Identifying polycyclic aromatic hydrocarbon-degrading bacteria in oil-contaminated surface waters at Deepwater Horizon by cultivation, stable isotope probing and pyrosequencing

2011 ◽  
Vol 10 (4) ◽  
pp. 301-305 ◽  
Author(s):  
Tony Gutierrez
Keyword(s):  
Stable Isotope ◽  
Polycyclic Aromatic Hydrocarbon ◽  
Aromatic Hydrocarbon ◽  
Surface Waters ◽  
Deepwater Horizon ◽  
Stable Isotope Probing ◽  
Degrading Bacteria ◽  
Polycyclic Aromatic

scholarly journals Stable Isotope Probing of an Algal Bloom To Identify Uncultivated Members of the Rhodobacteraceae Associated with Low-Molecular-Weight Polycyclic Aromatic Hydrocarbon Degradation

2011 ◽  
Vol 77 (21) ◽  
pp. 7856-7860 ◽  
Author(s):  
Tony Gutierrez ◽  
David R. Singleton ◽  
Michael D. Aitken ◽  
Kirk T. Semple
Keyword(s):  
Stable Isotope ◽  
Polycyclic Aromatic Hydrocarbon ◽  
Aromatic Hydrocarbon ◽  
Algal Bloom ◽  
Stable Isotope Probing ◽  
Gene Copy ◽  
Rrna Gene ◽  
Content Type ◽  
Degrading Bacteria ◽  
Polycyclic Aromatic

ABSTRACTPolycyclic aromatic hydrocarbon (PAH)-degrading bacteria associated with an algal bloom in Tampa Bay, FL, were investigated by stable isotope probing (SIP) with uniformly labeled [13C]naphthalene. The dominant sequences in clone libraries constructed from13C-enriched bacterial DNA (from naphthalene enrichments) were identified as uncharacterized members of the familyRhodobacteraceae. Quantitative PCR primers targeting the 16S rRNA gene of these uncultivated organisms were used to determine their abundance in incubations amended with unlabeled naphthalene and phenanthrene, both of which showed substantial increases in gene copy numbers during the experiments. As demonstrated by this work, the application of uniformly13C-labeled PAHs in SIP experiments can successfully be used to identify novel PAH-degrading bacteria in marine waters.

scholarly journals Heterologous Expression of Polycyclic Aromatic Hydrocarbon Ring-Hydroxylating Dioxygenase Genes from a Novel Pyrene-Degrading Betaproteobacterium

2012 ◽  
Vol 78 (10) ◽  
pp. 3552-3559 ◽  
Author(s):  
David R. Singleton ◽  
Jing Hu ◽  
Michael D. Aitken
Keyword(s):  
Polycyclic Aromatic Hydrocarbon ◽  
Aromatic Hydrocarbon ◽  
Enrichment Culture ◽  
Metagenomic Library ◽  
Stable Isotope Probing ◽  
Content Type ◽  
Close Proximity ◽  
Polycyclic Aromatic ◽  
Dominant Member ◽  
Pah Metabolism

ABSTRACTA betaproteobacterium within the familyRhodocyclaceaepreviously identified as a pyrene degrader via stable-isotope probing (SIP) of contaminated soil (designated pyrene group 1 or PG1) was cultivated as the dominant member of a mixed bacterial culture. A metagenomic library was constructed, and the largest contigs were analyzed for genes associated with polycyclic aromatic hydrocarbon (PAH) metabolism. Eight pairs of genes with similarity to the α- and β-subunits of ring-hydroxylating dioxygenases (RHDs) associated with aerobic bacterial PAH degradation were identified and linked to PG1 through PCR analyses of a simplified enrichment culture. In tandem with a ferredoxin and reductase found in close proximity to one pair of RHD genes, six of the RHDs were cloned and expressed inEscherichia coli. Each cloned RHD was tested for activity against nine PAHs ranging in size from two to five rings. Despite differences in their predicted protein sequences, each of the six RHDs was capable of transforming phenanthrene and pyrene. Three RHDs could additionally transform naphthalene and fluorene, and these genotypes were also associated with the ability of theE. coliconstructs to convert indole to indigo. Only one of the six cloned RHDs was capable of transforming anthracene and benz[a]anthracene. None of the tested RHDs were capable of significantly transforming fluoranthene, chrysene, or benzo[a]pyrene.

Selective enumeration of aromatic and aliphatic hydrocarbon degrading bacteria by a most-probable-number procedure

1996 ◽  
Vol 42 (3) ◽  
pp. 252-258 ◽  
Author(s):  
Brian A. Wrenn ◽  
Albert D. Venosa
Keyword(s):  
Polycyclic Aromatic Hydrocarbon ◽  
Aromatic Hydrocarbon ◽  
Oxidation Products ◽  
Most Probable Number ◽  
Probable Number ◽  
Microtiter Plates ◽  
Degrading Bacteria ◽  
Polycyclic Aromatic ◽  
Pure Cultures ◽  
Plate Counts

A most-probable-number (MPN) procedure was developed to separately enumerate aliphatic and aromatic hydrocarbon degrading bacteria, because most of the currently available methods are unable to distinguish between these two groups. Separate 96-well microtiter plates are used to estimate the sizes of these two populations. The alkane-degrader MPN method uses hexadecane as the selective growth substrate and positive wells are detected by reduction of iodonitrotetrazolium violet, which is added after incubation for 2 weeks at 20 °C. Polycyclic aromatic hydrocarbon degraders are grown on a mixture of phenanthrene, anthracene, fluorene, and dibenzothiophene in a second plate. Positive wells turn yellow to greenish-brown from accumulation of the partial oxidation products of the aromatic substrates and they can be scored after a 3-week incubation period. These MPN procedures are accurate and selective. For pure cultures, heterotrophic plate counts on a nonselective medium and the appropriate MPN procedure provide similar estimates of the population density. Bacteria that cannot grow on the selective substrates do not produce false positive responses even when the inoculum density is very high. Thus, this method, which is simple enough for use in the field, provides reliable estimates for the density and composition of hydrocarbon-degrading microbial populations.Key words: most probable number, polycyclic aromatic hydrocarbon, alkane, hydrocarbon, bacteria.

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