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1.
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).  相似文献   

2.
The purpose of this study was to investigate the mechanism by which phytoremediation systems promote hydrocarbon degradation in soil. The composition and degradation capacity of the bulk soil microbial community during the phytoremediation of soil contaminated with aged hydrocarbons was assessed. In the bulk soil, the level of catabolic genes involved in hydrocarbon degradation (ndoB, alkB, and xylE) as well as the mineralization of hexadecane and phenanthrene was higher in planted treatment cells than in treatment cells with no plants. There was no detectable shift in the 16S ribosomal DNA (rDNA) composition of the bulk soil community between treatments, but there were plant-specific and -selective effects on specific catabolic gene prevalence. Tall Fescue (Festuca arundinacea) increased the prevalence of ndoB, alkB, and xylE as well as naphthalene mineralization in rhizosphere soil compared to that in bulk soil. In contrast, Rose Clover (Trifolium hirtum) decreased catabolic gene prevalence and naphthalene mineralization in rhizosphere soil. The results demonstrated that phytoremediation systems increase the catabolic potential of rhizosphere soil by altering the functional composition of the microbial community. This change in composition was not detectable by 16S rDNA but was linked to specific functional genotypes with relevance to petroleum hydrocarbon degradation.  相似文献   

3.
4.
Plant-bacterial combinations can increase contaminant degradation in the rhizosphere, but the role played by indigenous root-associated bacteria during plant growth in contaminated soils is unclear. The purpose of this study was to determine if plants had the ability to selectively enhance the prevalence of endophytes containing pollutant catabolic genes in unrelated environments contaminated with different pollutants. At petroleum hydrocarbon contaminated sites, two genes encoding hydrocarbon degradation, alkane monooxygenase (alkB) and naphthalene dioxygenase (ndoB), were two and four times more prevalent in bacteria extracted from the root interior (endophytic) than from the bulk soil and sediment, respectively. In field sites contaminated with nitroaromatics, two genes encoding nitrotoluene degradation, 2-nitrotoluene reductase (ntdAa) and nitrotoluene monooxygenase (ntnM), were 7 to 14 times more prevalent in endophytic bacteria. The addition of petroleum to sediment doubled the prevalence of ndoB-positive endophytes in Scirpus pungens, indicating that the numbers of endophytes containing catabolic genotypes were dependent on the presence and concentration of contaminants. Similarly, the numbers of alkB- or ndoB-positive endophytes in Festuca arundinacea were correlated with the concentration of creosote in the soil but not with the numbers of alkB- or ndoB-positive bacteria in the bulk soil. Our results indicate that the enrichment of catabolic genotypes in the root interior is both plant and contaminant dependent.  相似文献   

5.
AIMS: A molecular tool for extensive detection of prokaryotic alkane hydroxylase genes (alkB) was developed. AlkB genotypes involved in the degradation of short-chain alkanes were quantified in environmental samples in order to assess their occurrence and ecological importance. METHODS AND RESULTS: Four primer pairs specific for distinct clusters of alkane hydroxylase genes were designed, allowing amplification of alkB-related genes from all tested alkane-degrading strains and from six of seven microcosms. For the primer pair detecting alkB genes related to the Pseudomonas putida GPo1 alkB gene and the one targeting alkB genes of Gram-positive strains, both involved in short-chain alkane degradation (相似文献   

6.
In total, 435 pure bacterial strains were isolated from microtherm oil-production water from the Karamay Oilfield, Xinjiang, China, by using four media: oil-production water medium (Cai medium), oil-production water supplemented with mineral salt medium (CW medium), oil-production water supplemented with yeast extract medium (CY medium), and blood agar medium (X medium). The bacterial isolates were affiliated with 61 phylogenetic groups that belong to 32 genera in the phyla Actinobacteria, Firmicutes, and Proteobacteria. Except for the Rhizobium, Dietzia, and Pseudomonas strains that were isolated using all the four media, using different media led to the isolation of bacteria with different functions. Similarly, nonheme diiron alkane monooxygenase genes (alkB/alkM) also clustered according to the isolation medium. Among the bacterial strains, more than 24 % of the isolates could use n-hexadecane as the sole carbon source for growth. For the first time, the alkane-degrading ability and alkB/alkM were detected in Rhizobium, Rhodobacter, Trichococcus, Micrococcus, Enterococcus, and Bavariicoccus strains, and the alkM gene was detected in Firmicutes strains.  相似文献   

7.
While bioremediation of total petroleum hydrocarbons (TPH) is in general a robust technique, heterogeneity in terms of contaminant and environmental characteristics can impact the extent of biodegradation. The current study investigates the implications of different soil matrix types (anthropogenic fill layer, peat, clay, and sand) and bioavailability on bioremediation of an aged diesel contamination from a heterogeneous site. In addition to an uncontaminated sample for each soil type, samples representing two levels of contamination (high and low) were also used; initial TPH concentrations varied between 1.6 and 26.6 g TPH/kg and bioavailability between 36 and 100 %. While significant biodegradation occurred during 100 days of incubation under biostimulating conditions (64.4–100 % remediation efficiency), low bioavailability restricted full biodegradation, yielding a residual TPH concentration. Respiration levels, as well as the abundance of alkB, encoding mono-oxygenases pivotal for hydrocarbon metabolism, were positively correlated with TPH degradation, demonstrating their usefulness as a proxy for hydrocarbon biodegradation. However, absolute respiration and alkB presence were dependent on soil matrix type, indicating the sensitivity of results to initial environmental conditions. Through investigating biodegradation potential across a heterogeneous site, this research illuminates the interplay between soil matrix type, bioavailability, and bioremediation and the implications of these parameters for the effectiveness of an in situ treatment.  相似文献   

8.
Total community DNA from 29 noncontaminated soils and soils impacted by petroleum hydrocarbons and chloro-organics from Antarctica and Brazil were screened for the presence of nine catabolic genes, encoding alkane monooxygenase or aromatic dioxygenases, from known bacterial biodegradation pathways. Specific primers and probes targeting alkane monooxygenase genes were derived from Pseudomonas putida ATCC 29347 (Pp alkB), Rhodococcus sp. strain Q15 (Rh alkB1, Rh alkB2), and Acinetobacter sp. ADP-1 (Ac alkM). In addition, primers and probes detecting aromatic dioxygenase genes were derived from P. putida ATCC 17484 (ndoB), P. putida F1 (todC1), P. putida ATCC 33015 (xylE and cat23), and P. pseudoalcaligenes KF707 (bphA). The primers and probes were used to analyze total community DNA extracts by using PCR and hybridization analysis. All the catabolic genes, except the Ac alkM, were detected in contaminated and control soils from both geographic regions, with a higher frequency in the Antarctic soils. The alkane monooxygenase genes, Rh alkB1 and Rh alkB2, were the most frequently detected alk genes in both regions, while Pp alkB was not detected in Brazil soils. Genes encoding the aromatic dioxygenases toluene dioxygenase (todC1) and biphenyl dioxygenase (bphA) were the most frequently detected in Antarctica, and todC1 and catechol-2,3-dioxygenase (cat23) were the most frequent in Brazil soils. Hybridization analysis confirmed the PCR results, indicating that the probes used had a high degree of homology to the genes detected in the soil extracts and were effective in detecting biodegradative potential in the indigenous microbial population.  相似文献   

9.
Abstract The biodegradation of a mixture of several creosote-related compounds, p-cresol, phenanthrene, fluorene, and carbazole was examined in columns containing aquifer sands. The aquifer material, itself, had an effect on the migration of the test compounds, with p-cresol being retarded the least, followed by carbazole, then fluorene, and finally phenanthrene. The biodegradation of all the compounds was greatly enhanced by the inclusion of p-cresol (10 ppm) in the substrate mixture. Associated with this enhanced degradation was a 100-fold increase in the total culturable bacterial population, and increases in the xylE- and ndoB-positive bacterial populations of more than three orders of magnitude. The products of these two genes are involved in the degradation of monocyclic and polycyclic aromatic compounds, respectively. In columns that did not receive p-cresol, there was no significant change in either the total culturable bacterial population density or the xylE-positive bacterial population, but there were significant increases of one to two orders of magnitude in the ndoB-positive bacterial populations. The results suggest that the ndoB gene probe can detect bacteria capable of utilizing phenanthrene, carbazole, and possibly fluorene. Received: 26 January 1996; Accepted: 20 June 1996  相似文献   

10.
Abstract The enrichment of several genes (xylE, nahAcd, todC1C2BA, tmoABCDE, alkB) that encode enzymes responsible for key steps in the degradation of hydrocarbons, and one gene specific to rRNA group I of the genus Pseudomonas, was studied in DNA extracted from a fuel oil–contaminated field site, and in laboratory microcosms (with the exception of alkB). Toluene, ethylbenzene, xylene, and naphthalene concentrations were related to the extent of hybridization of the genes in the field studies. Significant differences were observed in the extent of hybridization of some of the genes between contaminated and noncontaminated samples. In the microcosm studies, gasoline at rates ranging from 0.5 mg to 125 mg gasoline/g of soil as applied to soils, and the changes in hybridization intensity of these genes monitored with time. The lower threshold of gene enrichment of these genes in response to gasoline addition was below 0.5 mg/g soil. Small increases were observed at the 0.5-mg exposure level, but hybridization intensity quickly decreased to levels below detection 6–8 days after addition of the gasoline. A dose-response effect was observed from treatments with gasoline concentrations ranging from 0.5 to 35 mg/g soil. Inhibition by toxic components in gasoline was observed at 75 and 125 mg/g soil levels. Hybridization of the Pseudomonas group 1 probe to field DNA was not significantly enriched in the contaminated field site, although these sequences were enriched in the microcosm studies. Among the genes tested, xylE was the most sensitive indicator of low levels of fuel oil contamination. Received: 23 July 1996; Accepted 9 October 1996  相似文献   

11.
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.  相似文献   

12.
The alkane monooxygenase AlkB, which is encoded by the alkB gene, is a key enzyme involved in bacterial alkane degradation. To study the alkB gene within bacterial communities, researchers need to be aware of the variations in alkB nucleotide sequences; a failure to consider the sequence variations results in the low representation of the diversity and richness of alkane-degrading bacteria. To minimize this shortcoming, the use of a combination of three alkB-targeting primers to enhance the detection of the alkB gene in previously isolated alkane-degrading bacteria was proposed. Using this approach, alkB-related PCR products were detected in 79% of the strains tested. Furthermore, the chosen set of primers was used to study alkB richness and diversity in different soils sampled in Carmópolis, Brazil and King George Island, Antarctica. The DNA extracted from the different soils was PCR amplified with each set of alkB-targeting primers, and clone libraries were constructed, sequenced and analyzed. A total of 255 alkB phylotypes were detected. Venn diagram analyses revealed that only low numbers of alkB phylotypes were shared among the different libraries derived from each primer pair. Therefore, the combination of three alkB-targeting primers enhanced the richness of alkB phylotypes detected in the different soils by 45% to 139%, when compared to the use of a single alkB-targeting primer. In addition, a dendrogram analysis and beta diversity comparison of the alkB composition showed that each of the sampling sites studied had a particular set of alkane-degrading bacteria. The use of a combination of alkB primers was an efficient strategy for enhancing the detection of the alkB gene in cultivable bacteria and for better characterizing the distribution of alkane-degrading bacteria in different soil environments.  相似文献   

13.
Succinoyl trehalose lipids (STLs) are promising glycolipid biosurfactants produced from n-alkanes that are secreted by Rhodococcus species bacteria. These compounds not only exhibit unique interfacial properties but also demonstrate versatile biochemical actions. In this study, three novel types of genes involved in the biosynthesis of STLs, including a putative acyl coenzyme A (acyl-CoA) transferase (tlsA), fructose-bisphosphate aldolase (fda), and alkane monooxygenase (alkB), were identified. The predicted functions of these genes indicate that alkane metabolism, sugar synthesis, and the addition of acyl groups are important for the biosynthesis of STLs. Based on these results, we propose a biosynthesis pathway for STLs from alkanes in Rhodococcus sp. strain SD-74. By overexpressing tlsA, we achieved a 2-fold increase in the production of STLs. This study advances our understanding of bacterial glycolipid production in Rhodococcus species.  相似文献   

14.
An analysis of the effect of an oil spill on mangrove sediments was carried out by contamination of mesocosms derived from two different mangroves, one with a history of contamination and one pristine. The association between N2 fixers and hydrocarbon degradation was assessed using quantitative PCR (qPCR) for the genes rrs and nifH, nifH clone library sequencing and total petroleum hydrocarbon (TPH) quantification using gas chromatography. TPH showed that the microbial communities of both mangroves were able to degrade the hydrocarbons added; however, whereas the majority of oil added to the mesocosm derived from the polluted mangrove was degraded in the 75 days of the experiment, there was only partially degradation in the mesocosm derived from the pristine mangrove. qPCR showed that the addition of oil led to an increase in rrs gene copy numbers in both mesocosms, having almost no effect on the nifH copy numbers in the pristine mangrove. Sequencing of nifH clones indicated that the changes promoted by the oil in the polluted mangrove were greater than those observed in the pristine mesocosm. The main effect observed in the polluted mesocosm was the selection of a single phylotype which is probably adapted to the presence of petroleum. These results, together with previous reports, give hints about the relationship between N2 fixation and hydrocarbon degradation in natural ecosystems.  相似文献   

15.
Unraveling functional genes related to biodegradation of organic compounds has profoundly improved our understanding of biological remediation processes, yet the ecology of such genes is only poorly understood. We used a culture-independent approach to assess the abundance and diversity of bacteria catalyzing the degradation of n-alkanes with a chain length between C5 and C16 at a forest site co-contaminated with mineral oil hydrocarbons and metals for nearly 60 years. The alkB gene coding for a rubredoxin-dependent alkane monooxygenase enzyme involved in the initial activation step of aerobic aliphatic hydrocarbon metabolism was used as biomarker. Within the area of study, four different zones were evaluated: one highly contaminated, two intermediately contaminated, and a noncontaminated zone. Contaminant concentrations, hydrocarbon profiles, and soil microbial respiration and biomass were studied. Abundance of n-alkane-degrading bacteria was quantified via real-time PCR of alkB, whereas genetic diversity was examined using molecular fingerprints (T-RFLP) and clone libraries. Along the contamination plume, hydrocarbon profiles and increased respiration rates suggested on-going natural attenuation at the site. Gene copy numbers of alkB were similar in contaminated and control areas. However, T-RFLP-based fingerprints suggested lower diversity and evenness of the n-alkane-degrading bacterial community in the highly contaminated zone compared to the other areas; both diversity and evenness were negatively correlated with metal and hydrocarbon concentrations. Phylogenetic analysis of alkB denoted a shift of the hydrocarbon-degrading bacterial community from Gram-positive bacteria in the control zone (most similar to Mycobacterium and Nocardia types) to Gram-negative genotypes in the contaminated zones (Acinetobacter and alkB sequences with little similarity to those of known bacteria). Our results underscore a qualitative rather than a quantitative response of hydrocarbon-degrading bacteria to the contamination at the molecular level.  相似文献   

16.
Rhodococcus sp. and Pseudomonas sp. bioremediation experiments were carried out using free and immobilized cells on natural carrier material (corncob powder) in order to evaluate the feasibility of its use in the bioremediation of hydrocarbon-contaminated soils. Terminal restriction fragment length polymorphism analysis was performed on the 16S rRNA gene as molecular fingerprinting method in order to assess the persistence of inoculated strains in the soil over time. Immobilized Pseudomonas cells degraded hydrocarbons more efficiently in the short term compared to the free ones. Immobilization seemed also to increase cell growth and stability in the soil. Free and immobilized Rhodococcus cells showed comparable degradation percentages, probably due to the peculiarity of Rhodococcus cells to aggregate into irregular clusters in the presence of hydrocarbons as sole carbon source. It is likely that the cells were not properly adsorbed on the porous matrix as a result of the small size of its pores. When Rhodococcus and Pseudomonas cells were co-immobilized on the matrix, a competition established between the two strains, that probably ended in the exclusion of Pseudomonas cells from the pores. The organic matrix might act as protective agent, but it also possibly limited cell density. Nevertheless, when the cells were properly adsorbed on the porous matrix, the immobilization became a suitable bioremediation strategy.  相似文献   

17.
18.
Remediation of contaminated soils is often studied using fine-textured soils rather than low-fertility sandy soils, and few studies focus on recontamination events. This study compared aerobic and anaerobic treatments for remediation of freshly introduced used motor oil on a sandy soil previously phytoremediated and bioacclimated (microorganisms already adapted in the soil environment) with some residual total petroleum hydrocarbon (TPH) contamination. Vegetated and unvegetated conditions to remediate anthropogenic fill containing residual TPH that was spiked with nonaqueous phase liquids (NAPLs) were evaluated in a 90-day greenhouse pot study. Vegetated treatments used switchgrass (Panicum virgatum). The concentration of aerobic bacteria were orders of magnitude higher in vegetated treatments compared to unvegetated. Nevertheless, final TPH concentrations were low in all saturated soil treatments, and high in the presence of switchgrass. Concentrations were also low in unvegetated pots with fertilizer. Acclimated indigenous microbial communities were shown to be more effective in breaking down hydrocarbons than introducing microbes from the addition of plant treatments in sandy soils. Remediation of fresh introduced NAPLs on pre-phytoremediated and bioacclimated soil was most efficient in saturated, anaerobic environments, probably due to the already pre-established microbial associations, easily bioavailable contaminants, and optimized soil conditions for microbial establishment and survival.  相似文献   

19.
Environmental microbial communities are key players in the bioremediation of hydrocarbon pollutants. Here we assessed changes in bacterial abundance and diversity during the degradation of Tunisian Zarzatine oil by four indigenous bacterial consortia enriched from a petroleum station soil, a refinery reservoir soil, a harbor sediment and seawater. The four consortia were found to efficiently degrade up to 92.0% of total petroleum hydrocarbons after 2 months of incubation. Illumina 16S rRNA gene sequencing revealed that the consortia enriched from soil and sediments were dominated by species belonging to Pseudomonas and Acinetobacter genera, while in the seawater-derived consortia Dietzia, Fusobacterium and Mycoplana emerged as dominant genera. We identified a number of species whose relative abundances bloomed from small to high percentages: Dietzia daqingensis in the seawater microcosms, and three OTUs classified as Acinetobacter venetianus in all two soils and sediment derived microcosms. Functional analyses on degrading genes were conducted by comparing PCR results of the degrading genes alkB, ndoB, cat23, xylA and nidA1 with inferences obtained by PICRUSt analysis of 16S amplicon data: the two data sets were partly in agreement and suggest a relationship between the catabolic genes detected and the rate of biodegradation obtained. The work provides detailed insights about the modulation of bacterial communities involved in petroleum biodegradation and can provide useful information for in situ bioremediation of oil-related pollution.  相似文献   

20.

The impact of long-term crude oil pollution on soil microbial community structure in Bodo West Community, Ogoniland, Nigeria, was investigated to determine the amenability of the soil to microbial mediated remediation. Crude oil-polluted and pristine soil samples were collected approximately from 0 to 30 cm depth for both chemical and microbiological analyses. Total petroleum hydrocarbons (TPH) and polycyclic aromatic hydrocarbons (PAH) were determined using gas chromatograph–mass spectrophotometer (GC-MS). The soil microbiome was determined using the Illumina MiSeq platform. Results from this study were then compared with publicly available data from other oil-polluted sites. Taxonomic biomarkers and pathways associated with oil-polluted soils were detected using bioinformatics pipelines. TPH in the polluted and pristine soils were 7591 mg/kg and 199.70 mg/kg respectively, while the values of PAHs were significantly higher (p < 0.05) in the oil-polluted soil. Predictive functional and biomarker analysis demonstrated that microbes detected in the oil-polluted environment were involved in different metabolic pathways for degradation of a broad set of xenobiotic aromatic compounds. Established hydrocarbon degraders belonging to the families Alcanivoracaceae and Oceanospirillaceae were mostly detected in the oil-polluted soils. Sneathiella, Parvibaculum, Sphingobium, and Oceanicaulis were among biomarker taxa. The bacterial families Acidithiobacillaceae and Desulfobacteraceae were differentially more abundant in Bodo West spill site than any other site used for comparison. Furthermore, differentially represented species in our study site and other oil-polluted sites ranged from 21 to 42 bacterial families. The findings from this study revealed the bacterial community had a strong dependence on hydrocarbons and that acid-tolerant bacterial families can as well contribute significantly to biodegradation in the site and other polluted sites in Ogoniland usually known to have an acidic pH. Further research on Bodo West spill site will reveal the novel enzymes and pathways for enhanced microbial mediated eco-restoration.

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