Microbial population dynamics associated with crude-oil biodegradation in diverse soils

Soil bacterial population dynamics were examined in several crude-oil-contaminated soils to identify those organisms associated with alkane degradation and to assess patterns in microbial response across disparate soils. Seven soil types obtained from six geographically distinct areas of the United States (Arizona, Oregon, Indiana, Virginia, Oklahoma, and Montana) were used in controlled contamination experiments containing 2% (wt/wt) crude oil spiked with [1-(14)C]hexadecane. Microbial populations present during hydrocarbon degradation were analyzed using both 16S rRNA gene sequence analysis and by traditional methods for cultivating hydrocarbon-oxidizing bacteria. After a 50-day incubation, all seven soils showed comparable hydrocarbon depletion, where >80% of added crude oil was depleted and approximately 40 to 70% of added [(14)C]hexadecane was converted to (14)CO(2). However, the initial rates of hydrocarbon depletion differed up to 10-fold, and preferential utilization of shorter-chain-length n-alkanes relative to longer-chain-length n-alkanes was observed in some soils. Distinct microbial populations developed, concomitant with crude-oil depletion. Phylogenetically diverse bacterial populations were selected across different soils, many of which were identical to hydrocarbon-degrading isolates obtained from the same systems (e.g., Nocardioides albus, Collimonas sp., and Rhodococcus coprophilus). In several cases, soil type was shown to be an important determinant, defining specific microorganisms responding to hydrocarbon contamination. However, similar Rhodococcus erythropolis-like populations were observed in four of the seven soils and were the most common hydrocarbon-degrading organisms identified via cultivation.     

Molecular Analysis of Surfactant-Driven Microbial Population Shifts in Hydrocarbon-Contaminated Soil

We analyzed the impact of surfactant addition on hydrocarbon mineralization kinetics and the associated population shifts of hydrocarbon-degrading microorganisms in soil. A mixture of radiolabeled hexadecane and phenanthrene was added to batch soil vessels. Witconol SN70 (a nonionic, alcohol ethoxylate) was added in concentrations that bracketed the critical micelle concentration (CMC) in soil (CMC′) (determined to be 13 mg g⁻¹). Addition of the surfactant at a concentration below the CMC′ (2 mg g⁻¹) did not affect the mineralization rates of either hydrocarbon. However, when surfactant was added at a concentration approaching the CMC′ (10 mg g⁻¹), hexadecane mineralization was delayed and phenanthrene mineralization was completely inhibited. Addition of surfactant at concentrations above the CMC′ (40 mg g⁻¹) completely inhibited mineralization of both phenanthrene and hexadecane. Denaturing gradient gel electrophoresis of 16S rRNA gene segments showed that hydrocarbon amendment stimulated Rhodococcus and Nocardia populations that were displaced by Pseudomonas and Alcaligenes populations at elevated surfactant levels. Parallel cultivation studies revealed that the Rhodococcus population can utilize hexadecane and that the Pseudomonas and Alcaligenes populations can utilize both Witconol SN70 and hexadecane for growth. The results suggest that surfactant applications necessary to achieve the CMC alter the microbial populations responsible for hydrocarbon mineralization.     

UAF radiorespirometric protocol for assessing hydrocarbon mineralization potential in environmental samples

Following the EXXOn Valdez oil spill, a radiorespirometric protocol was developed at the University of Alaska Fairbanks (UAF) to assess the potential for microorganisms in coastal waters and sediments to degrade hydrocarbons. The use of bioremediation to assist in oil spill cleanup operations required microbial bioassays to establish that addition of nitrogen and phosphorus would enhance biodegradation. A technique assessing 1-¹⁴C-n-hexadecane mineralization in seawater or nutrient rich sediment suspensions was used for both of these measurements. Hydrocarbon-degradation potentials were determined by measuring mineralization associated with sediment microorganisms in sediment suspended in sterilized seawater and/or marine Bushnell-Haas broth. Production of ¹⁴CO₂ and CO₂ was easily detectable during the first 48 hours with added hexadecane levels ranging from 10 to 500 mg/l of suspension and dependent on the biomass of hydrocarbon degraders, the hydrocarbon-oxidation potential of the biomass and nutrient availability. In addition to assessment of the hydrocarbon-degrading potential of environmental samples, the radiorespirometric procedure, and concomitant measurement of microbial biomass, has utility as an indicator of hydrocarbon contamination of soils, aqueous sediments and water, and can also be used to evaluate the effectiveness of bioremediation treatments.     

Molecular analysis of surfactant-driven microbial population shifts in hydrocarbon-contaminated soil

We analyzed the impact of surfactant addition on hydrocarbon mineralization kinetics and the associated population shifts of hydrocarbon-degrading microorganisms in soil. A mixture of radiolabeled hexadecane and phenanthrene was added to batch soil vessels. Witconol SN70 (a nonionic, alcohol ethoxylate) was added in concentrations that bracketed the critical micelle concentration (CMC) in soil (CMC') (determined to be 13 mg g(-1)). Addition of the surfactant at a concentration below the CMC' (2 mg g(-1)) did not affect the mineralization rates of either hydrocarbon. However, when surfactant was added at a concentration approaching the CMC' (10 mg g(-1)), hexadecane mineralization was delayed and phenanthrene mineralization was completely inhibited. Addition of surfactant at concentrations above the CMC' (40 mg g(-1)) completely inhibited mineralization of both phenanthrene and hexadecane. Denaturing gradient gel electrophoresis of 16S rRNA gene segments showed that hydrocarbon amendment stimulated Rhodococcus and Nocardia populations that were displaced by Pseudomonas and Alcaligenes populations at elevated surfactant levels. Parallel cultivation studies revealed that the Rhodococcus population can utilize hexadecane and that the Pseudomonas and Alcaligenes populations can utilize both Witconol SN70 and hexadecane for growth. The results suggest that surfactant applications necessary to achieve the CMC alter the microbial populations responsible for hydrocarbon mineralization.     

Diversity and Functional Analysis of Bacterial Communities Associated with Natural Hydrocarbon Seeps in Acidic Soils at Rainbow Springs, Yellowstone National Park

In this paper we describe the bacterial communities associated with natural hydrocarbon seeps in nonthermal soils at Rainbow Springs, Yellowstone National Park. Soil chemical analysis revealed high sulfate concentrations and low pH values (pH 2.8 to 3.8), which are characteristic of acid-sulfate geothermal activity. The hydrocarbon composition of the seep soils consisted almost entirely of saturated, acyclic alkanes (e.g., n-alkanes with chain lengths of C₁₅ to C₃₀, as well as branched alkanes, predominately pristane and phytane). Bacterial populations present in the seep soils were phylogenetically characterized by 16S rRNA gene clone library analysis. The majority of the sequences recovered (>75%) were related to sequences of heterotrophic acidophilic bacteria, including Acidisphaera spp. and Acidiphilium spp. of the α-Proteobacteria. Clones related to the iron- and sulfur-oxidizing chemolithotroph Acidithiobacillus spp. were also recovered from one of the seep soils. Hydrocarbon-amended soil-sand mixtures were established to examine [¹⁴C]hexadecane mineralization and corresponding changes in the bacterial populations using denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene fragments. Approximately 50% of the [¹⁴C]hexadecane added was recovered as ¹⁴CO₂ during an 80-day incubation, and this was accompanied by detection of heterotrophic acidophile-related sequences as dominant DGGE bands. An alkane-degrading isolate was cultivated, whose 16S rRNA gene sequence was identical to the sequence of a dominant DGGE band in the soil-sand mixture, as well as the clone sequence recovered most frequently from the original soil. This and the presence of an alkB gene homolog in this isolate confirmed the alkane degradation capability of one population indigenous to acidic hydrocarbon seep soils.     

Potential for biodegradation of hydrocarbons in soil from the Ross Dependency, Antarctica

Oil spills occur in the Antarctic when fuel oils such as JP8 jet fuel are moved or stored. Hydrocarbons, both n-alkanes and aromatic compounds, have been detected in oil-contaminated soils of the Ross Dependency. In such areas hydrocarbon-degrading microbes, if naturally occurring, could be used for clean-up. Soil samples from oil-impacted and control sites were analysed for hydrocarbon-degrading microbes and for a range of parameters known to limit biodegradative activity. Soils were analysed for water content, pH, concentrations of nutrients (N and P) and electrical conductivity. Numbers of culturable heterotrophic bacteria and hydrocarbon degraders were greater in some of the oil-contaminated samples. Mineralisation studies with ¹⁴C-radiolabelled hexadecane and naphthalene demonstrated that nitrogen amendments significantly enhanced hydrocarbon mineralisation rates in an oil-impacted soil. Received: 30 June 1997 / Received revision: 15 September 1997 / Accepted: 19 September 1997     

Hydrocarbon bioremediation potential of an unimpacted Kuwaiti oil-field environment

Seasonal variations in the hydrocarbon-degrading potential of soil samples from an unimpacted site in the Kuwaiti Burgan oil field environment were studied under mesophilic conditions. Hydrocarbon-degrading microorganisms occurred but varied all-year-round, and their numbers ranged from 1.3 x 10(7) to 9.3 x 10(7) CFU g(-1) dry soil, while hydrocarbon-degrading fungi ranged from 3.0 x 10(4) - 3.8 x 10(5) CFU g(-1) dry soil, depending on the sampling period. These hydrocarbon-degraders also comprised variable but generally high proportions of the total aerobic heterotrophic organisms (2 to > 98%) for bacteria and lower levels (7-9%) for fungi. The crude oil-degrading capacity of the oil-degrading populations (bacteria and fungi) ranged from 80-95% of the hexane-extractable fractions. Differential inhibition studies carried out on soil samples showed that bacteria were the greater contributors to hydrocarbon degradation (79-92%) than fungi. Pure hydrocarbon substrates, hexadecane and phenanthrene, were degraded to near completion after a 28-day incubation by both the bacterial and fungal portions of the soil flora.     

Identification of nitrogen-incorporating bacteria in petroleum-contaminated arctic soils by using [15N]DNA-based stable isotope probing and pyrosequencing

Arctic soils are increasingly susceptible to petroleum hydrocarbon contamination, as exploration and exploitation of the Arctic increase. Bioremediation in these soils is challenging due to logistical constraints and because soil temperatures only rise above 0°C for ∼2 months each year. Nitrogen is often added to contaminated soil in situ to stimulate the existing microbial community, but little is known about how the added nutrients are used by these microorganisms. Microbes vary widely in their ability to metabolize petroleum hydrocarbons, so the question becomes: which hydrocarbon-degrading microorganisms most effectively use this added nitrogen for growth? Using [¹⁵N]DNA-based stable isotope probing, we determined which taxonomic groups most readily incorporated nitrogen from the monoammonium phosphate added to contaminated and uncontaminated soil in Canadian Forces Station-Alert, Nunavut, Canada. Fractions from each sample were amplified with bacterial 16S rRNA and alkane monooxygenase B (alkB) gene-specific primers and then sequenced using lage-scale parallel-pyrosequencing. Sequence data was combined with 16S rRNA and alkB gene C quantitative PCR data to measure the presence of various phylogenetic groups in fractions at different buoyant densities. Several families of Proteobacteria and Actinobacteria that are directly involved in petroleum degradation incorporated the added nitrogen in contaminated soils, but it was the DNA of Sphingomonadaceae that was most enriched in ¹⁵N. Bacterial growth in uncontaminated soils was not stimulated by nutrient amendment. Our results suggest that nitrogen uptake efficiency differs between bacterial groups in contaminated soils. A better understanding of how groups of hydrocarbon-degraders contribute to the catabolism of petroleum will facilitate the design of more targeted bioremediation treatments.     

Trophic Structure of Amoeba Communities Near Roots of Medicago sativa After Contamination with Fuel Oil No. 6

Root exudation increases microbial activity, selecting bacterial and fungal communities that metabolize organic matter such as hydrocarbons. However, a strong contamination pulse of hydrocarbons around plant roots may reorganize the soil's microbial trophic structure toward amoebae feeding on bacteria. We conducted a microcosm experiment to elucidate the effect of Medicago sativa on the trophic structure of naked amoebae after a strong pulse of pollution (50,000 ppm of fuel oil no. 6, which is a mixture of long chains ranging from C10 to C28). Plants were seeded 24 h after contamination and species of amoebae in the microcosms were identified at 1, 30, and 60 days after pollution. Several species from three trophic groups of naked amoeba were still alive 24 h after the hydrocarbon pulse. Non-planted microcosms harbored three trophic groups after 60 days, while planted ones nourished four groups. The bacterivore group was the most diverse in all microcosms, followed by protist-eaters and omnivores. The quantity of amoebae was significantly higher (3.4×10³ organisms/g soil) in the planted pots than in the non-planted ones (1.3×10³ organisms/g soil after 30 days of pollution (P?≤?0.01). The shortest hydrocarbon chains (C10–C14) disappeared or diminished in all microcosms, and the longest ones increased in the planted ones. M. sativa thus exerted a positive effect on species richness, quantity, and the composition of amoebae trophic groups in contaminated soil. This indirect effect on bacterial predators is another key factor underlying hydrocarbon assimilation by living organisms during phytoremediation.     

Hydrocarbon-degrading bacteria enriched by the Deepwater Horizon oil spill identified by cultivation and DNA-SIP

The massive influx of crude oil into the Gulf of Mexico during the Deepwater Horizon (DWH) disaster triggered dramatic microbial community shifts in surface oil slick and deep plume waters. Previous work had shown several taxa, notably DWH Oceanospirillales, Cycloclasticus and Colwellia, were found to be enriched in these waters based on their dominance in conventional clone and pyrosequencing libraries and were thought to have had a significant role in the degradation of the oil. However, this type of community analysis data failed to provide direct evidence on the functional properties, such as hydrocarbon degradation of organisms. Using DNA-based stable-isotope probing with uniformly ¹³C-labelled hydrocarbons, we identified several aliphatic (Alcanivorax, Marinobacter)- and polycyclic aromatic hydrocarbon (Alteromonas, Cycloclasticus, Colwellia)-degrading bacteria. We also isolated several strains (Alcanivorax, Alteromonas, Cycloclasticus, Halomonas, Marinobacter and Pseudoalteromonas) with demonstrable hydrocarbon-degrading qualities from surface slick and plume water samples collected during the active phase of the spill. Some of these organisms accounted for the majority of sequence reads representing their respective taxa in a pyrosequencing data set constructed from the same and additional water column samples. Hitherto, Alcanivorax was not identified in any of the previous water column studies analysing the microbial response to the spill and we discuss its failure to respond to the oil. Collectively, our data provide unequivocal evidence on the hydrocarbon-degrading qualities for some of the dominant taxa enriched in surface and plume waters during the DWH oil spill, and a more complete understanding of their role in the fate of the oil.     

Insight into heterogeneity in cell-surface hydrophobicity and ability to degrade hydrocarbons among cells of two hydrocarbon-degrading bacterial populations

The sequential bacterial adherence to hydrocarbons (BATH) of successive generations of hydrophobic fractions of Paenibacillus sp. R0032A and Burkholderia cepacia gave rise to bacterial populations of increasing cell-surface hydrophobicity. Thus, hydrophobicity of the first generation (H1) was less than that of the second generation (H2), which was less than that of the third generation (H3). Beyond H3, the hydrophobic populations became less stable and tended to lyse in hexadecane after violent (vortex) agitation, resulting in an apparent decline in BATH value. The exhaustively fractionated aqueous-phase population (L) was very hydrophilic. The overall cell-surface distribution of the population was L < parental strain < H1 < H2 < H3. The ability to degrade crude oil, hexadecane, or phenanthrene matched the degree of cell-surface hydrophobicity: L < P < H1 < H2 < H3. Thus, in natural populations of hydrocarbon-degrading Paenibacillus sp. R0032A and B. cepacia, there is a heterogeneity in the hydrophobic surface characteriistics that affects the ability of cells to use various hydrocarbon substrates.     

A simple strategy for investigating the diversity and hydrocarbon degradation abilities of cultivable bacteria from contaminated soil

The use of indigenous bacterial strains is a valuable bioremediation strategy for cleaning the environment from hydrocarbon pollutants. The isolation and selection of hydrocarbon-degrading bacteria is therefore crucial for obtaining the most promising strains for site decontamination. Two different media, a minimal medium supplemented with a mixture of polycyclic aromatic hydrocarbons and a MS medium supplemented with triphenyltetrazolium chloride, were used for the isolation of bacterial strains from two hydrocarbon contaminated soils and from their enrichment phases. The hydrocarbon degradation abilities of these bacterial isolates were easily and rapidly assessed using the 2,6-dichlorophenol indophenol assay. The diversity of the bacterial communities isolated from these two soil samples and from their enrichment phases was evaluated by the combination of a bacterial clustering method, fluorescence ITS-PCR, and bacterial identification by 16S rRNA sequencing. Different PCR-based assays were performed in order to detect the genes responsible for hydrocarbon degradation. The best hydrocarbon-degrading bacteria, including Arthrobacter sp., Enterobacter sp., Sphingomonas sp., Pseudomonas koreensis, Pseudomonas putida and Pseudomonas plecoglossicida, were isolated directly from the soil samples on minimal medium. The nahAc gene was detected only in 13 Gram-negative isolates and the sequences of nahAc-like genes were obtained from Enterobacter, Stenotrophomonas, Pseudomonas brenneri, Pseudomonas entomophila and P. koreensis strains. The combination of isolation on minimal medium with the 2,6-dichlorophenol indophenol assay was effective in selecting different hydrocarbon-degrading strains from 353 isolates.     

Fate of 14C-labeled anthracene and hexadecane in compost-manured soil

Experiments were carried out to evaluate the impact of the addition of ripe compost on the degradation of two ¹⁴C-labeled hydrocarbon model compounds (anthracene and hexadecane) in soil. The addition of mature compost (20 % dry wt./dry wt.) stimulated significantly the disappearance of the extractable fraction of both compounds. With compost, 23 % of the labeled anthracene was transformed into ¹⁴CO₂ and 42 % was fixed to the soil matrix irreversibly. In the unsupplemented control reactor more than 88 % of the original anthracene could be recovered by either of two applied organic extraction procedures. The formation of non-extractable bound residues was less significant with [¹⁴C] hexadecane since only 21 % of the labeled carbon had become non-extractable after 103 days. The results presented show that compost could stimulate the depletion of hydrocarbons by either mineralization or the formation of unextractable bound residues (humification). The latter process might be a significant route of depletion in soil especially, for those hydrocarbons that are mineralized only slowly. The meaning of this finding for the assessment of soil bioremediation is discussed.These authors contributed equally to the presented work and should therefore both be considered as first authors     

Rhodococcus lactonase with organophosphate hydrolase (OPH) activity and His6-tagged OPH with lactonase activity: evolutionary proximity of the enzymes and new possibilities in their application

Decontamination of soils with complex pollution using natural strains of microorganisms is a matter of great importance. Here we report that oil-oxidizing bacteria Rhodococcus erythropolis AC-1514D and Rhodococcus ruber AC-1513D can degrade various organophosphorous pesticides (OP). Cell-mediated degradation of five different OP is apparently associated with the presence of N-acylhomoserine lactonase, which is pronouncedly similar (46–50 %) to the well-known enzyme organophosphate hydrolase (OPH), a hydrolysis catalyst for a wide variety of organophosphorous compounds. Additionally, we demonstrated the high lactonase activity of hexahistidine-tagged organophosphate hydrolase (His₆-OPH) with respect to various N-acylhomoserine lactones, and we determined the catalytic constants of His₆-OPH towards these compounds. These experimental data and theoretical analysis confirmed the hypothesis about the evolutionary proximity of OPH and lactonases. Using Rhodococcus cells, we carried out effective simultaneous biodegradation of pesticide paraoxon (88 mg/kg) and oil hydrocarbon hexadecane (6.3 g/kg) in the soil. Furthermore, the discovered high lactonase activity of His₆-OPH offers new possibilities for developing an efficient strategy of combating resistant populations of Gram-negative bacterial cells.     

Rhamnolipid Stimulates Uptake of Hydrophobic Compounds by Pseudomonas aeruginosa

The biodegradation of hexadecane by five biosurfactant-producing bacterial strains (Pseudomonas aeruginosa UG2, Acinetobacter calcoaceticus RAG1, Rhodococcus erythropolis DSM 43066, R. erythropolis ATCC 19558, and strain BCG112) was determined in the presence and absence of exogenously added biosurfactants. The degradation of hexadecane by P. aeruginosa was stimulated only by the rhamnolipid biosurfactant produced by the same organism. This rhamnolipid did not stimulate the biodegradation of hexadecane by the four other strains to the same extent, nor was degradation of hexadecane by these strains stimulated by addition of their own biosurfactants. This suggests that P. aeruginosa has a mode of hexadecane uptake different from those of the other organisms. Rhamnolipid also enhanced the rate of epoxidation of the aliphatic hydrocarbon α,ω-tetradecadiene by a cell suspension of P. aeruginosa. Furthermore, the uptake of the hydrophobic probe 1-naphthylphenylamine by cells of P. aeruginosa was enhanced by rhamnolipid, as indicated by stopped-flow fluorescence experiments. Rhamnolipid did not stimulate the uptake rate of this probe in de-energized cells. These results indicate that an energy-dependent system is present in P. aeruginosa strain UG2 that mediates fast uptake of hydrophobic compounds in the presence of rhamnolipid.     

Taxonomic diversity of aerobic organotrophic bacteria from clean vietnamese soils and their capacity for oxidation of petroleum hydrocarbons

The dominant species and abundance of the cultured aerobic organotrophic bacteria were determined in the clean soils of the Republic of Vietnam. The total number of organotrophs varied from 2.0 × 10⁵ to 5.8 × 10⁸ CFU/g soil. A considerable fraction of the bacterial population (1.1 × 10⁵–9.5 × 10⁶ CFU/g soil) was able to utilize petroleum hydrocarbons as the sole carbon and energy source. Most of the organisms obtained in pure cultures were gram-positive bacteria; over 70% were hydrocarbon-oxidizing organisms. Analysis of 16S rRNA gene sequences resulted in tentative determination of the taxonomic position of 22 strains, with 12 belonging to the Firmicutes, 4, to the Proteobacteria, and 6 to the Actinobacteria. The most common bacteria capable of hydrocarbon oxidation belonged to the genera Acinetobacter, Bacillus, Brevibacillus, Chromobacterium, Cupriavidus, Gordonia, Microbacterium, Mycobacterium, and Rhodococcus. Some of the isolated Bacillus and Staphylococcus strains, as well as one Pseudomonas and one Sinomonas strain, did not utilize hydrocarbons. Gram-positive degraders, especially members of the order Actinomycetales, which exhibited high hydrocarbon-oxidizing activity, gained competitive advantage in the presence of hydrocarbons. This microbial group probably plays an important role in hydrocarbon degradation in tropical soils. Thus, Vietnamese soils, which had no history of petroleum contamination, support numerically significant and taxonomically diverse populations of h ydrocarbon-oxidizing bacteria.     

Distribution of Hydrocarbon-Utilizing Microorganisms and Hydrocarbon Biodegradation Potentials in Alaskan Continental Shelf Areas

Hydrocarbon-utilizing microorganisms were enumerated from Alaskan continental shelf areas by using plate counts and a new most-probable-number procedure based on mineralization of ¹⁴C-labeled hydrocarbons. Hydrocarbon utilizers were ubiquitously distributed, with no significant overall concentration differences between sampling regions or between surface water and sediment samples. There were, however, significant seasonal differences in numbers of hydrocarbon utilizers. Distribution of hydrocarbon utilizers within Cook Inlet was positively correlated with occurrence of hydrocarbons in the environment. Hydrocarbon biodegradation potentials were measured by using ¹⁴C-radiolabeled hydrocarbon-spiked crude oil. There was no significant correlation between numbers of hydrocarbon utilizers and hydrocarbon biodegradation potentials. The biodegradation potentials showed large seasonal variations in the Beaufort Sea, probably due to seasonal depletion of available nutrients. Non-nutrient-limited biodegradation potentials followed the order hexadecane > naphthalene pristane > benzanthracene. In Cook Inlet, biodegradation potentials for hexadecane and naphthalene were dependent on availability of inorganic nutrients. Biodegradation potentials for pristane and benzanthracene were restricted, probably by resistance to attack by available enzymes in the indigenous population.     

Nutrient Amendments of Inorganic Fertiliser and Oil Palm Empty Fruit Bunch and Their Influence on Bacterial Species Dominance and Degradation of the Associated Crude Oil Constituents

The effects of inorganic commercial fertiliser (N:P:K = 8:8:1) and oil palm empty fruit bunch (EFB) as nutrient amendments for crude oil degradation and microbial population shift by a microbial consortium [Pseudomonas sp. (UKMP-14T), Acinetobacter sp. (UKMP-12T), Trichoderma sp. (TriUKMP-1M and TriUKMP-2M)] were assessed. The bacterial populations present during crude oil degradation were analysed by spread plate method and 16S rRNA sequences, whereas the presence of fungi was assessed by growth on potato dextrose agar. Crude oil degradation analysed using gas chromatography-flame ionisation detection showed total petroleum hydrocarbon reduced between 70 and 100%, depending on the type of amendments compared to control (≈55%) after 30 days of incubation. Nutrient amendments using NPK fertiliser or EFB were found to influence the domination of different bacterial species, which in turn preferentially utilised different hydrocarbons. This study suggested different nutrient amendments could be used to preferentially select bacteria to degrade different components of crude oil, particularly pertaining to the recalcitrant phytane. This information is very useful for application of in situ bioremediation of soil hydrocarbon contamination.     

Characterization of Hydrocarbon-Degrading Microbial Populations in Contaminated and Pristine Alpine Soils

Biodegradation of petroleum hydrocarbons in cold environments, including Alpine soils, is a result of indigenous cold-adapted microorganisms able to degrade these contaminants. In the present study, the prevalence of seven genotypes involved in the degradation of n-alkanes (Pseudomonas putida GPo1 alkB; Acinetobacter spp. alkM; Rhodococcus spp. alkB1, and Rhodococcus spp. alkB2), aromatic hydrocarbons (P. putida xylE), and polycyclic aromatic hydrocarbons (P. putida ndoB and Mycobacterium sp. strain PYR-1 nidA) was determined in 12 oil-contaminated (428 to 30,644 mg of total petroleum hydrocarbons [TPH]/kg of soil) and 8 pristine Alpine soils from Tyrol (Austria) by PCR hybridization analyses of total soil community DNA, using oligonucleotide primers and DNA probes specific for each genotype. The soils investigated were also analyzed for various physical, chemical, and microbiological parameters, and statistical correlations between all parameters were determined. Genotypes containing genes from gram-negative bacteria (P. putida alkB, xylE, and ndoB and Acinetobacter alkM) were detected to a significantly higher percentage in the contaminated (50 to 75%) than in the pristine (0 to 12.5%) soils, indicating that these organisms had been enriched in soils following contamination. There was a highly significant positive correlation (P < 0.001) between the level of contamination and the number of genotypes containing genes from P. putida and Acinetobacter sp. but no significant correlation between the TPH content and the number of genotypes containing genes from gram-positive bacteria (Rhodococcus alkB1 and alkB2 and Mycobacterium nidA). These genotypes were detected at a high frequency in both contaminated (41.7 to 75%) and pristine (37.5 to 50%) soils, indicating that they are already present in substantial numbers before a contamination event. No correlation was found between the prevalence of hydrocarbon-degradative genotypes and biological activities (respiration, fluorescein diacetate hydrolysis, lipase activity) or numbers of culturable hydrocarbon-degrading soil microorganisms; there also was no correlation between the numbers of hydrocarbon degraders and the contamination level. The measured biological activities showed significant positive correlation with each other, with the organic matter content, and partially with the TPH content and a significant negative correlation with the soil dry-mass content (P < 0.05 to 0.001).