INTRODUCTION

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Contamination of estuarine sediments by highly hydrophobic, low-availability, toxic, organic contaminants has become a significant environmental concern. For example, industrial activities in the New Jersey/New York Harbor estuary have resulted in widespread contamination, and the management of contaminated sediments has become a significant problem. Polycyclic aromatic hydrocarbons (PAHs) are of particular concern because of their toxic, mutagenic, and carcinogenic properties (32). PAHs have also been found to bioaccumulate in aquatic organisms (29). Although PAHs are persistent, mainly due to their high hydrophobicity, a variety of microorganisms (bacteria and fungi) can degrade certain PAHs (for reviews, see references 6 and 44). There is thus a significant interest in studying microorganisms present in contaminated environments as a means for bioremediation.

The fate of PAHs and other organic contaminants in the environment is associated with both abiotic and biotic processes, including volatilization, photooxidation, chemical oxidation, bioaccumulation, and microbial transformation. Microbial activity has been deemed the most influential and significant cause of PAH removal (6). Numerous studies have been conducted on microbial consortia and enrichment, and several diverse genera of bacteria have been isolated (for a review, see reference 6). Recent work has indicated that the stimulation of microbial activity in the rhizosphere of plants can also stimulate biodegradation of various toxic organic compounds (1, 4, 37, 39, 41, 45, 47). This general "rhizosphere effect" is well known in terrestrial systems. The rhizosphere soil has been described as the zone of soil under the direct influence of plant roots and usually extends a few millimeters from the root surface and is a dynamic environment for microorganisms (7). The rhizosphere microbial community is comprised of microorganisms with different types of metabolism and adaptive responses to variation in environmental conditions. The production of mucilaginous material and the exudation of a variety of soluble organic compounds by the plant root play an important part in root colonization and maintenance of microbial growth in the rhizosphere. Microbial activity is thus generally higher in the rhizosphere due to readily biodegradable substrates exuded from the plant (35). Recent studies indicate that degradation of PAHs and polychlorinated biphenyls (PCBs) in soils can be enhanced by plants (37, 47). In the case of PCBs, plant compounds have been shown to induce the cometabolic degradation of PCBs (15, 16).

The activities of microbial communities in sediments and tidal marshes are important in improving sediment quality (pollution degradation), in the biological transformations of major nutrients influencing their availability for plant growth, and in their role in binding sediment particles to improve rooting medium structure. Wetlands have a higher biological activity than many other ecosystems, and they support enhanced biotransformation of toxic chemicals (25). Experiments with Spartina salt marsh mesocosms indicated that biodegradation of oil was influenced by flooding and fertilization conditions (28, 49).

There is extensive information on PAH degradation by both gram-positive and gram-negative bacteria, mostly isolated from soil environments. There is, however, little information on the microbial communities in salt marsh ecosystems and their role in the biodegradation of toxic organic contaminants. In addition, little is known about the ability of these microorganisms to degrade polyaromatic compounds. In the present study, we isolated PAH-degrading bacteria from the salt marsh rhizosphere. We were particularly interested in isolating spore-forming bacteria for two reasons. First, the ability of a bacterium to produce spores and therefore survive adverse conditions would be an attractive feature for remediation of contaminated sites. Secondly, only a few studies have examined the degradation of PAHs by spore-forming bacteria, and none, to our knowledge, involve plant-microbe interactions. Therefore, isolation of spore-forming, PAH-degrading bacteria and subsequent genetic studies of their degradation pathways may lead to the discovery of novel genes involved. In addition, we wanted to evaluate the biodegradation potential of select isolates and determine if reinoculation of the rhizosphere would enhance degradation of contaminated marine sediment.

	MATERIALS AND METHODS

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Plant and sediment samples. Salt marsh plant samples were obtained at two sites. The first site, considered to be uncontaminated, was the University of Delaware Marine Station at Lewes, Del. A total of five different plant samples were collected and included Distichlis spicata, Juncus gerardi, Phragmites australis, Spartina alterniflora, and Sporobolus airoides. A minimum of two plants of each species was obtained. The second site was Piles Creek, a contaminated tributary of Arthur Kill, in Linden, N.J. Estuarine sediment and S. alterniflora plant samples were obtained from the intertidal area. A total of three sediment and three plant samples were taken from separate areas within 50 feet of each other. The plant and sediment samples were transported on ice back to the laboratory where they were stored at 4°C until analyzed.

Bacterial strains, plasmids and media. Pseudomonas putida NCIB 9816-4 (9), Comamonas testosteroni GZ42 (18), Sphingomonas yanoikuyae B1 (14), and Mycobacterium sp. strain PY01 (Y.-S. Oh and G. J. Zylstra, unpublished data) were chosen as representatives of different genotypes of PAH-degrading organisms. The genes have previously been cloned from these strains into Escherichia coli, and the cloned fragments were used as gene probes for Southern hybridization experiments. Plasmid pDTG112 contains the cloned nah genes from P. putida NCIB 9816-4 (40). A 3.6-kb SalI fragment contains three of the four genes for naphthalene dioxygenase: nahAa (reductase), nahAb (ferredoxin), and nahAc (dioxygenase large subunit) (42). Plasmid pGJZ1822, cloned from C. testosteroni GZ42, has a 6.7-kb SstI-XhoI fragment that contains the genes necessary for the first few steps in PAH degradation (19). Plasmid pGJZ1512, cloned from S. yanoikuyae B1, has a 567-bp SphI fragment that contains bphC encoding a meta-ring cleavage enzyme involved in PAH degradation (26). A 600-bp fragment containing a gene encoding a dioxygenase large subunit involved in PAH degradation by Mycobacterium sp. strain PY01 (J. F. Cigolini and G. J. Zylstra, Abstr. 99th Gen. Meet. Am. Soc. Microbiol., abstr. Q-180, 1999) was PCR amplified with the primers 5'-GTTGACCCGTGACGC-3' and 5'-CTCACTCAAGGCCGG-3'. The PAH-degrading strain C. testosteroni GZ39 was used as a control in hybridization experiments (18).

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Enrichment and isolation of PAH-degrading bacteria. Bacterial enrichment cultures were set up in cotton-plugged Erlenmeyer flasks containing 50 ml of MSB medium using naphthalene, phenanthrene, or biphenyl as the sole source of carbon. Plant rhizosphere and contaminated estuarine sediment were used as sources of the inoculum for the enrichment cultures. Rhizosphere samples were obtained by splitting open the plant root mass, so that the inner roots that had not come in contact with sampling or storage devices could be aseptically cut and collected. One gram of root material was gently shaken to remove loose sediment material and was added to each enrichment culture. Sediment samples were added directly to each enrichment culture using a 1-g subsample. A total of two enrichment cultures for each substrate were set up with each rhizosphere and sediment sample. One set of enrichment cultures was placed directly in a 28°C environmental chamber and shaken at 150 rpm. The second set of enrichments was heat treated for 10 min at 80°C before incubation to enhance isolation of spore-forming bacteria.

Phenotypic characterizations. Phase-contrast microscopy (×1,000 magnification) was used for the morphologic examination of bacterial cells. The strains to be tested were inoculated into 2 ml of TS broth and incubated on a rotary wheel at 37°C. The cells were examined microscopically for motility and morphology at 24, 48, and 72 h and were examined again microscopically after Gram staining.

FAME analysis. Whole-cell fatty acid analyses were performed on all of the PAH-degrading isolates by growing the cells at 28°C for 24 h on TSA plates. Cellular fatty acids were saponified, methylated, extracted, and analyzed by gas chromatography following the procedures given for the Sherlock Microbial Identification System (MIDI, Inc., Newark, Del.). Identification and comparison were made to the Aerobe (TSBA version 3.9) database of the Sherlock Microbial Identification System. The Dendrogram program of the MIDI software package was used to produce unweighted-pair matchings based on fatty acid compositions.

DNA preparation. Bacterial strains were grown on either TSA or MSB agar plates supplemented with naphthalene, phenanthrene, or biphenyl. Total genomic DNA was extracted as described by Wilson (48), with the exception of pretreatment with 1.5 mg of lysozyme for 2 h. With the exception of plasmid pDTG112, plasmid DNA was obtained using the QIAprep Spin Miniprep Kit (Qiagen Inc., Santa Clarita, Calif.) according to the manufacturer's directions. For plasmid pDTG112, the plasmid DNA was purified from the total DNA using equilibrium centrifugation in a CsCl-ethidium bromide gradient as described by Sambrook et al. (38). Restriction enzyme digests were performed on the isolated plasmids, and the desired fragments were purified using the Qiaquick gel extraction kit (Qiagen).

Restriction fragment length polymorphism (RFLP). Total genomic restriction enzyme digests were performed on all of the PAH-degrading isolates using either the BamHI, EcoRI, HindIII, or NotI enzyme as recommended by the supplier (Gibco-BRL, Madison, Wis.). Approximately 2 to 3 µg of each isolate was digested. Electrophoretic analysis of the digested DNA was done on a 0.8% agarose gel at 25 V for 16 to 20 h. The restriction enzyme digest patterns were visualized by UV light following ethidium bromide staining.

16S rDNA analysis. Sequence analyses of 16S ribosomal DNA (rDNA) were performed on several of the isolates by amplifying the 16S rRNA genes by PCR using the eubacterial primers 27f and 1522r (24). The PCR mixtures (100 µl) contained 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl₂, a 200 µM concentration of each deoxynucleoside triphosphate, 50 pmol of each primer, 2.5 U of Taq DNA polymerase (Gibco-BRL, Grand Island, N.Y.), and ~10 ng of DNA template. PCR amplification was performed using a Perkin-Elmer GeneAmp PCR System 2400 thermocycler (Applied Biosystems, Foster City, Calif.) programmed as follows: 1 min of denaturation at 94°C; followed by 25 cycles of 96°C for 1 min, 55°C for 1 min, and 72°C for 1 min; with a final extension at 72°C for 10 min. The amplified 1.5-kb PCR products were excised from a 1% agarose gel and purified using the QIAquick gel extraction kit (Qiagen) according to the manufacturer's instructions.

Southern hybridization using PAH gene probes. Southern blots were performed on all unique isolates, as determined by RFLP, using gene probes for four different types of PAH dioxygenases. This was accomplished by digesting 2 to 3 µg of genomic DNA from each of the tested isolates with EcoRI according to the manufacturers' directions. In addition, genomic DNA obtained from P. putida NCIB 9816-4, C. testosteroni GZ42, C. testosteroni GZ39, S. yanoikuyae B1, and Mycobacterium sp. strain PY01 was digested and used as positive and negative controls for each hybridization. The digests were run on 0.8% agarose gels in 40 mM Tris-20 mM acetate-2 mM EDTA buffer at 25 V for 16 to 20 h, stained with ethidium bromide, and visualized under UV light prior to transferring onto positively charged nylon membranes (Boehringer Mannheim, Indianapolis, Ind.) using a VacuGene XL Vacuum Blotting System (Pharmacia Biotech, Piscataway, N.J.) The transferred DNA was fixed to the nylon membranes by exposure to a UV cross-linker (Spectronics Corp., Westbury, N.Y.) using a 2× optimal cross-link (120 mJ cm²). The Southern hybridization protocol was performed using nonradioactive digoxigenin (Boehringer Mannheim) as recommended by the supplier. Gene probes were prepared from the PAH dioxygenase gene fragments by a nonradioactive random primed DNA-labeling method with alkali-labile digoxigenin 11-dUTP. Prehybridization and hybridization were performed at 37°C. Following hybridization, the nylon membrane was washed under low-stringency conditions (0.1× SSC [1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate] and 0.1% sodium dodecyl sulfate) at room temperature.

Pure culture transformation of PAHs in liquid medium. Several of the isolates were analyzed for their ability to utilize a variety of aromatic compounds, including naphthalene, phenanthrene, fluorene, pyrene, and biphenyl, individually and as a mixture, in MSB medium. The isolates tested included the gram-negative pseudomonads PR-N7, PR-N9, PR-N10, and PS-P2; the gram-positive non-spore formers PR-N15 and PR-P3; and the spore former PR-P1. Each isolate was tested in duplicate for each substrate or substrates over a time course of 11 days. Cells were grown in 100 ml of MSB containing 5 mM succinate, pelleted by centrifugation (4,000 × g, 10 min), washed with MSB, pelleted again, and suspended in fresh MSB to give a cell density of approximately 10⁷ cells ml¹ for use in transformation experiments. Sterile 7-ml serum vials containing the appropriate carbon source at a final concentration of 100 µM were added from hexane stock solutions, and the material was allowed to evaporate prior to the addition of 2 ml of washed cell suspension. In addition, two controls were added to the experimental design and included a no-cell control (MSB medium only) and a dead-cell control (MSB + 4% formaldehyde + cells). The samples were incubated at 28°C on a rotary shaker at 90 rpm, and the amount of substrate remaining was analyzed over time. At each time point two vials were taken, were extracted with 1 ml of hexane, and were analyzed for the appropriate aromatic substrate using a Hewlett-Packard 5890 Series II Gas Chromatograph using an HP-5MS column and equipped with a 5971 series mass selective detector. Prior to extraction, 20 µM 2-methylnaphthalene was added from a hexane stock solution as an internal standard. Results are reported as the percentage remaining relative to uninoculated controls after 11 days of incubation.

Microbial-sediment slurry biotransformation experiment. An aerobic slurry system was designed to determine the rate and extent of microbial transformation of contaminated sediment. The rhizosphere-associated phenanthrene-degrading strains PR-P1 and PR-P3 were grown to mid-log phase in TS broth and were harvested by centrifugation at 4,000 × g for 10 min. The cells were washed and pelleted by centrifugation twice using 1/10 salts (8) as the diluent, diluted into 1/10 salts, and inoculated at three different cell concentrations into a PAH-spiked sediment slurry.

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Nucleotide sequence accession numbers. The 16S rRNA sequences for the PAH-degrading isolates have been deposited in GenBank under accession numbers AF353676 to AF353705.

	RESULTS

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Isolation of PAH-degrading bacterial strains. The PAH-degrading strains isolated in this study are listed in Table 1. A total of 42 isolates were obtained from S. alterniflora plant and sediment samples collected from contaminated Piles Creek in New Jersey, using either naphthalene, phenanthrene, or biphenyl as the sole source of carbon. A total of nine isolates were obtained from the four different plant rhizospheres collected from Lewes, Del. However, no PAH-degrading isolates were obtained from the rhizosphere of P. australis from this site. The lower number and diversity of bacteria isolated from this site may be expected due to its uncontaminated nature. Heat treatment prior to enrichment and isolation was included for each of the substrates tested, which resulted in isolation of several spore-forming bacteria. The isolates were characterized on the basis of their morphologic and phenotypic properties and by using a combination of molecular techniques.

FAME analysis. The tentative identification and similarity index (SI) of all of the isolates using the aerobic (TSBA) library version 3.9 of the Microbial Identification System is shown in Table 1. A pairwise-comparison Euclidean-distance dendrogram based on whole-cell fatty acid compositions of the PAH-degrading isolates (except isolate PS-P1) was constructed, revealing that the isolates fall within three main groups (Fig. 1). Group 1 included the endospore-forming Paenibacillus, group 2 included the gram-positive nocardioforms, and group 3 included the gram-negative pseudomonads. One of the isolates, PR-P12, falling outside these groups was tentatively identified as Flavobacterium resinovorum (SI = 0.79) (Table 1). Isolate PR-P3 clustered within the Paenibacillus group but had the closest match to Arthrobacter oxydans (SI = 0.46). In addition, one of the isolates, PS-P1, was not identified on several attempts by the MIDI Microbial Identification System but was considered a member of the nocardioform group based on the presence of 10-methyl 18:0 fatty acid (tuberculostearic acid).

View larger version (20K): [in this window] [in a new window]   FIG. 1.   Euclidean-distance dendrogram based on the whole-cell fatty acid compositions of the sediment and rhizosphere PAH-degrading isolates. Sediment and rhizosphere isolates are denoted as PS and PR, respectively. In addition, strains isolated using naphthalene are indicated by an N, while phenanthrene- and biphenyl-enriched isolates are denoted by a P and B, respectively. *, isolate PR-P3 is an Arthrobacter sp.

Genomic restriction enzyme digests. RFLP analysis of total genomic DNA digested with BamHI, EcoRI, HindIII, and NotI enzymes was used as a screening tool to determine unique isolates. A large number of gels were performed encompassing all of the cultured isolates. The gels were examined visually, and an isolate was considered unique based on the presence of a unique banding pattern. Isolates which shared a banding pattern and had similar FAME profiles were grouped together, and a single isolate from the group was chosen as a representative strain for sequencing and Southern hybridization experiments. The RFLP data are summarized below. All of the six nocardioform isolates were found to be unique. Of the 12 original pseudomonad isolates, 10 were found to have different restriction enzyme banding patterns. Shared RFLP patterns were seen between the pseudomonad isolates PS-N4 and PS-N5 and between PS-P2a and PS-P2b. The large Paenibacillus group, containing 31 isolates, was found to have 19 isolates exhibiting unique RFLP patterns. Shared banding patterns were seen between the mucoid colony morphology Paenibacillus isolates PR-N5, PR-N11, PR-N2, and PR-N12. In addition, shared banding patterns were seen between the nonmucoid colony morphology Paenibacillus isolates PR-B2 and PR-B1; JG-N1, DS-N1, and JG-N3; PR-N19 and PR-N20; PR-N21 and PR-N22; PR-P1, PR-P2, and PR-P6; and PR-N13, PR-N3, and PR-N4.

16S rRNA gene analyses. Several of the unique isolates from each of the three major groups were further identified by partial sequencing of their 16S rRNA gene. In general, the 16S rDNA sequence analysis confirmed the identification based on fatty acid profiles. Within the Paenibacillus group the isolates identified as P. validus by FAME also had a high sequence similarity to P. validus. The smaller subgroup of isolates having a low SI to P. alvei, P. pabuli, or P. validus by FAME had a low similarity to P. validus based on their partial 16S rRNA gene sequence (Table 1). Strain PR-P3, which was an outlier within the Paenibacillus group, was identified with the closest match by both FAME and 16S rDNA analysis to A. oxydans. The norcardioform isolates PR-N14 and PR-N15 were identified as belonging to the genera Rhodococcus and Tsukamurella, respectively. The identification of the Pseudomonas isolates as P. putida and P. stutzeri by FAME was confirmed by 16S rDNA phylogenetic analysis. The outlying isolate PR-P12 was identified with a high 16S rDNA sequence similarity to Sphingomonas subarctica.

Southern hybridization with PAH gene probes. All of the unique isolates, as determined by RFLP, were tested for the presence of previously described genes for PAH dioxygenases. A large number of Southern hybridizations were performed, with much of the data revealing no hybridization results. Therefore, we present the data in text form with one figure exemplifying the nah gene hybridization results for isolates representing the three major bacterial groups (pseudomonads, nocardiofoms, and Paenibacillus).

View larger version (59K): [in this window] [in a new window]   FIG. 2.   Southern hybridization of selected isolates with the nah genes for naphthalene degradation from P. putida NCIB 9816-4. EcoRI-digested genomic DNA was probed with a 3.5-kb SalI fragment from P. putida NCIB 9816-4 containing the genes for naphthalene dioxygenase. M1 and M2 are the digoxigenin-labeled and high-molecular-weight markers, respectively. Lanes 1 to 4 are the pseudomonad isolates PR-N7, PR-N9, PR-N10, and PS-P2; lane 5 is the sphingomonad isolate PR-P12; lanes 6 and 7 are the nocardioform isolates PR-N14 and PR-N15; lane 8 is Arthrobacter sp. strain PR-P3; and lanes 9 and 10 represent the mucoid and nonmucoid Paenibacillus isolates PR-N5 and PR-P1, respectively. P is the positive control P. putida NCIB 9816-4, and N1 and N2 are the negative controls C. testosteroni GZ39 and S. yanoikuyae B1, respectively.

Transformation studies using pure cultures. Isolates representing each group (pseudomonad, nocardioform, and Paenibacillus) and the Arthrobacter isolate PR-P3 were tested for the ability to degrade PAHs in cell suspensions. The isolates were tested for the ability to utilize PAHs individually or as a mixture containing naphthalene, fluorene, phenanthrene, pyrene, and biphenyl. Table 2 lists the amount (percent) of PAH remaining after 11 days of incubation. Results from cultures containing a mixture of PAHs were similar to those of cultures containing a single PAH (data not shown). None of the tested isolates was able to utilize pyrene in liquid suspension within the 11-day incubation time; however, all of the isolates were able to utilize naphthalene completely or partially (PS-P2 and PR-N10). In general, the strains isolated on phenanthrene (PS-P2, PR-P3, and PR-P1) were able to utilize a greater number of PAHs than the strains isolated on naphthalene (Table 2).

Sediment slurry transformation studies. Two gram-positive PAH-degrading isolates, PR-P1 (P. validus) and PR-P3 (A. oxydans), were inoculated at cell densities ranging from 10⁵ to 10⁹ cells ml¹ into an aerobic marine sediment slurry containing a mixture of PAHs (naphthalene, fluorene, phenanthrene, and pyrene) and biphenyl at a concentration of 50 ppm for each substrate. The transformation experiment was designed to follow the disappearance of a mixture of PAHs over time, and the results would determine whether specifically inoculated rhizosphere bacteria could enhance transformation over the indigenous microbial community. Uninoculated and inoculated sediment slurries both showed loss of the two- and three-ringed PAHs within 2 weeks of incubation (data not shown). However, pyrene transformation was only observed in treatments inoculated with strain PR-P1 or PR-P3 at 10⁹ cells ml¹ (Fig. 3). The sterile slurry microcosms showed no loss of pyrene, indicating that pyrene transformation was microbially mediated. In addition, the indigenous bacteria present in the nonsterile slurry microcosms were not responsible for the loss of pyrene seen in the inoculated microcosms. Pyrene transformation was not observed in the microcosms inoculated to a cell density of 10⁵ or 10⁷ cells ml¹. Microcosms inoculated with the spore-forming P. validus strain PR-P1 showed a more rapid loss of pyrene than did microcosms inoculated with the non-spore-forming A. oxydans strain PR-P3 (Fig. 3). A large variation was seen between the triplicate samples analyzed for the inoculated microcosms. A two-sample t test was performed on the pyrene transformation data. The t test indicated that the pyrene concentrations in the PR-P1- and PR-P3-inoculated microcosms were, by day 35, significantly different (95% confidence, P < 0.05) from the sterile and nonsterile control microcosms. By day 50 the PR-P1 and PR-P3 high inoculum density microcosms were found to contain mean concentrations of 3 and 11 ppm of pyrene, respectively (not significantly different from each other).

View larger version (22K): [in this window] [in a new window]   FIG. 3.   Loss of pyrene in contaminated sediment slurry inoculated with either the Paenibacillus sp. strain PR-P1 or the Arthrobacter sp. strain PR-P3. Data points represent the means of triplicate samples. Error bars indicate the standard deviation. Where no error bar is present, the value is less than the height of the symbol.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

We present a study of the culturable PAH-degrading bacteria associated with the rhizosphere of several salt marsh plant species in contaminated and uncontaminated estuarine sediments. In addition, a pasteurization method was successful in isolating spore-forming bacteria. Numerous studies have demonstrated the importance of the rhizosphere effect on degradation of organic contaminants. Most of these studies have examined terrestrial plants and agricultural chemicals (1, 2, 27); few have looked at the influence of plant-associated microorganisms on the fate of PCBs (15, 16) and PAHs (34, 39). There have been a limited number of studies on PAH degradation involving wetland or salt marsh ecosystems, but none have studied the diversity of PAH-degrading microorganisms present (28, 30, 49).

Recently, more studies have focused on PAH degradation in marine and estuarine ecosystems (3, 11, 12, 13, 17, 20, 21, 46). No studies have been conducted on the PAH-degrading microorganisms associated with salt marsh plants. In the present study, we isolated a variety of PAH-degrading microorganisms from the rhizosphere of the salt marsh grasses S. alterniflora, J. gerardi, D. spicata, and S. airoides. We found both gram-positive (predominantly nocardioform) and gram-negative (predominantly pseudomonad) bacteria, and a pasteurization technique prior to enrichment resulted in the isolation of spore-forming (exclusively Paenibacillus sp.) bacteria.

The rhizosphere has been reported to be predominantly associated with gram-negative bacteria (7); however, this view has been changing with more studies using molecular tools (5). Indeed, the culturable diversity of PAH-degrading microorganisms in this study would lend evidence to this trend. Many of the bacterial species and genera that we isolated have been reported to degrade PAHs previously. Few reports have been made regarding Paenibacillus and its ability to degrade PAHs. Pichinoty et al. (36) described Bacillus gordonae sp. nov., later to be emended as P. validus (22), which was able to utilize p-hydroxybenzoate, phthalate, isophthalate, protocatechuate, trimellitate, quinate, phenol, p-cresol, and naphthalene. Recently, Meyer et al. (33) reported the isolation of a PAH-degrading tentative Paenibacillus sp. from tar oil-contaminated soil.

We wanted to determine the ability of the rhizosphere and estuarine sediment isolates to degrade a variety of PAHs, individually and in combination, in order to determine their biodegradation and bioremediation potentials. The pure culture transformation experiments showed that phenanthrene-enriched isolates were able to utilize a greater variety of other PAHs than the naphthalene-enriched isolates, although none degraded pyrene in minimal media. The microbial slurries demonstrated a greater transformation activity than did the minimal-medium dense-cell experiments. The greater activity in the sediment slurries indicates that the organic or inorganic constituents or the extant microbial community of the sediment stimulated the activity of the inoculated strains. Siciliano and Germida (41) reported that inoculants required an unknown soil factor to degrade 2-chlorobenzoic acid in the rhizosphere of Dahurian wild rye. It should be noted that both the pure culture and microbial slurry experiments were performed under highly oxygenated conditions and that the limited diffusion of oxygen into organic-rich sediments has been found to restrict PAH biodegradation in the natural environment (10). The anoxic conditions found in most sediments, however, may not be the case in tidal and salt marsh ecosystems where plants provide oxygen to their deeper roots. For example, Spartina is known to pump oxygen into the rhizosphere (23). Indeed, when the plants were collected at low tide, highly oxidized areas were observed around the root system in comparison to the bulk nonrhizosphere sediment. Of specific interest is evaluating the role of oxygen cycling by roots of salt marsh plants in the enhancement of PAH biodegradation by rhizosphere-associated microorganisms.

Many different types of PAH oxygenase genes have been described (for a review, see reference 50). Recent studies using known PAH degradation genes as probes indicate that previously undescribed genes are present (3). In our study, 75% of the pseudomonad isolates hybridized to the classical nah gene from P. putida NCIB 9816-4, and approximately the same number hybridized to the nag genes cloned from C. testosteroni GZ42. The absence of homology of the Paenibacillus isolates to all of the tested gene probes indicates the possibility of novel PAH degradation genes.

Based on our results (morphologic and phenotypic characterization), there is a wide variation between the PAH-degrading isolates, indicating that the rhizosphere of S. alterniflora contains a diverse population of PAH-degrading bacteria. In addition, vegetated salt marsh ecosystems may hold promise as a means of remediation of contaminated coastal environments.