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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 incubations with [14C]-naphthalene (NAP), phenanthrene (PHE), anthracene (ANT), fluoranthene (FLU), pyrene (PYR), or benz[a]anthracene (BaA) by surface (0–4 cm) sediment cores 4571-2 (solid bar) and 4567-24 (open bar) during incubation at 21°C for 12 days, as compared to acid-killed controls. Bars are the averages and SD from triplicate incubations. *values for cumulative 14CO2 recovered were equal or below that of the respective control incubations.
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Figure 2: Cumulative 14CO2 recovered from incubations with [14C]-naphthalene (NAP), phenanthrene (PHE), anthracene (ANT), fluoranthene (FLU), pyrene (PYR), or benz[a]anthracene (BaA) by surface (0–4 cm) sediment cores 4571-2 (solid bar) and 4567-24 (open bar) during incubation at 21°C for 12 days, as compared to acid-killed controls. Bars are the averages and SD from triplicate incubations. *values for cumulative 14CO2 recovered were equal or below that of the respective control incubations.

Mentions: 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 (<0.5% mineralized of total hydrocarbon; data not shown). As shown in Figure 2, 14C incubations at 21°C using the 4571-2 sediment as inoculum revealed that significant levels of NAP had been mineralized (20.3 ± 1.1% cumulative 14CO2 captured of total initial 14C), whereas low mineralization levels (<3.5% cumulative 14CO2 captured of total initial 14C) were measured for FLU and BaA. PHE, ANT, and PYR were not mineralized by the bacterial community in the 4571-2 sediment sample. Conversely, all six of these PAHs were mineralized by the 4567-24 sediment sample at 21°C, with highest levels of cumulative 14CO2 captured from PHE (42.0 ± 7.0%) and NAP (28.8 ± 1.0%) of total initial 14C for each of these compounds. Whilst the oil-rich core 4571-2 was expected to have yielded higher mineralization levels than the quite oxidized 4567-24 sediment core, the converse which was measured may be attributed to the microbial community of core 4567-24 having been more amenable to aerobic conditions than the sulfide-adapted microbial inhabitants of core 4571-2. Cultured strains and uncultured clones of aromatic-degrading sulfate reducing bacteria, mainly belonging to the Desulfobacteraceae, have been described from Guaymas sediments (Phelps et al., 1998; Kniemeyer et al., 2003), and most likely play a significant role in the anaerobic oxidation and complete degradation of aromatic hydrocarbons at Guaymas. For SIP, achieving sufficient incorporation of the labeled carbon under short incubation times is desirable in order to minimize the potential for cross-feeding (see below). Hence, based on the significant mineralization of PHE measured with the 4567-24 sediment sample at 21°C, SIP experiments were subsequently conducted using this PAH, sediment sample and incubation temperature.


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 incubations with [14C]-naphthalene (NAP), phenanthrene (PHE), anthracene (ANT), fluoranthene (FLU), pyrene (PYR), or benz[a]anthracene (BaA) by surface (0–4 cm) sediment cores 4571-2 (solid bar) and 4567-24 (open bar) during incubation at 21°C for 12 days, as compared to acid-killed controls. Bars are the averages and SD from triplicate incubations. *values for cumulative 14CO2 recovered were equal or below that of the respective control incubations.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Cumulative 14CO2 recovered from incubations with [14C]-naphthalene (NAP), phenanthrene (PHE), anthracene (ANT), fluoranthene (FLU), pyrene (PYR), or benz[a]anthracene (BaA) by surface (0–4 cm) sediment cores 4571-2 (solid bar) and 4567-24 (open bar) during incubation at 21°C for 12 days, as compared to acid-killed controls. Bars are the averages and SD from triplicate incubations. *values for cumulative 14CO2 recovered were equal or below that of the respective control incubations.
Mentions: 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 (<0.5% mineralized of total hydrocarbon; data not shown). As shown in Figure 2, 14C incubations at 21°C using the 4571-2 sediment as inoculum revealed that significant levels of NAP had been mineralized (20.3 ± 1.1% cumulative 14CO2 captured of total initial 14C), whereas low mineralization levels (<3.5% cumulative 14CO2 captured of total initial 14C) were measured for FLU and BaA. PHE, ANT, and PYR were not mineralized by the bacterial community in the 4571-2 sediment sample. Conversely, all six of these PAHs were mineralized by the 4567-24 sediment sample at 21°C, with highest levels of cumulative 14CO2 captured from PHE (42.0 ± 7.0%) and NAP (28.8 ± 1.0%) of total initial 14C for each of these compounds. Whilst the oil-rich core 4571-2 was expected to have yielded higher mineralization levels than the quite oxidized 4567-24 sediment core, the converse which was measured may be attributed to the microbial community of core 4567-24 having been more amenable to aerobic conditions than the sulfide-adapted microbial inhabitants of core 4571-2. Cultured strains and uncultured clones of aromatic-degrading sulfate reducing bacteria, mainly belonging to the Desulfobacteraceae, have been described from Guaymas sediments (Phelps et al., 1998; Kniemeyer et al., 2003), and most likely play a significant role in the anaerobic oxidation and complete degradation of aromatic hydrocarbons at Guaymas. For SIP, achieving sufficient incorporation of the labeled carbon under short incubation times is desirable in order to minimize the potential for cross-feeding (see below). Hence, based on the significant mineralization of PHE measured with the 4567-24 sediment sample at 21°C, SIP experiments were subsequently conducted using this PAH, sediment sample and incubation temperature.

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