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Microbial degradation of petroleum hydrocarbons is a crucial process for the clean-up of oil-contaminated environments. Cycloclasticus spp. are well-known polycyclic aromatic hydrocarbon (PAH) degraders that possess PAH-degradation marker genes including rhd3α, rhd2α, and pahE. However, it remains unknown if the expression of these genes can serve as an indicator for active PAH degradation. Here, we determined transcript-to-gene (TtG) ratios with (reverse transcription) qPCR in cultures of Cycloclasticus pugetii strain PS-1 grown with naphthalene, phenanthrene, a mixture of these PAHs, or alternate substrates (i.e., no PAHs). Mean TtG ratios of 1.99 × 10-2, 1.80 × 10-3, and 3.20 × 10-3 for rhd3α, rhd2α, and pahE, respectively, were measured in the presence or absence of PAHs. The TtG values suggested that marker-gene expression is independent of PAH degradation. Measurement of TtG ratios in Arctic seawater microcosms amended with water-accommodated crude oil fractions, and incubated under in situ temperature conditions (i.e., 1.5°C), only detected Cycloclasticus spp. rhd2α genes and transcripts (mean TtG ratio of 4.15 × 10-1). The other marker genes - rhd3α and pahE - were not detected, suggesting that not all Cycloclasticus spp. carry these genes and a broader yet-to-be-identified repertoire of PAH-degradation genes exists. The results indicate that the expression of PAH marker genes may not correlate with PAH-degradation activity, and transcription data should be interpreted cautiously., Deutsche Forschungsgemeinschaft

The application of chemical dispersants during marine oil spills can affect the community composition and activity of marine microorganisms. Several studies have indicated that certain marine hydrocarbon-degrading bacteria, such as Marinobacter spp., can be inhibited by chemical dispersants, resulting in lower abundances and/or reduced biodegradation rates. However, a major knowledge gap exists regarding the mechanisms underlying these physiological effects. Here, we performed comparative proteomics of the Deepwater Horizon isolate Marinobacter sp. TT1 grown under different conditions. Strain TT1 received different carbon sources (pyruvate vs. n‑hexadecane) with and without added dispersant (Corexit EC9500A). Additional treatments contained crude oil in the form of a water-accommodated fraction (WAF) or chemically-enhanced WAF (CEWAF, with Corexit). For the first time, we identified the proteins associated with alkane metabolism and alginate biosynthesis in strain TT1, report on its potential for aromatic hydrocarbon biodegradation and present a protein-based proposed metabolism of Corexit components as carbon substrates. Our findings revealed that Corexit exposure affects hydrocarbon metabolism, chemotactic motility, biofilm formation, and induces solvent tolerance mechanisms, like efflux pumps, in strain TT1. This study provides novel insights into dispersant impacts on microbial hydrocarbon degraders that should be taken into consideration for future oil spill response actions.

Additional file 4: Supplementary Figure S2. Summary of significant predictive parameters (i.e., treatments and incubation days) determined by regression analysis based on unsupervised machine learning for two alpha diversity measures (Richness and Shannon index), NTI, NRI, and Local contribution to beta diversity (LCBD). The parameters shown here are from the optimal model for each metric, where blue represent negatively and red – positively influencing variables, respectively. FSC is the in-situ baseline microbial community, WAF - seawater and oil only, BEWAF – seawater, crude oil and biosurfactant, CEWAF – seawater, crude oil and synthetic dispersant, SW - seawater only, SWBS - seawater and biosurfactant, and SWD – seawater and synthetic dispersant.

Additional file 6: Supplementary Figure S3. Differential heat trees showing the key (significant) differential taxa (DESeq2; Wilcoxon p-value test adjusted with multiple comparison) in seawater-only control treatment (SW). The top 3 subsets with the highest correlation with the full ASV table considering Bray-Curtis distance (PERMANOVA) are listed for each treatment. The grey tree is the taxonomy key for the smaller unlabelled coloured trees. The colour of each taxon represents the log-10 ratio of median proportions of reads observed in each treatment. The size of tree nodes shows the number of ASVs (here labelled as OTUs) present in each sample.

Additional file 8: Supplementary Figure S5. Heatmap showing the scaled log abundance (color key on top left) of aliphatic and aromatic degradation, and biosurfactant synthesis pathways. Pathways are shown along the y-axis and BEWAF (seawater, crude oil, and biosurfactant), CEWAF (seawater, crude oil, and synthetic dispersant), and WAF (seawater and crude oil only) samples along the x-axis. The two color-coded bars on top of the heatmap indicate their treatments and incubation days status. Hierarchical clustering of the samples (top) is based on the correlation between samples’ predicted gene expression.

Additional file 9: Supplementary Figure S6. Differences in aliphatic and polycyclic aromatic hydrocarbon biomarker ratios of three different treatments amended with crude oil: BEWAF (seawater, crude oil, and biosurfactant), CEWAF (seawater, crude oil, and synthetic dispersant), and WAF (seawater and crude oil only), over time in days (grey boxes): nC17/pristane (nC17/pri), nC18/phytane (nC18/phy), Phenanthrene/9-methylphenanthrene (P/9MP), (3+2)-methylphenanthrene/(9+1)-mehylphenanthrene (3+2MP/9+1MP), and 3-methylphenanthrene/9-methylphenanthrene (3MP/9MP). Values are the mean of three independent replicates (except for BEWAF day 0 (two replicates) and CEWAF day 28 (one replicate)) +/- standard deviation.

Additional file 1: Supplementary methods. Supplementary Methods and Materials used to produce the main results in this study.

Additional file 3: Supplementary Figure S1. Local contribution to beta diversity (LCBD) for Bray-Curits, Unweighted and Weighted UniFrac distance matrices. Colours represent incubation time: red – in situ FSC at time of sampling, olive green – day 0, green – day 3, blue – day 7, pink – day 14 and brown – day 28. FSC is the in-situ baseline microbial community, WAF - seawater and oil only, BEWAF – seawater, crude oil and biosurfactant, CEWAF – seawater, crude oil and synthetic dispersant, SW - seawater only, SWBS - seawater and biosurfactant, and SWD – seawater and synthetic dispersant. Statistically different treatments (pair-wise ANOVA) are annotated with bracket and the level of significance is shown above the bracket with a star sign: * is p < 0.05, ** is p < 0.01, and *** is p < 0.001.

Additional file 7: Supplementary Figure S4. (A) Predicted functional alpha diversity of microbial pathways (expressed as number of KEGG orthologs). Statistically different treatments (pair-wise ANOVA) are connected by bracket and the level of significance is shown with: *(p < 0.05), ** (p < 0.01), or *** (p < 0.001). (B) Principal coordinate analysis (PCoA) on beta diversity measured with Bray-Curtis dissimilarity distance matrix. In both (A) and (B) treatments are represented by shape (shown on graph) and incubation time by colour: red – baseline microbial community at time of seawater sampling, olive green – day 0, green – day 3, blue – day 7, pink – day 14, and brown – day 28. FSC is the in-situ baseline microbial community, WAF - seawater and oil only, BEWAF – seawater, crude oil and biosurfactant, CEWAF – seawater, crude oil and synthetic dispersant, SW - seawater only, SWBS - seawater and biosurfactant, and SWD – seawater and synthetic dispersant.

Additional file 1: Fig. S1. Effect of various sea salt concentrations, pH, temperature and nitrogen source on the growth of Marinobacter sp. MCTG107b over a period of 96 h. Growth curves show the OD600 of strain MCTG107b during growth at different (A) salinities (5.0 to 40 g L−1), (B) pH values (4.0 to 8.5), or (C) temperatures (25 °C, 28 °C, 30 °C, 37 °C). Fig. S2. HPLC–MS chromatogram for rhamnolipids produced by Marinobacter sp. MCTG107b. The MS was operated in the negative mode. Main intensities in the chromatogram were Rha-Rha-C10-C10 and Rha-Rha-C10-C10-CH3. Table S1. Full 22 factorial design with pH and salt as independent variables using surface tension and biosurfactant yield as response variables. Surface tension and biosurfactant yield according to full factorial design after 96 h of shake-flask study of Marinobacter sp. MCTG107b. Table S2. Analysis of variance (ANOVA) for response variables surface tension and biosurfactant yield by Marinobacter sp. MCTG107b.

As oil reserves in established basins become depleted, exploration and production moves towards relatively unexploited areas, such as deep waters off the continental shelf. The Faroe-Shetland Channel (FSC, NE Atlantic) and adjacent areas have been subject to increased focus by the oil industry. In addition to extreme depths, metocean conditions in this region characterise an environment with high waves and strong winds, strong currents, complex circulation patterns, sharp density gradients, and large small- and mesoscale variability. These conditions pose operational challenges to oil spill response and question the suitability of current oil spill modelling frameworks (oil spill models and their forcing data) to adequately simulate the behaviour of a potential oil spill in the area. This article reviews the state of knowledge relevant to deepwater oil spill modelling for the FSC area and identifies knowledge gaps and research priorities. Our analysis should be relevant to other areas of complex oceanography.

In this study we report the formation of marine oil snow (MOS), its associated microbial community, the factors influencing its formation, and the microbial response to crude oil in surface waters of the Faroe-Shetland Channel (FSC). The FSC is a subarctic region that is hydrodynamically complex located in the northeast Atlantic where oil extraction is currently occurring and where exploration is likely to expand into its deeper waters (>500 m). A major oil spill in this region may mirror the aftermath that ensued following the Deepwater Horizon (DWH) blowout in the Gulf of Mexico, where the massive influx of Macondo crude oil triggered the formation of copious quantities of rapidly sinking MOS and successional blooms of opportunistic oil-degrading bacteria. In laboratory experiments, we simulated environmental conditions in sea surface waters of the FSC using water collected from this site during the winter of 2015. We demonstrated that the presence of dispersant triggers the formation of MOS, and that nutrient amendments magnify this. Illumina MiSeq sequencing revealed the enrichment on MOS of associated oil-degrading (Cycloclasticus, Thalassolituus, Marinobacter) and EPS-producing (Halomonas, Pseudoalteromonas, Alteromonas) bacteria, and included major representation by Psychrobacter and Cobetia with putative oil-degrading/EPS-producing qualities. The formation of marine snow, in the absence of crude oil and dispersant, in seawater amended with nutrients alone indicated that the de novo synthesis of bacterial EPS is a key factor in MOS formation, and the glycoprotein composition of the MOS aggregates confirmed that its amorphous biopolymeric matrix was of microbial (likely bacterial) origin. The presence of dispersants and crude oil with/without nutrients resulted in distinct microbial responses marked by intermittent, and in some cases short-lived, blooms of opportunistic heterotrophs, principally obligate hydrocarbonoclastic (Alcanivorax, Cycloclasticus, Thalassolituus, Marinobacter) and EPS-producing (Halomonas, Alteromonas, Pseudoalteromonas) bacteria. Interestingly, members of the Vibrionales (principally the genus Vibrio) were strongly enriched by crude oil (with/without dispersant or nutrients), highlighting a putative importance for these organisms in crude oil biodegradation in the FSC. Our findings mirror those observed at DWH and hence underscore their broad relevance.

Emerging evidence shows that hydrocarbonoclastic bacteria (HCB) may be commonly found associated with phytoplankton in the ocean, but the ecology of these bacteria and how they respond to crude oil remains poorly understood. Here, we used a natural diatom-bacterial assemblage to investigate the diversity and response of HCB associated with a cosmopolitan marine diatom, S. costatum, to crude oil. Pyrosequencing analysis revealed a dramatic transition in the diatom-associated bacterial community, defined initially by a short-lived bloom of Methylophaga (putative oil-degraders) that was subsequently succeeded by a distinct groups of HCB (Marinobacter, Polycyclovorans, Arenibacter, Parvibaculum, Roseobacter clade), including putative novel phyla, as well as other groups with previously unqualified oil-degrading potential. Interestingly, these oil-enriched organisms contributed to the apparent and exclusive biodegradation of substituted and non-substituted polycyclic aromatic hydrocarbons (PAHs), thereby suggesting that the HCB community associated with the diatom is tuned to specializing in the degradation of PAHs. Furthermore, the formation of marine oil snow (MOS) in oil-amended incubations was consistent with its formation during the Deepwater Horizon oil spill. This work highlights the phycosphere of phytoplankton as an underexplored biotope in the ocean where HCB may contribute importantly to the biodegradation of hydrocarbon contaminants in marine surface waters.

The role of methylotrophic bacteria in the fate of the oil and gas released into the Gulf of Mexico during the Deepwater Horizon oil spill has been controversial, particularly in relation to whether organisms such as Methylophaga had contributed to the consumption of methane. Whereas methanotrophy remains unqualified in these organisms, recent work by our group using DNA-based stable-isotope probing coupled with cultivation-based methods has uncovered hydrocarbon-degrading Methylophaga. Recent findings have also shown that methylotrophs, including Methylophaga, were in a heightened state of metabolic activity within oil plume waters during the active phase of the spill. Taken collectively, these findings suggest that members of this group may have participated in the degradation of high-molecular-weight hydrocarbons in plume waters. The discovery of hydrocarbon-degrading Methylophaga also highlights the importance of considering these organisms in playing a role to the fate of oil hydrocarbons at oil-impacted sites.

A marine bacterium, designated strain MCTG13d, was isolated from a laboratory culture of the dinoflagellate Lingulodinium polyedrum CCAP1121/2 by enrichment with polycyclic aromatic hydrocarbons (PAHs) as the sole carbon source. Based on 16S rRNA gene sequence comparisons, the strain was most closely related to Porticoccus litoralis IMCC2115 T (96.5%) and to members of the genera Microbulbifer (91.4 to 93.7%) and Marinimicrobium (90.4 to 92.0%). Phylogenetic trees showed that the strain clustered in a distinct phyletic line in the class Gammaproteobacteria for which P. litoralis is presently the sole cultured representative. The strain was strictly aerobic, rod shaped, Gram negative, and halophilic. Notably, it was able to utilize hydrocarbons as sole sources of carbon and energy, whereas sugars did not serve as growth substrates. The predominant isoprenoid quinone of strain MCTG13d was Q-8, and the dominant fatty acids were C 16:1ω7c , C 18:1ω7c , and C 16:0 . DNA G+C content for the isolate was 54.9 ± 0.42 mol%. Quantitative PCR primers targeting the 16S rRNA gene of this strain showed that this organism was common in other laboratory cultures of marine phytoplankton. On the basis of phenotypic and genotypic characteristics, strain MCTG13d represents a novel species of Porticoccus , for which the name Porticoccus hydrocarbonoclasticus sp. nov. is proposed. The discovery of this highly specialized hydrocarbon-degrading bacterium living in association with marine phytoplankton suggests that phytoplankton represent a previously unrecognized biotope of novel bacterial taxa that degrade hydrocarbons in the ocean.

The massive influx of crude oil into the Gulf of Mexico during the Deepwater Horizon (DWH) disaster triggered dramatic microbial community shifts in surface oil slick and deep plume waters. Previous work had shown several taxa, notably DWH Oceanospirillales, Cycloclasticus and Colwellia, were found to be enriched in these waters based on their dominance in conventional clone and pyrosequencing libraries and were thought to have had a significant role in the degradation of the oil. However, this type of community analysis data failed to provide direct evidence on the functional properties, such as hydrocarbon degradation of organisms. Using DNA-based stable-isotope probing with uniformly 13C-labelled hydrocarbons, we identified several aliphatic (Alcanivorax, Marinobacter)- and polycyclic aromatic hydrocarbon (Alteromonas, Cycloclasticus, Colwellia)-degrading bacteria. We also isolated several strains (Alcanivorax, Alteromonas, Cycloclasticus, Halomonas, Marinobacter and Pseudoalteromonas) with demonstrable hydrocarbon-degrading qualities from surface slick and plume water samples collected during the active phase of the spill. Some of these organisms accounted for the majority of sequence reads representing their respective taxa in a pyrosequencing data set constructed from the same and additional water column samples. Hitherto, Alcanivorax was not identified in any of the previous water column studies analysing the microbial response to the spill and we discuss its failure to respond to the oil. Collectively, our data provide unequivocal evidence on the hydrocarbon-degrading qualities for some of the dominant taxa enriched in surface and plume waters during the DWH oil spill, and a more complete understanding of their role in the fate of the oil.

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.

Marine hydrocarbon-degrading bacteria perform a fundamental role in the oxidation and ultimate removal of crude oil and its petrochemical derivatives in coastal and open ocean environments. Those with an almost exclusive ability to utilize hydrocarbons as a sole carbon and energy source have been found confined to just a few genera. Here we used stable isotope probing (SIP), a valuable tool to link the phylogeny and function of targeted microbial groups, to investigate hydrocarbon-degrading bacteria in coastal North Carolina sea water (Beaufort Inlet, USA) with uniformly labeled [13C]n-hexadecane. The dominant sequences in clone libraries constructed from 13C-enriched bacterial DNA (from n-hexadecane enrichments) were identified to belong to the genus Alcanivorax, with ≤98% sequence identity to the closest type strain—thus representing a putative novel phylogenetic taxon within this genus. Unexpectedly, we also identified 13C-enriched sequences in heavy DNA fractions that were affiliated to the genus Methylophaga. This is a contentious group since, though some of its members have been proposed to degrade hydrocarbons, substantive evidence has not previously confirmed this. We used quantitative PCR primers targeting the 16S rRNA gene of the SIP-identified Alcanivorax and Methylophaga to determine their abundance in incubations amended with unlabeled n-hexadecane. Both showed substantial increases in gene copy number during the experiments. Subsequently, we isolated a strain representing the SIP-identified Methylophaga sequences (99.9% 16S rRNA gene sequence identity) and used it to show, for the first time, direct evidence of hydrocarbon degradation by a cultured Methylophaga sp. This study demonstrates the value of coupling SIP with cultivation methods to identify and expand on the known diversity of hydrocarbon-degrading bacteria in the marine environment.