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Isolation of adherent polycyclic aromatic hydrocarbon (PAH)-degrading bacteria using PAH-sorbing carriers.

ABSTRACT: 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.

SUBMITTER: Bastiaens L

PROVIDER: S-EPMC101420 | biostudies-literature | 2000 May

REPOSITORIES: biostudies-literature

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Isolation of adherent polycyclic aromatic hydrocarbon (PAH)-degrading bacteria using PAH-sorbing carriers.

Bastiaens L L Springael D D Wattiau P P Harms H H deWachter R R Verachtert H H Diels L L

Applied and environmental microbiology 20000501 5

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 a ...[more]

PMID: 10788347

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Project description:The characterization of native polycyclic aromatic hydrocarbon (PAH)-degrading bacteria is significant for understanding the PAH degradation process in the natural environment and developing effective remediation technologies. Most previous investigations of PAH-degrading bacteria in environmental samples employ pahAc, which encodes the ?-subunit of PAH ring-hydroxylating dioxygenase, as a functional marker gene. However, the poor phylogenetic resolution and nonspecificity of pahAc result in a misestimation of PAH-degrading bacteria. Here, we propose a PAH hydratase-aldolase-encoding gene, pahE, as a superior biomarker for PAH-degrading bacteria. Comparative phylogenetic analysis of the key enzymes involved in the upper pathway of PAH degradation indicated that pahE evolved dependently from a common ancestor. A phylogenetic tree constructed based on PahE is largely congruent with PahAc-based phylogenies, except for the dispersion of several clades of other non-PAH-degrading aromatic hydrocarbon dioxygenases present in the PahAc tree. Analysis of pure strains by PCR confirmed that pahE can specifically distinguish PAH-degrading bacteria, while pahAc cannot. Illumina sequencing of pahE and pahAc amplicons showed more genotypes and higher specificity and resolution for pahE Novel reads were also discovered among the pahE amplicons, suggesting the presence of novel PAH-degrading populations. These results suggest that pahE is a more powerful biomarker for exploring the ecological role and degradation potential of PAH-degrading bacteria in ecosystems, which is significant to the bioremediation of PAH pollution and environmental microbial ecology.IMPORTANCE PAH contamination has become a worldwide environmental issue because of the potential toxic effects on natural ecosystems and human health. Biotransformation and biodegradation are considered the main natural elimination forms of PAHs from contaminated sites. Therefore, the knowledge of the degradation potential of the microbial community in contaminated sites is crucial for PAH pollution bioremediation. However, the nonspecificity of pahAc as a functional marker of PAH-degrading bacteria has resulted neither in a reliable prediction of PAH degradation potential nor an accurate assessment of degradation. Here, we introduced pahE encoding the PAH hydratase-aldolase as a new and better functional marker gene of PAH-degrading bacteria. This study provides a powerful molecular tool to more effectively explore the ecological role and degradation potential of PAH-degrading bacteria in ecosystems, which is significant to the bioremediation of PAH pollution.

Project description:Polycyclic aromatic hydrocarbon (PAH)-degrading bacteria were isolated from contaminated estuarine sediment and salt marsh rhizosphere by enrichment using either naphthalene, phenanthrene, or biphenyl as the sole source of carbon and energy. Pasteurization of samples prior to enrichment resulted in isolation of gram-positive, spore-forming bacteria. The isolates were characterized using a variety of phenotypic, morphologic, and molecular properties. Identification of the isolates based on their fatty acid profiles and partial 16S rRNA gene sequences assigned them to three main bacterial groups: gram-negative pseudomonads; gram-positive, non-spore-forming nocardioforms; and the gram-positive, spore-forming group, Paenibacillus. Genomic digest patterns of all isolates were used to determine unique isolates, and representatives from each bacterial group were chosen for further investigation. Southern hybridization was performed using genes for PAH degradation from Pseudomonas putida NCIB 9816-4, Comamonas testosteroni GZ42, Sphingomonas yanoikuyae B1, and Mycobacterium sp. strain PY01. None of the isolates from the three groups showed homology to the B1 genes, only two nocardioform isolates showed homology to the PY01 genes, and only members of the pseudomonad group showed homology to the NCIB 9816-4 or GZ42 probes. The Paenibacillus isolates showed no homology to any of the tested gene probes, indicating the possibility of novel genes for PAH degradation. Pure culture substrate utilization experiments using several selected isolates from each of the three groups showed that the phenanthrene-enriched isolates are able to utilize a greater number of PAHs than are the naphthalene-enriched isolates. Inoculating two of the gram-positive isolates to a marine sediment slurry spiked with a mixture of PAHs (naphthalene, fluorene, phenanthrene, and pyrene) and biphenyl resulted in rapid transformation of pyrene, in addition to the two- and three-ringed PAHs and biphenyl. This study indicates that the rhizosphere of salt marsh plants contains a diverse population of PAH-degrading bacteria, and the use of plant-associated microorganisms has the potential for bioremediation of contaminated sediments.

Project description:Bacteria play an important role in the removal of polycyclic aromatic hydrocarbons (PAHs) from polluted environments. In marine environments, Cycloclasticus is one of the most prevalent PAH-degrading bacterial genera. However, little is known regarding the degradation mechanisms for multiple PAHs by CycloclasticusCycloclasticus sp. strain P1 was isolated from deep-sea sediments and is known to degrade naphthalene, phenanthrene, pyrene, and other aromatic hydrocarbons. Here, six ring-hydroxylating dioxygenases (RHDs) were identified in the complete genome of Cycloclasticus sp. P1 and were confirmed to be involved in PAH degradation by enzymatic assays. Further, five gene clusters in its genome were identified to be responsible for PAH degradation. Degradation pathways for naphthalene, phenanthrene, and pyrene were elucidated in Cycloclasticus sp. P1 based on genomic and transcriptomic analysis and characterization of an interconnected metabolic network. The metabolic pathway overlaps in many steps in the degradation of pyrene, phenanthrene, and naphthalene, which were validated by the detection of metabolic intermediates in cultures. This study describes a pyrene degradation pathway for Cycloclasticus. Moreover, the study represents the integration of a PAH metabolic network that comprises pyrene, phenanthrene, and naphthalene degradation pathways. Taken together, these results provide a comprehensive investigation of PAH metabolism in CycloclasticusIMPORTANCE PAHs are ubiquitous in the environment and are carcinogenic compounds and tend to accumulate in food chains due to their low bioavailability and poor biodegradability. Cycloclasticus is an obligate marine PAH degrader and is widespread in marine environments, while the PAH degradation pathways remain unclear. In this report, the degradation pathways for naphthalene, phenanthrene, and pyrene were revealed, and an integrated PAH metabolic network covering pyrene, phenanthrene, and naphthalene was constructed in Cycloclasticus This overlapping network provides streamlined processing of PAHs to intermediates and ultimately to complete mineralization. Furthermore, these results provide an additional context for the prevalence of Cycloclasticus in oil-polluted marine environments and pelagic settings. In conclusion, these analyses provide a useful framework for understanding the cellular processes involved in PAH metabolism in an ecologically important marine bacterium.

Dataset Information

Isolation of adherent polycyclic aromatic hydrocarbon (PAH)-degrading bacteria using PAH-sorbing carriers.

Publications

Isolation of adherent polycyclic aromatic hydrocarbon (PAH)-degrading bacteria using PAH-sorbing carriers.

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