Alteromonas As a Key Agent of Polycyclic Aromatic Hydrocarbon

Article pubs.acs.org/est

Alteromonas As a Key Agent of Polycyclic Aromatic Hydrocarbon Biodegradation in Crude Oil-Contaminated Coastal Sediment Hyun Mi Jin,† Jeong Myeong Kim,† Hyo Jung Lee,† Eugene L. Madsen,‡ and Che Ok Jeon*,† †

School of Biological Sciences, Chung-Ang University, 84, HeukSeok-Ro, Seoul, 156-756, Republic of Korea Department of Microbiology, Cornell University, Ithaca, New York 14853-8101, United States

‡

S Supporting Information *

ABSTRACT: Following the 2007 oil spill in South Korean tidal flats, we sought to identify microbial players influencing the environmental fate of released polycyclic aromatic hydrocarbons (PAHs). Two years of monitoring showed that PAH concentrations in sediments declined substantially. Enrichment cultures were established using seawater and modified minimal media containing naphthalene as sole carbon source. The enriched microbial community was characterized by 16S rRNA-based DGGE profiling; sequencing selected bands indicated Alteromonas (among others) were active. Alteromonas sp. SN2 was isolated and was able to degrade naphthalene, phenanthrene, anthracene, and pyrene in laboratory-incubated microcosm assays. PCR-based analysis of DNA extracted from the sediments revealed naphthalene dioxygenase (NDO) genes of only two bacterial groups: Alteromonas and Cycloclasticus, having gentisate and catechol metabolic pathways, respectively. However, reverse transcriptase PCR-based analysis of field-fixed mRNA revealed in situ expression of only the Alteromonas-associated NDO genes; in laboratory microcosms these NDO genes were markedly induced by naphthalene addition. Analysis by GC/MS showed that naphthalene in tidal-flat samples was metabolized predominantly via the gentisate pathway; this signature metabolite was detected (0.04 μM) in contaminated field sediment. A quantitative PCR-based two-year data set monitoring Alteromonas-specific 16S rRNA genes and NDO transcripts in sea-tidal flat field samples showed that the abundance of bacteria related to strain SN2 during the winter season was 20-fold higher than in the summer season. Based on the above data, we conclude that strain SN2 and its relatives are site natives--key players in PAH degradation and adapted to winter conditions in these contaminated sea-tidal-flat sediments.

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INTRODUCTION Polycyclic aromatic hydrocarbons (PAHs) are a group of compounds composed of two or more fused aromatic rings that are important components of crude oil, creosote, and coal tar.1 PAHs are the cause of great environmental concern because of their persistence, toxicity, mutagenicity, and carcinogenicity; they are on ″priority-pollutant″ lists in most countries.2−4 Bioremediation employing microorganisms that can degrade PAHs has proven to be a nondisruptive, cost-effective, and highly efficient method of safely breaking down environmentally persistent compounds, including PAHs.5−8 The technology of bioremediation relies upon many disciplines (from engineering to hydrology to geochemistry to microbiology) addressing issues ranging from ecotoxicology and bioavailability (e.g., Luthy et al. 1997),9 to isolation of bacteria, to characterization of their genetic, biochemical, and physiological traits, to molecular microbial biomarkers that document populations and their gene expression in field sites. The scope of the present study is limited to the latter three topics. Molecular ecological studies of laboratory enrichments have helped to identify bacterial populations and genes that are functionally important at PAH-contaminated sites.10−14 PAHdegrading bacteria that may be key players in PAH © 2012 American Chemical Society

biodegradation at contaminated environmental sites have been isolated primarily from terrestrial habitats.15−20 Although PAH-degrading bacteria from marine habitats have been less extensively studied, reports of PAH-degrading bacteria such as Cycloclasticus,21−25 Neptunomonas,26 Halomonas,27 Pseudoalteromonas,28 Novosphingobium,29 Pseudomonas,30 and Alteromonas31,32 have appeared. The majority of PAH-degrading bacteria characterized to date are Gram-negative and it is well recognized that naphthalene catabolic genes utilized by these hosts also attack phenanthrene and anthracene;33 thus the single most insightful genetic biomarker for PAH biodegradation is naphthalene dioxygenase (NDO). Transcription of NDO (detected as mRNA transcripts) in field samples is an indication of in situ metabolic activity--contributing evidence for in situ cellular responses to PAHs. But mRNA transcripts, alone, do not prove in situ biodegradation,6 nor do NDO transcripts specify the particular PAH likely being metabolized. Over the last two decades, investigations into naphthalene Received: Revised: Accepted: Published: 7731

May 8, 2012 May 26, 2012 June 18, 2012 June 18, 2012 dx.doi.org/10.1021/es3018545 | Environ. Sci. Technol. 2012, 46, 7731−7740

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reported PCR primer sets, NahAc-114F/NahAc-596R, NDOF200/NDO-F201, NDO-F201/NDO-F202, P8073/P9047, and pPAH-F/pPAH-NR700 (Table S1), were applied to amplify the naphthalene dioxygenase (NDO) gene of strain SN2 using the PCR conditions described previously.26,43 The product amplified by pPAH-F/pPAH-NR700 was ligated into the pCR2.1 vector and then sequenced. A neighbor-joining tree showing the phylogenetic relationships of the resulting oxygenase gene was constructed using the PROTDIST of the Phylip package with the Jones-Taylor-Thornton matrix.44 Two PCR primer sets, AltNDO199F/AltNDO554R and CycNDO29F/CycNDO461R, were designed for targeting the dioxygenase genes of two groups of marine naphthalene degrading bacteria, Alteromonas sp., and its relatives and Cyclasticus sp., respectively, with the aid of the IDT Web site (http://eu.idtdna.com/Scitools/Applications/Primerquest/). PCR Amplification of NDO Genes from Genomic DNA and mRNA Extracted from the Contaminated Sea-Tidal Flat. All PCR primer sets used in this study to analyze NDO genes from genomic DNA and mRNA extracted from the contaminated sea-tidal flat are listed in Table S1. Additional details of NDO analyses are described in the Supporting Information. Sediment Microcosms: GC-MS Analysis of Naphthalene Metabolites (Catechol and Gentisate) and Nucleic Acid Extraction. Fresh sediment was sampled in June, 2011, from the contaminated sea-tidal flat. This sediment was utilized for two purposes: direct extraction and analysis of potential metabolites (catechol, gentisate) present in the field and establishing microcosm assays of both metabolite appearance and quantitative PCR (qPCR)-based measurement of Alteromonas-specific 16S rRNA genes and NDO transcripts. Details of these analyses are described in the Supporting Information. Enumeration of Total Bacteria and Alteromonas Species and Expressional Analysis of the Naphthalene Catabolic Gene of Alteromonas sp. and Its Relatives. The population densities of the total bacteria and Alteromonas species (strain SN2) in the above slurry systems and in the contaminated sea-tidal flat during a two-year monitoring period were enumerated based on 16S rRNA gene copy numbers using quantitative real time-PCR as described in the Supporting Information. Real time-reverse transcriptase PCR (real time RT-PCR) was used to analyze the naphthalene catabolic gene expressions of strain SN2 and its relatives in the above slurry systems and in the contaminated sea-tidal flat as described in the Supporting Information.

metabolism have established that there are only two biochemical pathways; these feature the nah or nag dissimilatory operons (catechol or gentisate pathways) featuring catechol and gentisate as key intermediary metabolites, respectively.34−38 Sea-tidal flats are broad, low-gradient coastal marsh, or muddy coast areas that experience exposure and flooding by seawater between low and high tides. The west and southwest coast of the Korean peninsula largely consists of sea-tidal flats, called getbol, which play an important role in the Korean fishing industry because of the huge numbers of mollusks, worms, and crabs living there. On December seventh, 2007, the oil tanker MV Hebei Spirit collided with a crane near the Taean coast of the Yellow Sea in South Korea, and an estimated 12,547,000 L of crude oil were released, which was recorded as one of the largest local tanker spills in recent years.39 Due to the oil spill accident, sea-tidal flats near the Taean coast were heavily contaminated and crude oil compounds including PAHs may be transferred to humans through seafood consumption. Sea-tidal flats are characterized by high primary production and nutrient cycling rates, which may rely upon high microbial abundance and diversity.40,41 We hypothesized that the Taean tidal flat sediments may also harbor diverse PAH-degrading microbial communities. The present study therefore aimed to investigate (i) the diversity and composition of the PAHdegrading bacterial populations enriched from the contaminated sea-tidal flat on the Taean coast; (ii) the isolation of PAH biodegrading bacteria from the enriched bacterial consortia and their PAH degradation abilities; and (iii) monitoring of the abundance of Alteromonas populations and in situ transcription of Alteromonas-specific dioxygenase genes in the contaminated sea-tidal flat over time.

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MATERIALS AND METHODS Sampling of Crude Oil-Contaminated Sea-Tidal Flat Sediment and Monitoring of PAH Compounds. For the enrichment and naphthalene degrading bacteria isolation, crude oil-contaminated tidal flat sediment samples (depth less than 5 cm) were obtained from the Taean coast (36°48′50.82″N,126°11′09.56″E) in South Korea in December 2007, twenty-two days after the oil spill accident. Additional details of sampling, storage, and PAH analyses are described in the Supporting Information. Enrichment Cultures and Denaturing Gradient Gel Electrophoresis (DGGE) Analysis. Approximately 500 mg of naphthalene pellets were added directly to each of two cottonplugged 500-ml Erlenmeyer flasks containing 10 g of the contaminated tidal flat sample and either 100 mL of 0.2 μmfiltered seawater or 100 mL of modified minimal salts basal (modified MSB, MSB,42 supplemented with 2% (w/v) NaCl) medium. Additional details regarding DGGE analysis are provided as the Supporting Information. Isolation of Naphthalene Degrading Bacteria from Enrichment Cultures and PAH Transformations Tests. Bacteria were isolated from the final enrichment cultures, and their naphthalene degradation abilities were tested as described in the Supporting Information. The PAH (naphthalene, phenanthrene, anthracene, and pyrene) biodegradation ability of strain SN2 was evaluated in serum bottles containing each individual PAH compound in seawater or modified MSB as described in the Supporting Information. Analysis of the Naphthalene Dioxygenase (NDO) Gene of Strain SN2 and PCR Primer Designs. Previously

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RESULTS Monitoring of PAH cOmpounds in the Crude OilContaminated Tidal Flat Sediments. Changes in concentration of PAH compounds in the surface tidal flat sediment (less than 5 cm deep) were monitored for approximately two years after the crude oil spill accident. The results showed that total PAHs, naphthalene, and phenanthrene decreased sharply (Figure 1). At the initial time of the contamination incident (December 2007), the total PAH concentration was ∼1,656 mg/kg-sediment, and this decreased to ∼16 mg/kg-sediment after two years (December 2009). Tidal flats are unique marine habitats alternatively undergoing flooding with seawater and exposure to the atmosphere. Thus, both physical transport and volatilization likely played major roles in the removal of PAHs from the surface sediment, in addition to the biodegradation of the contaminants. Figure 1 shows that naphthalene concen7732

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(Figure 2) and comparing them to available sequences (Table 1). The predominant band (# 8) in the seawater enrichment Table 1. Most Closely Matching Type Strains and Sequence Similarities of Ten 16S rRNA Gene-Based DGGE Bands Shown in Figure 2c band no. a

1 2 3 4 5 6 7

Figure 1. Time-dependent PAH, naphthalene, and phenanthrene concentrations in the crude oil-contaminated tidal flat sediment. Total PAH concentrations and the concentration of naphthalene and phenanthrene were determined by scanning fluorescence spectroscopy and gas chromatography, respectively. The values are averages of triplicates, and the error bars indicate one standard deviation.

8 9 10

tration (with its high degradability, solubility, and volatility) decreased faster than phenanthrene. Enrichment Cultures and DGGE Analysis. Enrichments of naphthalene degrading bacteria from the contaminated seatidal flat were prepared in seawater and modified MSB medium containing naphthalene pellets. Changes in the microbial communities within these enrichments were monitored for eight weeks (subcultures transferred biweekly) using a 16S rRNA gene-based PCR-DGGE approach. The results showed that the number of DGGE bands in the culture using seawater decreased dramatically as the enrichment progressed, and finally two predominant bands appeared in the consortium (Figure 2). In contrast, in the enrichment culture using modified MSB, many clear DGGE bands (band # 1−7) remained even after four transfers. Microbial populations of the DGGE bands were assessed by obtaining the 16S rRNA gene sequences from selected bands

closest type strain (accession no.) Pseudomonas mohnii Ipa-2T Pseudomonas taiwanensis BCRC 17751T Alteromonas stellipolaris LMG 21861T Thalassospira lucentensis DSM 14000T Marinobacterium lutimaris AN9T Gallaecimonas pentaromativorans CEE_131T Alteromonas stellipolaris LMG 21861T Thalassospira lucentensis DSM 14000T Maritimibacter alkaliphilus HTCC2654T Alteromonas stellipolaris LMG 21861T Desulf uromusa succinoxidans DSM 8270T

similarity (%)

frequencyb

95.0 98.6 100.0 96.1 99.6 96.1 100.0 98.4 97.0 100.0 94.8

(2/9) (3/9) (4/9)

a

The band could not be successfully cloned and sequenced. bClone inserts derived from bands 1−9 showed single fragment patterns in RFLP analysis. However, inserts from band #10 exhibited RFLP patterns indicative of 3 bacteria. cAfter separation, approximately 430 bp were sequenced. Closest relatives were assessed via EzTaxon 2.165.

culture represented Alteromonas and the next dominant band (# 9) belonged to Thalassospira. The intensity of the Alteromonas band was relatively constant, while the intensity of the Thalassospira band increased as the subculture progressed (Figure 2). Sequence analyses of the DGGE bands from the modified MSB enrichment culture showed that the bands represented an unidentified organism (band 1), Pseudomonas (bands 2 and 3), Alteromonas (band 4), Thalassospira (band 5), Marinobacter (band 6), and Gallaecimonas (band 7). Among these, the DGGE bands representing Alteromonas and Thalassospira were observed in both types of media. The DGGE band (# 10) of the original sample (at time zero), which corresponded to later bands 4 and 8, belonging to Alteromonas, was found to represent three different bacterial groups: Alteromonas, Desulf uromusa, and Maritimibacter. Thus, Alteromonas dominated the enrichment cultures and also was present initially in the crude oil-contaminated tidal sediment (Table 1). Isolation of Bacteria from Enrichment Cultures and Their Naphthalene Degradation Abilities. Bacterial strains from the two enrichment cultures were isolated by spreading the enrichment samples onto MA. The 16S rRNA genes of isolates grown on MA were PCR-amplified, double-digested with a mixture of HaeIII and HhaI, and sorted by their RFLP fragment patterns. 16S rRNA genes with unique RFLP patterns were sequenced and analyzed (Table 2). The enrichment culture from modified MSB yielded nine different bacterial groups, which corresponded largely with those retrieved from the DGGE bands. In contrast, the enrichment culture in seawater yielded two dominant bacterial groups: Alteromonas species (14 of 20 colonies) and two Thalassospira species (6 of 20 colonies) -- matching the bacterial groups obtained from the DGGE analysis (Table 2). Naphthalene degradation abilities of all strains isolated from the two enrichment cultures were evaluated in sterile 160-mL

Figure 2. DGGE profiles of partial 16S rRNA gene fragments from enrichment cultures in modified MSB and seawater with naphthalene pellets. The enrichment cultures were transferred into fresh modified MSB or filter-sterilized seawater with naphthalene pellets every two weeks. The transfer time (weeks) for the samples is listed above the lanes. The arrows indicate the bands that were excised and sequenced. Band numbers shown here correspond to partial 16S rRNA gene sequences shown in Table 1. 7733

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serum bottles. Surprisingly, only the Alteromonas species (strains AN5 and SN2) exhibited naphthalene degradation abilities (data not shown). Phylogenetic analysis using the almost complete 16S rRNA gene sequence (GenBank no. GU166736), indicated that strain SN2 shared 99.5% sequence similarity to Alteromonas stellipolaris LMG 21861T (Figure S1). The Alteromonas16S rRNA gene sequence retrieved from the unenriched sea-tidal flat (Figure 2, band 10) was identical to that of strain SN2. Biodegradation of PAHs by Alteromonas sp. Strain SN2 in Liquid Media and Slurry Systems. The PAH degradation ability of strain SN2 was evaluated in filtered seawater and modified MSB medium containing 30 mg/L of each individual PAH compound (naphthalene, phenanthrene, anthracene, and pyrene). Figure 3 shows that the individual PAH concentration profiles with respect to incubation time; PAH losses in uninoculated control experiments were negligible. Strain SN2 was able to degrade all of the tested PAH compounds, naphthalene, phenanthrene, anthracene, and even pyrene (Figure 3). For a given PAH compound, degradation rates were consistently more rapid in filtersterilized seawater than in the modified MSB medium. As expected, PAH compounds with more rings were degraded more slowly, thus pyrene degradation was very slow (Figure 3d). These results demonstrate that seawater was conducive for strain SN2 enrichment and PAH biodegradation in seawater by strain SN2 occurred without the addition of nutrients. PAH biodegradation tests were also conducted in slurry systems to better simulate in situ sea-tidal flat conditions. When strain SN2 cells were inoculated into sterile slurries, biodegradation of the four compounds occurred (Figure S2). More significantly, in nonsterile treatments, PAH degradation

Table 2. Identities and Abundances of Bacteria Isolated on Marine Agar from Enriched Microcosms Using Modified MSB and Seawater with Naphthalene enrichment media modified MSB

isolates AN1 AN2 AN3 AN4 AN5 AN6 AN7 AN8 AN9

seawater

SN1 SN2 SN3

closest type strain (accession no.) Galbibacter mesophilus Mok-17T Pseudomonas brassicacearum ATCC 49054 T Microbacterium maritypicum DSM 12512T Serratia odorifera DSM 4582T Alteromonas stellipolaris LMG 21861T Microbacterium aerolatum V-73T Thalassospira xiamenensis M-5T Pseudoalteromonas nigrifaciens NCIMB 8614T Marinobacterium litorale IMCC1877T Thalassospira lucentensis DSM 14000T Alteromonas stellipolaris LMG 21861T Thalassospira xiamenensis M-5T

similarity (%)

frequencya

91

1/50

99

4/50

91

3/50

99

4/50

100.0

6/50

99

1/50

99

10/50

99

2/50

96

19/50

98

5/20

100.0

14/20

99

1/20

a

Abundance of colony type found on modified MSB (50 colonies total) and seawater (20 colonies total).

Figure 3. Biodegradation of naphthalene (a), phenanthrene (b), anthracene (c), and pyrene (d) by strainSN2 added to seawater and modified MSB. Initial inoculum cell densities of strain SN2 were approximately 106 cells/mL for naphthalene degradation and 107 cells/mL for phenanthrene, anthracene, and pyrene degradations, respectively. Symbols in the figures are as follows: modified MSB inoculated with strain SN2 (●), filtersterilized seawater inoculated with strain SN2 (○), modified MSB uninoculated (▼), and filter-sterilized seawater uninoculated (Δ). The symbols indicate the averages of triplicate experiments; the error bars indicate one standard deviation. 7734

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Using Microcosm Slurry Incubations To Refine Metabolite and Nucleic Acid (Enumeration and Gene Expression) Biomarker Assays for Alteromonas-Catalyzed Naphthalene Degradation. Catechol and gentisate are key intermediary metabolites that distinguish the two primary naphthalene catabolic pathways (catechol versus gentisate) from one another. Using authentic standards, we developed a GC/MS protocol to detect and quantify catechol and gentisate in laboratory microcosms containing sediment slurries amended with naphthalene (100 ppm). In these microcosms containing freshly gathered sediment, gentisate was readily detected after 7 days of incubation (retention time 14.8 min; peak area corresponding to 0.57 μM), while catechol was undetectable (Figure S4a). In microcosms inoculated with strain SN2 (∼106 cells/mL), naphthalene loss was accelerated: after 17 h, the gentisate peak area found (Figure S4b) corresponded to 26.8 μM. Prior genomic analyses of strain SN2 45,46 have indicated that strain SN2 operates on naphthalene via the gentisate pathway. The elevated gentisate concentration found in the SN2-inoculated microcosms (Figure S4b) confirms that Alteromonas strain SN2 metabolizes naphthalene through the gentisate pathway and provides evidence that gentisate may be a metabolic biomarker supporting (at least consistent with) the naphthalenebiodegradation activity of Alteromonas-related bacteria in field samples. To confirm and strengthen the link between naphthalene loss in sediments and strain SN2 (and its relatives), we completed qPCR assays enumerating both total bacteria and Alteromonas-related species, while measuring transcription levels of Alteromonas-specific NDO genes in the microcosms described above (sediment slurries plus 100 ppm naphthalene with and without an inoculum of strain SN2 cells; Figure 4). Exact determination of bacterial cell numbers in microbial communities by quantitative real time-PCR is impossible because the copy number of chromosomal 16S rRNA gene operons varies with species type;47 however, the qPCR approach does allow valid comparison of relative abundance. The copy numbers of native Alteromonas-related species increased from 105 to 106/mL over 7 days in the serum bottles with 100 ppm naphthalene (Figure 4a), suggesting that native Alteromonas-related species metabolized naphthalene for their cell growth. In the parallel microcosms inoculated with strain SN2 cells, as expected, total Alteromonas cells were 1−2 orders of magnitude higher and the populations only grew slightly during the assay. Reverse transcriptase qPCR using the Alteromonas-specific primer set for mRNA transcripts showed that the transcriptional levels of native Alteromonas-related NDO genes increased markedly in these naphthalene-amended serum bottles (Figure 4b). Clearly the native Alteromonasrelated populations responded to naphthalene and this response was reflected by the NDO mRNA pool. As expected in the positive-control microcosms inoculated with strain SN2, the quantity of NDO transcripts also increased dramatically during the incubation. Applying the Nucleic-Acid and Metabolite Biomarker Assays to the Sea-Tidal Flat Field Site: Quantitative Seasonal Monitoring of Total Bacteria and Alteromonas Species and Alteromonas-Related NDO Gene Expression. The above series of laboratory-enrichment, pure culture, and sediment-based-microcosm experiments provided us with clear evidence that bacteria very closely related to strain SN2 occur naturally in the sea-tidal-flat habitat. This population

rates were enhanced by addition of SN2 cells (Figure S2). The naphthalene and phenathrene degradation rates in the slurry systems were much faster than in the liquid-media systems; strain SN2 completely degraded naphthalene within four hours (Figures S2ab). However, for the more hydrophobic PAH compounds (anthracene and pyrene) degradation was slower in the slurry system than liquid-media system (Figures S2 cd). This may stem from the propensity of anthracene and pyrene to adsorb onto sediment particles and limit compound bioavailability. These data establish that strain SN2 is metabolically active in PAH degradation under conditions mimicking those (nutritionally, physiologically, and ecologically) found in situ in the tidal-flat habitat. PCR Primer Design for NDO Genes and Their Application To Strain SN2. Using GenBank data, we created an alignment of gene sequences encoding known naphthalene dioxygenases (Figure S3). Noting that the clade containing the gene from Alteromonas (and Pseudoalteromonas) was adjacent to the corresponding clade from Cycloclasticus, we felt that an ability to discriminate between the two would be useful; thus, specific PCR primer sets (AltNDO199F/AltNDO554R and CycNDO29F/CycNDO461R) were designed to target the NDO genes of either Alteromonas sp. or Cycloclasticus sp., respectively (Table S1). Next, to characterize the NDO gene of strain SN2, the above two primer sets and five previously reported degenerate PCR primer sets (Table S1) were applied to SN2 genomic DNA for PCR amplification. A PCR amplicon was successfully produced only with AltNDO199F/AltNDO554R and pPAH-F/pPAH-NR700 primer sets (data not shown); the amplicon from the latter was sequenced. The resultant NDO gene sequence from strain SN2 (GenBank no. JF329708) clustered, as expected, closely with dioxygenases of the Neptumonas, Pseudoalteromonas, and Alteromonas groups (>98%) (Figure S3). Analysis of the NDO Gene Sequences from Genomic DNA and mRNA Extracted from the Contaminated SeaTidal Flat. Six PCR primer sets (NahAc-114F/NahAc-596R, NDO-F200/NDO-F201, NDO-F201/NDO-F202, P8073/ P9047, AltNDO199F/AltNDO554R, and CycNDO29F/CycNDO461R) were applied to genomic DNA and mRNA that were extracted from the contaminated sea-tidal flat in order to attempt to amplify a broad diversity of NDO genes. Amplification was successful with only two primer sets: AltNDO199F/AltNDO554R and CycNDO29F/CycNDO461R, which targeted Alteromonas sp. and its relatives and Cycloclasticus sp., respectively (data not shown). The resulting sequences were aligned, and their relationships to known NDO genes are presented in Figure S3. The naphthalene dioxygenase gene sequence retrieved by AltNDO199F/AltNDO554R from the genomic DNA was identical to that of strain SN2. The naphthalene dioxygenase gene sequence retrieved by CycNDO29F/CycNDO461R was very closely related (>99%) to that of known Cycloclasticus strains. Reverse transcriptase-PCR (RT-PCR) using the six primer sets and mRNA templates from the sea-tidal flat sample was conducted, but only the AltNDO199F/AltNDO554R primer set produced a RT-PCR product (data not shown). PCR without reverse transcriptase treatment did not produce any PCR product. The retrieved sequence from the mRNA transcripts was also identical to that of strain SN2 (Figure S3). Thus, in our broad survey of field-expressed NDO genes, only the gene hosted by strain SN2 in the tidal-flat sediments was detected. 7735

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Figure 5. Seasonal changes of the 16S rRNA gene copy numbers for total bacteria and Alteromomas species (strain SN2) and naphthalene catabolic gene transcription of Alteromomas-related species (strain SN2) in the crude oil-contaminated sea-tidal flat during the two years since the oil-spill accident. All measurements were performed independently in triplicate. The relative transcription ratios (Y-axis) were calculated as percentages of the naphthalene dioxygenase gene transcription level of the sea-tidal flat sample at the onset of the spill (December 2007). Error bars represent standard deviations.

Figure 4. Quantifying Alteromonas abundance and naphthalene dioxygenase (NDO) gene transcription in laboratory-incubated (10% sediment) microcosms. Shown are the 16S rRNA gene copy numbers for total bacteria and Alteromomas species (strain SN2) (a) and naphthalene catabolic gene transcription of strain SN2-related species (b). All measurements were performed independently in triplicate. The relative transcription ratios (Y-axis) were calculated as percentages of the transcription level of serum bottles (day 0) without SN2 inoculation. Error bars represent standard deviations.

tidal-flat ecosystem (carbon inputs and fluxes are just one example) that drastically influence heterotrophic activity. To focus in on metabolic pathways likely manifest during NDO gene expressed in situ, we analyzed field-fixed sediments for the presence of gentisate using the GC/MS procedure tested in microcosms. A sea-tidal flat sample (June, 2011) was obtained from sediments in which PAH compounds had almost disappeared (naphthalene concentration