Phylogenetic and Functional Metagenomic Profiling for Assessing Microbial Biodiversity in Environmental Monitoring

Environmental management decisions have to base on holistic understanding of biodiversity and functional capability in ecosystems. Environmental metagenomics is an emerging and powerful approach allowing rapidly and reliably determine and compare microbial biodiversity and functional profiles. Advances in next generation sequencing technologies and bioinformatic tools allow set up analysis pipelines which are useful for well designed and targeted monitoring exercises already today. In the paper we demonstrate how direct sequencing of the total community DNA and analysis of the data are applicable to assess anthropogenic impact on the coastal marine ecosystems.JRC.H.1-Water Resource

Evaluation of EDXRF for the Determination of Elements in PM10 Filter

Energy Dispersive X-ray Fluorescence (EDXRF) was compared to Inductively Coupled Plasma Mass Spectrometer (ICP-MS) for the measurements of elements in particulate matter (PM10). Lead (Pb), arsenic (As), nickel (Ni) and cadmium (Cd)) and the ones included in the EMEP programme, namely copper, chromium and zinc were tested. Other elements such as magnesium, aluminium, silicon, sulphur, chloride, potassium, calcium, titanium, vanadium, iron, cobalt, manganese, bromide, strontium, molybdenum, tin and antimony, which are essential for source apportionment studies, were also tested. PM10 samples collected at different sites on several types of filter were analyzed using both EDXRF and ICP-MS. As the overall result of this study, EDXRF can be considered as an alternative method to ICP-MS for measurements of PM-bound elements, particularly on Teflon filters. The European legislative requirements of Pb could be met for whatever filters type. The European legislative requirements of Ni and As are likely be met, but, it is unknown for Cd. The EMEP requirements can be met for Cu and Zn, but not for Cr. EDXRF can also be used to measure elements for source apportionment purposesJRC.H.2-Climate change and air qualit

Intercomparison Exercise for Heavy Metals in PM10

The Joint Research Centre (JRC) has carried out an Intercomparison Exercise (IE) for the determination of heavy metals in particulate matter (PM10). The IE focussed on Lead (Pb), Arsenic (As), Nickel (Ni) and Cadmium (Cd), the heavy metals regulated by the 1st and 4th Daughter Directives for Air Pollution. Copper (Cu), Chromium (Cr) and Zinc (Zn), the elements included in the EMEP programme together with Aluminium (Al), Cobalt (Co), Iron (Fe), Manganese (Mn) and Vanadium (V) were also tested. Fourteen Laboratories, generally members of the Network of Air Quality Reference Laboratories (AQUILA), participated in the IE. The participants mainly used microwave digestion with nitric acid and hydrogen peroxide and Inductively Coupled Plasma Mass Spectrometry (ICP-MS) or Graphite Furnace Atomic Absorption Spectrometry (GF-AAS) for analysis as recommended in the reference method (EN 14902). However, a few participants used other methods: Energy Dispersive X-ray Fluorescence (EDXRF), Atomic Emission Spectrometry (ICP-AES) and Voltammetry for analysis and vaporisation on hot plate before microwave digestion, Soxhlet extraction, high pressure or cold Hydrogen Fluoride methods for digestion. Each participant received 5 samples to be analysed: a liquid sample prepared by dilution of a Certified Reference Material (CRM), a solution of a dust CRM sample digested by the JRC13F, a sub-sample of a dust CRM that each participating laboratory had to digest and analyse, a solution prepared by JRC after digestion of an exposed filter and a pair of filters (one blank filter and one exposed filter) to be digested and analysed by each participant. For 89 % of all types of samples, the DQOs of the 1st and 4th European Directives (uncertainty of 25 % for Pb and 40 % for As, Cd and Ni) were met. All together, this is a very good score. The best results were obtained for the liquid CRM, dust CRM digested by JRC, dust CRM and filter digested by JRC with 92, 90, 96 and 93 % of DQOs being met, respectively. It was found that the DQOs were not met if the difference of acidity between test samples and participant calibration standards was high. Conversely, only 76 % of DQOs were met for the filter to be digested by each participant with (about 85 % for Cd and Ni, 73/64 % for Pb and As, the most difficult element to determine). The worst results were associated with special events: explosion in microwave oven during digestion for two participants, a wrong dilution factor used by one participant and a huge contamination in the blank filter for another participant. Among the two explosions, one of them was probably the effect of a lack of temperature control in the digestion vessel. For the other explosion, the microwave digestion and the digestion program advised by EN 14902 is to be questioned. Moreover, satisfactory results were obtained using Soxhlet extraction, high pressure method and cold Hydrogen Fluoride digestion methods which are not presented in EN 14902. The DQOs of As and Cd could not be met with EDXRF whose limit of detection was too high for these two elements and for Cd using Voltammetry which suffered a strong interference for this element. Regarding the methods of analysis, apart the points mentioned just before about EDXRF and Voltammetry, good results were observed using ICP-OES for Cd, Ni and Pb. A few discrepancies were also registered for GF-AAS and ICP-MS but they were created by the special events or acidity problem mentioned before. This shows that even though GF-AAS and ICP-MS are found suitable, the implementation by each participant may be responsible for important mistakes.JRC.H.4-Transport and air qualit

Phylogenetic and Functional Metagenomic Profiling for Assessing Microbial Biodiversity in Environmental Monitoring

<div><p>Decisions guiding environmental management need to be based on a broad and comprehensive understanding of the biodiversity and functional capability within ecosystems. Microbes are of particular importance since they drive biogeochemical cycles, being both producers and decomposers. Their quick and direct responses to changes in environmental conditions modulate the ecosystem accordingly, thus providing a sensitive readout. Here we have used direct sequencing of total DNA from water samples to compare the microbial communities of two distinct coastal regions exposed to different anthropogenic pressures: the highly polluted Port of Genoa and the protected area of Montecristo Island in the Mediterranean Sea. Analysis of the metagenomes revealed significant differences in both microbial diversity and abundance between the two areas, reflecting their distinct ecological habitats and anthropogenic stress conditions. Our results indicate that the combination of next generation sequencing (NGS) technologies and bioinformatics tools presents a new approach to monitor the diversity and the ecological status of aquatic ecosystems. Integration of metagenomics into environmental monitoring campaigns should enable the impact of the anthropogenic pressure on microbial biodiversity in various ecosystems to be better assessed and also predicted.</p> </div

Phylogenetic clustering.

<p>MG-RAST heatmaps representing the phylogenetic diversity of the four samples at the phylum (left) or class (right) level. Differences between PolS1 and PolS2 are more pronounced than between PriS1/PriS2 sample pairs. Red and green colours indicate low and high abundance, respectively.</p

Phylogenetic community diversity.

<p>Diversity represented at the class level as determined using GreenGenes. Values reported represent the percentage fraction present in each sample. Gammabacteria are enriched in the polluted samples PolS1/PolS2 whereas Actinobacteria dominate in the pristine samples PriS1/PriS2.</p

Sample site location.

<p>Geographic location of the sampling sites in the Mediterranean Sea. PolS – polluted site at Genoa Port; PriS – pristine site at the Montecristo Island.</p

Overview of raw sequence read output, processing and annotation of metagenomic profiles.

<p>Overview of raw sequence read output, processing and annotation of metagenomic profiles.</p