms (A) and oil-degrading microorganisms counts of the soil microcosms (B). Error bars are standard errors of the mean (n 3).

roughly 91e100%), whereas proles from different treatments and the control clustered at lower similarity levels. Treatments 1 and 3 were the most similar between treatments and formed cluster 1 at 91% similarity. Treatments 2 and 4 formed cluster 2 at 84% similarity, and together with samples of treatment 6 formed cluster 3 at 68% similarity. Cluster 3 presented <65% similarity with control samples. Replicates of treatment 5 did not group with high similarity to the other microcosms but had the highest similarity was with the control samples, forming a low similarity cluster 4 at 69% similarity. Cluster analyses of total community ngerprints showed that pronounced changes occurred in the diversity of the dominant members of the bacterial community in the treatment samples when compared with the control samples. However, soils that received hydrocarbons in equal concentration (with or without chromium), represented by treatments 1 and 3 (cluster 1) and treatments 2 and 4 (cluster 2) showed the highest similarity levels. In addition, hydrocarbon dosages would have to be responsible for the bacterial community discrepancies between T5 and T6 (<70% of similarity). These results suggested that hydrocarbon dosage had a greater effect on microbial community structure than chromium. These data also suggest that a combination of crude oil and metals selected populations able to tolerate stress from synergistic effects caused by both types of contaminants. Overall, the DGGE proles

We show that relatively simple enzyme assays can be used to efciently evaluate the metabolic activity of soil microbial communities with either crude oil contamination or crude oil and Cr and/or Cu contamination. The presence of crude oil and heavy metals in soil was shown to signicantly alter microbial enzyme and respiratory activities and community structure. Three quantiably different microbial soil communities were observed as follows: 1) uncontaminated, with high biomass and genetic diversity without dominant groupings, low respiration, and moderate enzyme activity; 2) crude oil contaminated, with moderate biomass with a genetic diversity containing dominant groupings, moderate respiration, and high enzyme activity; 3) crude oil and Cr and Cu contaminated, with low microbial biomass with a genetic diversity containing dominant groupings, high respiration rate, and low enzyme activity. Acknowledgments The authors would like to thank The Brazilian Council of Research (CNPq) for the PhD scholarship and nancial support for this research. References
Al-Saleh, E.S., Obuekwe, C., 2005. Inhibition of hydrocarbon bioremediation by lead in a crude oil-contaminated soil. International Biodeterioration and Biodegradation 56, 1e7.

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