Final Report | Patterns and Prediction: Molecular Analyses of PAH-Degrading Microbial Populations and Their Function in Real Contaminant Mixture Environment| Research Project Database | NCER | ORD

Final Report: Patterns and Prediction: Molecular Analyses of PAH-Degrading Microbial Populations and Their Function in Real Contaminant Mixture Environment

EPA Grant Number: R829357
Title: Patterns and Prediction: Molecular Analyses of PAH-Degrading Microbial Populations and Their Function in Real Contaminant Mixture Environment
Investigators: Inskeep, William P. , Colores, Gregory C. , Hamamura, Natsuko , Ward, David M.
Institution: Montana State University - Bozeman
EPA Project Officer: Fields, Nigel
Project Period: October 1, 2001 through September 30, 2004 (Extended to September 30, 2005)
Project Amount: $634,430
RFA: Complex Chemical Mixtures (2000)
Research Category: Hazardous Waste/Remediation

Description:

Objective:

Complex petroleum hydrocarbon mixtures, including crude oil, diesel fuel, and creosote, consist of varying concentrations of n- and branched alkanes, cycloalkanes, phenolics, aromatics, and polycyclic aromatic hydrocarbons (PAHs). Although these mixtures contain similar constituents, the relative abundance of mixture components and toxic compounds (e.g., heterocyclics, chlorophenols) vary considerably, and these variations potentially are important in determining which microbial populations are involved in biodegradation. The physical/chemical/biological properties of soils (e.g., temperature, pH, conductivity, nutrient status, texture, biota) are expected to influence further the selection of adapted microbial populations. The fate of individual constituents within complex hydrocarbon mixtures may vary across soil types in part because of the selection of different microbial populations that may be adapted to a specific set of physical-chemical properties. Conversely, the selection pressure imparted by contamination with a particular complex mixture, such as crude oil, may result in the selection of similar microbial populations across widely different soil environments.

The primary goal of this research project was to link the biodegradation patterns of complex petroleum hydrocarbon mixtures with the distribution and function of specific microbial populations important in contaminated soil environments. We examined four general types of environmental determinants for their importance in selecting specific hydrocarbon-degrading microbial populations: contaminant type, soil type, temperature, and time. To elucidate functional diversity, we developed group-specific polymerase chain reaction (PCR) and PCR-denaturing gradient gel electrophoresis (DGGE) primers targeting phylogenetically distinct groups of alkane hydroxylase genes(alkB). The expression of alkB genotypes in hydrocarbon- contaminated soils was examined by reverse- transcription PCR (RT-PCR) followed by DGGE analysis. The functional gene approach enabled us to identify functionally active populations and to link biodegradation activity with specific microbial populations.

Summary/Accomplishments (Outputs/Outcomes):

Patterns of Microbial Population Distribution and Selection in Diverse Soil Types

We examined soil bacterial population dynamics associated with the biodegradation of a single contaminant mixture, crude oil, to assess patterns in the selection of microbial populations across seven different soil types from geographically diverse locations.

Seven soil types obtained from geographically distinct areas (Montana, Arizona, Oregon, Indiana, Oklahoma, Virginia) (Table 1) were examined for biodegradation activity. Biodegradation experiments were conducted in soil batch vessels contaminated with 2 percent (w/w) crude oil spiked with [1-¹⁴C] hexadecane. Chemical changes were monitored by gas chromatography-mass spectrometry (GC-MS) and ¹⁴CO₂ analysis as described previously (Hamamura, et al., 2005). Microbial populations were analyzed using 16S rDNA-based DGGE. DGGE has been used successfully to reveal the distribution of dominant microbiota in many habitats (Ferris, et al., 1996; Rooney-Varga, et al., 1999; West and Scanlan, 1999; Norris, et al., 2002; Koizumi, et al., 2004; Sun, et al., 2004), which most often are not the microorganisms that are readily cultivated from such habitats. We intended to sample a broad subset of microorganisms potentially involved in hydrocarbon degradation, or other processes secondarily associated with hydrocarbon degradation, and to observe differences in the patterning of responses among soil types by using PCR primers that have been employed successfully in the amplification of 16S rRNA gene fragments from a wide range of subdomain lineages of bacteria representatives (Ferris, et al., 1996; Colores, et al., 2000; Norris, et al., 2002; Macur, et al., 2004). In addition, hydrocarbon-degrading bacteria were cultivated by direct plating from serially diluted samples from each contaminated soil, and their relevance was confirmed by comparing 16S rRNA gene sequences of isolates to those observed in crude oil contaminated soils.

Table 1. Physical and Chemical Characteristics of Soils Used in This Study

Soil	Location	Series name	Texture^a	pH	Organic C (%)	H₂O content (%)^b	Concentrations (mg/kg) of^c:
Soil	Location	Series name	Texture^a	pH	Organic C (%)	H₂O content (%)^b	NO₃-N	K	P
AZ	Arizona	Casa Grande	SaL	8.8	0.2	12.0	8.1	192.0	4.5
OR	Oregon	Jory	C	5.4	5.7	38.9	0.2	452.0	11.7
IN	Indiana	Chalmers	SCL	6.1	2.2	27.1	2.0	260.0	6.0
VA	Virginia	Groseclose	SL	7.4	4.5	35.8	17.2	260.0	12.8
OK	Oklahoma	Konowa	SaL	7.1	3.3	15.8	51.6	350.0	31.9
MT-N	Montana	Beaverton	L	7.6	3.9	29.9	61.4	1732.0	75.9
MT-O	Montana	Brocko	L	8.4	1.2	21.6	52.4	968.0	22.9

^aSaL, sandy loam; C, clay; SCL, silty clay loam; SL, silty loam; L, loam.

^bWater content determined at corresponding water potential of 33 kPa (0.33 bar).

^cNO₃-N, K and P extracted with KCl, NH₄-acetate, and NaHCO₃, respectively.

Crude Oil Amendment Assays. In all seven soils, greater than 80 percent of the added crude oil was depleted within the 50-day incubation period (see Figure 1) . Among the soils, diverse patterns in utilization of various chain-length n-alkanes were observed. A pattern of sequential disappearance of shorter before longer chain-length n-alkanes was observed in five soils (AZ, OR, VA, OK, and MT-O).

Figure 1. Degradation of Crude Oil Components in Different Soils. Depletion of n-alkanes of C₁₂ to C₂₄ chain-length is shown in larger figures. Mineralization of [1-¹⁴C]hexadecane to ¹⁴CO₂ is shown in inset figures with autoclaved soil as a control. Each point represents the average of triplicate bottles, and the error bars correspond to standard deviations; where absent, error bars are smaller than symbol size.

This pattern was most obvious in the VA soil, which showed initial loss of n-alkanes with chain lengths < C₁₅ followed by later loss of n-alkanes with chain lengths > C₁₆. OK and MT-O soils showed greater chain-length-dependent depletion than OR and AZ soils. In contrast, all n-alkane components were depleted at approximately the same rate regardless of chain length in the IN and MT-N soils. The fastest depletion rate and shortest lag period was observed with the MT-N soil, where approximately 81 percent of total n-alkanes disappeared by day 10. Only approximately 40 percent depletion was observed by day 13 in the IN soil, likely as a result of a longer lag period (approximately 7 days).

Concomitant measurement of ¹⁴CO₂ evolution from [1-¹⁴C] hexadecane was conducted to confirm the capability of indigenous microbial populations to mineralize crude oil components in the soils. Although the rates and extent of recovery of ¹⁴CO₂ varied among the soils, approximately 40-70 percent of added ¹⁴C-hexadecane was mineralized after the 50-day incubation period. The total ¹⁴C recovery (¹⁴CO₂ + residual soil ¹⁴C) from the amendment systems averaged 97.3 ± 4.6 percent. Control treatments with autoclaved soils showed no production of ¹⁴CO₂, confirming the biological mineralization of hexadecane in these experiments.

Molecular Analysis of Bacterial Community Dynamics. All seven soils showed the emergence of prominent DGGE bands with distinct banding patterns during crude oil depletion. No obvious changes in DGGE banding patterns were observed in the uncontaminated control soils during the incubation period. Consequently, the populations contributing prominent DGGE bands that emerged in treatments containing 2 percent crude oil clearly were the result of hydrocarbon amendments rather than other experimental factors (e.g., nutrient addition).

The DNA sequences of prominent DGGE bands corresponding to bacterial populations selected during crude oil degradation are reported along with their closest phylogenetic relative. Phylogenetically diverse populations related to β- and γ-Proteobacteria, Actinobacteria, and candidate division TM7 were identified across the set of amended soils. B ands N1 and R1, however, were detected in multiple soils; band N1 was detected in four soils (IN, OK, MT-N, and MT-O), and band R1 was detected in two soils (OR and IN). The sequences corresponding to band N1 were 100 percent identical to the partial 16S rRNA gene sequence of Rhodococcus erythropolis NRRL B-16531. All DGGE band sequences obtained from the MT-N and MT-O soils were affiliated with Gram-positive organisms, whereas all sequences obtained from the OR and VA soils were affiliated with Gram-negative organisms. AZ, IN, and OK soils contained DGGE band sequences affiliated with both Gram-positive and Gram- negative organisms. All the Gram-positive-related DGGE band sequences clustered with their relatives within the Nocardia-Rhodococcus-Nocardioides complex, some of which are known to degrade hydrocarbons (Vomberg and Klinner, 2000), whereas the Gram-negative-related DGGE band sequences were represented by phylogenetically diverse groups.

Isolation of Alkane-Degrading Bacteria. At least one of the prominent populations identified by DGGE in the amendments was cultivated successfully from six of the seven soils, confirming their involvement in alkane metabolism in situ. In many cases, the populations observed in amended soils were recovered most frequently during attempts to isolate alkane-degrading microorganisms. Isolates include known alkane-degrading genera, Rhodococcus spp., and Nocardioides spp., as well as a Collimonas sp., which has not been recognized previously as an alkane-degrader but rather for its chitinase activity and ability to grow on living fungal hyphae (de Boer, et al., 2004). We also obtained numerous isolates that did not correspond to prominent DGGE bands in amended soils; many of these isolates were closely related to known hydrocarbon-degrading organisms, including Variovorax, Burkholderia, Ralstonia, Nocardia, Gordonia, Acinetobacter, and Pseudomonas.

Contamination of soils with crude oil results in the emergence of dominant microbial populations, but there is remarkable diversity in the types of organisms selected across geographically diverse soil types. The results demonstrated that soil type may function as an important determinant of microbial populations that respond to hydrocarbon amendment and that may be involved in biodegradation processes. Our observation has important implications for contaminant bioremediation, as it is apparently the case that in situ biodegradation in different soil environments involves different microbial populations, which presumably have unique hydrocarbon-degrading capabilities (e.g., substrate range, kinetic properties).

Effects of Hydrocarbon Mixture Types on Microbial Population Selection

We examined the effect of different hydrocarbon mixture types on microbial population selection in a subset of soils examined above. We chose diesel fuel and kerosene as other mixture types to examine specifically the effect of different chain lengths of alkane components in the mixtures. The crude oil used in the above study (Conoco Corp., Billings, Montana) consisted of n-alkanes with chain lengths of C₉-C₃₁: 80 percent of the total n-alkanes ranged from C₁₂ to C₂₄ (30%: C₁₂-C₁₅, 26%: C₁₆-C₁₉, and 26%: C₂₀-C₂₄), whereas the diesel fuel and kerosene consisted mainly of n-alkanes with chain length of C₁₀-C₂₂ and C₁₀-C₁₆, respectively. In addition, a commonly used additive (e.g., wood preservative), pentachlorophenol (PCP), also was tested to examine the effect of chlorinated compound addition to the hydrocarbon mixture (diesel fuel).

Biodegradation experiments were conducted in soil batch vessels contaminated with 2 percent (w/w) diesel fuel spiked with [1-¹⁴C] hexadecane, kerosene spiked with either [1-¹⁴C] hexadecane or [1-¹⁴C] tridecane, or diesel fuel containing 600 ppm PCP spiked with either [1-¹⁴C] hexadecane or [UL-¹⁴C] PCP. Chemical changes were monitored by GC-MS and ¹⁴CO₂ analysis as described above. Microbial populations were analyzed using DGGE as described above.

Effect of Mixture Type on Alkane Biodegradation. Diesel fuel and kerosene biodegradation were examined using three selected soils (MT-N, OR, and AZ soils). The MT-N and OR soils showed degradation activities toward all three hydrocarbon mixture types, with longer lag times observed for the shorter chain-length alkane mixtures. In kerosene- contaminated MT-N and OR soils, 40 and 30 percent of added [1-¹⁴C] tridecane was mineralized to ¹⁴CO₂, respectively. The AZ soil showed different degradation activities toward three mixture types; although it showed degradation of crude oil comparable to other soil types , only approximately 5 percent of added [1-¹⁴C] hexadecane in diesel fuel or kerosene was converted to ¹⁴CO₂ during the 50-day incubation period. Chemical changes monitored by GC- MS analysis confirmed that other hydrocarbon components in diesel fuel and kerosene were not depleted in AZ soil amendments.

The effect of PCP addition to diesel fuel was examined on MT-N and OR soils. The two soil types exhibited different responses to the PCP addition. In the MT-N soil, substantial decrease (approximately 50% decrease in 50-day incubation period) in [1-¹⁴C] hexadecane mineralization activity was observed with PCP addition. In contrast, no change in [1-¹⁴C] hexadecane mineralization rates were observed in the OR soil as a result of PCP addition; however, there was a second phase of ¹⁴CO₂ evolution after day 50 in PCP- amended OR soil. This second phase of ¹⁴CO₂ evolution possibly could be caused by the emergence of an additional hexadecane-degrading population after day 50, or predation of ¹⁴C incorporated into microbial populations by other organisms. In both MT-N and OR soils, the evolution of ¹⁴CO₂ from [UL-¹⁴C] PCP was less than 10 percent after an 80-day incubation period. The result suggested that PCP was not degraded substantially even after hydrocarbon components in diesel fuel were depleted, indicating the absence of PCP-degrading populations or very low PCP-degrading activities in these soils.

Bacterial Community Dynamics. Contamination of the same soil type with different mixture types (crude oil versus diesel fuel, kerosene, or diesel plus PCP [diesel + PCP]) resulted in similar microbial population patterns in the MT-N soil. The R. erythropolis-like N1-type population was detected consistently from the amendments of all four mixture types. Diesel fuel and kerosene amendments exhibited an additional population, DN2, which was not detected as a prominent population in crude oil treatment. Thus, this Achromobacter-like DN2 population may be tolerant to shorter chain alkane toxicity or adjusted to degrade shorter chain alkane fractions. The successional changes in DGGE patterns were observed in the diesel + PCP amendment. Pseudomonas putida GPo1-like PN1 population emerged at day 19, followed by the emergence of N1 and DN2 populations at day 40. P. putida GPo1 is a well- characterized alkane-degrader and known to oxidize C₅ to C₁₂ alkanes. It is noteworthy that the rate and extent of ¹⁴C-hexadecane mineralization varied among different mixture types, although the same N1-type population was detected as a dominant population in all the treatments.

In the OR soil, no single population was detected consistently in all four mixture types. The diesel-fuel amendments showed similar population patterns observed in crude oil, with an additional DR2 population emerging on day 27. This Burkholderia-like DR2 population also was detected in the kerosene amendments, suggesting that it may be a shorter chain alkane- adapted population. Two additional populations, Sphingomonas-like KR1 and a Burkholderia-like PR2, were detected in kerosene and diesel + PCP amendments, respectively. It is interesting that the addition of PCP shifted rather diverse Gram-negative bacteria populations detected in diesel fuel amendment to a Burkholderia spp. dominant community. The DGGE banding patterns in the diesel + PCP treatment showed no changes in bacterial community structure after day 50, when the second phase of ¹⁴CO₂ evolution was observed. Thus, this second phase is not the result of the emergence of additional hexadecane-degrading bacterial population after day 50, but rather predation of ¹⁴C incorporated into microbial populations by other organisms or the emergence of hexadecane-degrading archeal populations that would not be detected by the primers used in this study.

Different degradation activity toward the shorter chain alkane mixtures (diesel fuel and kerosene) resulted in different microbial population patterns in the AZ soil. The DNA sequences of prominent DGGE bands showed that band DN2 was detected in both MT-N and AZ soils, although the degradation activities of those two soils were dramatically different.

In MT-N and OR soils, many of the sequences detected by molecular analysis corresponded to alkane-degrading isolates obtained from crude oil amended soils by the direct plating method (described above). It is noteworthy that those alkane-degrading isolates, which did not correspond to prominent bands in crude oil amended soils, became important under different mixture environments (DN2, DR2, PN1, and PR1). This suggests that the soils harbor various hydrocarbon-degrading microbial populations that may become potentially important under different environmental conditions with different niches.

Effects of Temperature on Hydrocarbon-Degrading Microbial Population Selection

Temperature plays a significant role in controlling microbial metabolism and thus microbial distribution and diversity in nature. We examined the importance of temperature as an environmental determinant in selecting cold-adapted hydrocarbon- degrading populations. Previous studies have shown the biodegradation of petroleum hydrocarbons in a variety of terrestrial and marine cold ecosystems including Arctic and Antarctic soils (Margesin and Schinner, 2001). The indigenous psychrotrophic microorganisms, which are capable of hydrocarbon-degradation, become dominant after a contamination event. It is not well understood, however, how microbial populations in a soil respond to a hydrocarbon contamination at different temperatures under conditions in which other parameters were held constant. Thus, we examined crude oil degradation by the MT-N soil under the same amendment conditions (as described above) except for altered incubation temperatures (10 and 4°C).

Crude Oil Degradation at Low Temperature. The MT-N soil amendments at 10 and 4°C incubation temperatures showed approximately 40 percent of added ¹⁴C-hexadecane mineralizations during a 50-day period, which was substantially lower (approximately 37%) compared to the activity at 25°C incubation. Longer lag periods were observed at lower incubation temperatures. The microbial population analysis by 16S rDNA-DGGE showed the emergence of DGGE band 2 in addition to band 1, which was observed at 25°C crude oil amendment as a dominant R. erythropolis-like N1-type population. The sequence of band 2 was 99 percent identical to the 16S rRNA gene sequence of a well-studied alkane-degrading P. putida GPo1.

Functional Gene Analysis. To elucidate functional diversity among cold-adapted hydrocarbon-degrading bacteria, we developed group-specific PCR primers targeting phylogenetically distinct groups of alkB. Alkane hydroxylase catalyzes initial oxidation of n-alkanes to n-alcohols and is prevalent among alkane-degrading Gram-negative as well as Gram-positive bacteria (Smits, et al., 1999). We applied all alkB primer sets on DNA extracts from the amended soils above incubated 10 and 4°C. The primer sets targeted for alkB from Rhodococcus spp. and P. putida GPo1 groups (R1~R4, and GP) showed positive PCR amplifications, which is consistent with detection of both Rhodococcus and P. putida GPo1-like populations by 16S rDNA-based molecular analysis. The alkB primers allow us to link phylogenetically defined populations (e.g., 16S rDNA sequences) and their potential functions in situ (e.g., alkane degradation). This result confirms the utility of the functional gene primers developed in this study for detection and characterization of dominant hydrocarbon-degrading bacterial groups in situ.

The alkB genes amplified with group-specific primers from the amended soil incubated at 4°C were analyzed further by cloning and subsequent sequencing. The diverse genotypes were detected from clones of Rhodococcus alkB groups (R1~R4), whereas only one genotype was detected in the P. putida GPo1 alkB group. Whyte, et al. (1998) has characterized the psychrotrophic Rhodococcus sp. strain Q15 for its ability to degrade n-alkanes and diesel fuel at low temperatures, suggesting the presence of various Rhodococcus spp. adapted to cold environments. The alkB sequences obtained from the amended soil incubated at 4°C with Rhodococcus group primers were highly similar (approximately 99% nucleotide identity) to known alkB sequences from other Rhodococcus spp. in GenBank, as well as other alkB sequences detected from the amended soil incubated at 25°C (discussed below). This result suggests that Rhodococcus-like N1 populations in MT-N soil play a major role in hydrocarbon degradation at a wide range of temperature conditions. In contrast, only one sequence type was obtained with the P. putida GPo1 alkB primer, and its sequence was more distantly related (approximately 88% nucleotide identity) to other sequences in the GP clade. This alkane hydroxylase gene, GPc10, was detected only in low- temperature incubations, and thus may possibly be a unique cold-adapted enzyme in Pseudonomas spp.

Molecular Analysis of Alkane Hydroxylase (alkB) Genes

We further developed the functional gene approach to identify functionally active populations that might be indistinguishable at the 16S rDNA level of molecular resolution and to link biodegradation activity with specific microbial populations. We compared the functional gene diversities in two Montana soils (MT-N and MT-O) that showed the same R. erythropolis N1-type populations being dominant during crude oil degradation by using the group-specific alkB PCR-DGGE primers. All four alkB primer sets targeting Rhodococcus alkB-groups (R1~R4) yielded positive PCR products. It was shown that the R1 primer set yielded greater resolution for detecting diverse genotypes than the other three primer sets; thus, we conducted further analysis using the R1 primer set.

Functional Gene Diversity. Both MT-N and MT-O soils showed the presence diverse alkB R1 genotypes elucidated by DGGE. Hydrocarbon-degrading R. erythropolis-likeN1-type isolates obtained from the crude oil amendments also were analyzed for alkB R1 genotypes. It is shown that N1-type isolates with identical 16S rDNA sequences possess distinct alkB genotypes, some of which correspond to the prominent alkB genotypes detected from the soil amendments (e.g., isolate 15 and R. erythropolis populations corresponded to SB2A and SB2C, respectively). DGGE profiles of alkB R1-PCR products from both MT-N and MT-O soil amendments showed similar banding patterns, although the two amendments exhibited different degradation patterns in utilization of various chain-length alkane components.

Gene Expressions of Diverse alkB Genotypes. To elucidate functionally active alkB genotypes in situ, alkB gene expression within Rhodococcus spp. in crude oil-amended MT-N and MT-O soils was examined by RT-PCR with the alkB R1 primer set. The expression of alkB by the Rhodococcus sp. was confirmed via detection of alkB mRNA from the MT-N amendment on day 6, as well as from the MT-O amendment on days 6, 13, and 27. The expressed alkB genotypes were examined further by DGGE analysis of RT-PCR products. Distinct alkB DGGE banding patterns were observed in RT-PCR products compared to DNA-template DGGE banding patterns in both MT-N and MT-O amendments. In MT-N amendment, RT-PCR products showed the presence of diverse genotypes that were observed as many rather indistinguishable bands, whereas no obvious prominent bands were observed. In contrast, MT-O amendment showed the presence of many prominent genotypes, and some distinct bands were observed on days 6 and 13. Interestingly, the successional patterns of several distinct alkB genotypes detected in the MT-O amendment corresponded to changes in hydrocarbon chemistry during crude oil degradation. These results indicate that the analysis of RNA, not DNA, from amendments is critical to identify functionally active populations, as only some of the alkB genotypes identified by DNA-based PCR-DGGE analysis were expressed actively (e.g., SB2A and SB2C).

The sequences of prominent alkB DGGE bands were determined. A prominent alkB band, AR1RTO1, was detected only in RT-PCR DGGE analysis and shown to be identical to alkB R1 genotype of isolate 121. Among the seven hydrocarbon-degrading N1-type isolates obtained, five isolates contained alkB genotypes (one isolate with 15-type and four isolates with 121-type) that were identified as functionally active alkB populations in situ. The utility of a functional gene approach was demonstrated for linking biodegradation activity with specific functionally active microbial populations.

Diversity and Functional Analysis of Bacterial Communities Associated With Natural Hydrocarbon Seeps

We have applied similar geochemical and molecular approaches to characterize microbial communities in geochemically unique soil environments (e.g., low pH, hydrocarbon seeps, geothermal activities) and elucidated functional attributes of phylogenetically identified microbial populations in situ (Hamamura, et al., 2005). Contamination of acidic environments with hydrocarbons is not uncommon (e.g., contamination of acidic wastewaters from mine drainage and industries by oil spills), and there has been significant interest in biodegradation of hydrocarbons in extreme environments.

In this study, we have described the diversity and potential function of bacterial populations associated with natural hydrocarbon seeps in acidic soils at Rainbow Springs, Yellowstone National Park. The hydrocarbons of the seep soils consisted almost entirely of saturated, acyclic alkanes with chain lengths of C₁₅ to C₃₀, as well as branched alkanes, predominately pristine and phytane. Bacterial community analysis with 16S rRNA gene cloning and sequencing revealed that the majority of sequences were related to heterotrophic acidophilic bacteria in Acetobacteraceae. Hydrocarbon-amended seep soil and sand mixtures exhibited mineralization of ¹⁴C-hexadecane to ¹⁴CO₂, which was accompanied by detection of heterotrophic acidophile-related populations being dominant in the amendments. A unique acidophilic alkane-degrading isolate was cultivated from the seep soil, whose relevance in situ was confirmed by the molecular analysis, and an alkB homolog was identified in this isolate.

This study of natural hydrocarbon seeps provided insight into the biodegradation potential of indigenous bacterial populations in acidic environments. It also confirmed the utility of our geochemical and molecular analysis combined with cultivation approaches to identify functionally active populations in hydrocarbon degradation in various soil environments.

Conclusions

To achieve the goal of our project, we have applied modern methods of molecular analysis to elucidate the spatio temporal dynamics and functions of the microbial populations associated with biodegradation of real contaminant mixtures in real soil environments. Our original focus was to examine PAH degradation in hydrocarbon mixtures. We have invested a substantial amount of effort into developing functional gene- targeted approaches for PAH-degrading organisms; however, the considerable sequence divergence among functional genes of these types has been so extensive (Laurie and Lloyd-Jones, 1999; Moser and Stahl, 2001; Baldwin, et al., 2003) that it is difficult to design PCR primers to detect all such genes. We also realized that degenerate PCR primers, which some other groups (Baldwin, et al., 2003) have developed to compensate for such divergent sequences, would not work in conjunction with DGGE, which we have planned to use to track specific sequence variants. Considering these limitations, we have then centered our focal areas on aliphatic hydrocarbons, which are a major constituent of various hydrocarbon mixtures (e.g., crude oil, diesel, and kerosene), with specific emphasis on alkane-degrading microorganisms and the alkB gene coding for alkane hydroxylase enzyme as targets for a functional gene approach.

We have successfully developed novel molecular methods that enable us to link the biodegradation of alkanes with specific microbial populations in situ. We have demonstrated the utility of the novel methods combined with physico chemical analysis to study microbial populations in complex mixture environments and applied these approaches to examine four general types of environmental determinants for their importance in selecting specific hydrocarbon-degrading microbial populations: soil type, contaminant type, temperature, and time. Our observations show that in situ biodegradation of alkane mixtures in different soil environments often involves different microbial populations, but many soils demonstrate a consistent pattern in the types of microorganisms that respond to alkane perturbation. The most consistent pattern observed in the current study was the emergence of R. erythropolis-like microorganisms (DGGE bands N1 2) in four of seven contrasting soil types subjected to crude oil contamination. The mixture types and temperature also influenced the microbial population selections, although same or similar population structures still were detected. It is noteworthy that many of the prominent populations detected under various mixture types and temperature conditions were isolated as alkane-degraders from the crude oil amended soils, although they did not correspond to prominent bands in crude oil amendments. This may indicate the presence of indigenous hydrocarbon-degrading populations in the soil environments with potential importance in biodegradation activities under different environmental conditions (mixture types, temperature, moisture level).

The underlying mechanistic basis for population selection in soils and natural waters contaminated with complex hydrocarbon mixtures is of significant interest for predicting and managing in situ bioremediation. Observations across many additional soil types may be necessary to identify patterns and possible causes, but this approach likely will be necessary to understand why soils of different character exhibit unique population responses to hydrocarbon contamination, and why, in other cases, similar genera are selected.

References:

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de Boer W, Leveau JHJ, Kowalchuk GA, Gunnewiek PJAK, Abeln ECA, Figge MJ, Sjollemak K, Janse JD, van Veen JA. Collimonas fungivorans gen. nov., sp. nov., a chitinolytic soil bacterium with the ability to grow on living fungal hyphae. International Journal of Systematic and Evolutionary Microbiology 2004;54(3):857-864.

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Laurie AD, Lloyd-Jones G. The phn genes of Burkholderia sp. strain RP007 constitute a divergent gene cluster for polycyclic aromatic hydrocarbon catabolism. Journal of Bacteriology 1999;181(2):531-540.

Macur RE, Jackson CR, Botero LM, McDermott TR, Inskeep WP. Bacterial populations associated with the oxidation and reduction of arsenic in an unsaturated soil. Environmental Science & Technology 2004;38(1):104-111.

Margesin R, Schinner F. Biodegradation and bioremediation of hydrocarbons in extreme environments. Applied Microbiology and Biotechnology 2001;56(5-6):650-663.

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Norris TB, Wraith JM, Castenholz RW, McDermott TR. Soil microbial community structure across a thermal gradient following a geothermal heating event. Applied and Environmental Microbiology 2002;68(12):6300-6309.

Rooney-Varga JN, Anderson RT, Fraga JL, Ringelberg D, Lovley DR. Microbial communities associated with anaerobic benzene degradation in a petroleum-contaminated aquifer. Applied and Environmental Microbiology 1999;65(7):3056-3063.

Smits THM, Röthlisberger M, Witholt B, van Beilen JB. Molecular screening for alkane hydroxylase genes in Gram-negative and Gram-positive strains. Environmental Microbiology 1999;1(4):307-317.

Sun HY, Deng SP, Raun WR. Bacterial community structure and diversity in a century-old manure-treated agroecosystem. Applied and Environmental Microbiology 2004;70(10):5868-5874.

Vomberg A, Klinner U. Distribution of alkB genes within n-alkane-degrading bacteria. Journal of Applied Microbiology 2000;89(2):339-348.

West NJ, Scanlan DJ. Niche-partitioning of Prochlorococcus populations in a stratified water column in the eastern North Atlantic Ocean. Applied and Environmental Microbiology 1999;65(6):2585-2591.

Whyte LG, Hawari J, Zhou E, Bourbonnière L, Inniss WE, Greer CW. Biodegradation of variable-chain-length alkanes at low temperatures by a psychrotrophic Rhodococcus sp. Applied and Environmental Microbiology 1998;64(7):2578-2584.

Journal Articles on this Report: 2 Displayed | Download in RIS Format

Other project views: All 9 publications 2 publications in selected types All 2 journal articles

Type Citation Project Document Sources

Journal Article Hamamura N, Olson SH, Ward DM, Inskeep WP. Diversity and functional analysis of bacterial communities associated with natural hydrocarbon seeps in acidic soils at Rainbow Springs, Yellowstone National Park. Applied and Environmental Microbiology 2005;71(10):5943-5950. R829357 (Final)
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Journal Article Hamamura N, Olson SH, Ward DM, Inskeep WP. Microbial population dynamics associated with crude-oil biodegradation in diverse soils. Applied and Environmental Microbiology 2006;72(9):6316-6324. R829357 (Final)
not available

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Type	Citation	Project	Document Sources
Journal Article	Hamamura N, Olson SH, Ward DM, Inskeep WP. Diversity and functional analysis of bacterial communities associated with natural hydrocarbon seeps in acidic soils at Rainbow Springs, Yellowstone National Park. Applied and Environmental Microbiology 2005;71(10):5943-5950.	R829357 (Final)	not available
Journal Article	Hamamura N, Olson SH, Ward DM, Inskeep WP. Microbial population dynamics associated with crude-oil biodegradation in diverse soils. Applied and Environmental Microbiology 2006;72(9):6316-6324.	R829357 (Final)	not available

chemical transport, ecological effects, bioavailability, toxics, bacteria, biology, ecology, environmental chemistry, petroleum, ecosystem protection, environmental exposure and risk, waste, water, chemistry, contaminated sediments, ecological risk assessment, ecology and ecosystems, environmental chemistry, environmental monitoring, fate and transport, hazardous waste, chemical mixtures, PAH metal mixtures, analytical models, biodegradation, biogeochemical partitioning, chemical contaminants, chemical kinetics, chemical transport, complex mixtures, contaminant biodegradation rates, contaminant transport models, contaminated sediment, contaminated soils, creosote, crude oil, environmental transport and fate, fate, hazardous chemicals, hazardous organic substances, microbial degradation, , Ecosystem Protection/Environmental Exposure & Risk, Water, Geographic Area, Scientific Discipline, Waste, RFA, chemical mixtures, Ecological Risk Assessment, Chemistry, Hazardous Waste, EPA Region, Fate & Transport, Environmental Chemistry, Contaminated Sediments, Hazardous, Ecology and Ecosystems, Environmental Monitoring, contaminant transport models, hazardous organic substances, biodegradation, fate, fate and transport, microbial degradation, fate and transport , crude oil, environmental transport and fate, chemical transport, creosote, chemical kinetics, contaminated sediment, analytical models, Region 8, contaminant biodegradation rates, contaminated soils, molecular biology, biogeochemical partitioning, hazardous chemicals, chemical contaminants, complex mixtures

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Final Report: Patterns and Prediction: Molecular Analyses of PAH-Degrading Microbial Populations and Their Function in Real Contaminant Mixture Environment

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