Limits...
Cultivation-dependent and cultivation-independent characterization of hydrocarbon-degrading bacteria in Guaymas Basin sediments.

Gutierrez T, Biddle JF, Teske A, Aitken MD - Front Microbiol (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Environmental Sciences and Engineering, Gillings School of Global Public Health, University of North Carolina at Chapel Hill, Chapel Hill, NC USA ; School of Life Sciences, Heriot-Watt University, Edinburgh UK.

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 [(13)C]-phenanthrene (PHE). The dominant sequences in clone libraries constructed from (13)C-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.

No MeSH data available.


Related in: MedlinePlus

Cumulative 14CO2 recovered from the incubations with 14C-labeled PHE (circles) that were run in parallel to the stable-isotope probing (SIP) incubations, and the respective removal of this polycyclic aromatic hydrocarbon (PAH) in incubations with the corresponding unlabeled substrates as measured by HPLC (squares). Each data point is the mean of results from triplicate flasks ± SD. Filled symbols represent live (uninhibited) cultures; open symbols represent acid-inhibited controls. Some error bars are smaller than the symbol.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4493657&req=5

Figure 3: Cumulative 14CO2 recovered from the incubations with 14C-labeled PHE (circles) that were run in parallel to the stable-isotope probing (SIP) incubations, and the respective removal of this polycyclic aromatic hydrocarbon (PAH) in incubations with the corresponding unlabeled substrates as measured by HPLC (squares). Each data point is the mean of results from triplicate flasks ± SD. Filled symbols represent live (uninhibited) cultures; open symbols represent acid-inhibited controls. Some error bars are smaller than the symbol.

Mentions: Careful attention must be employed in the design and execution of SIP in order for it to yield interpretable, unambiguous results. One of the main challenges in SIP is obtaining sufficient incorporation of the 13C into biomass, which in the case for DNA-SIP, its enrichment into DNA. Whilst the extent of labeling can be increased with longer incubation times, this can lead to the 13C becoming distributed among other members of the microbial community – i.e., those not necessarily directly capable of metabolizing the isotopically labeled substrate – by cross-feeding on 13C-labeled metabolic byproducts, intermediates, or dead cells (Lueders et al., 2004). To avert this, we had set up several 12C and 14C incubations that ran in parallel to the 13C incubations in order to tractably measure the degradation (by GC-MS) and mineralization (by scintillation counting) of the PHE to help guide our selection of the point at which to terminate the 13C incubations (endpoint of experiment) whereby sufficient 13C incorporation had been achieved with minimal cross-feeding. As shown in Figure 3, complete removal of the PHE occurred after day 9, whereas mineralization of the 14C substrate appeared to reach an asymptote by day 11. Based on these results, the endpoint selected for extraction of DNA from 13C incubations was 11 days. DNA extractions were performed on each of the duplicate 13C incubations for subsequent isopycnic ultracentrifugation to isolate the 13C-enriched ‘heavy’ DNA for analysis.


Cultivation-dependent and cultivation-independent characterization of hydrocarbon-degrading bacteria in Guaymas Basin sediments.

Gutierrez T, Biddle JF, Teske A, Aitken MD - Front Microbiol (2015)

Cumulative 14CO2 recovered from the incubations with 14C-labeled PHE (circles) that were run in parallel to the stable-isotope probing (SIP) incubations, and the respective removal of this polycyclic aromatic hydrocarbon (PAH) in incubations with the corresponding unlabeled substrates as measured by HPLC (squares). Each data point is the mean of results from triplicate flasks ± SD. Filled symbols represent live (uninhibited) cultures; open symbols represent acid-inhibited controls. Some error bars are smaller than the symbol.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4493657&req=5

Figure 3: Cumulative 14CO2 recovered from the incubations with 14C-labeled PHE (circles) that were run in parallel to the stable-isotope probing (SIP) incubations, and the respective removal of this polycyclic aromatic hydrocarbon (PAH) in incubations with the corresponding unlabeled substrates as measured by HPLC (squares). Each data point is the mean of results from triplicate flasks ± SD. Filled symbols represent live (uninhibited) cultures; open symbols represent acid-inhibited controls. Some error bars are smaller than the symbol.
Mentions: Careful attention must be employed in the design and execution of SIP in order for it to yield interpretable, unambiguous results. One of the main challenges in SIP is obtaining sufficient incorporation of the 13C into biomass, which in the case for DNA-SIP, its enrichment into DNA. Whilst the extent of labeling can be increased with longer incubation times, this can lead to the 13C becoming distributed among other members of the microbial community – i.e., those not necessarily directly capable of metabolizing the isotopically labeled substrate – by cross-feeding on 13C-labeled metabolic byproducts, intermediates, or dead cells (Lueders et al., 2004). To avert this, we had set up several 12C and 14C incubations that ran in parallel to the 13C incubations in order to tractably measure the degradation (by GC-MS) and mineralization (by scintillation counting) of the PHE to help guide our selection of the point at which to terminate the 13C incubations (endpoint of experiment) whereby sufficient 13C incorporation had been achieved with minimal cross-feeding. As shown in Figure 3, complete removal of the PHE occurred after day 9, whereas mineralization of the 14C substrate appeared to reach an asymptote by day 11. Based on these results, the endpoint selected for extraction of DNA from 13C incubations was 11 days. DNA extractions were performed on each of the duplicate 13C incubations for subsequent isopycnic ultracentrifugation to isolate the 13C-enriched ‘heavy’ DNA for analysis.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Environmental Sciences and Engineering, Gillings School of Global Public Health, University of North Carolina at Chapel Hill, Chapel Hill, NC USA ; School of Life Sciences, Heriot-Watt University, Edinburgh UK.

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 [(13)C]-phenanthrene (PHE). The dominant sequences in clone libraries constructed from (13)C-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.

No MeSH data available.


Related in: MedlinePlus