Unicellular cyanobacteria of the genus Synechococcus are a major component of the picophytoplankton and make a substantial contribution to primary productivity in the oceans. Here we provide evidence that supports the hypothesis that virus infection can play an important role in determining the success of different Synechococcus genotypes and hence of seasonal succession. In a study of the oligotrophic Gulf of Aqaba, Red Sea, we show a succession of Synechococcus genotypes over an annual cycle. There were large changes in the genetic diversity of Synechococcus, as determined by restriction fragment length polymorphism analysis of a 403- bp rpoC1 gene fragment, which was reduced to one dominant genotype in July. The abundance of co-occurring cyanophage capable of infecting marine Synechococcus was determined by plaque assays and their genetic diversity was determined by denaturing gradient gel electrophoresis analysis of a 118-bp g20 gene fragment. The results indicate that both abundance and genetic diversity of cyanophage covaried with that of Synechococcus. Multivariate statistical analyses show a significant relationship between cyanophage assemblage structure and that of Synechococcus. These observations are consistent with cyanophage infection being a major controlling factor in picophytoplankton succession.

Phylum-and class-specific PCR primers were tested for the production of clone libraries and for denaturing gradient gel electrophoresis (DGGE) analysis of complex bacterial communities. Primers were designed to specifically amplify 16S rRNA gene fragments of the phyla Bacteroidetes, Planctomycetes and Firmicutes, of three classes of the phylum Proteobacteria, the Alphaproteobacteria, Betaproteobacteria and Gammaproteobacteria, and of the Cyanobacteria (including chloroplast 16S rRNA genes). The specificity of the seven primer pairs was tested by producing clone libraries from environmental DNA samples from mesotrophic (Norwegian coastal) and oligotrophic (Northern Atlantic Gyre) environments. Five of the seven primer pairs specifically amplified target 16S rRNA gene sequences. Exceptions were the Betaproteobacteria-and Firmicutes-specific primers, which were relatively successful with coastal water mesocosm samples but less so with the Northern Atlantic Gyre sample. Phylogenetic analysis of sequences from the Gammaproteobacteria clone library revealed that the coastal sample yielded a number of clones that clustered within clades that belong to the oligotrophic marine Gammaproteobacteria (OMG) group, indicating that this group is not confined exclusively to the oligotrophic environment. Comparison of the bacterial diversity of the environmental DNA sample from the coastal and the open ocean using a two-or three-step nested PCR-DGGE process revealed significant differences in the bacterial communities. The application of the group-specific primers provides a higher resolution genetic fingerprinting approach than existing DGGE primer sets.

How microbial communities change over time in response to the environment is poorly understood. Previously a six-year time series of 16S rRNA V6 data from the Western English Channel demonstrated robust seasonal structure within the bacterial community, with diversity negatively correlated with day-length. Here we determine whether metagenomes and metatranscriptomes follow similar patterns. We generated 16S rRNA datasets, metagenomes (1.2 GB) and metatranscriptomes (157 MB) for eight additional time points sampled in 2008, representing three seasons (Winter, Spring, Summer) and including day and night samples. This is the first microbial ‘multi-omic’ study to combine 16S rRNA amplicon sequencing with metagenomic and metatranscriptomic profiling. Five main conclusions can be drawn from analysis of these data: 1) Archaea follow the same seasonal patterns as Bacteria, but show lower relative diversity; 2) Higher 16S rRNA diversity also reflects a higher diversity of transcripts; 3) Diversity is highest in winter and at night; 4) Community-level changes in 16S-based diversity and metagenomic profiles are better explained by seasonal patterns (with samples closest in time being most similar), while metatranscriptomic profiles are better explained by diel patterns and shifts in particular categories (i.e., functional groups) of genes; 5) Changes in key genes occur among seasons and between day and night (i.e., photosynthesis); but these samples contain large numbers of orphan genes without known homologues and it is these unknown gene sets that appear to contribute most towards defining the differences observed between times. Despite the huge diversity of these microbial communities, there are clear signs of predictable patterns and detectable stability over time. Renewed and intensified efforts are required to reveal fundamental deterministic patterns in the most complex microbial communities. Further, the presence of a substantial proportion of orphan sequences underscores the need to determine the gene products of sequences with currently unknown function.

This review paper reports the consensus of a technical workshop hosted by the European network, NanoImpactNet (NIN). The workshop aimed to review the collective experience of working at the bench with manufactured nanomaterials (MNMs), and to recommend modifications to existing experimental methods and OECD protocols. Current procedures for cleaning glassware are appropriate for most MNMs, although interference with electrodes may occur. Maintaining exposure is more difficult with MNMs compared to conventional chemicals. A metal salt control is recommended for experiments with metallic MNMs that may release free metal ions. Dispersing agents should be avoided, but if they must be used, then natural or synthetic dispersing agents are possible, and dispersion controls essential. Time constraints and technology gaps indicate that full characterisation of test media during ecotoxicity tests is currently not practical. Details of electron microscopy, dark-field microscopy, a range of spectroscopic methods (EDX, XRD, XANES, EXAFS), light scattering techniques (DLS, SLS) and chromatography are discussed. The development of user-friendly software to predict particle behaviour in test media according to DLVO theory is in progress, and simple optical methods are available to estimate the settling behaviour of suspensions during experiments. However, for soil matrices such simple approaches may not be applicable. Alternatively, a Critical Body Residue approach may be taken in which body concentrations in organisms are related to effects, and toxicity thresholds derived. For microbial assays, the cell wall is a formidable barrier to MNMs and end points that rely on the test substance penetrating the cell may be insensitive. Instead assays based on the cell envelope should be developed for MNMs. In algal growth tests, the abiotic factors that promote particle aggregation in the media (e.g. ionic strength) are also important in providing nutrients, and manipulation of the media to control the dispersion may also inhibit growth. Controls to quantify shading effects, and precise details of lighting regimes, shaking or mixing should be reported in algal tests. Photosynthesis may be more sensitive than traditional growth end points for algae and plants. Tests with invertebrates should consider non-chemical toxicity from particle adherence to the organisms. The use of semi-static exposure methods with fish can reduce the logistical issues of waste water disposal and facilitate aspects of animal husbandry relevant to MMNs. There are concerns that the existing bioaccumulation tests are conceptually flawed for MNMs and that new test(s) are required. In vitro testing strategies, as exemplified by genotoxicity assays, can be modified for MNMs, but the risk of false negatives in some assays is highlighted. In conclusion, most protocols will require some modifications and recommendations are made to aid the researcher at the bench.

SummaryThe potential for using marine microbes for biodiscovery is severely limited by the lack of laboratory cultures. It is a long‐standing observation that standard microbiological techniques only isolate a very small proportion of the wide diversity of microbes that are known in natural environments from DNA sequences. A number of explanations are reviewed. The process of establishing laboratory cultures may destroy any cell‐to‐cell communication that occurs between organisms in the natural environment and that are vital for growth. Bacteria probably grow as consortia in the sea and reliance on other bacteria for essential nutrients and substrates is not possible with standard microbiological approaches. Such interactions should be considered when designing programmes for the isolation of marine microbes. The benefits of novel technologies for manipulating cells are reviewed, including single cell encapsulation in gel micro‐droplets. Although novel technologies offer benefits for bringing previously uncultured microbes into laboratory culture, many useful bacteria can still be isolated using variations of plating techniques. Results are summarized for a study to culture bacteria from a long‐term observatory station in the English Channel. Bacterial biodiversity in this assemblage has recently been characterized using high‐throughput sequencing techniques. Although Alphaproteobacteria dominated the natural bacterial assemblage throughout the year, Gammaproteobacteria were the most frequent group isolated by plating techniques. The use of different gelling agents and the addition of ammonium to seawater‐based agar did lead to the isolation of a higher proportion of Alphaproteobacteria. Variation in medium composition was also able to increase the recovery of other groups of particular interest for biodiscovery, such as Actinobacteria.

Recent advancements in the nanotechnology industry have seen a growing interest in integrating silver nanoparticles (Ag-NPs) into consumer and medical products. To date, there has been little research into the toxicological impact that Ag-NPs will have when they enter the marine realm. Of particular concern are the possible effects of Ag-NPs on natural bacterial assemblages, given the antimicrobial activity of silver. In this study, estuarine sediment samples were dosed in triplicate for 20 days from a stock solution of Ag-NPs, with a final cumulative treatment of either 0 microg L(-1) (control), 25 microg L(-1) or 1000 microg L(-1). The experimental tanks were left for a further 10 days to allow for any recovery. Inductively coupled plasma-optical emission spectrometery (ICP-OES) of water and sediment samples confirmed that the Ag-NPs concentration in the aqueous phase decreased after each dosing and were transported to accumulate in the surface layer of the sediment (approximately top 3 mm). The overall concentration of Ag-NPs in the water column, however, increased steadily during the 20 days of dosing but decreased rapidly during the following 10 days without dosing. Nevertheless, the Ag-NPs did not have any impact on the prokaryotic abundance in the water column over the incubation period (ANOVA, P < 0.05). Environmental DNA was extracted from sediment samples and a two-step nested PCR-denaturing gradient gel electrophoresis (DGGE) approach, using PCR primers specific to the phylum Bacteria, was adopted to assess their diversity. Multivariate statistical analyses of presence/ absence matrices produced from DGGE profiles revealed negligible differences in bacterial diversity between treatments, suggesting that, under the selected experimental regime, Ag-NPs present little or no impact on estuarine sediment bacterial diversity. Possible reasons for this could include environmental factors, in particular the chloride ions in estuary water affecting the chemistry and behavior of Ag-NPs.

Acid mine drainage (AMD), associated with active and abandoned mining sites, is a habitat for acidophilic microorganisms that gain energy from the oxidation of reduced sulfur compounds and ferrous iron and that thrive at pH below 4. Members of the recently proposed genus “Ferrovum” are the first acidophilic iron oxidizers to be described within the Betaproteobacteria. Although they have been detected as typical community members in AMD habitats worldwide, knowledge of their phylogenetic and metabolic diversity is scarce. Genomics approaches appear to be most promising in addressing this lacuna since isolation and cultivation of “Ferrovum” has proven to be extremely difficult and has so far only been successful for the designated type strain “Ferrovum myxofaciens” P3G. In this study, the genomes of two novel strains of “Ferrovum” (PN-J185 and Z-31) derived from water samples of a mine water treatment plant were sequenced. These genomes were compared with those of “Ferrovum” sp. JA12 that also originated from the mine water treatment plant, and of the type strain (P3G). Phylogenomic scrutiny suggests that the four strains represent three “Ferrovum” species that cluster in two groups (1 and 2). Comprehensive analysis of their predicted metabolic pathways revealed that these groups harbor characteristic metabolic profiles, notably with respect to motility, chemotaxis, nitrogen metabolism, biofilm formation and their potential strategies to cope with the acidic environment. For example, while the “F. myxofaciens” strains (group 1) appear to be motile and diazotrophic, the non-motile group 2 strains have the predicted potential to use a greater variety of fixed nitrogen sources. Furthermore, analysis of their genome synteny provides first insights into their genome evolution, suggesting that horizontal gene transfer and genome reduction in the group 2 strains by loss of genes encoding complete metabolic pathways or physiological features contributed to the observed diversification.

The impact of ocean acidification and carbonation on microbial community structure was assessed during a large-scale in situ costal pelagic mesocosm study, included as part of the EPOCA 2010 Arctic campaign. The mesocosm experiment included ambient conditions (fjord) and nine mesocosms with <i>p</i>CO₂ levels ranging from ~145 to ~1420 μatm. Samples for the present study were collected at ten time points (<i>t</i>&ndash;1, <i>t</i>1, <i>t</i>5, <i>t</i>7, <i>t</i>12, <i>t</i>14, <i>t</i>18, <i>t</i>22, <i>t</i>26 to <i>t</i>28) in seven treatments (ambient fjord (~145), 2 × ~185, ~270, ~685, ~820, ~1050 μatm) and were analysed for "small" and "large" size fraction microbial community composition using 16S RNA (ribosomal ribonucleic acid) amplicon sequencing. This high-throughput sequencing analysis produced ~20 000 000 16S rRNA V4 reads, which comprised 7000 OTUs. The main variables structuring these communities were sample origins (fjord or mesocosms) and the community size fraction (small or large size fraction). The community was significantly different between the unenclosed fjord water and enclosed mesocosms (both control and elevated CO₂ treatments) after nutrients were added to the mesocosms, suggesting that the addition of nutrients is the primary driver of the change in mesocosm community structure. The relative importance of each structuring variable depended greatly on the time at which the community was sampled in relation to the phytoplankton bloom. The sampling strategy of separating the small and large size fraction was the second most important factor for community structure. When the small and large size fraction bacteria were analysed separately at different time points, the only taxon <i>p</i>CO₂ was found to significantly affect were the Gammaproteobacteria after nutrient addition. Finally, <i>p</i>CO₂ treatment was found to be significantly correlated (non-linear) with 15 rare taxa, most of which increased in abundance with higher CO₂