INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

Terrestrial oil or petroleum deposits, which often contain high levels of sulfurous hydrocarbons, are being increasingly employed for the production of fuels. However, concerns regarding the emission of sulfur dioxide to the atmosphere, causing acid rain, have stimulated oil refiners to develop strategies for the reduction of the levels of sulfur in their products. Efforts have thus been made to develop an effective oxidative biocatalytic desulfurization approach in order to decrease the sulfur moiety in fuels through a specific nondestructive ring cleavage pathway (20, 28).

Soil sites contaminated with oil are a concern for both industry and regulators. Crude oil contains polycyclic aromatic hydrocarbons with up to 5% organically bound sulfur (42). The response of bacterial populations in soil to the stress imposed by the presence of such compounds and their intrinsic capacity to degrade them have been relatively unexplored, even though a recent study addressed the hydrocarbon degradation response of microbial communities in beach areas (25). Bacterial communities in soil are likely to respond to pollution with sulfurous oil by changing their structure to one that favors organisms that are able to thrive under the new selective conditions (18) at the expense of other organisms that are suppressed (25). Due to the selective pressure exerted by sulfurous hydrocarbons and given the possible need of microbial consortia in soil for sources of sulfur, bacterial populations with an enhanced presence of desulfurization genes may be selected.

A classical system for the study of microbial desulfurization of hydrocarbons has made use of Rhodococcus erythropolis strain IGTS8 because of its ability to specifically remove the sulfur atom from the model compound dibenzothiophene (DBT) (20). The plasmid-borne sox (7) or dsz (24, 33) gene, responsible for the oxidation of sulfur in DBT, has been cloned and sequenced (6, 7, 33). These desulfurization genes have been well studied, and considerable knowledge of the properties of the corresponding enzymes has been gained. It was shown that the dszC gene encodes a sulfide/sulfoxide mono-oxygenase (7, 23, 28), which catalyzes the stepwise S oxidation of DBT via DBT 5-oxide (DBTO) to DBT sulfone (DBTO2). DBTO2 is subsequently converted to 2-(2'-hydroxyphenyl)benzene (HBP) sulfinate (HBPSi-) by the product of the dszA gene, after which HBPSi- is desulfinated to give HBP and sulfite by the aromatic sulfinic acid hydrolase encoded by dszB (28).

The desulfurization genes have often been shown to be conserved among Rhodococcus species (5). Several strains of R. erythropolis, i.e., SY1 (29, 30), D-1 (16), Ni-36, Ni-43, and QIA-22 (53), have been reported to perform a nondegradative DBT-desulfurizing reaction, probably through the same metabolic pathway as that of strain IGTS8. Also, species from other bacterial genera, including Brevibacterium sp. strain DO (49), strains identified as Arthrobacter spp. (22, 40), thermophilic Paenibacillus strains (21), and, more recently, Gordona sp. strain CYKS1 (34), are able to convert DBT to HBP. However, the exact desulfurization mechanisms used by these organisms have remained relatively unexplored (40).

In spite of the recent knowledge about the desulfurization ability of pure and mixed cultures obtained from oil-polluted environments (Duarte et al., unpublished observations), no studies regarding the presence and diversity of oil desulfurization genes or microbial populations in polluted-soil habitats have been performed. This study aimed to determine the bacterial community structure as well as the prevalence and diversity of dsz genes in soil samples polluted with sulfurous oil in the open environment as well as in microcosms containing soil treated with petroleum supplemented with DBT. The bacterial community structure of the soil samples was analyzed by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rDNA fragments generated from soil DNA (14, 15, 25, 27) to obtain information about the effects of different levels of sulfurous oil on soil bacterial communities. Furthermore, the occurrence and diversity of the dszA, dszB, and dszC genes in oil-polluted soil samples were assessed via PCR with specific primers, in conjunction with DGGE analysis and sequencing. Finally, the two data sets were linked via the detection of dsz genes in isolates from enrichment cultures that matched the responders identified by molecular means in DGGE profiles.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Bacterial strains and media. Cells of R. erythropolis strain IGTS8 were maintained in basal salts medium (BSM) (7) containing (per liter) 2 g of glycerol, 4 g of NaH₂PO₄ · H₂O, 4 g of K₂HPO₄ · 3H₂O, 2 g of NH₄Cl, 0.2 g of MgCl₂.6H₂O, 0.001 g of CaCl₂ · 2H₂O, and 0.001 g of FeCl₃ · 6H₂O) supplemented with 0.1 mM DBT as the sole sulfur source (added from a stock containing 10 mM DBT in ethanol). For plating, solidified (1.5% agar) BSM-DBT was used. Cells of Escherichia coli DH5 and Paenibacillus azotofixans ATCC 35681 were grown overnight in Luria-Bertani medium (38) at 30°C (200 rpm) and in TBN medium (39) at 32°C, respectively.

Field soil samples. Oil-contaminated soil samples (A soil; silt loam, pH 5.0) were collected from a grass-covered petroleum storage area in the harbor of Amsterdam, The Netherlands, with an oil pollution history of 50 years. The crude oil that had entered the soil contained sulfur levels of up to 5% (R. Veenstra, personal communication). Sampling was done randomly in blocks (52). Four random samples were taken from the top layer (10 cm) of each of six plots in the area, which had different degrees of oil contamination (four characterized as low and two characterized as high). The individual samples from each plot were bulked and mixed, generating a total of four composite samples for low-pollution sites and two for high-pollution sites. The levels of oil in the low-pollution sites, denoted LP1 to LP4, averaged 710 mg kg¹ of soil, and those in the polluted sites, P1 and P2, averaged 5,500 mg kg¹ of soil (R. Veenstra, personal communication). The average total S and sulfate S contents determined by standard soil analysis techniques (Analytico, Lunteren, The Netherlands; NEN norms 7322 and 6654) were 840 and 240 mg kg¹, respectively, in the low-pollution soil and 920 and 350 mg kg¹, respectively, in the high-pollution samples, whereas 426 mg of organic S kg¹ was found in an adjacent, non-oil-polluted area of the soil. Hence, 600 and 670 mg of organic S kg¹ were estimated to be present in the low-pollution and high-pollution sites, respectively, of which, respectively, 174 and 246 mg kg¹ were hydrocarbon bound.

Soil microcosm experiment. The effect of DBT-containing petroleum in FSL soil on the indigenous bacterial communities was assessed in microcosms. Thus, to mimic a spill of sulfurous oil, duplicate 100-g FSL soil portions with soil moisture contents of around 20% were treated with 5 ml of petroleum (containing <0.4% S) supplemented with 200 mg of DBT and the soil-petroleum mixture was carefully homogenized using a sterile spatula. Control soil portions were left untreated. All soil portions were placed in plastic cups to yield individual soil microcosms. The microcosms were incubated in the dark at 20°C and sampled periodically, i.e., after zero (3 h), 7, 14, 30, 60, and 90 days. Samples were subjected to cultivation-based and direct molecular analyses of bacterial diversity and the prevalence of dsz genes, as described below.

Enrichment cultures from the polluted field and treated microcosm soils. Soil samples from the highly polluted field sites (A soil; P1 and P2 plots) as well as samples of the petroleum-DBT-treated FSL soil microcosms after 15 days of incubation were selected for enrichment of DBT degraders. Both types of sample showed a prevalence of dszB genes by direct molecular assessment of soil DNA. Five-gram portions of soil were added to flasks containing 95 ml of BSM supplemented with 1 mM DBT as the sole source of sulfur. Flasks were incubated on a rotary shaker (150 rpm) at 20°C for 3 days or until turbid. Then, fresh flasks containing BSM-DBT were inoculated with 10% (first transfer) or 1% inocula (subsequent transfers), and the procedure was repeated (total of three transfers). Samples from each enrichment culture were subjected to analyses of culturable bacteria, of DBT utilizers, of dsz genes, and of the molecular community profile based on directly extracted DNA.

Enumeration of culturable bacterial communities in soil and screening for the presence of dsz genes. To assess the size of the culturable bacterial populations in soil, 10-g portions of each soil sample were mixed with 95 ml of 0.1% sodium pyrophosphate and 10 g of gravel and shaken for 20 min at 200 rpm. Serial 10-fold dilutions of these suspensions were plated on 10% strength tryptone soy agar (0.1×TSA; BioMérieux, Marcy l'Etoile, France) supplemented with cycloheximide (50 µg/ml) to enumerate total indigenous bacterial CFUs and onto BSM agar (7) supplemented with 0.1% DBT to count bacteria able to grow with DBT as the sole sulfur source. Plates were incubated at 27°C, and colonies were enumerated after 48 (0.1× TSA) or 72 h (BSM-DBT).

Isolation of strains and analysis of isolates. Colonies from BSM-DBT plates that revealed the presence of dsz genes were purified by streaking on the same medium. Stocks for storage at 80°C were then prepared from single colonies. Isolates thus obtained were subjected to analyses of their 16S rDNA sequence, to fatty acid methyl ester (FAME) analysis using the MIDI microbial identification system, and to assays of their DBT-desulfurizing capacity. For the last analysis, a presumptive assay performed in microtiter plates assessed the release of fluorescent compounds from DBT (o,o'-biphenol or 2-hydroxybiphenyl) (53). After 24 h of incubation in BSM-DBT at 30°C, cultures that were positive for fluorescence under UV were analyzed by gas chromatography and high-pressure liquid chromatography (HPLC), as described by Duarte et al. (submitted for publication). Briefly, 25-ml cultures in BSM-DBT grown for 3 to 5 days at 30°C were spun down as described previously (16). The culture broth was then acidified to pH 2.0 and extracted with 0.8 volume of ethyl acetate. An aliquot of the ethyl acetate layer was removed and centrifuged, and 5 µl of supernatant was analyzed by gas chromatography (GC-14A; Shimadzu, Kyoto, Japan). HPLC analysis was performed using a 4-µm-pore-size phenyl Novapak column (Waters, Milford, Mass.); DBT was detected at 233 nm.

DNA extraction from strains. An aliquot (1.5 ml) of an overnight culture was centrifuged for 2 min (12,000 × g), washed with Tris-EDTA (TE) (38) buffer, and centrifuged again, and the pellet was resuspended in 500 µl of TE buffer plus 0.5 g of glass beads (0.1 mm in diameter). After bead beating for 30 s, 30 µl of 10% sodium dodecyl sulfate (SDS) and 500 µl of Tris-buffered phenol (pH 8) (38) were added. The mixture was then centrifuged for 10 min in an Eppendorf centrifuge at 14,000 rpm, and the aqueous phase was extracted once with phenol-chloroform-isoamyl alcohol (25:24:1) and once with chloroform-isoamyl alcohol (24:1). Finally, the DNA was precipitated with 0.6 volume of isopropanol (5 min, room temperature), centrifuged (12,000 × g; 10 min), and washed with 70% ethanol. The pellet was air dried and resuspended in 50 µl of TE buffer. DNA concentrations in the purified extract were determined with a GeneQuant apparatus (Pharmacia, Uppsala, Sweden).

DNA extraction from soil and enrichment cultures. Direct extractions of total microbial DNA from soil samples and from enrichment cultures were performed using a rapid bead beating protocol (51). Briefly, 2 g of soil, or the bacterial pellet from a 95-ml enrichment culture, plus 5 ml of 120 mM phosphate buffer and 3 g of glass beads (0.11 mm in diameter) were subjected to three 90-s bead beating steps followed by the addition of 180 µl of SDS (20%). The resulting suspensions were subjected to extractions with equal volumes of Tris-buffered phenol and chloroform-isoamyl alcohol (24:1), precipitation with ethanol, and resuspension of the pellet in TE buffer (38). Subsequent steps consisted of purification with CsCl (51) to precipitate impurities, followed by precipitation of the DNA with isopropanol, precipitation with potassium acetate (20 µl of an 8 M solution per 100 µl), precipitation with 0.6 volume of isopropanol, and, finally, purification over resin spin columns (Wizard DNA cleanup system; Promega, Madison, Wis.). Soil DNA was visualized on 0.8% (wt/vol) agarose gels (38) to check its purity and molecular size. The final DNA extracts obtained from all soils were amplifiable by PCR using 1 µl of extract (10 to 20 ng of DNA) per 50-µl reaction volume.

PCR. All PCR amplifications were performed in a thermal cycler (model 480; Perkin-Elmer/Cetus, Nieuwerkerk, The Netherlands). The PCR mixtures were prepared with 1 µl of target DNA (ca. 10 to 20 ng), 5 µl of 10× Stoffel buffer (10 mM Tris-HCl [pH 8.3], 10 mM KCl), 200 µmol of each deoxyribonucleoside triphosphate, 3.75 mM MgCl₂, 20 pmol of each of the appropriate primers, 1% (vol/vol) formamide, 0.25 µg of T4 gene 32 protein (Boehringer, Ingelheim, Germany), 5 U of Taq DNA polymerase, and the Stoffel fragment (Perkin-Elmer/Cetus) in a 50-µl final volume. T4 gene 32 protein was added since it improved the efficiency of amplification of targets in soil DNA (45). The reaction mixtures were overlaid with mineral oil (Sigma, Zwijndrecht, The Netherlands). A hot-start procedure (5 min, 94°C) was used before the enzyme was added to prevent aspecific annealing of the primers (4). Negative controls (PCR mixture without added target DNA) were included in all PCRs.

DGGE analysis. Denaturing gels (6% polyacrylamide) were prepared and run with 0.5× TAE buffer (20 mM Tris-acetate [pH 7.4], 10 mM sodium acetate, 0.5 mM disodium EDTA) using a phorU2 apparatus (Ingeny, Leiden, The Netherlands). These gels contained 25 to 75% or 35 to 65% gradients of urea and formamide solution (100% is 7 M urea plus 40% [vol/vol] formamide deionized with AG501-X8 mixed-bed resin [Bio-Rad, Veenendaal, The Netherlands]) for products generated with the bacterial 16S rDNA-based primers or with the dszA, dszB, and dszC primer pairs, respectively. DGGE was performed for 16 h at 60°C and 100 V. After electrophoresis, gels were stained for 30 min with 1 µl of SYBR Green I (Molecular Probes, Leiden, The Netherlands) stock solution diluted in 10 ml of 1× TAE (pH 8.3) and photographed under UV ilumination.

Cloning and sequencing of 16S rDNA and dsz PCR fragments. PCR products obtained from soil, enrichment culture, or bacterial DNA were resolved via DGGE, and selected bands were either cloned directly or reamplified following scratching or a crush-soak protocol (38) (only bands from denaturing gels), purified, and cloned into the pCRII cloning vector (TA cloning kit, Invitrogen, Leek, The Netherlands). The nature of the bands obtained from DGGE gels was confirmed using PCR-DGGE. Competent E. coli Inv-alfa cells were transformed according to the protocol of the cloning kit. Plasmid DNA was then extracted from randomly picked recombinant clones as described previously (38) and purified using Wizard resin spin columns (Promega). This DNA was used as the template in sequencing reactions by applying the Thermo-Sequenase fluorescently labeled primer cycle sequencing kit, with 7-deaza-dGTP (Amersham, `s Hertogenbosch, The Netherlands) and an automatic sequence analyzer (ALF DNA sequencer; Pharmacia).

Analysis of sequence data. DNA sequence data were analyzed by using FastA of the GCG (University of Wisconsin Genetics Computer Group) (8) software via the CAOS/CAMM center (University of Nijmegen). New sequences obtained were further aligned to database sequences by using BLAST-N (1). The sequences were also checked for chimeric molecules by using the CHECK_CHIMERA tool of the Ribosomal Database Project (26).

Nucleotide sequence accession numbers. The sequences obtained in this study are available from GenBank under accession no. AF165524 through AF165526 (three selected bands from DGGE of A soil); AF322027 through AF322032 (six bands sequenced from soil enrichments); and AF322033 through AF322042 (10 of 37 sequences representative of groups I, II, and III).

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Enumeration of bacterial populations in oil-polluted field soil. The numbers of culturable bacteria detected in the field soil samples with different degrees of oil pollution are shown in Table 2. Total viable counts averaged 2.5 × 10⁶ CFU g¹ of soil, and there were no significant differences between counts obtained from the plots with different oil pollution levels. In contrast, the CFU counts on BSM agar supplemented with DBT were 10- to 100-fold lower than the total CFU counts, suggesting that organisms able to utilize DBT as a sulfur source made up only 1 to 10% of the total culturable bacterial populations. There was no significant difference in the counts of DBT utilizers between the plots with high and low oil pollution levels.

PCR-DGGE profiling of bacterial diversity in oil-polluted field soil. About 10 to 15 µg of DNA per g of soil was obtained for all samples from the A soil. Following purification, this DNA was amplifiable with the 16S rDNA-based bacterial primers. The 16S rDNA amplicons generated from the field soil samples were then subjected to DGGE profiling (Fig. 1A). A decrease in the number of dominant bacterial types detectable by DGGE was observed with increasing oil content, with a consistent concentration of amplicons in a few bands. The soil samples with oil contents averaging 710 mg of oil kg¹ of soil (lanes 1 to 4) had very similar profiles, with about 20 dominant bacterial types per lane. Samples P1 (lane 5) and P2 (lane 6), with oil contents averaging 5,500 mg of oil kg¹ of soil, revealed lower numbers of dominating bacterial types (respectively, about 14 and 10 bands). The clustering of the patterns obtained clearly indicated the existence of two clusters, one containing all plots with a low pollution level and the other one encompassing the two plots with a high pollution level (Fig. 1B).

View larger version (176K): [in this window] [in a new window]   FIG. 1.   PCR-DGGE analysis of the bacterial communities in the oil-polluted A soil samples. (A) Molecular community profiles on denaturing gel (25 to 65% denaturants; SybrGold stained). Lanes: 1 to 4, profiles obtained from plots LP1 through LP4, respectively (710 mg of oil per kg of soil); 5 and 6, profiles from plots P1 and P2, respectively (5,500 mg of oil per kg of soil). Numbers 1 to 3 to the side indicate bands 1 to 3, which were selected for sequence analysis. (B) Dendrogram of gel obtained via 1/0 clustering using UPGMA (Dice coefficient of similarity) followed by treeing using the Treecon program.

Enumeration of bacterial populations in soil microcosms treated with DBT-containing petroleum. The numbers of culturable bacteria detected in the FSL microcosms over time are shown in Table 3. The total CFU counts obtained on 0.1× TSA were generally on the order 10⁶ to 10⁷ CFU g¹ of dry soil. These counts were roughly stable over time, and they did not change significantly as a result of the treatment with petroleum-DBT. Counts on BSM agar supplemented with 0.1% DBT were also relatively stable over time in the untreated microcosms and were generally 5- to 20-fold lower than those obtained on 0.1× TSA. In the treated microcosms, these counts increased to roughly 50% of the total CFU counts (30 days), after which they dropped again to levels equalling those in the untreated microcosms. Unfortunately, direct-colony PCR and hybridization of colonies grown on the plates with 0.1× TSA and BSM plus DBT and the appropriate dsz gene-specific primer-probe combinations failed to unequivocally identify organisms that carried the dszA, dszB, or dszC gene.

PCR-DGGE profiling of bacterial diversity in FSL soil microcosms treated with DBT-containing petroleum. DGGE analysis of the PCR-amplified 16S rDNA fragments from the microcosm study provided a picture of relative stability of the bacterial community profiles both over time and between the treatments. Thirty to 45 bands were visible in all community profiles. Figure 2 shows the dendrogram obtained after the clustering of the profiles, revealing their relatedness. The profiles generated with the untreated soils were all very similar to each other, at >90% similarity, whereas the profiles generated with the treated soils fell in a separate cluster at roughly 10% difference. This cluster showed a divergent branch encompassing all day-60 and day-90 samples from the treated soil. Furthermore, there was no clear indication of a decreased "diversity" (richness and evenness of dominant bacterial types) over time due to the treatment, as a reduction in the numbers of bands was not found.

View larger version (20K): [in this window] [in a new window]   FIG. 2.   Dendrogram of PCR-DGGE analysis of the bacterial communities in FSL soil treated with petroleum-DBT in microcosms. The dendrogram was constructed by UPGMA (Dice coefficient of similarity), applied to a 1/0 matrix based on the primary gel. The tree was inferred using the Treecon program. t, time; d, days.

Screening for dsz genes in control and oil-polluted soils. The prevalence of dszA, dszB, and dszC genes was first assessed in a suite of soils with no known history of pollution with sulfurous oil. Total microbial community DNA from the ELS, FOS, C, V, and SP soils did not yield any product with the dszA, dszB, or dszC primer pair, whereas genomic DNA of R. erythropolis strain IGTS8 (10² to 10³ genome equivalents per reaction) was readily amplified with the respective primers in these soil DNA backgrounds. However, products of the same size as those obtained with DNA from R. erythropolis IGTS8, i.e., about 422 bp, were consistently obtained with the dszB detection system when applied to the unpolluted FSL soil. This dszB product hybridized under high-stringency conditions (indicating >90% similarity) with the dszB probe.

Enrichment cultures: selection of bacterial types and presence of dsz genes. Enrichment cultures were set up using the A (sample P2) and treated FSL soils (15 days) in BSM-DBT to more strongly select for DBT-desulfurizing organisms. Analysis of the bacterial populations in these enrichment cultures via PCR-DGGE clearly showed the selection of a limited number (5 to 10) of dominant bacterial types from the highly complex soil bacterial communities (Fig. 3; results for FSL soil enrichments are shown). Whereas the communities selected still shifted their compositions along the first two enrichments, there was considerable stability in the community profiles between enrichment cultures 3 and 4. As judged from the community profiles, the selection of dominant bands was further quite consistent among the replicate enrichment cultures (Fig. 3) and partly consistent among soil sample types (not shown). Six major bands from the profiles of the fourth cultures were successfully isolated for sequence determinations (Fig. 3). Analysis of the sequences of these bands provided evidence for the putative selection of the following bacterial types (from top to bottom; the closest neighbors from the database are indicated in the figure legend [similarity levels, >95%]): Buttiauxella brennerae DSM9396, Stenotrophomonas maltophilia, Pseudomonas rhodesiae, Pseudomonas putida, Pseudomonas aureofaciens, and R. erythropolis.

View larger version (98K): [in this window] [in a new window]   FIG. 3.   PCR-DGGE analysis of the bacterial diversity in enrichment cultures of FSL soils treated with petroleum supplemented with DBT (duplicate day-15 microcosms used). Lanes: M, marker (products of, from top to bottom, Enterobacter cloacae BE1, Listeria innocua ALM105, Rhizobium leguminosarum bv. trifolii R62, Arthrobacter sp., Burkholderia cepacia P2); 1, FSL soil, untreated; 2, FSL soil, treated, time zero; 3, FSL soil, treated, 15 days (replicate 1); 4 to 7, enrichment culture from FSL, treated, 15 days (replicate 1), from first to fourth culture, respectively; 8, FSL, treated, 15 days (replicate 2); 9 to 12, enrichment culture from FSL, treated, 15 days (replicate 2), from first to fourth culture, respectively; 13, band 3 (sequence similarity to R. erythropolis); 14, band 7 (similar to P. aureofaciens); 15, band 2 (similar to P. putida); 16, band 6 (similar to P. rhodesiae); 17, band 5 (similar to S. maltophilia); 18, band 4 (similar to B. brennerae DSM9396); 19, product of strain M39; 20, product of strain M41; 21, product of strain A36; 22, product of strain A69; 23, product of strain A96.

View larger version (14K): [in this window] [in a new window]   FIG. 4.   Cluster analysis of 37 dszA sequences obtained from the A and treated FSL soils, as well as from enrichment cultures and isolates obtained from these soils. I, II, and III, clusters I, II, and III, as described in the text. Bar, 0.05 distance. IGT8, canonical sequence of R. erythropolis IGTS8; A96 and A69, strains obtained from A soil; M39, M41, and M42, strains obtained from treated FSL soil; A1 and A2, sequences directly amplified from A soil (replicate sample number/sequence number); AE/d, sequences directly amplified from enrichment cultures of A soil (number, enrichment culture; d, days of incubation); F+DBT/w, sequences obtained from petroleum-DBT-treated FSL soil incubated 1 or 2 weeks (w); FE/d, sequences obtained from enrichment cultures from treated FSL soil.

Screening for and analysis of dsz genes obtained from soils, enrichments, and isolates. Initial analysis of the sequences of the PCR products generated with the dszA, dszB, and dszC primers using total microbial community DNA from the A soil sample (plot P2) revealed that all three products (one clone per gene analyzed) from this soil showed 98% similarity to their counterpart sequences from R. erythropolis IGTS8 (not shown).

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

An understanding of the ecology of microbial communities in soil requires knowledge on how these communities are structured under prevailing and changing environmental conditions. Since chemical pollutants can exert drastic effects on microbial diversity in soil, there is a need to understand the response of the indigenous communities to this trigger with respect to (i) putative shifts in the community structure caused by selective pressure and (ii) the selection of functional genes important for the catabolism of soil pollutants. The approach taken in this study represents an attempt to simultaneously address the possible selective effects of sulfurous hydrocarbon compounds in soil on bacterial community structure and on the prevalence of selected genes involved in hydrocarbon desulfurization pathways, i.e., those of the dsz operon. With respect to the sources of sulfur apparently available to bacteria in the soil systems studied (in particular the A soil), it is evident that organically (oil-) bound S made up a substantial part of the total S measured in the system. However, the data also showed that this fraction was not the only available source of S. Aspects of spatial location of bacteria in the soil, in particular in relation to the oil moiety, can have a drastic effect on the selective pressure sensed by the bacterial communities that are locally present. Hence, it is difficult to ascertain the relative contributions of the oil- or organically bound S to the in situ bacterial S provision. Nevertheless, it was thought to be likely that, within the bacterial communities with affinity to the ecological niche offered by oil, organisms that had the capacity to desulfurize DBT might prevail. It is obvious that by focusing on the dsz genes, this study excluded organisms with other desulfurizing capacities.

PCR-DGGE using primers based on conserved regions of the 16S rRNA sequence yields complex molecular profiles when used to analyze the soil microbiota (10, 14, 15, 25). The profiles thus obtained reflect the composition of the dominant soil microbiota, which includes the nonculturable fractions (10, 14). The DGGE patterns obtained in this study with total community DNA from soils with different levels of oil are to be interpreted as a first approximation to showing shifts in the composition of the dominant bacterial populations related to the oil pollution levels. Whereas the numbers of culturable bacteria present in these samples were not drastically affected, the molecular profiling data (Fig. 1) indicated that specific bacterial populations were selected with increasing oil levels, most likely by increasing their numbers at the expense of other (sensitive) bacterial groups. The analysis of the sequences of three selected DGGE bands revealed that these were <97% similar to any database sequence, suggesting that none was typical for any known bacterial species (43). However, the closest relatives often fell in bacterial groups, such as Arthrobacter, Rhodococcus, and Beggiatoa spp. and bacterium SRang2.5, which are potentially able to grow and metabolize in contaminated environments and to desulfurize DBT (16, 20, 22, 30, 53). These direct molecular data thus provide an indication as to the nature of the bacterial communities selected under long-term sulfurous hydrocarbon pressure in the A soil without conclusively showing the involvement of the putative organisms in any steps of the dissimilatory pathways.

The response of the bacterial community in soil to sulfurous hydrocarbons was evident in the soil microcosm experiment. In the first place, the counts of putative desulfurizing organisms were, albeit temporarily, affected as a result of the treatment, indicating a shift to growth in these communities. Although it is theoretically possible that the petroleum in soil affected plate counts by influencing desorption of cells from soil, from an analysis of the data it was felt that this effect was probably minor. In the second place, the molecular bacterial community profiles obtained from the FSL soil also showed an effect of the petroleum-DBT treatment, as evidence for the growth of an organism responding to the treatment was already found after 7 and 15 days. The effect of the treatment became more apparent over time, as the community profiles showed increasing divergence from those in untreated systems. However, a considerable level of overall similarity between all profiles persisted even after 90 days of incubation. This high similarity was illustrative of the difficulty often encountered in PCR-DGGE profiling with eubacterial primers in assessing short-term (short-lived) bacterial community shifts in soil (14). The discrepancy between the inferences made from the molecular community profiles observed in the A soil, with over 50 years of pressure by sulfurous oil, and the inferences made from those observed in the treated FSL soil, with a maximum of 90 days of such pressure, can also be related to this time factor.

This study further showed that the genes of the dsz operon are not easily found in (agricultural) soils with no known history of pollution with sulfurous hydrocarbons (Table 4). This suggests that agricultural soils do not commonly select organisms by virtue of their carrying the dsz operon. The finding of evidence for the occurrence of dszB in the pristine FSL soil was, therefore, surprising. A possible explanation is that natural, or even anthropogenic, sulfur-containing organic substrates, such as sulfonates and sulfate esters (19), might have selected for organisms carrying dsz genes, as suggested for organisms carrying tfd genes, which were also found to be present in soils of noncontaminated ecosystems (13, 17, 48).

On the other hand, relatively conserved dsz genes were shown to occur in the two soils containing sulfurous hydrocarbons obtained from geographically distinct regions in The Netherlands. dszA, dszB, and dszC gene sequences were amplified from DNA obtained from the field (A) soil and petroleum-DBT-treated microcosm (FSL soil) samples (albeit after enrichment). The conservation of these genes in A soil and the resemblance of their sequences to the canonical dszA, dszB, and dszC sequences were initially indicated by DGGE analysis of the PCR fragments generated from soil. PCR-DGGE applied to these genes was a first approach to analyzing their diversity in soil, as it allows the rapid, sequencing-independent detection of similarities and differences between sequences of the same functional genes (36, 54). The conservative nature of the genes was then confirmed by sequencing three clones, representative of the dszA, dszB, and dszC genes, obtained directly from the highly polluted A soil, as these showed >98% similarity with the respective genes from R. erythropolis IGTS8. Moreover, a more thorough study of the diversity of the dszA gene also showed it to be relatively conserved among 37 sequences recovered from soils, enrichments in BSM-DBT, and strains. Sequences obtained from the A and FSL soils, from soil enrichments, and from bacterial isolates fell into three groups with >90% internal similarity. Surprisingly, the highly polluted A soil also contained a group of sequences with limited but internally highly consistent divergence from the canonical (IGTS8) sequence. This potentially novel member group of the dszA gene family clearly warrants further study. An analysis of the putative amino acid sequence of the gene product of one representative sequence already indicated that only four changes seemed to have taken place, with virtually no change in protein hydrophobicity, compared to the canonical sequence. At this point, one can only speculate about the significance of the finding of these divergent putative gene products of dszA sequences directly from polluted soil, possibly originating from a difficult-to-culture, as yet uncultured, or even nonculturable organisms. Clearly, there is a need to obtain isolates carrying these genes for a study of their functionality.

The conserved nature of the dsz genotype has previously also been reported for Rhodococcus strains isolated from different geographic locations (5). In this study, we extend this observation, albeit in a nuanced form, to soils with oil pollution pressure. The results indicate that populations of bacteria related to Rhodococcus and harboring the dsz operon are present and selected in oil-contaminated soils. These bacteria can be isolated via enrichments in BSM-DBT, and some of them show vigorous desulfurization of DBT. In addition, evidence for the presence of uncultured organisms with divergent dszA gene sequences in the oil-polluted A soil was found.

The data obtained with isolates from the enrichment cultures clearly provided a link between the direct molecular observations on bacterial community structure in the polluted soils and enrichments from these and the data on the prevalence of genes of the dsz gene cluster. A number of key bacterial isolates whose specific 16S rDNA PCR products matched bands in the directly obtained molecular community profiles that represented putative responders were thus found to contain the dsz operon and to be capable of vigorously desulfurizing DBT. These isolates all fell into the R. erythropolis cluster. On the other hand, other isolates selected by the treatment, without clear desulfurizing capacity, fell into other taxons. It is thus likely that the treatment applied to soil and enrichment cultures selected for bacterial consortia in which the key desulfurization function was represented by the R. erythropolis-like organisms, whereas other functions were present in other coselected taxa. The presence of S. maltophilia as a consistent member of the selected consortia is remarkable, as this organism can be regarded as an avid colonizer of disturbed or stressed ecosystems and thus might play a role in hydrocarbon degradation processes in soils. S. maltophilia was also a member of two consortia with DBT-desulfurizing capacity that have previously been obtained from crude oil fields in Brazil (G. F. Duarte et al., submitted for publication). Even without a full understanding of all possible interactions between the members of the consortia, it can be said that those identified likely play an important role in hydrocarbon degradation and desulfurization processes in soil. Given the data provided in this paper, future work in this area should more specifically address the population dynamics and functional aspects of the response by these organisms to sulfurous oil in soil.