# Discovery pipelines for marine resources: an ocean of opportunity for biotechnology?

D. Smith, A. Buddie, R. Goss, J. Overmann, C. Lepleux, M. Brönstrup, B. Kloareg, T. Meiners, P. Brennecke, A. Ianora, et al.

► To cite this version:

D. Smith, A. Buddie, R. Goss, J. Overmann, C. Lepleux, et al. Discovery pipelines for marine resources: an ocean of opportunity for biotechnology?. World Journal of Microbiology and Biotechnology, 2019, 35 (7), 10.1007/s11274-019-2685-y. http://02326230

HAL Id: hal-02326230

https://hal.science/hal-02326230

Submitted on 30 May 2023

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>
# Discovery pipelines for marine resources: an ocean of opportunity for biotechnology?

D. Smith¹ Ⓓ A. G. Buddie¹ Ⓓ R. J. M. Goss² Ⓓ J. Overmann³ Ⓓ C. Lepleux³ Ⓓ M. Brönstrup⁴ Ⓓ B. Kloareg⁵ ·
T. Meiners⁶ Ⓓ P. Brennecke⁶ · A. Ianora⁷ Ⓓ F.-Y. Bouget⁸ · P. Gribbon⁹ Ⓓ M. Pina¹⁰

Received: 16 May 2019 / Accepted: 27 June 2019 / Published online: 2 July 2019
© The Author(s) 2019

## Abstract

Marine microbial diversity offers enormous potential for discovery of compounds of crucial importance in healthcare, food security and bioindustry. However, access to it has been hampered by the difficulty of accessing and growing the organisms for study. The discovery and exploitation of marine bioproducts for research and commercial development requires state-of-the-art technologies and innovative approaches. Technologies and approaches are advancing rapidly and keeping pace is expensive and time consuming. There is a pressing need for clear guidance that will allow researchers to operate in a way that enables the optimal return on their efforts whilst being fully compliant with the current regulatory framework. One major initiative launched to achieve this, has been the advent of European Research Infrastructures. Research Infrastructures (RI) and associated centres of excellence currently build harmonized multidisciplinary workflows that support academic and private sector users. The European Marine Biological Research Infrastructure Cluster (EMBRIC) has brought together six such RIs in a European project to promote the blue bio-economy. The overarching objective is to develop coherent chains of high-quality services for access to biological, analytical and data resources providing improvements in the throughput and efficiency of workflows for discovery of novel marine products. In order to test the efficiency of this prototype pipeline for discovery, 248 rarely-grown organisms were isolated and analysed, some extracts demonstrated interesting biochemical properties and are currently undergoing further analysis. EMBRIC has established an overarching and operational structure to facilitate the integration of the multidisciplinary value chains of services to access such resources whilst enabling critical mass to focus on problem resolution.

**Keywords** Marine · Microorganism · Discovery pipeline · microbial domain Biological Resource Centres (mBRC)

## Introduction

The marine environment provides a huge and, as yet, under-tapped resource for the biotechnology industry (Blasiak et al. 2018). However, whilst individual enzymes/products or their host organisms have been exploited for commercial products, to date there has not been a concerted effort to make use of marine organisms in a more holistic way. Indeed, a recent study highlighted the unevenness of commercial activities in this sector, as may be seen from patents derived from marine organisms: of almost 13,000 sequences of marine origin assigned to patents at the time of writing, 47% were filed by a single multinational company (see

Blasiak et al. 2018). Marine science now has a wealth of tools to overcome key bottlenecks in access to and releasing the potential of marine organisms. It has been estimated that less than 1% of novel marine genetic resources will make it to the market place (Royal Society 2017). The massive growth in opportunities afforded by new high throughput technologies enables understanding of the biological and chemical make-up of novel compounds and screening for them in the first place. This has been aided by a similar expansion in computing power and analytical tools in order to put such discoveries into context.

The European Marine Biological Research Infrastructure Cluster (EMBRIC-https://www.embric.eu/) brought six European Research Infrastructures together (Brennecke et al. 2018) and aimed to fulfil one of the key rules for ‘pragmatic blue growth’ described in a recent policy paper by being a ‘well-designed institution’ (Burgess et al. 2018) although

<figure><img src="image_2.png" /></figure>

<footnote>D. Smith
d.smith@cabi.org</footnote>
marine biotechnology, per se, was notable by its absence in a recent study of the management of ocean resources (Klinger et al. 2018). The European Strategy Forum for Research Infrastructures (ESFRI-https://ec.europa.eu/research/infrastructures/index_en.cfm?pg=home) have encouraged the establishment of Research infrastructures (RIs): facilities, resources and services used by the science community to conduct research and foster innovation.

## Materials and methods overview

There are numerous routes that may be taken in the discovery process. The first step is to select where to sample, followed by the prioritisation of organisms and selective sampling to enrich for organisms with the desired properties. Obviously, in order to invest time and effort wisely, these choices are critical, because the oceans are vast and their potential enormous (Royal Society 2017), but the organisms are difficult to isolate and when they are, they are generally slow-growing. A typical workflow begins with the source of organism, where there are two options:(1) isolation directly from the environment; or (2) selection from those already isolated; the latter for example, may be taken from *ex situ* biological resource collections such as the microbial domain Biological Resource Centres (mBRC—OECD 2007). A number of such mBRCs are coordinated either by the Microbial Resource Research Infrastructure (MIRRI; www.mirri.org/) or the European Marine Biological Research Centre (EMBRC-ERIC) (https://www.embrc.eu/). MIRRI brings together 31 international mBRCs which, together, hold a total of > 330 K microorganisms, ranging from bacteria (including cyanobacteria) to yeasts, filamentous fungi and algae. EMBRC brings together marine biological stations and institutes organised through nine national nodes.

At each level of the discovery process there are technical issues to be addressed. EMBRIC has begun the process of mapping current mechanisms available to help facilitate how the potential of novel organisms is identified and the organisms with the most potential targeted (Brennecke et al. 2018). Options to be considered are for example using metagenomics to assess sample content; using proteomics and metabolomics to identify chemical targets; analysing information on chemical interactions (e.g. allelopathic, grazer defence metabolites) to identify bioactive compounds; and using metabarcoding technology to characterise the species compositions of mass samples of environmental DNA. Bottlenecks could also revolve around compliance with regulation for example obtaining permissions to collect (Kamau et al. 2010; Overmann and Scholz 2017; Smith et al. 2017). Despite numerous attempts to explore the microbial diversity of the planet, to date the majority has yet to be isolated and cultured and consequently has remained

largely untapped, especially with regard to bioprospecting (Overmann and Smith 2017). Up to 95% of marine life (by weight) has been estimated to be microbial in nature (Vierros et al. 2016). The failure to recover phylogenetically novel microbial lineages of bacteria and fungi can be attributed to current isolation methodology that is often found to be inadequate (Overmann 2012). Phenotypically novel types of bacteria often have unknown growth requirements and, are highly fastidious, for example: *Myxobacteria*, *Acidobacteria* and *Dehalococcoides ethenogenes* (Foesel et al. 2013; Maymó-Gatell et al. 1997; Sanford et al. 2002). There is a need to employ improved methodology and undertake a systematic approach to culturing microorganisms such as harnessing “culturomics” (Lagier et al. 2012). Dedicated isolation protocols are required, e.g. selective media: to date, less than ca. 1% of all microorganisms can be cultivated (Pan Ming Huang et al. 2012; Overmann et al. 2017). Development of novel approaches to eliciting enhanced production—media development, use of small molecule elicitors, targeted heterologous expression and promoter refactoring. Growth/fermentation technologies need to be expanded, allowing bulking up for production (e.g. Hewitt and Nienow 2010). Once the organism is grown access to the most appropriate methodologies and technologies is needed:

* Immunological ID, Next Generation Sequencing (whole sample; e.g. Chun and Rainey 2014);

* Sequencing; genome, proteins; metabolomics (e.g. Nacache et al. 2014);

* Population/environmental metagenomics (e.g. Lorenz and Eck 2005);

* Bioinformatic tools, data interoperability; Common tools and software (e.g. Okonechnikov et al. 2012);

* Identification of bioactive compounds (e.g. Kim and Mendis 2006).

In identifying and isolating promising metabolites loss of resources and time by rediscovery of already known compounds must be avoided by using optimal de-replication protocols (e.g. Dias et al. 2012). Supporting technologies are:

* Genome mining of biosynthetic potential (e.g. Wagner-Döbler et al. 2002);

* Bioassay technologies (e.g. Delamarche et al. 2005);

* Routes from crude extract assay hits to scaling, identifying, purifying and structural characterising bioactive compounds (e.g. Li and Vederas 2009) utilising, where possible, genome guided characterisation in complement with spectroscopic guided characterisation.

Improved routes from novel bioactive organisms and bioactive extracts, identifying and purifying key bioactives as either a single compound or a synergistic mixture of compounds is needed. In response to this issue EMBRIC
is facilitating the search by exploring ways to integrate and mine metabolomic/genomic/metagenomic data. There is also a critical need to bridge the gap between ‘feature identification’ and compound chemical characterisation. Data analysis tools to integrate genome reading with metabolite information are seen as a crucial success factor. Thus, an essential first step is combining parallel, complementary streams to speed discovery (see Fig. 1).

An additional problem is that the majority of microorganisms in the environment are yet to be cultured and this is compounded further by the fact that those from marine environments are renowned slow growers (Joint et al. 2010). In order to address this, DSMZ researchers have been developing culturing technologies for less tractable microbes. Methods under consideration currently include applications already demonstrated in the field e.g. iChip technologies (Nichols et al. 2010) and the single-dilution MicroDrop® technique (Bruns et al. 2003). Nichols et al. (2010) designed an isolation chip (iChip) composed of several hundred, miniature diffusion chambers—each inoculated with a single environmental cell. They showed that microbial recovery in the iChip exceeds many-fold that afforded by standard cultivation, and the species thus obtained are of significant phylogenetic novelty. The method allows access to a large and diverse array of previously inaccessible microorganisms and is well suited for both fundamental and applied research. (e.g. Sherpa et al. 2015). As such, the iChip is a high-throughput platform for parallel cultivation and isolation of previously uncultivated microbial species from a variety of environments. Using a microdispenser system, Bruns et al. (2003) developed a high-throughput innovative technique for the isolation of bacteria in liquid. The MicroDrop® microdispenser system enables the dispensing of highly reproducible volumes (0.5 µl to 2.5 ml) in microtiter plates in less than

<figure><img src="image_3.png" /><figcaption>**Fig. 1** Combining parallel, complementary streams to speed discovery from marine resources</figcaption></figure>

1 min per plate allowing the generation of large numbers of bacterial enrichments for downstream cultivation and analysis. Using this technique Gich et al. (2005) obtained 570 bacterial cultures from which the majority was closely related to previously uncultured bacteria. It is necessary to bridge the gap between compound identification and compound chemical characterisation and, ultimately, to gain access to the most appropriate methodologies and technologies. Current “state of the art” approaches utilise High Resolution Mass Spectrometry (MS) and database comparison, coupled with genome analysis. Cloning and heterologous expression has potential but needs considerable further work. Bouslimani et al. (2014) described technologies in mass spectrometry of natural products. A key strength being that very small quantities of sample are required allowing much structural information to be gained from a single mass spectrum. This approach is particularly relevant with the slow-growing marine organisms and shows why such methods are being used to generate information on the selected test strains in EMBRIC’s component laboratories currently. Mass spectrometry structural analysis workflows are emerging that allows faster de-replication and structural elucidation of metabolites, facilitating the screening of new biologically active compounds.

## EMBRIC microbial product discovery pipeline output

To date, the work undertaken has involved the strategic isolation of biotechnologically useful microbes using a DSMZ biofilm method (Gich et al. 2012) and selection of the most promising microbes for extract analysis. DSMZ collected samples of sea water, sediment and sponges from the Pacific Ocean and from over 900 samples isolated, found 248 species considered rare and difficult to grow. These included strains from the phyla Actinobacteria, Bacteroidetes, Proteobacteria and Rhodothermaeota. Two new species of *Arco bacter* have been found and there may be more new taxa to be reported, as characterisation is ongoing. The partners at the University of St Andrews have cloned a large number of gene clusters and used promoter refactoring in attempts to awaken “sleeping clusters”. As a result, they detected novel metabolites demonstrated through the Global Natural Products Social Molecular Networking (GNPS) web-platform.

Helmholtz-Zentrum für Infektionsforschung (HZI) have received 156 extracts from DSMZ in total comprising pellets, supernatant and resin extracts of 25 strains which have been analysed. The analysis of the LC-MS/MS measurements revealed a total of 557 features detected in at least one of four strains; the number of features was reduced to 312 after discarding features that were found also in the control sample (KM14 growth medium). In-house library
matching identified 13 of the 312 metabolites found. Tyramine and Adenine are the metabolites found in each strain with the highest intensity; Glycocholate is present in three strains with very high intensity; 2-Deoxyadenosine is found with the highest intensity in one strain being present only in traces in the other strains (very low intensity); see deliverables on EMBRIC website https://www.embric.eu/deliverables specifically: D6.1 EMBRIC showcases: prototype pipelines from the microorganism to product discovery (Revised 2019) at https://www.embric.eu/sites/default/files/deliverables/EMBRIC%20Deliverable%20D6.1b.pdf. Further analysis of the more recently received strains is ongoing. An overview of the routes to discovery within the EMBRIC project are shown in Fig. 2. Extensive analysis of over 150 cell pellet extracts selected from the 248 strains was carried out via a combination of MS/MS in-house library matching and manual dereplication (using commercial and online opensource databases) allowed the team to assign between 10 and 25% of the detected features to known metabolites.

There were a number of outputs of significance from the microbial prototype pipeline:

* Flow cytometry and biofilm technologies were used for selection of marine organisms in samples that had greatest potential as candidates for biomass production;

* Combined cosmid and BAC library heterologous expression approach developed;

* Genome informed use of isotopes to track new metabolites;

* USTAN generated cosmid libraries for a series of more tractable and sequenced test strains. Biosynthetic gene cluster analysis prioritized nine clusters according to novelty. Cosmids containing them were taken into strains of *Streptomyces coelicolor* and *Streptomyces lividans* for heterologous expression. Fermentation broth extracts revealed heterologously produced metabolites;

* Larger biosynthetic gene clusters were accessed via a complementary BAC library approach, removing the need for cluster stitching prior to heterologous expression;

* Original bottlenecks in getting sufficient DNA for analysis were overcome:

* Expression level: addressed by introduction of promoters, utilization of different heterologous hosts, and utilization of small molecule elicitors;

* Metabolite de-replication and assignment: addressed with cluster analysis of LC-MS/MS data aiding the identification of series of closely related compounds;

* Cyclic peptides difficult to characterize: new approaches explored by pre-incubation with an enzyme;

* HZI have optimized analysis for different metabolite classes: e.g. short-chain fatty acids to larger polar and nonpolar compounds of primary and secondary metabolism. Lessons learned from the metabolome analysis of microalgae in EMBRIC work package 7 (https://www.embric.eu/WP) accelerated their application to the bacteria;

* Use of metabolite clustering analysis with a tool made available as an R package to assist in the identification of novel suites of compounds;

* Use of molecular networking tool from GNPS combined with manual dereplication and matching with in-house library allowed the faster and more efficient annotation of known metabolites. The visualization of the chemical space at the molecular level gave insights on structural relationship between chemical entities in the whole data set, pointing towards potential novel compounds (unknown metabolites);

Raw data and detailed results are to be found on the EMBRIC web site under work package 6 deliverables (https://www.embric.eu/deliverables).

## Discussion

The coordinated activities of the EMBRIC project has demonstrated that workflows across different ESFRI research infrastructures can improve our ability to isolate rare organisms and access their metabolites. The EMBRIC project examined the completeness of the necessary workflows for key types of marine bioproducts and identified some crucial bottlenecks that currently present obstacles to the exploitation of this vast resource. Where possible, strategies to overcome the gaps and problems identified in ways that may benefit the user community have been determined.

When seeking active molecules for use as an effective product, the first step is to identify where the potential solution might be. Are there already organisms, derivatives or even purified compounds that are available (i.e. de-replication) and how might more be discovered in the marine environment? This first step, therefore, may be considered to be *identifying the potential*. Twenty collections registered with the World Data Centre for Microorganisms (WDCM) hold microorganisms sourced from the marine environment; amongst them, they house over 334,000 strains. Even within the EMBRIC partners, there are collections that are not listed on the WDCM; additionally, there are many organisms held by scientists in non-registered collections around the world that are not visible to the researcher. The rest of the 700+ collections in the WDCM hold over two million strains of microorganism, many of which may well originate from the marine environment but there is no single place

<figure><img src="image_4.png" /></figure>
<table><caption>Figure 2. Overview of routes to discovery</caption><thead><tr><th>Source Research Infrastructure</th><th>Microbial Prototype partners</th><th>Pipeline</th><th>Best Practice</th><th>Legal Frame work</th></tr></thead><tbody><tr><td rowspan="2">EMBRC; MIRRI</td><td rowspan="2">DSMZ; CAB International (CABI)</td><td><strong>Sourcing organisms</strong><br>Ex situ - microbial domain Biological Resource centres (mBRC)<br><br>Selected from holdings<br><br>In situ - targeted isolation programmes from the marine environment and identification<br><br>Baiting and biofilm techniques; Flow cytometry selection</td><td rowspan="2">OECD (2007); ISO Standards<br><br>Institutional Methods<br><br>Genomic Standards Consortium</td><td rowspan="2">Intellectual Property Rights (IPR); Nagoya Protocol<br><br>Health & safety</td></tr><tr><td><strong>Cultivation and biomass production</strong><br>Biomass generated or DNA extracted for sequenced bioactive microorganisms<br><br>Cosmid libraries generated; genome analysis: selection of strains for growth</td></tr><tr><td>MIRRI and EMBRC</td><td>DSMZ; CABI<br>DSMZ; University of St Andrews (USTAN)</td><td><strong>Determination of organism potential</strong><br>Biosynthetic gene cluster analysis Larger clusters complementary Bac library approach<br><br>Cosmids containing them were incorporated into strains for heterologous expression</td><td></td><td>Nagoya Protocol</td></tr><tr><td>ELIXIR and EU-OPENSCREEN</td><td>USTAN;</td><td><strong>Extract production and exploration</strong><br>Fermentation in complementary media<br><br>Broths fractionated according to polarity<br><br>Bioassay Guided fractionation<br><br>LC-MSMS profiling</td><td>Institutional Methods</td><td>Health & safety; Nagoya Protocol</td></tr><tr><td>EU-OPENSCREEN; extracts from mBRCs or commercial sources</td><td>DSMZ</td><td><strong>Rapid identification of new compounds ensuring mitigation against wasted resource on compound rediscovery</strong><br>Enzyme mining<br>short-chain fatty acids to larger polar and nonpolar compounds of primary and secondary metabolism</td><td>Comparison of datasets<br><br>Institutional Methods</td><td></td></tr><tr><td>EU-OPENSCREEN</td><td>HZI</td><td><strong>Purification of characterised compounds</strong><br>Extracts and compounds to/from partners -IP - Compound Progression<br><br>up-scaled fermentation, extraction and purification<br><br>full chemical characterisation assisted by genome reading<br><br>biological assessment of compound bioassay</td><td>Institutional Methods</td><td>Health & safety</td></tr><tr><td>MIRRI - mBRCs; EU-OPENSCREEN libraries</td><td>DSMZ<br>HZI<br>CABI</td><td><strong>Protection of investment and future use</strong><br>Strains cryopreserved and catalogued; Related extracts and purified compounds stored; Related data and strains and compounds catalogued<br>Legal clarity for use: compliance with regulatory environment</td><td>OECD (2007); ISO Standards; EU-OPEN-SCREEN and MIRRI</td><td>IPR; Nagoya Protocol</td></tr></tbody></table>

<caption>Fig. 2 Overview of routes to discovery</caption>
where all catalogues can be searched and these strains easily
revealed. At present, each single collection catalogue would
need to be searched and only 85 collections (holding a total
of 356,889 strains) are included in the Global Catalogue of
Microorganisms (GCM), with only minimal data made avail-
able (https://gcm.wfcc.info/). A concerted effort, therefore,
is needed to make these strains more readily accessible: a
task that can be undertaken by the MIRRI and EMBRIC
consortia.

When the species coverage is examined it is very easy
to see that there are large gaps between what is known and
what is available. There are also estimates that less than 1%
of the microorganisms that are in the environment have been
discovered, to date. Thus, the question remains: how can this
vast resource be tapped? Targeting the organisms and estab-
lishing sampling regimes for the chemistry that is needed, is
not straightforward. New approaches such as “culturomics”
are being harnessed; this entails the exhaustive application
of culture media and growth conditions for the maximum
recovery of cultivable microorganisms from a biological
sample (Lagier et al. 2012). It may also be possible to add
to the traditional methods of “baiting” environments with
substrates to select specific potential based on chemistry or
utilising molecular tools or probes and cloning genes. The
danger of simply employing normal isolation techniques
to extract and identify the organisms and to get them into
axenic mono-culture is that the faster growing and/or more
common organisms will be isolated repeatedly whilst com-
pletely missing the slow-growing or yet-to-be-cultured ones.

Genomic DNA analysis using next generation sequenc-
ing approaches to examine total sample DNA for the yet
uncultured is notable way forward but accessing the relevant
expertise and facilities for this is not always obvious. Char-
acterisation technologies, data analysis and applications are
available and have been identified through this process. The
key function of the research infrastructures is to give access
to the resources needed for a researcher to make discov-
eries: e.g. the EMBRIC web tool. ELIXIR, a distributed
infrastructure for life-science information: https://www.
elixir-europe.org/tools tools offer different ways to find and
analyse data to enable such compounds to be identified.
The remaining elements in discovery pipelines, scaling-up,
extraction, purification and delivery of chemically defined
compounds are also elements that have been designed for
some specific cases. EU-OPENSCREEN (https://www.eu-
openscreen.eu/) in particular has pulled together much of
the best practice for chemical screening, and offers long-
standing expertise regarding the setup and implementation
of chemical screens and subsequent hit-to-lead optimiza-
tion. EU-OPENSCREEN works closely with collaborating
chemists and biologists and provides support throughout the
entire screening workflow—from the initial idea to the opti-
mized hit. In the context of EMBRIC, newly isolated marine

compounds can be tested against a variety of biological
assays using EU-OPENSCREEN in order to obtain bioactiv-
ity profiles of newly isolated compounds (e.g., antimicrobial
or antiproliferative activities). Moreover, compounds will
be transferred into the EU-OPENSCREEN comprehensive
compound library, thus increasing European visibility. All
of these aspects are organised in such a way that no loss of
intellectual property occurs.

A fundamental difficulty in assisting bioindustry to access
the most appropriate targets is getting specific information
from companies to enable delivery of their needs. To date,
resource centres and service providers have tried to second-
guess what a company needs, or wants, as many companies
are reluctant to let others know what they are working on.
Enabling companies to access and utilise information in
open databases anonymously misses potential and lessens
the opportunity to get the collections and their networks to
make more targets available. Appropriate business models
with non-disclosure agreements are needed and a common
agreement enabled for infrastructure partners to allow others
to act on their behalf.

By pooling expertise, a critical mass can be focused on
specific user problems, such as, the development of dedi-
cated isolation protocols (e.g. selective media to access the
microorganisms that cannot yet be cultivated). In terms of
the bottlenecks associated with specific active molecules,
gaps between the identification and chemical characteriza-
tion of compounds can be addressed and appropriate bio-
logical assay technology to comprehensively test for bioac-
tivities is available already. Routes from crude extract assay
hits to identifying and purifying bioactive compounds are
facilitated across the partner research infrastructures. This
may be done through access to the most appropriate meth-
odologies and technologies: e.g. common tools and soft-
ware. By accessing the research infrastructures, the loss of
resources and time, by rediscovery of known and already
patented compounds, can be avoided (i.e. de-replication).
Additionally, this approach ensures compliance with regula-
tions, ensuring that permissions to collect are secured where
needed and legal obligations are met. Experts in technol-
ogy transfer offices across the research infrastructures can
also help with expertise or simple information on routes to
market, licensing, product marketing and even production in
some cases. Above all, research infrastructures must ensure
that, in creating such platforms and gateways to technologies
and collaborations, they strive for high quality. Information
should be collated and incorporated into a web-based tool
that helps users negotiate the pipeline through the RI facili-
ties in a manner tailored to user needs. In other words, they
must provide the operational tools that meet community best
practice with appropriate quality checks and accreditation.

The work of the European Research Infrastructure clus-
ters will continue to evolve through reaction to user needs in

<figure><img src="image_5.png" /></figure>
order to serve the European Research Area better. Researchers are encouraged to use the resources, expertise and facilities available from the European Research Infrastructures (https://www.esfri.eu/roadmap-2016). In closing, therefore, we note the scale of the task facing stakeholders in the blue bio-economy and commend the multi-partner, international approach encompassed in EMBRIC, as a potential toolkit for enabling the use of the largely untapped marine microbial ecosystem as a source of novel bioactives in a sustainable and responsible manner.

**Acknowledgements** This project has received funding from the European Union's Horizon 2020 research and innovation programme under Grant agreement No 645884. CABI is an international intergovernmental organisation, and we gratefully acknowledge the core financial support from our member countries (and lead agencies) including the United Kingdom (Department for International Development), China (Chinese Ministry of Agriculture), Australia (Australian Centre for International Agricultural Research), Canada (Agriculture and Agri-Food Canada), Netherlands (Directorate-General for International Cooperation), and Switzerland (Swiss Agency for Development and Cooperation). See https://www.cabi.org/about-cabi/who-we-work-with/key-donors/ for full details.

**Open Access** This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

## References

Blasiak R, Jouffray J-B, Wabnitz CCC, Sundström E, Österblom H (2018) Corporate control and global governance of marine genetic resources. Sci Adv 4:5237

Bouslimani A, Sanchez LM, Garg N, Dorrestein PC (2014) Mass spectrometry of natural products: current, emerging and future technologies. Nat Prod Rep 31:718. https://doi.org/10.1039/c4np0044g

Brennecke P, Ferrante MI, Johnston IA, Smith D (2018) A collaborative European approach to accelerating translational marine science. J Mar Sci Eng 6: 81. https://doi.org/10.3390/jmse6030081

Bruns A, Hoffelner H, Overmann J (2003) A novel approach for high throughput cultivation assays and the isolation of planktonic bacteria. FEMS Microbiol Ecol 45:161–171. https://doi.org/10.1016/S0168-6496(03)00133-8

Burgess MG, Clemence M, McDermott GR, Costello C, Gaines SD (2018) Five rules for pragmatic blue growth. Mar Policy 87:331–339

Chun J, Rainey FA (2014) Integrating genomics into the taxonomy and systematics of the bacteria and archaea. Int J Syst Evol Microbiol 64:316–324. https://doi.org/10.1099/ijs.0.054171-0

Delamarche E, Juncker D, Schmid H (2005) Microfluidics for processing surfaces and miniaturizing biological assays. Adv Mater 17:2911–2933. https://doi.org/10.1002/adma.200501129

Dias DA, Urban S, Roessner U (2012) A historical overview of natural products in drug discovery. Metabolites 2:303–336. https://doi.org/10.3390/metabo2020303

Foesel BU, Rohde M, Overmann J (2013) *Blastocatella fastidiosa* gen. nov., sp. nov., isolated from semiarid savanna soil – the first described species of Acidobacteria subdivision 4. Syst Appl Microbiol 36:82–89

Gich F, Schubert K, Bruns A, Hoffelner H, Overmann J (2005) Specific detection, isolation, and characterisation of selected, previously uncultured members of the freshwater bacterioplankton community. Appl Environ Microbiol 17(10):5908–5919. https://doi.org/10.1128/AEM.71.10.5908-5919.2005

Gich F, Janys MA, König M, Overmann J (2012) Enrichment of previously uncultured bacteria from natural complex communities by adhesion to solid surfaces. Environ Microbiol 14(11):2984-2997. https://doi.org/10.1111/j.1462-2920.2012.02868.x

Hewitt CJ, Nienow AW (2010) The scale-up of microbial batch and fed-batch fermentation processes. In: Berovic M, Enfors S (eds) Comprehensive Bioprocess Engineering. University of Ljubljana, Ljubljana, pp 295–320

Huang Pan Ming, Li Yuncong, Malcolm E (eds) (2012) Handbook of soil sciences: properties and processes, 2nd edn. CRC Press, London

Joint I, Mühling M, Querellou J (2010) Culturing marine bacteria—an essential prerequisite for biodiscovery. Microb Biotechnol 3:564–575. https://doi.org/10.1111/j.1751-7915.2010.00188.x

Kamau EC, Fedder B, Winter G (2010) The Nagoya protocol on access to genetic resources and benefit sharing: what is new and what are the implications for provider and user countries and the scientific community? LEAD J 6(3):246

Kim S-K, Mendis E (2006) Bioactive compounds from marine processing byproducts—a review. Food Res Int 39:383–393

Klinger DH, Eikeset AM, Davíðsdóttir B, Winter A-M, Watson JR (2018) The mechanics of blue growth: management of oceanic natural resource use with multiple, interacting sectors. Mar Policy 87:356–362

Lagier JC, Armougom F, Million M, Hugon P, Pagnier I, Robert C, Bittar F et al (2012) Microbial culturomics: paradigm shift in the human gut microbiome study. Clin Microbiol Infect 18:1185–1193. https://doi.org/10.1111/1469-0691.12023

Li JW, Vederas JC (2009) Drug discovery and natural products: end of an era or an endless frontier? Science 325:161–165. https://doi.org/10.1126/science.1168243

Lorenz P, Eck J (2005) Metagenomics and industrial applications. Nat Rev Microbiol 3:510–516

Maymó-Gatell X, Y-t Chien, Gossett JM, Zinder SH (1997) Isolation of a bacterium that reductively dechlorinates tetrachloroethene to ethene. Science 276:1568–1571

Naccache SN, Federman S, Veeraraghavan N, Zaharia M, Lee D, Samayoa E, Bouquet J et al (2014) A cloud-compatible bioinformatics pipeline for ultrarapid pathogen identification from next-generation sequencing of clinical samples. Genome Res 24:1180–1192. https://doi.org/10.1101/gr.171934.113

Nichols D, Cahoon N, Trakhtenberg EM, Pham L, Metha A, Belanger A, Kanigan T, et al. (2010) Use of iChip for high-throughput *in situ* cultivation of “uncultivable” microbial species. Appl Environ Microbiol 76: 2445-2450. https://doi.org/10.1128/AEM.01754-09

OECD (2007) “Best Practice Guidelines for Biological Resource Centers. Organisation for Economic Cooperation and Development (OECD) Publishing, Paris, p 115

Okonechnikov K, Golosova O, Fursov M (2012) Unipro UGENE: a unified bioinformatics toolkit. Bioinformatics 28:1166–1167. https://doi.org/10.1093/bioinformatics/bts091

Overmann J (2012) Principles of enrichment, isolation, cultivation, and preservation of bacteria. In: Rosenberg E, Stackebrandt E, DeLong EF, Thompson F, Lory S (eds.) The prokaryotes, 4th edition, Springer, New York

Overmann J, Scholz AH (2017) Microbiological research under the Nagoya Protocol: facts and fiction. Trends Microbiol 25:85–88
Overmann J, Smith D (2017) Microbial resource centers contribute to bioprospecting of bacteria and filamentous microfungi. In: Topics in Biodiversity and Conservation", Volume 16. In: Paterson RRM, Lima N (eds) Bioprospecting—successes, potential and constraints. Springer, New York, pp 51–79

Overmann J, Abt B, Sikorski J (2017) The significance and future of cultivation. Annu Rev Microbiol 71:711–730

Royal Society (2017) Future ocean resources: metal-rich minerals and genetics—evidence pack. The Royal Society, London, p 64

Sanford RA, Cole JR, Tiedje JM (2002) Characterization and description of Anaeromyxobacter dehalogenans gen. nov., sp. nov., an aryl-halorespiring facultative anaerobic myxobacterium. Appl Environ Microbiol 68:893–900

Sherpa RT, Reese CJ, Aliabadi HM (2015) Application of iChip to grow “uncultivable” microorganisms and its impact on antibiotic discovery. J Pharm Pharm Sci 18(3):303–315

Smith D, da Silva M, Jackson J, Lyal C (2017) Explanation of the Nagoya Protocol on access and benefit sharing and its implication

for microbiology. Microbiology 163:289–296. https://doi.org/10.1099/mic.0.000425

Vierros M, Suttle CA, Harden-Davies H, Burton G (2016) Who owns the ocean? Policy issues surrounding marine genetic resources. Limnol Oceanogr Bull 25:29–35

Wagner-Döbler I, Beil W, Lang S, Meiner M, Laatsch H (2002) Integrated approach to explore the potential of marine microorganisms for the production of bioactive metabolites. In: K. Schügerl et al. (eds) Tools and applications of biochemical engineering science. Advances in Biochemical Engineering/Biotechnology, vol 74. Springer

**Publisher's Note** Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

## Affiliations

D. Smith¹ • A. G. Buddie¹ • R. J. M. Goss² • J. Overmann³ • C. Lepleux³ • M. Brönstrup⁴ • B. Kloareg⁵

T. Meiners⁶ • P. Brennecke⁶ • A. Ianora⁷ • F.-Y. Bouget⁸ • P. Gribbon⁹ • M. Pina¹⁰

R. J. M. Goss
rjmg@st-andrews.ac.uk

J. Overmann
joerg.overmann@dsmz.de

M. Brönstrup
mark.broenstrup@helmholtz-hzi.de

B. Kloareg
kloareg@sb-rostoff.fr

P. Brennecke
philip.brennecke@eu-openscreen.eu

A. Ianora
adrianna.ianora@szn.it

F.-Y. Bouget
francois-yves.bouget@obs-banyuls.fr

P. Gribbon
Philip.Gribbon@ime.fraunhofer.de

M. Pina
mery.pina-hinojosa@pasteur.fr

¹ CAB International (CABI), Bakeham Lane, Egham TW20 9TY, Surrey, UK

² School of Chemistry, Biomedical Sciences Research Complex, University of St Andrews, Fife KY169ST, UK

³ Leibniz-Institut DSMZ-Deutsche Sammlung Von Mikroorganismen Und Zellkulturen GmbH, Inhoffenstraße 7 B, 38124 Braunschweig, Germany

⁴ Department of Chemical Biology, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, 38124 Braunschweig, Germany

⁵ Station Biologique, CS 90074, 29688 Roscoff CEDEX, France

⁶ Leibniz-Institute for Molecular Pharmacology, Robert-Roessle-Strasse 10, 13125 Berlin, Germany

⁷ Stazione Zoologica “A. Dohrn” Villa Comunale, 80121 Naples, Italy

⁸ Laboratoire D’Océanographie Microbienne (LOMIC) - UMR 7621 CNRS-UPMC, Avenue Fontaulé, 66650 Banyuls sur mer, France

⁹ Fraunhofer IME, ScreeningPort, Schnackenburgallee 114, 22525 Hamburg, Germany

¹⁰ CRBIP, Institut Pasteur, 25-28 rue du Dr. Roux, 75015 Paris, France