Rapidly and repeatedly ascertaining the health status of coral reefs is an ever more pressing issue as part of activities to understand and monitor the damaging impacts of climate change. A combination of increasing ocean temperatures, acidity and frequency of extreme storm events continues to alter the marine environment beyond what sensitive organisms, such as coral, can cope with. It is therefore vital to establish technologies and validated methods to provide a metric or indication into the health of these organisms. There are currently many surveys and techniques used by coral scientists to uncover insights into the status and assessment of coral reefs, from colour wheels to multispectral satellite surveys. Here we outline an array of current techniques and methods focused specifically on coral monitoring and health diagnosis, ranging across the length scales from simple diver-based surveyance to satellite remote sensing. The technique of using hyperspectral fluorescence imaging is also introduced as a viable novel addition to aid and extend the current toolbox of available technologies.

The development of low-cost, open-source Remotely Operated Vehicle (ROV) systems has provided almost unrestricted access for researchers looking to monitor the marine environment in ever greater resolution. Sampling microbial communities from the marine environment, however, still usually relies on Niskin-bottle sampling (ROV or CTD based), a method which introduces an inaccuracy and variability that is incompatible with metatranscriptomic analysis. Here, we describe a versatile, easily-replicated platform which achieves in situ mRNA preservation, via the addition of RNAlater to filtered microbial cells, to enhance ROV or CTD functionality.

AbstractMicrobes and their associated viruses are key drivers of biogeochemical processes in marine and soil biomes. While viruses of phototrophic cyanobacteria are well-represented in model systems, challenges of isolating marine microbial heterotrophs and their viruses have hampered experimental approaches to quantify the importance of viruses in nutrient recycling. A resurgence in cultivation efforts has improved the availability of fastidious bacteria for hypothesis testing, but this has not been matched by similar efforts to cultivate their associated bacteriophages. Here, we describe a high-throughput method for isolating important virus-host systems for fastidious heterotrophic bacteria that couples advances in culturing of hosts with sequential enrichment and isolation of associated phages. Applied to six monthly samples from the Western English Channel, we first isolated one new member of the globally dominant bacterial SAR11 clade and three new members of the methylotrophic bacterial clade OM43. We used these as bait to isolate 117 new phages including the first known siphophage infecting SAR11, and the first isolated phage for OM43. Genomic analyses of 13 novel viruses revealed representatives of three new viral genera, and infection assays showed that the viruses infecting SAR11 have ecotype-specific host-ranges. Similar to the abundant human-associated phage ΦCrAss001, infection dynamics within the majority of isolates suggested either prevalent lysogeny or chronic infection, despite a lack of associated genes; or host phenotypic bistability with lysis putatively maintained within a susceptible subpopulation. Broader representation of important virus-host systems in culture collections and genomic databases will improve both our understanding of virus-host interactions, and accuracy of computational approaches to evaluate ecological patterns from metagenomic data.

ABSTRACTThe methylotrophic OM43 clade are Gammaproteobacteria that comprise some of the smallest free-living cells known and have highly streamlined genomes. OM43 represents an important microbial link 0between marine primary production and remineralisation of carbon back to the atmosphere. Bacteriophages shape microbial communities and are major drivers of microbial mortality and global marine biogeochemistry. Recent cultivation efforts have brought the first viruses infecting members of the OM43 clade into culture. Here we characterize a novel myophage infecting OM43 called Melnitz. Melnitz was isolated independently on three separate occasions (with isolates sharing >99.95% average nucleotide identity) from water samples from a subtropical ocean gyre (Sargasso Sea) and temperate coastal (Western English Channel) systems. Metagenomic recruitment from global ocean viromes confirmed that Melnitz is globally ubiquitous, congruent with patterns of host abundance. Bacteria with streamlined genomes such as OM43 and the globally dominant SAR11 clade use riboswitches as an efficient method to regulate metabolism. Melnitz encodes a two-piece tmRNA (ssrA), controlled by a glutamine riboswitch, providing evidence that riboswitch use also occurs for regulation during phage infection of streamlined heterotrophs. Virally encoded tRNAs and ssrA found in Melnitz were phylogenetically more closely related to those found within the alphaproteobacterial SAR11 clade and their associated myophages than those within their gammaproteobacterial hosts. This suggests the possibility of an ancestral inter-class host transition event between SAR11 and OM43. Melnitz and a related myophage that infects SAR11 were unable to infect hosts of the SAR11 and OM43, respectively, suggesting host transition rather than a broadening of host range.IMPORTANCEIsolation and cultivation of viruses is the foundation on which the mechanistic understanding of virus-host interactions and ground-truthing is based. This study isolated and characterised the first myophage known to infect the OM43 clade, expanding our knowledge of this understudied group of microbes. The near-identical genomes of four strains of Melnitz isolated from different marine provinces and global abundance estimations from metagenomic data suggest that this viral population is globally ubiquitous. Genome analysis revealed several unusual features in Melnitz and related genomes recovered from viromes, such as a curli operon and virally encoded tmRNA controlled by a glutamine riboswitch, neither of which are found in the host. Further phylogenetic analysis of shared genes indicates that this group of viruses infecting the gammaproteobacterial OM43 shares a recent common ancestor with viruses infecting the abundant alphaproteobacterial SAR11 clade. Host ranges are affected by compatible cell surface receptors, successful circumvention of superinfection exclusion systems and the presence of required accessory proteins, which typically limits phages to singular narrow groups of closely related bacterial hosts. This study provides intriguing evidence that for streamlined heterotrophic bacteria, virus-host transitioning is not necessarily restricted to phylogenetically related hosts, but is a function of shared physical and biochemical properties of the cell.

AbstractMarine viruses impact global biogeochemical cycles via their influence on host community structure and function, yet our understanding of viral ecology is constrained by limitations in culturing of important hosts and the lack of a ‘universal’ gene to facilitate community surveys. Short-read viral metagenomic studies have provided clues to viral function and first estimates of global viral gene abundance and distribution. However, short-read assemblies are confounded by populations with high levels of strain evenness and nucleotide diversity (microdiversity), limiting assembly of some of the most abundant viruses on Earth. Assembly across genomic islands which likely contain niche-defining genes that drive ecological speciation is also challenging. While such populations and features are successfully captured by single-virus genomics and fosmid-based approaches, both techniques require considerable cost and technical expertise. Here we established a low-cost, low-input, high throughput alternative method for improving assembly of viral metagenomics using long read technology. Named ‘VirION’ (Viral, long-read metagenomics via MinION sequencing), our sequencing approach and complementary bioinformatics pipeline (i) increased number and completeness of assembled viral genomes compared to short-read sequencing methods; (ii) captured populations of abundant viruses with high microdiversity missed by short-read methods and (iii) captured more and longer genomic islands than short-read methods. Thus, VirION provides a high throughput and cost-effective alternative to fosmid and single-virus genomic approaches to more comprehensively explore viral communities in nature.

The methylotrophic OM43 clade are Gammaproteobacteria that comprise some of the smallest free-living cells known and have highly streamlined genomes. OM43 represents an important microbial link between marine primary production and remineralization of carbon back to the atmosphere. Bacteriophages shape microbial communities and are major drivers of mortality and global marine biogeochemistry. Recent cultivation efforts have brought the first viruses infecting members of the OM43 clade into culture. Here, we characterize a novel myophage infecting OM43 called Melnitz. Melnitz was isolated independently from water samples from a subtropical ocean gyre (Sargasso Sea) and temperate coastal (Western English Channel) systems. Metagenomic recruitment from global ocean viromes confirmed that Melnitz is globally ubiquitous, congruent with patterns of host abundance. Bacteria with streamlined genomes such as OM43 and the globally dominant SAR11 clade use riboswitches as an efficient method to regulate metabolism. Melnitz encodes a two-piece tmRNA (ssrA), controlled by a glutamine riboswitch, providing evidence that riboswitch use also occurs for regulation during phage infection of streamlined heterotrophs. Virally encoded tRNAs and ssrA found in Melnitz were phylogenetically more closely related to those found within the alphaproteobacterial SAR11 clade and their associated myophages than those within their gammaproteobacterial hosts. This suggests the possibility of an ancestral host transition event between SAR11 and OM43. Melnitz and a related myophage that infects SAR11 were unable to infect hosts of the SAR11 and OM43, respectively, suggesting host transition rather than a broadening of host range. IMPORTANCE Isolation and cultivation of viruses are the foundations on which the mechanistic understanding of virus-host interactions and parameterization of bioinformatic tools for viral ecology are based. This study isolated and characterized the first myophage known to infect the OM43 clade, expanding our knowledge of this understudied group of microbes. The nearly identical genomes of four strains of Melnitz isolated from different marine provinces and the global abundance estimations from metagenomic data suggest that this viral population is globally ubiquitous. Genome analysis revealed several unusual features in Melnitz and related genomes recovered from viromes, such as a curli operon and virally encoded tmRNA controlled by a glutamine riboswitch, neither of which are found in the host. Further phylogenetic analysis of shared genes indicates that this group of viruses infecting the gammaproteobacterial OM43 shares a recent common ancestor with viruses infecting the abundant alphaproteobacterial SAR11 clade. Host ranges are affected by compatible cell surface receptors, successful circumvention of superinfection exclusion systems, and the presence of required accessory proteins, which typically limits phages to singular narrow groups of closely related bacterial hosts. This study provides intriguing evidence that for streamlined heterotrophic bacteria, virus-host transitioning may not be necessarily restricted to phylogenetically related hosts but is a function of shared physical and biochemical properties of the cell.

Monitoring the health of coral reefs is essential to understanding the damaging impacts of anthropogenic climate change as such non-invasive methods to survey coral reefs are the most desirable. Optics-based surveys, ranging from simple photography to multispectral satellite imaging are well established. Herein, we review these techniques, focusing on their value for coral monitoring and health diagnosis. The techniques are broadly separated by the primary method in which data are collected: by divers and/or robots directly within the environment or by remote sensing where data are captured above the water’s surface by planes, drones, or satellites. The review outlines a new emerging technology, low-cost hyperspectral imagery, which is capable of simultaneously producing hyperspectral and photogrammetric outputs, thereby providing integrated information of the reef structure and physiology in a single data capture.

Massive and recurrent strandings of pelagic Sargassum biomass have become the new norm in the Caribbean and the Western Africa since 2011, and there is no sign of this abating. These Sargassum events have negative environmental, socioeconomic and health impacts in the affected countries. In the meantime, various processing techniques and applications have been suggested for valorisation of this biomass. However, variability in quantity, quality and location creates substantial uncertainty for the development of reliable and robust industrial processes. As part of ongoing efforts to better characterise seasonal and geographical variations in the biochemical and elemental composition of the pelagic Sargassum biomass across the Caribbean, we analysed samples from Mexico, Jamaica and the Dominican Republic harvested during summer 2020 and winter 2021. Different degrees of variation were observed in the contents of ash, metals and metalloids, vitamins, fatty acids, amino acids and biogenic amines, and monosaccharides. Our results indicate that biomass is of highly variable quality depending on season and location. In this context, we suggest that biorefinery approaches geared towards controlled metal removal and focused on the extraction and purification of amino acids, fatty acids and vitamins should be prioritised to assess the potential valorisation of pelagic Sargassum biomass into standardised and high-value outputs.

Flavodoxins are small electron transport proteins that are involved in a myriad of photosynthetic and non-photosynthetic metabolic pathways in Bacteria (including cyanobacteria), Archaea and some algae. The sequenced genome of 0305φ8-36, a large bacteriophage that infects the soil bacterium Bacillus thuringiensis, was predicted to encode a putative flavodoxin redox protein. Here we confirm that 0305φ8-36 phage encodes a FMN-containing flavodoxin polypeptide and we report the expression, purification and enzymatic characterization of the recombinant protein. Purified 0305φ8-36 flavodoxin has near-identical spectral properties to control, purified Escherichia coli flavodoxin. Using in vitro assays we show that 0305φ8-36 flavodoxin can be reconstituted with E. coli flavodoxin reductase and support regio- and stereospecific cytochrome P450 CYP170A1 allyl-oxidation of epi-isozizaene to the sesquiterpene antibiotic product albaflavenone, found in the soil bacterium Streptomyces coelicolor. In vivo, 0305φ8-36 flavodoxin is predicted to mediate the 2-electron reduction of the β subunit of phage-encoded ribonucleotide reductase to catalyse the conversion of ribonucleotides to deoxyribonucleotides during viral replication. Our results demonstrate that this phage flavodoxin has the potential to manipulate and drive bacterial P450 cellular metabolism, which may affect both the host biological fitness and the communal microbiome. Such a scenario may also be applicable in other viral-host symbiotic/parasitic relationships.

Investigation of virus-induced microalgal host lysis and the associated infection dynamics typically requires sampling of infected cultures at multiple timepoints, visually monitoring the state of infected cells, or determining virus titration within the culture media. Such approaches require intensive effort and are prone to low sensitivity and high error rates. Furthermore, natural physiological variations can become magnified by poor environmental control, which is often compounded by variability in virus stock efficacy and relatively long infection cycles. We introduce a new method that closely monitors host health and integrity to learn about the infection strategy of Chloroviruses. Our approach combines aspects of spectrometry, plaque assays, and infection dose assessment to monitor algal cells under conditions more representative of the natural environment. Our automated method exploits the continuous monitoring of infected microalgae cultures in highly controlled lab-scale photobioreactors that provide the opportunity for environmental control, technical replication, and intensive culture monitoring without external intervention or culture disruption. This approach has enabled the development of a protocol to investigate molecular signalling impacting the virus life cycle and particle release, accurate determination of virus lysis time under multiple environmental conditions, and assessment of the functional diversity of multiple virus isolates.

Rising oceanic temperatures create more frequent coral bleaching events worldwide and as such there exists a need for rapid, non-destructive survey techniques to gather greater and higher definition information than that offered by traditional spectral based monitoring systems. Here, we examine thermally induced laboratory bleaching of Montipora capricornis and Montipora confusa samples, utilising hyperspectral data to gain an understanding of coral bleaching from a spectral standpoint. The data revealed several characteristic spectral peaks that can be used to make health determinations. The fluorescence peaks are attributed to fluorescent proteins (FPs) and Chlorophyll-a fluorescence. The reflectance peaks can be attributed to Chlorophyll absorption and accessory pigments such as Peridinin and Diadinoxanthin. Each characteristic spectral peak or ‘marker’ allows for observation of each aspect of coral health and hence, simultaneous monitoring of these markers using hyperspectral imaging techniques provides an opportunity to better understand the processes occurring during bleaching and the rates at which they occur relative to one another.

Suitable immobilisation of microorganisms and single cells is key for high-resolution topographical imaging and study of mechanical properties with atomic force microscopy (AFM) under physiologically relevant conditions. Sample preparation techniques must be able to withstand the forces exerted by the Z range-limited cantilever tip, and not negatively affect the sample surface for data acquisition. Here, we describe an inherently flexible methodology, utilising the highresolution three-dimensional based printing technique of multiphoton polymerisation to rapidly generate bespoke arrays for cellular AFM analysis. As an example, we present data collected from live Emiliania huxleyi cells, unicellular microalgae, imaged by contact mode High-Speed Atomic Force Microscopy (HS-AFM), including one cell that was imaged continuously for over 90 min.

Historically, algae have stimulated significant economic interest particularly as a source of fertilizers, feeds, foods and pharmaceutical precursors. However, there is increasing interest in exploiting algal diversity for their antiviral potential. Here, we present an overview of 50-years of scientific and technological developments in the field of algae antivirals. After bibliometric analysis of 999 scientific references, a survey of 16 clinical trials and analysis of 84 patents, it was possible to identify the dominant algae, molecules and viruses that have been shaping and driving this promising field of research. A description of the most promising discoveries is presented according to molecule class. We observed a diverse range of algae and respective molecules displaying significant antiviral effects against an equally diverse range of viruses. Some natural algae molecules, like carrageenan, cyanovirin or griffithsin, are now considered prime reference molecules for their outstanding antiviral capacity. Crucially, while many algae antiviral applications have already reached successful commercialization, the large spectrum of algae antiviral capacities already identified suggests a strong potential for future expansion of this field.

Marine macroalgae offers a promising third generation feedstock for the production of fuels and chemicals, avoiding competition with conventional agriculture and potentially helping to improve eutrophication in seas and oceans. However, an increasing amount of plastic is distributed into the oceans, and as such contaminating macroalgal beds. One of the major plastic contaminants is nylon 6 derived from discarded fishing gear, though an increasing amount of alternative nylon polymers, derived from fabrics, are also observed. This study aimed to assess the effect of these nylon contaminants on the hydrothermal liquefaction of Fucus serratus. The hydrothermal liquefaction (HTL) of macroalgae was undertaken at 350◦ C for 10 min, with a range of nylon polymers (nylon 6, nylon 6/6, nylon 12 and nylon 6/12), in the blend of 5, 20 and 50 wt.% nylon to biomass; 17 wt.% biocrude was achieved from a 50% blend of nylon 6 with F. serratus. In addition, nylon 6 completely broke down in the system producing the monomer caprolactam. The suitability of converting fishing gear was further demonstrated by conversion of actual fishing line (nylon 6) with the macroalgae, producing an array of products. The alternative nylon polymer blends were less reactive, with only 54% of the nylon 6/6 breaking down under the HTL conditions, forming cyclopentanone which distributed into the biocrude phase. Nylon 6/12 and nylon 12 were even less reactive, and only traces of the monomer cyclododecanone were observed in the biocrude phase. This study demonstrates that while nylon 6 derived from fishing gear can be effectively integrated into a macroalgal biorefinery, alternative nylon polymers from other sectors are too stable to be converted under these conditions and present a real challenge to a macroalgal biorefinery.

In recent years there has been a significant effort from food, nutraceutical, cosmeceutical, pharmaceutical, and biomedical industries to discover and develop new natural ingredients. Microalgae have been recognised as potential sources of high value chemicals, with most attention focused upon antioxidants, pigments, and specialty oils. An under-exploited group of biochemicals produced by microalgae are extracellular polymeric substances (EPS). Lobochlamys segnis (formerly called Chlamydomonas segnis) was previously reported to produce a large amount of capsular polysaccharide (CPS) closely related to hyaluronan (HA) under stress conditions. In this work, the purified CPS was characterised and shown to have an average molecular mass (Mn) of about 3.7 MDa, and displayed a highly branched random coil structure unlike the simple repeating linear HA polysaccharide. Chemical analysis showed the presence of galactose, glucuronic acid, and glucose sugars confirming that the product has a different composition to that of HA. Mixotrophic growth and stress conditions were identified and improved upon with a pH control system using acetic acid solution to induce efficient production of CPS. Extraction and purification conditions were also identified exploiting the high Mn of the product. The CPS showed thickening properties and both significant antioxidant capacity and reducing power, which could have commercial applications. This is the first report on the characterization of this novel polysaccharide with non-Newtonian properties similar to HA.

For some professionally, vocationally, or technically oriented careers, curricula delivered in higher education establishments may focus on teaching material related to a single discipline. By contrast, multidisciplinary, interdisciplinary, and transdisciplinary teaching (MITT) results in improved affective and cognitive learning and critical thinking, offering learners/students the opportunity to obtain a broad general knowledge base. Chemistry is a discipline that sits at the interface of science, technology, engineering, mathematics, and medicine (STEMM) subjects (and those aligned with or informed by STEMM subjects). This article discusses the significant potential of inclusion of chemistry in MITT activities in higher education and the real-world importance in personal, organizational, national, and global contexts. It outlines the development and implementation challenges attributed to legacy higher education infrastructures (that call for creative visionary leadership with strong and supportive management and administrative functions), and curriculum design that ensures inclusivity and collaboration and is pitched and balanced appropriately. It concludes with future possibilities, notably highlighting that chemistry, as a discipline, underpins industries that have multibillion dollar turnovers and employ millions of people across the world.

Harvesting microalgal cultures is often energetically intensive and costly. To improve efficiencies, a two-step harvesting method utilising alkaline flocculation and sedimentation to pre-concentrate cultures can be used prior to centrifugation. When applied to the microalga Chromochloris zofingiensis, high rates of sedimentation (>90%) were found at low concentrations of base (

Oleaginous microalgae and yeast are of increasing interest as a renewable resource for single cell oils (SCOs). These have applications in fuels, feed and food products. In order to become cost competitive with existing terrestrial oils, a biorefinery approach is often taken where several product streams are valorised alongside the SCO. Whilst many life cycle assessment (LCA) and Techno-economic (TEA) studies have employed this biorefinery approach to SCO production, a systematic analysis of their implications is missing. This review evaluates the economic and environmental impacts associated with the use of coproducts. Overall, protein production plays the greatest role in determining viability, with coproduct strategy crucial to considering in the early stages of research and development.

In this investigation a novel synergistic approach for the bioremediation of metal-contaminated water and bioenergy production was developed. Two microalgae, Chlorella vulgaris and Arthrospira platensis (Spirulina), and two macroalgae, Ulva lactuca and Sargassum muticum, were used as passive bioremediation agents for the metals Ni(ii), Zn(ii), Cd(ii) and Cu(ii). The metals were added singularly and in combination between 10-150 mM. The metal contaminated biomass was then processed through hydrothermal liquefaction to yield four phases: a bio-crude oil, an aqueous phase, solid residue and gas. Both C. vulgaris and A. platensis gave high bio-crude yields of 39 and 31 wt% respectively, while U. lactuca and S. muticum gave 14% and 9% respectively. Initial studies demonstrated that the addition of up to 150 mM of the target metal sulfates to the biomass feedstock did not significantly affect bio-crude production, and, for microalgae, over 99% of the target metals were partitioned to the solid phase products predominantly as phosphates or oxides. Subsequently, bioremediation of waste water and HTL were successfully coupled, with over 80% of a 10 mM solution of the metals biosorbed, though efficacy depended heavily on the algal species. Upon HTL of the remediating biomass, the yield and composition of the bio-crude were not changed significantly. For the microalgae, the aqueous phase contained significant nitrogen, potassium and phosphate levels, and the majority of the target metals deposited in the solid phase, with over 99.5% metal recovery for Spirulina when all four metals were used. The macroalgal species were not as effective in this process, with limited phosphate recovery in the aqueous phase (albeit with extensive potassium recovery) and with less than 50% of the target metals depositing in the solid residue for the Ulva species examined, presumably due to the affinity of the metals to proteinous species rather than polysaccharide in this species. Combining microalgal bioremediation with hydrothermal liquefaction is therefore a potentially highly effective method of remediating contaminated waste waters, whilst a macroalgae based process may offer a cheaper alternative, albeit with substantially reduced efficacy. The recovery of the target metals and multiple product formation improves the economic viability of the process, thereby valorising the bioremediation process and subsidising environmental clean-up.

Seaweeds contain many varied and commercially valuable components, from individual pigments and metabolites through to whole biomass, and yet they remain an under cultivated and underutilised commodity. Currently, commercial exploitation of seaweeds is predominantly limited to whole biomass consumption or single product extracts for the food industry. The development of a seaweed biorefinery, based around multiple products and services, could provide an important opportunity to exploit new and currently underexplored markets. Here, we assessed the native and invasive seaweeds on the South West coast of the UK to determine their characteristics and potential for exploitation through a biorefinery pipeline, looking at multiple components including pigments, carbohydrates, lipids, proteins and other metabolites.

Macroalgal blooms are environmentally problematic and costly to remediate, but they also represent a vast untapped resource for the production of renewable chemicals and fuels. The responsible exploitation of such marine resources will become increasingly prominent in the transition away from the crude oil economy that currently dominates global productivity. However, crude oil-derived plastic pollution is now a ubiquitous presence in the marine environment, which hampers the effective conversion of marine feedstocks. If the full potential of macroalgae is to be realized, any large-scale industrial process will need to accommodate the presence of this plastic. This study, for the first time, aimed to assess the effect of several common marine plastic pollutants on the hydrothermal liquefaction (HTL) of four UK macroalgae species and determine the impact on the major HTL products and biocrude oil quality. Co-liquefaction of polyethylene and polypropylene with L. digitata, U. lactuca, F. serratus, and S. muticum led to modest synergistic effects for plastic conversion. Under hydrothermal conditions, polyethylene underwent fragmentation to olefinic species, as well as oxidative depolymerization to form ketones. Modest synergistic effects on biocrude production were also observed for polypropylene, which depolymerized more readily in the presence of biomass to form gaseous propylene as well as oil-phase products. In both cases, the presence of plastics increased total biocrude carbon content, decreased nitrogen, and boosted higher heating value (HHV), constituting an overall improvement in biocrude fuel properties. Alternatively, nylon-6, typically originating from fisheries debris, depolymerized almost entirely under HTL conditions to form caprolactam, which partitioned mainly to the aqueous phase. While this is not favorable for biocrude production, the reclamation of marine nylon debris for hydrothermal processing to monomers may present a promising revenue stream in future biorefineries. The results demonstrate that plastic contaminants may well represent an opportunity, rather than a threat, to the successful development of an HTL macroalgal biorefinery.

Plant triterpenoids constitute a diverse class of organic compounds that play a major role in development, plant defence and environmental interaction. Several triterpenes have demonstrated potential as pharmaceuticals. One example is betulin, which has shown promise as a pharmaceutical precursor for the treatment of certain cancers and HIV. Major challenges for triterpenoid commercialization include their low production levels and their cost-effective purification from the complex mixtures present in their natural hosts. Therefore, attempts to produce these compounds in industrially relevant microbial systems such as bacteria and yeasts have attracted great interest. Here, we report the production of the triterpenes betulin and its precursor lupeol in the photosynthetic diatom Phaeodactylum tricornutum, a unicellular eukaryotic alga. This was achieved by introducing three plant enzymes in the microalga: a Lotus japonicus oxidosqualene cyclase and a Medicago truncatula cytochrome P450 along with its native reductase. The introduction of the L. japonicus oxidosqualene cyclase perturbed the mRNA expression levels of the native mevalonate and sterol biosynthesis pathway. The best performing strains were selected and grown in a 550-L pilot-scale photobioreactor facility. To our knowledge, this is the most extensive pathway engineering undertaken in a diatom and the first time that a sapogenin has been artificially produced in a microalga, demonstrating the feasibility of the photo-bio-production of more complex high-value, metabolites in microalgae.

In all biorefinery systems, excess water represents a key challenge, and its removal by drying is often a necessary and crucial pre-treatment. Second-generation feedstocks have often fallen at this hurdle, particularly microalgae-derived biomasses, which require extensive (and costly) dewatering. Hydrothermal liquefaction (HTL) has gained increasing attention in recent years as a technology that uses the water present in the feedstock as a versatile reaction medium, which functions as a solvent, reactant, and catalyst for a cascade of organic reactions. Converting organic biomasses into oil, aqueous, solid, and gas fractions, the development of HTL provides the opportunity to exploit previously unsuitable biomasses in a versatile bio-refinery approach. Marine macroalgae (seaweeds) offer a sustainable source of renewable biomass, which require no land or freshwater to cultivate or harvest. With 70% of the surface of the planet covered in seawater and levels of eutrophication increasing, seaweeds are an underutilized resource with excellent potential for relieving the pressure on fossil resources. Hitherto, this exploitation has been hindered by a lack of suitable and economical processing tools. Here we review the potential for applying HTL to processing marine macroalgae and discuss the potential products and services that can be derived from this potential biorefinery system. © 2019 Society of Chemical Industry and John Wiley & Sons, Ltd.

Fluorescent proteins are a crucial visualisation tool in a myriad of research fields including cell biology, microbiology and medicine. Fluorescence is a result of the absorption of electromagnetic radiation at one wavelength and its reemission at a longer wavelength. Coral communities exhibit a natural fluorescence which can be used to distinguish between diseased and healthy specimens, however, current methods, such as the underwater visual census, are expensive and time-consuming constituting many manned dive hours. We propose the use of a remotely operated vehicle mounted with a novel hyperspectral fluorescence imaging (HyFI) “payload” for more rapid surveying and data collection. We have tested our system in a laboratory environment on common coral species including Seriatopora spp. Montipora verrucosa, Montipora spp. Montipora capri-cornis, Echinopora lamellose, Euphyllia ancora, Pocillopora damicornis and Montipora confusa. With the aid of hyperspectral imaging, the coral speci-mens’ emission wavelengths can be accurately assessed by capturing the emission spectra of the corals when excited with light emitting diodes (395–405 and 440 nm). Fluorescence can also provide an indicator of coral bleaching as shown in our bleaching experiment where we observe fluorescence reduction alongside coral bleaching.

Electro-coagulation floatation (ECF) is a foam-floatation dewatering method that has been shown to be a highly effective, rapid, and scalable separation methodology. In this manuscript, an in-depth analysis of the gas and flocculant levels observed during the process is provided, with microbubbles observed in the 5–80 μm size range at a concentration of 10 2 –10 3 bubbles mL −1. Electrolysis of microalgae culture was then observed, demonstrating both effective separation using aluminium electrodes (nine microalgal species tested, 1–40 μm size range, motile and non-motile, marine and freshwater), and sterilisation of culture through bleaching with inert titanium electrodes. Atomic force microscopy was used to visualise floc formation in the presence and absence of algae, showing nanoscale structures on the magnitude of 40–400 nm and entrapped microalgal cells. Improvements to aid industrial biotechnology processing were investigated: protein-doping was found to improve foam stability without inducing cell lysis, and an oxalate buffer wash regime was found to dissolve the flocculant whilst producing no observable difference in the final algal lipid or pigment profiles, leaving the cells viable at the end of the process. ECF separated microalgal culture had an algal biomass loading of 13% and as such was ideal for direct down-stream processing through hydrothermal liquefaction. High bio-crude yields were achieved, though this was reduced slightly on addition of the Al(OH) 3 after ECF, with carbon being distributed away to the aqueous and solid residue phases. The amenability and compatibility of ECF to integration with, or replacement of, existing centrifugation and settling processes suggests this process may be of significant interest to the biotechnology industry.

Macroalgae (seaweeds) represent an emerging resource for food and the production of commodity and specialty chemicals. In this study, a single-step microwave process was used to depolymerise a range of macroalgae native to the United Kingdom, producing a growth medium suitable for microbial fermentation. The medium contained a range of mono- and polysaccharides as well as macro- and micronutrients that could be metabolised by the oleaginous yeast Metschnikowia pulcherrima. Among twelve macroalgae species, the brown seaweeds exhibited the highest fermentation potential, especially the kelp Saccharina latissima. Applying a portfolio of ten native M. pulcherrima strains, yeast growth kinetics, as well as production of lipids and 2-phenylethanol were examined, with productivity and growth rate being strain dependent. On the 2 L scale, 6.9 g L−1 yeast biomass – a yield of 0.14 g g−1 with respect to the supplied macroalgae – containing 37.2% (w/w) lipid was achieved through utilisation of the proteins, mono- and polysaccharides from S. latissima, with no additional enzymes. In addition, the yeast degraded a range of fermentation inhibitors released upon microwave processing at high temperatures and long holding times. As macroalgae can be cultured to food grade, this system offers a novel, potentially low-cost route to edible microbial oils as well as a renewable feedstock for oleochemicals.

Intense anthropogenic activity continues to expose the natural environment to heavy metal contamination. Whilst a number of physical and chemical solutions for remediation exist, the use of higher plants and algae for clean-up of contaminated landscapes, termed ‘phytoremediation’ and ‘phycoremediation’, respectively, offer an attractive and sustainable alternative. However, these remediation processes will always lead to a high-moisture, heavy metal-contaminated biomass, which must be further processed to partition, or render inert, the metal contaminants. Conversion of this metal-rich biomass into second-generation biofuels offers a useful route to subsidise the economics of remediation activities. Here we briefly review the various methods for bioremediation of heavy metals, and discuss the potential to produce bioenergy from these biomass sources. Coupling the bioremediation activity to bioenergy production gives far-reaching social and economic benefits; however, established processes such as direct combustion and anaerobic digestion risk releasing heavy metals back into the environment. Alternatively, thermochemical conversions such as pyrolysis or hydrothermal liquefaction (HTL) offer significant advantages in terms of the segregation of metals into a relatively inert and compact solid phase while producing a biocrude oil for bioenergy production. In addition, preliminary work suggests that the HTL process can also be used to partition essential macronutrients, such as N, P and K, into an aqueous medium, allowing additional nutrient recycling. © 2019 Society of Chemical Industry.

Marine macroalgae (seaweed) has many advantages over terrestrial crops as a source of renewable biomass but is severely underutilised at present, especially within Europe. In particular, macroalgae has elevated poly- and monosaccharide content, making it an ideal feedstock as a heterotrophic fermentation sugar source for the production of higher value chemicals. Recent reports have detailed the suitability of seaweeds as a feedstock for the production of single-cell oils (SCOs) which have application in food, oleochemicals and fuels. It is proposed that a biorefinery system based on the production of SCOs alongside other secondary metabolites, has the potential to provide a sustainable replacement to terrestrial oils such as palm oil. This work therefore evaluates, for the first time, the environmental and economic sustainability of a production process for SCOs from seaweed Saccharina latissima using the oleaginous yeast Metschnikowia pulcherrima. Two alternative fermentation systems were considered, and uncertainties associated with the seasonal variation in seaweed carbohydrate yield and fermentation performance were integrated into the analysis. From an environmental perspective, the work indicates that seaweed derived SCO lipids and fats can be comparable to a terrestrial oil mix, with a potential climate change impact ranging between 2.5 and 9.9 kg CO2 eq. kg−1 refined SCO. Interestingly and of particular significance, environmental impacts are mainly dominated by energy demand within fermentation and upstream processing steps. From an economic perspective, a break-even selling price for the oil was determined as between €5,300-€31,000 tonne−1 refined SCO, which was highly dependent on cost of the seaweed feedstock. Overall, we demonstrate that key uncertainties relating to seaweed cultivation costs and hydrolysate fermentation at scale result in a large range in values for environmental impact and economic return on investment. Yet even within the constraints and limitations of current knowhow, seaweed already offers a viable proposition for the competitive production of exotic oils similar to cocoa or shea butter in price and nature.

There is clear scientific evidence that emissions of greenhouse gases (GHG), arising from fossil fuel combustion and land-use change as a result of human activities, are perturbing the Earth's climate. Microalgae-derived biofuels have been chased since the 1980swithout success but, lately, a new biorefinery concept is receiving increasing attention. Here, we discuss the possible solutions to the many problems that make this process unrealised to date, considering also the possibility of including genetically modified (GM) organisms to improve the productivity and process economics. Currently, unless coupled to a service or higher value product production, biofuels derived from microalgae fail to achieve economic reality. However, provided sufficient development of new technologies, potentially including new or improved organisms to lower both production and processing costs, as well as looking at the utility of distributed versus centralised production models, algae biofuels could achieve an impact, off-setting our heavy reliance on petroleum-based liquid fuels.

© 2018 by the authors. Licensee MDPI, Basel, Switzerland. Visualization of algal viruses has been paramount to their study and understanding. The direct observation of the morphological dynamics of infection is a highly desired capability and the focus of instrument development across a variety of microscopy technologies. However, the high temporal (ms) and spatial resolution (nm) required, combined with the need to operate in physiologically relevant conditions presents a significant challenge. Here we present a short history of virus structure study and its relation to algal viruses and highlight current work, concentrating on electron microscopy and atomic force microscopy, towards the direct observation of individual algae–virus interactions. Finally, we make predictions towards future algal virus study direction with particular focus on the exciting opportunities offered by modern high-speed atomic force microscopy methods and instrumentation.

Present study consists the incidence of enteric bacteria in the Kapıdağ Peninsula. Nine sampling stations were selected and taken seasonally from 2011-2012. Presence of Escherichia coli was noted at all stations, except K1 and K6, throughout the sampling period irrespective of season. K1 and K6 stations (utilised as public beaches during the summer months) and depths below 30m displayed distinct genetic profile suggestive of diverse community of enteric bacteria other than E. coli. Analysis of uidA gene showed temporal and spatial genetic homogeneity in these E. coli populations. Ability to characterize E. coli communities might not only facilitate the mitigation of fecal pollution on Kapıdağ Peninsula, but also address the impact of environmental disturbances and seasonal and spatial variation on pathogen indicators.

The development of low-cost, open-source Remotely Operated Vehicle (ROV) systems has provided almost unrestricted access for researchers looking to monitor the marine environment in ever greater resolution. Sampling microbial communities from the marine environment, however, still usually relies on Niskin-bottle sampling (ROV or Conductivity-Temperature-Depth sampler (CTD) based), a method which introduces an inaccuracy and variability that is incompatible with metatranscriptomic analysis, for example. Here, we describe a versatile, easily-replicated platform which achieves in situ mRNA preservation, via the addition of RNAlater to filtered microbial cells, to enhance ROV or CTD functionality.

Coccolithoviruses (Phycodnaviridae) infect and lyse the most ubiquitous and successful coccolithophorid in modern oceans, Emiliania huxleyi. So far, the genomes of 13 of these giant lytic viruses (i.e. Emiliania huxleyi viruses—EhVs) have been sequenced, assembled, and annotated. Here, we performed an in‐depth comparison of their genomes to try and contextualize the ecological and evolutionary traits of these viruses. The genomes of these EhVs have from 444 to 548 coding sequences (CDSs). Presence/absence analysis of CDSs identified putative genes with particular ecological significance, namely sialidase, phosphate permease, and sphingolipid biosynthesis. The viruses clustered into distinct clades, based on their DNA polymerase gene as well as full genome comparisons. We discuss the use of such clustering and suggest that a gene‐by‐gene investigation approach may be more useful when the goal is to reveal differences related to functionally important genes. A multi domain “Best BLAST hit” analysis revealed that 84% of the EhV genes have closer similarities to the domain Eukarya. However, 16% of the EhV CDSs were very similar to bacterial genes, contributing to the idea that a significant portion of the gene flow in the planktonic world inter‐crosses the domains of life.

Algae have been proposed as a source of biofuels and high value chemical products, but if this potential is to be fully realised, it is crucial to understand the factors affecting the suspension rheology. Suspensions of three algae species, Tetraselmis chuii, Chlorella sp. and Phaeodactylum tricornutum, were sheared in a rotational rheometer in order to characterise their rheology and examine the effects of cell concentration, motility and morphology. The volume fraction ranged from 0.05 to 0.2, and the shear rate from 20 to 200 s−1. The rheology measurements are fitted to the Herschel-Bulkley model, and the intrinsic viscosity is estimated using both Einstein’s equation and the Krieger-Dougherty model, which are found to perform well for low concentrations. The intrinsic viscosity of T. chuii suspensions is shown not to be constant, but decreases with strain rate, indicating that the suspension viscosity is less sensitive to the cell concentration at high strain rates. The rate of decline is constant for strain rates below approximately 100 s−1, after which it continues to decline linearly, but at a slower rate. It is speculated that this transition at 100 s−1 is related to the appearance of flocculation at low strain rates. The effect of the cell motility on the rheology of T. chuii suspensions is investigated by comparing the rheology of motile and passive cells. The shear-thinning behaviour is absent and the effective viscosity is considerably lower for the passive cell suspensions, indicating that the motility of the T. chuii cells causes them to align to resist the flow. In contrast, the Chlorella sp. suspensions exhibit shear-thickening behaviour, which has not previously been reported. Finally, the influence of the effective aspect ratio on the cell suspensions is examined by comparing the intrinsic viscosity of all three species. The algal species with the largest aspect ratio, P. tricornutum, has the largest intrinsic viscosity, while the smallest aspect ratio strain, Chlorella sp. has the smallest viscosity. However, it is shown that the increase in viscosity of motile compared to non-motile T. chuii suspensions cannot be attributed to a change in the effective aspect ratio of individual cells due to the motion of the flagella alone.

The genetic modification of microalgal strains for enhanced or modified metabolic activity shows great promise for biotechnological exploitation. However, of key concern for many is the safety of genetic modification technology and genetically modified organisms with regard to both the environment and human health, and how these concerns are met will play a key role in ensuring how successful commercialisation of genetically modified (GM) algae is achieved. Commercialisation opportunities for GM microalgae will inevitably require translation from laboratory to industrial settings, on scales beyond those typically associated with the current biotechnology sector. Here we provide an overview of the current situation with regards to genetic modification techniques and legislation, and the implications of large-scale cultivation with regards to developing a safe and effective risk assessment system for contained and uncontained activities. We discuss the rationale and options for modification and the implications for risks associated with scale up to human health and the environment, current grey areas in political/technical legislation, the use of contained/uncontained production systems, deliberate release and monitoring strategies. We conclude that while existing procedures are not entirely sufficient for accurate and exhaustive risk assessment, there exists a substantial knowledge base and expertise within the existing aquaculture, fermentation and (algal) biotechnology industries that can be combined and applied to ensure safe use in the future.

© 2017 by the authors. Viruses play a crucial role in the marine environment, promoting nutrient recycling and biogeochemical cycling and driving evolutionary processes. Tiny marine phytoplankton called prasinophytes are ubiquitous and significant contributors to global primary production and biomass. A number of viruses (known as prasinoviruses) that infect these important primary producers have been isolated and characterised over the past decade. Here we review the current body of knowledge about prasinoviruses and their interactions with their algal hosts. Several genes, including those encoding for glycosyltransferases, methyltransferases and amino acid synthesis enzymes, which have never been identified in viruses of eukaryotes previously, have been detected in prasinovirus genomes. The host organisms are also intriguing; most recently, an immunity chromosome used by a prasinophyte in response to viral infection was discovered. In light of such recent, novel discoveries, we discuss why the cellular simplicity of prasinophytes makes for appealing model host organism–virus systems to facilitate focused and detailed investigations into the dynamics of marine viruses and their intimate associations with host species. We encourage the adoption of the prasinophyte Ostreococcus and its associated viruses as a model host–virus system for examination of cellular and molecular processes in the marine environment.

© the Royal Society of Chemistry 2017. Biorefineries have been established since the 1980s for biofuel production, and there has been a switch lately from first to second generation feedstocks in order to avoid the food versus fuel dilemma. To a lesser extent, many opportunities have been investigated for producing chemicals from biomass using by-products of the present biorefineries, simple waste streams. Current facilities apply intensive pre-treatments to deal with single substrate types such as carbohydrates. However, most organic streams such as municipal solid waste or algal blooms present a high complexity and variable mixture of molecules, which makes specific compound production and separation difficult. Here we focus on flexible anaerobic fermentation and hydrothermal processes that can treat complex biomass as a whole to obtain a range of products within an integrated biorefinery concept.

Hydrothermal liquefaction (HTL) is a promising biomass conversion method that can be incorporated into a biorefinery paradigm for simultaneous production of fuels, aqueous fertilisers and potential remediation of municipal or mariculture effluents. HTL of aquatic crops, such as marine macro- or microalgae, has significant potential for the UK owing to its extensive coastline. As such, macroalgae present a particularly promising feedstock for future UK biofuel production. This study aimed to bridge the gaps between previous accounts of macroalgal HTL by carrying out a more comprehensive screen of a number of species from all three major macroalgae classes, and examining the correlations between biomass biochemical composition and HTL reactivity. HTL was used to process thirteen South West UK macroalgae species from all three major classes (Chlorophyceae, Heterokontophyceae and Rhodophyceae) to produce bio-crude oil, a bio-char, gas and aqueous phase products. Chlorophyceae of the genus Ulva generated the highest bio-crude yields (up to 29.9% for U. lactuca). Aqueous phase phosphate concentrations of up to 236 mg L−1 were observed, obtained from the Rhodophyta, S. chordalis. Across the 13 samples, a correlation between increasing biomass lipids and increasing bio-crude yield was observed, as well as an increase in biomass nitrogen generally contributing to bio-crude nitrogen content. A broader range of macroalgae species has been examined than in any study previously and, by processing using identical conditions across all feedstocks, has enabled a more cohesive assessment of the effects of biochemical composition.

The hydrothermal liquefaction (HTL) of algal biomass is a promising route to viable second generation biofuels. In this investigation HTL was assessed for the valorisation of algae used in the remediation of acid mine drainage (AMD). Initially the HTL process was evaluated using Arthrospira platensis (Spirulina) with additional metal sulphates to simulate metal remediation. Optimised conditions were then used to process a natural algal community (predominantly Chlamydomonas sp.) cultivated under two scenarios: high uptake and low uptake of metals from AMD. High metal concentrations appear to catalyse the conversion to bio-oil, and do not significantly affect the heteroatom content or higher heating value of the bio-oil produced. The associated metals were found to partition almost exclusively into the solid residue, favourable for potential metal recovery. High metal loadings also caused partitioning of phosphates from the aqueous phase to the solid phase, potentially compromising attempts to recycle process water as a growth supplement. HTL was therefore found to be a suitable method of processing algae used in AMD remediation, producing a crude oil suitable for upgrading into hydrocarbon fuels, an aqueous and gas stream suitable for supplementing the algal growth and the partitioning of most contaminant metals to the solid residue where they would be readily amenable for recovery and/or disposal.

Microalgae are of increasing interest due to their occurrence in the environment as harmful algal blooms and as a source of biomass for the production of fine and bulk chemicals. A method for the low cost disruption of algal biomass for environmental remediation or bioprocessing is desirable. Naturally-occurring algal lytic agents from bacteria could provide a cost-effective and environmentally desirable solution. A screen for algal lytic agents against a range of marine microalgae has identified two strains of algicidal bacteria isolated from the coastal region of the Western English Channel. Both strains (designated EC-1 and EC-2) showed significant algicidal activity against Skeletonema sp. and were identified as members of Alteromonas sp. and Maribacter sp. respectively. Characterisation of the two bioactivities revealed that they are small extracellular metabolites displaying thermal and acid stability. Purification of the EC-1 activity to homogeneity and initial structural analysis has identified it as a putative peptide with a mass of 1266. amu.

A polyhydroxybutyrate (PHB) producing cyanobacteria was converted through hydrothermal liquefaction (HTL) into propylene and a bio-oil suitable for advanced biofuel production. HTL of model compounds demonstrated that in contrast to proteins and carbohydrates, no synergistic effects were detected when converting PHB in the presence of algae. Subsequently, Synechocystis cf. salina, which had accumulated 7.5 wt% PHB was converted via HTL (15% dry weight loading, 340°C). The reaction gave an overall propylene yield of 2.6%, higher than that obtained from the model compounds, in addition to a bio-oil with a low nitrogen content of 4.6%. No propylene was recovered from the alternative non-PHB producing cyanobacterial strains screened, suggesting that PHB is the source of propylene. PHB producing microorganisms could therefore be used as a feedstock for a biorefinery to produce polypropylene and advanced biofuels, with the level of propylene being proportional to the accumulated amount of PHB.

Viruses are a major cause of coccolithophore bloom demise in both temperate and sub-temperate oceanic regions. Most infection studies on coccolithoviruses have been conducted with a single virus strain, and the effect of intragenus competition by closely related coccolithoviruses has been ignored. Here we conducted combined infection experiments, infecting Emiliania huxleyiCCMP 2090 with two coccolithoviruses: EhV-86 and EhV-207 both simultaneously and independently. EhV-207 displayed a shorter lytic cycle and increased production potential than EhV-86 and was remarkably superior under competitive conditions. Although the viruses displayed identical adsorption kinetics in the first 2h post infection, EhV-207 gained a numerical advantage as early as 8h post infection. Quantitative polymerase chain reaction (PCR) revealed that when infecting in combination, EhV-207 was not affected by the presence of EhV-86, whereas EhV-86 was quickly out-competed, and a significant reduction in free and cell-associated EhV-86 was seen as early as 2 days after the initial infection. The observation of such clear phenotypic differences between genetically distinct, yet similar, coccolithovirus strains, by flow cytometry and quantitative real-time PCR allowed tentative links to the burgeoning genomic, transcriptomic and metabolic data to be made and the factors driving their selection, in particular to the de novo coccolithovirus-encoded sphingolipid biosynthesis pathway. This work illustrates that, even within a family, not all viruses are created equally, and the potential exists for relatively small genetic changes to infer disproportionately large competitive advantages for one coccolithovirus over another, ultimately leading to a few viruses dominating the many.

Despite the increasing use of Decentralised Waste Water Systems (DEWATS) in the developing world, which effectively dewater sludge, the problem of preventing the pathogen-laden water produced by these systems from re-entering the food chain constitutes a continuing burden on developing countries, which hinders subsequent advancements. We report on a swirl flow reactor generating high mixing areas which in conjunction with Cu/alginate beads effectively reduces Escherichia coli numbers by five orders of magnitude in 10. min. The system is simple, low cost, portable and modular; it can be assembled with simple plastic plumbing parts available in most areas and, once further developed, may represent a useful adjunct for both existing and new DEWATS facilities.

It is estimated that approximately 1.1 billion people globally drink unsafe water. We previously reported both a novel copper-alginate bead, which quickly reduces pathogen loading in waste streams and the incorporation of these beads into a novel swirl flow bioreactor (SFB), of low capital and running costs and of simple construction from commercially available plumbing pipes and fittings. The purpose of the present study was to trial this system for pathogen reduction in waste streams from an operating Dewats system in Hinjewadi, Pune, India and in both simulated and real waste streams in Seattle, Washington, USA. The trials in India, showed a complete inactivation of coliforms in the discharged effluent (Mean Log removal Value (MLRV) = 3.51), accompanied by a total inactivation of E. coli with a MLRV of 1.95. The secondary clarifier effluent also showed a 4.38 MLRV in viable coliforms during treatment. However, the system was slightly less effective in reducing E. coli viability, with a MLRV of 1.80. The trials in Seattle also demonstrated the efficacy of the system in the reduction of viable bacteria, with a LRV of 5.67 observed of viable Raoultella terrigena cells (100%).

The marine diatom Phaeodactylum tricornutum can accumulate up to 30% of the omega-3 long chain polyunsaturated fatty acid (LC-PUFA) eicosapentaenoic acid (EPA) and, as such, is considered a good source for the industrial production of EPA. However, P.Tricornutum does not naturally accumulate significant levels of the more valuable omega-3 LCPUFA docosahexaenoic acid (DHA). Previously, we have engineered P.Tricornutum to accumulate elevated levels of DHA and docosapentaenoic acid (DPA) by overexpressing heterologous genes encoding enzyme activities of the LC-PUFA biosynthetic pathway. Here, the transgenic strain Pt-Elo5 has been investigated for the scalable production of EPA and DHA. Studies have been performed at the laboratory scale on the cultures growing in up to 1 L flasks a 3.5 L bubble column, a 550 L closed photobioreactor and a 1250 L raceway pond with artificial illumination. Detailed studies were carried out on the effect of different media, carbon sources and illumination on omega-3 LC-PUFAs production by transgenic strain Pt-Elo5 and wild type P.Tricornutum grown in 3.5 L bubble columns. The highest content of DHA (7.5% of total fatty acids, TFA) in transgenic strain was achieved in cultures grown in seawater salts, Instant Ocean (IO), supplemented with F/2 nutrients (F2N) under continuous light. After identifying the optimal conditions for omega-3 LC-PUFA accumulation in the small-scale experiments we compared EPA and DHA levels of the transgenic strain grown in a larger fence-style tubular photobioreactor and a raceway pond. We observed a significant production of DHA over EPA, generating an EPA/DPA/DHA profile of 8.7%/4.5%/12.3% of TFA in cells grown in a photobioreactor, equivalent to 6.4 μg/mg dry weight DHA in a mid-exponentially growing algal culture. Omega-3 LC-PUFAs production in a raceway pond at ambient temperature but supplemented with artificial illumination (110 μmol photons m-2s-1) on a 16:8h light:dark cycle, in natural seawater and F/2 nutrients was 24.8% EPA and 10.3% DHA. Transgenic strain grown in RP produced the highest levels of EPA (12.8%) incorporated in neutral lipids. However, the highest partitioning of DHA in neutral lipids was observed in cultures grown in PBR (7.1%). Our results clearly demonstrate the potential for the development of the transgenic Pt-Elo5 as a platform for the commercial production of EPA and DHA.

We show in this study that the combination of a swirl flow reactor and an antimicrobial agent (in this case copper alginate beads) is a promising technique for the remediation of contaminated water in waste streams recalcitrant to UV-C treatment. This is demonstrated by comparing the viability of both common and UV-C resistant organisms in operating conditions where UV-C proves ineffective - notably high levels of solids and compounds which deflect UV-C. The swirl flow reactor is easy to construct from commonly available plumbing parts and may prove a versatile and powerful tool in waste water treatment in developing countries.

Most marine scientists have a love-hate relationship with dolphins, sharks, whales and turtles. They tend to get all the public's attention. We have pictures of them on our walls, own cuddly toys in their likeness and amuse our children with animated tales about their fictional adventures. Unfortunately, they distract from the real beauty and interest in the marine environment: the microbes, and in particular, the viruses.

Despite the global oceanic distribution and recognised biogeochemical impact of coccolithoviruses (EhV), their diversity remains poorly understood. Here we employed a metagenomic approach to study the occurrence and progression of natural EhV community genomic variability. Analysis of EhV metagenomes from the early and late stages of an induced bloom led to three main discoveries. First, we observed resilient and specific genomic signatures in the EhV community associated with the Norwegian coast, which reinforce the existence of limitations to the capacity of dispersal and genomic exchange among EhV populations. Second, we identified a hyper-variable region (approximately 21. kbp long) in the coccolithovirus genome. Third, we observed a clear trend for EhV relative amino-acid diversity to reduce from early to late stages of the bloom. This study validated two new methodological combinations, and proved very useful in the discovery of new genomic features associated with coccolithovirus natural communities.

Flavin-containing monooxygenases (FMOs) catalyse asymmetric oxidation reactions that have potential for preparative organic synthesis, but most use the more expensive, phosphorylated nicotinamide cofactor NADPH to reduce FAD to FADH2 prior to formation of the (hydro)peroxy intermediate required for substrate oxygenation. A comparison of the structures of NADPH-dependent FMO from Methylophaga aminisulfidivorans (mFMO) and SMFMO from Stenotrophomonas maltophilia, which is able to use both NADPH and NADH, suggested that the promiscuity of the latter enzyme may be due in part to the substitution of an Arg-Thr couple in the NADPH phosphate recognition site in mFMO, for a Gln-His couple in SMFMO (Jensen et al. 2012, Chembiochem, 13, 872-878). Natural variation within the phosphate binding region, and its influence on nicotinamide cofactor promiscuity, was explored through the cloning, expression, characterisation and structural studies of FMOs from Cellvibrio sp. BR (CFMO) and Pseudomonas stutzeri NF13 (PSFMO), which possess Thr-Ser and Gln-Glu in the putative phosphate recognition positions, respectively. CFMO and PSFMO displayed 5- and 1.5-fold greater activity, respectively, than SMFMO for the reduction of FAD with NADH, and were also cofactor promiscuous, displaying a ratio of activity with NADH:NADPH of 1.7:1 and 1:1.3, respectively. The structures of CFMO and PSFMO revealed the context of the phosphate binding loop in each case, and also clari.ed the structure of the mobile helix-loop-helix motif that appears to shield the FAD-binding pocket from bulk solvent in this class of FMOs, a feature that was absent from the structure of SMFMO.

Coccolithoviruses infect the marine coccolithophorid microalga Emiliania huxleyi. Here, we describe the genomes of four new coccolithoviruses isolated from UK coastal locations. of particular interest, EhV-18 and EhV-145 encode serine palmitoyltransferase function via two distinct genes, whereas all other coccolithoviruses have SPT as a gene fusion of LCB1/LCB2 domains. © 2014 Elsevier B.V.

The prevalence of faecal coliform bacteria and to detect Escherichia coli via the uidA gene, to identify the associated faecal pollution sources and to predict faecal contamination in an active seaside area of the Marmara Sea. Systematic microbiological testing and PCR/DGGE analysis of subsequent isolates. Most probable number (MPN) analysis was performed to monitor the E. coli population in seawater over a period of 24 months (January 2009-December 2010) and the genetic heterogeneity was determined using uidA PCR/DGGE analysis of subsequent isolates. The prevalence of total coliform bacteria ranged from a mean MPN value of 101-104 per 100 ml. In this study, in addition to E.coli other enteric bacteria species were also detected at Stations 4 and 5. It could be concluded that post bloom waters contain secondary metabolite compounds exhibiting anti-bacterial activity against E.coli. Analysis of uidA gene using BLAST analysis showed a temporal and spatial genetic homogeneity in E. coli marine populations. We concluded that the enteric bacteria levels observed at the stations sampled, are a likely consequence of the discharge of untreated wastewater to the Ayamama Stream and Kucukcekmece Lagoon on the coastal line. It is interesting to note that there were no remedial activities, even though the anthropogenic pollutants were above levels considered a threat to human health. The high density of faecal coliform bacteria and prevalence of E. coli in the area under study suggests a potential public health risk of water-borne outbreaks.

During viral infection of Emiliania huxleyi, laboratory studies have shown that photo - system (PS) II efficiency declines during the days post-infection and is thought to be associated with viral-induced interruption of electron transport rates between photosystems. However, measuring the impact of viral infection on PSII function in E. huxleyi populations from natural, taxo nomically diverse phytoplankton communities is difficult, and whether this phenomenon occurs in nature is presently unknown. Here, chlorophyll fluorescence analysis was used to assess changes in PSII efficiency throughout an E. huxleyi bloom during a mesocosm experiment off the coast of Norway. Specifically, we aimed to determine whether a measurable suppression of the efficiency of PSII photochemistry could be observed due to viral infection of the natural E. huxleyi populations. During the major infection period prior to bloom collapse, there was a significant reduction in PSII efficiency with an average decrease in maximum PSII photochemical efficiency (Fv/Fm) of 17% and a corresponding 75% increase in maximum PSII effective absorption crosssection (sPSII); this was concurrent with a significant decrease in E. huxleyi growth rates and an increase in E. huxleyi virus (EhV) production. As E. huxleyi populations dominated the phytoplankton community and potentially contributed up to 100% of the chlorophyll a pool, we believe that the variable chlorophyll fluorescence signal measured during this period was derived predominantly from E. huxleyi and, thus, reflects changes occurring within E. huxleyi cells. This is the first demonstration of suppression of PSII photochemistry occurring during viral infection of natural coccolithophore populations. © Inter-Research 2014.

Although the bactericidal effect of copper has been known for centuries, there is a current resurgence of interest in the use of this element as an antimicrobial agent. During this study the use of dendritic copper microparticles embedded in an alginate matrix as a rapid method for the deactivation of Escherichia coli ATCC 11775 was investigated. The copper/alginate produced a decrease in the minimum inhibitory concentration from free copper powder dispersed in the media from 0.25 to 0.065 mg/ml. Beads loaded with 4% Cu deactivated 99.97% of bacteria after 90 minutes, compared to a 44.2% reduction in viability in the equivalent free copper powder treatment. There was no observed loss in the efficacy of this method with increasing bacterial loading up to 10(6) cells/ml, however only 88.2% of E. coli were deactivated after 90 minutes at a loading of 10(8) cells/ml. The efficacy of this method was highly dependent on the oxygen content of the media, with a 4.01% increase in viable bacteria observed under anoxic conditions compared to a >99% reduction in bacterial viability in oxygen tensions above 50% of saturation. Scanning electron micrographs (SEM) of the beads indicated that the dendritic copper particles sit as discrete clusters within a layered alginate matrix, and that the external surface of the beads has a scale-like appearance with dendritic copper particles extruding. E. coli cells visualised using SEM indicated a loss of cellular integrity upon Cu bead treatment with obvious visible blebbing. This study indicates the use of microscale dendritic particles of Cu embedded in an alginate matrix to effectively deactivate E. coli cells and opens the possibility of their application within effective water treatment processes, especially in high particulate waste streams where conventional methods, such as UV treatment or chlorination, are ineffective or inappropriate.

Cytochrome b5 is a ubiquitous electron transport protein. The sequenced viral OtV-2 genome, which infects Ostreococcus tauri, was predicted to encode a putative cytochrome b5 enzyme. Using purified OtV-2 cytochrome b5 we confirm this protein has identical spectral properties to purified human cytochrome b5 and additionally that the viral enzyme can substitute for yeast cytochrome b5 in yeast cytochrome P450 51 mediated sterol 14α-demethylation. The crystal structure of the OtV-2 cytochrome b5 enzyme reveals a single domain, comprising four β sheets, four α helices and a haem moiety, which is similar to that found in larger eukaryotic cytochrome proteins. As a product of a horizontal gene transfer event involving a subdomain of the host fumarate reductase-like protein, OtV-2 cytochrome b5 appears to have diverged in function and is likely to have evolved an entirely new role for the virus during infection. Indeed, lacking a hydrophobic C-terminal anchor, OtV-2 encodes the first cytosolic cytochrome b5 characterised. The lack of requirement for membrane attachment (in contrast to all other microsomal cytochrome b5s) may be a reflection of the small size of the host cell, further emphasizes the unique nature of this virus gene product and draws attention to the potential importance of cytochrome b5 metabolic activity at the extremes of cellular scale.

The cosmopolitan calcifying alga Emiliania huxleyi is one of the most abundant bloom forming coccolithophore species in the oceans and plays an important role in global biogeochemical cycling. Coccolithoviruses are a major cause of coccolithophore bloom termination and have been studied in laboratory, mesocosm and open ocean studies. However, little is known about the dynamic interactions between the host and its viruses, and less is known about the natural diversity and role of functionally important genes within natural coccolithovirus communities. Here, we investigate the temporal and spatial distribution of coccolithoviruses by the use of molecular fingerprinting techniques PCR, DGGE and genomic sequencing. The natural biodiversity of the virus genes encoding the major capsid protein (MCP) and serine palmitoyltransferase (SPT) were analysed in samples obtained from the Atlantic Meridional Transect (AMT), the North Sea and the L4 site in the Western Channel Observatory. We discovered nine new coccolithovirus genotypes across the AMT and L4 site, with the majority of MCP sequences observed at the deep chlorophyll maximum layer of the sampled sites on the transect. We also found four new SPT gene variations in the North Sea and at L4. Their translated fragments and the full protein sequence of SPT from laboratory strains EhV-86 and EhV-99B1 were modelled and revealed that the theoretical fold differs among strains. Variation identified in the structural distance between the two domains of the SPT protein may have an impact on the catalytic capabilities of its active site. In summary, the combined use of 'standard' markers (i.e. MCP), in combination with metabolically relevant markers (i.e. SPT) are useful in the study of the phylogeny and functional biodiversity of coccolithoviruses, and can provide an interesting intracellular insight into the evolution of these viruses and their ability to infect and replicate within their algal hosts. © 2013 Wuhan Institute of Virology, CAS and Springer-Verlag Berlin Heidelberg.

The flavoprotein monooxygenase (FPMO) from Stenotrophomonas maltophilia (SMFMO, Uniprot: B2FLR2) catalyses the asymmetric oxidation of thioethers and is unusual amongst FPMOs in its ability to use the non-phosphorylated cofactor NADH, as well as NADPH, for the reduction of the FAD coenzyme. In order to explore the basis for cofactor promiscuity, structure-guided mutation of two residues in the cofactor binding site, Gln193 and His194, in SMFMO were performed in an attempt to imitate the cofactor binding site of the NADPH-dependent FMO from Methylophaga aminisulfidivorans sp. SK1 (mFMO), in which structurally homologous residues Arg234 and Thr235 bind the NADPH 2'-ribose phosphate. Mutation of His194 to threonine proved most significant, with a switch in specificity from NADH to NADPH [(kcat/Km NADH)/kcat/Km NADPH) from 1.5:1 to 1:3.5, mostly as a result of a reduced Km for NADPH of approximately sevenfold in the His194Thr mutant. The structure of the Gln193Arg/His194Thr mutant revealed no substantial changes in the backbone of the enzyme or orientation of side chains resulting from mutation. Mutation of Phe52, in the vicinity of FAD, and which in mFMO is an asparagine thought to be responsible for flavin hydroperoxide stabilisation, is, in SMFMO, a determinant of enantioselectivity in sulfoxidation. Mutation of Phe52 to valine resulted in a mutant that transformed para-tolyl methyl sulfide into the (S)-sulfoxide with 32% e.e. compared to 25% (R)- for the wild type. These results shed further light both on the cofactor specificity of FPMOs, and their determinants of enantioselectivity, with a view to informing engineering studies of FPMOs in the future. © 2013 the Authors.

A gene from the marine bacterium Stenotrophomonas maltophilia encodes a 38.6 kDa FAD-containing flavoprotein (Uniprot B2FLR2) named S. maltophilia flavin-containing monooxygenase (SMFMO), which catalyses the oxidation of thioethers and also the regioselective Baeyer-Villiger oxidation of the model substrate bicyclo[3.2.0]hept-2-en-6-one. The enzyme was unusual in its ability to employ either NADH or NADPH as nicotinamide cofactor. The K M and k cat values for NADH were 23.7±9.1 μM and 0.029 s -1 and 27.3±5.3 μM and 0.022 s -1 for NADPH. However, k cat/K M value for the ketone substrate in the presence of 100 μM cofactor was 17 times greater for NADH than for NADPH. SMFMO catalysed the quantitative conversion of 5 mM ketone in the presence of substoichiometric concentrations of NADH with the formate dehydrogenase cofactor recycling system, to give the 2-oxa and 3-oxa lactone products of Baeyer-Villiger reaction in a ratio of 5:1, albeit with poor enantioselectivity. The conversion with NADPH was 15%. SMFMO also catalysed the NADH-dependent transformation of prochiral aromatic thioethers, giving in the best case, 80% ee for the transformation of p-chlorophenyl methyl sulfide to its R enantiomer. The structure of SMFMO reveals that the relaxation in cofactor specificity appears to be accomplished by the substitution of an arginine residue, responsible for recognition of the 2′-phosphate on the NADPH ribose in related NADPH-dependent FMOs, with a glutamine residue in SMFMO. SMFMO is thus representative of a separate class of single-component, flavoprotein monooxygenases that catalyse NADH-dependent oxidations from which possible sequences and strategies for developing NADH-dependent biocatalysts for asymmetric oxygenation reactions might be identified. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The Coccolithoviridae are a group of viruses which infect the marine coccolithophorid microalga Emiliania huxleyi. The Emiliania huxleyi viruses (known as EhVs) described herein have 160- to 180-nm diameter icosahedral structures, have genomes of approximately 400 kbp, and consist of more than 450 predicted coding sequences (CDSs). Here, we describe the genomic features of four newly sequenced coccolithoviruses (EhV-88, EhV-201, EhV-207, and EhV-208) together with their draft genome sequences and their annotations, highlighting the homology and heterogeneity of these genomes to the EhV-86 model reference genome. © 2012, American Society for Microbiology.

Emiliania huxleyi virus 202 (EhV-202) is a member of the Coccolithoviridae, a group of viruses that infect the marine coccolithophorid Emiliania huxleyi. EhV-202 has a 160- to 180-nm-diameter icosahedral structure and a genome of approximately 407 kbp, consisting of 485 coding sequences (CDSs). Here we describe the genomic features of EhV-202, together with a draft genome sequence and its annotation, highlighting the homology and heterogeneity of this genome in comparison with the EhV-86 reference genome. © 2012, American Society for Microbiology.

Stenotrophomonas maltophilia PML168 was isolated from Wembury Beach on the English Coast from a rock pool following growth and selection on agar plates. Here we present the permanent draft genome sequence, which has allowed prediction of function for several genes encoding enzymes relevant to industrial biotechnology, including a novel flavoprotein monooxygenase. © 2012, American Society for Microbiology.

Coccolithoviruses are giant dsDNA viruses that infect Emiliania huxleyi, the most ubiquitous marine microalga. Here, we present the genome of the latest coccolithovirus strain to be sequenced, EhV-99B1, and compare it with two other coccolithovirus genomes (EhV-86 and EhV-163). EhV-99B1 shares a pairwise nucleotide identity of 98% with EhV-163 (the two strains were isolated from the same Norwegian fjord but in different years), and just 96.5% with EhV-86 (isolated in the same spring as EhV-99B1 but in the English Channel). We confirmed and extended the list of relevant genomic differences between these EhVs from the Norwegian fjord and EhVs from the English Channel, namely the removal/insertions of: a phosphate permease, an endonuclease, a transposase, and two specific tRNAs. As a whole, this study provided new clues and insights into the diversity and mechanisms driving the evolution of these large oceanic viruses, in particular those processes involving selfish genetic elements. Copyright © 2012 S. Karger AG, Basel.

The identification of inteins in viral genomes is becoming increasingly common. Inteins are selfish DNA elements found within coding regions of host proteins. Following translation, they catalyse their own excision and the formation of a peptide bond between the flanking protein regions. Many inteins also display homing endonuclease function. Here, the newly identified coccolithovirus intein is described and is predicted to have both self-splicing and homing endonuclease activity. The biochemical mechanism of its protein splicing activity is hypothesised, and the prevalence of the intein among natural coccolithovirus isolates is tested. © 2010.

Emiliania huxleyi is a single celled, marine phytoplankton with global distribution. As a key species for global biogeochemical cycling, a variety of strains have been amassed in various culture collections. Using a library consisting of 52 strains of E. huxleyi and an 'in house' enzyme screening program, we have assessed the functional biodiversity within this species of fundamental importance to global biogeochemical cycling, whilst at the same time determining their potential for exploitation in biocatalytic applications. Here, we describe the screening of E. huxleyi strains, as well as a coccolithovirus infected strain, for commercially relevant biocatalytic enzymes such as acid/alkali phosphodiesterase, acid/alkali phosphomonoesterase, EC1.1.1-type dehydrogenase, EC1.3.1-type dehydrogenase and carboxylesterase.

The Coccolithoviridae is a recently discovered group of viruses that infect the marine coccolithophorid Emiliania huxleyi. Emiliania huxleyi virus 84 (EhV-84) has a 160 -180 nm diameter icosahedral structure and a genome of approximately 400 kbp. Here we describe the structural and genomic features of this virus, together with a near complete draft genome sequence (~99%) and its annotation. This is the fourth genome sequence of a member of the coccolithovirus family.

The Coccolithoviridae are a recently discovered group of viruses that infect the marine coccolithophorid Emiliania huxleyi. Emiliania huxleyi virus 203 (EhV-203) has a 160- to 180-nm-diameter icosahedral structure and a genome of approximately 400 kbp, consisting of 464 coding sequences (CDSs). Here we describe the genomic features of EhV-203 together with a draft genome sequence and its annotation, highlighting the homology and heterogeneity of this genome in comparison with the EhV-86 reference genome. © 2011, American Society for Microbiology.

Ostreococcus tauri, a unicellular marine green alga, is the smallest known free-living eukaryote and is ubiquitous in the surface oceans. The ecological success of this organism has been attributed to distinct low-and high-light-adapted ecotypes existing in different niches at a range of depths in the ocean. Viruses have already been characterized that infect the high-light-adapted strains. Ostreococcus tauri virus (OtV) isolate OtV-2 is a large double-stranded DNA algal virus that infects a low-light-adapted strain of O. tauri and was assigned to the algal virus family Phycodnaviridae, genus Prasinovirus. Our working hypothesis for this study was that different viruses infecting high- versus low-light-adapted O. tauri strains would provide clues to propagation strategies that would give them selective advantages within their particular light niche. Sequence analysis of the 184,409-bp linear OtV-2 genome revealed a range of core functional genes exclusive to this low-light genotype and included a variety of unexpected genes, such as those encoding an RNA polymerase sigma factor, at least four DNA methyltransferases, a cytochrome b5, and a high-affinity phosphate transporter. It is clear that OtV-2 has acquired a range of potentially functional genes from its host, other eukaryotes, and even bacteria over evolutionary time. Such piecemeal accretion of genes is a trademark of large double-stranded DNA viruses that has allowed them to adapt their propagation strategies to keep up with host niche separation in the sunlit layers of the oceanic environment. © 2011, American Society for Microbiology.

The Prymnesiophyceae coccolithophore Emiliania huxleyi is one of the most abundant alga in our oceans and therefore plays a central role in marine foodwebs. E. huxleyi is notable for the synthesis and accumulation of the omega-3 long chain polyunsaturated fatty acid docosahexaenoic acid (DHA; 22:6Δ4,7,10,13,16,19, n - 3) which is accumulated in fish oils and known to have health-beneficial properties to humans, preventing cardiovascular disease and related pathologies. Here we describe the identification and functional characterisation of the five E. huxleyi genes which direct the synthesis of docosahexaenoic acid in this alga. Surprisingly, E. huxleyi does not use the conventional Δ6-pathway, instead using the alternative Δ8-desaturation route which has previously only been observed in a few unrelated microorganisms. Given that E. huxleyi accumulates significant levels of the Δ6-desaturated fatty acid stearidonic acid (18:4Δ6,9,12,15, n - 3), we infer that the biosynthesis of DHA is likely to be metabolically compartmentalised from the synthesis of stearidonic acid. © 2011 Elsevier Ltd. All rights reserved.

Vibrio tubiashii NCIMB 1337 is a major and increasingly prevalent pathogen of bivalve mol-lusks, and shares a close phylogenetic relationship with both V. orientalis and V. coralliilyti-cus. It is a Gram-negative, curved rod-shaped bacterium, originally isolated from a moribund juvenile oyster, and is both oxidase and catalase positive. It is capable of growth under both aerobic and anaerobic conditions. Here we describe the features of this organism, together with the draft genome and annotation. The genome is 5,353,266 bp long, consisting of two chromosomes, and contains 4,864 protein-coding and 86 RNA genes.

The Earth's oceans are teeming with life. Despite our obsession with the terrestrial environment we inhabit, it is the oceans that first gave rise to life 2.9 billion years ago, and, with an extra billion years or so of evolution behind them, it is the oceans that harbour the greatest genetic and metabolic diversity on the planet. Yet we know surprisingly little about the biochemistry of our oceans. Microbial life forms covering the three cellular kingdoms (Archaea, Bacteria and Eukarya) represent by far the largest fraction of the total biomass existent in the oceans. However, a poorly studied and, until recently, largely ignored other kingdom also exists: the viral kingdom. Entirely dependent on the three cellular kingdoms of life, the viral kingdom rules above with an iron fist, ruthlessly and mercilessly destroying their hosts in order to replicate and produce their own progeny. © 2011 the Biochemical Society.

Lytic viruses have been implicated in the massive cellular lysis observed during algal blooms, through which they assume a prominent role in oceanic carbon and nutrient flows. Despite their impact on biogeochemical cycling, the transcriptional dynamics of these important oceanic events is still poorly understood. Here, we employ an oligonucleotide microarray to monitor host (Emiliania huxleyi) and virus (coccolithovirus) transcriptomic features during the course of E. huxleyi blooms induced in seawater-based mesocosm enclosures. Host bloom development and subsequent coccolithovirus infection was associated with a major shift in transcriptional profile. In addition to the expected metabolic requirements typically associated with viral infection (amino acid and nucleotide metabolism, as well as transcription- and replication-associated functions), the results strongly suggest that the manipulation of lipid metabolism plays a fundamental role during host-virus interaction. The results herein reveal the scale, so far massively underestimated, of the transcriptional domination that occurs during coccolithovirus infection in the natural environment. © 2011 Federation of European Microbiological Societies.

Mike Allen and Willie Wilson examine their work on the coccolithoviruses, some of the most abundant and widespread organisms in the oceans. These photosynthetic microscopic algae form the base of the oceans' food chain and play a major role in the global carbon cycle, drawing down vast volumes of carbon dioxide from the atmosphere. Analysis of these viruses' genetic make-up revealed large double-stranded DNA viruses with genomes of about 410,000 base pairs. These viruses belong to the newly-created genus Coccolithovirus. Emiliania huxleyi seems to use an intriguing strategy to avoid viral infection. It switches to an entirely different state in its life cycle so that an attacking virus can not get a foothold. When the virus infects a coccolithophore, it quickly turns on this sphingolipid pathway and expresses it to a level two orders of magnitude greater than the normal Emiliania huxleyi pathway.

The cosmopolitan coccolithophore Emiliania huxleyi forms frequent massive blooms and thus is important for global climate and the carbon cycle. Lytic viral infection of this alga leads to termination of blooms and therefore influences global climate. To understand the host-virus interaction of E. huxleyi an expressed sequence tag (EST) approach was used to determine changes in gene expression during viral infection. Three cDNA libraries, generated 6, 12 and 24 h after viral infection, were compared to a library from an uninfected culture by sequencing, clustering and manual annotation of 1100-1500 ESTs per library. To verify the gene expression results of the ESTs we used two-colour oligonucleotide microarrays. A total of 4480 ESTs were assembled into 1871 clusters, of which, 223 are of viral origin. Microarray expression analysis indicated that 231 out of 565 oligonucleotides of E. huxleyi changed their expression level for at least at one time point in response to viral infection. Results suggest that viral infection affects the following processes: photosynthesis, transcription and translation, carbohydrate and lipid metabolism (particularly glycolysis), metabolism, and signal transduction. Results of this study provide insights into the gene expression of E. huxleyi during infection by the virus EhV-86. © 2010 British Phycological Society.

Ostreococcus tauri virus (OtV-1) is a large double-stranded DNA virus and a prospective member of the family Phycodnaviridae, genus Prasinovirus. OtV-1 infects the unicellular marine green alga O. tauri, the smallest known free-living eukaryote. Here we present the 191 761 base pair genome sequence of OtV-1, which has 232 putative protein-encoding and 4 tRNA-encoding genes. Approximately 31% of the viral gene products exhibit a similarity to proteins of known functions in public databases. These include a variety of unexpected genes, for example, a PhoH-like protein, a N-myristoyltransferase, a 3-dehydroquinate synthase, a number of glycosyltransferases and methyltransferases, a prolyl 4-hydroxylase, 6-phosphofructokinase and a total of 8 capsid proteins. A total of 11 predicted genes share homology with genes found in the Ostreococcus host genome. In addition, an intein was identified in the DNA polymerase gene of OtV-1. This is the first report of an intein in the genome of a virus that infects O. tauri. Fifteen core genes common to nuclear-cytoplasmic large dsDNA virus (NCLDV) genomes were identified in the OtV-1 genome. This new sequence data may help to redefine the classification of the core genes of these viruses and shed new light on their evolutionary history. © 2009 Society for Applied Microbiology and Blackwell Publishing Ltd.

Interactions between viruses and phytoplankton, the main primary producers in the oceans, affect global biogeochemical cycles and climate. Recent studies are increasingly revealing possible cases of gene transfers between cyanobacteria and phages, which might have played significant roles in the evolution of cyanobacteria/phage systems. However, little has been documented about the occurrence of horizontal gene transfer in eukaryotic phytoplankton/virus systems. Here we report phylogenetic evidence for the transfer of seven genes involved in the sphingolipid biosynthesis pathway between the cosmopolitan eukaryotic microalga Emiliania huxleyi and its large DNA virus EhV. PCR assays indicate that these genes are prevalent in E. huxleyi and EhV strains isolated from different geographic locations. Patterns of protein and gene sequence conservation support that these genes are functional in both E. huxleyi and EhV. This is the first clear case of horizontal gene transfer of multiple functionally linked enzymes in a eukaryotic phytoplankton-virus system. We examine arguments for the possible direction of the gene transfer. The virus-to-host direction suggests the existence of ancient viruses that controlled the complex metabolic pathway in order to infect primitive eukaryotic cells. In contrast, the host-to-virus direction suggests that the serial acquisition of genes involved in the same metabolic pathway might have been a strategy for the ancestor of EhVs to stay ahead of their closest relatives in the great evolutionary race for survival. © 2009 by Cold Spring Harbor Laboratory Press.

The interactions between viruses and phytoplankton play a key role in shaping the ecological and evolutionary dynamics of oceanic ecosystems. One of the most fascinating examples of horizontal gene transfer between a eukaryotic host and its virus is a de novo sphingolipid biosynthesis pathway (SBP) found in the genomes of both Emiliania huxleyi and its coccolithovirus EhV-86. Here, we focus on a natural E. huxleyi/coccolithovirus system off the coast of Norway and investigate the dynamics of host and virus homologous gene expression for two of the most important sphingolipid biosynthesis enzymes, serine palmitoyl transferase (SPT) and dihydroceramide desaturase (DCD). Transcriptional dynamics display three defined stages along E. huxleyi bloom formation and decline, with the coccolithovirus transcripts taking over and controlling the SBP in stages 2 and 3. The observed patterns fit the hypothesis according to which viral sphingolipids are involved in the timing and physical processes of virion release from the host cells. This study provides a unique insight into the transcriptional interplay of homologous metabolic pathways between virus and host during temporal progression of oceanic E. huxleyi blooms. © 2009 Society for Applied Microbiology and Blackwell Publishing Ltd.

Viruses are arguably the simplest form of life yet they play a crucial role in regulating planetary processes. From shuttling genes to 'lubricating' microbial loop dynamics, viruses are integral in shaping microbial ecology. In every environment on Earth the role of viruses goes far beyond the simple infect-replicate-kill cycle. Their enormous abundance and seemingly infinite diversity provide the vital clues to the true function of viruses. New 'omic' approaches are now allowing researchers to gain extraordinary insights into virus diversity and inferred function, particularly within aquatic environments. The development of molecular markers and application of techniques including microarrays, metagenomic sequencing and proteomic analysis are now being applied to virus communities. Despite this shift towards culture-independent approaches it has proved difficult to derive useful information about infection strategies since so much of the sequence information has no database matches. Future advances will involve tools such as microarrays to help determine the functionality of unknown genes. Sequence information should be considered as a starting point for asking questions and developing hypotheses about the role of viruses. It is an exciting new era for virus ecology and when used in combination with more traditional approaches, virus genomics will give us access to their ecological function on an unprecedented scale. © 2008 Elsevier Ltd. All rights reserved.

Background: Emiliania huxleyi virus 86 (EhV-86) is the type species of the genus Coccolithovirus within the family Phycodnaviridae. The fully sequenced 407,339 bp genome is predicted to encode 473 protein coding sequences (CDSs) and is the largest Phycodnaviridae sequenced to date. The majority of EhV-86 CDSs exhibit no similarity to proteins in the public databases. Results: Proteomic analysis by 1-DE and then LC-MS/MS determined that the virion of EhV-86 is composed of at least 28 proteins, 23 of which are predicted to be membrane proteins. Besides the major capsid protein, putative function can be assigned to 4 other components of the virion: two lectin proteins, a thioredoxin and a serine/threonine protein kinase. Conclusion: This study represents the first steps toward the identification of the protein components that make up the EhV-86 virion. Aside from the major capsid protein, whose function in the virion is well known and defined, the nature of the other proteins suggest roles involved with viral budding, caspase activation, signalling, anti-oxidation, virus adsorption and host range determination. © 2008 Allen et al; licensee BioMed Central Ltd.

The coccolithophore Emiliania huxleyi is one of the most successful eukaryotes in modern oceans. The two phases in its haplodiploid life cycle exhibit radically different phenotypes. The diploid calcified phase forms extensive blooms, which profoundly impact global biogeochemical equilibria. By contrast, the ecological role of the noncalcified haploid phase has been completely overlooked. Giant phycodnaviruses (Emiliania huxleyi viruses, EhVs) have been shown to infect and lyse diploid-phase cells and to be heavily implicated in the regulation of populations and the termination of blooms. Here, we demonstrate that the haploid phase of E. huxleyi is unrecognizable and therefore resistant to EhVs that kill the diploid phase. We further show that exposure of diploid E. huxleyi to EhVs induces transition to the haploid phase. Thus we have clearly demonstrated a drastic difference in viral susceptibility between life cycle stages with different ploidy levels in a unicellular eukaryote. Resistance of the haploid phase of E. huxleyi provides an escape mechanism that involves separation of meiosis from sexual fusion in time, thus ensuring that genes of dominant diploid clones are passed on to the next generation in a virus-free environment. These "Cheshire Cat" ecological dynamics release host evolution from pathogen pressure and thus can be seen as an opposite force to a classic "Red Queen" coevolutionary arms race. In E. huxleyi, this phenomenon can account for the fact that the selective balance is tilted toward the boom-and-bust scenario of optimization of both growth rates of calcifying E. huxleyi cells and infectivity of EhVs. © 2008 by the National Academy of Sciences of the USA.

Blooms of the coccolithophorid Emiliania huxleyi can be infected by viruses, which can lead to bloom-termination. This pilot study used an expressed sequence tag (EST) approach to get a first view of gene-expression changes that occur during viral infection of E. huxleyi. cDNA libraries were constructed from uninfected cultures and 6, 12, and 24 h after infection with E. huxleyi-specific virus 86 (EhV-86). From each library 60-90 ESTs were randomly selected and annotated manually with PhyloGena. Viral genes were identified using BLAST-Search of the known viral genome. The data of this study show, that 6 h after viral infection the algal transcriptome changed significantly although few viral transcripts were present. At this point, changes mainly concerned transcripts related to photosynthesis and protein metabolism. However, after 24 h viral transcripts were most abundant. Viral transcripts found at this stage of viral infection encode proteins involved in protein degradation, nucleic acid degradation, transcription and replication. © 2007 Elsevier B.V. All rights reserved.

The diversity among the coccolithovirus strains held in the Plymouth Virus Collection (PVC) was assessed using three complementary techniques: phylogeny based on DNA polymerase and major capsid protein gene sequence; host range; and a new, microarray-based genome-wide approach. The PVC is composed of three groups of strains that are geographically and temporally distinct. Virus strains clustered according to these groups in all three diversity assessments. Furthermore, the microarray approach based on genomic content showed that two strains, previously considered as identical to others in the PVC, are actually distinct. These results show the importance of genome-wide surveys for assessing strain diversity. Not only has the microarray provided an alternative to the phylogeny-derived pattern for virus evolution, it has also begun to provide some clues to the genes that may be responsible for the different phenotypes displayed by these viruses. © 2007 the Authors.

We recently determined the genome sequence of the Coccolithoviridae strain Emiliania huxleyi virus 86 (EhV-86), a giant double-stranded DNA (dsDNA) algal virus from the family Phycodnaviridae that infects the marine coccolithophorid E. huxleyi. Here, we determine the phylogenetic relationship between EhV-86 and other large dsDNA viruses. Twenty-five core genes common to nuclear-cytoplasmic large dsDNA virus genomes were identified in the EhV-86 genome; sequence from eight of these genes were used to create a phylogenetic tree in which EhV-86 was placed firmly with the two other members of the Phycodnaviridae. We have also identified a 100-kb region of the EhV-86 genome which appears to have transferred into this genome from an unknown source. Furthermore, the presence of six RNA polymerase subunits (unique among the Phycodnaviridae) suggests both a unique evolutionary history and a unique lifestyle for this intriguing virus. © the Author 2005. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved.

The genus Coccolithovirus is a recently discovered group of viruses that infect the globally important marine calcifying microalga Emiliania huxleyi. Surprisingly, the viral genome contains a cluster of putative sphingolipid biosynthetic genes not found in other viral genus. To address the role of these genes in viral pathogenesis, the ehv050 gene predicted to encode a serine palmitoyltransferase (SPT), the first and rate-limiting enzyme of sphingolipid biosynthesis, was expressed and characterized in Saccharomyces cerevisiae. We show that the encoded protein is indeed a fully functional, endoplasmic reticulum-localized, single-chain SPT. In eukaryotes SPT is a heterodimer comprised of long chain base 1 (LCB1) and LCB2 subunits. Sequence alignment and mutational analysis showed that the N-terminal domain of the viral protein most closely resembled the LCB2 subunit and the C-terminal domain most closely resembled the LCB1 subunit. Regardless of whether the viral protein was expressed as a single polypeptide or as two independent domains, it exhibited an unusual preference for myristoyl-CoA rather than palmitoyl-CoA. This preference was reflected by the increased presence of C16-sphingoid bases in yeast cells expressing the viral protein. The occurrence of a single-chain SPT suggested to us that it might be possible to create other fusion SPTs with unique properties. Remarkably, when the two subunits of the yeast SPT were thus expressed, the single-chain chimera was functional and displayed a novel substrate preference. This suggests that expression of other multisubunit membrane proteins as single-chain chimera could provide a powerful approach to the characterization of integral membrane proteins. © 2006 by the American Society for Biochemistry and Molecular Biology, Inc.

Background: the Coccolithoviridae is a recently discovered family of viruses that infect the marine coccolithophorid Emiliania huxleyi. Following on from the sequencing of the type strain EhV-86, we have sequenced a second strain, EhV-163. Results: We have sequenced approximately 80% of the EhV-163 genome, equating to more than 200 full length CDSs. Conserved and variable CDSs and a gene replacement have been identified in the EhV-86 and EhV-163 genomes. Conclusion: the sequencing of EhV-163 has provided a wealth of information which will aid the re-annotating of the EhV-86 genome and identified a gene insertion in EhV-163. © 2006Allen et al; licensee BioMed Central Ltd.

Emiliania huxleyi virus strain 86 is the largest algal virus sequenced to date and is unique among the Phycodnaviridae since its genome is predicted to contain six RNA polymerase subunit genes. We have used a virus microarray to profile the temporal transcription strategy of this unusual virus during infection. There are two distinct transcription phases to the infection process. The primary phase is dominated by a group of coding sequences (CDSs) expressed by 1 h postinfection that are localized to a subregion of the genome. The CDS of the primary group have no database homologues, and each is associated with a unique promoter element. The remainder of the CDSs are expressed in a secondary phase between 2 and 4 hours postinfection. Compartmentalized transcription of the two distinctive phases is discussed. We hypothesize that immediately after infection the nucleic acid of the virus targets the host nucleus, where primary-phase genes are transcribed by host RNA polymerase which recognizes the viral promoter. Secondary-phase transcription may then be conducted in the cytoplasm. Copyright © 2006, American Society for Microbiology. All Rights Reserved.

EhV-86 is a large double stranded DNA virus with a 407,339 base pair circular genome that infects the globally important microalga Emiliania huxleyi. It belongs to a new genus of viruses termed the Coccolithoviridae within the algal virus family Phycodnaviridae. By plotting the EhV-86 genome against itself in a dot-plot analysis we revealed three families of distinctly different repeat sequences throughout its genome, designated Family A, B and C. Family a repeats are non-coding, found immediately upstream of 86 predicted coding sequences (CDSs) and are likely to play a crucial role in controlling the expression of the associated CDSs. Family B repeats are GC rich, coding and correspond to possible calcium binding sites in 22 proline-rich domains found in the protein products of eight predicted EhV-86 CDSs. Family C repeats are AT-rich, non-coding and are likely to form part of the origin of replication. We suggest that these repeat regions are of fundamental importance during virus propagation being involved with transcriptional control (Family A), virus adsorption/release (Family B) and DNA replication (Family C). © Springer-Verlag 2005.

The Coccolithoviridae is a recently discovered family of giant algal viruses. Here, we review the genomic and transcriptomic characterization of this family based on the results generated from a coccolithovirus microarray. The microarray has been used to aid the annotation of the genome, to investigate the infection process at the transcriptional level and to assess the diversity in genomic content within the family. © 2006 Oxford University Press.

The global response of Escherichia coli to the broad-spectrum biocide polyhexamethylene biguanide (PHMB) was investigated using transcriptional profiling. The transcriptional analyses were validated by direct determination of the PHMB-tolerance phenotypes of derivatives of E. coli MG1655 carrying either insertionally inactivated genes and/or plasmids expressing the cognate open reading frames from a heterologous promoter in the corresponding chromosomally inactivated strains. The results showed that a wide range of genes was altered in transcriptional activity and that all of the corresponding knockout strains subsequently challenged with biocide were altered in tolerance. of particular interest was the induction of the rhs genes and the implication of enzymes involved in the repair/binding of nucleic acids in the generation of tolerance, suggesting a novel dimension in the mechanism of action of PHMB based on its interaction with nucleic acids. © 2006 SGM.

The genus Coccolithovirus is a recently discovered group of viruses that infect the globally important marine calcifying microalga Emiliania huxleyi. Among the 472 predicted genes of the 407,339-base pair genome are a variety of unexpected genes, most notably those involved in biosynthesis of ceramide, a sphingolipid known to induce apoptosis. Uniquely for algal viruses, it also contains six RNA polymerase subunits and a novel promoter, suggesting this virus encodes its own transcription machinery. Microarray transcriptomic analysis reveals that 65% of the predicted virus-encoded genes are expressed during lytic infection of E. huxleyi.

The interaction between the broad-spectrum antimicrobial agent, polyhexamethylene biguanide (PHMB), and various nucleic acids was investigated. Titration of either single- or double-stranded 100-bp DNA, or mixed-molecular weight marker DNA, or tRNA with PHMB caused precipitation of a complex between nucleic acid and PHMB in which the nucleotide/biguanide ratio was always close to unity. Binding of PHMB was highly cooperative, with apparent Hill coefficients 10.3-14.6. When a fluorescent derivative of PHMB was titrated with increasing amounts of nucleic acid, all four forms of nucleic acid caused strong polarisation of fluorescence, demonstrating the association with PHMB. The intensity and broad-spectrum binding of PHMB to all forms of nucleic acid has significant implications for the mechanism of action of this biocide. © 2004 Elsevier Inc. All rights reserved.