Environ. Sci. Technol. 2008, 42, 7580–7586
Assessing Soil Microbial Populations Responding to Crude-Oil Amendment at Different Temperatures Using Phylogenetic, Functional Gene (alkB) and Physiological Analyses N A T S U K O H A M A M U R A , * ,†,§ MANABU FUKUI,‡ DAVID M. WARD,† AND WILLIAM P. INSKEEP† Department of Land Resources and Environmental Sciences, Montana State University, Bozeman, Montana 59717, and Institute of Low Temperature Science, Hokkaido University, Sapporo, Hokkaido 060-0819, Japan
Received January 4, 2008. Revised manuscript received July 28, 2008. Accepted August 7, 2008.
The effect of temperature as a determinant for selecting microbial populations associated with alkane-degradation was examined in crude oil-amended soil microcosms. After a 30day incubation, >95% of n-alkane components in the crudeoil were depleted and approximately 40 and 60% of added [14C] hexadecane was converted to 14CO2 at 4-10 and 25 °C, respectively. Concomitant with crude-oil depletion, 16S rRNA gene sequence analysis revealed the emergence of a prominent Rhodococcus-like 16S rRNA sequence at all temperatures and a prominent Pseudomonas-like sequence at 4 and 10 °C. The diversity of alkane hydroxylase genes (alkB) associated with the amendments was examined using group-specific alkBPCR primers targeting phylogenetically distinct groups of alkanedegrading bacteria and subsequent cloning, denaturing gradient gel electrophoresis and sequencing analyses. Diverse Rhodococcus-alkB genes were detected at all temperatures, while a single prominent Pseudomonas-alkB genotype was detected only at lower temperatures. Two isolates obtained from the microcosms were shown to have 16S rRNA and alkB genes identical to those observed and were used to examine growth as a function of temperature. The Pseudomonas isolate exhibited a substantially higher growth rate at 4 and 10 °C than the Rhodococcus isolate, consistent with the inference that differences in adaptation to low temperature explain the observed shift in populations. High resolution analysis of alkB genes enabled the differentiation of distinct alkane-degrading populations responding to crude-oil amendment from other closely related, well-studied strains with different temperature adaptations.
* Corresponding author phone: (503) 564-4167; fax: (503) 7253888; e-mail:
[email protected]. † Montana State University. ‡ Hokkaido University. § Current address: Department of Biology, Portland State University, SB2 Rm246, 1719 SW 10th Ave, Portland OR 97201. 7580
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Introduction Environmental factors such as temperature, nutrient status, and water potential influence the microbial response to hydrocarbon perturbation in soil environments and the potential for in situ bioremediation. Temperature affects not only the solubility and bioavailability of petroleum components, but also the physiology of indigenous microorganisms. At low temperatures, the increase in oil viscosity reduces distribution and diffusion rates of petroleum components, while decreases in volatilization and solubility of some hydrocarbons affect toxicity and bioavailability (1, 2). Previous studies have reported the biodegradation of petroleum hydrocarbons in a variety of terrestrial ecosystems including Arctic and Antarctic soils (2-6), and Alpine soils (7-9). These cold habitats possess indigenous psychrotolerant and psychrophilic microorganisms that are capable of hydrocarbondegradation. However, changes in temperature at the same location, which are expected in temperate environments, may influence the selection of hydrocarbon-degrading populations. In a recent study, Coulon et al. (10) examined changes in microbial community structure in temperate marine environments after crude-oil addition. Molecular analysis of oil-degrading microcosms showed that 16S rRNA gene sequences closely related to those of known or proposed hydrocarbon-degrading bacteria were detected at both 4 and 20 °C, while sequences related to that of a psychrophilic alkane-degrader, Oleispira antarctica (98% 16S rRNA similarity) were dominant in the 4 °C microcosms (10). Although their observations demonstrated the selection of both versatile psychrotolerant and specialized psychrophilic hydrocarbon-degrading bacteria in low-temperature marine environments, it is hard to draw definite conclusions about the physiological function of specific microorganisms based solely on 16S rRNA gene sequence information. Molecular methods targeting key functional genes coding for hydrocarbon-degrading enzymes have been exploited to assess biodegradation potential and activity in situ, and to resolve population diversity at higher molecular resolution (11-16). Alkane hydroxylases catalyze the first step in the aerobic oxidation of n-alkanes, a major component in petroleum mixtures. The gene coding for alkane hydroxylase (alkB) is widely distributed among both gram-positive and gram-negative bacteria (11-14, 17). Due to the highly conserved topologies of alkB and 16S rRNA gene phylogenetic trees, a targeted primer approach allows reasonable linkage of alkB with 16S rRNA phylotypes. Oligonucleotide primers and DNA probes targeting alkB genes have been developed previously (11-14, 17), and have been used to detect alkB genotypes from various environments including cold habitats such as Arctic, Antarctic, and Alpine soils (7, 12, 18). Several of the primers and/or probes were designed to target specific lineages of alkB genes (11-13, 17), including known alkaneoxidizing Pseudomonas spp., Acinetobacter spp., and Rhodococcus spp. However, the specificity of previously available primer sets may not be sufficient to resolve the diversity of alkB genotypes present in hydrocarbon-contaminated soils. For instance, some of the group-specific alkB primers developed by Kohno et al. (13) or Heiss-Blanquet et al. (17) were designed to target a wide-range of alkB genes from multiple phylogenetic groups (e.g., Rhodococcus, Gordonia, and Nocardioides) and thus may not be useful in associating alkB genotypes with specific 16S rRNA phylotypes. Consequently, new primers were designed and used in the current study to detect a broader range of alkB lineages, to enhance primer specificity for phylogenetically distinct alkB se10.1021/es800030f CCC: $40.75
2008 American Chemical Society
Published on Web 09/10/2008
TABLE 1. Group-Specific alkB Primers primer
target group
sequence (5′ to 3′)
Tm (°C)
product size (bp)
R1f438 R1r835 R2f468 R2r894 R3f585 R3r863 R4f634 R4r869 NAf498 NAr825 P1f485 P1r851 GPf519 GPr744 ACf532 ACr872 BCf577 BCr837
alkB R1
5′-CGTCGAGCGCTGGTTGTCC 5′-GACGTAGGAGTCCGTAGTGC 5′-GGCGCAGTCGTTTTACGG 5′-CCAACTGTGCTCCGGTGC 5′-GCCCCGCAGCGTATTCGG 5′-CTGCCGTCTTGTCTTCGC 5′-CGCTCGTCTGAGGCGTCAG 5′-CGGGCGAGCACCGGAC 5′-CGAGCACAACCGGGGCC 5′-CCGTAGTGCTCCATGTAG 5′-CGCGGGGTTCAAGGTCGAGC 5′-GGTCCGCTCGTAGCGCCCG 5′-CACCGTGATGTIGCTACACCG 5′-GGAACACCAGCATCTTIGG 5′-GCGACICCTGAAGATCC 5′-TTCCAICTATGCTCIGGC 5′-CGTCGAGCACAACCGCGG 5′-GTGTACGGCGCGTCGC
54
417
Rhodococcus spp.
54
444
Rhodococcus spp.
59
296
Rhodococcus spp.
59
235
Rhodococcus spp.
57
327
Nocardioides/Prauserella
59
366
Pseudomonas aeruginosa
54
225
Pseudomonas spp.
54
340
Acinetobacter spp.
54
286
Burkholderia spp.
alkB R2 alkB R3 alkB R4 alkB NA alkB P1 alkB GP alkB AC alkB BC
quences, and to elucidate alkB variants. Although this approach may not detect novel alkB genes with low homology to known sequences, it is useful for screening known alkB phylogenetic types and for resolving in situ genotypic diversity within the alkB lineages. The primary objectives of this study were to (i) examine how temperature influences microbial population responses to hydrocarbon perturbation in a temperate soil environment when other parameters (e.g., soil type, nutrients, water potential, aeration) were held constant, and (ii) assess the utility of functional gene approaches to detect distinct alkanedegrading populations. Cultivation-independent 16S rRNA gene sequence analysis was used to monitor bacterial population changes following crude-oil amendment in a temperate loam soil (from Montana) at different temperatures (4, 10, and 25 °C). Diversity among hydrocarbon-degrading bacteria enriched at various temperatures was further elucidated using group-specific PCR primers targeting phylogenetically distinct groups of alkane hydroxylase genes. In addition, the effect of temperature on growth rate was examined for hydrocarbon-degrading bacterial isolates with 16S rRNA and alkB sequences identical to those detected using molecular approaches. This comprehensive approach provided genotypic and phenotypic identification of microbial populations selected under different temperatures, and our results indicate the utility of functional gene analysis for detecting alkane-degrading populations with distinct physiological traits.
Materials and Methods Soil. Crude-oil contamination experiments were conducted with the surface horizon of a Beaverton loam soil collected from Gallatin County, Montana, with no history of crude-oil contamination (confirmed by gas chromatography/mass spectrometry (GC-MS) analysis, which showed no detectable hydrocarbons). The soil was passed through a 2 mm sieve, and stored field moist at 4 °C. The soil contained 3.9% organic carbon, 61.4, 1732.0, and 75.9 mg/kg of extractable NO3-N, K, and P, respectively (19). The pH and gravimetric water content at 33 kPa were determined to be 7.6 and 29.9%, respectively (19). Crude-Oil Amended Soil Microcosms. Crude-oil amendment assays were conducted in duplicate at 25, 10, and 4 ( 2 °C in the dark without shaking, using 150 mL serum bottles containing 30 g (dry wt) soil as described previously (19). Uncontaminated control soils were prepared and treated identically. Sterile soils were prepared by autoclaving three times for 1 h within 24 h intervals, and used as abiotic controls.
phylogenetic affiliation
Crude-oil (Conoco Corp., Billings, MT) was mixed with 300 000 dpm [1-14C]hexadecane (>98% purity, specific activity ) 2.6 mCi mmol-1, Sigma Chemical, St. Louis, MO), then added to each bottle to achieve a final concentration of 2% (wt/wt). To eliminate the possibility of nutrient limitation, we supplemented soils with a nutrient solution (ref 20; Supporting Information (SI)) and periodically purged the headspace with air (see below). The total volume of sterile deionized water and nutrient solution added to the soil was calculated to achieve an equivalent matric potential of 33 kPa (29.9% water content). The depletion of individual hydrocarbon components was assessed by monitoring changes in chemical composition by capillary GC-MS (ref 21; SI). The degradation of [1-14C]hexadecane was determined simultaneously by measuring the evolution of 14CO2 as described previously (19). The amendments were purged twice a week with humidified CO2-free air to trap 14CO2, which also served to keep the system aerobic. After completion of the experiment, mass balance was determined based on the sum of evolved 14CO2 plus residual soil 14C measured using total combustion (21), and resulted in average recoveries of 98.2 ( 4.2%. Design of Alkane Hydroxylase Gene (alkB) GroupSpecific Primers. DNA sequences of representative alkane hydroxylase genes were retrieved from GenBank, aligned using ClustalX (version 1.81) (22) and edited manually. Primer sets (Table 1) were designed to amplify phylogenetically distinct groups of alkB genes from each lineage by examining the alignment for conserved regions. PCR amplification with group-specific alkB primers was conducted using the following protocol: 10 min at 94 °C; then 32 cycles of 45 s at 94 °C, 45 s at Tm shown in Table 1, and 90 s at 72 °C, followed by final extension of 7 min at 72 °C. The specificity of each primer was confirmed by comparison to the available sequences in the GenBank database by BLAST search (23), and by PCR amplification with DNA from a positive control strain in the targeted alkB lineage as well as negative control strains from all other alkB lineages. The reference strains from each lineage group were Rhodococcus erythropolis NRRLB-16531, Prauserella rugosa, Burkholderia cepacia ATCC25416, Pseudomonas putida GPo1, P. aeruginosa PG201, and Acinetobacter calcoaceticus EB104 (as gifts from Dr. J.B. van Beilen). With a single exception involving the R4 primer (SI), all primer sets amplified products of the predicted size from positive control strains as well as in Rhodococcus and Pseudomonas strains obtained in a companion study of MT soils (ref 19; described below). DNA sequences of the positive products were verified. VOL. 42, NO. 20, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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Molecular Analysis. Bacterial populations in the crudeoil amended microcosms were monitored by denaturing gradient gel electrophoresis (DGGE) analysis of PCR-amplified 16S rRNA and alkB gene fragments from each treatment. DNA was extracted from 0.5 g (dry wt) subsamples after bead beating, and 16S rRNA gene fragments were PCR-amplified using Bacteria-specific primer 1070F and the universal primer 1392R containing a GC-clamp, followed by separation of PCR products using DGGE, and sequencing of individual dominant bands as described previously (24). For alkane hydroxylase gene analysis, alkB-PCR was conducted using group-specific primers as described above and the same GCclamp on the reverse primer. Positive PCR products were purified with a QIAquick gel extraction kit (Qiagen, Chatsworth, CA) and cloned into the pGEM-T Easy vector (Promega, Madison, WI) following the directions of the manufacturer. Randomly selected clone inserts (total of 35 clones) were sequenced with vector primers using the ABI Prism BigDye Terminator cycle-sequencing reaction kit and an ABI 310 DNA sequencer (Applied Biosystems, Foster City, CA) as described previously (21). Sequences were assembled using Sequencher 4.1 (Gene Codes Corporation, Ann Arbor, MI) and compared to the GenBank database using BLAST (23). Alignments were performed by ClustalX (version 1.81) using default values (22) and edited manually. Distance analysis was performed using the Jukes and Cantor correction (25), followed by phylogenetic tree construction using the neighbor-joining method (26) of PAUP*4.0 software (Sinauer Associates, Sunderland, MA). The nucleotide sequences reported in this paper have been deposited in the GenBank database under accession numbers DQ983831 to DQ983843 (alkB clone sequences) and DQ149098 and DQ983844 (16S rRNA gene DGGE band sequences). Growth Temperature of Two Relevant Alkane-Degrading Isolates. Alkane-degrading isolates obtained in a companion study of MT soils (ref 19; SI) were grown in liquid Xm medium containing 1% (vol/vol) n-hexadecane as a sole C source by inoculation from a single colony. A 50 µL inoculum of hexadecane-grown cells (early stationary phase culture) was transferred in triplicate to 60 mL vials containing 10 mL Xm medium with 1% (vol/vol) n-hexadecane (0.3 mM final concentration) or 1% (wt/vol) glucose (0.5 mM final concentration) as a substrate and 0.05% (wt/vol) yeast extract and incubated at 25, 10, and 4 ( 2 °C with constant shaking in the dark. Optical density at 600 nm was determined periodically from aseptically collected subsamples.
Results and Discussion Crude-Oil Mineralization at Low Temperatures. The hydrocarbon mineralization capacity of indigenous microbial populations in a Montana soil incubated at 4, 10, and 25 °C was assessed by measuring 14CO2 production from 14Chexadecane added with crude-oil (2% (wt/wt)) (Figure 1). Control treatments with autoclaved soils showed no production of 14CO2, confirming the biological mineralization of hexadecane in these experiments. Although the rates of hexadecane mineralization were comparable from 4 to 25 °C (0.14, 0.12, and 0.12% 14CO2 day-1 g dry wt soil-1, respectively), longer lag periods (5 days and 8-15 days at 25 °C and 4-10 °C, respectively) and lower extents of CO2 evolution (68 and 45% at 25 °C and 4-10 °C, respectively) were observed at lower temperatures, consistent with previous results of 14Cdodecane mineralization by psychrotrophic Rhodococcus sp. strain Q15 (27). Differences in the extent of 14CO2 evolution from 4 to 25 °C may also reflect differences in the ratio of respiration versus incorporation of carbon into biomass. The complete depletion of all n-alkane components (chain-length of C10-C30) in crude-oil was observed at all temperatures examined (4, 10, and 25 °C) (SI Figure S1) and was consistent 7582
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FIGURE 1. Mineralization of [1-14C]hexadecane added with 2% (wt/wt) crude-oil to soil and incubated at 25, 10, and 4 °C. Autoclaved soil was used as a control. Each point represents the mean from duplicate samples; the error bars represent the standard error. Where absent, error bars are smaller than symbol size.
FIGURE 2. DGGE profiles of 16S rRNA gene fragments (322 bp) from crude-oil contaminated soils incubated at 25, 10, and 4 °C (A) and uncontaminated control soil (B). Gels were poured with denaturant gradient of 40-70%. The nucleotide sequences of bands 1 and 2 were determined to be Rhodococcus sp. and Pseudomonas sp., respectively. with results from 14C-hexadecane mineralization. There were no obvious patterns of preferential utilization of different n-alkanes at 25 °C, while sequential disappearance of shorterbefore longer-chain-length n-alkanes was observed to a limited extent at 4 and 10 °C (SI Figure S1). The differences in hydrocarbon degradation observed at 4, 10, and 25 °C (Figure 1 and SI Figure S1) may be due to the presence of different microbial populations, and or the effect of temperature on hydrocarbon-degrading population(s). Bacterial Community Dynamics. To assess mechanisms responsible for changes in hydrocarbon degradation as a function of temperature, bacterial populations present in soil microcosms were examined directly using PCR-amplified 16S rRNA gene fragments separated via DGGE (Figure 2). Prominent DGGE bands emerged concomitant with crudeoil degradation at all temperatures (Figure 2A). Conversely, no obvious changes in DGGE banding patterns were observed in the uncontaminated control soil at 4 °C (Figure 2B). Sterile soils amended with crude-oil and used as abiotic controls (Figure 1) showed no amplification products of 16S rRNA genes. Thus, the population shifts corresponding to the prominent DGGE bands in soils treated with crude-oil were clearly due to hydrocarbon perturbation. Band 1 was observed at all temperatures, while an additional prominent band (Band 2) was observed only at 10 and 4 °C. The DNA sequence of Band 1 was 100% identical to the 16S rRNA gene sequence of a known alkane-degrading actinobacterium, Rhodococcus erythropolis NRRL B-16531 (28, 29). Conversely, the sequence of Band 2 was 100% identical to the 16S rRNA gene sequence of a γ-Proteobacterium, Pseudomonas frederiksbergensis, a phenanthrenedegrading isolate obtained from soil at a coal gasification site (30), and 99% identical to the well-characterized alkanedegrader, P. putida GPo1 (31). Thus, the 16S rRNA gene sequence analysis confirmed a distinct shift in microbial community composition as a function of temperature. The detection of single predominant Rhodococcus and Pseudomo-
FIGURE 3. Phylogenetic analysis of alkane hydroxylase genes (alkB). Nine group specific alkB primers and phylogenetic affiliation of each lineage are indicated on the right. Putative alkB sequences cloned from crude-oil contaminated 4 °C soil (day-19) and relevant isolates (Rhodococcus isolate R121 and Pseudomonas isolate P146) are indicated by boldface type (numbers in parentheses indicate clones obtained for each sequence type). alkB sequences with an asterisk were also detected in DGGE profiles (Figure 4). The neighbor-joining tree was generated based on partial gene sequences (430 bp) of previously described and putative alkane hydoxylases. (The tree was rooted with the xylM sequence of P. putida (M64747) (not shown); bootstrap values per 100 trials of major branch points are shown; bar ) 0.05 substitutions per nucleotide position). nas 16S rRNA genotypes in crude-oil contaminated soils at temperatures of 4 and 10 °C is consistent with previous studies conducted on cold hydrocarbon-contaminated ecosystems, including Arctic and Antarctic soils (12, 32), and Alpine soils (7, 9). Detection and Characterization of Alkane Hydroxylase Genes. To study alkane-degrading bacteria identified by 16S rRNA gene analysis at a higher level of molecular resolution, we also evaluated the importance of different alkB genotypes across treatments. A phylogenetic analysis of alkB from gramnegative and gram-positive bacteria showed nine distinct clusters consistent with 16S rRNA gene sequence phylogeny (Figure 3). Hence, we designed nine lineage-specific, nondegenerate PCR primer sets targeting each phylogenetically distinct alkB group based on conserved regions found among representatives within each lineage (Figure 3 and Table 1). We included four alkB paralogs known to occur in Rhodococcus spp. to capture as much diversity as possible. The function of the alkB paralogs has not been shown except that alkB2 genes from Rhodococcus sp. strains NRRL B-16531 and Q15 were associated with oxidation of C12 to C16 n-alkanes when expressed heterologously in Pseudomonas sp. (33). Consequently, different alkB paralogs within the same
organism may be responsible for oxidation of alkanes with different chain-lengths (29, 33). The suite of lineage-specific alkB primers was used on DNA extracts from crude-oil amendments across temperature treatments to examine the presence of alkane-degrading genotypes. Positive PCR products of the expected sizes were observed at 4 and 10 °C using primers targeting alkB from Rhodococcus spp. and P. putida GPo1 lineages (R1-R4 and GP, respectively; Figure 3); no PCR products were obtained with other alkB primers (SI Figure S2). At 25 °C, the only positive PCR products were obtained using alkB primers targeting the Rhodococcus spp. (R1-R4). Only minor PCR products were obtained from alkB R1 and R2 primers using soil DNA from Day 0 (data not shown), indicating that the copy number of these genes increased after crude-oil contamination. The alkB phylotypes are consistent with the bacterial populations identified using 16S rRNA gene sequence analysis (Figure 2A), indicating the successful application of our primer sets to detect alkane-degrading genotypes in the soil amendments. The screening of environmental samples by lineage-specific alkB primers thus provides a useful tool to identify dominant alkane-degrading phylotypes without 16S rRNA gene characterization. Further, VOL. 42, NO. 20, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 4. DGGE profiles of alkB PCR amplification products from crude-oil contaminated soils compared to alkB profiles of positive control strains: (A) sequences obtained with GP-specific primer sets compared to the P. putida str. GPo1 alkB sequence, and (B) sequences obtained with the R1-specific primer sets compared to the R. erythropolis str. NRRLB-16531 alkB1 sequence. The DGGE bands whose nucleotide sequences were 100% identical to the alkB sequences of the isolates (R121 and P146), the R1 clone (R1c1, 3, 4, 8, and 9) and the GP clone (GPc10) (from Figure 3) are labeled. The DGGE bands whose nucleotide sequences were determined and confirmed to be unique alkB sequences are indicated with asterisks (gels were poured with a denaturant gradient of 45-65%). application of these primer sets would improve assessment of bioremediation potential and monitoring of hydrocarbondegrading populations in contaminated environments. The alkB PCR products amplified from a 4 °C sample (day-19) were further analyzed by cloning and sequencing (a total of 35 clones were analyzed), and included representatives of both the Rhodococcus R1-R4 lineages and the Pseudomonas GP lineage (Figure 3). This result is consistent with phyla identified using 16S rRNA gene analysis. Among the alkB clones examined, multiple alkB genotypes were detected in lineages R1-R4, whereas only one genotype was detected within the GP lineage. The multiple genotypes detected with in the R1-R4 lineage were highly similar to one another (>95% nucleotide identity), as well as to other alkB sequences within these lineages. In contrast, the single genotype obtained within the GP lineage (GPc10) was more distantly related (∼83-87% nucleotide identity) to other GP alkB sequences. 7584
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alkB DGGE Profiles during Crude-Oil Degradation at Various Temperatures. To monitor alkB genotypes during crude-oil degradation, the lineage-specific alkB primers were used to amplify alkB sequences that were further separated using DGGE (Figure 4). The DGGE profile of GP-type alkB PCR products from 4 and 10 °C showed the presence of only one prominent band throughout the incubation period, whose sequence corresponded to that of clone GPc10 (Figure 4A). Among the four primer sets targeting alkB paralogs within the Rhodococcus-group (R1-R4), the R1 primer set exhibited greater resolution for detecting various genotypes than primer sets R2, R3 and R4 (Figure 3 and DGGE analysis of R2-R4 PCR products (data not shown)). Thus, we conducted further analysis of the alkB Rhodococcus-group using the R1 primer set. DGGE profiles of alkB R1-PCR products (Figure 4B) showed the enrichment of diverse R1-type alkB genotypes, consistent across different sampling times (33-day incubation) and temperatures (25, 10, and 4 °C), suggesting the presence of the same Rhodococcus alkB-genotypes at all temperatures. The expression of these alkB variants was also confirmed by conducting reverse-transcriptase PCR with RNA extracts from the same soils (data not shown). Fourteen prominent alkB R1-DGGE bands were sequenced and confirmed to be unique alkB sequences. Several of these bands corresponded to the sequences of alkB clones obtained from the 4 °C treatments (Figures 3 and 4B). While DGGE bands R1c3 and R1c8 (as well as R1c1 and R1c9) appear to constitute single intense bands, they are each actually two closely migrating bands that were separated on different denaturant gradient gels (40-70%). Upon crude-oil enrichment and degradation, considerable diversity in Rhodococcus alkB genotypes was observed within the population, in contrast to the single prominent Pseudomonas alkB genotype. The greater Rhodococcus alkB diversity could have several possible causes. First, the presence of alkB variants could be explained in part by multiple paralogous genes within individual hydrocarbon-degrading organisms. However, the observation that all of the Rhodococcus isolates we obtained (21 isolates; ref 19), as well as previously described isolates (29), had a single R1 genotype argues against this. Second, alkB variants could be associated with a single population of ecologically distinct, yet ecologically interchangeable individuals (ecotypes). Here, alkB sequence variation would be functionally neutral (34-36), having simply accumulated as an ecotype (defined by adaptations in other genes) evolved. Third, the alkB variants could represent ecologically interchangeable members of more than one ecotype population. Again, alkB variants need not themselves be adaptive either within or between different ecotypes. The Pseudomonas alkB genotype apparently represents a population that is either more clonal or that contains a highly conserved alkB gene. More information is needed to resolve the relationship between the Rhodococcus alkB diversity we detected and possible ecological diversity. The Pseudomonas population was preferentially enriched at 4 and 10 °C, as compared to 25 °C, suggesting that it is relatively more fit at lower temperatures. Effects of Temperature on Growth of Two Relevant Hydrocarbon-Degrading Isolates. Among several isolates obtained from the 25 °C crude-oil amended soil (19), two isolates were particularly relevant based on the molecular results obtained at different temperatures. Specifically, Rhodococcus sp. strain R121 and Pseudomonas sp. strain P146 exhibit 16S rRNA and alkB gene sequences identical to those detected in our temperature studies (Figures 2 and 4). This provided an opportunity to test the relative fitness of strains of known genetic relevance to genotypes enriched at different temperatures. The growth of each isolate was examined at 4, 10, and 25 °C with hexadecane or glucose as a growth substrate (Figure 5). Generation times of Rhodococcus sp.
organisms and pathways for the microbiological degradation of alkanes during in situ bioremediation.
Acknowledgments
FIGURE 5. Generation times of Rhodococcus sp. strain R121 and Pseudomonas sp. strain P146 at growth temperatures of 4, 10, and 25 °C. Each point represents the average of triplicate experiments; the error bars indicate standard deviations. Where absent, error bars are smaller than symbol size. strain R121 increased nearly 5-fold at lower temperatures (∼3 h at 25 °C to ∼14 h at 4 °C). In contrast, the generation times of Pseudomonas sp. strain P146 increased only 2.5fold at 4 °C compared to 25 °C (∼2 h at 25 °C to ∼5 h at 4 °C). In addition, Rhodococcus sp. strain R121 reached higher final cell density at 25 °C than at 10 and 4 °C, whereas the Pseudomonas strain P146 reached higher final cell densities at 10 and 4 °C. Hexadecane and glucose-grown cells of both strains showed similar responses to changes in temperature (Figure 5), indicating that the decrease in growth rates at low temperature was not caused by decreased hexadecane solubility. Clearly, Pseudomonas sp. strain P146 is better adapted to rapid growth at lower temperatures (e.g., 4-10 °C) than is Rhodococcus sp. strain R121. The relative distribution of Rhodococcus and Pseudomonas populations detected using 16S rRNA and alkB gene analysis is consistent with the observed growth patterns of the relevant isolates, where Pseudomonas sp. strain P146 showed better fitness at lower temperature than Rhodococcus sp. strain R121 (Figure 5). It remains to be seen whether the different temperature responses of these individual strains is representative of the native Rhodococcus and Pseudomonas populations. The absence of the Pseudomonas sp. 16S rRNA and alkB genotypes in soil amendments incubated at 25 °C, when Pseudomonas sp. strain P146 exhibited comparable growth rates to Rhodococcus sp. strain R121 in pure culture at this temperature, suggests that additional factors influence the relative competitive fitness of these organisms, such that the realized niche is narrower than the fundamental niche for the Pseudomonas strain. Initial population sizes of either the Pseudomonas or Rhodococcus-like organisms in soils may also contribute to the apparent competitive advantage observed for the Rhodococcus-like population at 25 °C. The temperature responses exhibited by the relevant isolates obtained from the soil amendments could indicate potentially important physiological adaptation, considering that two known alkane-degrading isolates used as positive controls (R. erythropolis NRRL B-16531 and P. putida GPo1) showed comparable generation times at 25 °C (3.3 ( 0.2 and 3.8 ( 0.1 h, respectively), but no significant growth at either 10 or 4 °C with hexadecane as a growth substrate. Interestingly, these control isolates had alkB genes distinct from those observed in the amendments (Figures 3 and 4, R1 and GP lineages), indicating that the greater molecular resolution provided by alkB enables detection of ecologically distinct populations with identical or nearly identical 16S rRNA sequences. The lineagespecific functional gene approach described here can thus provide a useful means of assessing functionally important
We thank Dr. Jan B. van Beilen greatly for providing us with six reference strains. We also appreciate technical assistance from Mary Bateson, Rich Macur, Yuriko Higashioka, and Yusuke Odake. We are indebted to the Conoco refinery (Billings, MT) for providing us with crudeoil. This work was supported by the U.S. Environmental Protection Agency (project no. 829357-01-0) and the Montana Agricultural Experiment Station (projects 911398, 911300, and 911352), and partly supported by the Grant for Joint Research Program of the Institute of Low Temperature Science, Hokkaido University (proposal nos. 05-30 and 06-36).
Supporting Information Available Additional methods details, Figure S1: Depletion of nalkane components of crude-oil added to soil and incubated at 25, 10, and 4 °C, Figure S2: Detection of alkane hydroxylase genes in crude-oil contaminated soil incubated at 4 °C (Day 19). This material is available free of charge via the Internet at http://pubs.acs.org.
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