Marine Microbial Biodiversity, Bioinformatics and Biotechnology
Micro B3 is based on a strong user- and data basis from ongoing European sampling campaigns to long-term ecological research sites. For the first time a strong link between oceanographic and molecular microbial research will be established to integrate global marine data with research on microbial biodiversity and functions. The Micro B3 Information System will provide innovative open source software for data-processing, -integration, -visualisation, and -accessibility. Interoperability will be the key for seamless data transfer of sequence and contextual data to public repositories.
Micro B3 will allow taking full advantage of current sequencing technologies to efficiently exploit large-scale sequence data in an environmental context. Micro B3 will create integrated knowledge to inform marine ecosystems biology and modelling. Moreover, it will facilitate detecting candidate genes to be explored by targeted laboratory experiments for biotechnology and for assigning potential functions to unknown genes. Micro B3 will develop clear IP agreements for the protection and sustainable use of pre-competitive microbial genetic resources and their exploitation in high potential commercial applications.
To underline the translational character of Micro B3, outreach and training activities for diverse stakeholders are planned as well as an Ocean Sampling Day to transparently make project results accessible and gain valuable user feedback.
JACOBS UNIVERSITY BREMEN GGMBH
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Grant agreement ID: 287589
1 January 2012
31 December 2015
€ 11 496 409,35
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JACOBS UNIVERSITY BREMEN GGMBH
Bioinformatics boosts biotechnology applications
FOOD AND NATURAL RESOURCES
CLIMATE CHANGE AND ENVIRONMENT
Grant agreement ID: 287589
1 January 2012
31 December 2015
€ 11 496 409,35
€ 8 987 491
JACOBS UNIVERSITY BREMEN GGMBH
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Final Report Summary - MICRO B3 (Marine Microbial Biodiversity, Bioinformatics and Biotechnology)
Micro B3: Marine Microbial Biodiversity, Bioinformatics and Biotechnology
Technological advances in the fields of 'Omics have enabled marine scientists to realise projects they only dreamt of 10 years ago. Large amounts of next generation sequencing data stand in contrast to the small amount of data management infrastructure with integrated analysis software currently available. The Micro B3 Project improved Europe’s capacity for bioinformatics and marine microbial data integration for the benefit of a variety of disciplines in biosciences, technology, computing and law.
Work has focused on mobilizing the larger marine research community for sampling of the world’s ocean, which was accomplished by two global Ocean Sampling Days (OSD) on 21 June 2014 and 2015 (www.oceansamplingday.org). OSD included the citizen science campaign MyOSD (www.my-osd.org). MyOSD is the first citizen science campaign which enabled citizens to perform microbial sampling of the ocean across the globe. This direct participation raised awareness for marine microbes and their important ecological roles.
A data-flow was implemented between four European infrastructures, which allows for direct sharing of metadata and provides access to a wealth of contextual data. The published Micro B3 Standard Operating Procedures and Reporting Standards together with the Micro B3-inspired MIBiG specification (Minimum Information about a Biosynthetic Gene cluster) support marine microbial ecological research, as well as biotechnology for industrial applications.
The Micro B3 Information System is operational for integrated data queries, especially of the OSD data sets, and enables seamless processing, integration, visualisation of and accessibility to huge amounts of marine data. Biodiversity research in Micro B3 has led to many novel results, based on new tools like annotation pipelines, network and statistical analysis in microbial ecology, including interactive guidance for a community-led and -curated service.
For biotechnological applications several bioinformatics tools were developed and tested: the 3DM database system, developed by an SME-academia collaboration, uses dedicated workflows for computational predictions of substrate selectivity of enzymes. A novel tool uses co-occurrence networks for determining hypothetical functions of unknown genes found in marine microbes. Lately, this was extended to create the ultra-fast Biosynthetic Gene Cluster recruiter, which is generating biotechnological relevant enzyme targets for wet-lab testing from metagenomes. Diverse results from genome mining for anti-tumour compounds, enzyme toolboxes, libraries and new expression systems for experimental screening were tested, applied and are now available.
On the legal side, an Access and Benefit Sharing model agreement (ABS-MA) and a data policy were developed, tested during training courses and utilised during the two OSDs. This directly implements the Nagoya Protocol, entered into force on 12 October 2014, on access to genetic resources and the fair and equitable sharing of benefits arising from their utilization to the Convention on Biological Diversity (CBD).
Further cross-cutting activities included a training pipeline enhancing interdisciplinary oceanographic, biodiversity, bioinformatics and biotechnological knowledge. The courses, stakeholder and industry expert workshops integrated many partners and related projects in knowledge exchange and technology transfer with academic and industrial stakeholders.
Overall, Micro B3 particularly fostered (1) Research on ‘environmental intelligence’ of European open ocean and coastal marine ecosystems for better understanding of their diversity and functions they contain. (2) Community interaction, technology transfer and data sharing between science and the bio-economy to overcome fragmentation in European marine research as well as to promote product and service developments. (3) Public awareness of the importance of marine research and the fundamental role of marine microbes in ecosystem functioning.
Project Context and Objectives:
Summary description of project context and objectives
The 9 million Euro project Micro B3, led by Dr. Frank Oliver Glöckner, Professor of Bioinformatics at Jacobs University gGmbH in Bremen, involved 32 partners from 14 European countries and two international organisations. It formed nine interdisciplinary teams of experts in bioinformatics, computer science, biology, ecology, oceanography, bioprospecting, biotechnology, ethics and law. From January 1, 2012 until the end of 2015 Micro B3 enabled integrated access to biodiversity, genomic, oceanographic and earth-observation databases building on global standards for sampling and data processing.
Marine ecosystems research has undergone a paradigm shift by moving on from a single experiment science towards a data intensive endeavour. The wealth of new technologies ranging from improved laboratory systems and automation, to next-generation sequencing technologies has started to contribute to a better understanding of fundamental questions like the impact of man and global change on our ocean. The following set of obstacles and bottlenecks, limiting the usability of the ever increasing datasets for marine biodiversity research and biotechnological applications, have set the scene and objectives of the research performed within Micro B3.
1. Absence of quality management and standards ranging from field sampling to data acquisition (sequencing, environmental parameters), storage and processing. A huge amount of data that has been and is being generated by marine researchers worldwide is virtually lost because they are either incomplete, stored in hand-written laboratory notebooks, or on individual computers. What is finally published represents only a small subset of the original data and is often only ‘human readable’ and unable to be assimilated into structured databases. These practices greatly hamper any kind of Europe-wide electronic data exchange and integration.
2. Limited comprehensive bioinformatics approaches and capacities in Europe to deal with the already existing and upcoming flood of molecular and environmental data. The major problems are (1) dealing with the computational aspects of data representation, analysis and visualization of thousands of millions of DNA sequences which are produced and released to databases, (2) addressing the large scale integration and interpretation of molecular data in their environmental context and finally (3) opening up the still untapped option to explore the accumulated bioinformatics knowledge for commercial products.
3. Limited integrated approaches to link ecosystems biology with targeted lab experiments to explore biotechnological applications and the commercial potential of the marine ecosystem. Although projects exists that make use of metagenomics for new biochemical activities and pathways, there is no concerted action available that consequently uses integrated datasets to nail down the function of enzymes for commercial applications and use this information for a better understanding of the marine microbial diversity.
4. Absence of an overarching legal framework for access and benefit sharing in the marine environment. The complex and heterogeneous IPR issues involved in getting legal access to materials and data especially from Exclusive Economic Zones hamper not only commercialization but also pre-competitive fundamental research.
5. Absence of models for the marine ecosystem that include the influence of marine microbes as a ‘biological factor’. To achieve this, it is necessary to link biological investigations with global oceanographic data layers comprising of remote sensing and profile data. A baseline needs to be found as a reference to monitoring and assessing changes. A missing registry of marine stations and accumulated data is currently hampering this process.
6. Limited training capacities in bioinformatics and data management. Data management and bioinformatic data analysis have become a must for marine researchers these days, mostly based on ad hoc training. Appropriate workshops and training entities tailored to the needs of the users are needed to make full use of the data. Interdisciplinary workshops will also foster a new communication culture between the disciplines.
Description of the main S&T results/foregrounds
In order to address the issues mentioned above, Micro B3 delivered on the following targeted objectives:
1. A comprehensive registry of European study sites and data sources for data safeguarding and acquisition.
2. A set of community agreed standards for sampling and data acquisition (lab protocols), storage and exchange of data to reach a new level of interoperability and data integration across disciplines.
3. Innovative software approaches for quality management, data processing, data integration, accessibility and visualisation.
4. Ecosystem models for selected sites in the marine system to provide a predictive understanding of the contributions of functional microbial biodiversity to marine ecosystems functioning, with a special focus on the role of microbes to climate change and the effect of climate change on microbial communities.
5. A series of new biocatalytic processes, enzymes, biosynthetic pathway and bioactive compounds for use in biotechnological applications.
6. An innovative legal framework and model contracts for the protection and sustainable use of marine genetic resources.
7. A series of workshops, outreach and training entities as well as an Ocean Sampling Day to establish a baseline and make the project results accessible for researchers, the industry, the public and policy makers.
8. A new generation of marine microbial researchers well trained in oceanography, data management, omics technologies and biodiversity policy.
9. A new communication culture with bilateral understanding between computer scientists, biologists, biochemists, bioinformaticians, oceanographers as well as philosophy and judiciary.
10. Awareness of the importance of the marine system by implementing the citizen science campaign MyOSD 2014 and 2015.
Micro B3 organised the Ocean Sampling Day and the citizen science campaign MyOSD mobilizing the marine research community for global sampling of the world’s ocean. This was successfully accomplished by two Ocean Sampling Days (OSDs), on June 21 2014 and 2015. Over 190 marine stations as well as nearly 200 citizen scientists took part and delivered samples and environmental data. The OSDs and MyOSDs were major cross-cutting activites, joining almost all work packages and partners in sampling, analysis, safeguarding of legal requirements, training and dissemination. Dissemination was aided by a dedicated OSD/MyOSD movie and the Micro B3 documentary films, shown at many public events.
Micro B3 developed an innovative, transparent and user-friendly Information system for seamless, integration, and visualisation of huge amount of marine data from past and on-going biodiversity sampling campaigns like the Global Ocean Sampling (GOS) and Tara Oceans expeditions, long-term ecological research stations, and from Micro B3’s novel Ocean Sampling Days and MyOSD, which performed global snapshots of marine microbial plankton diversity.
Micro B3 developed innovative solutions on ABS and related IP aspects which were strongly promoted to many stakeholders in preparation for the OSDs and through workshops with national representatives of the CBD/Nagoya Protocol, through collaborations with several FP7 projects on marine biotechnology and at the final Micro B3 conference.
The combined expertise within Micro B3 facilitated all the way to integrated data analysis, utilizing sound statistic methods and testing of applications within selected biotechnology value chains. The innovative joint analysis of genetic and ecological information generated knowledge, new perspectives for the modelling and exploration of marine microbial communities, and new targets for biotechnological applications. Most of Micro B3’s results are documented in 110 peer-reviewed publications with full texts available through an OpenAire repository.
A comprehensive registry of European study sites and data sources for data safeguarding and acquisition
The ocean is the largest ecosystem on Earth and yet we know very little about it. This is particularly true for the microbes that drift within. These organisms are at least as important for the Earth system as the forests on land. Because they are invisible to the naked eye they are largely uncharacterized, even though they form the base of marine food webs.
An Ocean Sampling Day (OSD) Network of 191 registered sites was created for sampling microbe and environmental data. Samples were taken at 153 marine locations worldwide during the two OSD events. Work led to the adoption of many standards (see below), as well as an OSD Data Policy and it is continuing towards creation of a sustainable OSD Consortium that could extend this reference data set to the decadal level.
In addition to the Micro B3-funded sequencing of 500 amplicon profiles and 150 metagenomes, eukaryotic samples from 33 sites were sequenced through a Life Watch fund under the lead of Micro B3 partner Stazione Zoologica in Naples. Furthermore, the company Pacific Biosciences performed sequencing runs on two samples from the December 2013 OSD pilot, and an additional five samples from OSD 2014 at no extra cost. Also a partnership with the Smithsonian’s Global Genome Initiative was started early in the project to bioarchive up to 10,000 OSD samples (as an in kind contribution).
The final focus was (and still is) to coordinate the OSD Analysis Consortium, which began as an OSD Analysis Core Group with a Micro B3 Team of 25 experts, and transformed into a global Consortium of 132 experts. This was achieved after an open invitation to the entire OSD Community to take part in the collective analysis of OSD 2014 data. Led by Dr Mesude Bicak and Dr Francesca Malfatti (Institute of Oceanography and Experimental Geophysics, Italy, no Micro B3 partner), this initiative resulted in 45 proposals to analyse OSD 2014 data, which led to three categories for investigation: (1) diversity, (2) insights into metabolic functions with focus on human impact and their role in the ecosystems, and (3) towards an understanding of broad-scale ecological patterns. Ongoing collective community analysis of OSD 2014 data is a significant milestone in the demonstration of the real benefit and value of OSD and Micro B3 for years to come.
To increase our understanding of the marine ecosystem, Micro B3 focused its efforts on specific programmes such as Tara Oceans, which was formed around the 110-ft research schooner Tara. From 2009-2013 Tara sampled plankton at more than 210 sites and multiple depth layers in all the major oceanic regions. The on board scientific sampling followed protocols developed to capture the entire morpho-genetic complexity of the plankton community across seven orders of organismal size magnitude (from 0.01 µm to a few cm), together with an extensive range of physico- chemical information. Besides the sampling, a dry lab on board contained a range of online instruments and microscopes to monitor the content of the samples as they were being collected. Guided by remote satellite and in-situ real-time sensing, Tara and its crew also targeted specific features throughout the expeditions, such as mesoscale eddies, upwelling areas, low-pH water bodies or Oxygen Minimum Zones. In addition to being used for genomics and oceanography, many samples were collected for other analyses such as high-throughput imaging and flow cytometry. The samples collected on board formed the basis for extensive processing and data integration on land.
Micro B3 scientists participated in the preparation and publication of five research articles in Science describing the first foundational resources from the project (based on a first data freeze from 579 samples at 75 stations) and their initial analyses. Three resource papers describe the diversity of viruses, prokaryotes and eukaryotes across a significant part of the world’s Ocean. They provide the first estimates about the extent of eukaryote species inhabiting the oceans, in particular that of single-celled protists (de Vargas et al. 2015). They establish a reference gene catalogue comprising more than 40 million genes, serving as a resource for ocean microbial community studies (Sunagawa et al. 2015), and they show how viral communities are passively transported by oceanic currents and structured by local environmental conditions (Brum et al. 2015). Two additional papers use these resources to derive species interaction networks, identifying many interactions within and between the three domains of life and viruses (Lima-Mendez et al. 2015), and to study the dispersal of plankton from Indo-Pacific to the Atlantic basin at a critical ocean circulation chokepoint (Villar et al. 2015).
Taken together, the work illustrates several aspects of the Micro B3 ecosystems biology approach, and provides unique resources for several scientific disciplines, capturing biodiversity of a wide range of organisms that are rarely studied together, exploring interactions between them and integrating them with environmental conditions to further our understanding of life in the ocean and beyond in the context of ongoing climate changes. Thanks to the infrastructures generated by the Micro B3 project, all Tara Oceans data is in the public domain (www.ebi.ac.uk/services/tara-oceans-data) and additional published papers have reported the resources (Pesant et al. 2015) and interoperability standards generated by the project (Ten Hoopen et al. 2015). Such efforts have been particularly useful for establishing an operative framework for the OSD.
A set of community agreed standards for sampling and data acquisition (lab protocols), storage and exchange of data to reach a new level of interoperability and data integration across disciplines
A large scientific team within Micro B3 coordinated the development of best practice guidelines on marine sample collection, logistics and bioinformatics for marine sampling stations and cruises, focusing primarily on the principal Micro B3 marine sampling campaign – the Ocean Sampling Day in June 2014. Micro B3 Standard Operating Procedures and Reporting Standards, available in the Ocean Sampling Day Handbook and associated online tutorials were agreed by experts across oceanographic, biodiversity and molecular domains and allow data to be collected in and orchestrated way and consistently with national and international legal commitments in the countries involved, see: https://www.microb3.eu/sites/default/files/osd/OSD_Handbook_June_2015.pdf .
The Micro B3-developed data reporting standard and interoperability structures were published as the Marine Microbial Biodiversity, Bioinformatics and Biotechnology (M2B3) data reporting and service standards (ten Hoopen et al., 2015). The M2B3 data reporting standard represents minimum information about a marine microbial sample collected from the epipelagic zone and is targeted to a community of marine laboratories with microbial sampling programmes. The M2B3 interoperability structures are relevant mostly to data management centres dealing with marine biological data. This harmonisation allows multiple use of each sample, meaningful comparison between samples and integration of molecular data generated from these samples with a rich environmental context.
An intensive data management support of the Ocean Sampling Day and Tara Oceans throughout the Micro B3 project has led to well organised reference datasets. A checklist management environment within the EMBL-EBI/ENA data resource was made available to support sustainable maintenance of marine reporting standards to facilitate future compliance and continued support for compliance with M2B3 standards across relevant data resources. Thus, the Micro B3-developed data reporting standards and interoperability structures will support also other marine microbial sampling enterprises well into the future.
Innovative software approaches for quality management, data processing, data integration, accessibility and visualisation
The complete Micro B3 Information System (Micro B3-IS) is a set of modular and interoperable software components which together implement the innovative “bioinformatics scientific discovery workflow” from data generation to gaining new insights. The Micro B3-IS was successfully used for Micro B3’s Ocean Sampling Day (OSD) and the accompanying MyOSD citizen science campaigns. The modular and interoperable approach of Micro B3-IS builds on shoulders of existing European infrastructures such as EMBL-EBI’s European Nucleotide Archive (ENA) Archive and SeaDataNet to not re-invent existing components. However, within Micro B3 ENA developed and deployed software that simplifies reporting of marine nucleotide data and associated information to the archive and software that improves discoverability of the archived data. While marine microbial research was the direct use case for this development many of the enhancements will serve the needs of the whole environmental genomics, the metagenomics community and beyond.
The capability to also use one or more of the Micro B3-IS components for different purposes other than OSD or MyOSD is another advantage of this modular approach. If researchers want to perform own sampling campaigns with the aim to follow the “Marine microbial biodiversity, bioinformatics and biotechnology (M2B3) data reporting and service standards” (M2B3) they can build on the OSD Registry sample registration component and also make use of the OSD Smartphone App. Each bioinformatics pipeline like e.g. EBI’s Metagenome Portal can be used independently of all other components. The PostBIS database extension is directly usable for any kind of DNA or Protein data. The ProX tool for large-scale network-visualization can be used for many kinds of different large-scale biological networks. The tools for the ecological analysis of microbial diversity data GUSTAME and MASAME are usable stand-alone. These are just examples to illustrate the value of the modular architecture of Micro B3-IS.
Additionally, all but one component are open source with a permissive license (Apache 2 License). This allows everyone to make free use of every component for all kind of purposes. First, it allows scientists to scrutinize the software w.r.t. scientific quality. Second, any commercial entity can use any component for free on a legally clear and save basis for any business goal.
Oceanographic databases, earth observation and monitoring, as well as data management
A large team of data managers and scientists have developed and implemented a model for organising data flows for marine environmental biodiversity and genomic data. Interoperable structures were established for submission, storage and exchange of these data between the established archives SeaDataNet, EurOBIS, the European Nucleotide Archive ENA, and the Micro B3 Information System (Micro B3-IS). A model for organising the data flows, for sharing metadata and giving access to related data was developed. Geographical pilot areas (use cases) were selected as matching locations with genomic and oceanographic data available. Features are operational OGC WMS-WFS and OpenSearch services for integrated query of data from these infrastructures as managed by MARIS, VLIZ, EMBL-EBI and MPIMM.
The list of Micro B3 use cases were expanded by IFREMER with Ocean Sampling Day stations as matching locations with genomic and oceanographic data available. The new list was used by MARIS to configure a Micro B3 filter for compiling a dynamic buffer with data sets. This buffer was made accessible for the MB3-IS. The metadata and data in this buffer were harvested from the SeaDataNet distributed data centres and are now maintained by the SeaDataNet robot harvesting system. Data resources were complemented by IFREMER with data sets from the GOSUD programme and by ICES.
Furthermore data collected during the Tara Oceans expedition and the Ocean Sampling Day (OSD 2014) were organised and included by UniHB in the PANGAEA system with metadata, controlled vocabularies and DOIs for data publishing. UniHB has populated SeaDataNet with the Tara Oceans and OSD 2014 entries from PANGAEA, making these oceanographic data sets part of the dynamic Micro B3 buffer. The present SeaDataNet Micro B3 buffer contains almost 48,000 records divided between 36 data centres and 136 originators from 20 different countries and covering 37 parameter groups. In addition sequence data sets from Tara Oceans and OSD 2014 were loaded into the ENA infrastructure, while taxonomic data were shared with EurOBIS.
Ecosystem models for selected sites in the marine system to provide a predictive understanding of the contributions of functional microbial biodiversity to marine ecosystems functioning, with a special focus on the role of microbes to climate change and the effect of climate change on microbial communities
In order to transfer the wealth of sequence data prioritized by Micro B3 partners into knowledge of marine microbial ecosystem function for interpretation of biological information in its environmental context, work aimed to reach the following detailed objectives:
• To explore whole microbial ecosystems, including viruses and giruses, Bacteria and Archaea, picoeukaryotes, nanoplankton, and microplankton from both biodiversity and systems biology perspectives.
• To unite the fields of molecular microbiology and ecology to develop an understanding of community function by relating biodiversity with the functional structure of the ecosystem.
• To extend data analysis by modelling experimental validation, where necessary.
• To enrich studies at other sampling sites through contributing to the OSD, and to provide new leads for biotechnology.
In addition to Tara Oceans, case studies chosen included another spatial monitoring done (the Malaspina expeditions), temporal monitoring programmes of long-term sampling sites (Plymouth (L4), Barcelona (Blanes Bay), Naples, and Heraklion), and the OSD campaigns. An additional case study focused on an analysis of sediment samples (AWI).
Annotation pipelines have been generated and applied to identify and functionally characterize DNA sequences derived from viruses, prokaryotes, and eukaryotes. Results are now available from several integrative studies, e.g. www.ebi.ac.uk/services/tara-oceans-data.
Concerning viruses, Micro B3 generated an annotation pipeline to identify viral sequences in metagenomic reads or contigs. These annotation strategies have been used for two ecological studies, one aimed at identifying nucleo-cytoplasmic large DNA viruses in Tara Oceans pyrosequence data, the other aimed at examining transport of large DNA viruses through the Agulhas leakage off South Africa. For both studies the diversity, abundance and biogeography of marine viruses in 17 metagenomes were characterized, derived from microbial samples (0.2–1.6 μm size range) collected during the Tara Oceans expedition. The pipeline was used to understand the role of Agulhas rings in virus biogeography, in particular transport from the Indian Ocean to the South Atlantic Ocean.
For prokaryotes and eukaryotes, a combination of metabarcoding approach, i.e. massive sequencing of short PCR-amplified regions of markers genes such as 18S rRNA (V4 or V9 region) and of metagenomics, i.e. massive random sequencing of environmental DNA with global sampling surveys, was applied to map specific groups of prokaryotic and eukaryotic microbes at oceanic scale. For example, metagenomes and metatranscriptomes from prokaryotic fractions derived from Tara Oceans and Malaspina have been used to extract single marker genes to investigate the taxonomic composition and variation of Bacteria and Archaea among samples.
Using these very large data sets from the ensemble of organisms within marine microbial communities, network analysis approaches have been developed to deepen the understanding of community diversity. These took into account the impact of varying taxonomic resolution and a range of contextual data accompanying the community data. Altogether, this first-order analysis of marine microbial community structure and dynamics serves as a reference framework to interpret all the metagenomics and metatranscriptomics data analysed in the Micro B3 project, and will orient the choice of high-throughput technologies to further explore highly complex marine eukaryotic metagenomes and metatranscriptomes in the future. In one case study, a marine microbial sediment community was also evaluated.
From these integrative studies, an interactive ecological analysis guide (called GUSTAME) and accompanying Ecological Analysis Tools for Microbial Ecology (called MASAME) were developed, evaluated, and synthesised. A range of methods available in the MASAME package were further used to explore ecological factors underlying different rank-abundance patterns observed in different samples. These resources are being exploited by Micro B3 participants to comprehensively explore plankton community diversity, and also serve as a key foundation for the diversity analyses that are ongoing in the context of the OSD data sets.
Work within Micro B3 has also focused on applying uni- and multivariate statistics to correlate species composition with functional composition, and to correlate both of these to environmental oceanographic parameters. These have been published in Lima-Mendes et al. 2015.
An integrated approach to biodiversity studies that potentially can link abiotic and biotic parameters and their inclusion within a conceptual modelization of ecosystem biodiversity has been developed. It is based on the pros and cons of various numerical modelling approaches, a review of meta-omics approaches for marine biodiversity assessments, standardization of the experimental procedures, accurate reference databases and the impact of meta-omics tools.
A series of new biocatalytic processes, enzymes, biosynthetic pathway and bioactive compounds for use in biotechnological applications
An important objective for the biotechnological work within Micro B3 was to develop, validate, and apply bioinformatics-based tools and laboratory-screening protocols for the discovery, description, and exploitation of new gene functions, such as enzyme reactions, biosynthetic pathways, and synthesis of bioactive compounds. In order to contribute to this objective, the University of Groningen (UGRO) developed a workflow for the computational prediction of substrate selectivity of homologues of genes with known activities. It consists of high-throughput ligand-assisted homology modelling and docking simulations of query ligand(s) into the predicted active site. The system should rank homologous protein sequences according to their likelihood of being active with a certain target compound. This was applied to predict aminotransferase selectivity, and subsequently tested with expression and characterization of a set of marine sequences encoding for aminotransferase that should be useful in the synthesis of green polymer precursors. MPI Bremen established and tested a system to identify networks of co-occurring unknown genes. The network was constructed using sequences of a number of marine sites. If, within the network, correlations to genes with known functions are found, this hints at a hypothetical function of the unknown genes.
Using samples collected at the Atlantic ocean and Icelandic beaches with tidal thermal fluctuations, MATIS, in collaboration with PharmaMar, and BioMerit have isolated a range of microbial cultures and subjected the most promising ones to further partially high-throughput processing. The partners also constructed metagenomic libraries from OSD 62 and OSD 62-2013 material, as well as from river sediments under tidal influences and from deep-sea sediments.
Libraries have also been constructed from marine samples at Univ. Bangor, IAMC, Bio-Iliberis, and at BIOMERIT. These partners also constructed new expression systems employing broad host range vectors for cloning large DNA inserts. Thus, two novel broad-host range shuttle fosmids were constructed for functional screening of for example esterases, other hydrolases, and oxygenases. Univ. Bangor and IAMC constructed metagenomic libraries from marine samples taken at a hypersaline brine lake at the bottom of the Mediterranean Sea.
Screening assays for metagenomic library analysis became available at UGRO (use of compounds as nitrogen source by E. coli transformants), and, in a more extensive way at Bangor for a range of plate assays for the enzymes mentioned below. Also screening assays were implemented at BIOMERIT for anti-bacterial compounds, and for compounds influencing quorum sensing, at Bio-Iliberis (SME) for transformation of xenobiotics compounds, oxidative biotransformations, and phosphate production assays, and at MATIS. Some approaches were considered less effective, for example SIGEX-based screening reported in the literature provided more problems than solutions.
The SME partner Bio-Prodict extended a second system for three-dimensional protein structure prediction and in total established seven new databases for protein families, for example for two phylogenetically unrelated classes of epoxide hydrolases. The databases and accompanying 3DM software support tailoring of enzymes for use in applied biocatalysis. PharmaMar, the large industry partner within Micro B3, focused on genome mining for specific sequences of putative biosynthetic gene clusters of antitumor compounds. This included analysis of sponge-associated microorganisms.
Screening methods and metagenomic libraries were explored for the presence of genes encoding enzymes useful for applied biocatalysis. In metagenomic libraries investigated by Bio-Iliberis within Micro B3, genes encoding different enzymes for mineralization of organic compounds were identified and the most promising enzymes were overexpressed for further characterized (the activities are still confidential). Bio-Iliberis has additionally explored new enzymatic activities useful for the solubilisation of phosphate in soils. This is particularly important because of the limitations imposed by the EU to restrict the use of microorganisms as growth supporters. BANGOR in collaboration with IAMC, have cloned and biochemically characterized 147 new enzymes including esterases, proteases, lipases, glucosidases, dehydrogenases and dehalogenases. Furthermore, 16 crystal structures were resolved. Positive lipases and esterase clones were sent to MATIS for further analysis, whereas cellulases and different other activities were further analysed in Bangor. Enzyme characterization at IAMC focused on haloarchaeal glycosyl hydrolases (celullases and chitinases), sulfur transferases (rhodaneses) and polysulfide reductases. Several predictions based on functional screening and sequence analysis were confirmed experimentally.
In view of the exposure of marine microorganisms to various amines and the expected robustness of marine enzymes, UGRO examined the application of marine aminotransferases and amino acid decarboxylases for key steps in the synthesis of non-proteinogenic amino acids, such as γ-aminobutyrate, β-phenylalanine and 6-aminohexanoate, which can be intermediates in the synthesis of useful nitrogen compounds, including polyamides like nylon. Other PLP-dependent enzymes decarboxylated amino acids, also producing useful amines. UGRO also performed initial engineering studies on a decarboxylase from the thermophilic marine bacterium Thermotoga maritima based on protocols for molecular docking and MD simulations developed within Micro B3. The results provided a toolbox of marine enzyme variants for aminotransferase and decarboxylase reactions.
Novel and interesting enzymes were also obtained at MATIS. In total 53 putative genes were selected for expression, aiming at beta-glucan enzymes, beta-galactosidases, alginases, chitinases, xylanases, carragenases, amylases, hyaluronidases, chondroitin sulfate lyases, sulfatases, esterases and lipases. Out of them, 27 proteins were successfully cloned and expressed and 16 enzymes with different activities were characterized.
At BIOMERIT both culture-based and genomic/metagenomic approaches were used to discover novel enzymes produced by marine sponge-derived microorganisms. Functional screens for proteases, lipases and transaminases were performed. Two putative ω-transaminase genes were identified, of which one appeared to be functional. BIOMERIT also identified two novel lipases with novel activity and enantioselectivity against commercially challenging substrates. Metagenomic screening also identified a unique serine protease gene.
For screening for bioactive compounds using cultivation-dependent methods, a range of microbial cultures has been isolated (PharmaMar, BIOMERIT, MATIS, and IAMC). PharmaMar selected sponges that could produce interesting bioactive compounds (polyketides and/or peptides) and used bio guided-chromatographic purification, mass spectrometry and NMR as analytical methods to identify new compounds. PharmaMar also used protocols for high-throughput screening of cell extracts for cytotoxicity. Three new antitumor compounds have been identified from sponge associated bacteria for preclinical assays, in addition to the identification of 30 families of cytotoxic compounds whose chemical scaffolds are polyketides or non-ribosomal peptides. Biodiversity analysis led to the identification of bacteria intimately associated with specific sponges and improved in silico genomic mining for polyketides and non-ribosomal peptides. The cultures at BIOMERIT were subjected to high-throughput analysis for the production of quorum-sensing and anti-bacterial compounds.
Most deliverables resulting from the work described above have restricted or confidential status, but many publications were done by the partners, which can be found via the Micro B3 website.
An innovative legal framework and model contracts for the protection and sustainable use of marine genetic resources
The legal team developed and circulated Micro B3 model agreements on access to marine microorganisms and benefit sharing (ABS-MA) to be used for pre-competitive utilization and research, competitive research, and hybrid situations. The draft model was extensively discussed with stakeholders, e.g. during a dedicated stakeholder workshop with invited scientific, legal and industry representatives in 2013.
The final model agreements are characterized by an innovative distinction between research and development for the public domain and for proprietary purposes. A viral license clause for transfer of genetic materials to third parties was introduced. A successful multi-stakeholder workshop discussed tracking and tracing of ABS-relevant material and data, as they travel through databases and analysis pipelines.
A broad consultation of industry partners and a literature review lead to a policy brief on open innovation models for out-licensing the final marketable research results: this last policy brief complemented the analysis of the IP issues by providing options for IP management at the end of the pipeline.
A Data Policy for the Ocean Sampling Day was developed in collaboration with WP 2 and circulated to guarantee that the OSD dataset will be a reference data set and as widely accessible and used to support downstream research as possible. It specifies that all the data extracted from the samples of the OSD are released to the public domain, respecting the Ft. Lauderdale Principles, which entitle the data producers to make the first presentation and publish the first genome-wide analysis of the data.
Micro B3 legal experts maintained close collaboration with the EU-funded project PharmaSea participating in its Advisory Panel of Policy and Legal Experts (APPLE). This panel addresses the key policy and legal barriers that currently hinder progress in innovative marine biotechnology in Europe.
A book was edited by Prof. G. Winter from the Micro B3 legal team (Chege Kamau, E., Stoll, P.-T. and Winter, G. (eds.) Research and Development on Genetic Resources. Public Domain Approaches in Implementing the Nagoya Protocol. Routledge, June 2015). It includes several chapters based on Micro B3 results dealing with specific definitions of non-commercial versus commercial research, with a comparison of the Nagoya Protocol/CBD with UNCLOS, with points to consider for implementing the Nagoya Protocol and with the MicroB3 model agreement plus commentary.
A final paper by Prof. T. Dedeurwaerdere, et al. covers further research- and IPR-relevant aspects, dealing with the global scientific research commons under the Nagoya Protocol: Towards a collaborative economy model for the sharing of basic research assets (Environmental Science & Policy, 55: 1–10, 2016; Open Access).
A series of workshops, outreach and training entities as well as an Ocean Sampling Day to establish a baseline and make the project results accessible for researchers, the industry, the public and policy makers.
A detailed Dissemination & Training Plan, generated in the first year of the project and based on an extensive stakeholder questionnaire which targeted marine scientists as well as industry representatives, communication work ensured the active and timely dissemination of relevant information on marine microbial diversity, bioinformatics and biotechnology and in particular on the OSDs and MyOSD to diverse stakeholder groups.
Outreach, knowledge and technology transfer aspects aimed for the public, the biotechnological industry and policy makers. Dissemination included magazine articles, four roll-up banners, a multi-language press kit and many further publications. Based on the kit, intense world-wide press work was conducted for the OSDs, enabling local and international communications and a large media response. The MyOSD element ensured outreach and active involvement especially of young citizens. Several films were produced covering the OSD and MyOSD initiative.
OSD Teaser http://youtu.be/FEw5AS1qo-o
OSD Movie Trailer https://youtu.be/hBGOkB-EImc?list=PLgacjRIHqvMC39eKYdGH0HAM68YszmbuJ
OSD Movie http://youtu.be/yUm7SsSe-cw?list=PLgacjRIHqvMC39eKYdGH0HAM68YszmbuJ
Best of OSD 2014/2015 https://youtu.be/5vpKlkzusE8
OSD Sites http://youtu.be/7QOWfogzeKs
OSD Citizen Science App Tutorial http://youtu.be/1lhDdPbzuTs
NOAA OSD http://youtu.be/7whot0vBTUQ
Two stakeholder workshops for policy and biotechnology target groups and three Industry Expert Workshops were held for knowledge and technology transfer, the last one together with the final Micro B3 conference. Thus closer contacts of many Micro B3 partners with users of marine knowledge from industry, policy and society have been established.
The first Micro B3 Stakeholder Workshop had a strong legal focus and was held under the lead of UCL with input from the full legal team of WP 8. It took place in Brussels on 27 and 28 February 2013 with 37 participants. The full title of the workshop was ‘Towards a Model Agreement on Access and Benefit Sharing (ABS) for Marine Genetic Resources (with a focus on marine micro-organisms) - proposed best practices to access MGRs and support metagenomic science for utilization in data-driven global research collaborations based on the Convention on Biological Diversity, taking into account the Nagoya Protocol’. Six providing countries participated in the workshop with representatives from scientific, legal and ministerial sectors, ten Micro B3 partners including industry representatives, as well as of the marine KBBE projects (BlueGenics, PharmaSea, SeaBioTech) and of the research infrastructure on microbial collections, MIRRI. Legal and scientific experts from Micro B3 and invited speakers introduced the framework of Micro B3’s Access and Benefit Sharing Model Agreement and its synergies with research and development, and two practical case studies. Then stakeholders analysed and discussed the core clauses of the agreement, dealing with access to genetic material and with data management. Finally, their input was integrated into an improved version.
The second Micro B3 Stakeholder Workshop encompassed the OSD and the formal signature of the CIESM Charter on Access and Benefit Sharing by the Micro B3 coordinator Prof. F. O. Glöckner and F. Briand, the head of CIESM, as performed in Monaco in summer 2014. The Charter applies to parties engaging in the collection and exploitation of Marine Genetic Resources. The OSD activity helped to spread the information on the charter to a wide scientific network. It covers the sharing of scientific knowledge with concerted handling of data, traceability, nature conservation and environmental respect. It lists nine ethical guidelines aimed at providers, enquirers and end-users regarding the use of marine resources, thus maintaining and protecting the access to knowledge to everyone and preventing abuses of the ocean global commons. The CIESM Charter goes beyond biological approaches and extends beyond the strict perimeter of the Mediterranean/ Black Sea Region. It is applicable to large scientific initiatives such as oceanographic campaigns in the world oceans and was to date signed by 398 individual scientists from 49 countries (for more see www.ciesm.org/marine/charter/index.php).
The first Industrial Expert Workshop, titled ‘Harvesting Environmental Genomes for the Development of Biocatalysts’, was organized at the University Groningen, on 14 and 15 October 2013. It was a joint endeavour of the Micro B3 and MetaExplore FP7-supported projects. A total of 72 participants from biotechnological industry and academia from 16 different European countries met for this two-day workshop. Four lecture sessions were held and one final panel discussion as a more interactive element, all striking a balance between academic and industry input. The experts exchanged knowledge on current genome-based approaches for discovery of new bioactive compounds and on how new, especially bioinformatic tools can support the understanding of complex genomic and proteomic data for use in engineering of new enzymes and production of small molecules.
The second Industrial Expert Workshop had been organized, under the lead of Micro B3, by EMPA, Jacobs University, PharmaMar and Bio-Iliberis in collaboration with the coordinators of the MaCuMBA and PharmaSea EU projects. It was titled ‘Marine Micr'Omics for Biotech Applications’, and was held at the headquarter of the company PharmaMar (Colmenar Viejo, Spain), on the 30 and 31 March 2015. A total of 75 participants (including 9 PharmaMar employees) from industry and academia from seven different European countries met for this two-day workshop. They discussed current approaches on the discovery of new bioactive compounds and how new tools especially in the fields of metagenomics and bioinformatics could support the understanding of complex data for identifying new pharmaceuticals and for producing new or better enzymes and other small molecules. A newsletter reporting on the workshop, including interviews with the three project coordinators is available here: https://www.microb3.eu/sites/default/files/pdf/Marine Microbes Research Update_June 2015_Web.pdf
The third Micro B3 Industry Expert Workshop was embedded in the Micro B3 Final Conference. It was titled ‘Collaboration between Academia, SMEs, and Industry - Transforming Inventions into Innovations in the Fields of Bioinformatics and Biotechnology’, and was held in Brussels, at the Flemish Academy of Science and the Arts, on third November 2015. A total of 47 participants from industry and academia met for this half-day workshop. It brought together managers of various backgrounds and from both within and outside of the Micro B3 consortium to illuminate different strategies for knowledge and technology transfer between the public and private sector and corresponding experiences, based on use cases in bioinformatics and (marine) biotechnology. Examples were shown on how EU-funded collaboration can be used to gain access to external expertise and technology, as well as alternative strategies.
A five and a 20-min version of a documentary film on the entire Micro B3 project was produced, sub-titled in six languages and shown at many occasions. It is available for viewing in streaming mode under www.vimeo.com/121480945 and www.vimeo.com/115160161 for the 5 min version.
A new generation of marine microbial researchers well trained in oceanography, data management, omics technologies and biodiversity policy
The Micro B3 training pipeline included four one-week courses and a two-week summer school.
1. Bioinformatics for Beginners (2012, Bremen, DE, 11 trainees)
2. Biodiversity Statistics (June 2013, Bremen, DE, 21 trainees)
3. Marine Metagenomics Bioinformatics (Spring 2014, EBI Training Centre, UK, 24 trainees)
4. Multi-disciplinary Summer School (May/June 2014, Crete, GR, 21 trainees, four local tutors)
5. Micro B3 OSD Analysis Workshop/Jamboree (March 2015 at EBI Training Centre, UK, 30 trainees)
6. KyroBio Masterclass (Spring 2015, Univ. Groningen, NL, 28 trainees)
The first three training courses focused on bioinformatics, on the Guide to Statistical Analysis in Microbial Ecology and on EBI’s metagenomic pipeline. The summer school enhanced interdisciplinary training of oceanography, biodiversity and bioinformatics researchers for OSD 2014, including legal and biotechnological components. The fifth course, an OSD data analysis jamboree, was held at EBI in March 2015 in order to analyse the OSD 2014 data, concluding the planned training pipeline. For the summer school a short summary is provided below as an example, more detail including all course evaluations can be found directly on the website www.microb3.eu under ‘workshops’.
The Micro B3 summer school, held at the HCMR marine station on Crete, took place from 26 May to 6 June 2014. 25 participants attended for the full duration, with one additional Norwegian candidate visiting for the first week, all from OSD participating parties. The first week dealt with standardized sampling and sample extraction, as well as covering the legal aspects of ABS-conform sample taking. In the second week the OSD handbook was tested and refined, including real data submission and details of bioinformatic and statistical analyses of amplicon and metagenomics data analyses practiced, with a view to the upcoming OSD 2014.
Additionally to the original training plan, a sixth course called 'Masterclass Computational Approaches for Discovery and Engineering of Enzymes for Biocatalysis and Synthetic Biology' was held in Groningen from April 20-24, 2015. It aimed mainly at teaching biochemists and biotechnologists to become more familiar with and use bioinformatic approaches to facilitate their laboratory work. It was done under the auspices of the KyroBio project with support from the BE Basic and the Micro B3 projects by Prof. D. Janssen from partner Univ. Groningen. Overall, Micro B3 substantially helped to establish a new generation of marine microbial researchers well trained in oceanography, bioinformatics (Omics), biotechnology and biodiversity policy.
A new communication culture with bilateral understanding between computer scientists, biologists, biochemists, bioinformaticians, oceanographers as well as philosophy and judiciary
The management and coordination team performed professional management of the project in a collaborative working environment until the end of Micro B3 on 31 December 2015. Key scientists maintained close contacts with many related projects, initiatives and with a high-level advisory board, linking Micro B3 to major research infrastructures and initiatives.
The advisory board consisting of European and US-American members has aided Micro B3 throughout its duration with scientific, infrastructural and legal advice by attending many project meetings and the final conference, providing talks and giving specific advice.
Also Micro B3 was collaborating with other European funded projects and relevant international initiatives:
• MaCuMBA, an emerging large EU-project on cultivating marine microbes
• EMODNet, an initiative of DG MARE related to marine knowledge
• BioVel, biodiversity virtual e-laboratory
• MAMBA, marine metagenomics for new biotechnological applications
• INMARE, innovative screening and expression platforms to discover and use the functional protein diversity from the sea
• BlueGenics, PharmaSea, SeaBioTech three marine biotech projects within KBBE
• KyroBio and BioNexGen as related biotechnological EU projects.
• MIRRI, microbial resource research infrastructure
• ELIXIR, a distributed infrastructure for life-science information
• The Genomics Standards Consortium (GSC) an international initiative
• Mirada Long Term Environmental Research (Part of NSF’s US LTER Program)
• Earth Microbiome Project
• Genomic Observatories initiative
• Oceanomics, a large French marine genomics project
Micro B3 was unique with respect to the interdisciplinarity of its consortium hosting a broad range of expertise. And therefore was able to achieve a new communication culture crossing traditional boundaries. Improved communication between wide-ranging disciplines significantly enhanced Europe’s ability to make use of the Petabytes of data produced to generate better understanding of the microbial elements of the marine ecosystem.
Knowledge transfer and dissemination work to enhance Micro B3’s visibility beyond the activities detailed above, was done during many international and EU-wide external events, for example at several Genome Standard Consortium yearly meetings, during European and national Maritime Days, Ocean of Tomorrow & ASLO conferences, EMBO workshops; BioMarine, EFIB, ECMNP business conventions, etc.. Micro B3 scientists gave lectures, some in Micro B3-related sessions, presented the roll-ups, manned booths and were invited as panellists. About 170 dissemination events have been organized or input provided in the form of sessions, posters, panels or presentations, including articles for the general public, press releases and films as compiled in the dissemination table (see A2 table).
Also the Micro B3 scientific groups were very active in publishing their findings. 110 peer-reviewed publications have already been published and more manuscripts are in the process of publication (see A1 table).
A highlight was the final Micro B3 Final Conference in 2015 with contributions by all members of the executive board and invited guests, including the advisory board. It was held in Brussels, at the Flemish Academy of Science and the Arts, from 2nd to 5th November 2015. The objective of the Micro B3 Final Conference was to bring together policy and biodiversity stakeholders from Europe and overseas to present the project results, discuss on the project perspectives and provide key recommendations and inputs from Micro B3 researchers to the European Commission.
Finally, 31 exploitable results have been compiled and entered into the SESAM database as part of the Plan for Use and Dissemination of Knowledge, documenting the ongoing interest by many partners to continue research, development and innovation. Many of the documented software tools and databases, as well as standards and metagenomics training resources are available in open access for interested users. Work on patenting a few of the biotechnological results is still in progress, as some further research and upscaling is needed.
Awareness of the importance of the marine system by implementing the citizen science campaign MyOSD 2014 and 2015
Citizen science (CS) involves the participation of the lay community in scientific research activities and often centers on the collection of data. Over the last few decades, CS has grown in popularity and it is now regularly featured in conservation science. Besides data collection, CS is also a means to enhance the scientific literacy of the lay community and influence their attitude towards environmental issues.
Already in 2014 OSD was not only popular among scientists, but also the general public was interested and contribute to OSD via the CS project called MyOSD. In 2014, MyOSD participants were asked to measure environmental parameters such as salinity or water temperature, which have crucial impact on microbial community structures. In 2015, OSD and MyOSD went into the second round. Given that some MyOSD participants from the previous event in 2014 had requested to also be able to sample for marine microorganisms a special MyOSD sampling kit was developed. The kit keeps the sample procedure straightforward and simple, while still producing adequate scientific samples. Hence, the MyOSD citizen science project expanded the OSD 16S ribosomal RNA data set of 2015 even further resulting in an even higher resolution of the marine microbial community snapshot OSD is aiming for. However, it also gives citizen scientists the possibility to investigate the prokaryotic life in the waters of their interest and the chance to learn more about the importance of marine microbial life with hands-on experience.
In total 250 sampling kits have been distributed via the OSD hubs. 192 kits have been returned and 319 DNA extractions have been performed. The MyOSD sequence and contextual data, just like the OSD data, will be made publicly available to everyone via the International Nucleotide Sequence Database Collaboration (INSDC) umbrella study PRJEB512926 and at PANGAEA. A survey performed among the MyOSD participants clearly indicated that over 95% of the participants felt more engaged with ocean related issues after participating in MyOSD.
Potential impact and the main dissemination activities and exploitation of results
Micro B3 has set out to improve Europe’s capacity for bioinformatics and marine microbial data integration to the benefit of a variety of disciplines in bioscience, technology, computing and law. The size of the consortium with 32 participants from universities, institutes and companies reflected the spirit of European researchers to jointly address pressing challenges in marine science. Micro B3 led to integration of expertise from sampling to supporting storage, analysis and downstream use of resulting environmental and bioinformatics data with the objective to create new ecological and biotechnological knowledge, as well as to the establishment of long-lasting and interoperable structures and resources for data mining.
Technologies were fully tested through the submission, storage and exchange of Micro B3 data between three established archives, hosting molecular (ENA), oceanographic (SeaDataNet) and biodiversity databases (EurOBIS). The Micro B3 Information System now facilitates access to marine environmental and genomic data sets, to information and bioinformatics tools.
Micro B3 contributed to promote long-term collaborations, also with the United States of America. Micro B3/OSD aligns nicely with the discussions on creating an Unified Microbiome Initiative (UMI) or an International Microbiome Initiative (IMI), with both proposals just published in the journals Science and Nature (October 29/30 2015).
Impacts on European Policy
Micro B3 addressed marine microbial biodiversity, bioinformatics and biotechnology, reflecting the three main topics the project impacted upon. Its interdisciplinary and intersectoral teams of excellent European researchers overcame current obstacles in marine biodiversity research and blue biotechnology and fully addressed impacts from the Ocean of Tomorrow Call Topic OCEAN.2011-2: ‘Marine microbial diversity – new insights into marine ecosystems functioning and its biotechnological potential’ through its cross-thematic and multi-disciplinary approach. Bio- and other informaticians, marine biologists, molecular ecologists, oceanographers, modellers and biotechnology developers joined forces with legal policy and communication experts and worked in an integrated way to achieve maximum outputs and impacts. Micro B3’s multi-sectoral partnership included basic researchers as well as applied ones, SMEs and other companies from software, biotech, data management and diversity sectors; legal experts and organisations like CIESM, IUCN and ICES.
Relevant end-users, esp. from governmental agencies and further representatives of biotechnology sectors, were addressed in several ways (as highlighted in the results section above) to get their input early on and increase awareness of marine microbial diversity and its utilization in the European society. Furthermore, the advisory board, representing high-level outward links for all three pillars (biodiversity, bioinformatics and biotechnology) enabled contacts to different user groups worldwide, especially in the USA. This enhanced early uptake and helped to guarantee wide accessibility of outputs. Micro B3’s two Ocean Sampling Days served outreach purposes very well, engaging the European public and the marine scientific community.
Addressing EC policy goals
Due to its cross-thematic research Micro B3 strongly improved our understanding and the predictive capacity of marine ecosystems' responses, inter alia the microbial loop, primary production, the biological carbon pump, energy flow in the food chain, to a combination of natural and anthropogenic factors, such as global warming, biodiversity loss and ocean acidification. Micro B3 scientists have been actively involved in raising awareness about the critical links between ocean and climate, and about how marine microbes affect and are affected by climate change, for example during the 21st Conference of Parties meeting in Paris (COP21) that resulted in an international agreement to address and limit the consequences of future climate change. Micro B3 has also fostered innovations in understanding and utilizing gene functions to make the most of the almost unexplored marine microbial diversity for biotechnological applications, as part of the genetic resources in the sea.
Micro B3 also addressed the novel European industrial policy for the globalisation era (MEMO/10/532, October 2010) contributing to strengthen the single market through European harmonisation of legal guidelines and IPR approaches, in our case for marine microbial diversity utilisation and through improving scientific research communication infrastructures, as well as contributing to standardisation. It also helped through forming innovative links between marine researchers and the industrial biotechnology sector, one of the key technologies mentioned. And it made natural sciences and engineering more interesting disciplines, including technological and skill- intensive activities, as well as applied research and SME aspects. Thus Micro B3 contributed to the ‘Agenda for new skills and jobs’, especially in the fields of environmental bioinformatics and marine or blue biotechnology and helped to counteract the perceived ‘widening of the innovation gap’.
The EurOcean conference held in autumn 2010 during the Belgian presidency confirmed in its final declaration that ‘the Seas and Oceans are one of the Grand Challenges for the 21st Century’.
Micro B3 partner have strongly contributed to three prioritised initiatives named in the declaration, i.e. ‘Innovation’ as addressed below under biotechnology; ‘Training and Career Development’ as described in the results section and under environmental bioinformatics capacity building below; and ‘International Cooperation’. The Micro B3 project as a whole focussed on world-wide coverage and citizen engagement for the two OSDs, on Mediterranean countries through CIESM, the summer school, as well as on cooperation, especially in the context of access and benefit sharing of marine biodiversity for its protection and sustainable use as detailed below.
Convention on Biological Diversity (CBD)
Micro B3 contributed to the implementation of the Convention on Biological Diversity by developing model agreements for access and benefit sharing of marine genetic resources. In particular, terms and modalities for innovative intellectual property management were developed to be used by various public, private and non-profit partners involved in Micro B3, but also beyond, in other European and international contexts. The proposed strategies will promote innovation, while at the same time contributing to benefit sharing with provider states in a mutually agreed manner, through measures such as sharing research results, building research skills and other capacities for source countries.
Many of the Micro B3 partners were involved in collecting as well as using genetic resources at later stages of the innovation chain. By gathering the main science and industry stakeholders in European marine research, together with representatives of user communities of the source countries, and science policy officials, Micro B3 defined a common approach to implementing the obligations under the Nagoya protocol, which entered into force during the project’s lifetime, in a manner facilitating large-scale collaborative research. This is a tool to overcome the high transaction costs of case-by-case bargaining which so far has slowed down the research and innovation process. The explicit goal was to work towards a sectoral Access and Benefit Sharing agreement for marine science that finding wide approval, while fulfilling a major objective of EU environmental policy. Thus, tight cooperation for example with related marine biotechnological projects like PharmaSea, but also close communication with national and international CBD-related bodies (in particular national focal points of countries that were consulted during the preparation of the Ocean Sampling Day) led to them acknowledging the Micro B3 Model Agreement as useful for their purposes.
Impacts of Micro B3 on better understanding of marine biodiversity
Improved assessment of marine microbial biodiversity
Although global initiatives to study and archive marine biodiversity were underway when Micro B3 started, they focused almost exclusively on macroscopic organisms. Despite representing 98% of the biomass in the oceans, the microscopic life was largely ignored. Micro B3 scientists addressed this critical issue by combining the ecosystems expertise of marine ecologists with appropriate bioinformatics approaches in order to close the widening gap between technology providers and field researchers. In this manner, Micro B3 scientists contributed to establishing a standardized baseline for molecular studies of marine microbial biodiversity.
Impacts which became already apparent during the lifetime of the project included comparative analyses of different biodiversity assessment technologies for viruses, bacteria, archaea, and protists and biodiversity assessments for entire microbial assemblages spanning five orders of magnitude in size, from viruses to protists (see several articles from May 2015 10.1126/science.aac5605).
Micro B3 also will have further impacts over longer terms as it provided transparent, accessible, and robust standards, best practice solutions, and protocols which were thoroughly tested for operability during the Ocean Sampling Day and MyOSD; a comprehensive registry of European marine microbial study sites and new standardised interoperability of data sources and databases for data acquisition and safeguarding. OSD and MyOSD have initiated a new kind of global but site-based research activities which is already followed by others.
Improved assessments of the role of marine microbes in driving biogeochemical cycles
Many of the metabolic transformations performed by marine microbes drive major biogeochemical cycles, notably for nitrogen, carbon, phosphorus, sulphur, and silicon. To understand further the role of marine microbial ecosystems in the cycling of macro- and micro-nutrients, we need more information about the metabolic capacities within the different organismal groups. Micro B3 has helped generate metabolic maps from different organisms, based on individual genome and transcriptome sequences, and extrapolated them to whole communities by probing metagenomic and metatranscriptomic sequences from a range of contextualized environments. The project has brought a wealth of gene sequences from all plankton groups, making it possible to position them on phylogenetic trees and metabolic pathways in order to understand who is doing what in each sampled ecosystem (e.g. see several articles from May 2015 10.1126/science.aac5605).
The combined knowledge within Micro B3 of gene function in representative organisms such as Prochlorococcus, Synechococcus, Prasinophytes and diatoms, which was combined with the bioinformatics and oceanographic competence, already had profound impact for improving our understanding of major biogeochemical cycles during the lifetime of the project. With additional study sites explored using the methodologies, tools and knowledge developed by Micro B3, e.g. as part of OSD and MyOSD, more impacts will arise in the near future.
Improved understanding of the feedbacks between marine microbes and climate
Although a range of geochemical models are in use to predict climate variations as a function of CO2 increases in the atmosphere, they usually do so in the absence of biological factors. A coarse grain assumption of CO2 recycling by ocean life was developed as a function of various scenarios of CO2 accumulation in the coming 350 years (Sarmiento et al. (1996) Science 274: 1346-1350). This is an important step forward, but in these models the treatment of organisms is highly oversimplified for the obvious reason that there is not enough knowledge of ocean life and its impact on carbon cycling to build it into models.
Furthermore, although powerful modelling tools are emerging to address the complexity and functioning of marine microscopic ecosystems, correlative data about the spatio-temporal distribution of plankton organisms in relation to environmental parameters required to test and feed such models are dramatically absent. Micro B3 collected these data, made them interoperable and initiated interdisciplinary work to use complex datasets in order to progress towards an integrated understanding of world-wide plankton ecology. Using Micro B3’s advanced modelling approaches our capacity to predict the dynamic evolution of marine microbial ecosystems in response to climate change has been improved significantly. This new generation of modelling combined with the large data sets provided by Micro B3 has a profound impact on the understanding of evolutionary and functional relationships of planktonic organisms in relation to ecosystem services and can reveal the potential consequences of anthropogenic activities over different scales.
Impacts of Micro B3 on improving environmental bioinformatics capacity building in Europe
Micro B3 created and implemented the M2B3 Standard which is based on the already existing “Minimum about any (x) Sequence” (MIxS) checklist standards of the Genomic Standards Consortium. Moreover, Micro B3 actively participated and implemented the new Minimum Information about Biosynthetic Gene Clusters (MIBiG) which achieved widespread support in biotechnology community. These standards are already adopted beyond Micro B3 and continue to do so.
The Micro B3 Information System improved European capacity in environmental bioinformatics by implementing an interoperable “scientific discovery workflow” from initial data gathering to generating new insights. The Information System and workflow is based on the new standards and was successfully used for Micro B3’s Ocean Sampling Day (OSD) and the accompanying MyOSD citizen science campaigns. The capability to use one or more of the Micro B3-IS components for different purposes other than OSD or MyOSD is an advantage of the modular approach of the Micro B3-IS. Hence, several components are going to be picked up and used by ELXIR, EMBRC and new European projects such as INMARE and AtlantOS and will form the basis for the emerging network of Genomic Observatories.
Increased bioinformatic training capacities
A final survey of the training pipeline was done end of 2015 to estimate the medium-term impacts with participants of more than one course. It highlighted the network building aspects of a training pipeline, i.e. when trainees had the chance to take part in more than one course and when the courses build on each other, at least partially. Many of the Postdocs, who attended more than one course, did mention papers done or in planning for which the new knowledge obtained through the trainings helped. In total six training courses were held and 135 trainees profited formally and directly. They spread the information obtained widely, through embedding it in courses or seminars, as well as personally, to at least further 161 scientists.
Overall 30 Micro B3 partners were involved in dissemination and training and many trainers were teaching in several courses, which was very helpful for the training pipeline concept. At least 18 out of 32 partners sent trainees, some to almost all of the courses. Repetitions of some courses are planned. This also helped with the achievement of the next objective, through spreading the standards widely.
Optimum integration of marine microbial diversity research and oceanographic research by adopting standards and establishing interoperability with the leading oceanographic data infrastructure initiatives in Europe
With Micro B3 a new communication culture crossing traditional boundaries was implemented. Improved communication is significantly enhancing Europe’s ability to make use of the petabytes of marine ecological and genetic data. Data is now available for in-silico modelling of key microbial ecosystem components to enhance ecosystems biology and biotechnology. The biodiversity research in Micro B3 has led to standardized protocols for sampling and metadata acquisition as well as sample preparation and analysis as described in detail in the OSD handbook.
Joining European initiatives like SeaDataNet and EmodNet within Micro B3 and using large genomic data sets from the Tara Oceans expedition, from long-term monitoring and study sites as well as newly generated data from the Ocean Sampling Day, Micro B3 significantly contributed to increased interoperability and data quality between the sectors of oceanographic and contextual data as well as in biodiversity and meta(genomic) data. It has enabled the linking of molecular changes to changes in the marine environment. Key genes have been identified as well as genomic responses to stressors like temperature changes, eutrophication and others.
Micro B3 addressed all three objectives named in the Communication on Marine Knowledge 2020 (COM(2010) 461 final), namely (1) reducing operational costs and delays for those who use marine data and therefore helping private industry compete in the global economy and meet the challenge of sustainability, improving the quality of public decision-making at all levels and strengthening marine scientific research; (2) increasing competition and innovation amongst users and re-users of marine data by providing wider access to quality-checked, rapidly available coherent marine data and (3) reducing uncertainty in knowledge of the oceans and the seas and so providing a sounder basis for managing future changes.
Micro B3 scientists have established an agreed model for the data flow from the field (cruises, monitoring stations) via data centres like NODCs, ICES, PANGAEA, to European overarching data infrastructures for marine environmental data (SeaDataNet), marine biodiversity data (EurOBIS), and genomic data (EMBL-EBI/ENA database). This model allows for sharing metadata and giving access to related data also with the Micro B3 Information System. This major achievement will have a long term effect and pave the way providing a common access for users to marine data from the diverse domains and infrastructures.
Micro B3 is in constant communication with TARA Oceans as well as upcoming Microbiome initiatives like MetaSub to sample microbiomes of major cities in the US and the River Sampling Day in Europe. In 2016, a high-resolution citizen science project MyOSD Germany is organized, targeting the microbial diversity of rivers as well as that of the North- and Baltic Seas.
Impacts on exploitation of marine microbial diversity and functional potential for biotechnological applications
MicroB3's diverse biotechnological approaches based on marine (meta)genomic information have socio-economic impacts on industrial, pharmaceutical and other applications. The foci were on using co-occurrence networks for determining hypothetical functions of the many unknown genes; on determining the functional factors for similarities in microbial community structure in remote locations and on determining the anthropogenic influence that triggers microbial resistance (resistomes) in coastal waters. By screening for biosynthetic gene clusters, additional putative NRPS and polyketide clusters have been found, as well as hot spots for potential new antibiotics and anti-cancer agents: three new antitumor compounds have been identified from sponge-associated bacteria for preclinical assays. Work also yielded many novel enzymes available for further testing in biotechnological applications, such as enantioselective biocatalysis, degradation of pollutants, and biomass processing.
The bioinformatics and computational tools that have been developed will continue to be useful for focusing and reducing laboratory work both in the stages of discovery and of tailoring enzymes for practical applications beyond Micro B3. To facilitate genome mining for bioactive compounds with potential medical applications, micro-scale cultures and sequence information have been used and also will act in the future as a resource for further exploitation of marine biodiversity. The transition to a bio-based economy and the growing use of green chemistry depends on novel and improved biocatalysts for key reaction steps. The Micro B3 project directly contributed to the bio-based economy by training scientists in the use of resource-efficient computational and bioinformatics approaches, which can replace or reduce high-cost and labour-intensive laboratory work. This not only leads to quicker development times for new processes, it also supports the competitiveness of the European R&D effects in synthetic biology, applied biocatalysis and green chemistry.
Tools explored in Micro B3 will continue to be used in bilateral and national research projects and results also continue to improve new EU research and innovation projects, such as ROBOX, where enzyme robustness engineering is of key relevance.
Libraries will continue to be used in R&D projects aimed at enzyme discovery and in the search for new active agents; further resources (software, data, samples, teaching and legal tools) are now available in open access to allow for maximum socio-economic and policy impacts.
Micro B3 scientists explored different extreme marine environments, from brine lakes on the bottom of the Mediterranean to coastal springs with large tidal variations in temperatures for new enzymes to be applied in industrial biocatalysis; from polluted sites as well as microbiota from marine sponges. This helped to cover a very different spectrum of microbial activities in view of physico-chemical conditions, input of resources, and dominant physiological processes.
Using genome sequences, and metagenomic expression libraries, as well as high-throughput sequencing, many new activities were discovered for industrially important reactions, such as bioprocessing enzymes for polymers, enzymes involved in production and modification of secondary metabolites, and stereospecific enzymes acting on C-N and C-O bonds.
Micro B3 has thus created a marine-microbe-derived multi-enzyme platform for the production of enzymes with potential applications in industrial biocatalysis, food/feed technology and bioprocessing of raw renewable materials. Such platforms are needed as addressed in the Recommendations of the recent ESF paper on Marine Biotechnology.
Findings of the biotechnological team within Micro B3 can be potentially used in the following fields: food processing, cleaning, bioactive peptide synthesis; biodegradation, bioremediation, enantioselective catalysis, carbohydrate processing, the fine chemicals industry, green polymers, biomass processing, protein hydrolysis, food industry, peptide synthesis and carbohydrate hydrolysis (for an overview see table in the attachment).
Plan for the use and dissemination of foreground
Most of the 31 results documenting exploitable foreground in this plan were shortly described in the results section above. For more detail see table B2.
Dissemination and outreach aspects of Micro B3
A dissemination plan for Micro B3 was followed, including outreach, training and capacity building elements based on a questionnaire spread widely to diverse user communities to elicit training and dissemination needs and was revised after the first year. A three-fold approach was implemented on top of an informative website, factsheets, newsletter contributions, several films and sessions and a multi-user final conference: (1) workshops for the interested public, including policy-makers to inform them about marine microbial diversity, the genomic revolution, its application options and aspects of sustainable use of marine genetic resources.
(2) Dedicated short workshops for interaction with industry: Selected specific topics were chosen based on the questionnaire and especially on what the consortium had to offer. Intense exchanges of knowledge and technology transfer with industrial partners took place, especially with SME within the white or industrial biotechnology sector, but also with larger company representatives. Their needs were discussed, with a view especially to bioinformatics, and knowledge shared on the exciting areas of marine (meta)genomics and blue biotechnology.
(3) Scientific training, which was organized within a training pipeline, including one summer school: It provided bioinformatics expertise to young Micro B3 scientists, and to the participants of the Ocean Sampling Day. The trained new generation has become the seed for disseminating Micro B3 standards and tools to the wider scientific community. Some of the courses and modules serve as models for later repetition, and thus were thoroughly documented. Several multipliers were selected to attend in order to enable future training on smaller e.g. regional scales and in other languages in order to generate more long-term sustainability in capacity building.
The Ocean Sampling Day was instrumental in establishing a baseline, joint understanding of sampling approaches and adoption of standards. A group of young marine scientists was trained throughout the project, mainly in bioinformatics, but also hands-on in sampling and analysis techniques, in the organisation and full implementation of the Ocean Sampling Day. This new generation of marine and bioinformatic scientists, learning also about IPR, ABS and biodiversity issues, add impact to the European capacity needed to fully exploit the plethora of data related to marine genetic resources being generated right now and in the years, maybe even decades to come. They, as well as many of the lecturers who trained them during Micro B3 are available for repetitions or similar courses performed on a local or regional scale, which may be organized by OSD participants, some of which have already stated their interest.
To establish a flexible workshop and training environment in a highly collaborative effort was one of the long term goals of Micro B3 and it was a very positive development that the EMBL-EBI hosted two of the training pipeline activities, marine metagenomics bioinformatics and the OSD annotation jamboree, documenting fully the course resources on metagenomics bioinformatics, thus facilitating its further use and a possible repetition. The Micro B3 scientists, together with the participants of the Ocean Sampling Day became ambassadors for promoting the new European guidelines for marine microbial sampling, bioinformatic analyses, exploration, knowledge generation, and finally exploitation.
IPR issues related to the exploitation and protection of marine genetic resources and large-scale genomic data
An appropriate legal framework and model contracts were elaborated with CIESM representing Mediterranean interests, and with input from several biotech companies. The outcomes were tested whether they are acceptable to researchers, policy makers and companies including also representatives from sequence databases and culture collections, which had pertinent experience and may play key roles in ABS IPR issues, and whether they guarantee access to samples and data as well as proper benefit sharing with owners of biodiversity.
Micro B3 contributed to developing IPR agreements that promote the development of downstream applications from marine biodiversity, while generating new funding streams for conservation through appropriate benefit sharing. More specifically, in relation to IPR, Univ. Catholique de Louvain produced industry-university models for the management of IP rights based on an adaptation of open-collaboration models and models for corporate-sponsored research initiatives. This work was based on discussions with the main industry and policy stakeholders and is now available as a short policy brief.
Open access and open source models which generate spill-overs for the commercial sector
For data and information, Micro B3 implemented the principles of the Fort Lauderdale Agreement for fair use of data of large-scale genome sequencing projects, which have already successfully been used in other large-scale genomic projects in microbial science, such as the international human microbiome consortium (www.human-microbiome.org). Micro B3 promoted the rapid pre-publication release of sequence data, a practice which has shown to generate tremendous benefit to the scientific research community in general. On the other hand strategies and tools for companies were developed, with the aim to generate commercial spill overs from these open access resources, by offering services, especially tailor-made research services, and training in the use of the data portals and services.
Bio-Prodict, for example, offered in-house training in the use of protein structure databases, which serve as tools to predict and improve enzyme properties and also contributed to two masterclasses offered at the University of Groningen. Both training schemes provided background information on the foundations and uses of the 3DM protein structure analysis platform and also hands-on training to discover networks of functional interactions in enzymes. This stimulated application of novel bioinformatics tools in a laboratory environment. This SME profited strongly from their involvement and work in Micro B3, which led to new customers and new project partnerships.
Almost all software in Micro B3 is open source, e.g. the Micro B3 Information System and its many software modules. A commercially friendly licensing model was developed based upon two-tiered open source license models under an Apache 2 and/or CC-BY license). The advantages of this approach are that scientists can openly publish their results and companies gain competitive advantage by commercially exploiting the lead time gained through immediate access to new tools and applications developed within the consortium (see exploitation plan for further detail on all software tools).
Further freestanding software tools are available in open access and were developed by the scientific partners CNRS and AWI. Examples include Swarms, an amplicon clustering tool for biodiversity studies, Wisescaffolder, an algorithm for the semi-automatic scaffolding of Next Generation Sequencing data, Cyanorak, Cyanolyase, and Micro B3 Viral DB, which are databases on picocyanobacterial genomes, on phycobiliprotein synthesis and on viral genomes with host annotations.
List of Websites:
Address of the project public website
It offers access to all public deliverables (https://www.microb3.eu/work-packages/public-deliverables) background information, news, events and diverse materials for the media, especially on the OSDs and the final conference. Micro B3-related peer-reviewed publications (for Open Access full articles, for Restricted Access abstracts) are available through an OpenAire repository, and are also directly accessible here:
Grant agreement ID: 287589
1 January 2012
31 December 2015
€ 11 496 409,35
€ 8 987 491
JACOBS UNIVERSITY BREMEN GGMBH
Deliverables not available
Grant agreement ID: 287589
1 January 2012
31 December 2015
€ 11 496 409,35
€ 8 987 491
JACOBS UNIVERSITY BREMEN GGMBH
Grant agreement ID: 287589
1 January 2012
31 December 2015
€ 11 496 409,35
€ 8 987 491
JACOBS UNIVERSITY BREMEN GGMBH