APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 2001, p. 2683–2691 Vol. 67, No. 6 0099-2240/01/$04.00ϩ0 DOI: 10.1128/AEM.67.6.2683–2691.2001 Copyright © 2001, American Society for Microbiology. All Rights Reserved.

Isolation and Characterization of Polycyclic Aromatic Hydrocarbon-Degrading Associated with the Rhizosphere of Salt Marsh Plants

1 1 1,2 1,2 L. L. DAANE, † I. HARJONO, G. J. ZYLSTRA, AND M. M. HA¨ GGBLOM * Biotechnology Center for Agriculture and the Environment1 and Department of Biochemistry and Microbiology,2 Cook College, Rutgers University, New Brunswick, New Jersey 08901

Received 25 September 2000/Accepted 12 March 2001

Polycyclic aromatic hydrocarbon (PAH)-degrading bacteria were isolated from contaminated estuarine sediment and salt marsh rhizosphere by enrichment using either naphthalene, phenanthrene, or biphenyl as the sole source of carbon and energy. Pasteurization of samples prior to enrichment resulted in isolation of gram-positive, spore-forming bacteria. The isolates were characterized using a variety of phenotypic, morpho- logic, and molecular properties. Identification of the isolates based on their fatty acid profiles and partial 16S rRNA gene sequences assigned them to three main bacterial groups: gram-negative pseudomonads; gram- positive, non-spore-forming nocardioforms; and the gram-positive, spore-forming group, Paenibacillus. Genomic digest patterns of all isolates were used to determine unique isolates, and representatives from each bacterial group were chosen for further investigation. Southern hybridization was performed using genes for PAH degradation from Pseudomonas putida NCIB 9816-4, Comamonas testosteroni GZ42, Sphingomonas yanoikuyae B1, and Mycobacterium sp. strain PY01. None of the isolates from the three groups showed homology to the B1 genes, only two nocardioform isolates showed homology to the PY01 genes, and only members of the pseudomonad group showed homology to the NCIB 9816-4 or GZ42 probes. The Paenibacillus isolates showed no homology to any of the tested gene probes, indicating the possibility of novel genes for PAH degradation. Pure culture substrate utilization experiments using several selected isolates from each of the three groups showed that the phenanthrene-enriched isolates are able to utilize a greater number of PAHs than are the naphthalene-enriched isolates. Inoculating two of the gram-positive isolates to a marine sediment slurry spiked with a mixture of PAHs (naphthalene, fluorene, phenanthrene, and pyrene) and biphenyl resulted in rapid transformation of pyrene, in addition to the two- and three-ringed PAHs and biphenyl. This study indicates that the rhizosphere of salt marsh plants contains a diverse population of PAH-degrading bacteria, and the use of plant-associated microorganisms has the potential for bioremediation of contaminated sediments.

Contamination of estuarine sediments by highly hydropho- significant cause of PAH removal (6). Numerous studies have bic, low-availability, toxic, organic contaminants has become a been conducted on microbial consortia and enrichment, and significant environmental concern. For example, industrial ac- several diverse genera of bacteria have been isolated (for a tivities in the New Jersey/New York Harbor estuary have re- review, see reference 6). Recent work has indicated that the sulted in widespread contamination, and the management of stimulation of microbial activity in the rhizosphere of plants contaminated sediments has become a significant problem. can also stimulate biodegradation of various toxic organic com- Polycyclic aromatic hydrocarbons (PAHs) are of particular pounds (1, 4, 37, 39, 41, 45, 47). This general “rhizosphere concern because of their toxic, mutagenic, and carcinogenic effect” is well known in terrestrial systems. The rhizosphere properties (32). PAHs have also been found to bioaccumulate soil has been described as the zone of soil under the direct in aquatic organisms (29). Although PAHs are persistent, influence of plant roots and usually extends a few millimeters mainly due to their high hydrophobicity, a variety of microor- from the root surface and is a dynamic environment for mi- ganisms (bacteria and fungi) can degrade certain PAHs (for croorganisms (7). The rhizosphere microbial community is reviews, see references 6 and 44). There is thus a significant comprised of microorganisms with different types of metabo- interest in studying microorganisms present in contaminated lism and adaptive responses to variation in environmental con- environments as a means for bioremediation. ditions. The production of mucilaginous material and the ex- The fate of PAHs and other organic contaminants in the udation of a variety of soluble organic compounds by the plant environment is associated with both abiotic and biotic pro- root play an important part in root colonization and mainte- cesses, including volatilization, photooxidation, chemical oxi- nance of microbial growth in the rhizosphere. Microbial activ- dation, bioaccumulation, and microbial transformation. Micro- ity is thus generally higher in the rhizosphere due to readily bial activity has been deemed the most influential and 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 * Corresponding author. Mailing address: Department of Biochem- the case of PCBs, plant compounds have been shown to induce istry and Microbiology, Cook College, Rutgers University, 76 Lipman Dr., New Brunswick, NJ 08901-8525. Phone: (732) 932-9763, ext. 326. the cometabolic degradation of PCBs (15, 16). Fax: (732) 932-8965. E-mail: [email protected]. The activities of microbial communities in sediments and † Present address: Avon Products, Inc., Suffern, NY 10901-5605. tidal marshes are important in improving sediment quality

2683 2684 DAANE ET AL. APPL.ENVIRON.MICROBIOL.

(pollution degradation), in the biological transformations of degradation by Mycobacterium sp. strain PY01 (J. F. Cigolini and G. J. Zylstra, major nutrients influencing their availability for plant growth, Abstr. 99th Gen. Meet. Am. Soc. Microbiol., abstr. Q-180, 1999) was PCR amplified with the primers 5Ј-GTTGACCCGTGACGC-3Ј and 5Ј-CTCACTCA and in their role in binding sediment particles to improve AGGCCGG-3Ј. The PAH-degrading strain C. testosteroni GZ39 was used as a rooting medium structure. Wetlands have a higher biological control in hybridization experiments (18). activity than many other ecosystems, and they support en- Mineral salts basal (MSB) medium (43) was used for enrichment cultures and hanced biotransformation of toxic chemicals (25). Experiments isolation. Solid minimal medium contained 2% noble agar (Difco Laboratories, Ϫ1 with Spartina salt marsh mesocosms indicated that biodegra- Detroit, Mich.). Stock concentrations (100 mg ml ) of naphthalene, biphenyl, and phenanthrene were dissolved in dimethyl formamide and were added to Ϫ dation of oil was influenced by flooding and fertilization con- liquid medium at a final concentration of 1 mg ml 1. Naphthalene and biphenyl ditions (28, 49). were added in the vapor phase as crystals in the petri dish lid for solid medium. There is extensive information on PAH degradation by both Phenanthrene was added as a 2% noble agar overlayer onto MSB agar plates at Ϫ gram-positive and gram-negative bacteria, mostly isolated a final concentration of 1 mg ml 1. All PAHs were obtained from Aldrich from soil environments. There is, however, little information Chemical Co. (Milwaukee, Wis.) and were a minimum of 98% pure. Bacterial strains were grown on Trypticase soy (Beckton Dickinson & Co., on the microbial communities in salt marsh ecosystems and Cockeysville, Md.) agar (TSA) plates for various phenotypic and fatty acid their role in the biodegradation of toxic organic contaminants. methyl ester (FAME) analyses. For plasmid preparations, bacterial strains were Ϫ In addition, little is known about the ability of these microor- grown in Luria-Bertani medium (38) containing either kanamycin (50 ␮gml 1; ␮ Ϫ1 ganisms to degrade polyaromatic compounds. In the present Sigma Chemical Co., St. Louis, Mo.) or ampicillin (100 gml ; Sigma Chemical Co.) added from filter-sterilized stock solutions. All bacterial strains and isolates study, we isolated PAH-degrading bacteria from the salt marsh were routinely grown aerobically at 30°C, except for E. coli strains, which were rhizosphere. We were particularly interested in isolating spore- grown at 37°C. Strains were maintained on minimal medium at 4°C, and long- forming bacteria for two reasons. First, the ability of a bacte- term storage was in 50% glycerol at Ϫ80°C. rium to produce spores and therefore survive adverse condi- Enrichment and isolation of PAH-degrading bacteria. Bacterial enrichment tions would be an attractive feature for remediation of cultures were set up in cotton-plugged Erlenmeyer flasks containing 50 ml of MSB medium using naphthalene, phenanthrene, or biphenyl as the sole source contaminated sites. Secondly, only a few studies have examined of carbon. Plant rhizosphere and contaminated estuarine sediment were used as the degradation of PAHs by spore-forming bacteria, and none, sources of the inoculum for the enrichment cultures. Rhizosphere samples were to our knowledge, involve plant-microbe interactions. There- obtained by splitting open the plant root mass, so that the inner roots that had fore, isolation of spore-forming, PAH-degrading bacteria and 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 subsequent genetic studies of their degradation pathways may material and was added to each enrichment culture. Sediment samples were lead to the discovery of novel genes involved. In addition, we added directly to each enrichment culture using a 1-g subsample. A total of two wanted to evaluate the biodegradation potential of select iso- enrichment cultures for each substrate were set up with each rhizosphere and lates and determine if reinoculation of the rhizosphere would sediment sample. One set of enrichment cultures was placed directly in a 28°C enhance degradation of contaminated marine sediment. 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. MATERIALS AND METHODS The cultures were monitored for the presence of microorganisms by micros- Plant and sediment samples. Salt marsh plant samples were obtained at two copy and gram staining and were subcultured (1:50) into fresh MSB medium sites. The first site, considered to be uncontaminated, was the University of containing the selected PAH when growth was detected. After three to four Delaware Marine Station at Lewes, Del. A total of five different plant samples subcultures, the cultures were plated onto MSB agar plates containing the same were collected and included Distichlis spicata, Juncus gerardi, Phragmites australis, substrate as the enrichment. Piles Creek sediment and S. alterniflora rhizosphere Spartina alterniflora, and Sporobolus airoides. A minimum of two plants of each isolates are denoted as PS and PR, respectively. Delaware isolates obtained from was obtained. The second site was Piles Creek, a contaminated tributary the rhizosphere of S. airoides, S. alterniflora, D. spicata, J. gerardi,orP. australis of Arthur Kill, in Linden, N.J. Estuarine sediment and S. alterniflora plant are denoted as SA, Salt, DS, JG, or PA, respectively. In addition, strains isolated samples were obtained from the intertidal area. A total of three sediment and using naphthalene are indicated by an N, while phenanthrene- and biphenyl- three plant samples were taken from separate areas within 50 feet of each other. enriched isolates are denoted by P and B, respectively. The plant and sediment samples were transported on ice back to the laboratory Phenotypic characterizations. Phase-contrast microscopy (ϫ1,000 magnifica- where they were stored at 4°C until analyzed. tion) was used for the morphologic examination of bacterial cells. The strains to Contaminated sediment for the microbial-sediment slurry biotransformation be tested were inoculated into 2 ml of TS broth and incubated on a rotary wheel experiment was obtained from Newtown Creek in the New York Harbor, Brook- at 37°C. The cells were examined microscopically for motility and morphology at lyn, N.Y. Material was collected by the Army Corps of Engineers, and a chemical 24, 48, and 72 h and were examined again microscopically after Gram staining. analysis showed the dredged sediment to contain 2 to 7 ppm of the PAHs FAME analysis. Whole-cell fatty acid analyses were performed on all of the naphthalene, anthracene, and phenanthrene. The sediment texture consisted of PAH-degrading isolates by growing the cells at 28°C for 24 h on TSA plates. 50% sand, 41% silt, and 9% clay, as determined by the Soil Testing Laboratory Cellular fatty acids were saponified, methylated, extracted, and analyzed by gas at the New Jersey Agricultural Experiment Station. The structure is considered chromatography following the procedures given for the Sherlock Microbial Iden- a silt loam, organic matter was 3.7%, and the pH was 7.90. tification System (MIDI, Inc., Newark, Del.). Identification and comparison were Bacterial strains, plasmids and media. Pseudomonas putida NCIB 9816-4 (9), made to the Aerobe (TSBA version 3.9) database of the Sherlock Microbial Comamonas testosteroni GZ42 (18), Sphingomonas yanoikuyae B1 (14), and My- Identification System. The Dendrogram program of the MIDI software package cobacterium sp. strain PY01 (Y.-S. Oh and G. J. Zylstra, unpublished data) were was used to produce unweighted-pair matchings based on fatty acid composi- chosen as representatives of different genotypes of PAH-degrading organisms. tions. The genes have previously been cloned from these strains into Escherichia coli, DNA preparation. Bacterial strains were grown on either TSA or MSB agar and the cloned fragments were used as gene probes for Southern hybridization plates supplemented with naphthalene, phenanthrene, or biphenyl. Total experiments. Plasmid pDTG112 contains the cloned nah genes from P. putida genomic DNA was extracted as described by Wilson (48), with the exception of NCIB 9816-4 (40). A 3.6-kb SalI fragment contains three of the four genes for pretreatment with 1.5 mg of lysozyme for 2 h. With the exception of plasmid naphthalene dioxygenase: nahAa (reductase), nahAb (ferredoxin), and nahAc pDTG112, plasmid DNA was obtained using the QIAprep Spin Miniprep Kit (dioxygenase large subunit) (42). Plasmid pGJZ1822, cloned from C. testosteroni (Qiagen Inc., Santa Clarita, Calif.) according to the manufacturer’s directions. GZ42, has a 6.7-kb SstI-XhoI fragment that contains the genes necessary for the For plasmid pDTG112, the plasmid DNA was purified from the total DNA using first few steps in PAH degradation (19). Plasmid pGJZ1512, cloned from S. equilibrium centrifugation in a CsCl-ethidium bromide gradient as described by yanoikuyae B1, has a 567-bp SphI fragment that contains bphC encoding a Sambrook et al. (38). Restriction enzyme digests were performed on the isolated meta-ring cleavage enzyme involved in PAH degradation (26). A 600-bp frag- plasmids, and the desired fragments were purified using the Qiaquick gel extrac- ment containing a gene encoding a dioxygenase large subunit involved in PAH tion kit (Qiagen). VOL. 67, 2001 PAH-DEGRADING BACTERIA FROM SALT MARSH RHIZOSPHERE 2685

Restriction fragment length polymorphism (RFLP). Total genomic restriction Microbial-sediment slurry biotransformation experiment. An aerobic slurry enzyme digests were performed on all of the PAH-degrading isolates using either system was designed to determine the rate and extent of microbial transforma- the BamHI, EcoRI, HindIII, or NotI enzyme as recommended by the supplier tion of contaminated sediment. The rhizosphere-associated phenanthrene-de- (Gibco-BRL, Madison, Wis.). Approximately 2 to 3 ␮g of each isolate was grading strains PR-P1 and PR-P3 were grown to mid-log phase in TS broth and digested. Electrophoretic analysis of the digested DNA was done on a 0.8% were harvested by centrifugation at 4,000 ϫ g for 10 min. The cells were washed agarose gel at 25 V for 16 to 20 h. The restriction enzyme digest patterns were and pelleted by centrifugation twice using 1/10 salts (8) as the diluent, diluted visualized by UV light following ethidium bromide staining. into 1/10 salts, and inoculated at three different cell concentrations into a PAH- 16S rDNA analysis. Sequence analyses of 16S ribosomal DNA (rDNA) were spiked sediment slurry. performed on several of the isolates by amplifying the 16S rRNA genes by PCR Approximately equal volumes (500 ml) of estuarine sediment material, ob- using the eubacterial primers 27f and 1522r (24). The PCR mixtures (100 ␮l) tained from Newtown Creek, and water were mixed under forced aeration for 1 ␮ week prior to bacterial inoculation of slurry microcosms. The microcosms con- contained 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, a 200 M concentration of each deoxynucleoside triphosphate, 50 pmol of each primer, 2.5 sisted of 50-ml glass serum bottles containing 5 ml of aerated slurry spiked with UofTaq DNA polymerase (Gibco-BRL, Grand Island, N.Y.), and ϳ10 ng of a mixture of PAHs (naphthalene, fluorene, phenanthrene, and pyrene) and DNA template. PCR amplification was performed using a Perkin-Elmer Gene- biphenyl from an acetone stock at a concentration of approximately 50 ppm of Amp PCR System 2400 thermocycler (Applied Biosystems, Foster City, Calif.) each. Each bacterial isolate was added to the spiked slurry at 3 inoculum den- 5 7 9 Ϫ1 programmed as follows: 1 min of denaturation at 94°C; followed by 25 cycles of sities, 10 ,10,or10 cells ml . Two controls were used consisting of an 96°C for 1 min, 55°C for 1 min, and 72°C for 1 min; with a final extension at 72°C uninoculated nonsterile slurry control and a sterile (autoclaved three times over for 10 min. The amplified 1.5-kb PCR products were excised from a 1% agarose 3 days) slurry control. All the bottles were sealed with Teflon stoppers pierced gel and purified using the QIAquick gel extraction kit (Qiagen) according to the with an 18-gauge cotton-plugged needle, and the cultures were incubated in a manufacturer’s instructions. 30°C environmental chamber at 90 rpm. The loss of PAHs and biphenyl was Partial DNA sequences using the primers 27f and 685r (24) were determined analyzed over time. directly from the purified PCR products with automated fluorescent Taq cycle Entire microcosms were sacrificed in triplicate and extracted for PAH analyses sequencing using the ABI 373A Sequencer (Applied Biosystems). at nine time points over a 56-day period (7-day sampling interval). Twenty parts per million of 2-methylnaphthalene was added as an internal standard, and the Partial 16S rRNA sequences (ranging from 301 to 1,500 bp) of each isolate content of the entire vial was extracted overnight with 4 ml of hexane and 2 ml were analyzed using the Ribosomal Database Project Sequence Match and of acetone by shaking on a wrist-action shaker at 200 rpm. The samples were Similarity Matrix programs (31) to obtain the most closely matched species. centrifuged for 5 min at 300 ϫ g, and the organic phase was removed. The slurry Southern hybridization using PAH gene probes. Southern blots were per- was further extracted using 2 ml of hexane for4hbyshaking at 200 rpm. The formed on all unique isolates, as determined by RFLP, using gene probes for organic phases were combined and analyzed for PAH and biphenyl content by four different types of PAH dioxygenases. This was accomplished by digesting 2 gas chromatography, as described above. Statistical analyses were performed to 3 ␮g of genomic DNA from each of the tested isolates with EcoRI according using the Minitab Software Package Release 11.21. to the manufacturers’ directions. In addition, genomic DNA obtained from P. Nucleotide sequence accession numbers. The 16S rRNA sequences for the putida NCIB 9816-4, C. testosteroni GZ42, C. testosteroni GZ39, S. yanoikuyae B1, PAH-degrading isolates have been deposited in GenBank under accession num- and Mycobacterium sp. strain PY01 was digested and used as positive and neg- bers AF353676 to AF353705. ative 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 RESULTS positively charged nylon membranes (Boehringer Mannheim, Indianapolis, Ind.) using a VacuGene XL Vacuum Blotting System (Pharmacia Biotech, Piscataway, Isolation of PAH-degrading bacterial strains. The PAH- N.J.) The transferred DNA was fixed to the nylon membranes by exposure to a degrading strains isolated in this study are listed in Table 1. A UV cross-linker (Spectronics Corp., Westbury, N.Y.) using a 2ϫ optimal cross- Ϫ link (120 mJ cm 2). The Southern hybridization protocol was performed using total of 42 isolates were obtained from S. alterniflora plant and nonradioactive digoxigenin (Boehringer Mannheim) as recommended by the sediment samples collected from contaminated Piles Creek in supplier. Gene probes were prepared from the PAH dioxygenase gene fragments New Jersey, using either naphthalene, phenanthrene, or biphe- by a nonradioactive random primed DNA-labeling method with alkali-labile nyl as the sole source of carbon. A total of nine isolates were digoxigenin 11–dUTP. Prehybridization and hybridization were performed at obtained from the four different plant rhizospheres collected 37°C. Following hybridization, the nylon membrane was washed under low- stringency conditions (0.1ϫ SSC [1ϫ SSC is 0.15 M NaCl plus 0.015 M sodium from Lewes, Del. However, no PAH-degrading isolates were citrate] and 0.1% sodium dodecyl sulfate) at room temperature. obtained from the rhizosphere of P. australis from this site. The Pure culture transformation of PAHs in liquid medium. Several of the isolates lower number and diversity of bacteria isolated from this site were analyzed for their ability to utilize a variety of aromatic compounds, in- may be expected due to its uncontaminated nature. Heat treat- cluding naphthalene, phenanthrene, fluorene, pyrene, and biphenyl, individually and as a mixture, in MSB medium. The isolates tested included the gram- ment prior to enrichment and isolation was included for each negative pseudomonads PR-N7, PR-N9, PR-N10, and PS-P2; the gram-positive of the substrates tested, which resulted in isolation of several non-spore formers PR-N15 and PR-P3; and the spore former PR-P1. Each spore-forming bacteria. The isolates were characterized on the isolate was tested in duplicate for each substrate or substrates over a time course basis of their morphologic and phenotypic properties and by of 11 days. Cells were grown in 100 ml of MSB containing 5 mM succinate, using a combination of molecular techniques. pelleted by centrifugation (4,000 ϫ g, 10 min), washed with MSB, pelleted again, Ϫ FAME analysis. and suspended in fresh MSB to give a cell density of approximately 107 cells ml 1 The tentative identification and similarity for use in transformation experiments. Sterile 7-ml serum vials containing the index (SI) of all of the isolates using the aerobic (TSBA) appropriate carbon source at a final concentration of 100 ␮M were added from library version 3.9 of the Microbial Identification System is hexane stock solutions, and the material was allowed to evaporate prior to the shown in Table 1. A pairwise-comparison Euclidean-distance addition of 2 ml of washed cell suspension. In addition, two controls were added dendrogram based on whole-cell fatty acid compositions of the to the experimental design and included a no-cell control (MSB medium only) and a dead-cell control (MSB ϩ 4% formaldehyde ϩ cells). The samples were PAH-degrading isolates (except isolate PS-P1) was con- incubated at 28°C on a rotary shaker at 90 rpm, and the amount of substrate structed, revealing that the isolates fall within three main remaining was analyzed over time. At each time point two vials were taken, were groups (Fig. 1). Group 1 included the endospore-forming extracted with 1 ml of hexane, and were analyzed for the appropriate aromatic Paenibacillus, group 2 included the gram-positive nocardio- substrate using a Hewlett-Packard 5890 Series II Gas Chromatograph using an forms, and group 3 included the gram-negative pseudomonads. HP-5MS column and equipped with a 5971 series mass selective detector. Prior ␮ One of the isolates, PR-P12, falling outside these groups was to extraction, 20 M 2-methylnaphthalene was added from a hexane stock ϭ solution as an internal standard. Results are reported as the percentage remain- tentatively identified as Flavobacterium resinovorum (SI ing relative to uninoculated controls after 11 days of incubation. 0.79) (Table 1). Isolate PR-P3 clustered within the Paenibacil- 2686 DAANE ET AL. APPL.ENVIRON.MICROBIOL.

TABLE 1. PAH-degrading isolates obtained from contaminated estuarine sediment and salt marsh plant rhizosphere and comparison of bacterial identification using FAME and partial 16S rRNA gene sequences of several of the strains

FAME analysisc Partial 16S rRNA gene sequenced Isolatea Source PAHb Best match SI Best match % Similarity DS-N1 D. spicata N Brevibacillus brevis 0.45 P. validus 99.6 DS-N2 D. spicata N P. validuse 0.92 P. validus 96.7 DS-N3 D. spicata N P. validus 0.81 P. validus 97.9 JG-N1 J. gerardi N P. validus 0.80 NDf JG-N2 J. gerardi N P. validus 0.81 P. validus 96.0 JG-N3 J. gerardi N P. validus 0.87 ND SA-N1 S. airoides N Paenibacillus sp. Poor P. validus 96.0 Salt-N1 S. alterniflora N Pseudomonas fluorescens 0.59 ND Salt-N2 S. alterniflora N P. putida 0.80 ND PR-N1 S. alterniflora N Paenibacillus sp. Poor P. validus 97.3 PR-N2 S. alterniflora N Paenibacillus sp. Poor ND PR-N3 S. alterniflora N P. validus 0.87 ND PR-N4 S. alterniflora N P. validus 0.86 P. validus 98.0 PR-N5 S. alterniflora N Paenibacillus sp. Poor P. validus 96.9 PR-N6 S. alterniflora N Pseudomonas chlororaphis 0.85 ND PR-N7 S. alterniflora N P. stutzeri 0.88 P. stutzeri 99.0 PR-N8 S. alterniflora N P. putida 0.87 ND PR-N9 S. alterniflora N P. putida 0.87 P. putida 99.0 PR-N10 S. alterniflora N P. putida 0.82 P. putida 98.0 PR-N11 S. alterniflora N Paenibacillus sp. Poor ND PR-N12 S. alterniflora N Paenibacillus sp. Poor ND PR-N13 S. alterniflora N P. validus 0.81 P. validus 98.2 PR-N14 S. alterniflora N N. asteroides 0.77 Rhodococcus ruber 97.1 PR-N15 S. alterniflora N N. brasiliensis 0.21 Tsukamurella wratislaviensis 99.8 PR-N16 S. alterniflora N Paenibacillus sp. Poor P. validus 97.3 PR-N17 S. alterniflora N N. asteroides 0.57 ND PR-N18 S. alterniflora N Nocardia sp. 0.14 ND PR-N19 S. alterniflora N P. validus 0.93 P. validus 97.3 PR-N20 S. alterniflora N P. validus 0.84 P. validus 98.0 PR-N21 S. alterniflora N P. validus 0.75 P. validus 98.9 PR-N22 S. alterniflora N P. validus 0.81 P. validus 98.5 PR-P1 S. alterniflora P P. validus 0.85 P. validus 98.5 PR-P2 S. alterniflora P P. validus 0.90 ND PR-P3 S. alterniflora P A. oxydans 0.46 A. oxydans 95.4 PR-P6 S. alterniflora P P. validus 0.88 ND PR-P9 S. alterniflora P P. validus 0.91 P. validus 99.5 PR-P10 S. alterniflora P P. validus 0.91 P. validus 99.8 PR-P11 S. alterniflora P P. validus 0.70 P. validus 99.5 PR-P12 S. alterniflora P F. resinovorum 0.79 S. subarctica 99.3 PR-P13 S. alterniflora P P. validus 0.91 P. validus 97.9 PR-B1 S. alterniflora B P. validus 0.87 ND PR-B2 S. alterniflora B B. brevis 0.34 P. validus 98.4 PS-N1 Sediment N P. pabuli 0.26 P. validus 97.1 PS-N2 Sediment N P. fluorescens 0.47 ND PS-N3 Sediment N N. asteroides 0.71 ND PS-N4 Sediment N P. stutzeri 0.89 ND PS-N5 Sediment N P. stutzeri 0.86 ND PS-N6 Sediment N P. validus 0.33 P. validus 96.4 PS-P1 Sediment P Nocardioform NDg ND PS-P2a Sediment P Alcaligenes xylosoxidans 0.65 Alcaligenes faecalis 98.4 PS-P2b Sediment P A. xylosoxidans 0.56 ND

a DS, JG, SA, and salt isolates obtained from plants collected from Lewes, Del. PR and PS designates isolates obtained at Piles Creek, N.J. b PAH carbon source in enrichment culture. N, naphthalene; P, phenanthrene; and B, biphenyl. c FAME identification was performed using the aerobic (TSBA) library version 3.9 of the Microbial Identification System (MIDI, Inc.). SI values below 0.2 are listed as poor, and the match is given only to the genus. d 16S rRNA genes were amplified by PCR using universal primers. The PCR products were partially sequenced (ϳ500 bp, ranging from 292 to 1,488 bp) and were compared to the Ribosomal Database Project database to obtain the most closely matched species. e P. validus listed as the invalid synonym P. gordonae in MIDI library. f ND, not determined. g Identification based on presence of fatty acid 10-methyl 18:0 (TSBA). lus group but had the closest match to Arthrobacter oxydans form group based on the presence of 10-methyl 18:0 fatty acid (SI ϭ 0.46). In addition, one of the isolates, PS-P1, was not (tuberculostearic acid). identified on several attempts by the MIDI Microbial Identi- The dendrogram (Fig. 1) also shows subclusters within each fication System but was considered a member of the nocardio- of these groups. In the spore-forming Paenibacillus group there VOL. 67, 2001 PAH-DEGRADING BACTERIA FROM SALT MARSH RHIZOSPHERE 2687

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 .isolate PR-P3 is an Arthrobacter sp ,ء .by an N, while phenanthrene- and biphenyl-enriched isolates are denoted by a P and B, respectively

were two subgroups. The members of the smaller subgroup, tled colony morphology when grown on solid media, and with consisting of isolates PS-N1, PR-N16, PR-N5, PR-N11, PR-N2, the exception of PR-B2 and DS-N1, had a high SI to P. validus PR-N12, PS-N6, PR-N1, and SA-N1, all exhibited a mucoid (Table 1). The five isolates found within the nocardioform colony morphology when grown on solid media and had a low group also formed two subgroups. Isolates PR-N18 and PR- SI to either Paenibacillus alvei, Paenibacillus pabuli,orPaeni- N15 both had a cream-colored, waxy colony morphology and bacillus validus (Table 1). In contrast, the larger subgroup were identified, with low SIs, as Nocardia asteroides and No- within the Paenibacillus cluster exhibited a nonmucoid, mot- cardia brasiliensis, respectively (Table 1). The remaining three 2688 DAANE ET AL. APPL.ENVIRON.MICROBIOL. nocardioforms had an orange, waxy colony morphology and were identified with a high SI as N. asteroides (Table 1). In the pseudomonad group, the three isolates PR-N7, PS-N4, and PS-N5 were identified with a high match to Pseudomonas stutzeri (Table 1). These three isolates cluster closely together in relation to the remaining pseudomonad isolates. Of the P. stutzeri strains, PS-N5 and PR-N7 exhibit a small, crinkled, tan colony morphology, while PS-N4 exhibits a small, tan colony morphology. The crinkled, tan morphology can give rise to noncrinkled colonies resembling PS-N4. This dual colony mor- phology is commonly seen in P. stutzeri (Norberto Palleroni, personal communication). 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 FIG. 2. Southern hybridization of selected isolates with the nah all of the cultured isolates. The gels were examined visually, genes for naphthalene degradation from P. putida NCIB 9816-4. and an isolate was considered unique based on the presence of EcoRI-digested genomic DNA was probed with a 3.5-kb SalI fragment a unique banding pattern. Isolates which shared a banding from P. putida NCIB 9816-4 containing the genes for naphthalene pattern and had similar FAME profiles were grouped together, dioxygenase. M1 and M2 are the digoxigenin-labeled and high-molec- ular-weight markers, respectively. Lanes 1 to 4 are the pseudomonad and a single isolate from the group was chosen as a represen- isolates PR-N7, PR-N9, PR-N10, and PS-P2; lane 5 is the sphin- tative strain for sequencing and Southern hybridization exper- gomonad isolate PR-P12; lanes 6 and 7 are the nocardioform isolates iments. The RFLP data are summarized below. All of the six PR-N14 and PR-N15; lane 8 is Arthrobacter sp. strain PR-P3; and lanes nocardioform isolates were found to be unique. Of the 12 9 and 10 represent the mucoid and nonmucoid Paenibacillus isolates PR-N5 and PR-P1, respectively. P is the positive control P. putida original pseudomonad isolates, 10 were found to have different NCIB 9816-4, and N1 and N2 are the negative controls C. testosteroni restriction enzyme banding patterns. Shared RFLP patterns GZ39 and S. yanoikuyae B1, respectively. were seen between the pseudomonad isolates PS-N4 and PS-N5 and between PS-P2a and PS-P2b. The large Paenibacil- lus group, containing 31 isolates, was found to have 19 isolates with much of the data revealing no hybridization results. exhibiting unique RFLP patterns. Shared banding patterns Therefore, we present the data in text form with one figure were seen between the mucoid colony morphology Paenibacil- exemplifying the nah gene hybridization results for isolates lus isolates PR-N5, PR-N11, PR-N2, and PR-N12. In addition, representing the three major bacterial groups (pseudomonads, shared banding patterns were seen between the nonmucoid nocardiofoms, and Paenibacillus). colony morphology Paenibacillus isolates PR-B2 and PR-B1; All of the unique pseudomonad isolates with the exception JG-N1, DS-N1, and JG-N3; PR-N19 and PR-N20; PR-N21 and of PS-P2 hybridized to the classical nah genes from P. putida PR-N22; PR-P1, PR-P2, and PR-P6; and PR-N13, PR-N3, and NCIB 9816-4. The unique pseudomonad isolates PR-N10, PS- PR-N4. N2, PR-N6, Salt-N1, and Salt-N2 gave the same pattern of 16S rRNA gene analyses. Several of the unique isolates from hybridization as the control strain P. putida NCIB 9816-4 (Fig. each of the three major groups were further identified by 2). This double-banding pattern was a result of an EcoRI partial sequencing of their 16S rRNA gene. In general, the 16S digestion of the genomic DNA. The positive bands were at 18.5 rDNA sequence analysis confirmed the identification based on and 3.7 kb. Pseudomonad isolate PR-N7 also gave a double- fatty acid profiles. Within the Paenibacillus group the isolates banding pattern to the nah gene probe; however, the bands identified as P. validus by FAME also had a high sequence were at 9.0 and 1.9 kb. In addition, all of the pseudomonad similarity to P. validus. The smaller subgroup of isolates having isolates, except for PS-P2 and PS-N5, hybridized to the nag a low SI to P. alvei, P. pabuli,orP. validus by FAME had a low genes cloned from C. testosteroni GZ42. Of the pseudomonad similarity to P. validus based on their partial 16S rRNA gene isolates hybridizing to the nag gene, six isolates (Salt-N1, Salt- sequence (Table 1). Strain PR-P3, which was an outlier within N2, PR-N6, PS-N2, PR-N10, and PR-N7) gave the same single- the Paenibacillus group, was identified with the closest match band pattern, while the remaining two isolates (PR-N8 and by both FAME and 16S rDNA analysis to A. oxydans. The PR-N9) gave a different single-band pattern distinguishable norcardioform isolates PR-N14 and PR-N15 were identified as from the other six isolates. However, the nocardioforms, Paeni- belonging to the genera Rhodococcus and Tsukamurella, re- bacillus isolates, Arthrobacter isolate PR-P3, or the sphin- spectively. The identification of the Pseudomonas isolates as P. gomonad isolate PR-P12 did not hybridize to either of these putida and P. stutzeri by FAME was confirmed by 16S rDNA PAH dioxygenase genes (Fig. 2). None of the tested PAH- phylogenetic analysis. The outlying isolate PR-P12 was identi- degrading isolates hybridized to the gene probe from S. fied with a high 16S rDNA sequence similarity to Sphingomo- yanoikuyae B1, and only norcardioform isolates PR-N18 and nas subarctica. PS-P1 hybridized to the PAH oxygenase gene cloned from Southern hybridization with PAH gene probes. All of the Mycobacterium sp. strain PY01. unique isolates, as determined by RFLP, were tested for the Transformation studies using pure cultures. Isolates repre- presence of previously described genes for PAH dioxygenases. senting each group (pseudomonad, nocardioform, and Paeni- A large number of Southern hybridizations were performed, bacillus) and the Arthrobacter isolate PR-P3 were tested for the VOL. 67, 2001 PAH-DEGRADING BACTERIA FROM SALT MARSH RHIZOSPHERE 2689

TABLE 2. Abilities of several isolates to utilize a variety of PAHs spore-forming A. oxydans strain PR-P3 (Fig. 3). A large vari-

% PAH remainingb ation was seen between the triplicate samples analyzed for the Isolatea inoculated microcosms. A two-sample t test was performed on Naphthalene Biphenyl Fluorene Phenanthrene Pyrene the pyrene transformation data. The t test indicated that the Pseudomonads pyrene concentrations in the PR-P1- and PR-P3-inocu- PR-N7 0 85 100 85 90 lated microcosms were, by day 35, significantly different (95% PR-N9 0 69 78 71 90 confidence, P Ͻ 0.05) from the sterile and nonsterile control PR-N10 40 90 97 89 100 PS-P2 44 51 79 28 100 microcosms. By day 50 the PR-P1 and PR-P3 high inoculum density microcosms were found to contain mean concentra- Gram-positive non- tions of 3 and 11 ppm of pyrene, respectively (not significantly spore formers different from each other). PR-N15 0 84 80 68 100 PR-P3 0 84 42 1 100 DISCUSSION Spore former PR-P1 0 0 29 8 100 We present a study of the culturable PAH-degrading bacte- a Substrate utilization experiments were performed using dense-cell suspen- ria associated with the rhizosphere of several salt marsh plant sions in MSB medium containing the appropriate PAH at a final concentration of 100 ␮M. Samples were incubated at 28°C at 90 rpm, were extracted with species in contaminated and uncontaminated estuarine sedi- hexane, and were analyzed by gas chromatography. ments. In addition, a pasteurization method was successful in b Percentage remaining was calculated relative to uninoculated controls after isolating spore-forming bacteria. Numerous studies have dem- 11 days of incubation. onstrated the importance of the rhizosphere effect on degra- dation of organic contaminants. Most of these studies have examined terrestrial plants and agricultural chemicals (1, 2, ability to degrade PAHs in cell suspensions. The isolates were 27); few have looked at the influence of plant-associated mi- tested for the ability to utilize PAHs individually or as a mix- croorganisms on the fate of PCBs (15, 16) and PAHs (34, 39). ture containing naphthalene, fluorene, phenanthrene, pyrene, There have been a limited number of studies on PAH degra- and biphenyl. Table 2 lists the amount (percent) of PAH re- dation involving wetland or salt marsh ecosystems, but none maining after 11 days of incubation. Results from cultures have studied the diversity of PAH-degrading microorganisms containing a mixture of PAHs were similar to those of cultures present (28, 30, 49). containing a single PAH (data not shown). None of the tested Recently, more studies have focused on PAH degradation in isolates was able to utilize pyrene in liquid suspension within marine and estuarine ecosystems (3, 11, 12, 13, 17, 20, 21, 46). the 11-day incubation time; however, all of the isolates were No studies have been conducted on the PAH-degrading mi- able to utilize naphthalene completely or partially (PS-P2 and croorganisms associated with salt marsh plants. In the present PR-N10). In general, the strains isolated on phenanthrene study, we isolated a variety of PAH-degrading microorganisms (PS-P2, PR-P3, and PR-P1) were able to utilize a greater from the rhizosphere of the salt marsh grasses S. alterniflora, J. number of PAHs than the strains isolated on naphthalene gerardi, D. spicata, and S. airoides. We found both gram-posi- (Table 2). tive (predominantly nocardioform) and gram-negative (pre- Sediment slurry transformation studies. Two gram-positive dominantly pseudomonad) bacteria, and a pasteurization tech- PAH-degrading isolates, PR-P1 (P. validus) and PR-P3 (A. oxydans), were inoculated at cell densities ranging from 105 to Ϫ 109 cells ml 1 into an aerobic marine sediment slurry contain- ing 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 inocu- lated rhizosphere bacteria could enhance transformation over the indigenous microbial community. Uninoculated and inoc- ulated 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 Ϫ ml 1 (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 transfor- mation was not observed in the microcosms inoculated to a cell FIG. 3. Loss of pyrene in contaminated sediment slurry inoculated 5 7 Ϫ1 with either the Paenibacillus sp. strain PR-P1 or the Arthrobacter sp. density of 10 or 10 cells ml . Microcosms inoculated with strain PR-P3. Data points represent the means of triplicate samples. the spore-forming P. validus strain PR-P1 showed a more rapid Error bars indicate the standard deviation. Where no error bar is loss of pyrene than did microcosms inoculated with the non- present, the value is less than the height of the symbol. 2690 DAANE ET AL. APPL.ENVIRON.MICROBIOL. nique prior to enrichment resulted in the isolation of spore- contains a diverse population of PAH-degrading bacteria. In forming (exclusively Paenibacillus sp.) bacteria. addition, vegetated salt marsh ecosystems may hold promise as The rhizosphere has been reported to be predominantly a means of remediation of contaminated coastal environments. associated with gram-negative bacteria (7); however, this view has been changing with more studies using molecular tools (5). ACKNOWLEDGMENTS Indeed, the culturable diversity of PAH-degrading microor- This work was supported by a grant from The State of New Jersey ganisms in this study would lend evidence to this trend. Many Commission on Science and Technology. G.J.Z. acknowledges the of the bacterial species and genera that we isolated have been support of the NSF under grant MCB-9723921. reported to degrade PAHs previously. 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