Occurrence and phylogenetic diversity of Sphingomonas strains in soils contaminated with polycyclic aromatic hydrocarbons.
ABSTRACT: Bacterial strains of the genus Sphingomonas are often isolated from contaminated soils for their ability to use polycyclic aromatic hydrocarbons (PAH) as the sole source of carbon and energy. The direct detection of Sphingomonas strains in contaminated soils, either indigenous or inoculated, is, as such, of interest for bioremediation purposes. In this study, a culture-independent PCR-based detection method using specific primers targeting the Sphingomonas 16S rRNA gene combined with denaturing gradient gel electrophoresis (DGGE) was developed to assess Sphingomonas diversity in PAH-contaminated soils. PCR using the new primer pair on a set of template DNAs of different bacterial genera showed that the method was selective for bacteria belonging to the family Sphingomonadaceae.Single-band DGGE profiles were obtained for most Sphingomonas strains tested. Strains belonging to the same species had identical DGGE fingerprints, and in most cases, these fingerprints were typical for one species. Inoculated strains could be detected at a cell concentration of 10(4) CFU g of soil(-1). The analysis of Sphingomonas population structures of several PAH-contaminated soils by the new PCR-DGGE method revealed that soils containing the highest phenanthrene concentrations showed the lowest Sphingomonas diversity. Sequence analysis of cloned PCR products amplified from soil DNA revealed new 16S rRNA gene Sphingomonas sequences significantly different from sequences from known cultivated isolates (i.e., sequences from environmental clones grouped phylogenetically with other environmental clone sequences available on the web and that possibly originated from several potential new species). In conclusion, the newly designed Sphingomonas-specific PCR-DGGE detection technique successfully analyzed the Sphingomonas communities from polluted soils at the species level and revealed different Sphingomonas members not previously detected by culture-dependent detection techniques.
Project description:Considering their key role for ecosystem processes, it is important to understand the response of microbial communities in unpolluted soils to pollution with polycyclic aromatic hydrocarbons (PAH). Phenanthrene, a model compound for PAH, was spiked to a Cambisol and a Luvisol soil. Total community DNA from phenanthrene-spiked and control soils collected on days 0, 21, and 63 were analyzed based on PCR-amplified 16S rRNA gene fragments. Denaturing gradient gel electrophoresis (DGGE) fingerprints of bacterial communities increasingly deviated with time between spiked and control soils. In taxon specific DGGE, significant responses of Alphaproteobacteria and Actinobacteria became only detectable after 63 days, while significant effects on Betaproteobacteria were detectable in both soils after 21 days. Comparison of the taxonomic distribution of bacteria in spiked and control soils on day 63 as revealed by pyrosequencing indicated soil type specific negative effects of phenanthrene on several taxa, many of them belonging to the Gamma-, Beta-, or Deltaproteobacteria. Bacterial richness and evenness decreased in spiked soils. Despite the significant differences in the bacterial community structure between both soils on day 0, similar genera increased in relative abundance after PAH spiking, especially Sphingomonas and Polaromonas. However, this did not result in an increased overall similarity of the bacterial communities in both soils.
Project description:Members of the Sphingomonas genus are often isolated from petroleum-contaminated soils due to their unique abilities to degrade polycyclic aromatic hydrocarbons (PAHs), which are important for in situ bioremediation. In this study, a combined phenotypic and genotypic approach using streptomycin-containing medium and Sphingomonas-specific PCR was developed to isolate and identify culturable Sphingomonas strains present in petroleum-contaminated soils in the Shenfu wastewater irrigation zone. Of the 15 soil samples examined, 12 soils yielded yellow streptomycin-resistant colonies. The largest number of yellow colony-forming units (CFUs) could reach 10(5)CFUsg(-1)soil. The number of yellow CFUs had a significant positive correlation (p<0.05) with the ratio of PAHs to total petroleum hydrocarbons (TPH), indicating that Sphingomonas may play a key role in degrading the PAH fraction of the petroleum contaminants at this site. Sixty yellow colonies were selected randomly and analyzed by colony PCR using Sphingomonas-specific primers, out of which 48 isolates had PCR-positive signals. The 48 positive amplicons generated 8 distinct restriction fragment length polymorphism (RFLP) patterns, and 7 out of 8 phylotypes were identified as Sphingomonas by 16S rRNA gene sequencing of the representative strains. Within these 7 Sphingomonas strains, 6 strains were capable of using fluorene as the sole carbon source, while 2 strains were phenanthrene-degrading Sphingomonas. To the best of our knowledge, this is the first report to evaluate the relationship between PAHs contamination levels and culturable Sphingomonas in environmental samples.
Project description:BACKGROUND: Copper mining has led to Cu pollution in agricultural soils. In this report, the effects of Cu pollution on bacterial communities of agricultural soils from Valparaiso region, central Chile, were studied. Denaturing gradient gel electrophoresis (DGGE) of the 16S rRNA genes was used for the characterization of bacterial communities from Cu-polluted and non-polluted soils. Cu-resistant bacterial strains were isolated from Cu-polluted soils and characterized. RESULTS: DGGE showed a similar high number of bands and banding pattern of the bacterial communities from Cu-polluted and non-polluted soils. The presence of copA genes encoding the multi-copper oxidase that confers Cu-resistance in bacteria was detected by PCR in metagenomic DNA from the three Cu-polluted soils, but not in the non-polluted soil. The number of Cu-tolerant heterotrophic cultivable bacteria was significantly higher in Cu-polluted soils than in the non-polluted soil. Ninety two Cu-resistant bacterial strains were isolated from three Cu-polluted agricultural soils. Five isolated strains showed high resistance to copper (MIC ranged from 3.1 to 4.7 mM) and also resistance to other heavy metals. 16S rRNA gene sequence analyses indicate that these isolates belong to the genera Sphingomonas, Stenotrophomonas and Arthrobacter. The Sphingomonas sp. strains O12, A32 and A55 and Stenotrophomonas sp. C21 possess plasmids containing the Cu-resistance copA genes. Arthrobacter sp. O4 possesses the copA gene, but plasmids were not detected in this strain. The amino acid sequences of CopA from Sphingomonas isolates (O12, A32 and A55), Stenotrophomonas strain (C21) and Arthrobacter strain (O4) are closely related to CopA from Sphingomonas, Stenotrophomonas and Arthrobacter strains, respectively. CONCLUSIONS: This study suggests that bacterial communities of agricultural soils from central Chile exposed to long-term Cu-pollution have been adapted by acquiring Cu genetic determinants. Five bacterial isolates showed high copper resistance and additional resistance to other heavy metals. Detection of copA gene in plasmids of four Cu-resistant isolates indicates that mobile genetic elements are involved in the spreading of Cu genetic determinants in polluted environments.
Project description:The distributions of endophytic bacteria in Alopecurus aequalis Sobol and Oxalis corniculata L. grown in soils contaminated with different levels of polycyclic aromatic hydrocarbons (PAHs) were investigated with polymerase chain reaction followed by denaturing gradient gel electrophoresis technology (PCR-DGGE) and cultivation methods. Twelve types of PAHs, at concentrations varying from 0.16 to 180 mg·kg(-1), were observed in the roots and shoots of the two plants. The total PAH concentrations in Alopecurus aequalis Sobol obtained from three different PAH-contaminated stations were 184, 197, and 304 mg·kg(-1), and the total PAH concentrations in Oxalis corniculata L. were 251, 346, and 600 mg·kg(-1), respectively. The PCR-DGGE results showed that the endophytic bacterial communities in the roots and shoots of the two plants were quite different, although most bacteria belonged to Firmicutes, Proteobacteria, Actinobacteria and Bacteroidetes. A total of 68 endophytic bacterial strains were isolated from different tissues of the two plants and classified into three phyla: Firmicutes, Proteobacteria and Bacteroidetes. In both plants, Bacillus spp. and Pseudomonas spp. were the dominant cultivable populations. With an increase in the PAH pollution level, the diversity and distribution of endophytic bacteria in the two plants changed correspondingly, and the number of cultivable endophytic bacterial strains decreased rapidly. Testing of the isolated endophytic bacteria for tolerance to each type of PAH showed that most isolates could grow well on Luria-Bertani media in the presence of different PAHs, and some isolates were able to grow rapidly on a mineral salt medium with a single PAH as the sole carbon and energy source, indicating that these strains may have the potential to degrade PAHs in plants. This research provides the first insight into the characteristics of endophytic bacterial populations under different PAH pollution levels and provides a species resource for the isolation of PAH-degrading endophytic bacteria.
Project description:This is the first investigation of the diversity and distribution of 16S rRNA and phenol monooxygenase (PHE) genes in endophytic and rhizosphere bacteria of plants at sites contaminated with different levels of PAHs. Ten PAHs at concentrations from 34.22 to 55.29 and 45.79 to 97.81?mg·kg(-1) were measured in rhizosphere soils of Alopecurus aequalis Sobol and Oxalis corniculata L., respectively. The diversity of 16S rRNA and PHE genes in rhizosphere soils or plants changed with varying PAH pollution levels, as shown based on PCR-DGGE data. Generally, higher Shannon-Weiner indexes were found in mild or moderate contaminated areas. A total of 82 different bacterial 16S rRNA gene sequences belonging to five phyla; namely, Acfinobacteria, Proteobacteria, Chloroflexi, Cyanophyta, and Bacteroidetes, were obtained from rhizosphere soils. For the 57 identified PHE gene sequences, 18 were excised from rhizosphere bacteria and 39 from endophytic bacteria. The copy numbers of 16S rRNA and PHE genes in rhizosphere and endophytic bacteria varied from 3.83?×?10(3) to 2.28?×?10(6) and 4.17?×?10(2) to 1.99?×?10(5), respectively. The copy numbers of PHE genes in rhizosphere bacteria were significantly higher than in endophytic bacteria. Results increase our understanding of the diversity of rhizosphere and endophytic bacteria from plants grown in PAH-contaminated sites.
Project description:Two different procedures were compared to isolate polycyclic aromatic hydrocarbon (PAH)-utilizing bacteria from PAH-contaminated soil and sludge samples, i.e., (i) shaken enrichment cultures in liquid mineral medium in which PAHs were supplied as crystals and (ii) a new method in which PAH degraders were enriched on and recovered from hydrophobic membranes containing sorbed PAHs. Both techniques were successful, but selected from the same source different bacterial strains able to grow on PAHs as the sole source of carbon and energy. The liquid enrichment mainly selected for Sphingomonas spp., whereas the membrane method exclusively led to the selection of Mycobacterium spp. Furthermore, in separate membrane enrichment set-ups with different membrane types, three repetitive extragenic palindromic PCR-related Mycobacterium strains were recovered. The new Mycobacterium isolates were strongly hydrophobic and displayed the capacity to adhere strongly to different surfaces. One strain, Mycobacterium sp. LB501T, displayed an unusual combination of high adhesion efficiency and an extremely high negative charge. This strain may represent a new bacterial species as suggested by 16S rRNA gene sequence analysis. These results indicate that the provision of hydrophobic sorbents containing sorbed PAHs in the enrichment procedure discriminated in favor of certain bacterial characteristics. The new isolation method is appropriate to select for adherent PAH-degrading bacteria, which might be useful to biodegrade sorbed PAHs in soils and sludge.
Project description:Quinones and other oxygenated polycyclic aromatic hydrocarbons (oxy-PAHs) are toxic and/or genotoxic compounds observed to be cocontaminants at PAH-contaminated sites, but their formation and fate in contaminated environmental systems have not been well studied. Anthracene-9,10-dione (anthraquinone) has been found in most PAH-contaminated soils and sediments that have been analyzed for oxy-PAHs. However, little is known about the biodegradation of oxy-PAHs, and no bacterial isolates have been described that are capable of growing on or degrading anthraquinone. PAH-degrading Mycobacterium spp. are the only organisms that have been investigated to date for metabolism of a PAH quinone, 4,5-pyrenequinone. We utilized DNA-based stable-isotope probing (SIP) with [U-(13)C]anthraquinone to identify bacteria associated with anthraquinone degradation in PAH-contaminated soil from a former manufactured-gas plant site both before and after treatment in a laboratory-scale bioreactor. SIP with [U-(13)C]anthracene was also performed to assess whether bacteria capable of growing on anthracene are the same as those identified to grow on anthraquinone. Organisms closely related to Sphingomonas were the most predominant among the organisms associated with anthraquinone degradation in bioreactor-treated soil, while organisms in the genus Phenylobacterium comprised the majority of anthraquinone degraders in the untreated soil. Bacteria associated with anthracene degradation differed from those responsible for anthraquinone degradation. These results suggest that Sphingomonas and Phenylobacterium species are associated with anthraquinone degradation and that anthracene-degrading organisms may not possess mechanisms to grow on anthraquinone.
Project description:Homologs of the glutathione S-transferase (GST)-encoding gene were identified in a collection of aromatic hydrocarbon-degrading Sphingomonas spp. isolated from New Zealand, Antarctica, and the United States by using PCR primers designed from the GST-encoding gene of Sphingomonas paucimobilis EPA505. Sequence analysis of PCR fragments generated from these isolates and of the GST gene amplified from DNA extracted from polycyclic aromatic hydrocarbon (PAH)-contaminated soil revealed a high degree of conservation, which may make the GST-encoding gene a potentially useful marker for PAH-degrading bacteria.
Project description:The potential for biodegradation of polycyclic aromatic hydrocarbons (PAHs)at low temperature and under anaerobic conditions is not well understood, but such biodegradation would be very useful for remediation of polluted sites. Biodegradation of a mixture of 11 different PAHs with two to five aromatic rings, each at a concentration of 10 micro g/ml, was studied in enrichment cultures inoculated with samples of four northern soils. Under aerobic conditions, low temperature severely limited PAH biodegradation. After 90 days, aerobic cultures at 20 degrees C removed 52 to 88% of the PAHs. The most extensive PAH degradation under aerobic conditions at 7 degrees C,53% removal, occurred in a culture from creosote-contaminated soil. Low temperature did not substantially limit PAH biodegradation under nitrate-reducing conditions. Under nitrate-reducing conditions,naphthalene, 2-methylnaphthalene, fluorene, and phenanthrene were degraded. The most extensive PAH degradation under nitrate-reducing conditions at 7 degrees C, 39% removal, occurred in a culture from fuel-contaminated Arctic soil. In separate transfer cultures from the above Arctic soil, incubated anaerobically at 7 degrees C, removal of 2-methylnaphthalene and fluorene was stoichiometrically coupled to nitrate removal. Ribosomal intergenic spacer analysis suggested that enrichment resulted in a few predominant bacterial populations,including members of the genera Acidovorax,Bordetella, Pseudomonas, Sphingomonas, and Variovorax. Predominant populations from different soils often included phylotypes with nearly identical partial 16S rRNA gene sequences (i.e., same genus) but never included phylotypes with identical ribosomal intergenic spacers (i.e., different species or subspecies). The composition of the enriched communities appeared to be more affected by presence of oxygen, than by temperature or source of the inoculum.
Project description:Degradative strains of fast-growing Mycobacterium spp. are commonly isolated from polycyclic aromatic hydrocarbon (PAH)-contaminated soils. Little is known, however, about the ecology and diversity of indigenous populations of these fast-growing mycobacteria in contaminated environments. In the present study 16S rRNA genes were PCR amplified using Mycobacterium-specific primers and separated by temperature gradient gel electrophoresis (TGGE), and prominent bands were sequenced to compare the indigenous Mycobacterium community structures in four pairs of soil samples taken from heavily contaminated and less contaminated areas at four different sites. Overall, TGGE profiles obtained from heavily contaminated soils were less diverse than those from less contaminated soils. This decrease in diversity may be due to toxicity, since significantly fewer Mycobacterium phylotypes were detected in soils determined to be toxic by the Microtox assay than in nontoxic soils. Sequencing and phylogenetic analysis of prominent TGGE bands indicated that novel strains dominated the soil Mycobacterium community. Mineralization studies using [(14)C]pyrene added to four petroleum-contaminated soils, with and without the addition of the known pyrene degrader Mycobacterium sp. strain RJGII-135, indicated that inoculation increased the level of degradation in three of the four soils. Mineralization results obtained from a sterilized soil inoculated with strain RJGII-135 suggested that competition with indigenous microorganisms may be a significant factor affecting biodegradation of PAHs. Pyrene-amended soils, with and without inoculation with strain RJGII-135, experienced both increases and decreases in the population sizes of the inoculated strain and indigenous Mycobacterium populations during incubation.