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 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. In 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.

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.

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.

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.

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.

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.

Exploration of marine bacterioplankton community assembly mechanisms during chemical dispersant and surfactant-assisted oil biodegradation.

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.

Response and oil degradation activities of a northeast Atlantic bacterial community to biogenic and synthetic surfactants.

BACKGROUND: Biosurfactants are naturally derived products that play a similar role to synthetic dispersants in oil spill response but are easily biodegradable and less toxic. Using a combination of analytical chemistry, 16S rRNA amplicon sequencing and simulation-based approaches, this study investigated the microbial community dynamics, ecological drivers, functional diversity and robustness, and oil biodegradation potential of a northeast Atlantic marine microbial community to crude oil when exposed to rhamnolipid or synthetic dispersant Finasol OSR52. RESULTS: Psychrophilic Colwellia and Oleispira dominated the community in both the rhamnolipid and Finasol OSR52 treatments initially but later community structure across treatments diverged significantly: Rhodobacteraceae and Vibrio dominated the Finasol-amended treatment, whereas Colwellia, Oleispira, and later Cycloclasticus and Alcanivorax, dominated the rhamnolipid-amended treatment. Key aromatic hydrocarbon-degrading bacteria, like Cycloclasticus, was not observed in the Finasol treatment but it was abundant in the oil-only and rhamnolipid-amended treatments. Overall, Finasol had a significant negative impact on the community diversity, weakened the taxa-functional robustness of the community, and caused a stronger environmental filtering, more so than oil-only and rhamnolipid-amended oil treatments. Rhamnolipid-amended and oil-only treatments had the highest functional diversity, however, the overall oil biodegradation was greater in the Finasol treatment, but aromatic biodegradation was highest in the rhamnolipid treatment. CONCLUSION: Overall, the natural marine microbial community in the northeast Atlantic responded differently to crude oil dispersed with either synthetic or biogenic surfactants over time, but oil degradation was more enhanced by the synthetic dispersant. Collectively, our results advance the understanding of how rhamnolipid biosurfactants and synthetic dispersant Finasol affect the natural marine microbial community in the FSC, supporting their potential application in oil spills. Video abstract.

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.

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.

Inter- and Intra-Annual Bacterioplankton Community Patterns in a Deepwater Sub-Arctic Region: Persistent High Background Abundance of Putative Oil Degraders.

Hydrocarbon-degrading bacteria naturally degrade and remove petroleum pollutants, yet baselines do not currently exist for these critical microorganisms in many regions where the oil and gas industry is active. Furthermore, understanding how a baseline community changes across the seasons and its potential to respond to an oil spill event are prerequisites for predicting their response to elevated hydrocarbon exposures. In this study, 16S rRNA gene-based profiling was used to assess the spatiotemporal variability of baseline bacterioplankton community composition in the Faroe-Shetland Channel (FSC), a deepwater sub-Arctic region where the oil and gas industry has been active for the last 40 years. Over a period of 2 years, we captured the diversity of the bacterioplankton community within distinct water masses (defined by their temperature and salinity) that have a distinct geographic origin (Atlantic or Nordic), depth, and direction of flow. We demonstrate that bacterioplankton communities were significantly different across water samples of contrasting origin and depth. Taxa of known hydrocarbon-degrading bacteria were observed at higher-than-anticipated abundances in water masses originating in the Nordic Seas, suggesting these organisms are sustained by an unconfirmed source of oil input in that region. In the event of an oil spill, our results suggest that the response of these organisms is severely hindered by the low temperatures and nutrient levels that are typical for the FSC.IMPORTANCE Oil spills at sea are one of the most disastrous anthropogenic pollution events, with the Deepwater Horizon spill providing a testament to how profoundly the health of marine ecosystems and the livelihood of its coastal inhabitants can be severely impacted by spilled oil. The fate of oil in the environment is largely dictated by the presence and activities of natural communities of oil-degrading bacteria. While a significant effort was made to monitor and track the microbial response and degradation of the oil in the water column in the wake of the Deepwater Horizon spill, the lack of baseline data on the microbiology of the Gulf of Mexico confounded scientists' abilities to provide an accurate assessment of how the system responded relative to prespill conditions. This data gap highlights the need for long-term microbial ocean observatories in regions at high risk of oil spills. Here, we provide the first microbiological baseline established for a subarctic region experiencing high oil and gas industry activity, the northeast Atlantic, but with no apparent oil seepage or spillage. We also explore the presence, relative abundances, and seasonal dynamics of indigenous hydrocarbon-degrading communities. These data will advance the development of models to predict the behavior of such organisms in the event of a major oil spill in this region and potentially impact bioremediation strategies by enhancing the activities of these organisms in breaking down the oil.

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.

Recent advances in biochar engineering for soil contaminated with complex chemical mixtures: Remediation strategies and future perspectives.

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.

Integrating micro-algae into wastewater treatment: A review.

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.

Specific enrichment of hydrocarbonclastic bacteria from diesel-amended soil on biochar particles.

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.

Comparative Proteomics of Marinobacter sp. TT1 Reveals Corexit Impacts on Hydrocarbon Metabolism, Chemotactic Motility, and Biofilm Formation.

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