Abstract
Marine hydrocarbon-degrading bacteria perform a fundamental role in the biodegradation of crude oil and its petrochemical derivatives in coastal and open ocean environments. However, there is a paucity of knowledge on the diversity and function of these organisms in deep-sea sediment. Here we used stable-isotope probing (SIP), a valuable tool to link the phylogeny and function of targeted microbial groups, to investigate polycyclic aromatic hydrocarbon (PAH)-degrading bacteria under aerobic conditions in sediments from Guaymas Basin with uniformly labeled [13C]-phenanthrene (PHE). The dominant sequences in clone libraries constructed from 13C-enriched bacterial DNA (from PHE enrichments) were identified to belong to the genus Cycloclasticus. We used quantitative PCR primers targeting the 16S rRNA gene of the SIP-identified Cycloclasticus to determine their abundance in sediment incubations amended with unlabeled PHE and showed substantial increases in gene abundance during the experiments. We also isolated a strain, BG-2, representing the SIP-identified Cycloclasticus sequence (99.9% 16S rRNA gene sequence identity), and used this strain to provide direct evidence of PHE degradation and mineralization. In addition, we isolated Halomonas, Thalassospira, and Lutibacterium sp. with demonstrable PHE-degrading capacity from Guaymas Basin sediment. This study demonstrates the value of coupling SIP with cultivation methods to identify and expand on the known diversity of PAH-degrading bacteria in the deep-sea.
Highlights
In deep-sea naturally oil-laden marine sediments, such as cold seeps, hydrocarbon-degrading microorganisms contribute importantly to the diagenesis, and biological transformation of hydrocarbons
Exposure of Sediment Samples to Labeled and Unlabeled polycyclic aromatic hydrocarbon (PAH) We determined the potential of the bacterial community in the two surface sediment core samples (4571-2 and 4567-24) to mineralize various 14C-labeled PAHs (NAP, PHE, ANT, FLU, PYR, or BaA), since these hydrocarbons have been shown to be present in oily surficial sediment samples at Guaymas (Bazylinski et al, 1988)
This was important to thereby inform our choice of the hydrocarbon(s) that would be most suitable for obtaining sufficient incorporation of the 13C into biomass, including DNA, since mineralization of a substrate can be suggestive of growth on that substrate. 14C-hydrocarbon incubations conducted at 4◦C with each of the six hydrocarbons and the two sediment samples yielded very low levels of mineralization (
Summary
In deep-sea naturally oil-laden marine sediments, such as cold seeps, hydrocarbon-degrading microorganisms contribute importantly to the diagenesis, and biological transformation of hydrocarbons. DNA-SIP and cultivation-based methods were used to identify PAH-degrading bacteria in the surficial sediment environment of Guaymas at ∼2000 m depth below the sea surface in order to expand current knowledge on the diversity of hydrocarbon-degrading microbial communities in Guaymas Basin oil-rich sediments.
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