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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. [ABSTRACT FROM AUTHOR]

Members of the Marinobacter genus play an important role in hydrocarbon degradation in the ocean – a topic of special significance in light of the recent Deepwater Horizon oil spill of 2010. The Marinobacter group has thus far lacked a genus level phylogenetic probe that would allow in situ identification of representative members. Here, we developed two new 16S rRNA-targeted oligonucleotide probes (Mrb-0625-a and Mrb-0625-b) to enumerate Marinobacter species by fluorescence in situ hybridization (FISH). In silico analysis of this probe set demonstrated 80% coverage of the Marinobacter genus. A competitor probe was developed to block hybridization by Mrb-0625-a to six Halomonas species with which it shared a one base pair mismatch. The probe set was optimized using pure cultures, and then used in an enrichment experiment with a deep sea oil plume water sample collected from the Deepwater Horizon oil spill. Marinobacter cells rapidly increased as a significant fraction of total microbial abundance in all incubations of original contaminated seawater as well as those amended with n -hexadecane, suggesting this group may be among the first microbial responders to oil pollution in the marine environment. The new probe set will provide a reliable tool for quantifying Marinobacter in the marine environment, particularly at contaminated sites where these organisms can play an important role in the biodegradation of oil pollutants. [ABSTRACT FROM AUTHOR]

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 keletonema costatum, to crude oil. Pyrosequencing analysis and q PCR revealed a dramatic transition in the diatom-associated bacterial community, defined initially by a short-lived bloom of M ethylophaga (putative oil degraders) that was subsequently succeeded by distinct groups of HCB ( M arinobacter, P olycyclovorans, A renibacter, P arvibaculum, R oseobacter 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. [ABSTRACT FROM AUTHOR]

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 TG409T) exhibited>99% sequence identity to three type strains of this genus. On the basis of phenotypic and genotypic characteristics, strain TG409T represents a novel species in the genus Arenibacter, for which the name Arenibacter algicola sp. nov. is proposed. Wereveal 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 forPAHmineralization 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 TG409T 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 hydrocarbondegrading bacteria found living in association with marine eukaryotic phytoplankton [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

A strictly aerobic, halotolerant, rod-shaped bacterium, designated strain TG408, was isolated from a laboratory culture of the marine diatom Skeletonema costatum (CCAPIO77/1C) by enrichment with polycyclic aromatic hydrocarbons (PAH5) as the sole carbon source. 16S rRNA gene sequence analysis placed this organism within the order Xanthomonadales of the class Gamma-proteobacteria. Its closest relatives included representatives of the Hydrocarboniphaga-Nevskia-Sinobacter dade (<92% sequence similarity) in the family Sinobacteraceae. The strain exhibited a narrow nutritional spectrum, preferring to utilize auphatic 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 metacleavage 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 C16:0, C16:1 w7c, and C18:1 w7c. 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. [ABSTRACT FROM AUTHOR]

A marine bacterium, designated strain MCTG13d, was isolated from a laboratory culture of the dinoflagellate Lingulodinium polyed rum CCAP1121/2 by enrichment with polycyclic aromatic hydrocarbons (PAHs) as the sole carbon source. Based on 1 6S rRNA gene sequence comparisons, the strain was most closely related to Porticoccus litoralis IMCC2115T (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 C16.1ω,7c, C18:1ω7c, and C16:0. 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. [ABSTRACT FROM AUTHOR]

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 13cjnaphthalene. The dominant sequences in clone libraries constructed from 13C-enriched bacterial DNA (from naphthalene enrichments) were identified as uncharacterized members of the family Rhodobaaeraceae. 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 identitSi novel PAH-degrading bacteria in marine waters. [ABSTRACT FROM AUTHOR]

In this study, we describe the isolation and characterization of a new exopolymer that exhibits high emulsifying activities against a range of oil substrates and demonstrates a differential capacity to desorb various mono-, di-, and trivalent metal species from marine sediment under nonionic and seawater ionic-strength conditions. This polymer, PE12, was produced by a new isolate, Pseudoalteromonas sp. strain TG12 (accession number EF685033), during growth in a modified Zobell's 2216 medium amended with 1% glucose. Chemical and chromatographic analysis showed it to be a high-molecular-mass (>2,000 kDa) glycoprotein composed of carbohydrate (32.3%) and protein (8.2%). PE12 was notable in that it contained xylose as the major sugar component at unusually high levels (27.7%) not previously reported for a Pseudoalteromonas exopolymer. The polymer was shown to desorb various metal species from marine sediment—a function putatively conferred by its high content of uronic acids (28.7%). Seawater ionic strength (simulated using 0.6 M NaCI), however, caused a significant reduction in PE12's ability to desorb the sediment-adsorbed metals. These results demonstrate the importance of electrolytes, a physical parameter intrinsic of seawater, in influencing the interaction of microbial exopolymers with metal ions. In summary, PE12 may represent a new class of Pseudoalteromonas exopolymer with a potential for use in biotechnological applications as an emulsifying or metal-chelating agent. In addition to the biotechnological potential of these findings, the ecological aspects of this and related bacterial exopolymers in marine environments are also discussed. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. • Identification of the oil-degrader Marinobacter associated with Lophelia pertusa. • Use of a bespoke vacuum seal sampler at depth to collect coral polyps. • CARD-FISH was coupled with histological methods to analyse the coral polyps. • Marinobacter detected in different anatomical sites of polyps from three reef sites. • Marinobacter may confer Lophelia with the ability to cope with toxic hydrocarbons. [ABSTRACT FROM AUTHOR]

Assembly processes in marine microbial communities amended with crude oil and chemical dispersant are poorly understood and even more so when biosurfactants are used. We set up a microcosm experiment in which microbiome structure was analyzed using 16S rRNA gene amplicon sequencing and six null models to better understand and quantify the mechanisms and patterns controlling the assembly of a marine crude oil degrading microbial community in the presence of chemical dispersant or rhamnolipid biosurfactant. Although each null model quantifies different aspects of the community assembly, there was a general agreement that neither purely stochastic nor purely deterministic processes dominated the microbial communities, and their influence was variable over time. Determinism was dominant in the early phase of incubation, while stochasticity was prevalent in the middle and late stages. There was faster recruitment of phylogenetically distant species in the dispersant‐amended community compared to oil‐only or rhamnolipid‐amended communities. This analysis provides important insights of how chemical dispersants and rhamnolipid influence microbial communities' dynamics and identified which groups may be excluded—an important consideration for biodegradation process and oil spill response. [ABSTRACT FROM AUTHOR]

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 rhdPS1β , 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 rhdPS1β genes might be potential target genes to monitor its environmental naphthalene-degradation activity. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Marine plastic pollution is rapidly colonized by a biofilm of microorganisms associated with the control of biogeochemical cycles, plastic biodegradation, and potentially pathogenic activities. An extensive number of studies have described the taxonomic composition of this biofilm, referred to as the 'plastisphere', and previous reviews have reported on the influence of location, plastic type, and plastic-biodegradation ability on plastisphere formation. However, few studies have investigated the metabolic activity of this complex biofilm and how microbial pathogenicity and bioremediation could be regulated in this ecosystem. In this review, we highlight the understudied molecular and abiotic factors influencing plastisphere formation and microbial functioning beyond taxonomic description. In this context, we critically discuss the impacts of (i) organism-organism interaction, (ii) microbial cell wall composition, and (iii) commonly encountered plastic-bound co-pollutants (heavy metals, persistent organic pollutants, UV filters). For the first time, we review the anticipated impact of lipophilic organic UV-filters – found in plastic additives and sunscreens – on the plastisphere due to their reported affinity for plastics, persistence, and co-location at high concentrations in touristic coastal environments. Herein, we integrate the findings of 34 global studies exploring plastisphere composition, 35 studies quantifying co-pollutant concentrations, and draw upon 52 studies of cell -cell and -substrate interaction to deduce the inferred, yet still unknown, metabolic interactions within this niche. Finally, we provide novel future directions for the advancement of functional plastisphere research applying advanced molecular tools to new, and appropriate research questions. Data was compiled from 97 plastispheres across 34 different studies and an additional 87 studies relating to the impacts of plastisphere components on microbial species. The biotic and abiotic factors influencing microbial adhesion to different plastic polymers, including cell wall composition, and plastisphere location are considered. The impacts of heavy metals and organic co-pollutants – and for the first time, organic UV-filters – on plastisphere formation and function are reviewed and discussed. A change in direction from novel research questions to the use of state-of-the-art methodologies are recommended for the advancement of functional plastisphere research. [ABSTRACT FROM AUTHOR]

Halomonas species are recognized for producing exopolysaccharides (EPS) exhibiting amphiphilic properties that allow these macromolecules to interface with hydrophobic substrates, such as hydrocarbons. There remains a paucity of knowledge, however, on the potential of Halomonas EPS to influence the biodegradation of hydrocarbons. In this study, the well-characterized amphiphilic EPS produced by Halomonas species strain TG39 was shown to effectively increase the solubilization of aromatic hydrocarbons and enhance their biodegradation by an indigenous microbial community from oil-contaminated surface waters collected during the active phase of the Deepwater Horizon oil spill. Three Halomonas strains were isolated from the Deepwater Horizon site, all of which produced EPS with excellent emulsifying qualities and shared high (97-100%) 16S rRNA sequence identity with strain TG39 and other EPS-producing Halomonas strains. Analysis of pyrosequence data from surface water samples collected during the spill revealed several distinct Halomonas phylotypes, of which some shared a high sequence identity (≥97%) to strain TG39 and the Gulf spill isolates. Other bacterial groups comprising members with well-characterized EPS-producing qualities, such as Alteromonas, Colwellia and Pseudoalteromonas, were also found enriched in surface waters, suggesting that the total pool of EPS in the Gulf during the spill may have been supplemented by these organisms. Roller bottle incubations with one of the Halomonas isolates from the Deepwater Horizon spill site demonstrated its ability to effectively produce oil aggregates and emulsify the oil. The enrichment of EPS-producing bacteria during the spill coupled with their capacity to produce amphiphilic EPS is likely to have contributed to the ultimate removal of the oil and to the formation of oil aggregates, which were a dominant feature observed in contaminated surface waters. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Marine plastic debris is well characterized in terms of its ability to negatively impact terrestrial and marine environments, endanger coastal wildlife, and interfere with navigation, tourism and commercial fisheries. However, the impacts of potentially harmful microorganisms and pathogens colonising plastic litter are not well understood. The hard surface of plastics provides an ideal environment for opportunistic microbial colonisers to form biofilms and might offer a protective niche capable of supporting a diversity of different microorganisms, known as the “Plastisphere”. This biotope could act as an important vector for the persistence and spread of pathogens, faecal indicator organisms (FIOs) and harmful algal bloom species (HABs) across beach and bathing environments. This review will focus on the existent knowledge and research gaps, and identify the possible consequences of plastic-associated microbes on human health, the spread of infectious diseases and bathing water quality. [ABSTRACT FROM AUTHOR]

One of the defining features of the Deepwater Horizon oil spill was the rapid formation and persistence of a hydrocarbon plume in deep water. Here we use 16S rRNA gene clone libraries and pyrosequencing of 16S rRNA gene fragments to outline the temporal dynamics of the bacterial community in the water column near the Macondo wellhead. Our timeline starts with the pre-spill (March 2010) status of the water column bacterial community, continues through the bacterial enrichments dominating the hydrocarbon plume after the blowout (DWH Oceanospirillales , Cycloclasticus , Colwellia in late May 2010), and leads towards post-spill bacterial communities with molecular signatures related to degradation of phytoplankton pulses (September and October 2010; July 2011) in the water column near the Macondo wellhead. We document a dramatic transition as the complex bacterial community before the oil spill was temporarily overwhelmed by a few specialized bacterial groups responding to the massive influx of hydrocarbons in May 2010. In September and October 2010, this bacterial bloom had been replaced by a diversified bacterial community which resembled its predecessor prior to the spill. Notably, the post-plume 16S rRNA gene clone libraries and pyrosequencing datasets illustrated the continued presence of oil-degrading bacteria in the water column near the Macondo wellhead which we posit to represent an inherent signature of hydrocarbon catabolic potential to the Gulf of Mexico. The pyroseqencing results detected and tracked minority bacterial populations that were not visible in the conventional 16S rRNA gene clone libraries and allowed us to identify natural reservoirs of the Deepwater Horizon Oceanospirillales within and outside of the Gulf of Mexico. [ABSTRACT FROM AUTHOR]

We studied long-term (64.5 weeks) biodegradation of diesel fuel, diesel/biodiesel blends (B10-B90) and biodiesel fuels in urban soil microcosms containing indigenous microorganisms, or indigenous microorganisms augmented with a hydrocarbon-degrading bacterial community. Mineralization extent (mmol of CO 2 per day) of B10-B30 blends was smaller compared with diesel fuel at both short- (28 days) and long-term (109 days), and increased with biodiesel content. Priming with hydrocarbon degraders accelerated mineralization in the short-term (by up to 140%), with highest influence using blends with lower biodiesel content, but did not significantly influence kinetics and mineralization extent in the long-term. Although the biodiesel fraction was degraded completely within 64.5 weeks, 3–12% of the total aromatic and aliphatic hydrocarbons remained in the microcosms. Barcoded 16S rRNA gene MiSeq sequencing analysis revealed a significant effect of blend type on the community structure, with a marked enrichment of Sphingobacteriia and Actinobacteria classes. However, no significant influence was determined in the long-term, suggesting that the inoculated bacterial community may not have survived. Our findings show that biodiesel is preferentially degraded in urban soil and suggest that the value of bioaugmentation for bioremediating biodiesel fuels with hydrocarbon-degrading bacteria is limited to short-term exposures to lower (B10-B30) blends. Unlabelled Image • Long-term (64.5 weeks) biodegradation of diesel/biodiesel in urban soil was studied. • 3–12% of the total aromatic and aliphatic hydrocarbons remained in the microcosms. • Effect of bioaugmentation was evaluated. • MiSeq sequencing analysis revealed a significant effect of blend type. • No significant influence of bioaugmentation was determined in the long-term. [ABSTRACT FROM AUTHOR]

Abstract Some studies have described the isolation and 16S rRNA gene sequence-based identification of hydrocarbon-degrading bacteria living associated with marine eukaryotic phytoplankton, and thus far the direct visual observation of these bacteria on micro-algal cell surfaces ('phycosphere') has not yet been reported. Here, we developed two new 16S rRNA-targeted oligonucleotide probes, PCY223 and ALGAR209, to respectively detect and enumerate the obligate hydrocarbonoclastic bacteria Polycyclovorans algicola and Algiphilus aromaticivorans by Catalyzed Reporter Deposition Fluorescence in situ Hybridization (CARD-FISH). To enhance the hybridization specificity with the ALGAR209 probe, a competitor probe was developed. These probes were tested and optimized using pure cultures, and then used in enrichment experiments with laboratory cultures of micro-algae exposed to phenanthrene, and with coastal water enriched with crude oil. Microscopic analysis revealed these bacteria are found in culture with the micro-algal cells, some of which were found attached to algal cells, and whose abundance increased after phenanthrene or crude oil enrichment. These new probes are a valuable tool for identifying and studying the ecology of P. algicola and A. aromaticivorans in laboratory and field samples of micro-algae, as well as opening new fields of research that could harness their ability to enhance the bioremediation of contaminated sites. Highlights • Two new 16S rRNA-targeted probes for CARD-FISH were developed to detect and enumerate P. algicola and A. aromaticivorans. • We show the direct visual association/attachment of these obligate hydrocarbonoclastic bacteria with micro-algae. • These probes will provide a valuable tool for identifying and examining these bacteria in lab and field samples. [ABSTRACT FROM AUTHOR]

Thraustochytrids were first discovered in 1934, and since the 1960's they have been increasingly studied for their beneficial and deleterious effects. This review aims to provide an enhanced understanding of these protists with a particular emphasis on their taxonomy, ecology and biotechnology applications. Over the years, thraustochytrid taxonomy has improved with the development of modern molecular techniques and new biochemical markers, resulting in the isolation and description of new strains. In the present work, the taxonomic history of thraustochytrids is reviewed, while providing an up-to-date classification of these organisms. It also describes the various biomarkers that may be taken into consideration to support taxonomic characterization of the thraustochytrids, together with a review of traditional and modern techniques for their isolation and molecular identification. The originality of this review lies in linking taxonomy and ecology of the thraustochytrids and their biotechnological applications as producers of docosahexaenoic acid (DHA), carotenoids, exopolysaccharides and other compounds of interest. The paper provides a summary of these aspects while also highlighting some of the most important recent studies in this field, which include the diversity of polyunsaturated fatty acid metabolism in thraustochytrids, some novel strategies for biomass production and recovery of compounds of interest. Furthermore, a detailed overview is provided of the direct and current applications of thraustochytrid-derived compounds in the food, fuel, cosmetic, pharmaceutical, and aquaculture industries and of some of the commercial products available. This review is intended to be a source of information and references on the thraustochytrids for both experts and those who are new to this field. [ABSTRACT FROM AUTHOR]

Thraustochytrids isolated from hot tropical and sub-tropical waters have been well studied for DHA and biodiesel production in the last decades. However, little research has been performed on the oils of cold water thraustochytrids, in particular from the North Sea region. In this study, thraustochytrid strains from British waters showed high relative levels of omega-3 long-chain (≥C) polyunsaturated fatty acids (LC-PUFA), including docosahexaenoic acid (DHA, 22:6ω3). The relative levels of DHA (as % of total fatty acids, TFA) in the different British strains are hitherto amongst the highest recorded from any thraustochytrid screening study, with strain TL18 reaching up to 67% DHA in modified Glucose-Yeast Extract-Peptone (GYP) medium. At this screening stage, low final biomass and fatty acid yield were observed in modified GYP and MarChiquita-Brain Heart Broth (MCBHB), while PUFA profiles (as % of PUFA) remained unaltered regardless of the culture medium used. Hence, optimizing the medium and culture conditions to improve growth and lipid content, without impacting the relative percentage of DHA, has the potential to increase the final DHA concentration. With this in mind, three strains were identified as promising organisms for the production of DHA. In the context of possible future industrial exploitation involving a winterization step, we investigated the recycling of the residual oil for biodiesel use. To do this, a mathematical model was used to assess the intrinsic properties of the by-product oil. The results showed the feasibility of producing primary DHA-rich oil, assuming optimized conditions, while using the by-product oil for biodiesel use. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: Variable inorganic nutrient concentrations were used to generate silicate (Si) or nitrogen (N) limited conditions in cultures of the marine diatom Skeletonema costatum. Dissolved organic matter (DOM) harvested in the nutrient limited phase of these cultures was added to a natural bacterial community. Enhanced bacterial abundance and bacterial production were observed, in comparison to un-supplemented controls, when Si-limited (Si-DOM) rather than N-limited (N-DOM) diatom derived DOM or inorganic nutrients was added. This indicates that the bacterial population was limited by organic rather than inorganic resources but only Si-DOM had sufficient lability to alleviate this. Within the bacterial assemblage, a notable increase in the proportion of γ-Proteobacteria was evident on receipt of only Si-DOM. Assessment of the composition of the added DOM suggested that the observed dynamics were related to organic matter composition rather than molecular size as, within the Si- and N-DOM, the proportions of low and high molecular weight compounds were similar, but the polysaccharide and protein signatures were different. [ABSTRACT FROM AUTHOR]

This study evaluated the performance of microalgae under static cultivation for primary settled municipal wastewater (PSW) treatment as a low energy treatment process. The availability of a suitable carbon substrate was determined to be the main limiting factor affecting the algal treatment performance. To overcome the material cost of applying commercial sources of organic carbon, we evaluated pot ale – a carbohydrate-rich by-product from the production of malt whiskey – as a carbon substrate to promote microalgae growth and the removal of nitrogen (NH 3 –N) and phosphate (PO 4 –P) in PSW. For this, the mixotrophic microalgal species Chlorella vulgaris was used in batch experiments of PSW enriched with pot ale. Characterisation of the wastewater in the microalgae treatments compared with the control treatments (WWC) and wastewater with pot ale (WWPA) highlighted that C. vulgaris was a key organism in the algal-bacterial consortium responsible in inorganic N and P removal. We also observed a high variability in the characteristics of PSW across independent batches enriched with pot ale, which resulted in variability in the N and P removal efficiency by the alga, from 99% to 58% at reducing NH 3 –N, and from 94% to 58% at reducing PO 4 –P. As an extension of these batch-wise operated treatments, we investigated removal of NH 3 –N and PO 4 –P under semi-continuous operation with pot ale enrichment and found this to be a viable system for potential further development. This work highlights the use of pot ale enrichment with microalgae as a promising application for enhancing the efficiency of inorganic nutrient removal from PSW. [Display omitted] • Pot ale enrichment with microalgae significantly enhances the inorganic nutrient (N & P) removal. • C. vulgaris played a key role in inorganic N and P removal from PSW. • Availability of the carbon substrate is a major factor affecting PSW treatment by micro-algae. • Removal of N and P under semi-continuous operation with pot ale is a viable option for development. [ABSTRACT FROM AUTHOR]

Heavy metal/metalloids (HMs) and polycyclic aromatic hydrocarbons (PAHs) in soil have caused serious environmental problems, compromised agriculture quality, and have detrimental effects on all forms of life including humans. There is a need to develop appropriate and effective remediation methods to resolve combined contaminated problems. Although conventional technologies exist to tackle contaminated soils, application of biochar as an effective renewable adsorbent for enhanced bioremediation is considered by many scientific researchers as a promising strategy to mitigate HM/PAH co-contaminated soils. This review aims to: (i) provide an overview of biochar preparation and its application, and (ii) critically discuss and examine the prospects of (bio)engineered biochar for enhancing HMs/PAHs co-remediation efficacy by reducing their mobility and bioavailability. The adsorption effectiveness of a biochar largely depends on the type of biomass material, carbonisation method and pyrolysis conditions. Biochar induced soil immobilise and remove metal ions via various mechanisms including electrostatic attractions, ion exchange, complexation and precipitation. PAHs remediation mechanisms are achieved via pore filling, hydrophobic effect, electrostatic attraction, hydrogen bond and partitioning. During last decade, biochar engineering (modification) via biological and chemical approaches to enhance contaminant removal efficiency has garnered greater interests. Hence, the development and application of (bio)engineered biochars in risk management, contaminant management associated with HM/PAH co-contaminated soil. In terms of (bio)engineered biochar, we review the prospects of amalgamating biochar with hydrogel, digestate and bioaugmentation to produce biochar composites. Unlabelled Image • Microbial-enhanced biochar to tackle complex chemical mixtures in soil • Biochar engineered with hydrogel, digestate and microbes for wider bioremediation options • Bioengineered biochar reviving bioremediation and sustainability • Field scale application and verification needs [ABSTRACT FROM AUTHOR]

Biochar has been proposed as a suitable biostimulant for the remediation of hydrocarbon contamination, and also has the potential to act as a carrier for hydrocarbonoclastic microorganisms which could bioaugment endogenous microbial communities. However, the evidence regarding the biostimulatory effects of biochars on hydrocarbon bioremediation is somewhat equivocal, possibly due to variability of the physicochemical properties of biochar and soil across studies. Here, we use standard biochars with defined properties produced from softwood pellets (SWP) and rice husk (RH) at pyrolysis temperatures of 550 °C or 700 °C to test the effects of biochar amendment on microbial community composition and hydrocarbon degradation in soil microcosms contaminated with diesel oil. Combining this approach for the first time with specific analysis of microbial community composition using amplicon sequence variants (ASVs), we find that oil contamination causes extreme short-term loss of soil microbial diversity, and highly-specific selection of a limited set of genera defined by 13 ASVs. Biochar ameliorates the short-term loss of diversity, and in the longer term (9 weeks), changes community composition in a type-specific manner. The majority of the 13 selected ASVs are further enriched on biochar particles, although SWP biochars perform better than RH biochar in enrichment of putative hydrocarbonoclastic Aquabacterium spp. However, complete degradation of normal (n) alkanes from the aliphatic hydrocarbon fraction is prevented in the presence of biochar amendment, possibly due to their adsorption onto the char surface. Furthermore, we show that putative hydrocarbon degraders released from diesel-amended soil can subsequently be enriched to high levels on SWP biochar particles in growth medium supplemented with diesel oil as the sole carbon source; these include selected ASVs representing the genera Rhodococcus , Aquabacterium , and Cavicella. This work suggests that use of biochar pre-enriched with endogenous, conditionally-rare hydrocarbon degrading bacteria is a promising strategy for bioaugmentation of diesel-contaminated soils. Unlabelled Image • Biochar was evaluated for the biostimulation of hydrocarbon contaminated soils. • Amplicon sequence variants (ASVs) reveal specific soil microbial responses to oil. • Oil amplifies 13 ASVs - putative hydrocarbon degraders - to high relative abundance. • Standard biochars reduce soil diversity loss and are enriched in oil-selected ASVs. • Biochar enriched with endogenous microbes is a promising approach to bioremediation. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Improving the ecological status of water sources is a growing focus for many developed and developing nations, in particular with reducing nitrogen and phosphorus in wastewater effluent. In recent years, mixotrophic micro-algae have received increased interest in implementing them as part of wastewater treatment. This is based on their ability to utilise organic and inorganic carbon, as well as inorganic nitrogen (N) and phosphorous (P) in wastewater for their growth, with the desired results of a reduction in the concentration of these substances in the water. The aim of this review is to provide a critical account of micro-algae as an important step in wastewater treatment for enhancing the reduction of N, P and the chemical oxygen demand (COD) in wastewater, whilst utilising a fraction of the energy demand of conventional biological treatment systems. Here, we begin with an overview of the various steps in the treatment process, followed by a review of the cellular and metabolic mechanisms that micro-algae use to reduce N, P and COD of wastewater with identification of when the process may potentially be most effective. We also describe the various abiotic and biotic factors influencing micro-algae wastewater treatment, together with a review of bioreactor configuration and design. Furthermore, a detailed overview is provided of the current state-of-the-art in the use of micro-algae in wastewater treatment. Wastewater treatment dates back to the 1800s when the first municipal water treatment plant was built in Scotland, and since then the process has become established throughout the world for treatment of municipal and other sewage. In addition to any preceding physical and mechanical treatment operations, the process fundamentally relies on the biological breakdown of organic matter and pollutants, driven by bacterial consortia. In recent years, mixotrophic micro-algae have received increased interest in implementing them as part of wastewater treatment. This is based on their ability to utilise organic and inorganic carbon, as well as inorganic nitrogen (N) and phosphorous (P) in wastewater for their growth, with the desired results of a reduction in the concentration of these substances in the water. The aim of this review is to provide a critical account of micro-algae as an important step in wastewater treatment for enhancing the reduction of N, P and the chemical oxygen demand (COD) in wastewater, whilst utilising a fraction of the energy demand of conventional biological treatment systems. Here, we begin with an overview of the various steps in the treatment process, followed by a review of the cellular and metabolic mechanisms that micro-algae use to reduce N, P and COD of wastewater with identification of when the process may potentially be most effective. We also describe the various abiotic and biotic factors influencing micro-algae wastewater treatment, together with a review of bioreactor configuration and design. Furthermore, a detailed overview is provided of the current state-of-the-art in the use of micro-algae in wastewater treatment. This review is intended to be a source of information and references for both experts and those who are new to this field, with the hope also that it will garner significant interest towards integrating micro-algae for the enhanced and cost-effective treatment of wastewater. Unlabelled Image • A critical overview of the role of micro-algae cultivation for wastewater treatment. • Efficient reduction of N, P, and COD by micro-algae in wastewater treatment discussed. • The energy demand of conventional biological treatment systems compared to micro-algae cultivation. • Economic challenges of microalgal cultivation in wastewater treatment reviewed. • Various abiotic and biotic factors influencing micro-algae discussed. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]