Environ. Sci. Technol. 2008, 42, 4099–4105
Effects of Enrichment with Salicylate on Bacterial Selection and PAH Mineralization in a Microbial Community from a Bioreactor Treating Contaminated Soil SABRINA N. POWELL, DAVID R. SINGLETON, AND MICHAEL D. AITKEN* Department of Environmental Sciences and Engineering, School of Public Health, CB#7431, 166 Rosenau Hall, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7431
Received December 2, 2007. Revised manuscript received March 5, 2008. Accepted March 13, 2008.
We investigated enrichment with salicylate as a method to stimulate the degradation of polycyclic aromatic hydrocarbons (PAHs) by a microbial community from a bioreactor treating PAHcontaminated soil. DNA-based stable isotope probing (SIP) was used to compare the effect of alternate methods of salicylate addition (spike vs slow, continuous addition) on the diversity of the enriched microbial community. After identification of salicylate degraders by SIP, real-time quantitative PCR (qPCR) primers were developed to quantify the abundances of three groups containing salicylate-utilizing organisms in the bioreactor community before and after enrichment. The different methods of salicylate addition were found to select for different microbial communities. Two groups containing salicylatedegrading bacteria increased in abundance substantially after enrichment by continuous addition of salicylate but did not increase in abundance in response to the spike addition, whereas a third group increased in abundance in response to both methods of salicylate addition. The initial rate of naphthalene mineralization increased significantly after enrichment by spike addition of salicylate, but neither phenanthrene nor benzo[a]pyrene mineralization rates were enhanced. Continuous addition of salicylate did not enhance the mineralization rate for any of the PAHs. These results suggest that enrichment with salicylate can select for naphthalene-degrading bacteria, but does not select for organisms responsible for degrading PAHs of higher molecular weight. Differences in microbial selection observed in this study that resulted from different rates of carbon source addition also have implications for the design of SIP experiments with water-soluble carbon sources.
Introduction Polycyclic aromatic hydrocarbons (PAHs) are among the most common pollutants found at contaminated industrial sites. Because many PAHs are susceptible to microbial degradation (1), bioremediation is an attractive treatment option for PAH* Corresponding author phone 919-966-1024; e-mail: mike_aitken@ unc.edu 10.1021/es703007n CCC: $40.75
Published on Web 04/19/2008
2008 American Chemical Society
contaminated systems (2). However, the extent of removal of the four-, five-, and six-ring PAHs during biological treatment of contaminated soil is often substantially lower than the removal of the lower-molecular-weight compounds (3), so that strategies to enhance the performance of bioremediation systems might be required to achieve cleanup goals. Strategies to increase the growth or sustain the activity of organisms responsible for degrading the more recalcitrant PAHs would be appropriate in situations in which the abundances of these organisms are low or when the PAHs are degraded primarily by cometabolism. Salicylate induces the metabolism of a range of PAHs in PAH-degrading bacteria (4–9), and its addition to contaminated soil or sediment as a selective carbon source has been proposed as a means of stimulating PAH degradation during bioremediation (6, 10). The addition of salicylate has been demonstrated to increase the populations of naphthalenedegrading bacteria in PAH-contaminated soil (10, 11) and to sustain naphthalene degradation by a microbial community derived from activated sludge (12). Salicylate has also been used as a selective carbon source to sustain populations of so-called biological control bacteria possessing genes for naphthalene metabolism in agricultural systems (13–15). Limited work has been done on the effects of salicylate on the degradation of PAHs other than naphthalene in complex microbial communities. In one study, the addition of salicylate to PAH-contaminated soils had no effect on phenanthrene or pyrene mineralization (16). Salicylate addition to uncontaminated soil spiked with pyrene and benzo[a]pyrene (BaP) enhanced pyrene mineralization but had no effect on BaP (17). The objective of the present study was to investigate whether enrichment with salicylate would select for PAHdegrading bacteria and correspondingly stimulate PAH degradation by the microbial community in a slurry-phase bioreactor treating PAH-contaminated soil. We hypothesized that the manner in which salicylate was added to the microbial community (continuous addition vs a single pulse, or spike) would influence the types and abundances of the organisms selected, and that differences in the selected organisms could correspond to differences in PAH degradation. DNA-based stable-isotope probing (SIP) was used as the basis for evaluating the diversity and abundance of the salicylate-consuming microorganisms. We then measured the effect of salicylate enrichment on initial rates of mineralization of naphthalene, phenanthrene, and benzo[a]pyrene in the soil slurry. Naphthalene and phenanthrene were selected because they are both known to be metabolized via pathways in which salicylate is an intermediate. Benzo[a]pyrene was selected as a high-molecular-weight (HMW) PAH that is degraded primarily by cometabolism (18), implying that organisms capable of cometabolizing BaP must grow on other carbon sources.
Materials and Methods Chemicals. [13C6]salicylic acid was synthesized from [13C6]phenol (Cambridge Isotope Laboratories, Andover, Mass.) as described previously (19). Natural abundance isotopomer sodium salicylate (Mallinckrodt) was American Chemical Society grade. [UL-14C]naphthalene, [9-14C]phenanthrene, and [7-14C]benzo[a]pyrene were purchased from Sigma-Aldrich (St. Louis, Missouri). The specific activities were 17.8 mCi/mmol, 8.3 mCi/mmol, and 26.6 mCi/ mmol, respectively. Samples. PAH-contaminated soil was obtained from a former manufactured-gas plant (MGP) site in Charlotte, NC VOL. 42, NO. 11, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
4099
and treated in a bench-scale, aerobic, slurry-phase bioreactor operated as described previously (19). Samples for all experiments were obtained directly from the bioreactor at the time of each experiment. Salicylate Enrichment Experiments. All incubations were performed in 125 mL sterilized screw-top Erlenmeyer flasks containing 10 mL of freshly sampled reactor slurry as inoculum and were shaken at 150 rpm and 23 °C. Salicylate (1 mg total) dissolved in phosphate buffer (10 mM, pH 7.0) was added to reactor slurry in one of two ways: an initial single-dose spike or continuously throughout the incubation. The total dose of salicylate was selected to ensure substantial growth of salicylate-degrading bacteria and to minimize accumulation of salicylate during continuous incubation over a 10-d period, as determined from preliminary experiments. A dose of 0.5 mL salicylate dissolved in phosphate buffer (2.0 mg/mL) was added to spike incubations at the beginning of the enrichment. At the same time, 0.5 mL of phosphate buffer (without salicylate) was added to continuous incubations. For continuous incubations, dilute salicylate (0.2 mg/mL) was then infused into each flask with a syringe pump (ColeParmer 79400 series; Vernon Hills, Ill.) at a rate of 0.5 mL/ day for 10 days. Spike incubations received phosphate buffer (without salicylate) delivered at the same rate. Salicylate concentrations in aliquots of slurry sampled during the enrichment were determined by HPLC as described previously (19). The estimated detection limit was 0.2 mg/L. Preliminary experiments showed no loss of added salicylate in uninoculated controls. SIP Incubations. Salicylate (1.0 mg, either 13C-labeled or unlabeled) dissolved in phosphate buffer was added to 10 mL reactor slurry either continuously or in spike form as described above. Incubations with 13C-labeled salicylate were performed in duplicate, and duplicate incubations with unlabeled salicylate were set up in parallel for extraction of DNA for quantitative real-time PCR (qPCR) analysis. Single spike and continuous incubations with unlabeled salicylate were also set up in parallel to monitor salicylate concentrations by HPLC. DNA was extracted as described previously (19), except a Mini-BeadBeater (Biospec Products, Bartlesville, OK) run for 2 min at 2500 rpm was used instead of vortexing. Separation of 13C-enriched DNA from unenriched DNA using cesium chloride density-gradient ultracentrifugation, collection of fractions, and purification of the DNA in each fraction were carried out as described previously (19). A total of 12 fractions of 400 µL each were collected for each sample. DNA recovered from each fraction was resuspended in a total of 75 µL of Tris-EDTA buffer (pH 8.0). Molecular Analyses. Fractions obtained after ultracentrifugation of extracted DNA from SIP incubations were analyzed by denaturing-gradient gel electrophoresis (DGGE) to confirm separation of 13C-labeled (“heavy”) and unlabeled DNA, as described previously (19). Screening of the heavy DNA for archaeal and fungal sequences, construction of bacterial 16S rRNA gene clone libraries, and construction of the phylogenetic tree were also performed as described previously (19). Randomly picked clones were partially sequenced by SeqWright (Houston, Texas) using primer 8f (20). Clones SalSp08, SalCon01, SalCon26, SalCon39, and SalCon44, which represented groups of frequently occurring, nearly identical sequences in the libraries, were fully sequenced using primers M13f, M13r, 338f, 338r, 907f, and 907r by the University of North CarolinasChapel Hill Genome Analysis Facility. Sequences were deposited in GenBank with accession numbers EF101781-EF101865. LIBSHUFF analyses of clone libraries were performed using 1000 comparisons (21, 22). Primer development for qPCR and quantification of targeted 16S rRNA genes in extracted DNA samples were performed using the protocols described in Singleton et al. (23). Primer sets used in this 4100
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 42, NO. 11, 2008
study are detailed in Table S1 of the Supporting Information. Standard curves for quantification were run in duplicate and experimental samples in triplicate. Mineralization Assays. Mineralization assays with [14C]naphthalene, [14C]phenanthrene, and [14C]benzo[a]pyrene were used to compare initial rates of degradation of these compounds by the reactor slurry microbial community under each of three conditions: unamended, amended with salicylate as a spike, or amended with salicylate continuously. Preliminary experiments were conducted with each compound to identify the time period over which the initial rate of 14CO2 recovery was linear in reactor slurry preincubated for 48 h after adding a spike of 100 mg/L salicylate (Figure S4 in the Supporting Information). Assays were carried out in triplicate for each condition in sterilized 40 mL EPA vials (Laboratory Supply Distributors, Mt. Laurel, NJ) with screwtop lids and septa that were lined with aluminum foil. Each assay consisted of adding 20 000 disintegrations per minute (dpm) of a 14C-labeled PAH to 5 mL of freshly sampled reactor slurry (unamended), reactor slurry amended with salicylate as a spike (48 h preincubation before adding 14C-PAH), or slurry amended with salicylate continuously over 10 days as described above for the SIP incubations. After adding the 14C-labeled PAH, incubation periods were 20 min for [14C]naphthalene, 20 min for [14C]phenanthrene, and 24 h for [14C]benzo[a]pyrene. The vials were shaken at 150 rpm and 23 °C. For killed controls, 20% H3PO4 was added to acidify the reactor slurry to pH < 2. The 14CO2 generated was captured in CO2 traps and counted as described elsewhere (24).
Results Preliminary Experiments. We first verified that salicylate was undetectable in freshly sampled reactor slurry. In preliminary experiments with spike addition of salicylate, the salicylate was consumed to below the detection limit in 24–48 h (data not shown) and the rate of salicylate degradation increased following enrichment of the reactor slurry with salicylate (Figure S1 in the Supporting Information). Based on increases in abundance of total bacterial 16S rRNA genes during a spike incubation with salicylate, the maximum specific growth rate µm was 0.23 h-1 and the yield coefficient Y was 1.6 × 109 gene copies/mg salicylate (see Supporting Information for details). These values represent collective growth of salicylate-degrading bacteria under batch growth conditions. SIP Incubations. We compared the organisms selected by adding 13C-salicylate to reactor slurry either continuously over a 10 day period or as a single spike. In the spike incubations, the salicylate concentration in the SIP experiment was below the detection limit within 24 h (Figure S2 in the Supporting Information). In continuous incubations, the concentration accumulated to 3.7 mg/L after 6 h of incubation but was below the detection limit by 18 h and for the remainder of the incubation (Figure S2). The minimal accumulation of salicylate in continuous incubations indicates that the microbial community was metabolizing salicylate as fast as it was being infused and that it was exposed to very low salicylate concentrations over virtually the entire incubation period. DNA yields from the spike and continuous incubations were comparable. No significant differences in the quantity or quality of DNA were observed between replicates or between incubations with unlabeled and 13C-labeled salicylate. PCR-DGGE was used to screen fractions collected from ultracentrifuge tubes after separation of the 13C-labeled DNA from unlabeled DNA and no differences were seen in DGGE banding patterns between duplicate incubations (data not shown). In samples with unlabeled salicylate, there were no PCR products in the fractions where 13C-enriched (“heavy”) DNA would be expected to be present. Domain-
FIGURE 1. Phylogenetic tree of 16S rRNA gene sequences recovered from heavy (13C-enriched) DNA in SIP incubations and selected close relatives. Clones from this study are shown in bold and follow the naming scheme of SalSp (salicylate spike) or SalCon (salicylate continuous) and a number assigned to each clone for identification purposes. GenBank accession numbers are shown in parentheses for all sequences. When a sequence represents similar sequences in an OTU, the number of clones in each library (S, spike; C, continuous) is indicated in brackets after the clone name. Bootstrap values are indicated on nodes with an open (O) and closed (b) circle representing >95 and >50% bootstrap support, respectively. Primer sets expected to amplify a particular group of sequences are denoted to the right side of the tree for all sequences that fall within the brackets with the exception of Polaromonas naphthalenivorans CJ2, which, based on sequence data, should not be amplified by the Sal Group 1 qPCR primers. specific PCR primers (25–27) used to screen heavy DNA from all incubations produced no archaeal or fungal amplicons. 16S rRNA Gene Clone Libraries. Clone libraries of 16S rRNA genes were constructed from the fractions containing heavy DNA from spike and continuous incubations. Partial sequences were obtained for 42 randomly selected clones derived from spike incubations and 42 randomly selected clones derived from the continuous incubations. Clones were grouped at the >99% similarity level to define eight operational taxonomic units (OTUs), and clones representing the five OTUs containing the most sequences in the clone library
were completely sequenced: SalSp08, SalCon01, SalCon26, SalCon39, and SalCon44. An additional six singleton clones were not grouped into OTUs. Phylogenetic relationships among the eight OTUs and six singleton sequences are illustrated in Figure 1. Sequences that were targeted by the qPCR primers developed in this study are designated as SAL Groups 1, 2, and 3 in Figure 1. The majority of 16S rRNA gene clones (78 of 84) were within these groups. SAL Groups 1 and 2 contained sequences recovered only from incubations in which salicylate was added continuously, whereas SAL Group 3 contained seVOL. 42, NO. 11, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
4101
quences recovered from both spike and continuous incubations. The remaining six sequences that did not cluster with these groups were found only in the library from the continuous incubation (SalCon28, SalCon31, and SalCon36) or only in the library from the spike incubation (SalSp15 and SalSp35). SAL Group 3 contained sequences that are similar to members of the Ralstonia genus of the β-Proteobacteria, which have been found previously in PAH-contaminated soils (28, 29), and contained 62% of the combined sequences from both libraries. Sequences from the continuous incubation that clustered in SAL Group 1 were closely related to β-proteobacteria in the Comamonadaceae family, as well as uncultured organisms from various sources (Figure 1). Clone SalCon23 shares some similarity to Polaromonas naphthalenivorans, which was first identified through stable-isotope probing with naphthalene (30), although this organism would not have been targeted by primers for SAL Group 1. All of the sequences in SAL Group 3, as well as singleton clones SalSp15 and SalSp35, were very similar to sequences recovered from our previous SIP study with salicylate (spike addition) and naphthalene on the bioreactor slurry (19). Clone SalSp15 was also closely related to two clones recovered from an SIP investigation with phenol at an agricultural field site (31). Clone SalCon01 (SAL Group 2) clustered with sequences from the genus Pseudomonas and shared high similarity to uncultured clones 14 and 42 from another SIP study of naphthalene-degrading organisms in a PAH-contaminated soil (32). The singleton sequence SalCon28 from the continuous incubation was closely related to a group of sequences previously found in the same bioreactor used as the inoculum source in this study (23), but which have not been associated with PAH degradation. Clone SalCon31 was related to uncultured bacterium clone LO13.11 from an SIP study examining methanotrophs in peat soil (33). LIBSHUFF analyses indicated that the clone library derived from the spike incubation was not significantly different from that derived from the continuous incubation (p ) 0.001). However, the clone library from the continuous incubation was significantly different from the clone library derived from the spike incubations (p ) 0.892). These results are consistent with the clustering of sequences in Figure 1, which indicates that although both libraries shared many similar sequences (SAL Group 3), the library from the continuous incubations contained a number of sequences not present in the library from the spike incubation (particularly SAL Groups 1 and 2). Abundance of Salicylate-Degrading Organisms before and after Salicylate Enrichment. Quantitative PCR primers were designed to quantify the increase in abundance of 16S rRNA genes from SAL Groups 1, 2, and 3 in response to amendment with unlabeled salicylate in incubations carried out in parallel with the SIP incubations. For spike addition of salicylate, separate incubations were run for 2 or 10 days to evaluate the effect of extended incubation time on the abundance of the targeted organisms. SAL Group 1 primers had no mismatches to 200 16S rRNA gene sequences in RDPII Release 9.57 (Table S1), 190 of which are in the family Comamonadaceae. SAL Group 2 primers matched nine sequences in RDP-II, all of which are Pseudomonas spp. SAL Group 3 primers matched 52 sequences in the family Burkholderiaceae, all of them in the genera Ralstonia or Cupriavidus. All three groups evaluated by qPCR were a minority of the initial reactor slurry microbial community (∼0.01% of total 16S rRNA genes; Figure 2). The spike addition of salicylate in the 2 day incubation provided a strong selection for members of SAL group 3 (Ralstonia spp.), whose sequences increased by 4 orders of magnitude relative to their con4102
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 42, NO. 11, 2008
centration in the initial slurry community (Figure 2a). The increase in 16S rRNA gene copies of SAL Group 3 sequences after 2 days (approximately 109 copies) is consistent with the yield of 1.6 × 109 copies of total bacterial 16S rRNA genes per mg salicylate determined in a separate 2 day incubation with spike addition of salicylate (see Supporting Information). However, the abundance of SAL group 3 sequences fell by 2 orders of magnitude by the end of the 10 day spike incubation (Figure 2a). SAL group 3 organisms comprised 28% of the total 16S rRNA genes present after the 2 day spike incubation, but only 9% after the 10 day spike incubation (Figure 2b). Thus, there appears to have been significant decay of the SAL group 3 organisms between 2 and 10 days. Sequences associated with SAL groups 1 and 2 (various β-Proteobacteria and select Pseudomonas spp., respectively) were not appreciably selected during spike addition of salicylate (Figure 2), consistent with their presence only in the clone library derived from the continuous incubation with salicylate during the SIP experiment. Conversely, after enrichment by continuous addition of salicylate these two groups combined represented approximately 15% of the total 16S rRNA genes (Figure 2b). Continuous addition also enriched SAL group 3, which, although not attaining the copy number observed with the spike addition, was still approximately 5% of the total 16S rRNA genes after enrichment (Figure 2b). These trends were confirmed in a followup experiment in which the abundances of SAL Groups 1, 2, and 3 were measured at various time points during spike and continuous enrichment with salicylate (Supporting Information, Figure S3). Most of the observed growth of SAL Group 3 organisms occurred between 24 and 48 h in the spike enrichment. The greatest relative increase in abundance of all three groups occurred over the first 3 days in the continuous enrichment (Figure S3). Effect of Salicylate Addition on PAH Mineralization. Preliminary experiments verified that enrichment of the bioreactor slurry with salicylate either as a spike or added continuously led to a substantial increase (approximately 2.5-fold) in the mineralization of 14C-labeled salicylate over a 1 h period compared to unenriched controls (data not shown). Spike enrichment with salicylate increased the initial rate of [14C]naphthalene mineralization by 50% relative to the unenriched sample (Figure 3), a difference that was significant according to a two-tailed Student’s t test (p ) 0.0014). Spike enrichment did not appreciably increase the mineralization rate of [14C]phenanthrene or [14C]benzo[a]pyrene. Continuous enrichment with salicylate did not increase the mineralization of any of the three PAHs tested.
Discussion The microorganisms selected by the addition of salicylate to slurry from a bioreactor treating PAH-contaminated soil depended on the manner in which salicylate was added. Continuous addition enriched a more diverse population of salicylate utilizers than spike addition, as determined from the clone libraries of “heavy” DNA from SIP incubations (Figure 1). In subsequent qPCR analyses, we could not determine whether all sequences that were amplified by the qPCR primers were from organisms capable of degrading salicylate. However, the order-of-magnitude increases in 16S rRNA gene copy number (from very low initial abundances; Figure 2) after salicylate addition strongly suggest that the sequences amplified during qPCR are associated with salicylate-degrading bacteria in the targeted groups. Clone libraries indicated that the organisms selected by spike addition of salicylate were largely a subset of the organisms selected by the continuous addition of salicylate (SAL Group 3, Figure 1). This conclusion was supported by the LIBSHUFF analyses. The organisms selected in the spike
FIGURE 2. Abundances of organisms represented by SAL Groups 1, 2, and 3 before and after enrichment with salicylate, expressed as (a) 16S rRNA gene copy number per mL reactor slurry and (b) percent relative abundance. Day 0 represents unenriched reactor slurry. Abundances measured 2 or 10 days after spike addition of salicylate were from separate incubations. Values are the means and standard deviations of triplicate qPCR reactions using the same DNA as template, performed on duplicate samples. For calculations of relative abundance, the number of 16S rRNA gene copies of a given group was normalized by the average number of total 16S rRNA gene copies in the sample as determined with general bacterial primers applied to the group-specific standard. incubation appear to be primarily Ralstonia and Pseudomonas spp. related to known or suspected naphthalene degraders, whereas sequences that were unique to the continuous incubations were generally not closely related to previously characterized PAH-degrading organisms. Ralstonia spp. were also the dominant sequences recovered in a recent study (24) of the effects of spike addition of phthalate, another intermediate of PAH degradation, on the same bioreactor slurry used in this study, suggesting that some Ralstonia spp. might be efficient degraders of aromatic acids. The observation that different rates of substrate addition can select for different organisms within a microbial community can have implications for the addition of supplemental carbon sources as a bioremediation strategy. It is a well-known principle in microbial ecology that some microorganisms are better adapted to conditions in which carbon and energy sources are always present at low concentrations (so-called K-strategists), whereas other organisms flourish by growing rapidly under conditions in which high substrate concentrations exist (so-called rstrategists). This principle has also been well-established in the study of bioreactors for waste treatment (34–36). The findings of this study also have implications for the design of SIP experiments. The application of SIP requires growth of a microbial community on a given carbon source, with a corresponding potential for enrichment bias (37) that
could depend on the rate at which the carbon source is made available to the community or on its instantaneous concentration in the medium. In our previous SIP studies with the reactor community (19, 23), in which sparingly soluble substrates such as phenanthrene and pyrene were added in crystalline form, dissolution of the crystals was more analogous to continuous incubation with a water-soluble substrate than to spike addition. In most other SIP experiments published in the literature, the [13C]substrate was delivered as single or multiple spikes. Researchers planning future SIP experiments with water-soluble substrates should carefully consider the concentrations and rates at which the substrate will be provided to the microbial community. Nevertheless, although the total dose and rate of addition of an exogenous substrate in an SIP experiment can introduce enrichment bias, the enrichment methods used in the present study were intended to represent a potential remediation strategy, not extant conditions in the bioreactor or in the original contaminated soil. To evaluate the extent to which organisms that grew on salicylate could also degrade PAHs, we measured the rate at which the bioreactor microbial community mineralized selected PAHs. Of the three PAHs tested, only naphthalene was mineralized at a higher initial rate following enrichment with salicylate, and only when it was added as a spike (Figure 3). This agrees with the SIP results in a prior study with this VOL. 42, NO. 11, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
4103
FIGURE 3. Mineralization of [14C]naphthalene, [14C]phenanthrene, and [14C]benzo[a]pyrene by reactor slurry before and after spike or continuous enrichment with salicylate. Spike enrichments were evaluated 2 days after adding salicylate, and continuous enrichments were evaluated at the end of the 10 day period over which salicylate was added continuously. Values are means and standard deviations of triplicate incubations. Incubations were terminated over time points within the period over which mineralization was approximately linear (20 min for naphthalene and phenanthrene, 24 h for BaP). bioreactor community (19), in which salicylate degraders selected after spike addition were closely related to naphthalene degraders but not to phenanthrene degraders. The uncharacterized salicylate degraders that were unique to enrichment by continuous addition of salicylate in this study (SAL Groups 1 and 2) do not appear to be able to mineralize any of the three PAHs tested at significant rates. Although the abundances of SAL Groups 1 and 2 increased by orders of magnitude in response to continuous salicylate addition (Figure 2), no increase in PAH mineralization was observed after continuous enrichment (Figure 3). Spike addition of salicylate to PAH-degrading microbial communities in previous studies has also led to increased naphthalene degradation rates (10, 12) but no effect on phenanthrene degradation (12, 16). The increase in naphthalene mineralization rate after spike enrichment with salicylate was modest (approximately 50% relative to unenriched controls). Although the mineralization and qPCR measurements were performed with different samples of reactor slurry, spike enrichment with salicylate led to similar periods over which the added salicylate was consumed in different experiments and to similarly high abundances of SAL Group 3 organisms (compare Figures 2 and S3). A substantial increase in concentration of salicylatedegrading bacteria that are phylogenetically related to naphthalene-degrading bacteria would have been expected to lead to a larger increase in naphthalene mineralization rate than we observed. It is possible that some of the salicylate degraders selected by spike enrichment are not capable of mineralizing naphthalene. Alternatively, there may have been a high background rate of naphthalene mineralization by organisms in the initial reactor slurry that are capable of mineralizing naphthalene but which might not grow on salicylate (or naphthalene) to an extent that would have been observed in an SIP experiment. For example, some of the sequences associated with naphthalene degradation in our earlier SIP study with the bioreactor slurry (19) were not associated with salicylate degradation in either that study or the present study. Salicylate has been observed to induce the degradation of naphthalene (4), phenanthrene (6, 7, 38), benz[a]anthracene (5, 6), chrysene (6), fluoranthene (6, 8), and BaP 4104
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 42, NO. 11, 2008
(6, 9) in various aerobic bacteria. Not all pathways for PAH metabolism include salicylate as an intermediate, but the inducers of such pathways are generally unknown. Although an ability to induce the metabolism or cometabolism of a target compound would be desirable for a selective carbon source added to contaminated soil as a bioremediation strategy, induction alone might be insufficient to enhance the degradation of the target compound. Substantial growth of the relevant organism(s) would also be required, implying that sufficient carbon must be added to the system to result in significant increases in organism abundance. In this study, it is clear that sufficient salicylate was added to lead to substantial growth of salicylate-degrading bacteria, but with only modest effect on naphthalene mineralization potential and no effect on mineralization of phenanthrene or BaP. In a similar study (24), we found that the addition of phthalate, an intermediate in both phenanthrene and pyrene metabolism by aerobic bacteria, also did not stimulate PAH degradation by the bioreactor slurry used in the present study. The extent to which the findings from this study can be extrapolated to other systems is not known. However, enrichment with salicylate might be a useful strategy for enhancing naphthalene removal in those systems in which naphthalene degradation is limited at least in part by the abundance of naphthalene-degrading organisms. For example, naphthalene is a principal contaminant of groundwater downgradient of source zones at PAH-contaminated sites. Enrichment with a selective carbon source to stimulate the growth of naphthalene degraders might have to be accompanied by increased oxygen supply to augment the generally low dissolved oxygen concentrations in the contaminated groundwater at these sites (39–41).
Acknowledgments This work was supported by the National Science Foundation (grant BES-0221836), the National Institute of Environmental Health Sciences (grant 5 P42 ES05948), and an Environmental Protection Agency Science to Achieve Results (STAR) graduate fellowship to SNP (grant U 91592101).
Supporting Information Available Details on qPCR primers, determining the gross maximum specific growth rate and yield coefficient, and results of preliminary experiments referred to in the text. This material is available free of charge via the Internet at http:// pubs.acs.org.
Literature Cited (1) Cerniglia, C. E. Biodegradation of polycyclic aromatic hydrocarbons. Biodegradation 1992, 3, 351–368. (2) USEPA Office of Solid Waste and Emergency Response. Treatment Technologies for Site Cleanup. Annual Status Report, 12th ed.; U. S. Environmental Protection Agency: Washington, DC, 2007. (3) Aitken, M. D.; Long, T. C. Biotransformation, biodegradation and bioremediation of polycyclic aromatic hydrocarbons. In Soil Biology, Volume 2: Biodegradation and Bioremediation; Singh, A., Ward, O. P., Eds.; Springer-Verlag: Heidelberg, Germany, 2004; pp 83–124. (4) Yen, K. M.; Serdar, C. M. Genetics of naphthalene catabolism in pseudomonads. CRC Crit. Rev. Microbiol. 1988, 15, 247–268. (5) Mahaffey, W. R.; Gibson, D. T.; Cerniglia, C. E. Bacterial oxidation of chemical carcinogens: formation of polycyclic aromatic acids from benz[a]anthracene. Appl. Environ. Microbiol. 1988, 54, 2415–2423. (6) Chen, S. H.; Aitken, M. D. Salicylate stimulates the degradation of high-molecular weight polycyclic aromatic hydrocarbons by Pseudomonas saccharophila P15. Environ. Sci. Technol. 1999, 33, 435–439. (7) Meyer, S.; Moser, R.; Neef, A.; Stahl, U.; Kampfer, P. Differential detection of key enzymes of polyaromatic-hydrocarbondegrading bacteria using PCR and gene probes. Microbiology 1999, 145, 1731–1741.
(8) Alemayehu, D.; Gordon, L. M.; O’Mahony, M. M.; O’Leary, N. D.; Dobson, A. D. W. Cloning and functional analysis by gene disruption of a novel gene involved in indigo production and fluoranthene metabolism in Pseudomonas alcaligenes PA-10. FEMS Microbiol. Lett. 2004, 239, 285–293. (9) Rentz, J. A.; Alvarez, P. J. J.; Schnoor, J. L. Benzo[a]pyrene degradation by Sphingomonas yanoikuyae JAR02. Environ. Pollut. 2008, 151, 669–677. (10) Ogunseitan, O. A.; Olson, B. H. Effect of 2-hydroxybenzoate on the rate of naphthalene mineralization in soil. Appl. Microbiol. Biotechnol. 1993, 38, 799–807. (11) Ogunseitan, A. O.; Delgado, I. L.; Tsai, Y. L.; Olson, B. H. Effect of 2-hydroxybenzoate on the maintenance of naphthalenedegrading pseudomonads in seeded and unseeded soil. Appl. Environ. Microbiol. 1991, 57, 2873–2879. (12) Cardinal, L. J.; Stenstrom, M. K. Enhanced biodegradation of polyaromatic hydrocarbons in the activated sludge process. Res. J. Water Pollut. Control Fed. 1991, 63, 950–957. (13) Colbert, S. F.; Schroth, M. N.; Weinhold, A. R.; Hendson, M. Enhancement of population densities of Pseudomonas putida PpG7 in agricultural ecosystems by selective feeding with the carbon source salicylate. Appl. Environ. Microbiol. 1993, 59, 2064–2070. (14) Wilson, M.; Lindow, S. E. Enhanced epiphytic coexistence of near-isogenic salicylate-catabolizing and non-salicylate-catabolizing Pseudomonas putida strains after exogenous salicylate application. Appl. Environ. Microbiol. 1995, 61, 1073–1076. (15) Ji, P.; Wilson, M. Enhancement of population size of a biological control agent and efficacy in control of bacterial speck of tomato through salicylate and ammonium sulfate amendments. Appl. Environ. Microbiol. 2003, 69, 1290–1294. (16) Carmichael, L. M.; Pfaender, F. K. The effect of inorganic and organic supplements on the microbial degradation of phenanthrene and pyrene in soils. Biodegradation 1997, 8, 1–13. (17) Vanderford, M. Influence of Metabolites and Co-contaminants on the Biodegradation of Polycyclic Aromatic Hydrocarbons in Soils. PhD Dissertation, University of North Carolina at Chapel Hill, Chapel Hill, NC, 2001. (18) Kanaly, R. A.; Harayama, S. Biodegradation of high-molecularweight polycyclic aromatic hydrocarbons by bacteria. J. Bacteriol. 2000, 182, 2059–2067. (19) Singleton, D. R.; Powell, S. N.; Sangaiah, R.; Gold, A.; Ball, L. M.; Aitken, M. D. Stable-isotope probing of bacteria capable of degrading salicylate, naphthalene or phenanthrene in a bioreactor treating contaminated soil. Appl. Environ. Microbiol. 2005, 71, 1202–1209. (20) Edwards, U.; Rogall, T.; Blöcker, H.; Emde, M.; Böttger, E. C. Isolation and direct complete nucleotide determination of entire genes. Characterization of a gene coding for 16S ribosomal RNA. Nucleic Acids Res. 1989, 17, 7843–7853. (21) Singleton, D. R.; Furlong, M. A.; Rathbun, S. L.; Whitman, W. B. Quantitative comparisons of 16S rRNA gene sequence libraries from environmental samples. Appl. Environ. Microbiol. 2001, 67, 4374–4376. (22) Singleton, D. R.; Rathbun, S. L.; Dyszynski, G. E.; Whitman, W. B. LIBSHUFF comparisons of 16S rRNA gene clone libraries. In Molecular Microbial Ecology Manual, 2nd ed.; Kowalchuk, G. A.; de Bruijn, F. J.; Head, I. M.; Akkermans, A. D. L.; van Elsas, J. D., Eds.; Kluwer Academic Publishers: Dordrecht, Netherlands, 2004; pp 1361–1371. (23) Singleton, D. R.; Sangaiah, R.; Gold, A.; Ball, L. M.; Aitken, M. D. Identification and quantification of uncultivated proteobacteria associated with pyrene degradation in a bioreactor treating PAHcontaminated soil. Environ. Microbiol. 2006, 8, 1736–1745. (24) Singleton, D. R.; Richardson, S. D.; Aitken, M. D. Effects of enrichment with phthalate on polycyclic aromatic hydrocarbon biodegradation in contaminated soil. Biodegradation. in press.
(25) Dojka, M. A.; Hugenholtz, P.; Haack, S. K.; Pace, N. R. Microbial diversity in a hydrocarbon- and chlorinated-solvent-contaminated aquifer undergoing intrinsic bioremediation. Appl. Environ. Microbiol. 1998, 64, 3869–3877. (26) Gardes, M.; Bruns, T. D. ITS primers with enhanced specificity for basidiomycetes - application to the identification of mycorrhizae and rusts. Mol. Ecol. 1993, 2, 113–118. (27) White, T. J.; Bruns, T. D.; Lee, S.; Taylor, J. W. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In PCR Protocols: a Guide to Methods and Applications, Innis, M. A., Gelfland, D. H., Sninsky, J. J., White, T. J., Eds.; Academic Press: New York, N.Y., 1990. (28) Dionisi, H. M.; Chewning, C. S.; Morgan, K. H.; Menn, F. M.; Easter, J. P.; Sayler, G. S. Abundance of dioxygenase genes similar to Ralstonia sp. strain U2 nagAc is correlated with naphthalene concentrations in coal tar-contaminated freshwater sediments. Appl. Environ. Microbiol. 2004, 70, 3988–3995. (29) Widada, J.; Nojiri, H.; Kasuga, K.; Yoshida, T.; Habe, H.; Omori, T. Molecular detection and diversity of polycyclic aromatic hydrocarbon-degrading bacteria isolated from geographically diverse sites. Appl. Microbiol. Biotechnol. 2002, 58, 202–209. (30) Jeon, C. O.; Park, W.; Padmanabhan, P.; DeRito, C.; Snape, J. R.; Madsen, E. L. Discovery of a bacterium, with distinctive dioxygenase, that is responsible for in situ biodegradation in contaminated sediment. Proc. Natl. Acad. Sci. USA 2003, 100, 13591–13596. (31) DeRito, C. M.; Pumphrey, G. M.; Madsen, E. L. Use of fieldbased stable isotope probing to identify adapted populations and track carbon flow through a phenol-degrading soil microbial community. Appl. Environ. Microbiol. 2005, 71, 7858–7865. (32) Yu, C. P.; Chu, K. H. A quantitative assay for linking microbial community function and structure of a naphthalene-degrading microbial consortium. Environ. Sci. Technol. 2005, 39, 9611– 9619. (33) Morris, S. A.; Radajewski, S.; Willison, T. W.; Murrell, J. C. Identification of the functionally active methanotroph population in a peat soil microcosm by stable-isotope probing. Appl. Environ. Microbiol. 2002, 68, 1446–1453. (34) Chudoba, J.; Cech, J. S.; Farkac, J.; Grau, P. Control of activated sludge filamentous bulking. Experimental verification of a kinetic selection theory. Water Res. 1985, 19, 191–196. (35) Chiesa, S. C.; Irvine, R. L.; Manning, J. F. Feast/famine growth environments and activated sludge population selection. Biotechnol. Bioeng. 1985, 27, 562–569. (36) Smith, R. C.; Oerther, D. B. Microbial community development in a laboratory-scale nitrifying activated sludge system with input from a side-stream bioreactor treating digester supernatant. Water Sci. Technol. 2006, 54 (1), 209–216. (37) Dumont, M. G.; Murrell, J. C. Stable isotope probingsLinking microbial identity to function. Nature Rev. Microbiol. 2005, 3, 499–504. (38) Tian, L.; Ma, P.; Zhong, J. J. Impact of the presence of salicylate or glucose on enzyme activity and phenanthrene degradation by Pseudomonas mendocina. Process Biochem. 2003, 38, 1125– 1132. (39) Madsen, E. L.; Mann, C. L.; Bilotta, S. Oxygen limitations and aging as explanation for the persistence of naphthalene in coaltar contaminated surface sediments. Environ. Toxicol. Chem. 1996, 15, 1876–1882. (40) Pitterle, M. T.; Andersen, R. G.; Novak, J. T.; Widdowson, M. A. Push-pull tests to quantify in situ degradation rates at a phytoremediation site. Environ. Sci. Technol. 2005, 39, 9317– 9323. (41) Bianchin, M.; Smith, L.; Barker, J. F.; Beckie, R. Anaerobic degradation of naphthalene in a fluvial aquifer: A radiotracer study. J. Contam. Hydrol. 2006, 84, 178–196.
ES703007N
VOL. 42, NO. 11, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
4105
