Toxicity Profiling of Biosurfactants Produced by Novel Marine Bacterial Strains.

Surface active agents (SAAs), currently used in modern industry, are synthetic chemicals produced from non-renewable sources, with potential toxic impacts on humans and the environment. Thus, there is an increased interest for the identification and utilization of natural derived SAAs. As such, the marine environment is considered a promising source of biosurfactants with low toxicity, environmental compatibility, and biodegradation compared to their synthetic counterparts. MARISURF is a Horizon 2020 EU-funded project aiming to identify and functionally characterize SAAs, derived from a unique marine bacterial collection, towards commercial exploitation. Specifically, rhamnolipids produced by Marinobacter MCTG107b and Pseudomonas MCTG214(3b1) strains were previously identified and characterized while currently their toxicity profile was assessed by utilizing well-established methodologies. Our results showed a lack of cytotoxicity in in vitro models of human skin and liver as indicated by alamar blue and propidium iodide assays. Additionally, the use of the single gel electrophoresis assay, under oxidative stress conditions, revealed absence of any significant mutagenic/anti-mutagenic potential. Finally, both 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonicacid) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) cell-free assays, revealed no significant anti-oxidant capacity for neither of the tested compounds. Consequently, the absence of significant cytotoxicity and/or mutagenicity justifies their commercial exploitation and potential development into industrial end-user applications as natural and environmentally friendly biosurfactants.

Production and characterisation of a marine Halomonas surface-active exopolymer.

During screening for novel emulsifiers and surfactants, a marine gammaproteobacterium, Halomonas sp. MCTG39a, was isolated and selected for its production of an extracellular emulsifying agent, P39a. This polymer was produced by the new isolate during growth in a modified Zobell's 2216 medium amended with 1% glucose, and was extractable by cold ethanol precipitation. Chemical, chromatographic and nuclear magnetic resonance spectroscopic analysis confirmed P39a to be a high-molecular-weight (~ 261,000 g/mol) glycoprotein composed of carbohydrate (17.2%) and protein (36.4%). The polymer exhibited high emulsifying activities against a range of oil substrates that included straight-chain aliphatics, mono- and alkyl- aromatics and cycloparaffins. In general, higher emulsification values were measured under low (0.1 M PBS) compared to high (synthetic seawater) ionic strength conditions, indicating that low ionic strength is more favourable for emulsification by the P39a polymer. However, as observed with other bacterial emulsifying agents, the polymer emulsified some aromatic hydrocarbon species, as well as refined and crude oils, more effectively under high ionic strength conditions, which we posit could be due to steric adsorption to these substrates as may be conferred by the protein fraction of the polymer. Furthermore, the polymer effected a positive influence on the degradation of phenanthrene by other marine bacteria, such as the specialist PAH-degrader Polycyclovorans algicola. Collectively, based on the ability of this Halomonas high-molecular-weight glycoprotein to emulsify a range of pure hydrocarbon species, as well as refined and crude oils, it shows promise for the bioremediation of contaminated sites.

Biosynthesis of rhamnolipid by a Marinobacter species expands the paradigm of biosurfactant synthesis to a new genus of the marine microflora.

BACKGROUND: In comparison to synthetically derived surfactants, biosurfactants produced from microbial culture are generally regarded by industry as being more sustainable and possess lower toxicity. One major class of biosurfactants are rhamnolipids primarily produced by Pseudomonas aeruginosa. Due to its pathogenicity rhamnolipid synthesis by this species is viewed as being commercially nonviable, as such there is a significant focus to identify alternative producers of rhamnolipids. RESULTS: To achieve this, we phenotypically screened marine bacteria for biosurfactant production resulting in the identification of rhamnolipid biosynthesis in a species belonging to the Marinobacter genus. Preliminary screening showed the strain to reduce surface tension of cell-free supernatant to 31.0 mN m-1. A full-factorial design was carried out to assess the effects of pH and sea salt concentration for optimising biosurfactant production. When cultured in optimised media Marinobacter sp. MCTG107b produced 740 ± 28.3 mg L-1 of biosurfactant after 96 h of growth. Characterisation of this biosurfactant using both HPLC-MS and tandem MS showed it to be a mixture of different rhamnolipids, with di-rhamnolipid, Rha-Rha-C10-C10 being the most predominant congener. The strain exhibited no pathogenicity when tested using the Galleria mellonella infection model. CONCLUSIONS: This study expands the paradigm of rhamnolipid biosynthesis to a new genus of bacterium from the marine environment. Rhamnolipids produced from Marinobacter have prospects for industrial application due to their potential to be synthesised from cheap, renewable feed stocks and significantly reduced pathogenicity compared to P. aeruginosa strains.

Enhanced crude oil biodegradative potential of natural phytoplankton-associated hydrocarbonoclastic bacteria.

Phytoplankton have been shown to harbour a diversity of hydrocarbonoclastic bacteria (HCB), yet it is not understood how these phytoplankton-associated HCB would respond in the event of an oil spill at sea. Here, we assess the diversity and dynamics of the bacterial community associated with a natural population of marine phytoplankton under oil spill-simulated conditions, and compare it to that of the free-living (non phytoplankton-associated) bacterial community. While the crude oil severely impacted the phytoplankton population and was likely conducive to marine oil snow formation, analysis of the MiSeq-derived 16S rRNA data revealed dramatic and differential shifts in the oil-amended communities that included blooms of recognized HCB (e.g., Thalassospira, Cycloclasticus), including putative novel phyla, as well as other groups with previously unqualified oil-degrading potential (Olleya, Winogradskyella, and members of the inconspicuous BD7-3 phylum). Notably, the oil biodegradation potential of the phytoplankton-associated community exceeded that of the free-living community, and it showed a preference to degrade substituted and non-substituted polycyclic aromatic hydrocarbons. Our study provides evidence of compartmentalization of hydrocarbon-degrading capacity in the marine water column, wherein HCB associated with phytoplankton are better tuned to degrading crude oil hydrocarbons than that by the community of planktonic free-living bacteria.

Response of the bacterial community associated with a cosmopolitan marine diatom to crude oil shows a preference for the biodegradation of aromatic hydrocarbons.

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, Skeletonema costatum, to crude oil. Pyrosequencing analysis and qPCR 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 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.

Polycyclic aromatic hydrocarbon degradation of phytoplankton-associated Arenibacter spp. and description of Arenibacter algicola sp. nov., an aromatic hydrocarbon-degrading bacterium.

Pyrosequencing of the bacterial community associated with a cosmopolitan marine diatom during enrichment with crude oil revealed several Arenibacter phylotypes, of which one (OTU-202) had become significantly enriched by the oil. Since members of the genus Arenibacter have not been previously shown to degrade hydrocarbons, we attempted to isolate a representative strain of this genus in order to directly investigate its hydrocarbon-degrading potential. Based on 16S rRNA sequencing, one isolate (designated strain TG409(T)) exhibited >99% sequence identity to three type strains of this genus. On the basis of phenotypic and genotypic characteristics, strain TG409(T) represents a novel species in the genus Arenibacter, for which the name Arenibacter algicola sp. nov. is proposed. We reveal for the first time that polycyclic aromatic hydrocarbon (PAH) degradation is a shared phenotype among members of this genus, indicating that it could be used as a taxonomic marker for this genus. Kinetic data for PAH mineralization rates showed that naphthalene was preferred to phenanthrene, and its mineralization was significantly enhanced in the presence of glass wool (a surrogate for diatom cell surfaces). During enrichment on hydrocarbons, strain TG409(T) emulsified n-tetradecane and crude oil, and cells were found to be preferentially attached to oil droplets, indicating an ability by the strain to express cell surface amphiphilic substances (biosurfactants or bioemulsifiers) as a possible strategy to increase the bioavailability of hydrocarbons. This work adds to our growing knowledge on the diversity of bacterial genera in the ocean contributing to the degradation of oil contaminants and of hydrocarbon-degrading bacteria found living in association with marine eukaryotic phytoplankton.

Identification of the bacterial community that degrades phenanthrene sorbed to polystyrene nanoplastics using DNA-based stable isotope probing.

In the Anthropocene, plastic pollution has become a new environmental biotope, the so-called plastisphere. In the oceans, nano- and micro-sized plastics are omnipresent and found in huge quantities throughout the water column and sediment, and their large surface area-to-volume ratio offers an excellent surface to which hydrophobic chemical pollutants (e.g. petrochemicals and POPs) can readily sorb to. Our understanding of the microbial communities that breakdown plastic-sorbed chemical pollutants, however, remains poor. Here, we investigated the formation of 500 nm and 1000 nm polystyrene (PS) agglomerations in natural seawater from a coastal environment, and we applied DNA-based stable isotope probing (DNA-SIP) with the 500 nm PS sorbed with isotopically-labelled phenanthrene to identify the bacterial members in the seawater community capable of degrading the hydrocarbon. Whilst we observed no significant impact of nanoplastic size on the microbial communities associated with agglomerates that formed in these experiments, these communities were, however, significantly different to those in the surrounding seawater. By DNA-SIP, we identified Arcobacteraceae, Brevundimonas, Comamonas, uncultured Comamonadaceae, Delftia, Sphingomonas and Staphylococcus, as well as the first member of the genera Acidiphilum and Pelomonas to degrade phenanthrene, and of the genera Aquabacterium, Paracoccus and Polymorphobacter to degrade a hydrocarbon. This work provides new information that feeds into our growing understanding on the fate of co-pollutants associated with nano- and microplastics in the ocean.

Genetic redundancy in the naphthalene-degradation pathway of Cycloclasticus pugetii strain PS-1 enables response to varying substrate concentrations.

Polycyclic aromatic hydrocarbon (PAH) contamination in marine environments range from low-diffusive inputs to high loads. The influence of PAH concentration on the expression of functional genes [e.g. those encoding ring-hydroxylating dioxygenases (RHDs)] has been overlooked in PAH biodegradation studies. However, understanding marker-gene expression under different PAH loads can help to monitor and predict bioremediation efficiency. Here, we followed the expression (via RNA sequencing) of Cycloclasticus pugetii strain PS-1 in cell suspension experiments under different naphthalene (100 and 30 mg L-1) concentrations. We identified genes encoding previously uncharacterized RHD subunits, termed rhdPS1α and rhdPS1ß, that were highly transcribed in response to naphthalene-degradation activity. Additionally, we identified six RHD subunit-encoding genes that responded to naphthalene exposure. By contrast, four RHD subunit genes were PAH-independently expressed and three other RHD subunit genes responded to naphthalene starvation. Cycloclasticus spp. could, therefore, use genetic redundancy in key PAH-degradation genes to react to varying PAH loads. This genetic redundancy may restrict the monitoring of environmental hydrocarbon-degradation activity using single-gene expression. For Cycloclasticus pugetii strain PS-1, however, the newly identified rhdPS1α and rhdPS1ß genes might be potential target genes to monitor its environmental naphthalene-degradation activity.

Characterization of the surface-active exopolysaccharide produced by Halomonas sp TGOS-10: Understanding its role in the formation of marine oil snow.

In this study, we characterize the exopolymer produced by Halomonas sp. strain TGOS-10 -one of the organisms found enriched in sea surface oil slicks during the Deepwater Horizon oil spill. The polymer was produced during the early stationary phase of growth in Zobell's 2216 marine medium amended with glucose. Chemical and proton NMR analysis showed it to be a relatively monodisperse, high-molecular-mass (6,440,000 g/mol) glycoprotein composed largely of protein (46.6% of total dry weight of polymer). The monosaccharide composition of the polymer is typical to that of other marine bacterial exopolymers which are generally rich in hexoses, with the notable exception that it contained mannose (commonly found in yeast) as a major monosaccharide. The polymer was found to act as an oil dispersant based on its ability to effectively emulsify pure and complex oils into stable oil emulsions-a function we suspect to be conferred by the high protein content and high ratio of total hydrophobic nonpolar to polar amino acids (52.7:11.2) of the polymer. The polymer's chemical composition, which is akin to that of other marine exopolymers also having a high protein-to-carbohydrate (P/C) content, and which have been shown to effect the rapid and non-ionic aggregation of marine gels, appears indicative of effecting marine oil snow (MOS) formation. We previously reported the strain capable of utilising aromatic hydrocarbons when supplied as single carbon sources. However, here we did not detect biodegradation of these chemicals within a complex (surrogate Macondo) oil, suggesting that the observed enrichment of this organism during the Deepwater Horizon spill may be explained by factors related to substrate availability and competition within the complex and dynamic microbial communities that were continuously evolving during that spill.

Polycyclovorans algicola gen. nov., sp. nov., an aromatic-hydrocarbon-degrading marine bacterium found associated with laboratory cultures of marine phytoplankton.

A strictly aerobic, halotolerant, rod-shaped bacterium, designated strain TG408, was isolated from a laboratory culture of the marine diatom Skeletonema costatum (CCAP1077/1C) by enrichment with polycyclic aromatic hydrocarbons (PAHs) as the sole carbon source. 16S rRNA gene sequence analysis placed this organism within the order Xanthomonadales of the class Gammaproteobacteria. Its closest relatives included representatives of the Hydrocarboniphaga-Nevskia-Sinobacter clade (<92% sequence similarity) in the family Sinobacteraceae. The strain exhibited a narrow nutritional spectrum, preferring to utilize aliphatic and aromatic hydrocarbon compounds and small organic acids. Notably, it displayed versatility in degrading two- and three-ring PAHs. Moreover, catechol 2,3-dioxygenase activity was detected in lysates, indicating that this strain utilizes the meta-cleavage pathway for aromatic compound degradation. Cells produced surface blebs and contained a single polar flagellum. The predominant isoprenoid quinone of strain TG408 was Q-8, and the dominant fatty acids were C(16:0), C(16:1) ω7c, and C(18:1) ω7c. The G+C content of the isolate's DNA was 64.3 mol% ± 0.34 mol%. On the basis of distinct phenotypic and genotypic characteristics, strain TG408 represents a novel genus and species in the class Gammaproteobacteria for which the name Polycyclovorans algicola gen. nov., sp. nov., is proposed. Quantitative PCR primers targeting the 16S rRNA gene of this strain were developed and used to show that this organism is found associated with other species of marine phytoplankton. Phytoplankton may be a natural biotope in the ocean where new species of hydrocarbon-degrading bacteria await discovery and which contribute significantly to natural remediation processes.

Effect of ocean acidification on the growth, response and hydrocarbon degradation of coccolithophore-bacterial communities exposed to crude oil.

Hydrocarbon-degrading bacteria, which can be found living with eukaryotic phytoplankton, play a pivotal role in the fate of oil spillage to the marine environment. Considering the susceptibility of calcium carbonate-bearing phytoplankton under future ocean acidification conditions and their oil-degrading communities to oil exposure under such conditions, we investigated the response of non-axenic E. huxleyi to crude oil under ambient versus elevated CO2 concentrations. Under elevated CO2 conditions, exposure to crude oil resulted in the immediate decline of E. huxleyi, with concomitant shifts in the relative abundance of Alphaproteobacteria and Gammaproteobacteria. Survival of E. huxleyi under ambient conditions following oil enrichment was likely facilitated by enrichment of oil-degraders Methylobacterium and Sphingomonas, while the increase in relative abundance of Marinobacter and unclassified Gammaproteobacteria may have increased competitive pressure with E. huxleyi for micronutrient acquisition. Biodegradation of the oil was not affected by elevated CO2 despite a shift in relative abundance of known and putative hydrocarbon degraders. While ocean acidification does not appear to affect microbial degradation of crude oil, elevated mortality responses of E. huxleyi and shifts in the bacterial community illustrates the complexity of microalgal-bacterial interactions and highlights the need to factor these into future ecosystem recovery projections.

Substrate-independent expression of key functional genes in Cycloclasticus pugetii strain PS-1 limits their use as markers for PAH biodegradation.

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.

Exploring novel alkane-degradation pathways in uncultured bacteria from the North Atlantic Ocean.

IMPORTANCE: Petroleum pollution in the ocean has increased because of rapid population growth and modernization, requiring urgent remediation. Our understanding of the metabolic response of native microbial communities to oil spills is not well understood. Here, we explored the baseline hydrocarbon-degrading communities of a subarctic Atlantic region to uncover the metabolic potential of the bacteria that inhabit the surface and subsurface water. We conducted enrichments with a 13C-labeled hydrocarbon to capture the fraction of the community actively using the hydrocarbon. We then combined this approach with metagenomics to identify the metabolic potential of this hydrocarbon-degrading community. This revealed previously undescribed uncultured bacteria with unique metabolic mechanisms involved in aerobic hydrocarbon degradation, indicating that temperature may be pivotal in structuring hydrocarbon-degrading baseline communities. Our findings highlight gaps in our understanding of the metabolic complexity of hydrocarbon degradation by native marine microbial communities.

Porticoccus hydrocarbonoclasticus sp. nov., an aromatic hydrocarbon-degrading bacterium identified in laboratory cultures of marine phytoplankton.

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.

Algiphilus aromaticivorans gen. nov., sp. nov., an aromatic hydrocarbon-degrading bacterium isolated from a culture of the marine dinoflagellate Lingulodinium polyedrum, and proposal of Algiphilaceae fam. nov.

A strictly aerobic, halotolerant, Gram-stain-negative, rod-shaped bacterium, designated strain DG1253(T), was isolated from a laboratory culture of the marine dinoflagellate Lingulodinium polyedrum (CCAP 1121/2). The strain was able to degrade two- and three-ring polycyclic aromatic hydrocarbons. It exhibited a narrow nutritional spectrum, preferring to utilize aliphatic and aromatic hydrocarbon compounds and small organic acids. Cells produced surface blebs and contained a single polar flagellum. The predominant isoprenoid quinone of strain DG1253(T) was Q-8. The fatty acid profile was dominated by C(18:1)ω7c. The mean DNA G+C content of strain DG1253(T) was 63.6 ± 0.25 mol%. 16S rRNA gene sequence analysis placed this organism within the order Xanthomonadales of the class Gammaproteobacteria. Its closest relatives included representatives of the Hydrocarboniphaga-Nevskia-Sinobacter clade (≤ 89.9% 16S rRNA gene sequence similarity) in the family Sinobacteraceae. On the basis of distinct phenotypic and genotypic characteristics, strain DG1253(T) is considered to represent a novel species of a new genus in the class Gammaproteobacteria, for which the name Algiphilus aromaticivorans gen. nov., sp. nov. is proposed. The type strain of the type species, Algiphilus aromaticivorans, is DG1253(T) (=ATCC BAA-2243(T)=DSM 24793(T)). In addition, a new family, Algiphilaceae fam. nov., is proposed to accommodate the genus Algiphilus.

Metal binding properties of the EPS produced by Halomonas sp. TG39 and its potential in enhancing trace element bioavailability to eukaryotic phytoplankton.

An emergent property of exopolysaccharides (EPS) produced by marine bacteria is their net negative charge, predominantly conferred by their high uronic acids content. Here, we investigated the EPS produced by an algal-associated marine bacterium, Halomonas sp. strain TG39, for its capacity to sequester trace metals and mediate their bioavailability to eukaryotic phytoplankton. Metal analysis of the purified EPS revealed that it contained high levels of K, Ca, Mg and several essential trace metals, including Zn, Cu, Fe and the metalloid Si. Desorption experiments with marine sediment showed that the EPS possessed a specific binding capacity for Ca, Si, Fe, Mn, Mg and Al. Depending on the ionic conditions, Fe was the third or fourth most highly-adsorbed metal out of 27 elements analyzed. Experiments employing Fe-limited synthetic ocean seawater showed that growth of the marine diatom Thalassiosira weissflogii (axenic strain) was enhanced when incubated in the presence of either purified EPS or EPS that had been pre-exposed to marine sediment, compared to non-EPS amended controls. This growth enhancement was attributed to the EPS binding and increasing the bioavailability of key trace metal elements, such as Fe(III). Since the bacterium used in this study was originally isolated from a marine micro-alga, this work highlights the possibility that bacterial associates of eukaryotic algae could be influencing the bioavailability of Fe(III) to phytoplankton via their production of polyanionic EPS. More widely, this work reinforces the potential importance of marine bacterial EPS in trace metal biogeochemical cycling.

Priorities to inform research on marine plastic pollution in Southeast Asia.

Southeast Asia is considered to have some of the highest levels of marine plastic pollution in the world. It is therefore vitally important to increase our understanding of the impacts and risks of plastic pollution to marine ecosystems and the essential services they provide to support the development of mitigation measures in the region. An interdisciplinary, international network of experts (Australia, Indonesia, Ireland, Malaysia, the Philippines, Singapore, Thailand, the United Kingdom, and Vietnam) set a research agenda for marine plastic pollution in the region, synthesizing current knowledge and highlighting areas for further research in Southeast Asia. Using an inductive method, 21 research questions emerged under five non-predefined key themes, grouping them according to which: (1) characterise marine plastic pollution in Southeast Asia; (2) explore its movement and fate across the region; (3) describe the biological and chemical modifications marine plastic pollution undergoes; (4) detail its environmental, social, and economic impacts; and, finally, (5) target regional policies and possible solutions. Questions relating to these research priority areas highlight the importance of better understanding the fate of marine plastic pollution, its degradation, and the impacts and risks it can generate across communities and different ecosystem services. Knowledge of these aspects will help support actions which currently suffer from transboundary problems, lack of responsibility, and inaction to tackle the issue from its point source in the region. Being profoundly affected by marine plastic pollution, Southeast Asian countries provide an opportunity to test the effectiveness of innovative and socially inclusive changes in marine plastic governance, as well as both high and low-tech solutions, which can offer insights and actionable models to the rest of the world.

Stable isotope probing of an algal bloom to identify uncultivated members of the Rhodobacteraceae associated with low-molecular-weight polycyclic aromatic hydrocarbon degradation.

Polycyclic 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 from ¹³C-enriched bacterial DNA (from naphthalene enrichments) were identified as uncharacterized members of the family Rhodobacteraceae. 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 uniformly ¹³C-labeled PAHs in SIP experiments can successfully be used to identify novel PAH-degrading bacteria in marine waters.

Detection of hydrocarbon-degrading bacteria on deepwater corals of the northeast Atlantic using CARD-FISH.

Recently, studies have begun to identify oil-degrading bacteria and host-taxon specific bacterial assemblages associated with the coral holobiont, including deep-sea cold-water corals, which are thought to provide metabolic functions and additional carbon sources to their coral hosts. Here, we describe the identification of Marinobacter on the soft tissue of Lophelia pertusa coral polyps by Catalyzed Reporter Deposition Fluorescence in situ Hybridization (CARD-FISH). L. pertusa samples from three reef sites in the northeast Atlantic (Logachev, Mingulay and Pisces) were collected at depth by vacuum seal to eliminate contamination issues. After decalcification, histological processing and sagittal sectioning of the soft coral polyp tissues, the 16S rRNA-targeted oligonucleotide HRP-labelled probe Mrb-0625-a, and Cyanine 3 (Cy3)-labelled tyramides, were used to identify members of the hydrocarbon-degrading genus Marinobacter. Mrb-0625-a-hybridized bacterial cell signals were detected in different anatomical sites of all polyps collected from each of the three reef sites, suggesting a close, possibly intimate, association between them, but the purpose of which remains unknown. We posit that Marinobacter, and possibly other hydrocarbon-degrading bacteria associated with Lophelia, may confer the coral with the ability to cope with toxic levels of hydrocarbons in regions of natural oil seepage and where there is an active oil and gas industry presence.

Biosurfactants and Their Applications in the Oil and Gas Industry: Current State of Knowledge and Future Perspectives.

Surfactants are a group of amphiphilic chemical compounds (i.e., having both hydrophobic and hydrophilic domains) that form an indispensable component in almost every sector of modern industry. Their significance is evidenced from the enormous volumes that are used and wide diversity of applications they are used in, ranging from food and beverage, agriculture, public health, healthcare/medicine, textiles, and bioremediation. A major drive in recent decades has been toward the discovery of surfactants from biological/natural sources-namely bio-surfactants-as most surfactants that are used today for industrial applications are synthetically-manufactured via organo-chemical synthesis using petrochemicals as precursors. This is problematic, not only because they are derived from non-renewable resources, but also because of their environmental incompatibility and potential toxicological effects to humans and other organisms. This is timely as one of today's key challenges is to reduce our reliance on fossil fuels (oil, coal, gas) and to move toward using renewable and sustainable sources. Considering the enormous genetic diversity that microorganisms possess, they offer considerable promise in producing novel types of biosurfactants for replacing those that are produced from organo-chemical synthesis, and the marine environment offers enormous potential in this respect. In this review, we begin with an overview of the different types of microbial-produced biosurfactants and their applications. The remainder of this review discusses the current state of knowledge and trends in the usage of biosurfactants by the Oil and Gas industry for enhancing oil recovery from exhausted oil fields and as dispersants for combatting oil spills.