Marine board pp17_microcean

Position Paper 17
Marine Microbial Diversity and
its role in Ecosystem Functioning
and Environmental Change
May 2012

Cover photograph credits:
Left: Research Vessel lowering a rosette with Niskin bottles to obtain seawater samples (courtesy, Lucas Stal)
Right from top to bottom: Fluorescence in situ hybridisation picture of a marine water sample stained with a probe specific
for Bacteria (courtesy, Frank Oliver Glöckner); Marine microbiologist working in the laboratory (courtesy, Frank Oliver Glöckner and
Anna Klindworth); Example of a microorganism culture on a laboratory Petri dish (courtesy, Frank Oliver Glöckner);
Image of an alga using Scanning Electron Microscopy (SEM). (courtesy, Ruth-Anne Sandaa).
Marine Board
The Marine Board provides a pan-European platform
for its member organisations to develop common pri
orities, to advance marine research, and to bridge the
gap between science and policy in order to meet future
marine science challenges and opportunities.
The Marine Board was established in 1995 to facilitate
enhanced cooperation between European marine sci
ence organisations (both research institutes and research
funding agencies) towards the development of a com
mon vision on the research priorities and strategies for
marine science in Europe. In 2012, the Marine Board
represents 34 Member Organisations from 20 countries.
The marine Board provides the essential components for
transferring knowledge for leadership in marine research
in Europe. Adopting a strategic role, the Marine Board
serves its member organisations by providing a forum
within which marine research policy advice to national
agencies and to the European Commission is developed,
with the objective of promoting the establishment of the
European Marine Research Area.
www.marineboard.eu

Marine Microbial Diversity and its role in Ecosystem
Functioning and Environmental Change
Marine Board-ESF Position Paper 17
Coordinating author
Frank Oliver Glöckner
Contributing authors
Frank Oliver Glöckner (Chair),
Lucas J. Stal (Coordinator Chapter 2),
Ruth-Anne Sandaa (Coordinator Chapter 3),
Josep Maria Gasol (Coordinator Chapter 4),
Fergal O’Gara, Francisco Hernandez,
Matthias M. Labrenz, Elena Stoica,
Marta Varela Rozados, Adriano Bordalo,
Paraskevi Pitta
Special contributions from
Frede Thingstad, Marcel Kuypers
Series Editor
Niall McDonough
Publication Editor
Jan-Bart Calewaert
This Position Paper is based on the activities of the Marine Board Working Group on Marine Microbial Diversity
(WG MICROCEAN) which convened in Brussels on 17 February 2010, 2-3 September 2010 and 10-11 February
2011, and in Oostende (Belgium) on 15-16 June 2011.
Suggested reference:
Glöckner F.O., Stal L.J., Sandaa R.-A., Gasol J.M., O’Gara F., Hernandez F., Labrenz M., Stoica E., Varela M.M.,
Bordalo A., Pitta P. (2012). Marine Microbial Diversity and its role in Ecosystem Functioning and Environmental
Change. Marine Board Position Paper 17. Calewaert, J.B. and McDonough N. (Eds.).
Marine Board-ESF, Ostend, Belgium.

Contents
Foreword 5
Executive summary 7
1 Introduction 11
2 Marine microbial diversity: current knowledge and gaps in our understanding 17
2.1 Introduction 17
2.2 Role and impact of marine microorganisms 17
2.3 Societal impacts and benefits 34
2.4 Concluding remarks 39
3 Technologies and research toolkits 42
3.1 Introduction 42
3.2 Classical microbiology 42
3.3 Novel culture-independent techniques 43
3.4 Why models? 50
3.5 Expected future developments in marine microbial cultivation and -omics studies 51
4 Marine microbiology observation and data infrastructures 54
4.1 Types of microbial deliverables 54
4.2 Origin of microbiological data 56
4.3 Current databases and data handling practices 58
4.4 From data to integration and interpretation 62
5 European status, research gaps and recommendations 64
5.1 Introduction 64
5.2 The status of European marine microbial research 64
5.3 Major research gaps and challenges for future research 67
5.4 Recommendations 70
5.5 Implementing the recommendations 73
References 77
List of abbreviations and acronyms 78
Annex I. Participating authors 80

Marine Microbial Diversity and its role in Ecosystem Functioning and Environmental Change | 5
Foreword
Several Marine Board foresight activities have previously
highlighted the importance of marine microbial diversity.
In 2007, the Marine Board published its Position Paper
9 on the Impacts of Climate Change on the European
Marine and Coastal Environment. This publication drew
attention to the importance of marine microorganisms
for the production of oxygen (approximately 50% of the
total photosynthesis on Earth), the removal of carbon
dioxide from the atmosphere and the regulation of our
climate. The Marine Board Special Report on Climate
Change and Marine Ecosystem Research (CLAMER)
published in 2011, also emphasized the key role of mi-
crobes in all global cycles of matter and energy and how
climate-induced changes in marine microbial communi-
ties and interactions may affect critical biogeochemical
cycles of elements such as carbon, nitrogen and iron.
Meanwhile, the enormous – but largely untapped – po-
tential of marine microorganisms for the development
of new biotechnology applications and products was
described in Marine Board position paper 15, Marine
Biotechnology: A new Vision and Strategy for Europe
(2010).
In recent years, there has been an increasing recogni-
tion of the importance of microbes, coupled with the
discovery of a vast microbial diversity. The key role that
microbes play in regulating Earth’s climate has also
emerged as a result of research in this area. However,
it is also likely that very rapid (geologically speaking) and
ongoing human-induced climate change will, and prob-
ably already has, altered the microbial diversity itself. As
a result of increasing sea water temperatures, acidifica-
tion of the ocean and salinity changes, to name just a
few environmental parameters of importance, dominant
Bacteria, Archaea and viruses may become dormant
and completely unknown species may become domi-
nant. Since we cannot currently predict these changes,
it has proven very difficult to predict the microbial influ-
ence on biogeochemical cycles and hence factor this
influence into predictions of climate change itself.
A number of major European and international projects
have made significant progress in addressing marine mi-
crobial biodiversity in recent years. With the completion
of the FP6 Networks of Excellence, Marine Genomics
Europe (MGE) and MarBEF (Marine Biodiversity and
Ecosystem Functioning), and large international pro-
grammes such as the Census of Marine Life (CoML) and
the International Census of Marine Microbes (ICoMM), it
is timely to reflect on where we are in the field of marine
microbial research and to make plans for the future of
European research effort in this area.
Realizing the societal importance of marine microbial
diversity and the need to advance and better coor-
dinate the European research effort in this area, the
Marine Board set-up a working group of experts (WG
MICROCEAN) in 2010 to (i) re-emphasize the impor-
tance and role of microbes in the marine environment;
(ii) indentify strategic areas for Europe in the realm of
marine microbial diversity research with specific refer-
ence to ecosystem functioning, biogeochemical cycling
and environmental change; (iii) provide recommenda-
tions to guide European research in the medium-term
(to 2020) to substantially increase our knowledge of
marine microbial diversity and its role in, and response
to, global change and to improve the competitiveness
of European research in this field.
The working group was tasked to deliver a strategic
position paper with recommendations designed to di-
rectly influence research agendas at both national and
European level. In the first instance, this paper is target-
ed towards those who determine and set the research
agenda, including European and national research fund-
ing organizations, programme managers and science
policy advisors/developers. At the same time, the paper
is intended to strengthen the marine microbial research
domain by stimulating scientific networking and devel-
oping common views of expert scientists, potentially
leading to new collaborative projects.
On behalf of the Marine Board, we sincerely thank all of
the members of the working group who generously gave
their time and expertise to support the production of this
important position paper. Their work has been crucial
to highlight the importance and central role of microbes
in the marine environment and in providing a clear set
of research priorities and recommendations to further
improve marine microbial research in Europe. Our
special thanks goes to the working group Chair, Frank
Oliver Glöckner, of the Max Planck Institute for Marine
Microbiology, Germany, and to Jan-Bart Calewaert of
the Marine Board Secretariat for his diligent support to
the working group and his work in finalizing this report.
We also thank our external reviewers for their comments
and invaluable suggestions, and Ivo Kostadinov for ide-
as and comments on some parts of the text.
Kostas Nittis,
Chair, Marine Board
Niall McDonough,
Executive Scientific Secretary, Marine Board

6 | Marine Microbial Diversity and its role in Ecosystem Functioning and Environmental Change

Executive summary
Seas and oceans cover more than 70% of the Earth’s
surface, host the majority of its biomass, and contribute
significantly to all global cycles of matter and energy. All
life on Earth most likely originated from microbes in the
sea. In today’s marine ecosystems, following billions of
years of evolution, microbes such as Bacteria, Archaea,
viruses, fungi and protists (including microalgae), domi-
nate the living biomass. Recent rapid developments in
molecular ecology, metagenomics and ecological mod-
elling illustrate that microbes represent the most impor-
tant biological group on Earth in terms of phylogenetic
and functional diversity. In addition, interdisciplinary
research has uncovered new and unexpected roles of
microbes in the biogeochemical cycling of carbon, ni-
trogen, silica and iron and many other (trace) elements
in our seas and oceans. Marine microorganisms pro-
duce the organic matter and oxygen required to sustain
life and facilitate the storage, transport, and turnover of
key biological elements. Thus, microorganisms are the
foundation of life and are of critical importance to the
habitability and sustainability of our planet (Executive
Summary Box A).
The enormous microbial diversity also gives rise to
a largely untapped amount of genetic information,
bioactive compounds and biomaterials which could
deliver important benefits and applications of societal
interest, for example, to improve medical treatments,
fisheries and aquaculture applications, the supply of
energy and for the development of industrial products
and processes. Yet, despite the clear importance of
marine microbes and the major opportunities they
present, very little is known about marine microbial
diversity, the enormous array of microbial types and their
ecological functions and interactions. Moreover, the vast
majority (90-99%) of marine microorganisms cannot be
cultured under standard laboratory conditions and their
growth and physiology cannot, therefore, be studied in
the way that has proven so successful throughout the
20th
century for medically-important microorganisms.
Addressing these knowledge-gaps will require a
significant increase in research investment in Europe,
coupled with a better coordination of European
researchers, projects, programmes and infrastructures.
However, since marine “bugs” fail to capture the public
imagination in the same way that, for example, whales
or turtles do, making a convincing political case for
increased support for marine microbial research can
be difficult. Not surprisingly, the non-scientific public
is generally unaware of the importance of microbial
communities in the functioning and health of our
environment. Instead, microbes are most commonly
associated with disease in humans, crops and livestock.
It is time to educate the public and research policy
makers about the importance of marine microbes and
marine microbiological research.
Europe has traditionally played a leading role in marine
microbial research. But the field is advancing rapidly,
driven by technological and scientific developments and
the problems of global change, population growth and
over exploitation of marine resources. With the advent of
single cell genomics and meta-omics technologies, we are
improving our capacity to assess the status of, and changes
in, marine microbial diversity (see also Executive Summary
Box B). The enormous expansion in the use of these
Executive Summary Box A.
Key facts highlighting the importance of marine
microorganisms
• Marine microorganisms occur in vast numbers
and represent a huge genetic diversity: ocean
water contains up to one million microorganisms
per milliliter and several thousand microbial
types. These numbers may be an order of
magnitude higher in coastal waters with their
higher productivity and higher load of organic
matter and nutrients;
• On the tree of life, the Eukarya (including
plants, animals and protists) comprise only a
tiny branch; Bacteria and Archaea encompass
virtually all genetic diversity, notwithstanding
their limited morphological diversity;
• Marine microorganisms are key to all biochemical
cycles and are crucial for the functioning of
marine ecosystems;
• Microbes are responsible for the degradation of
organic matter in the ocean and are thus key for
maintaining the balance between produced and
fixed carbon dioxide;
• Marine phototrophic microorganisms
(Cyanobacteria, diatoms and pico- and
nanophytoplankton) are responsible for more
than 50% of the oxygen production on Earth.
• Marine microorganisms represent a largely
untapped source of novel bioactive compounds
and metabolic pathways which could be
exploited for new biotechnological applications
and products;
• Marine microorganisms play an indispensable
role in ensuring a sustainable supply of seafood
products by occupying the critical bottom
trophic level in marine foodwebs and offering
solutions for regulating aquaculture processes
and bioremediation and waste management;
• Marine microorganisms and their activities
are, and will continue to be, affected by global
change and may also promote or alleviate
climate change.

techniques has already resulted in a significant increase
in the quantity of heterogeneous data which needs to be
integrated, analysed and interpreted. At present, Europe
has a much smaller capacity than the US in marine data
processing and bioinformatics. Furthermore, the existing
capacities are often not designed for interoperability
within the European research community. It is of crucial
importance, therefore, to urgently consider and put in place
the bioinformatics infrastructure and standards needed to
enable the next generation of European marine microbial
research.
Although unprecedented technological improvements
in the last years have provided us with a wealth of
knowledge about the dominant types of microorgan-
isms in the oceans and their activities, there are still
many research questions that have not been solved,
either because the appropriate methodologies have not
yet been developed or applied or, more often, because
answering these questions implies an amount of work
and resources that is out of the reach of most labora-
tories. In this position paper, the expert working group
identified a set of key societal and scientific questions
(Executive Summary Box C) and provided high-level
recommendations on key future research priorities and
needs (Executive Summary Box D) and proposed an
approach with solutions to addressing these questions
(Executive Summary Box E).
Executive summary
• 1977: Development of techniques to enumerate
microbes in the ocean reveals, for the first time, the
vast microbial abundance;
• 1979: Microbes discovered in hydrothermal vents;
• 1980: Bacteria in the ocean are shown to be
actively synthesizing DNA and RNA, growing and
reproducing with similar growth rates to those of
algae and with production rates about one third of
those of algae;
• 1982: Discovery that Bacteria are actively predated
by a group of highly specialized small protists, the
heterotrophic nanoflagellates;
• 1983: It is established that most primary production
in the ocean is carried out by microbes smaller than
2 µm;
• 1986-1988: Research shows that most predators of
Bacteria and Algae are indeed mixotrophic flagellates
and ciliates;
• 1989: Use of flow cytometry allows the discovery of
the picocyanobacteria, Prochlorococcus, the most
abundant photosynthetic organisms on Earth;
• 1990: First cultivation-independent assessment of
marine bacterial diversity through rRNA analysis
shows that they are highly diverse and that most
groups in the ocean were previously unknown;
• 1990: First phylogenetic stains used for marine
plankton;
• 1989-1990: The discovery of the vast numbers
of viruses (ten million per millilitre) in the ocean
and their role in recycling of nutrients, keeping in
check successful microorganisms and horizontal
gene transfer, all of which generates and maintains
microbial diversity;
Executive Summary Box B.
Major achievements of marine microbiology in the last decades
• 1992-1994: High abundances of Archaea in marine
plankton, even in cold, well-oxygenated waters;
• 1997: Bacterial activity in the ocean is found
to be so important that bacterial respiration in
the oligotrophic ocean is higher than primary
production;
• 2000: Discovery of photoheterotrophy in the sea
by metagenomic techniques (proteorhodopsine)
and by other techniques (AAP bacteria);
• 2001: Scientists find that a large unknown
diversity also exists among the smallest protists;
• 2002: Isolation of SAR11 in pure culture. This
is followed by the discovery and isolation of
many new marine microorganisms and new
metabolic pathways (Pelagibacter, the most
abundant bacterium in the ocean; temperate
Archaea such as the ammonium-oxidizing
Thaumarchaeota; anaerobic ammonium oxidizing
(anammox) Planctomycetes; unicellular N2-fixing
Cyanobacteria), etc.
• 2003: Genomes of environmentally
important microorganisms are sequenced
(e.g. Cyanobacteria)
• 2004: A metagenome is produced from the
Sargasso Sea revealing a large amount of
unknown microbial functions in the ocean
• 2006-2007: High-throughput sequencing
technologies introduced in marine microbial
ecology reveal that bacterial diversity is larger
than expected and that the ocean contains an
extremely large number of microbial genes,
most of them of unknown function.

Executive Summary Box C.
Key scientific and societal questions related to
marine microbial diversity research
Key societal questions:
1. What is the role of marine microbial communities
in relation to major societal challenges such
as global change, the sustainable supply of
healthy food and energy, human health and
environmental health?
2. If we have a better understanding of the role
of marine microbial communities for the above
functions, is it possible to manage them (better)?
If so, how?
3. What opportunities can marine microorganisms
provide for innovation in support of the
European bioeconomy?
High level scientific questions that will need to
be addressed:
1. What is the nature of microbial diversity in the
oceans?
2. Which taxa are most relevant in terms of
function across different ocean ecosystems?
3. What metabolic pathways exist?
4. What regulatory and signalling networks exist?
5. How do environmental factors influence
metabolism and regulation within and between
taxa?
6. How do microbial interactions influence ecology
and ecosystem functioning?
Executive Summary Box D.
Strategic research priorities and needs
I. Ensure appropriate and accurate data
acquisition to obtain more and better data;
II. Build a registry for samples and genetic
materials;
III. Develop innovative cultivation approaches and,
in turn, establish model organism databases
and genetic systems for marine organisms;
IV. Improve classical methods/approaches and
develop novel techniques and combine their
results with abiotic and biotic information.
By applying such an approach it will become
possible to generate a more precise picture of
the function, interaction and diversity within
marine microbial food-webs;
V. Generate a dense network of data on the
marine ecosystem by increasing both the
number and the technological capacities of
marine observatories;
VI. Contextualise sequence data with a set
of standardized parameters (metadata) of
habitats;
VII. Improve data management to be able to
coordinate sampling, sample analyses and
processing of data and make this data openly
available for users;
VIII. Ensure interoperability of multidisciplinary data
repositories;
IX. Implement well-curated and integrated
ecological/diversity/and genetic databases;
X. Fully implement a systems approach to
microbial ecology to understand ecosystem
functioning;
XI. Prepare European researchers to be able to
participate in, and benefit from, data delivered
by international projects and programmes.

Training
outreach
Freely available
data from
European sites
Tools for
sampling,
analysis and
storing of data,
cultures and
samples
Understand and
manage marine
microbial
communities in
relation to major
societal challenges
•Global change
•Sustainable food
and energy supply
•Human health
•Environmental
health
Fill gaps in scientiﬁc
knowledge
Communication outreach
Innovative solutions
in support of the
European
bioeconomy
Identity proﬁle
of European Marine
Microbiology
Research
European repository for
cultivated collections
reference library
Long term
pan-European
research
programme on
marine
microbiology
Integrated centre for marine
data management
analysis
Sampling
and
analysis
of key
European
sites
Develop
standard
operating
procedures
Develop and
populate
appropriate
databases
Create a
virtual
repository
Strengthen
local
culture
collections
Develop
standards
Executive summary
The high-level recommendations are:
• Recommendation 1:
Establish a coordinated pan-European research
programme on marine microbiology
Create a European repository for cultivated
collections and a reference library
Create an integrated, multidisciplinary European
Centre for marine data management and analysis
Promote interest in marine microbial research and
improve training and education
These four high-level recommendations are interde-
pendent, complimentary and positively reinforcing.
Creating better visibility for the importance of marine
microbial diversity and associated research will assist
in creating the necessary leverage for appropriate sci-
ence policy measures and funding of relevant capacities
and research activities. At the same time, the realiza-
tion of a major pan-European research programme on
marine microbiology will create a wealth of material and
opportunities for outreach and education which will un-
doubtedly raise the profile of marine microbial research
in Europe.
A multidisciplinary European Centre for marine data
management and analysis is necessary to prevent
the large-scale implementation of omics technologies
turning into a disruptive process. It will provide critical
support and capacity to deal with the wealth of sam-
ples and genetic information that will come on-stream
through the research in the framework of the pan-Eu-
ropean research programme on marine microbiology.
Likewise, material gathered during sampling campaigns
would be preserved and made available to a larger
community if the proposed European repository for
cultivated collections and a reference library becomes
reality. The combined set of recommendations, when
realized, will provide an enormous positive impact on
European marine microbial research and provide the
necessary push to improve Europe’s competitiveness in
this important research area (Executive Summary Box E)
Executive Summary Box E.
Schematic overview of main recommendations and their expected impacts in relation to addressing
the key societal and scientific challenges

Microorganisms are defined by their size: any organ-
ism that is too small to be observed in sufficient detail
by the unaided human eye is a microorganism. This in-
cludes basically any organism smaller than 0.1 mm. The
smallest well-known marine microorganism Candidatus
Pelagibacter ubique HTCC1062 measures only 0.5×0.15
µm and hence the size range of microorganisms com-
prises more than three orders of magnitude, more or
less the same as for macroorganisms. All three do-
mains of life (Figure 1.1) comprise microorganisms while
Bacteria and Archaea are comprised exclusively of mi-
croorganisms. All macroorganisms are Eukarya but the
vast majority of the eukaryotic phylogenetic groups are
nevertheless also microorganisms.
Bacteria and Archaea are unicellular organisms with
cell shapes basically varying from spherical to elon-
gated rods although a variety of other morphological
characteristics are also possible. Some of these micro-
organisms may produce multicellular forms and a few
are even capable of a simple form of cell differentiation.
Sometimes microscopic organisms produce macro-
1. Introduction
scopic appearances such as microbial mats and water
blooms. Microorganisms can also produce multicellular
structures that develop their own behaviour through cell-
to-cell communication and a distribution of tasks.
Although Bacteria and Archaea do not differ much in
appearance, they show an enormous range of metabolic
capacities. Eukarya, on the one hand, are limited in their
metabolic capacity to aerobic respiration of organic car-
bon (animals, protists) and to oxygenic photosynthesis
connected to the fixation of CO2 (plants, protists), and
basically cycle carbon between inorganic and organic
forms producing and consuming oxygen. The other two
domains, on the other hand, take care of the full plethora
of energy generating reactions that are possible by mov-
ing electrons from oxidized and reduced forms of e.g.
iron, nitrogen, sulfur, etc., including the aerobic respira-
tion and oxygenic photosynthesis that are characteristic
of Eukarya. Bacteria and Archaea therefore are exclu-
sively driving all other biogeochemical cycles while at the
same time taking an important part in the global carbon
and oxygen cycles.
Acidobacteria*
Verrucomicrobia*
OP11
Chloroflexi*
ε-proteobacteria*
δ-proteobacteria*
α-proteobacteria*
β-proteobacteria*
γ-proteobacteria*
Spirochaetes*
Archaea
Bacteria
Eukarya
Planctomycetes*
ChlamydiaeBacteroidetes
Chlorobi*
Aquificae*
Thermotogae
Root?
Firmicutes*
Tenericutes
Actinobacteria*Deinococcus/Therm
us
Cyanobacteria*
Plastids*
Acasid slime molds
Vahlkampfiid amoebas
Euglenids*
Trypanosomes
Leishm
anias
Core jakobids*
Parabasalids
Diplomonads
Oxymonads*
Cryptophytes*
Haptophytes*
Opalinids
Phaeophytes*
Chrysophytes*
Diatoms*
Labyrinthulids*
Oomycetes
Heterokonts
Apicom
plexa
Dinoflagellates*
Ciliates*
Syndinales*
M
arineGroup
I*
Redalgae*
Fungi
Nucleariids
*Animals
*Choanoflagellates
Mesomycetozoa
Plasmodial slime molds
Cellularslim
em
olds
*Am
oebas
Rhizaria
Plants
Alveolates
*Cercom
onads
Plasmodiophorids
*Radiolarians
*Chlorarachniophytes
*Radiolarians
*Foraminifera
Landplants*
Greenalgae*
Glaucophytealgae
*Greenalgae
Nanoarchaeota
Pyrococcus
*Methanothermus*Methanobacterium
*Methanopyrus
Methanospirillum
Methanosarcina
Pyrobaculum
*Halobacteria
ANME-1
*Thermoplasma
Archaeoglobus
Thermoproteales
Aeropyrum
Desulfurococcus
Sulfolobus
*Cenarchaeum
Korarchaeota
Euryarchaeota
Crenarchaeota
Thaumarchaeaota
Opisthokonts Disicristates
Excavates
Proteobacteria
Deferribacteres*
Gemmatimonadetes*
Lentisphaerae*
Nitrospirae*
Fibrobacteres
Figure 1.1. A tree of life including Archaea, Bacteria and Eukarya. The groups in black colour are mostly or completely microbial. The groups
in red are not. Groups with asterisks are marine, or include a large amount of marine organisms. (Figure adapted from Baldauf 2003
using the colouring scheme of Barton et al. 2007 and the archaeal groups following Brochier-Armanet et al. 2008).

1. Introduction
What about viruses?
Organisms are defined by their capacity of independent
cell division and therefore possess the full machinery
for the duplication of DNA and for the binary division of
the cell. Viruses, in contrast, require a living organism in
order to duplicate their genetic material and to synthe-
size the virus particle. They do not, therefore, belong to
any of the three domains of life. Nevertheless, viruses
are of prime importance for generating and maintaining
diversity amongst living organisms in all three domains
of life through the mediation of horizontal gene transfer.
They are also critical for the functioning and balance of
the microbial food web and biogeochemical cycles by
facilitating the production of nutrients for growth and
maintaining high diversity, all by preventing the domi-
nance of the most successful microorganisms. Viruses
not only occur in numbers exceeding an order of mag-
nitude those of microorganisms but also represent an
untold genetic diversity. In all microbial ecosystems, vi-
ruses represent a considerable amount of the particulate
organic matter. For these reasons we will consider the
viruses as part of the microbial world and discuss them
as such in this position paper.
What are marine microorganisms?
In general, the distinction between terrestrial microor-
ganisms and aquatic microorganisms is rather clear,
even though this is attributed in part to the traditional
and historical differences in the scientific approaches
between aquatic and soil microbiology. The distinction
between freshwater and marine microbiology is less
clear as it is based on the assumption that salinity is a
factor discriminating aquatic microbial life, i.e. micro-
organisms growing in freshwater do not occur in the
ocean and vice-versa. In fact this is not the case. Certain
microorganisms occur in a wide range of aquatic envi-
ronments with different levels of salinity.
For the purpose of this position paper, we will consider
any microorganism that grows in a marine environment
as a marine microorganism. This includes low-salinity
environments adjacent to the sea such as estuaries and
brackish-water environments (e.g. Baltic Sea) which are
traditionally considered “marine” and hypersaline envi-
ronments such as deep sea brines and solar salterns
receiving seawater.
Although traditionally not considered marine, what is
stated in this document will in many cases be relevant
to inland saline and non-saline waters as well. This is
because, in general, the scientific approaches in fresh-
Figure 1.2. Examples
of microorganisms from
key domains of the tree
of life: (a) Haloquadratum
walsbyi as an example
of the Archaea (©Henk
Bolhuis); (b) Synechococcus
(Cyanobacteria) as an
example of the Bacteria
(Modified from image by Ralf
Wagner, Denmark); and (c,
d) Pediastrum and Amphora
as examples of the Eukarya
(courtesy Lucas Stal)
(a)
(c)
(b)
(d)

water and marine aquatic microbiology do not differ
fundamentally, except for matters of scale: oceans and
seas cover a much wider area than freshwater bodies;
the depth (and hence pressure) is an order of magnitude
larger in the ocean; and there is basically one global
ocean that is connected through a complex system of
currents.
Microorganisms are those organisms that are too
small to be observed in sufficient detail by the unaid-
ed human eye which includes roughly any organism
smaller than 0.1 mm. For the purpose of this posi-
tion paper, we consider marine microorganisms
those microorganisms that can grow in marine en-
vironments, independent of whether they are more
abundant in other aquatic or terrestrial environ-
ments. Although traditionally not considered marine,
what is stated in this document will in many cases
also be relevant to inland saline and non-saline wa-
ters.
What is the problem?
Marine microorganisms are of critical importance to the
health of our environment and our well-being. They are
integral to all major biogeochemical cycles, fluxes and
processes occurring in marine systems where elements
move between oxidised and reduced forms. Microbes
are extremely abundant and diverse and produce and
release carbon products that are key in the regula-
tion of the Earth’s climate, particularly CO2 and CH4.
Marine microorganisms also provide essential goods
and services to our society in terms of production of
oxygen, supporting sustainable supply of food, regulat-
ing the health of the marine environment and providing
an largely untapped source of genetic information and
biomolecules for use in industrial and medical applica-
tions and products.
In spite of their importance, very little is known of marine
microbial diversity, how many types of microorganisms
are present in the oceans, and what the role of each of
them is – i.e. their ecological functions and interactions.
In fact, microbiologists are still uncertain about how to
define a microbial species. Moreover, the vast majority
(90-99%) of marine microorganisms cannot be cultured
under standard laboratory conditions and as such their
growth behaviour and physiology cannot be studied, an
approach that has proven so successful throughout the
20th
century in the case of medically-important micro-
organisms.
It was only with the development of a molecular toolbox
to detect and sequence DNA from the natural environ-
ment that information about the exceptional prokaryotic
diversity in the oceans began to accumulate. An exam-
ple of these molecular tools is the powerful Polymerase
Chain Reaction (PCR)-based methods that have been
established for direct amplification, cloning, and analysis
of ribosomal RNA (rRNA) genes from the environment.
Over the last 30 years two million rRNA gene sequences
have been added to public repositories and represent an
impressive hallmark of this development. The full cycle
rRNA approach makes use of these data for the design
of specific probes to label microorganisms using fluo-
rescent dyes. With the development of this Fluorescent
In Situ Hybridisation (FISH) technique it was, for the first
time, possible to visualize and enumerate specific mi-
crobes by preserving the microbial communities in their
natural habitats. The advent of the rRNA gene sequence
as a gold standard in analysing microbial diversity and
abundances has paved the way for microbiology to an-
swer the first of three basic ecological questions:
“What is out there?
While fundamental microbial functions have been dis-
covered through traditional approaches since studies
of Antony van Leeuwenhoek in the late 17th
century,
Figure 1.3. TEM micrographs of representatives of the most
common phage families in the surface ocean: Myoviridae,
represented by the cyanophage S-PM2 (above), Sipoviridae,
represented by the bacteriophage VP6 (below left) and Podoviridae,
represented by the bacteriophage H100/1 (below right). Scale bar
100 nm. (©Professor Hans-Wolfgang Achermann, Laval University,
Canada; from Sandaa, R-A. 2009).

1. Introduction
more recently, marine microbial genomics ranging from
the study of the genomes of model organisms to the
wealth of meta-omics approaches (e.g. metagenomics,
metatranscriptomics and metaproteomics) has proven
to be very successful to target the second basic eco-
logical question “What are they doing?” (see also
Chapter 3). The cultivation independent approaches, in
particular, have resulted in an explosion of information
on marine microbes. For example, the first part of the
Global Ocean Sampling (GOS) project, which sampled
the North Atlantic, Caribbean and a small part of the
Pacific Ocean, added DNA sequence information that
was equivalent to 50% of all protein-encoding sequenc-
es that had previously been deposited in GenBank. The
GOS project confirmed that marine microbes are di-
verse, revealing how little is known about the genetic
information of natural assemblages. At the same time,
this study also highlighted the difficulties of making
sense of metagenomic sequence data: a significant
proportion of the open reading frames (ORFs, which
are presumed to equate to genes) could not be char-
acterized because there were no similar sequences in
the databases. This demonstrates that even billions of
nucleotides do not automatically provide much informa-
tion when it comes to the understanding the function of
organism or a community.
Information Box 1. Basic questions of marine
microbial diversity research
While recent technological developments and scien-
tific discoveries have been substantial, we still lack a
major understanding at all levels of the basic ecologi-
cal questions in relation to the microorganisms in our
seas and oceans. These fundamental questions are:
1. What is out there? Which microorganisms are pre-
sent in our seas and oceans and in what numbers
do they occur?
2. What are they doing? What functions do each of
these microorganisms perform in the marine environ-
ment and how do they contribute to the global cycles
of energy and matter?
3. What are the factors that determine the presence
or absence of a microorganism and how do they
influence biodiversity and function and vice versa?
This lack of understanding limits our ability to assess
the precise relationship between the diversity and
function of marine microorganisms on the one hand,
and anthropogenic global changes on the other. For
example, currently we are neither able to answer the
question of how climate change influences the diver-
sity and function of microbes nor can we determine
the exact role and contribution of marine microor-
ganisms in moderating climatic changes. This lack
of knowledge also limits our ability to tap the full
potential of the marine realm to develop goods and
services using marine microorganisms.
More cultures of environmentally relevant marine
Bacteria, Archaea and viruses can backup these efforts.
Most culture collections are based on readily cultivated
microbes. When these organisms were isolated, there
were no techniques to establish if the isolate was abun-
dant in the natural environment or even if it had any
relevant function. Molecular biology has changed that,
and the isolation of new cultivable microbes can now
be based on their abundance and relevance in defined
marine habitats.
At the same time, there are also a number of novel
and innovative approaches to the isolation of new po-
tential model microorganisms. For example, in 2002
Rappé used a dilution approach to isolate one SAR11
representative, Candidatus Pelagibacter ubique, the
bacterium which can be found in almost all marine
biodiversity or metagenomic studies. Hence, methods
do exist for isolating useful model microbes from the
natural environment, but they remain labour-intensive.
Nevertheless, these methods are probably the only way
in which relevant bacteria can be brought into culture.
Having the relevant cultures in hand, the whole toolbox
Figure 1.4. Example of a microorganism culture on a laboratory
Petri dish. However, the vast majority of known marine
microorganisms cannot be cultured under standard laboratory
conditions. (Courtesy Frank Oliver Glöckner)

only a small subset of the original data and is often only
“human readable” and cannot be assimilated into data-
bases without major efforts in text recognition.
The lack of knowledge on the organism and habitat level
greatly hampers any kind of Europe-wide or even inter-
national medium to large scale data integration. To move
towards a better understanding of the marine ecosystem
as a whole, community efforts to improve data manage-
ment and standardization are a prerequisite.
The way forward
Over the past years, considerable progress has been
made to advance marine microbial research in Europe
through better coordination and collaboration of re-
search activities, strategies and infrastructures. Major
pan-European and macro-regional initiatives and pro-
jects such as the marine Networks of Excellence on
marine biodiversity (MarBEF) and marine genomics
(MGE) have contributed to the initial development of a
European Research Area (ERA) for marine sciences and
developed a strong momentum to further strengthen
Information Box 2. This Position Paper: objectives, underlying questions and approach
2. If we have a better understanding of the role of ma-
rine microbial communities for the above functions
and are able to measure this, is it possible to man-
age it (better)? If so, how?
3. What opportunities can marine microorganisms
provide for innovation in support of the European
bioeconomy?
The high level scientific questions that will need to
be addressed are:
1. What is the nature of microbial diversity in the oceans?
2. Which taxa are most relevant in terms of function
across different ocean ecosystems?
3. What metabolic pathways exist?
4. What regulatory and signalling networks exist?
5. How do environmental factors influence metabolism
and regulation within and between taxa?
6. How do microbial interactions influence ecology and
ecosystem functioning?
To answer the above societal and scientific questions,
this position paper assesses the current state of the
art in terms of scientific knowledge and research ca-
pacities available in Europe. It identifies what gaps
exist in terms of knowledge and available research
frameworks and highlights essential future research
priorities and requirements to substantially increase
our ability to address these issues.
of genomics, genetics, biochemistry and physiology can
be applied to start unravelling the function of hypotheti-
cal genes.
To address the functional potential of organisms, inte-
grated approaches are needed that take into account
the results of the third basic ecological question
“What are the environmental factors of relevance
and how do they influence biodiversity and func-
tion?
Full geo-referencing has the potential to be extremely
useful in this respect, especially for the open ocean,
where any kind of genomic data can be easily linked
with measured and remote sensing data based on lo-
cation, time and depth. Subsequently, the diversity and
abundances of organisms, the genomic information, as
well as gene expression information at the transcrip-
tome and proteome level can easily be integrated with
measured or interpolated environmental parameters.
Unfortunately, these so called contextual data or meta-
data of sampling campaigns are often incomplete and
stored in hand-written laboratory notebooks or on indi-
vidual computers. What is finally published represents
This publication is the result of the endeavours of a
Marine Board Working Group of experts set up to high-
light the importance of marine microbial biodiversity
research and put forward ways to address the current
lack of knowledge and barriers to progress. To this
end, the Working Group general objectives were to:
• Re-emphasise the importance and role of microbes
in the marine environment;
• Identify strategic areas for Europe in the realm of
marine microbial diversity research, with specific
reference to ecosystem functioning, biogeochemi-
cal cycling and environmental change;
• Provide recommendations and a roadmap to guide
European research in the medium term (to 2020)
to substantially increase our knowledge of marine
microbial diversity and its role in, and response to,
environmental change and to improve the competi-
tiveness of European research in this field;
To address the above objectives, the Working Group
formulated key underlying societal and scientific ques-
tions which will need to be tackled in future research
in the area of marine microbiology.
The three societal questions are:
1. What is the role of marine microbial communities in
relation to major societal questions such as global
change, the sustainable supply of healthy food and
energy, human health and environmental health?

European capacity on marine microbial research (see
also Chapter 5). Nevertheless, major gaps in our scien-
tific knowledge still exist and marine microbial research
remains largely uncoordinated and fragmented with the
majority of funding coming from national resources.
To deal with these issues and address the identified so-
cietal and scientific questions (see Information Box 2),
this position paper assesses the current state of the art
in terms of scientific knowledge and research capacities
available in Europe. It identifies what knowledge gaps
exist and highlights essential future research priorities
and requirements to substantially increase our ability to
address these issues. To this end, the position paper
starts with an overview of what we know, what we don’t
know, and what we should know about marine microbial
diversity which reveals the important role, impact and
societal benefits provided by marine microorganisms
(Chapter 2). Next it considers the most important ex-
isting and emerging technologies and research toolkits
which have shaped marine microbial research and will
continue to drive it forward (Chapter 3). Available re-
sources and gaps in marine microbiology observation
and data infrastructures are considered in Chapter 4.
Finally, Chapter 5 reflects on the European status of ma-
rine microbial diversity research and considers the main
research gaps which need to be addressed to answer
the basic scientific and societal questions and provides
science policy recommendations to bridge the gaps.
This paper is intended in first instance for those who
determine and develop research agendas, the Research
Funding Organisations (RFOs), programme managers
and science policy advisors and developers both at
the national and European level. At the same time, the
expected outcome is also intended to strengthen the
marine microbial research community by stimulating
networking and developing common views of expert
scientists and the major marine science organisations
in Europe.
1. Introduction

2.1 Introduction
Life on Earth was microbial for more than 3.2 billion years
and it developed and maintained every biogeochemi-
cal cycle that exists today. Microorganisms evolved the
plant-like photosynthesis that resulted in the enrichment
of the biosphere and atmosphere with oxygen. Microbes
shaped the Earth and made it habitable for macroscopic
life including humans. Microbes continuously changed
the environmental conditions on Earth and have adapted
to global environmental changes as they happened.
The oceans comprise the largest continuous ecosystem
on Earth. Marine microbial communities are an integrat-
ed part of the ocean and are responsible for the uptake
of a large part of the carbon dioxide that human society
emits into the atmosphere and that causes global warm-
ing. The biological pump transports CO2 to the seafloor,
a process entirely driven by microbes. The constantly
increasing level of atmospheric CO2 is causing the acidi-
fication of surface waters of the ocean. This may lead
to the dissolution of carbonates, and the change of the
carbonate equilibrium could eventually lead to an ocean
that would release CO2 into the atmosphere instead of
taking it up. Marine microbial communities respond to
these changes in hitherto unpredictable ways.
Marine microbial communities are the basis of the
ocean food web and, hence, produce the food for all
life in the ocean. Fisheries are supplying a major part of
the protein demand of human society and they depend
directly from adequately functioning microbial communi-
ties. This awareness has resulted in a growing interest
in sea-based aquaculture for sustainable production of
sea-food, as well as for the efficient recycling of nutrients
in these production plants ensuring a minimum of nega-
tive effects on the environment.
More recently, it has been realized that marine micro-
organisms may be relevant agents for the sustainable
production of energy. Sunlight at the ocean surface is
used by Cyanobacteria and microalgae to produce bio-
mass by fixing CO2 while producing O2. Fossil fuels such
as natural gas and oil were produced by the same organ-
isms and accumulated in the Earth’s crust for hundreds
of millions of years. Attempts are now being made to
apply this process directly so that the CO2 produced
from burning fuels could be moved from the atmosphere
to be fixed back as renewable fuel.
Society relies on marine microbial communities for its
own health as well as for the health of the environment.
Marine microbial communities are the source of a large
variety of bioactive compounds which may have medical
applications and, as such, contribute to human health.
Marine microbial communities also provide a variety of
services, such as bioremediation of polluted environ-
ments.
2.2 Role and impact of marine
microorganisms
2.2.1 Introduction
The environments in which marine microorganisms are
found are diverse. First of all, many microorganisms live
as plankton in the water column, while other types live
a benthic lifestyle on the seafloor as biofilms or as mi-
crobial mats (Figure 2.1).
A virtually unexplored marine environment is the deep
subsurface, the Earth material near but not exposed to
the sea bottom that can be found at about 50m below
the surface of the Earth’s crust, and which extends vari-
ably downward, up to 2.8km. Furthermore, we should
consider specific environments such as deep sea hot-
and cold hydrothermal vents, microorganisms that live
attached to marine plants (epiphytes), or animals, and in
fact on any submersed marine surface (including fouling
of ship hulls). Last but not least we must consider micro-
organisms that live inside and in symbiotic relationships
with marine organisms. Sponges and corals are well-
known examples of organisms which have symbiotic
relationships with microorganisms in the marine envi-
ronment, but any fish in the sea carries its own specific
microbiota.
As a rule of thumb we may say that ocean water contains
one million microorganisms per millilitre. This number
may be an order of magnitude higher in coastal waters
which have higher productivities and loads of organic
matter and nutrients. Likewise, the deep ocean may
contain one to two orders of magnitude less microor-
2. Marine microbial diversity: current knowledge and gaps
in our understanding
Figure 2.1. Microbial mat composed of Cyanobacteria and other
microorganisms (courtesy Lucas Stal)

Figure 2.2. Examples of symbiotic marine microorganisms: Bobtail squid (Euprymna berryi) from East Timor with light organs filled with
luminescent microorganisms (©Nick Hobgood) (above left); Gutless oligochaetes are a unique group of marine worms appearing bright white
because of the elemental sulfur stored in their bacteria endosymbionts (above right). Epifluorescence image of the symbiont-containing
region of the O. crassitunicatus worm’s body wall (middle left) (Courtesy Anna Blazejak); Corals have small symbiotic single-cell algae
residing in their tissue called Zooxanthellae (middle right) which photosynthesize in the presence of sunlight to make sugars which is given
to the coral animal and may account for up to 90% of the coral animal’s overall nutritional needs. Zooxanthellae give coral its coloration,
with the specific color depending on the particular clade. Coral bleaching is the loss of intracellular endosymbionts through either expulsion
or loss of algal pigmentation. Under stress (e.g. due to changes in temperature or occurrence of by pathogenic bacteria), corals may expel
their zooxanthellae, which leads to a lighter or completely white appearance, hence the term “bleached”, and ultimately the death of the coral
(bottom). (©Bruce Monger, NOAA).

Figure 2.3. Epifluorescence micrograph of prokaryotes and viruses
in a seawater sample stained with a fluorescent dye, SYBR Green
I. The dye specifically stains doubled-stranded DNA (dsDNA).
Smallest dots are viruses and larger ones are prokaryotes (Bacteria
or Archaea). With about 1 billion bacterial cells and 10 billion viral
particles per liter of seawater, bacteria and in particular viruses
are by far the most common biological entities in the marine
environment. (©Ruth-Ann Sandaa)
ganisms. Altogether, the number of microorganisms in
aquatic habitats is estimated at 1.2 × 1029
and in the
oceanic subsurface up to 3.5 × 1030
. The number of vi-
ruses in the ocean is estimated to be higher than 1030
.
Very little is known about the diversity of marine micro-
organisms. The number of species of microorganisms
has been estimated from as low as 104
– 105
to as high
as 106
– 107
, let alone that it is not always clear how to
define a microbial ‘species’.
2.2.2 Biogeochemical cycles
Oceans and coastal waters, including estuaries, harbour
a tremendous diversity of Bacteria, Archaea, viruses,
fungi, protists and microalgae able to transform C-, N-,
P- and S-containing compounds in ways that influence
their availability for biological production. Thus, the me-
tabolism of marine microorganisms maintains the major
biogeochemical cycles on Earth, including the signifi-
cant production of oxygen required for aerobic life and
the biological removal of carbon. The balance of all
these cycles and compounds controls the dynamics of
all ocean biomes. Thus, studying the ecology of marine
microbial communities is essential for an understanding
of ecosystem function.
Information Box 3. Biogeochemical cycles
in the sea
A biogeochemical cycle is a pathway by which a
chemical element or molecule moves through both
biotic (biosphere) and abiotic (lithosphere, atmos-
phere, and hydrosphere) compartments of Earth.
A cycle is a series of change which comes back to
the starting point and which can be repeated. The
term “biogeochemical” indicates that biological,
geological and chemical factors are involved in the
process. The circulation of water and chemical nu-
trients like carbon, oxygen, nitrogen, phosphorus,
calcium, etc. through the biological and physical
world is achieved through biogeochemical cycling.
In effect, these elements are recycled, although in
some cycles there may be places (called reservoirs)
where the element is accumulated or held for a long
period of time (such as the ocean or a lake for water).
Elements, chemical compounds, and other forms of
matter are passed from one organism to another and
from one part of the biosphere to another through
biogeochemical cycles.
In Figure 2.4, microorganisms in the box in the
middle represent the particulate organic car-
bon matter (POC) that recycles key elements (see
also Figure 2.10. on the microbial and viral loops).
Microorganisms include Cyanobacteria, microalgae,
Bacteria, Archaea, ciliates, flagellates, zooplankton,
and viruses. For simplicity, only the cycles of C, N,
P, Fe, O and H and a part of the sulfur cycle are de-
picted.
Sunlight is the main driver of the system through
photosynthetic CO2 fixation, although a consider-
able part of the CO2, especially in oxygen minimum
zones (OMZ) or hydrothermal vent systems, may be
fixed chemosynthetically. Photosynthesis results in
the production of oxygen from water and is used
for respiration (re-oxidation of reduced carbon, ni-
trogen, iron, sulfur) to become water again. Oxygen
and carbon dioxide exchange with the atmosphere.
The fixation of CO2 results in the formation of par-
ticulate organic carbon (POC) and dissolved organic
carbon (DOC) both of which are respired back to
CO2. Under anaerobic conditions, CO2 may be re-
duced to methane (by methanogenic bacteria) which
is oxidized back to CO2 by methanotrophic bacteria.
Iron cycles from oxidized ferric iron (Fe3+
) to reduced
ferrous iron (Fe2+
) and back catalyzed by bacteria as
well as by (abiotic) chemical processes. In seawa-
ter, virtually all iron is oxidized which is insoluble and
therefore this essential element is thought to be una-
vailable and to limit growth of microorganisms, even
though it is one of the most abundant elements. The

cycle of phosphorus is simple. Phosphate is taken
up and incorporated in the cell and is released as
dissolved organic phosphorus (DOP). Alkaline phos-
phatases cleave the DOP. Seawater contains a large
amount of sulfate which can be assimilated by micro-
organisms and serves as the source of sulfur. Other
organisms depend on organic sulfur, mainly in the
form of protein. Some algae produce DMSP (dime-
thyl sulfoniopropionate) which, when liberated, can be
transformed by some bacteria and other algae to DMS
(dimethylsulfide). This gas, which is responsible for the
“smell of the sea”, escapes to the atmosphere where
it may cause cloud albedo and thereby represents
a negative climate feedback (see also Figure 2.13).
Nitrogen is quantitatively one of the most important
elements. Most organisms depend on ammonium, ni-
trate or dissolved organic nitrogen (DON). Ammonium
can be converted anaerobically to N2 or aerobically to
nitrate, which is denitrified to N2. Incomplete denitrifi-
cation may lead to the formation of nitrous oxide (N2O)
which is a strong greenhouse gas. Only a few bacteria
are capable of fixing N2 and use it as a source of nitro-
gen. All these elements (and many more) are required
for the synthesis of structural cell material. Dead bio-
mass sinks to the ocean floor where part of it is stored
on the geological time scale away from the biosphere
(this process is called “the biological pump”).
N2
NH4
+

NO2
-‐
,
NO3
-‐

N2O
DON

ATMOSPHERE

CO2

POC,
DOC,

CH4

Fe3+
Fe2+

DOP

PO4
3-‐

S,
P

N

C

Fe

SEA
FLOOR

H2
O

O2

SO4
2-‐

DMSP
DMS

Figure 2.4. Biogeochemical cycles in the sea and the role of the microbial food webs (developed by Lucas Stal and JM Gasol)

2.2.2.1 Carbon
Inorganic carbon in seawater is in equilibrium with
atmospheric CO2. Carbon dioxide dissolves in the
seawater where it is protonated to bicarbonate and
carbonate that are in a pH dependent equilibrium.
The fixation of CO2 results in the shift of the carbon-
ate equilibrium towards carbonate and consequently
to an increase in pH. Atmospheric carbon dioxide dis-
solving in seawater will have the opposite effect on the
pH. Calcium carbonate precipitation in seawater is in
fact largely controlled by biology. Much of the calcium
carbonate will sink to the ocean floor and be recycled
over geological timescales. The increasing atmospheric
CO2 concentration causes a higher dissolution of car-
bonic acid and is expected to decrease the seawater pH
(causing ocean acidification). This would also affect cal-
cium carbonate precipitation or even cause dissolution
of the massive amounts of calcium carbonate (stored in
coral reefs, other carbonate platforms, shellfish, etc.).
The organic carbon produced by cyanobacterial and
microalgal CO2 fixation (and by sea grasses in shallow
coastal areas) enters the food web and is liberated as
particulate or dissolved organics through fecal pellets
produced by grazers and predators, death of organ-
isms, viral lysis and by various exudation processes.
Carbon cycles between the most oxidized form (carbon
dioxide) and the reduced forms: organic matter or in
the most reduced form, methane. Seawater contains
a remarkably high amount of dissolved or colloidal or-
ganic matter as well as particulate organic matter, which
are all subject to (partial) degradation by the microbial
community (Bacteria and Archaea). Part of the organic
matter sinks to the ocean floor and the part that is not
mineralized during that voyage is eventually buried and
stored over geological timescales during which diage-
netic processes transfer it to natural gas, petroleum,
kerogen and bitumen. Together with limestone and other
fixed carbonates, these pools represent by far the larg-
est source of carbon on Earth. Anthropogenic activities
speed up the recycling of a tiny part of these geological
carbon deposits, but this is sufficient to cause a signifi-
cant global change in the carbon cycle.
Atm CO2
Dissolved CO2
CO2 + H2O H2CO3
HCO3
-
CO3
=
H+
Carbonic acid Hydrogen ions
Atmosphere
Ocean
Carbonate saturation horizon
Carbonateions
Hydrogenocarbonate
CaCO3 Ca++ + CO3
= Dissolution
Figure 2.5. Chemical basis of the carbonate system in seawater. Calcareous shell-forming plants and animals (blue circles) can develop in
waters which depths are shallower than the carbonate saturation horizon. Reversely, calcium carbonate dissolution is active in waters below
this depth level.

2.2.2.2 Nitrogen
Nitrogen is an important element which may represent
up to 10% of the dry mass of living organisms. Nitrogen
occurs in amino acids (the building blocks of proteins),
nucleic acids, bacterial cell walls and chlorophylls.
Most organisms rely on combined forms as a source
of nitrogen such as ammonium, nitrate, urea, or organic
compounds (amino acids, proteins). A few specialized
Bacteria and Archaea are capable of utilizing atmos-
pheric dinitrogen (N2), which is the most abundant form
of nitrogen on Earth, while combined nitrogen is often
in low supply. The simplest form of nitrogen cycling is
the assimilation of any form of nitrogen for growth and
the subsequent release of ammonia (deamination) after
the death and mineralization of the organism. However,
this mini nitrogen cycle is interrupted because of mi-
croorganisms that oxidize ammonium to nitrite. In the
ocean this is done by Thaumarchaeota, a newly discov-
ered group of Archaea. Other bacteria may oxidize this
nitrite further to nitrate. Nitrate and nitrite are subject to
denitrification, a bacterial process that converts these
compounds to dinitrogen. Anammox (anaerobic am-
monium oxidation), a newly discovered process, also
converts ammonium and nitrite to dinitrogen. These pro-
cesses are important in anaerobic marine sediments,
in the ocean oxygen-minimum zones (OMZs) and also
in marine snow, faecal pellets and similar aggregates
in the water column. Finally, nitrite and nitrate can also
be reduced to ammonia in a dissimilatory process (dis-
similatory nitrite or nitrate reduction to ammonia, DNRA).
Thus, the nitrogen cycle is entirely microbially driven.
While most of the nitrogen is cycled rapidly by these
biological processes, part of the nitrogen is buried and
stored in the sediment (see also Figure 2.13).
2.2.2.3 Sulfur
With 28mM sulfate in seawater, the marine environment
is dominated by sulfur, which is a micronutrient, essential
as a component of a few amino acids and in iron-sulfur
clusters in enzymes. A simple sulfur cycle is one that
just includes uptake, assimilation and its liberation after
death and mineralization of organisms. Quantitatively
Precipitation
N2
N2
N2 NO3
-
NO3
-
NH4
+
Nitrogen fixation
Denitrification Nitrification
Vertical
mixing
Sedimentation
mixing
NH4
+
Atmosphere
Illuminatedpart
oftheOcean
Darkpart
oftheOcean
Figure 2.6. Schematic overview of the nitrogen cycle in the ocean (courtesy JM Gasol)

more important is dissimilatory sulfur metabolism.
Sulfate (and other oxidized sulfur compounds) can be
reduced to sulfide by anaerobic sulfate-reducing bac-
teria. Sulfide and other reduced sulfur compounds can
be used as an electron donor for anoxygenic photosyn-
thesis (particularly sulfide and elemental sulfur). Sulfide
can be oxidized aerobically with O2 or anaerobically
with nitrate as the terminal electron acceptor. Because
sulfide also reacts chemically with O2, the biological pro-
cess occurs usually at the aerobic-anaerobic interface
in ecosystems. Anoxygenic phototrophic bacteria often
produce elemental sulfur, which can be reduced back to
sulfide as an electron acceptor in anaerobic respiration
or as an electron sink in fermentation (sulfur reducing
bacteria). The oxidation of sulfide to elemental sulfur and
back to sulfide occurs rapidly in anaerobic ecosystems
and is also known as the “mini sulfur cycle”. Another
set of microbial transformations of sulfur compounds is
known as disproportionation or as sulfur fermentation.
Figure 2.7. Schematic overview of the the Sulfur Cycle. Sulfur
occurs in redox states of -2 (as in H2S, sulfide) and +6 (as in SO4
2-
,
sulfate) and in any state in between. Transitions from one redox
state into the other are catalyzed by microorganisms or in some
cases also by chemical reactions. Altogether these transitions
form the sulfur cycle. The ‘mini-sulfur cycle’ is the quick 2-electron
transition between sulfide and elemental sulfur (So
, redox state = 0).
Anoxygenic phototrophic (photosynthesis) or colorless sulfur
bacteria (chemosynthesis) oxidize sulfide to elemental sulfur and
anaerobic respiring bacteria reduce it back to sulfide. Eventually,
anoxygenic phototrophic or colorless sulfur bacteria oxidize
sulfur further to sulfate. Colorless sulfur bacteria may also oxidize
sulfide directly to sulfate. Sulfate can go three ways: (i) it can be
assimilated for synthesis of structural cell material, (ii) it can be
reduced to sulfide by anaerobic respiring sulfate-reducing bacteria,
and (iii) it may be deposited as organic (and inorganic) minerals
in sediments. Sulfide is re-mineralized from dead organic matter
(recognized by the smell of rotten eggs). During these sulfur
transitions several intermediate redox states may be formed.
Bacteria may use these intermediates (e.g. So
, SO3
2-
, S2O3
2-
) by
oxidizing part of it to sulfate and using the electrons generated to
reduce the same component to sulfide. This process is also known
as inorganic fermentation or disproportionation. (Courtesy Lucas
Stal)

2.2.2.4 Phosphorus
Phosphorus is an essential micronutrient in organisms.
It is an important component of nucleic acids (DNA and
RNA) and therefore central to cell division, transcrip-
tion and translation processes and protein synthesis.
With its energy-rich bond in nucleotides (ATP) it is also
central to cellular energy metabolism. Phosphorus
occurs in various redox states, but unlike carbon, ni-
trogen, sulfur and iron, it is not subject to dissimilatory
metabolism. Phosphorus occurs in the environment as
phosphate. It is taken up as such and occurs in the cell
also in the oxidized state, from which it is liberated after
cell lysis. Recently, phosphonate has been recognized
as a source of phosphorus in the marine environment.
There are various enzymes such as phosphatases, nu-
cleotidases and phosphonatases that are involved in the
degradation of P-rich organic compounds. Enzymatic
hydrolysis of organic phosphorus is an essential step in
the biogeochemical phosphorus cycle. Bacterioplankton
in low-P environments (e.g., the Sargasso Sea, the
Mediterranean Sea) are particularly frugal with P.
Phospholipids and nucleic acids appear to be the pri-
mary cellular reservoirs of P in the open sea. Recent
studies show that plankton have evolved mechanisms
to economize on their biochemical P requirements. For
example, Prochlorococcus and Synechococcus, the
picocyanobacteria that often dominate low-P environ-
ments, primarily synthesize sulfur-containing membrane
lipids rather than phospholipids. This switch from P- to
S-lipids obviously decreases the cellular demand for P
and may be an important adaptation to low-P environ-
ments. Phosphate moves quickly through the biosphere
but the processes that move it through sediments and
the water column are slow, making the phosphorus cy-
cle overall one of the slowest biogeochemical cycles.
Phosphates are often trapped in marine sediments as
iron or calcium minerals and may be liberated through
the activity of anaerobic bacteria.
2.2.2.5 Iron
Iron, one of the most abundant elements on Earth, is a
micronutrient essential for virtually all living organisms.
It occurs in iron-sulfur cofactors in various enzymes and
electron transport components (cytochromes). Oxidized
(ferric) iron is virtually insoluble except at very low pH.
Reduced (ferrous) iron, in contrast, is soluble but is
readily oxidized under aerobic conditions. Hence, bio-
available iron will be present at very low concentrations
in the water column of the ocean and is, therefore, sus-
pected to be the limiting nutrient for primary production.
Sahara dust containing large amounts of iron is regu-
larly transported by wind to the Atlantic Ocean and the
Mediterranean Sea and has been thought to fertilize
these marine environments stimulating primary produc-
tion and N2 fixation (see Information Box 4). It is possible
that UV light reduces dust ferric iron before it is taken
up by the phytoplankton. Alternatively, microorganisms
can excrete organic iron chelating compounds called
siderophores that bind iron and transport it into the cell.
Information Box 4. Sahara dust and marine
microbial diversity
Sandstorms, or dust storms, are usually the result of
atmospheric convection currents, which form when
warm, lighter air rises and cold, heavier air sinks.
Dust from the Sahara Desert can be transported
over thousands of kilometres by convection currents,
which also cause other meteorological conditions
such as thunderstorms (Figure 2.8). Sandstorms are
common over the Sahara, which is a major source of
mineral dust, transported to the Mediterranean, the
tropical Atlantic and the Caribbean. Saharan dust
plays an important role in the Mediterranean region
because it is the major source of mineral nutrients
for phytoplankton and bacteria. However, it is not
always beneficial. In the Caribbean, Saharan dust is
believed to infect coral reefs with the sea fan disease
and some studies suggest dust is linked to health
risks, such as increased incidences of paediatric
asthma attacks and epidemics of lethal meningitis
in the semi-arid sub-Saharan territory known as the
Sahel belt, although more research is needed to
confirm this.
Figure 2.8. ESA Satellite Envisat captures sand and dust from
the Sahara Desert blowing across the Atlantic Ocean along the
coasts of Mauritania (top), Senegal (middle) and Guinea Bissau
(bottom). The cloud-covered Cape Verde islands are visible off
the coast of Senegal. (©ESA)

Iron fertilization, the intentional introduction of
iron to the upper ocean to stimulate a phytoplank-
ton bloom, has long been proposed as a potential
way to geo-engineer the removal of carbon dioxide
from the atmosphere. This stems from the observa-
tion that growing plankton takes up carbon dioxide
during photosynthesis and iron is often the limiting
nutrient for phytoplankton growth in the open ocean.
When phytoplankton die, they sinks to the bottom
of the ocean locking away some of the carbon they
has absorbed from the atmosphere. Fertilization also
occurs naturally when upwelling brings nutrient-rich
water to the surface or when weather carries dust
long distances over the ocean, or iron-rich minerals
are carried into the ocean by glaciers, rivers and ice-
bergs.
Since the early 1990s, a number of research groups,
scientists and private organizations have been ex-
ploring iron fertilization as a means to stimulate
phytoplankton growth with the aim of sequestering
atmospheric carbon dioxide in the deep ocean. This
research has confirmed that large phytoplankton
blooms can be created by artificially supplying iron
to iron-deficient ocean waters. However, iron ferti-
lization remains highly controversial in terms of the
effectiveness of atmospheric CO2 sequestration and
the associated ecological effects. Computer models
suggest that iron fertilization of the oceans is much
less efficient at decreasing atmospheric carbon diox-
ide than initially hoped. Opponents of this proposed
geo-engineering strategy cite the risk of unintended
environmental impacts and argue that in addition,
such schemes could distract from efforts to decrease
anthropogenic carbon emissions.
Scientists acknowledge that much remains to be
learned about the efficiency of iron fertilization in
promoting long-term sequestration of carbon diox-
ide by the oceans, as well as its impact on marine
ecosystems. For this reason, a new impetus to iron
fertilization research has taken shape under the form
of the In situ Iron Studies (ISIS) consortium, an in-
ternational effort established in February 2011, to
assess the efficiency of ocean iron fertilisation (OIF)
and determine its potential impacts on marine eco-
systems. For more information see also http://noc.
ac.uk/news/international-consortium-study-impacts-
iron-fertilisation
Information Box 5. Iron fertilisation
Figure 2.9. Envisat satellite image showing green swirls of a
phytoplankton bloom in the North Sea off the coast of eastern
Scotland (©ESA)
Iron was proposed to be the nutrient limiting factor in
the so-called high nutrient low chlorophyll areas, such
as the Southern Ocean or the equatorial Pacific. To test
this hypothesis a series of large scale iron fertilization
experiments were designed to increase marine biologi-
cal productivity and test if atmospheric carbon dioxide
could be sequestered into the deep ocean. Results
confirmed that large phytoplankton blooms can be arti-
ficially created, at least temporarily.
Oxidized and reduced iron are subject to microbial
conversions. Oxidized iron can serve as an electron
acceptor for anaerobic respiration and a variety of mi-
croorganisms are capable of this reaction. It will be clear
that these microbial transformations and dissimilatory
iron metabolisms are confined to specific environments,
especially sediments, OMZs and certain geothermal
springs. Reduced iron is subject both to microbial and
chemical oxidation. Iron forms minerals with a variety of
other elements, particularly with sulfur and phosphate.
2.2.3 Ecosystem functioning
Although there are ecosystems that function inde-
pendently from sunlight, some of them in the marine
environment, the vast majority of life on Earth (including
the ocean) depends on photosynthetic primary pro-

The 1% rule does not exclude the possibility of primary
production at lower values.
Primary production is the fixation of CO2 into organic
carbon using sunlight as the source of energy and water
as the source of electrons, resulting in the evolution of
O2 (oxygenic photosynthesis). The organic carbon and
the nutrients (N, P, micro- and trace nutrients) that the
primary producers have accumulated form the basis of
the oceanic food web. Zooplankton graze on the primary
producers, are grazed themselves by other zooplankton
and fish and so up to higher and bigger predators. Each
of these groups produce fecal pellets which contain or-
ganic matter and nutrients that serve as a substrate for
Bacteria and Archaea that decompose it and recycle
the nutrients. Living and dead primary producers, fecal
duction. In the marine environment virtually all primary
production is attributed to microalgae (Eukarya) and
Cyanobacteria (Bacteria) (each roughly responsible for
half of the global oceanic primary production). In shallow
(coastal) waters we may find macroalgae and seagrasses
that locally contribute importantly to primary production
but globally, they are of minor importance in terms of
CO2 fixation.
Primary production depends on sunlight and is, there-
fore, restricted to the upper photic zone of the ocean,
usually defined as the depth at which sunlight is attenu-
ated to 1% of the surface incident intensity. The depth to
which it reaches depends on the absorption character-
istics of the water column and may range from 200m in
the open ocean to centimetres in some turbid estuaries.
Figure 2.10. Schematic representation of the ocean food web showing, on the left, the classic pathway of carbon and energy flow through
photosynthetic Eukarya, to herbivores and on to higher trophic levels. Depicted on the right is the microbial food web, which uses energy
stored in the non-living, detrital carbon pool to produce microbial biomass that can re-enter the classic pathway of carbon and energy flow.
Cell-associated ectoenzymes (Ecto) enable bacteria to use high-molecular-weight (HMW) dissolved organic carbon (DOC) in addition to the
more traditional low-molecular-weight (LMW) and gaseous carbon substances. Also shown in the microbial food web are viral particles and
Archaea. At the present time, there is only rudimentary knowledge of the role of Archaea in the oceanic food web. Shown at the bottom of
this diagram is the downward flux of particulate carbon (and energy), which is now thought to fuel most subeuphotic zone processes. The
classic algae-herbivore grazer pathway (left side) is most important in this regard. (From DeLong and Karl, Nature 437, 2005)

pellets and aggregates of debris, organic matter and
microorganisms sink and provide the food web below
the photic zone with substrate. Microorganisms may
also escape the food web by direct lysis through viral
infection (viral loop) or through exudation/excretion pro-
cesses through which the organic carbon and nutrients
become directly available to the microbial community.
Most of the biomass and biological activity in the ocean
is microscopic with microbes playing a central role in
Earth’s carbon, oxygen, sulfur, and nitrogen cycles.
Microbial communities are much more important in the
overall marine food web than was assumed until recent-
ly. Great numbers of micro-, nano- , and pico-plankton
make up the microbial food web which is linked in many
ways to the classical food chain with dissolved organic
carbon playing an important central role (Figure 2.10).
2.2.3.1 The role of viruses
Viruses are known to be the most abundant and diverse
organisms on Earth. Understanding their evolutionary
processes is, therefore, of fundamental importance.
Viruses contribute to biogeochemical cycles by lys-
ing up to 30-50% of the microbial biomass every day.
Carbon and nutrients are released upon viral lysis and,
hence, they mediate and enhance biogeochemical cy-
cling in the marine environment (Figure 2.11). In addition,
viruses directly affect the abundance and diversity of
host cell communities and contribute to microbial gene
exchange, which are important for the overall evolu-
tion of both the host and the viral community. We are
beginning to unravel the significance of viruses and vi-
ral-mediated processes in the ocean and considerable
effort has been targeted at uncovering viral diversity
e.g. through metagenome sequencing. Also, the use of
novel techniques in the study of virus-host interactions
in culture has uncovered new and unexpected results.
Virus-host interactions have revealed that viruses may
Figure 2.11. Microbial food web and virus-mediated carbon flow (the microbial and viral loops). The red arrows represent virus-mediated
pathways. The black arrows represent transport of dissolved organic carbon (DOC) in the microbial food web. The black dotted arrows show
the contribution to CO2 cycling and the blue lines the transport of mineral nutrients in the microbial food web. DOC is the largest biogenic
pool of carbon in the ocean. The dynamics of DOC may have an indirect impact on the global carbon cycling contributing to control of
atmospheric CO2. DOC is only accessible to microbes, primarily heterotrophic bacteria. When viruses lyse the host, carbon is transformed
from a particulate form (POC) into the dissolved form (DOC). (Modified from Sandaa, 2009)
Figure 2.11. Microbial food web and virus-mediated carbon flow (the microbial and viral loops). The red arrows represent virus-mediated
pathways. The black arrows represent transport of dissolved organic carbon (DOC) in the microbial food web. The black dotted arrows show
the contribution to CO2 cycling and the blue lines the transport of mineral nutrients in the microbial food web. DOC is the largest biogenic
pool of carbon in the ocean. The dynamics of DOC may have an indirect impact on the global carbon cycling contributing to control of
atmospheric CO2. DOC is only accessible to microbes, primarily heterotrophic bacteria. When viruses lyse the host, carbon is transformed
from a particulate form (POC) into the dissolved form (DOC). (Modified from Sandaa, 2009)
DOC
Fish
Bacteria Small algae Large algae
and diatoms
Heterotroph
icﬂagellates
Ciliates
Zooplankton
CO2
Mineral
nutrients
Viruses

also have a tight and mutualistic relationship with their
hosts. Indeed, ‘host’ genes have been uncovered in the
viral genome, such as those encoding components of
the photosynthetic apparatus, that are likely beneficial
to the viral infection process. At present, several genes
with hitherto unknown function have been identified that
might play a role in the virus-host relationship. This new
knowledge about virus-host interactions indicates that
viral genes could play important roles because they may
affect critical rate-limiting steps of the host metabolism.
Nevertheless, our understanding of the effect of viruses
on marine community structure and rates of nutrient cy-
cling is far from complete, and their role in the marine
ecosystem is still emerging as methodologies evolve.
2.2.3.2 The importance of chemosynthesis
in the ocean
There are several sites of chemosynthetic CO2 fixa-
tion in the ocean. Chemosynthetic microorganisms
(chemolithoautotrophs) use the oxidation of a reduced
compound to generate energy with which they fix CO2.
Although this process has been considered as of mi-
nor importance in the water column, Recent reports
indicate localized chemosynthesis may be more impor-
tant than previously believed. For instance, planktonic
Thaumarchaeotahave been shown to oxidize ammonium
and fix CO2 and such organisms may occur in consider-
able numbers. In oxygen minimum zones (OMZs), cold
and hot seeps and black smokers, life is formed through
chemosynthetic CO2 fixation at the expense of the oxi-
dation of sulfide, methane or other reduced compounds
(e.g. iron, manganese). These ecosystems are fully in-
dependent of light and organic matter which is unique
for marine systems.
2.2.3.3 The critical role of the fixation
of dinitrogen in the ocean
A critical nutrient for primary production is nitrogen.
Although dinitrogen represents by far the largest source
of nitrogen on Earth, it is unavailable to virtually all organ-
isms. This is due to the very stable triple bond between
the two nitrogen atoms rendering N2 almost inert. Only
some Bacteria and a few Archaea are capable of re-
ducing N2 to ammonia and assimilating it. Nitrogenase,
the enzyme responsible for N2, functions only under
anaerobic conditions, limiting its activity to anaerobic
environments, anaerobic microorganisms or to organ-
isms that provide low O2 conditions for the enzyme. In
the ocean, Cyanobacteria are the major N2-fixing (di-
azotrophic) organisms and they are responsible for 50%
of the global fixation of N2 (excluding anthropogenic
fertilizer production). As oxygen-evolving phototrophs,
Cyanobacteria have a problem with the incompatibility
of N2 fixation with O2.
The non-heterocystous, filamentous Cyanobacteria
Trichodesmium spp. was previously considered the ma-
jor diazotroph in the ocean. These organisms occur only
in the (sub)tropical ocean where the water temperature
is well above 25o
C. However, recently it was found that
unicellular diazotrophic Cyanobacteria may be equally
important in some oceanic regions. The most abundant
belong to the very small (~1 µm) uncultured ‘UCYN-A’
group which seems to lack the oxygenic photosystem II.
These organisms may live in a symbiotic relationship.
The apparent absence of N2 fixation from the temper-
ate and cold regions of the ocean is not fully explained.
It is generally assumed that the marine environment
is chronically depleted of combined nitrogen and we
would, therefore, expect that N2 fixation is important
here as well.
2.2.3.4 Conversion of bioavailable nitrogen
to dinitrogen
The traditional pathway of ammonium oxidation in the
marine (but also freshwater and terrestrial ecosystems)
nitrogen cycle follows aerobic oxidation in two steps
via nitrite to nitrate. Two specific groups of Bacteria
are responsible for each of these steps. The oxidation
of ammonium to nitrite is carried out by ammonium
oxidizing (nitritifying) Bacteria. However, there is com-
pelling evidence that in the marine environment, certain
Thaumarchaeota are in fact oxidizing the bulk of ammo-
nia to nitrite. The oxidation of nitrite to nitrate is carried
out by nitrite-oxidizing (nitratifying) Bacteria. It is not
clear how important this second oxidation step is in the
marine environment.
Nitrate can follow two routes. It can be used as a ni-
trogen source by photoautotrophic plankton (known
as ‘new’ or recycled nitrogen) or under anaerobic
conditions can be denitrified to N2 or reduced back to
ammonium (DNRA). We can anticipate that the micro-
bial conversions of nitrogenous compounds are quick.
While both denitrification, DNRA, or nitrate assimilation
go through nitrite as the first intermediate, it might not
be unreasonable to conceive that the oxidation of nitrite
to nitrate is of limited importance in the marine environ-
ment and that nitrite is readily used in these processes
before being oxidized to nitrate. It would also explain
why ammonium-oxidizing Thaumarchaeota are so abun-
dant while this is not so clear in the case of the oxidation
of nitrite to nitrate. Apart from being assimilated or used
in denitrification and DNRA, nitrite is also a substrate
in a relatively recently discovered process, anaerobic
oxidation of ammonium (anammox), in which it is used
as the oxidant. Anammox oxidizes one molecule of am-
monium with one molecule of nitrite, resulting in the
formation of one molecule of N2. It is, therefore, next to
denitrification, the second process leading to conver-

sion of ‘fixed’ and bioavailable nitrogen to the largely
inert atmospheric dinitrogen. Anammox bacteria belong
to the Planctomycetes.
Anammox has been discovered in marine ecosystems,
including sediments as well as in the water column in
OMZs, but its quantitative importance is under debate.
Only one genus, Scalindua, has been found in natural
marine environments. It seems that in sediments deni-
trification is more important but in the water column
of OMZs, anammox may be the dominant process.
In the marine suboxic water column anammox is not
particularly abundant and represents 4% of the mi-
crobial community. These organisms grow slowly with
a doubling time of ~11 days. Anammox is sensitive to
oxygen and also to sulfide, explaining its absence in en-
vironments dominated by sulfate reduction. Anammox
bacteria are autotrophic organisms that fix CO2 through
the acetyl-CoA pathway. This chemosynthetic CO2 fixa-
tion is probably not of quantitative importance for the
global C cycle.
2.2.3.5 Summary conclusions: marine microbial
research on ecosystem functioning
For decades scientists have invested much time and ef-
fort in investigating oceanic biogeochemical processes
and the microbes responsible for them. However, new
discoveries in the past few years, as a result of the
development of new genomic and geochemical tech-
nologies, have dramatically changed our understanding
of biogeochemical cycling processes at different levels.
Examples are the discovery of the processes of anaero-
bic ammonium oxidation (anammox), and the discovery
of ammonia oxidation by Archaea, recognized as two
new fundamental links in the global marine nitrogen cy-
cle. Thus, there is a need to move from the description
of organisms to functional analysis using methods that
measure and monitor microbial functions. Moreover, to
gain a full understanding of the contemporary and the
probable future state of the marine environment, we need
to understand the genetic basis of marine microbial bio-
geochemistry and ocean processes.
2.2.4 Global change
Current global change is associated with a range of
physical and chemical factors (rising air and water tem-
peratures, increasing CO2 uptake by the oceans and
seas causing acidification of the marine environment,
sea level rise, eutrophication, etc.) and it has been
shown that these processes have a profound impact
on marine ecosystems, an impact that is predicted to
increase (Figure 2.13).
Since the beginning of life, microorganisms have left a
notable fingerprint on the geochemistry of Earth and its
biogeochemical cycles. Earlier microbes have shaped
our atmosphere under which more complex life devel-
oped, paving the way for the advent of multicellullar
organisms and complex biological communities. While
microbial communities will adapt to global change, they
might modify ecosystem functioning in a way that we
find undesirable.
Climate change is usually referred to as the increase in
the average global temperature as the result of an in-
crease of greenhouse gases in the atmosphere (usually
emphasizing CO2) due to anthropogenic activities. While
climate changes are a natural phenomenon on Earth, the
change we are currently anticipating is unprecedented
both in the speed by which it is likely to occur and the
expected size of this change.
2.2.4.1 Consequences of a rise in temperature
An increase in global temperature will cause a rise in
sea surface temperature (SST). Temperature has a di-
rect effect on the growth rate and metabolic activity of
marine microorganisms. It may also select for different
microorganisms that are better adapted to higher tem-
peratures or existing microorganisms may disappear
when temperatures move outside their normal toler-
ance range. As an example of the type of processes
involved, respiration rates may change with tempera-
ture differentially compared to production rates, which
could drive the ecosystem towards a low nutrient status
(oligotrophy). Similarly, bacterial production and loss
rates to grazers might also be affected differentially by
temperature, so that an increase in temperature might
tighten predator control. Increasing temperatures will
also affect the density of the water and consequently
stratification and currents. This will, in turn, affect the
transport of nutrients. Another effect of the increase
of the global temperature is an expected increase in
the number and intensity of storms. These events may
cause deep mixing of the water column of oceans and
seas and may counteract stratification. The sum of
these processes is complex and difficult to predict.
2.2.4.2 Consequences of a rise in atmospheric CO2
The anthropogenic emissions of CO2 to the atmosphere
cause a rise in global temperature (greenhouse effect)
with a range of direct and indirect impacts on the marine
environment including changes in sea water tempera-
ture, sea level, geographic distribution and abundance
of species, ecosystem functions, sedimentation pro-
cesses, etc. In addition, the associated increased uptake
of CO2 has sparked ocean acidification (see below). The
effects and impacts of these changes on ocean micro-
bial processes are far from understood. Thus, one of the
challenges of the future research will be to understand
how climate changes may influence microbial life in the

ocean but also the other way around, i.e. how marine
microbes influence climate.
The increase in atmospheric CO2 concentrations may
affect marine microorganisms in various ways. In the
first place, it might stimulate primary producers, al-
though an increase in CO2 is the result of a complex
cascade of processes. For instance, an increase in
primary production would only be expected when CO2
is indeed the limiting factor. This is unlikely to be the
case for many primary producers and for many prevail-
ing environmental conditions. Indeed, even if primary
production were enhanced, the resulting phytoplankton
blooms, even if not toxic, often result in poor water
quality. Decomposition of the increased organic mat-
ter would lead to oxygen depletion and to anaerobic
processes that in the marine environment would lead
to the formation of toxic sulfide. The cascade of such
processes may in fact move an ecosystem state into
another, unwanted, stable state.
The oceans are a sink of CO2 by a process that is known
as the biological pump. The CO2 that dissolves in the
water is used by phytoplankton to be converted into
biomass. Dead biomass sinks and is decomposed by
bacteria which produces CO2 and the seawater at high
depths (with higher pressure) contains considerable
higher concentrations than surface water which is in
equilibrium with the atmosphere. Another (small) part
of the organic matter is eventually buried in the seafloor
and recycles on geological time scales. Calcium carbon-
ate skeletons of coccolithophoric algae (coccoliths) rain
down to the seafloor and also this inorganic carbon is
buried. The big unknown is how much CO2 can be ac-
commodated by the oceans and what would happen if
the CO2-rich water masses come to the surface.
2.2.4.3 Acidification
The other effect of increasing CO2 concentration is
known as ocean acidification. CO2 is an acid and its dis-
solution in seawater will decrease the pH. The effects of
small changes in pH on the growth and activity of marine
microorganisms are probably minor, except for calcifica-
tion which is negatively affected so that the dissolution
of calcium carbonate (such as that forming coral reefs)
may occur. Calcification is essential for shellfish and an
effect of acidification on shell formation has been dem-
onstrated. It is not clear whether coccolithophorid algae
are affected by acidification and although deformation
of the coccolith platelets has been demonstrated, it is
uncertain whether they are important for the organism
except for generating CO2 (which would be unnecessary
in case of high CO2). More problematic could be the
massive dissolution of calcium carbonate, which would
increase the dissolved CO2 and further decrease pH
generating a possible run-away effect.
2.2.4.4 Emission of nitrogenous greenhouse
gases
Microbial activity also affects atmospheric concentra-
tions of the greenhouse gas nitrous oxide (N2O). N2O
has been identified as the dominant ozone-depleting
compound and it is projected that it will continue to be
throughout the 21st
century. Thus, understanding the
processes controlling emissions of N2O from coastal
and marine systems is important for evaluating climate
change scenarios.
The source of N2O in the ocean is most likely incomplete
denitrification, although nitrification may also lead to the
formation of N2O. Denitrification occurs under anaero-
bic conditions, in aggregates (marine snow), OMZs and
sediments.
While we would anticipate that nitrate is in low supply
relative to the organic matter that is oxidized, it is unclear
why the nitrogen is not oxidized all the way to N2. It is
possible that other sources of N2O exist. For instance,
it has been suggested that dinitrogen-fixing organisms
may produce it. The only sink of N2O is the last step
in denitrification where it is reduced to N2. However, if
blocked, for example due to the presence of Dimethyl
Sulfide (DMS) or other sulfur components, N2O accumu-
lates. Once in the water column it is most likely emitted
to the atmosphere. Increasing the load of nitrogen by
dry and wet deposition, run-off and riverine discharge,
as well as by the fixation of dinitrogen, may lead to pro-
cesses that increase N2O emissions.
2.2.4.5 Dimethyl Sulfide (DMS)
Oceans emit considerable quantities of dimethylsulfide
(DMS), which is produced from the degradation of di-
methylsulfoniopropionate (DMSP) by Bacteria and
microbial Eukarya. DMSP is produced by some micro-
algae and serves as an osmoprotectant, allowing them
to grow at high salt concentrations (in seawater). The
emission of DMS into the atmosphere is thought to act
as a climate feedback regulation. The so-called CLAW
hypothesis states that DMS in the atmosphere is con-
verted to an aerosol form of sulfate (non-sea salt sulfate,
NNS) and that these aerosols act as cloud condensation
nuclei (CCN). Hence, the more DMS is emitted, the more
cloud albedo is formed, resulting in a greater reflection of
sunlight. This would subsequently decrease the growth
of microalgae and the production of DMSP and conse-
quently less DMS would be emitted. It is, however, likely
that this climate feedback is far more complex. DMS
may also be the transporter of sulfur from the oceans to
the continents were it precipitates as sulfuric acid with
rain. This may enhance rock weathering, securing the
transport of nutrients from the continents to the sea,
nutrients which support microalgal growth and primary

production. These are all interesting hypotheses that
emphasize the complexity of the biogeochemical cycles
on Earth.
2.2.4.6 Methane
Methane is a very potent greenhouse gas. There are
basically two sources of methane; one is geothermal
(natural gas, volcanic, hot springs, cold seeps, methane
gas hydrates) and the other is biological. Methanogenic
Archaea are anaerobic organisms that convert CO2 or
simple organic compounds (e.g. acetate) to methane. In
terrestrial environments these organisms are particularly
known from the rhizosphere of rice paddy fields and
from the intestines of a range of organisms such as cat-
tle and termites. Anthropogenic activities have increased
this source of methane considerably. Methanogenic
bacteria are also known from swamps and wetlands.
However, in the marine environment methanogenic bac-
teria compete with sulfate reducing bacteria which have
an advantage because of the very high concentration of
sulfate in seawater (28 mM). Therefore, methanogenic
bacteria may play a less prominent role in the marine
environment, even though there are non-competitive
substrates (which are not used by sulfate reducing
bacteria) and molecular surveys indicate their abun-
dant presence. Moreover, methanotrophic bacteria are
expected to utilize any methane that is formed. In the
water column aerobic methanotrophic bacteria would
consume methane, while under anaerobic conditions,
consortia of methanogenic bacteria and sulfate reduc-
ing bacteria oxidize methane (in terrestrial anaerobic
environments methane is oxidized by oxygen which the
organism generates from nitrite). Seafloor gas hydrates
are a possible source of methane also. Such massive
amounts of methane will travel through the water col-
umn and be released into the atmosphere without being
consumed. These are phenomena independent of hu-
man activities.
In conclusion, there are still many unknowns regarding
sources and fates of methane in the sea and the role of
marine microorganisms in methane regulation. Much of
our knowledge is based on old paradigms which may
need revision. The possible emission of methane from
coastal swamps and wetlands needs particular atten-
tion.
2.2.4.7 Freshwater inflow
Increasing global temperature is causing melting of the
polar icecaps resulting in sea level rise. Apart from the
problems this will cause in countries bordering the sea,
the melting of ice may have a range of other effects.
The sudden release of massive volumes of freshwater
Sun rediation (warmth, light, UV)
Temperature
Wind
Albedo
NCN
Sulfate aerosoles
DMS g
DMS ac
Marine trophic web
DMSPDMSP
Bacteria
Figure 2.12. Dimethyl sulfide (DMS) or methylthiomethane is an organosulfur compound with the formula (CH3)2S (right) which provides
the characteristic smell of seafoods and when cooking certain vegetables, notably maize, cabbage and beetroot. The production of
dimethylsulfide (DMS) in the oceans is driven by microorganisms (left; courtesy JM Gasol and R Simó). The emission of DMS into the
atmosphere is thought to act as a climate feedback regulation.

will cause salinity changes which may affect water cur-
rents and stratification. Obviously, salinity changes also
affect microorganisms and may cause dramatic shifts in
the composition of the microbial community. In addition,
with the melting of the ice, trapped organic matter, con-
taminants and microorganisms are released and these
could also cause shifts in local community composition.
2.2.4.8 Summary conclusions: marine
microorganisms and global change
Gas exchange between the ocean surface and atmos-
phere involves air (mostly oxygen and dinitrogen), and
the trace gases CO2 (carbon dioxide), N2O (nitrous ox-
ide or laughing gas), CH4 (methane), and dimethylsulfide
(DMS). Some of these gases are greenhouse gases
and considered to be responsible for global warming.
In addition, the increasing dissolution of CO2 leads to
acidification of seawater, which could cause the dis-
solution of carbonates. DMS may trigger cloud albedo
formation and cause acid rain. These gas exchanges are
the result of biogeochemical processes which depend
on the composition and activity of the marine microbial
community, which in turn respondss to and cause global
climate change. Unfortunately, our understanding of
these complex processes and interactions is limited. We
are just beginning to collect the necessary genetic data,
discover the relevant biochemical processes, and get a
grip on the untold diversity of marine microorganisms
and their interactions. This will be the basis of predictive
models which will help us to better manage the ocean
as one of our most precious and vulnerable resources.
Figure 2.13. Scheme illustrating the potential global change effects on the ocean. Globally increasing temperatures and concentrations
of greenhouse gases such as CO2 and CH4 will have significant impacts on microbial activities and communities, which may affect
biogeochemical cycles. (Developed by Matthias Labrenz)

Mediterranean Sea (Phaeocystis, dinoflagellates), which
in recent years have been subject to increased urbaniza-
tion (see Information Box 6 dealing with cyanobacterial
blooms in the Baltic Sea).
The spread or introduction of non-native species across
biogeographic ranges is a relevant case of biotic distur-
bance. The rapid proliferation of non-native macroalgal
species may derive from their fast growth and dispersal,
high competition with native organisms, and/or the sta-
tus of the invaded native ecosystems, altered in many
cases by human activities. The transmission of non-na-
tive species may drive changes in marine biodiversity
and the functioning of marine ecosystems, which may
decrease ecosystem resistance and resilience to distur-
bance. HABs are known to be particularly invasive, but
very little is known about particular Bacteria or Archaea
which are thought to have a universal distribution.
Increased or decreased freshwater runoff poses an
ecosystem threat as coastal water circulation patterns
might be affected and terrestrial runoff may be rich in
inorganic nutrients, refractory organic matter, or both.
This can affect the heterotrophic/autotrophic balance
of the ecosystem, which is mostly in place by virtue of
2.2.5 Local anthropogenic forcing
Aside from global changes, a number of human activi-
ties exert an important influence on marine microbial
life. Some examples are habitat modification, freshwater
runoff and river discharges, oil and other chemical spills.
Occasionally, these lead to unwelcome consequences
such as eutrophication and the occurrence of harmful
algal blooms (HABs). Humans change the coastline and
may also modify the characteristics of the substrates
thus affecting the role of microbes in the environment. A
typical case is the building of harbours which decrease
the seawater residence time. Under such conditions,
when turbulence is prevented and temperature tends
to increase, the development of HABs is favoured. These
HABs may comprise species which are a nuisance sim-
ply because they affect the colour and smell of the water
and are not appreciated by swimmers and tourists or,
more seriously, may pose a direct health hazard when
they produce neuro- or enterotoxins. While in some
cases a direct link between human intervention and
ecosystem responses can be demonstrated, more of-
ten such links remain elusive or indirect. This problem is
well known for the Baltic Sea (Cyanobacteria), but HABs
likewise affect the coastlines of the Atlantic Ocean and
Figure 2.14. A conceptual model of how microbial diversity in the ocean is expressed, i.e. a few organisms are relatively abundant and
participate in ecosystem functioning while the majority of microbes await more optimal growing conditions, as shown above in the large ‘tail
of biodiversity’. The image also indicates the level of diversity that can be assessed by each current methodology (Adaptation by Thomas
Pommier, CNRS, of an original figure by Pedrós-Alió, 2006)

microbial action. Similarly, dredging or habitat alteration
in the sediments might alter the functioning of sediment
microbes by modifying the concentration of terminal
electron acceptors (oxygen, nitrate, sulfate, ferric iron)
and thus modifying the dominant end product reactions.
The accidental release of man-made organic or inor-
ganic substances which are allochthonous to the marine
environment may produce interesting effects. Oil, for ex-
ample, is known to be degradable by bacteria subject
to the presence of sufficient oxygen (for hydrocarbon
degradation) and inorganic nutrients. Specialized bac-
teria are known to reside in the “rare biosphere” of
environments subjected occasionally to oil pollution
(such as harbours, rivers, marinas, but also including
the rhizosphere) and to grow and degrade most of the
oil components. Other chemicals which can be present
in urban waters can be degraded but a problem may
arise in the case of discharges of antibiotics in waste
water because it may lead to the development of resist-
ances which could eventually be transferred to human
pathogens.
2.3 Societal impacts and benefits
Marine microorganisms influence our lives more than we
can imagine. First of all, as illustrated in Section 2.2.3 of
this chapter on ecosystem functioning, marine microor-
ganisms provide critical ecosystem services in terms of
regulating marine food webs, producing oxygen (about
50% of all the oxygen on Earth), biogeochemical cy-
cling, assuring ecosystem integrity, and CO2 uptake and
buffering (carbonate system). Secondly, marine microor-
ganisms also provide important products and processes
which are of use to our societies. At the same time,
some marine microorganisms are responsible for po-
tentially negative impacts such as the spread of human
and fish diseases and the contamination of fisheries and
aquaculture products with toxic substances (e.g. result-
ing from HABs). This section will describe some of the
most important societal benefits which we may obtain
from marine microbial research in the future.
Eutrophication is one of the fundamental results of
human activity in the Baltic Sea. High nutrient loads,
predominantly from intensive agriculture or municipal
sewage, cause the formation of extensive blooms of
primary producers in spring and summer. The spring
bloom is dominated by diatoms and dinoflagellates
and leads to a decrease of nitrate and phosphorus
concentrations in the water. The summer bloom is
dominated by the cyanobacteria, Nodularia spumi-
gena, Aphanizomenon ﬂos-aquae, Anabaena sp.,
and Synechococcus spp., of which the first three are
often characterized by their ability to fix dinitrogen.
Dinitrogen-fixing primary producers become predom-
inant in the summer bloom when nitrogen availability
is limited but phosphorus is still available. The gen-
eral availability of phosphorus in the water column
is a specific feature of the central Baltic Sea and is
connected to the anoxic nature of the bottom waters
of this area as anoxia enhances the release of phos-
phorus from the sediment. The phosphorus stock in
the water column thus increases and feeds surface
primary production. As demonstrated by satellite im-
ages (Figure 2.15), cyanobacterial blooms may cover
wide areas of the whole Baltic Sea and since some
Cyanobacteria are potential cyanotoxin producers,
the blooms can be hazardous for higher life forms,
which in itself reveals the potential socio-economic
impact on Baltic riparian countries.
Figure 2.15. Satellite image, acquired on 31 July 2008, by
Envisat’s Medium Resolution Imaging Spectrometer (MERIS),
captures a cyanobacterial bloom in the Baltic Sea (provided by
H. Siegel, IOW). (©ESA)
Information Box 6. Cyanobacterial blooms in the Baltic Sea

2.3.1 The relationship between biodiversity
and environmental stability
A fundamental ecological tenet is that of the relation-
ship between biodiversity and environmental stability.
The idea behind such a tenet is that an ecosystem that
has a large number of potential solutions to problems
(i.e. a large number of species which can act in slightly
different ways) has a higher probability of surviving
environmental stress (e.g. the introduction of an alloch-
thonous toxic chemical, or a large increase of a nutrient)
than an environment which has fewer available choices.
It is always possible that a particular microorganism will
grow and replace a dominant organism which is affected
in such a way that the overall community performance
and ecosystem functioning remains mostly unaffect-
ed. This is the so-called “insurance-hypothesis” and
it arises from initial studies on this issue performed in
temperate grassland communities that demonstrated
that plant diversity affects ecosystem functioning such
as above-ground production, and ecosystem properties
such as resistance and resilience.
While we lack direct experimental evidence of this
phenomenon, some studies have shown that where
greater numbers of microorganisms exist in an environ-
ment, more organic C is degraded and the environment
fluctuates less. This suggests that maintenance of bio-
diversity, including microbial biodiversity, is essential for
ecosystem functioning and, by extension, the well-being
of human societies. Research that supports this concept
is limited by the available methodologies that, as dis-
cussed elsewhere in this document, reach only a small
fraction of the true microbial biodiversity present in the
marine ecosystem.
2.3.2 Biodiscovery and bioactive
compounds
The marine environment is emerging as a ‘gold mine’
for novel bioactive compounds with a staggering 1011
new compounds reported for 2009. Marine-derived
natural products present an enormous range of novel
chemical structures and provide an interesting and chal-
lenging blueprint for creating new entities via synthetic
chemistry. Marine invertebrates and plants, in particular,
represent an environment rich in microorganisms that
produce compounds with bioactive properties including
antibacterial, antifungal, antiviral, anticancer, antifoul-
ing and antibiofilm activities. However, only 1% of these
microorganisms can be isolated using traditional cul-
turing techniques, which has been a major bottleneck
when mining the marine environment for novel bioactive
molecules.
2.3.2.1 Antimicrobial and antifungal
The emergence of multidrug resistant bacteria and
fungi, the latter including certain Aspergillus fumigatus
and Candida albicans strains, drives the continuous
search for novel antibacterial and antifungal agents.
Most natural antibiotics used today originate from soil
actinomycetes. However, since the rate of discovery of
novel antibiotics of terrestrial origin is declining, other
ecological niches, including the marine environment,
are being exploited in the search for new antibiotics.
Classes of compounds with antibacterial and/or anti-
fungal activity which have been isolated from the marine
environment include peptides, sterols, terpenes, alka-
loids, and polyketides.
Three classes of antibiotic resistant bacterial patho-
gens are emerging as major threats to public health:
(i) methicillin-resistant Staphylococcus aureus (MRSA),
(ii) multidrug resistant Gram negative bacteria, includ-
ing Escherichia coli and Pseudomonas aeruginosa, and
(iii) multidrug resistant Mycobacterium tuberculosis.
Numerous compounds which could potentially combat
these classes of pathogen have been isolated from the
marine environment. These include structurally novel
compounds such as marinopyrrole A and abyssomicin
C with activity against MRSA, the alkaloid cyclostel-
lettamine F with activity against P. aeruginosa, and
trichoderins, novel aminolipopeptides with anti-myco-
bacterial activity. Compared with infections caused by
drug resistant bacteria, infections caused by resistant
fungal pathogens occur relatively infrequently. However,
Candida species are a common cause of hospital-
acquired bloodstream infection and kill 40% of those
patients, whereas disseminated Aspergillus infections
can kill up to 80% of affected patients. Compounds of
marine origin with activity against these fungal patho-
gens include the cyclic depsipeptide kahalalide F and
the alkaloid araguspongin C.
2.3.2.2 Antiviral
Viral diseases, such as HIV and influenza A subtype
H1N1, are a major threat to human health. Since viruses
can rapidly evolve and develop resistance to currently
used antiviral agents, discovering new antiviral drugs
is of paramount importance. Many classes of anti-
viral compounds have been isolated from the marine
environment, including nucleosides, terpenes, cyclic
depsipeptides, alkaloids, macrolides, and polysaccha-
rides. The first commercial antiviral drug, Ara-A, was
synthesised based on the structure of the nucleosides
spongothymidine and spongouridine which were iso-
lated from marine sponges. Although Ara-A and other
synthetic nucleosides have been used to treat herpes
simplex virus (HSV) and HIV, few marine antiviral com-
pounds have entered preclinical trials. Examples of such

compounds include the anti-HIV avarol, isolated from
the marine sponge Disidea avara, and Cyanovirin-N, iso-
lated from the cyanobacterium Nostoc ellipsosporum.
2.3.2.3 Anticancer
Molecules with anticancer properties comprise the
majority of bioactive molecules derived from marine
sources. However, relatively few compounds enter pre-
clinical and clinical trials and only a small group stems
from microbes. Although chemically synthesised, ara-
C (cytarabine) was designed based on the nucleosides
from spongothymidine and spongouridine, originally
isolated from the marine sponge, Tethya crypta, and is
in clinical use for more than 40 years. Today, Yondelis®,
a potent anticancer drug developed by Pharmamar from
compounds produced by the tunicate Ecteinascidia tur-
binata, is probably the most successful example of how
marine natural products can lead to anticancer treat-
ments.
2.3.2.4 Antifouling and antibiofilm properties
Biofouling, the undesirable accumulation of micro-
organisms, plants, algae, and/or animals on wetted
structures, is of great concern in a wide range of ap-
plications, ranging from food packaging/storage, water
purification systems, marine and industrial equipment,
to medical devices. The two main strategies that are
used to combat biofouling are to either prevent initial
attachment or to degrade fouling biofilms. Several coat-
ings are designed to prevent this initial attachment.
However, some applications of the antifouling coating
require certain requirements/restrictions. For example,
many countries have now imposed a ban on the most
effective antifouling coating (organotins) available for
marine applications, urging instead the use of non-toxic
novel biofouling compounds. In the health care sector,
antifouling coatings for medical applications require
compounds that are bactericidal and non-toxic to the
human body. Marine organisms, in particular seaweeds
and marine invertebrates, have proven to be a successful
source of antifouling compounds. However, these ma-
rine compounds are difficult to obtain in large quantities.
To overcome this problem, marine microorganisms are
being explored to identify novel biofouling molecules
which can be produced in larger quantities. Although
the mechanism is unknown, several fatty acids (e.g. 1-hy-
droxymyristic acid, 9-Z-oleic acid and 12-methylmyristic
acid) produced by marine microorganisms have antifoul-
ing properties. The most promising evidence came from
an experiment in which a coating consisting of 10% fatty
acids prevented attachment of micro- and macro-foul-
ing organisms on a panel which had been immersed in
the ocean for 1.5 years. Similar results were obtained
from a coating containing synthesised alkyl butenolide,
after alkylated butenolides isolated from a deep sea
Streptomyces species were found to exhibit antifouling
properties. Several other compounds show promise in-
cluding pyolipic acid, phenazine-1-carboxylic acid and
2-alkylquinol-4-ones and proteases, but their further
potential is yet to be examined.
Bacteria possess a cell-to-cell communication system
termed quorum sensing. This communication system
is dependent on the production of small molecules,
which when sensed in high concentrations lead to co-
ordinated behaviour by regulating several physiological
processes including bioluminescence, motility, antibiotic
resistance, virulence factor production and biofilm for-
mation. Biofilms (aggregates of microorganisms where
cells adhere to each other or to a surface) are of major
concern for treating bacterial infections, since bacteria
present in biofilms have increased antibiotic resistance
profiles compared to their sessile counterparts. Since
quorum sensing and biofilm formation are closely linked,
it is not surprising that quorum sensing inhibitors often
also exhibit antifouling/antibiofilm properties.
Figure 2.16. SEM picture of a marine biofilm growth on top of a
copper-based antifouling paint. The two biofilms shown in the top
pictures contain mainly Amphora sp. diatom cells settled onto
a TBT-SPC paint applied on a ship hull. A thick bacterial biofilm
grown onto a vinyl paint containing Cu2O and TPTF within 4 weeks
of exposure to sea water (14,000×, bottom). Pictures courtesy by
Dr. Maureen Callow (top left), Hempel A/S (top right) and Dr. Mike
Dempsey (bottom).

Recently, extracts from several microbial isolates from
a marine habitat were shown to contain antiquorum
sensing and antibiofilm activity against Pseudomonas
aeruginosa, the primary cause of morbidity and mor-
tality among cystic fibrosis patients. As such, quorum
sensing and biofilm inhibitors could provide novel treat-
ment options for treating bacterial infections, and these
are urgently needed due to the emergence of multidrug
resistant microorganisms.
2.3.2.5 Enzymes
Due to their immense genetic and biochemical diversity,
marine microorganisms are of interest as a promising
new source of enzymes with unique properties, includ-
ing salt tolerance, hyperthermostability, barophilicity,
and cold adaptation. Proteases represent an important
class of industrial enzymes, with applications in the de-
tergent, leather and pharmaceutical industries and many
have been isolated from marine microorganisms. Other
enzymes that have been isolated from marine microor-
ganisms include lipases (involved in the breakdown of
fats and oil), and a range of polysaccharide-degrading
enzymes, such as chitinases, alginate lyases, agarases,
carragenases, amilases, cellulases and lignocellulases.
These latter enzymes are of particular interest in the
production of bioethanol, since the development of
alternative energy sources is an urgent global priority.
Bioethanol derived from crops such as corn are the most
developed form of biofuel, but there are also initiatives
that aim at the direct production of ethanol by (marine)
Cyanobacteria. Metabolically enhanced Cyanobacteria
convert the fixed CO2 directly into ethanol. Concerns
with regard to world food shortages may restrict the
long-term viability of plant-biomass derived biofuels.
However, biofuel derived from marine microalgae is a
promising and clean alternative. Enzymes specific to the
breakdown of the complex polysaccharides present in
biomass may be isolated from the marine environment.
Marine enzymes also have potential applications in the
bioremediation of polluted waters.
2.3.2.6 Future prospects: potential
of metagenomic approaches
While culture-dependent techniques have been highly
successful in the identification of novel bioactive com-
pounds from marine microorganisms, the advent of
deep sequencing technologies such as pyrosequenc-
ing has revealed a huge diversity of microorganisms in
various niches in the marine environment, the majority
of which cannot be accessed by traditional culturing
techniques. Metagenomics involves the cloning of total
community DNA, including DNA from uncultured mi-
croorganisms, and subsequent sequence-based or
function-based analyses. Sequence-based metagen-
omic approaches rely on the comparison of cloned DNA
to known sequences from databases, while function-
based metagenomic approaches involve screening
library clones for a phenotype, such as antibacterial or
anti-quorum sensing activity. These metagenomic ap-
proaches allow access to natural products produced by
microorganisms which cannot be cultured, effectively in-
creasing the mining potential of the marine environment.
2.3.3 Bioremediation
Organic contaminants can undergo biodegradation as
a result of the activity of microorganisms resulting in
less toxic, less mobile and/or less bioavailable products.
This is the basis of bioremediation: the use of biological
agents, namely microorganisms, to remediate organic
contaminants in the environment. Bioremediation is con-
sidered an effective, low cost, preferred clean-up option
for moderately contaminated areas. Nevertheless, fur-
ther research is needed to find suitable combinations
of microorganisms and environmental characteris-
tics to improve bioremediation processes. In fact, for
a remediation process to be effective, the overall rate
of degradation needs to be accelerated above current
microbial processes. Accelerating the biodegradation
of organic contaminants is thus a major challenge to im-
prove the performance and acceptance of cost-saving
bioremediation techniques.
A systematic approach to select suitable microbial spe-
cies for a particular remediation application is crucial
to maximize the dissipation of the contaminant in the
environment. Hence, the successful application of biore-
mediation techniques is dependent on the identification
and isolation of appropriate microbial strains and their
subsequent survival and activity, once released into
the target habitat. Strain selection is founded on the
principle that certain microorganisms are better suited
for particular (catabolic) tasks and environments than
others. Increasing our knowledge of the relevant mi-
croorganisms and genes will thus be critical to improve
biotechnological applications.
2.3.4 Building with nature
Engineers recently begun to appreciate the potential
of microorganisms for engineering purposes. Erosion
and stabilization of coastal sediments such as intertidal
mudflats and sandy beaches may depend, to a consid-
erable extent, on the microorganisms that live in these
sediments. For example cyanobacteria form microbial
mats in the intertidal areas of sandy beaches. These
microbial mats are rigid structures that trap and bind
sediment particles and render stability to the sediment,
increasing its erosion threshold. Also, as a result of the
trapping and binding of sediment, microbial mats tend

to increase the profile of the tidal sediment and give
rise to the formation of a salt marsh and eventually new
land. Mudflats are characterized by biofilms of benthic
diatoms. These photosynthetic microalgae exude co-
pious amounts of extracellular polymeric substances
(EPS) which act as a glue for the mud particles, like-
wise increasing the erosion threshold. Thus, microbial
communities can have considerable effects on coastal
morphodynamics and enrich and improve the sediment.
This knowledge can be used for coastal protection as
well as for soil improvement in terrestrial and agricultural
environments. Another interesting application of micro-
organisms is found in so-called self-healing concrete.
This concrete is mixed with microorganisms and with
a substrate that contains calcium. When the concrete
shows cracks, water will enter. Normally this will cause
further deterioration of the concrete and oxidation of
the reinforcing bars. However, in this case the micro-
organisms become active, degrade their substrate and
precipitate calcite which fills up the cracks. It is obvious
that the potential of microorganisms as micro-engineers
is far from being fully explored and awaits our imagina-
tion and discovery.
2.3.5 Marine food from fisheries
and aquaculture
The oceans and seas are an important source of food for
a growing global population. Projections from the FAO
show an increase in the demand for seafood products
worldwide and in Europe towards 2030. The average per
capita consumption of 28 European countries included
in an FAO foresight analysis indicates an evolution from
22 kg/person/year in 1998 to 24 kg/person/year in 2030.
There is general agreement, however, that world fisher-
ies have reached their maximum sustainable yield and
many commercial stocks might collapse by 2030 if cur-
rent trends continue. To supply the growing demand
for healthy food from the sea, aquaculture production
has increased significantly over the last decades and
will need to become even more important in providing
sustainable sources of marine food in the future. Marine
microorganisms dominate the marine food web and are
the basis of the production of the food for all life in the
ocean. Not surprisingly, marine microorganisms are of
great importance to the fisheries and aquaculture sec-
tors and a better knowledge of their role will be critical
to secure the necessary food supply from marine living
resources in the coming decades.
Fisheries depend on the adequate functioning of mi-
crobial communities. Yet, despite their central role in
marine food webs, we still know very little about marine
microbial community functioning and how alterations,
e.g. as a result of pollution or climate change, might
affect commercial fish stocks. This needs to be recog-
nized and addressed.
To meet the challenge of supplying growing seafood
markets, aquaculture will need to become more efficient
and cost-effective, whilst simultaneously decreasing its
environmental impact. Microbiological research has al-
ready contributed to improving aquaculture production
systems. For example, microbial bioremediation, par-
ticularly in land-based mariculture, and microbial control
of intensive production systems have improved contain-
ment and environmental compatibility. Another critical
issue for the commercial aquaculture sector which has
been a key area for applied research relates to diseases
in cultured fish and shellfish stocks. Usually, bacterial
pathogens are treated with antibacterial agents, which
can eventually lead to antibacterial resistance with a
subsequent and direct impact on human health. A bet-
ter understanding of host-microbe interactions within
confined marine ecosystems may provide us with com-
pletely new tools to understand disease development
in general and how to fight new diseases in a more
ecological way. Disease management through the pre-
ventive use of probiotics and immunostimulants such
as yeasts is another approach which shows interesting
results. Research in this area has mainly concentrated
on pathogen exclusion, competition or microbiotama-
nipulation (see also Marine Board Position Paper 15 on
Marine Biotechnology). Microbial control could result in
an improved disease management, as well as having
positive effects on the environment.
New approaches and techniques such as Integrated
Multi-Trophic Aquaculture (IMTA) and Recirculating
Aquaculture Systems (RAS) are also becoming increas-
ingly important in the search for more environmentally
friendly and efficient production systems. In RAS, for
example, seafood production is combined with water
purification to maintain a healthy culture environment, of-
ten in a closed system. RAS are advanced and complex
aquaculture systems that rely on biological processes
which are primarily microbial and can, therefore, benefit
from advances in marine microbial ecology. In addition,
RAS open the possibility to integrate microalgal systems
into recirculating aquaculture systems and further down-
stream in the management of fish processing outflows.
Unfortunately, the ecology of microbial communities in
RAS and its interaction with the microbiota in the food
and gut of cultured organisms is still poorly understood.
In addition, the microbiota present during larval develop-
ment is highly variable and is thought to influence larval
viability and health.
Adequately functioning microbial communities are also
required to ensure that the produced sea-food is healthy
and to prevent the proliferation of unwanted and patho-
genic microorganisms. For example, about forty known

microalgal species produce potent toxins which, in the
case of explosions of growth (HABs), may harm marine
life and cause losses to aquaculture operations or a ban
on sales of the products for human health considera-
tions.
2.3.6 Human health
Several pathogenic organisms are ubiquitous to the
marine environment. Vibrio cholerae, a brackish water
microbe causes cholera, a disease that afflicts hun-
dreds of thousands of people worldwide. About 200
serogroups of V. cholerae are recognized, but only se-
rogroup O1 and the newly emerged O139 are associated
with cholera pandemics. V. cholerae has been well-
recognized for over 30 years to be endemic to aquatic
environments, but the proliferation of virulent V. cholerae
strains and the factors that affect disease dynamics in
aquatic systems are still incompletely understood.
Places where cholera has been absent for decades
have recently been plagued by cholera epidemics, par-
ticularly in West Africa. Riverine, estuarine and coastal
waters are reservoirs for V. cholerae, harbouring viru-
lence genes which may at any time revert to a readily
transmissible infectious state under certain nutrient, pH,
salinity and temperature conditions. The emergence of
V. cholerae outbreaks in some cases has been cor-
related to increasing surface water temperature and
global warming. Heating of oceanic surface waters,
especially those near tropical or subtropical coasts, in-
creases the abundance of copepods, a host for cholera
bacteria. Also, in temperate estuaries, the abundance
of V. cholerae in the environment is seasonal, and the
association with zooplankton may provide some protec-
tion from the relatively harsh environmental conditions
prevailing in aquatic habitats. In the temperate Baltic
Sea, Vibriovulnificus, which can cause wound infections,
gastroenteritis, or a syndrome known as primary septi-
caemia, was found to be responsible for some fatalities
in immune-compromised persons.
2.3.7 Energy
One of the major societal challenges we face is the long-
term supply of sustainable energy. Marine living sources
can contribute importantly to secure the growing de-
mand for energy in Europe and beyond. The two most
promising approaches to harvest energy from marine
living organisms are based on marine microbial life.
The first approach, Microbial Enhanced Oil Recovery
(MEOR), aims to utilize marine microbes to improve the
recovery rates of classical fossil oil reserves. This is
done by either decreasing the viscosity of oil and/or the
permeability of the rock material in which the oil resides.
The second one focuses on the production of biofuels
from microalgae. Both of these applications require an
in-depth knowledge of marine microbial diversity and
functionality in order to select useful strains and/or to
identify by-products that are of use in these produc-
tion systems. At the time of writing, the application of
MEOR and the production of biofuels from microalgae
is either not yet applied, or not yet commercially viable
and considerable research will be needed in the future
to develop operational systems at a commercial scale
which are controllable and environmentally sound. More
details about the state-of-the-art and research challeng-
es associated with these promising technologies can be
found in the 2010 Marine Board-ESF Position Paper 15
on Marine Biotechnology.
2.4 Concluding remarks
Ninety per cent of the biomass in the ocean is microbial
and represents an enormous living diversity. Relatively
few types of microorganisms are abundant and control
the major biogeochemical cycles. The vast majority are
rare but represent an almost infinite genetic pool from
which the microbial community can draw in order to
be able to respond to environmental changes, thereby
contributing to the stability of the ecosystem. The ocean
also contains a huge number of viruses, exceeding that
of Bacteria by an order of magnitude. Viruses may con-
trol microbial biomass and keep in check the successful
microorganisms by ‘killing the winner’. Viruses also gen-
erate and maintain biodiversity since they are the main
mediators of genetic exchange between organisms. The
balance of all these biogeochemical cycles and their
compounds controls the dynamics of all ocean biomes.
Thus, understanding the ecology of marine microbial
Figure 2.17. Recirculated fish tank with biofilter (drum on right),
which uses beneficial microorganisms to remove chemical wastes
from the water. (Courtesy Yonathan Zohar, COMB)

communities is essential for our understanding of the
oceanic system, for the sustainable use of the ocean
and to be able to anticipate changes in ecosystem
functioning. A major problem that we face in studying
changes in the ocean is that we are lacking a baseline of
microbial ecosystems that we can refer to for assessing
and quantifying the changes that we observe. In order
to accomplish this it is not only necessary to make an
inventory of which microorganisms currently live in the
ocean, but also to reconstruct what has lived in the past
ocean. Only then might we be able to predict what will
live in the future ocean.
Fishing is a major human activity in the ocean as it sup-
ports a major part of the protein supply for humans.
The production of fish depends mostly on primary pro-
duction by phototrophic microorganisms, which, in turn,
depends on the availability of nutrients. When nutrients
are in excess it may lead to the development of HABs.
Some of the species that produce HABs are toxic and
may cause serious health problems. Even if these algae
are not toxic they may reduce water quality and cause
anoxia. Organic matter produced by phototrophic mi-
croorganisms and its transmission through the food web
results in a huge amount of dissolved organic carbon
(DOC) which is decomposed through the microbial food
web, recycling nutrients which give rise to new biomass.
The viral loop speeds up the recycling of nutrients and
carbon. The microbial food web is responsible for the
biological pump, namely the transport of CO2 as organic
carbon to the abyssal ocean and ocean floor. It is un-
clear how much CO2 can be stored and what processes
might return it to the atmosphere.
Nitrogen is a major driver of primary production but it
is in short supply in vast areas of the ocean. Half of the
world fixation of N2 is anthropogenic (by the industrial
Haber-Bosch process) and a large part is discharged in
the marine environment through wet and dry deposition,
river discharge, and run-off. Half of the natural global
N2 fixation takes place in the ocean representing an im-
portant driver of primary production and CO2 fixation.
N2 fixation counteracts the losses of combined nitrogen
through denitrification and anaerobic ammonium oxida-
tion (anammox) although the contribution of the latter is
uncertain.
Current knowledge of community processes at the
genomic and biochemical level, taking into account the
vast range of microbial interactions, is insufficient for
the mechanistic and predictive understanding of the
global CO2, N2O, DMS and CH4 fluxes. These fluxes
have an impact on the atmospheric composition and
influence climate. Thus, while there is knowledge about
the processes that mediate the emission of such climate
change gases by microbes, it is unclear how these mi-
crobes influence climate. One of the reasons is the lack
of understanding of the controls on the fluxes and con-
centrations of these gases in the surface ocean.
Marine microorganisms have an untold potential for pro-
viding services and products for human society which is
not exploited to any significant extent. There is a world
of microorganisms to discover, understand and put to
good use.

1. Develop a census of marine microorganisms cur-
rently living in the ocean; develop a reconstruction
of what has lived in the past ocean; and develop
models which can predict what might live in the
future ocean.
2. Produce a comprehensive inventory of all microbi-
al metabolic pathways in the ocean including their
genetic basis and analyze them in the context of
marine microbial systems biology to better under-
stand ecosystem processes and functioning.
3. Research is required to elucidate the environmen-
tal factors which control and regulate microbial
metabolism and the ecological interactions (com-
petition, cooperation, symbiosis) between different
microorganisms, and how they influence ecosys-
tem functioning.
4. Increase the efforts to understand the fluxes,
sources and sinks of key components of the
oceanic biogeochemical cycles through process-
based measurements using innovative and novel
technologies.
5. Increase the efforts to elucidate the CO2 storage
capacity of the ocean and initiate research to bet-
ter understand the functioning of the biological
pump and the processes that potentially return the
stored CO2 back to the atmosphere.
6. Increase the efforts to improve the understand-
ing of the processes that control the efflux of the
greenhouse gases CO2, N2O, DMS and CH4 in the
surface ocean.
7. Increase the efforts to explore and exploit marine
microorganisms for biotechnological applications
and products in line with the recommendations of
Marine Board Position Paper 15 Marine Biotech-
nology : A New Vision and Strategy for Europe *
8. Analyze oceanic metagenomic databases to iden-
tify putative new metabolic pathways and facilitate
their discovery. In particular, target the under-
standing of signalling networks.
9. Drastically increase our knowledge of the biologi-
cal, physical and chemical aspects of the deep
(abyssal) ocean and ocean floor with relevance for
the occurrence, diversity and functions of microor-
ganisms.
10. Develop regulations and programmes for moni-
toring microbial changes as a result of human
activities such as deep-sea mining.
Summary Box 2.1. Key research priorities and recommendations
* Marine Board Position Paper 15 Marine Biotechnology:
A New Vision and Strategy for Europe is available for download from
www.marineboard.eu/publications/

3.1 Introduction
Microbial ecology is currently experiencing a renais-
sance spurred by the rapid development of molecular
techniques and the so called omics technologies in par-
ticular. These tools have allowed researchers to produce
a vast amount of information through in situ measure-
ments and analysis of natural microbial communities.
Such approaches are vital to unravel the interactions of
microbes with their environment and with one another.
Today, genomics of DNA and RNA (transcriptomics),
of proteins (proteomics) and of metabolites (metabo-
lomics), used alone or in combination with each other
and/or with more classical methods, are fields that are
rapidly transforming many areas of biological research.
In the future, however, it will be crucial to combine
results obtained by application of these techniques
together with information about the physical, chemical
and other biological parameters in order to form a ba-
sis for addressing the societal questions raised in this
position paper (see Chapter 1). Such multidisciplinary
approaches are necessary to understand and potentially
predict the complex role of marine microbial ecology in
global change, sustainable supply of healthy food and in
preserving human and environmental health. Likewise,
information from these combined techniques integrat-
ed with the measurements of various abiotic and biotic
factors, will give a better understanding of the complex
role and interplay of marine microbial communities.
Furthermore, such an integrative approach will almost
certainly reveal a wealth of new metabolic processes
and functions which could potentially support innova-
tion in biotechnology and, therefore, for the bioeconomy
as a whole.
Nevertheless, using cultivated microorganisms is still
the only way to get detailed information about micro-
bial characteristics and processes, thus highlighting
the need to further focus on culturing microorgan-
isms and developing better culturing techniques. This
chapter provides an overview of the most important
technologies and research toolkits available to marine
microbiologists today, highlighting the critical barriers
to progress.
3.2 Classical microbiology
3.2.1 Enrichment, isolation and cultivation
Traditionally, microbiology has relied mostly on the
enrichment of microorganisms from their natural en-
vironment, followed by their isolation into pure culture
allowing their growth and physiology to be studied.
Enrichment media and conditions were selected based
on the supposed physiology of the organism subject to
study. But it was soon realised that this way of selection
would probably enrich a microorganism with the desired
basic physiological properties but not necessarily the
organism that would prevail under natural conditions.
For example, high starting concentrations of growth
factors would select for an organism that grows at the
highest maximum specific growth rate while in nature
organisms often are competing for low substrate con-
centrations with low specific growth rates. In order to
enrich microorganisms at low substrate concentrations
and low specific growth rates, chemostats were applied
using sterile growth media with a chosen limiting sub-
strate.
To obtain pure (axenic) cultures, i.e. a clonal culture, an
enriched microorganism is isolated and purified away
from contaminating microorganisms. This is often done
by plating the culture on a Petri dish in such a way that
single cells are spatially separated, with the growth me-
dium solidified with an agent, or in a tube when isolating
anaerobic organisms. After incubation for a certain pe-
riod under appropriate conditions, the single cells will
have grown out to visible colonies (Figure 3.1).
Unfortunately, the approaches described above rarely
result in the isolation of the dominant and presumably
3. Technologies and research toolkits
Figure 3.1. Colonies of the bioluminescent marine bacterium Vibrio
fischeri. The photograph of colonies growing on agar (left) was
taken with an artificial light source. The photograph of colonies
on the right was taken using their own bioluminescence as a light
source. (© J.W. Hastings, Harvard University, through E. G. Ruby,
University of Hawaii; provided courtesy of the National Science
Foundation)

ecologically most important microorganism from the
environment. In addition, these approaches are time-
consuming, in particular the chemostat approach, and
other approaches have therefore been proposed. For
instance, the direct plating of a diluted seawater sample
on a Petri dish with a suitable growth medium would
yield colonies of varying microorganisms that can be
identified and further purified. However, this only yields
those microorganisms that are capable of growing on
the chosen medium and under the given incubation
conditions. Another approach to just obtain dominant
microorganisms is the dilution to extinction technique.
A sample is diluted with sterile seawater from the same
site as the sample from which the microorganisms are
to be isolated until 1-10 cells are left.
Larger microorganisms (e.g. filamentous or large uni-
cellular Cyanobacteria) can be directly isolated by
micromanipulation. Using a finely drawn glass capillary
and an appropriate microscope (e.g. a dissecting mi-
croscope), individual trichomes, cells or aggregates can
be picked out from a community and transferred to a
growth medium. A variety of other isolation techniques
have been developed for specific microorganisms or
samples. For example, the laser forceps technique in
combination with microscopy in which a single cell is
isolated from the rest. High throughput flow cytomet-
ric single-cell sorting (Figure 3.9) is another approach,
applied to particles as small as viruses up to large phy-
toplankton cells such as Cyanobacteria or unicellular
algae.
3.3 Novel culture-independent
techniques
Despite having a large variety of different microbiological
techniques at our disposal and the number of isolated
and described microorganisms steadily increasing
(31,000 Bacteria and Archaea), this number remains ex-
tremely low when compared to the estimated number
of microorganism types, which stands at over 1 million.
It is often stated, therefore, that we know less than 1%
of the existing types of Bacteria and Archaea. However,
that is not to say that more than 99% of the microorgan-
isms which remain undescribed and uncultivated are
by definition not culturable. In fact, it is more likely that
we simply do not know how to isolate and grow these
organisms for the following reasons. First of all, for many
of the uncultivated microorganisms, there has been no
serious attempt made to isolate them. Secondly, tradi-
tional techniques and procedures as described above
are not enough for purifying and describing the remain-
ing 99% in the coming decades, even if we knew how
to isolate all types of microorganisms. This is because
they are too time-consuming and unsuitable for high
throughput. Thirdly, it is possible that many, or even the
majority, of the microorganisms are unable to grow as
pure cultures in the laboratory. There are several exam-
ples of organisms that grow only in consortia with other
microorganisms, even demonstrating typical multicel-
lular behaviour and cell-to-cell communication.
Culturing new marine microorganisms will, however,
always be of importance as it remains the only way to
get detailed information about processes and func-
tions from studies in controlled laboratory experiments.
Culture-based techniques will also be the best approach
for linking genes with function. However, cultivation of
the vast majority of the uncultivated marine microorgan-
isms introduces a challenge not only because most of
the marine prokaryotes have not yet been cultivated but
also because cultivation techniques often yield bacteria
that are very rare in whole-community cloning studies.
Culture-independent methods make it possible to study
a whole microbial community and get a better under-
standing of the genetic diversity, population structure,
and ecological roles of the majority of microorganisms
in the marine environment. However, most of these
culture–independent (molecular) studies tend to find pri-
marily the most common organisms, like metagenomic
sequencing that only (partly) enables reconstructions
of the genomes of the most abundant members in the
community. Thus, cultured bacterial and archaeal iso-
lates that have yielded complete genome sequences
will be important for evaluating metagenomic data.
Recently, progress in the cultivation of microorganisms
has accelerated, and developments in technology have
Figure 3.2. Marine microbiologist working in the laboratory
(courtesy Frank Oliver Glöckner and Anna Klindworth)

been a major factor in these advances (see above). A
combination of methods now enables microbiologists to
screen large numbers of cultures and to manipulate cells
growing at low biomass densities that are characteristic
of those found in the ocean.
3.3.1 Omics technologies
Marine microbial ecology is currently undergoing a para-
digm shift, driven by the development and application of
the so-called ‘omics’ technologies. Omics is a term used
to denote approaches to analyze information embed-
ded in available molecular subsets, hence, genomics,
proteomics and metabolomics. Results from such stud-
ies have provided new and exciting insights in marine
microbial ecology by adding new information about the
complexity of the structure, interactions and function-
ing of marine microorganisms in the marine ecosystem.
While genomics can provide information regarding the
genetic potential of microbes, proteomics character-
izes the primary end-stage product, proteins, thereby
conveying functional information concerning microbial
activity.
Metabolomics is the “systematic study of the unique
chemical fingerprints that specific cellular processes
leave behind”. Specifically this means the study of their
small-molecule metabolite profiles (Figure 3.3). The me-
tabolome represents the collection of all metabolites
in a biological organism or sample, which are the end
products of cellular processes. Thus, while mRNA gene
expression data and proteomic analyses do not tell the
whole story of what might be happening in a cell, meta-
bolic profiling can give an instantaneous snapshot of the
physiology of a cell or given sample.
3.3.1.1 Genomics/metagenomics
Metagenomics is the culture-independent study of a
community of microorganisms. Early environmental
gene sequencing cloned specific genes (often the 16S
or 18S rRNA gene) to produce a profile of diversity in a
natural sample. Recent studies use “shotgun” Sanger
sequencing or massively parallel pyrosequencing to get
samples of all the genes from all members of the sam-
pled communities. Advances in these technologies now
permits two types of massive sequencing: (i) parallel py-
rosequencing of amplicons from different samples, also
called tag-sequencing, where the target gene often is
part of the 16S rRNA gene or another core gene of func-
tional or ecological importance; and (ii) high-throughput
random sequencing of short gene products of total mi-
crobial community DNA or RNA.
The recent advances in metagenomics have revealed
an enormous diversity of previously unknown, uncul-
tured microorganisms that predominate in the ocean.
However, these new techniques have also unravelled
some new challenges that have to be focused on future
research. One of the major challenges lies in the fact
that most of the sequences so far identified show no
similarity to previously described sequences, indicat-
ing an urgent need to develop novel research tools to
characterise and describe the functions and ecology of
the hereto unknown marine microorganisms.
3.3.1.2 Transcriptomics/metatranscriptomics
While the disciplines of genomics and metagenomics
allow the study of the genomic potential of a particu-
lar organism or a microbial community, respectively,
transcriptomics and metatranscriptomics deal with the
subset of genes that are transcribed under certain en-
vironmental conditions. Accordingly, transcriptomics
and metatranscriptomics are powerful tools to capture
snapshots of the genes essential for the survival of mi-
croorganisms under specific environmental conditions.
Recent studies have shown the potential of such meth-
ods to provide detailed information on metabolic and
biogeochemical responses of the microbial community
to environmental change.
One of the unexpected results of the first metatranscrip-
tomics studies was the detection of the high abundance
and diversity of small RNAs (sRNA) in the marine mi-
crobial community. Microbial sRNAs are untranslated
short transcripts that generally reside within intergenic
regions on microbial genomes. Oceanic microbial sR-
NAs are thought to be involved in gene regulation and
signalling processes related to environmental response.
However, both the function and diversity of these genes
are still unexplored.
Environmental transcriptomics protocols are technically
difficult and a lot of effort has been given to develop
Figure 3.3. Schematic representation of community-based
“omics” approaches. (from Zoetendal et al., 2008)

new approaches that would produce reliable and un-
biased data-sets. Further emphasis should be given to
the development of new metatranscriptomic methods
as this approach represents a powerful method for the
study of metabolic processes and ecological activities
within the microbial food-web. Such data, in conjunction
with controlled field and laboratory experiments, will al-
low linkage of environmental variation with changes in
the RNA pools and have the potential to provide new
insights into environmental sensing and response (sig-
nalling) in natural microbial communities.
3.3.1.3 Proteomics/metaproteomics
Metaproteomics, in combination with metagenomics
and bioinformatic binning methods, has the potential
to provide a high-resolution representation of the ac-
tive genotypic traits of distinct community members.
Advances in mass spectrometry instrumentation and
methodologies, along with bioinformatic approaches,
has brought this analytic chemistry technique to rel-
evance in the biological realm, thanks to its powerful
applications in proteomics. Mass spectrometry-enabled
proteomics, including “bottom-up” and “top-down”
approaches, is capable of supplying a wealth of bi-
ologically-relevant information from simple protein
cataloguing of the proteome of a microbial community
to the identification of post-translational modifications
of individual proteins. The techniques represent an
emerging field of study which have been limited to a
few studies so far, yet the potential of these techniques
for the advancement of marine microbiology has already
been clearly demonstrated.
All community proteomic methods face challenges
due to the large complexity of protein species and the
large dynamic range of protein levels. Current technolo-
gies can identify proteins of populations that comprise
at least 1% of the community and for which closely
related genomic sequences are available. Recent tech-
nological developments have improved the methods
for extraction, separation, and identification of proteins
from natural microbial communities, and have made
this method both more sensitive and more accurate.
However, these methods have so far been most suitable
when applied to microbial communities with limited di-
versity and/or those dominated by particular organisms.
Metaproteomics has significant potential for solving one
of the major challenges facing microbial ecologists in
providing a high-resolution representation of community
structure and function. The data generated will be im-
portant to provide crucial and fundamental information.
Once integrated and analysed alongside metagenomic
Figure 3.4. Improvements in the rate of DNA sequencing over the past 30 years and into the future.
(From MR Stratton et al., 2009, Nature 458, 719-724)

data, metaproteomic datasets will provide information
on the activity of genes and metabolic pathways that is
currently not available.
3.3.1.4 Metabolomics
Metabolomics is an approach used to define the small-
molecule diversity in the cell and to display differences in
small molecule abundance. Metabolomics shows many
advantages in terms of metabolic analyses because
metabolites are the functional entities within the cells
and their concentration levels vary as a consequence
of genetic or physiological changes. The technique is
one of the newest omics techniques and is, therefore,
less developed and used in marine research. To date,
most of the environmental metabolomics studies have
been performed on higher eukaryotic organisms to
measure biological, physical and chemical stressors
and no studies have been reported on marine Bacteria
or Archaea. In microbial ecology, the potential to employ
the method will be limited by the difficulties in discrimi-
nating between metabolites of the three domains of life
or even between the species within one of the domains.
However, a promising approach is based on isotope-
labelled intermediate metabolites combined with various
types of dynamic metabolic flux modelling and may be a
useful tool in investigations of complex and large-scale
metabolic systems. Nevertheless, improvements are still
needed in both instruments and data-analysing software
to improve identification coverage and accuracy in or-
der to make metabolomics compatible with other omics
technologies.
3.3.2 Coupling identity with function
To associate the identity of a microbe with its function
in the ecosystem there are several strategies. One of
them is the so-called single-cell approach, by which we
can use a series of probes to “interrogate” each cell at
a time. One such probe is the “identity probe”. Either
before observation (by microscopy) or after some sort of
physical separation (e.g. flow cytometry cell sorting), the
probes are applied to the sample to collect information
on its identity. Such probes include, for example, the
FISH probes to the required phylogenetic depth level,
but single-cell genomics screening with 16S rRNA PCR
is also an option.
“Functional” probes can be of different types: (i) tar-
geting the structure of the cell; and (ii) targeting the
physiology of the cell (such as the redox probe CTC
(5-cyano-2,3-ditolyl tetrazolium chloride) which indicates
which cells are expressing an active electron chain, the
probe ELF97 which indicates whether a cell is express-
ing phosphatase activity, or the probe PI (propidium
iodide) which reports on the state of the membrane); or
(iii) activity probes (such as radioactivity-labelled organic
matter or nutrients that, combined to micro-autoradiog-
raphy will indicate which cells are taking up the provided
substrate (See also the section on Nano-SIMS below)).
In any case, the power of the analyses is made larger
by combining different types of probes: identity plus
functional probes. For example, techniques such as (i)
stable-isotope probing, (ii) fluorescence in situ hybridi-
Figure 3.5. Shotgun
metaproteomics approach
used to identify microbial
proteins environmental
samples (Modified from
Verberkmoes et al., 2009)
Modified from Verberkmoes et al, ISME-09
Environmental
Sample
Differential
centrifugation
to extract
Prokaryotic cells
Environmental
Sample
Differential
centrifugation
to extract
Prokaryotic cells
Environmental
Sample
Differential
centrifugation
to extract
Prokaryotic cells

zation (FISH) micro-autoradiography (MAR-FISH), (iii)
isotope array, or (iv) FISH-secondary-ion mass spec-
trometry (Nano-SIMS) will allow substrate incorporation
and specific processes to be linked to microbial phylo-
types. These single-cell techniques have been used to
describe the environmental distribution and the func-
tional role of microorganisms in soil, water, sediments
and other ecosystems. The field has come a long way in
the past decade to answer two basic ecological ques-
tions: “Who is there and how many? and What are they
doing?”
3.3.2.1 Stable-isotope probing (SIP)
The goal of SIP lies in linking the identity of microbial
communities with their function in the environment. The
SIP technique involves introducing a stable isotope-
labelled substrate into a microbial community and
following the fate of the substrate by extracting molecu-
lar species such as fatty acids and nucleic acids from
the community, determining which specific molecules
have incorporated the isotope. Lipids and nucleic acids
are used as the biomarker molecules. In SIP of lipids
(PLFA-SIP) the approach is to expose a community of
interest to an isotopically labelled substrate (usually
13
C or 15
N), then isolate lipids and trace the labels to
specific molecules which can be characterized by gas
chromatography. In nucleic acids-SIP (DNA- RNA-
SIP) the community is exposed to a labelled substrate
(again usually 13
C). After labelling, either DNA or RNA
is isolated and subjected to buoyant density gradient
centrifugation. The nucleic acids of organisms that have
assimilated the 13
C have a higher buoyant density than
nucleic acids of organisms that have not. Following
centrifugation, those nucleic acids that have taken up
the substrate are amplified by PCR and its product
may subsequently be identified by fingerprinting tech-
niques or identified by sequencing. A major advantage
of lipid-SIP is the high sensitivity, in contrast to nucleic
acids-SIP where the synthesis of the labelled nucleic
acid must be sufficient for the separation and detec-
tion of labelled molecules. A potential disadvantage of
SIP studies is cross-feeding, requiring verification of the
initial community members that consume a particular
substrate and possible subsequent consumers of meta-
bolic by-products, which has been seen as a potential
problem for both DNA and RNA-SIP that require long
incubation times.
All SIP applications are concerned with the detection
of the assimilation of specific substrates. One exam-
ple is to study the direct uptake of naturally occurring
substrates by microorganisms or experiments to detect
the flow of C into microorganisms. There are, of course,
many possibilities for using this approach. Perhaps the
most exciting studies are those which start with informa-
tion from experiments of PLFA-SIP, where a particular
group of microorganisms is involved in assimilation of a
substrate. Following these studies, RNA-SIP from that
group may be selected from the sample and subcloned,
indicating which community members were assimilating
the substrate. Future studies will likely include analysis
with mRNA (mRNA-SIP) which would help in the study
of the expression of genes from different environments.
Figure 3.6. The single-cell
approach
The
single-‐cell
approach
methods
and
research
strategies
for
the
single-‐cell
analysis

of
natural
bacterioplankton
organisms

Iden9ty
probes

•Phylogene9c
probing
(FISH
or
HISH)

•Natural
pigment
probing
(iden9fica9on)

•Single-‐cell
whole
genome
amplifica9on

Physiology
probes

•Natural
pigment
probing
(ac9vity)

•Viability
probing
(membrane
status,
etc.)

•X-‐ray
single-‐cell
microanalysis

Ac9vity
probes

•Radioac9ve
substrates
(autoradiography)

•Stable
isotopes
(SIP,
nanoSIMS)

•Func9oning
probing
(respira9on)

•DNA
probing
(flow
cytometry
and
NA
stains)

The
power
lies
in
the

combina9on
of
methods

MAR-‐FISH

Flow
cytometry
sor4ng

HISH-‐SIMMS

3.3.2.2 Fluorescence in situ hybridization
(FISH)-microautoradiography (MAR-FISH)
Microautoradiography is one of the earliest single-cell
methods used in aquatic microbial ecology. In this
technique, microbial assemblages are incubated with
a radio-labelled substrate and cells are then placed in
contact with an autoradiographic emulsion. Subsequent
exposure of the emulsion to the radioactive emissions
produces silver grain deposits around the cells that
have incorporated the radioactive substrates. Acetate,
glucose, amino acids were used in the initial studies,
but recent studies have expanded to other substrates
such as 14
CO2 or the carbon excreted by phytoplank-
ton. It is possible to combine the microautoradiography
technique with fluorescence in situ hybridization (MAR-
FISH), providing information on the in situ single-cell
activity of specific microbial groups. The application
of MAR-FISH is limited to radio-isotopes with a suit-
able half-life and that can be obtained at high specific
activities. In addition, the biologically relevant elements
nitrogen and oxygen cannot be tracked by MAR-FISH,
because of the lack of proper isotopes. However, MAR-
FISH has the advantage that the active substrate uptake
can be related to individual cells and that single-cell
rates of substrate incorporation can be calculated. This
is not the case with most methods that use stable iso-
tope tracers.
3.3.2.3 Isotope array
Environmental arrays are gene fragments that are ar-
rayed on microscopic grids which allow simultaneous
monitoring of the diversity and substrate incorporation
of complex microbial communities. DNA or RNA extract-
ed from environments is hybridized with the gene array
and the hybridization is measured as an indication of the
presence of the active microorganism in the sample For
instance, the diversity and bicarbonate incorporation of
ammonia oxidizing bacteria (AOB) have been measured
using a prototype DNA microarray for AOB detection in
activated sludge samples. One advantage of microar-
rays compared to MAR-FISH is the ability to study the
uptake of several substrates and apply many probes in
Figure 3.7. Different types of AOM (Anaerobic Oxidation of Methane) consortia visualized by fluorescence in situ hybridization (FISH).
Red colours are ANME Archaea and green colours indicate sulfate-reducing bacteria. (Courtesy Dr. Katrin Knittel, MPI-Bremen)

parallel. Furthermore, the technique is neither radioac-
tive nor toxic. However, the main limitation of the isotope
array is its sensitivity and thus its application to samples
collected from ecosystems with not very active or dense
microbial communities, such as open ocean waters. In
addition, substrate cross-feeding must be considered
as it may cause problems in differentiating between pri-
mary substrate consumers and microorganisms which
live on the lysis products of the primary consumers,
particularly with prolonged substrate incubation time.
3.3.2.4 Secondary-ion mass spectrometry
(Nano-SIMS)
Coupling the identity of microbes with their activity in
the environment remains an important gap in our abil-
ity to explore microbial ecology. The development of
techniques to quantify the metabolic activity of single
microbial cells has been especially challenging, mostly
due to their small size. A particularly promising tech-
nique is nanoscale Secondary Ion Mass Spectrometry
(Nano-SIMS), a new high-resolution imaging method,
which can help decipher what individual microbes are
“doing” in the environment. High-resolution nanometer
scale secondary-ion mass spectrometry (SIMS) has
become established within environmental microbiology
and this opens up new possibilities for the coupling of
phylogenetic identity and metabolic function of single
cells in studies of mixed microbial communities from the
environment. Nano-SIMS is a surface analysis technique
which provides information about the spatial distribution
of any element and its isotopes as well as quantitative
information about the isotopic composition of a sample.
Nano-SIMS can determine the chemical, radioisotopic
and stable-isotopic composition of biological material
down to the submicrometer level. By exposing microbial
communities to substrates labelled with isotopes, Nano-
SIMS-based imaging allows visualization of metabolic
activity in single cells. Moreover, substrate uptake rates
and fluxes can be quantified.
The combination of Nano-SIMS with fluorescence in
situ hybridization (FISH) finally provides the link be-
tween the identity of microbial cells and their activity.
FISH uses fluorescent-labelled probes that are specific
to the organism of interest and that bind to the intra-
cellular 16S ribosomal RNA. The use of HRP-labelled
oligonucleotide probes and catalyzed reported deposi-
tion of halogenated tyramides (HISH) makes the method
very useful for the in situ analysis of a wide range of
environmental samples. Replacing fluorescent probes
with halogenated probes allows individual cells to be
directly identified (by probe hybridization to targets) by
Nano-SIMS.
However, one of the disadvantages of Nano-SIMS tech-
nology which limits its widespread use is the expense of
the equipment. Alternatives such as RAMAN spectros-
copy which is much cheaper and still allowing functional
information of single cells are, therefore, noteworthy.
In RAMAN spectroscopy, a spectroscopic technique
is used to study vibrational, rotational, and other low-
frequency modes in a system. It relies on inelastic
scattering, or Raman scattering, of monochromatic light,
usually from a laser in the visible, near infrared, or near
ultraviolet range.
Figure 3.8. Enrichment of elements and their stable isotopes in a trichome of the cyanobacterium Nodularia spumigena. The left-side image
shows the enrichment of carbon relative to nitrogen, indicating areas of active carbon fixation and storage. The middle image shows the
accumulation of the stable isotope 13
C, relative to 12
C after feeding the sample with 13
C labelled bicarbonate, indicating the location of carbon
fixation. The right-side image shows the same for nitrogen after supplying the organism with 15
N labelled N2. (Picture courtesy of M. Kuypers)

3.4 Why models?
The obvious challenge to the scientific community of ma-
rine microbial ecologists is to transform the avalanche of
omics data at the molecular scale into “understanding”
at the time and space scales of marine ecosystems. In a
traditional hypothetical-deductive perspective, science
is seen as a spiralling process where observations lead
to models (insights, hypotheses) that can be used to
construct new experiments that generate new data, and
where each turn brings us a step up along the spiral
towards a better “understanding”. In this generalized
context where “models” are the conceptual frameworks
we construct to relate observations and make predic-
tions, models are an integral and obligatory part of the
research process and data collection without models
remains just data collection, just as models without any
root in data remain just models. Although the possibility
for automating the whole process, hypothesis genera-
tion included, has been discussed (King et al. 2004) the
need to extract meaningful “understanding” from huge
sets of data is likely to increase the need for models.
Looking back at the last decades of marine micro-
bial ecology, an important refinement to this picture
becomes obvious: Journeys into new and unknown ter-
ritories requires open-minded and model-independent
observations to allow an unbiased observer to be able
to see “the unexpected” and thus create a new entrance
into the hypothetical-deductive spiral. You need to know
that there are ~109
bacteria and ~1010
virus L-1
seawater
before you can start looking for mechanisms behind and
relationships between such data.
A characteristic trait of a mature field of science is a
common theoretical framework within which observa-
tions can be organized and related, new observations
predicted, and therefore experiments planned to chal-
lenge the theory. Despite its progress, one can ask
whether marine microbiology scores particularly well
on such a criterion. Perhaps is it still to a large extent
in the initial descriptive phase where it gathers increas-
ingly detailed descriptions of the unknown? There is a
huge effort to measure microbial diversity, but to what
extent is this still the initial hunt for a correct number,
more than a hypothesis-driven search for the underlying
mechanisms and relationships producing a particular
diversity? The abundance of viruses is well known, but
there is no satisfactory theory explaining the order of
magnitude of this number. Meta-analyses can gener-
ate correlation plots between bacterial production and
primary production, but to what extent can these cor-
relations be matched with mechanistic explanations for
how the system works? Is the correct description of the
‘state-of-the-art’ that we have a lot of observations and
data on what the marine microbial ecosystem looks like
in terms of numbers, fluxes and even species, strains
and genes, but still a relatively vague idea of the mecha-
nisms relating these numbers? From this perspective,
the new wave of molecular data is flooding into a field
that is immature in the sense that it does not yet have
a mature theory within which these datasets can be ar-
ranged, analysed and interpreted.
One optimistic argument is that this is exactly what is
needed: with all this new data, there will finally be “all”
the pieces of the puzzle, and the “modeling” part of
the job, putting the pieces together, will then be trivial
(or at least easier). Such optimism is not necessarily
supported by the history of science. Compared to ex-
plaining microbial ecology from molecular data, the task
of deriving heliocentric models for planetary motion from
observing the night sky may (in hindsight) seem simple,
yet required an observational period lasting through
the history of mankind up to Copernicus and Kepler.
Relevant processes in microbial ecology cover a con-
tinuous spectrum from molecular reactions via cells and
food webs to global element cycles. For many of us,
the insight that climate is related to nanoscale micro-
bial interactions is part of our fascination for this field.
However, detailed low-level descriptions are required to
understand the systems’ behaviour at higher levels. The
traditional example is classical and statistical thermody-
namics where factors such as temperature, pressure,
volume and entropy could be related to each other
without the later insight that they represent average
properties based on the behaviour of a vast number of
colliding gas molecules. Since the essence of concep-
tual modelling is to “explain as much as possible with as
little as possible”, an overwhelming amount of detailed
molecular data may even create situations where “one
cannot see the forest because of all the beautiful trees.”
In conclusion, a maturation of the field of marine micro-
bial ecology is being hampered by a lack of “models”;
both those relating entities at the community level (phy-
toplankton, protists, prokaryotes, viruses), at the cellular
and molecular levels and, in particular, “models” con-
necting such levels.
3.5 Expected future developments
in marine microbial cultivation and
-omics studies
Understanding the complexity of the structure, interac-
tion and functioning of microorganisms in the marine
ecosystem, is the key to understand and thus to pro-
tect and sustain marine ecosystems. This understanding
is important for addressing the societal and scientific
questions pointed out in this position paper. In order to

achieve progress in the quest for a better scientific basis
for action, new developments in both classical and novel
techniques are essential. No single omics analysis will
fully unravel the complexity of fundamental microbial
biology. Therefore, integration of multiple layers of infor-
mation through multi-omics approaches in combination
with environmental data layers (e.g. environmental data
streams stemming from sampling, ocean observatories
and remote sensing) and classical techniques is re-
quired. By applying such approaches one will eventually
gain a more precise picture of the function, interaction
and diversity within the microbial food-web.
As a consequence of the rapid rate of discovery in the
omics era, there will probably be a resurgence in tra-
ditional ecological and functional approaches that will
be essential in order to place our new discoveries into
their proper environmental context. One example is the
development of genomics of uncultured single cells by
combining flow cytometry and sequencing (single-cell
genomics; see below). Another example of mutually
beneficial combinations of classical and novel technol-
ogy is the combination of metagenomic sequencing with
stable isotopic analysis of geochemical signatures. The
latter method provides information both about the iden-
tity and interplay between microorganisms and chemical
processes within microbial communities.
The single cell genomics method is based on the com-
bination of cell sorting by flow cytometry and whole
genome amplification (WGA), followed by sequenc-
ing (Figure 3.9). By using the DNA from single cells it
is thus possible to generate reference genomes of un-
cultured taxa from a complex microbial community of
marine microorganisms. Thus, the method allows for
the description of the functional diversity within the nu-
merically significant uncultured fraction of the marine
microbial community, avoiding the difficulties associ-
ated with cultivation. Additionally, the information can
also be beneficial to developing and improving culture
techniques of the so far uncultured fraction of the mi-
crobial community.
One limitation which is intrinsic to metagenome datasets
is the difficulty with linking genes to the organisms car-
rying the genes. Single cell techniques might be useful in
Figure 3.9. The single cell genomics pipeline: flow cytometry and sorting combined with single cell genomics. The WGA technique is a non-
PCR based DNA amplification technique that has proven efficient in the amplification of small amounts of DNA, including DNA from single
cells, resulting in a reasonable quantity for genomic analysis. Another alternative also includes flow cytometry cell sorting, but based on the
natural fluorescence of some microorganism (Figure by Ramunas Stepanauskas)
Metagenome
Metaproteome
Biogeochemical cues
Cultures
Genome
reconstruction
Targeted loci
DNA
archive
Cell lysis
Single cell
isolation
Environment
Physiology
Ecology
Evolution
Biotechnology
Whole genome
ampliﬁcation

overcoming this limitation. One example is data from the
GOS expedition showing a predominance of photorho-
dopsin genes in marine metagenome libraries. The gene
encodes a function of great importance to the marine
food web, as the gene is potentially involved in the light-
driven energy flux in ocean ecosystems. The dataset,
however, cannot be used to assign the photorhodopsin
gene to any specific organism. By combination of single
cell genomics and metagenomics it will be possible in
future research to analyze the genome content, meta-
bolic adaptation and biogeography of specific groups
of uncultured microorganisms and connect this infor-
mation to metabolic pathways, ecological niches and
evolutionary histories of marine microorganisms that
are important players in the marine microbial food-web.
With the omic technologies, it has proved difficult to de-
rive useful information about functions as so much of the
sequence information has no database matches. In the
future, unravelling the functions of all these sequences
will be of great importance for expanding the knowledge
about microbial processes and function in the marine
microbial food-web. Future advances involving a combi-
nation of tools, both classical and novel, might be useful
for uncovering the functional role of these unknown
genes. This might include a combination of methods
such as microarrays, metatranscriptomic, and proteom-
ics together with metagenomics. Ultimately though,
characterization of unknown genes requires the inte-
gration of classical methods, e.g. biochemical assay of
enzyme activity or characterization of gene knockouts.
To date, all omics studies at the metascale have been
carried out on bulk samples. However, microbial com-
munities that exhibit distinct organismal and functional
organization and particular enzyme variants may be
localized within distinct microniches. Future research
will need to resolve the functional significance of gene
and protein localization within microbial communities.
Hence, more advancement in fine-scale measurements
needs to be developed. Additionally, more knowledge
of low abundance community members is required, as
these organisms have been overlooked by present tech-
nologies. New low-cost, high-throughput sequencing
techniques and the technical developments in prot-
eomics will increase the range of detectability both at
the protein and nucleic acid level. These improvements
will, in turn, increase the possibility of discovering the
distributions and functions of rare organisms.
Advancements in marine microbiology have been tra-
ditionally driven by progress in other disciplines such
as medicine and biotechnology. Marine microbiologists
have not traditionally been trained to think about the
development of technological solutions for specific
problems. This is an issue that needs to be addressed
for future improvements within the field of marine mi-
crobial ecology. One example exists, however, where
medicine has benefited from both methodological de-
velopment and knowledge present in marine microbial
ecology. Community-wide sequencing (metagenomics)
of viruses and prokaryotes in the marine environment
has provided tools that have recently been applied to
human health. Recent studies have investigated the viral
community in human respiratory tract and faeces and
have provided important early insight into these sys-
tems. The viral communities found in hosts with cystic
fibrosis have been shown to differ greatly from com-
munities in a healthy host, suggesting that viruses may
play a major role in bacterial population control in the
human oral cavity and respiratory system. Thus, not only
the methodology but also the present microbial ecol-
ogy knowledge may be important for understanding the
processes and interactions between microorganisms in
other disciplines.
In order to achieve progress in the quest for a better
scientific basis to take measures and address the soci-
etal and scientific questions pointed out in this position
paper, there is a need for development in both classical
and novel techniques. For the future, it will be crucial to
combine results obtained by the application of differ-
ent techniques together with information combined with
abiotic and biotic information. Integration of multiple lay-
ers of information through the multi-omics approaches,
with classical methods, novel microscopy techniques
and environmental data is required. By applying such
an approach it will eventually be possible to get a more
precise picture of the function, interaction and diversity
within microbial foodwebs. There is a fundamental need
to focus on developing technology that is specifically
designed for studying marine microbial ecology.

1. Increase efforts to develop technological solutions
for specific marine microbial ecology problems.
Traditionally, advancements in marine microbiol-
ogy have been mostly driven by progress in other
disciplines e.g. medicine and biotechnology.
2. Strengthen the focus on culturing microorganisms
and developing better cultivation techniques as in-
formation from microorganisms in cultures is still
the best way to get detailed information about pro-
cesses and functions.
3. Advance single cell methods enabling the coupling
of microbe identity with activity.
4. Develop more low-cost, high-throughput se-
quencing techniques and technical developments
in proteomics which would increase the range of
detectability both at the protein and nucleic acid
level. More knowledge about low abundance com-
munity members needs to be collected, as these
organisms have been overlooked by current tech-
nologies.
5. Develop faster methods to unravel the functions
of unknown genes. With the omics technologies
it has been difficult to derive useful information
about functions since much of the sequence in-
formation has no database matches. In the future,
this will be of great importance for expanding the
knowledge about microbial processes and func-
tions in the marine microbial food web.
6. Improve bioinformatics tools to analyse genetic se-
quences.
7. Stimulate advancement in fine-scale measure-
ments and process-based measurements. Mi-
crobial communities exhibit distinct organism and
functional organization, and particular enzyme
variants may be localized within distinct micron-
iches. Future research has to resolve the functional
significance of gene and protein localization within
microbial communities.
8. Ensure a better integration of genomics, proteom-
ic, transcriptomic, and metabolomic information
together with physical, chemical and biological
parameters to address fundamental smaller-scale
scientific questions in areas such as microbial
ecology, biodiversity and evolution. To further
overcome current methodological limitations of the
separate omics technologies, attention should also
be given to combining these technologies both
with each other and with traditional classical and
novel techniques.

Marine microbial researchers are mostly interested in
obtaining the following two main types of data: (i) data
about microbe diversity; and (ii) data about ecosystem
functions driven by microbes. The tools and techniques
used by marine microbiologists for sampling, data col-
lection, management and analysis, are similar to those
used by other marine disciplines. However, the study
of microorganisms poses specific problems, which re-
quire additional, specialized procedures, techniques and
tools.
This chapter provides an overview of the current status
and practices in sampling and data collection processes
within the marine microbial research community. It de-
scribes the process of managing the results of marine
microbial research (data, information products, publi-
cations), and, in particular, marine microbial research
products (samples, cultures and sequences) which we
will collectively call Microbial Deliverables (MDs) for
the purpose of this position paper. These MDs can be
considered the raw data products generated by any re-
search program, but they are not necessarily the final
goal of research, which might be to understand the flow
of carbon or nutrients, the production of an antibiotic
product, the biodiversity patterns in a series of environ-
ments, or to isolate a not-yet cultivated organism etc.
MDs can be considered the raw data products gener-
ated by any research program. Finally, this chapter also
identifies key technological and organizational gaps, and
formulates possible solutions and recommendations to
improve future research.
4. Marine microbiology observation and data infrastructures
4.1 Types of microbial deliverables
Marine microbiology research generates products which
can be assigned to one of the following types (see also
Figure 4.1):
(i) Geographic and oceanographic data
Environmental descriptors are often referred to as
“ancillary data” in studies of marine microbial diver-
sity. This includes purely geographic data (position,
depth of sample origin), oceanographic (temperature,
salinity, density, etc.), chemical (oxygen, inorganic
and/or organic nutrient concentrations) or biologi-
cal (chlorophyll, prokaryote abundance) data, etc.
Data can also be split into state variables (concentra-
tions, abundances) and rates (leucine incorporation,
nitrogen fixation, etc.). Some of these data are ob-
tained while sampling via the ship GPS positioning
systems, or with a CTD (conductivity, temperature
and depth) probe or a cluster of sensors and Niskin
bottle samplers attached to a rosette. Others require
further laboratory work to obtain the precise meas-
urement of e.g. chlorophyll a, prokaryote abundance
or bacterial production.
(ii) Sorted and preserved isolates, cultures
of isolates (in liquid or solid media), or single-
cells.
In general, long-term maintenance can only be as-
sured by sample freezing.
(iii) Concentrates of microbial biomass
Microbial biomass concentrates can be obtained
with a specific prefiltration step and a specific fil-
ter type and pore size. The concentrates include
purified DNA, RNA, proteins and the rest of the
metabolome along with cell walls and other cellular
fragments. The concentrates might have the DNA or
RNA extracted (DNA or RNA extracts), and the RNA
might be converted to cDNA as this is more sta-
ble and the subject of most subsequent molecular
analyses. It is often the case that laboratories keep
a stock of the untreated original sample concentrate,
another one with the nucleic acids extracted, and a
third one with the cDNA products. All of these are
commonly kept frozen. When cloning is used, the E.
coli clones themselves are commonly stored for a
given amount of time.
(iv) Genetic sequences
Genetic sequences may result from (a) metagenom-
ic studies in which no PCR amplification has been
used; (b) PCR amplification of a single gene (e.g. 16S
rRNA or functional genes such as pufM). Data can
originate from either Sanger (typically sequences
of 500 bp) or short-read (60-400 bp) sequences
from 454 pyrosequencing, Illumina, Solexa, SOLiD,
etc. Additionally, proteomic studies produce protein

sequences either directly (e.g. ESI-MS: electron-
spray ionization mass spectrometry) or after a gel
electrophoresis step. When using PCR amplification,
the specific conditions used (primers, chemistry,
machine type, conditions) should somewhere be
explicitly stated. Also, it should be clear whether the
data have been cleaned or treated in any way, or
whether they are raw untreated data.
(v) Fingerprinting analyses
Fingerprinting analyses (e.g. ARISA, T-RFLP, DGGE)
are often performed with the amplified DNA of
marker genes like the ribosomal RNA. However, a
lack of standardization in specific analysis methods
between laboratories means that obtained gels and
datasets are not always documented or linked to the
rest of the data.
(vi) Original samples
Water samples can be filtered and stored to perform
subsequent quantitative single cell counting e.g. by
Fluorescence In Situ Hybridization (FISH). In these
cases the samples might be stored before or after
hybridization and/or after the results are recorded.
Ideally, data from the different types listed above should
be integrated so that, for example, an isolate can be
linked to a specific sampling position and correspond-
ing oceanographic and biological conditions and to the
isolate’s rRNA and other (meta)genomic data. Figure
4.1 illustrates these different types of data and the flow
between them.
The abovementioned products are obtained from three
basic types of studies which are further discussed in
section 4.2. These are
(i) Samples of opportunity;
(ii) Planned oceanographic cruises; and
(iii) Fixed time-series studies.
Figure 4.1. Types of data in studies of marine microbial diversity and function: (1) Geographic data; (2) Oceanographic data collected by the
CTD sensors; (3) Chemical and Biological ancillary data; (4) Isolates from a given sample; (5) Microbial biomass or DNA stocks; (6) DNA, RNA
and/or protein sequences; (7) Fluorescence In Situ Hybridization (FISH) samples; (8) fingerprints of the distribution of different sequences of
a specific gene. (Image by C. Ruiz-González and J.M. Gasol)
ATCGGCTAATTGCGCA
MGAAKLAFAVFLISCS
11
22
33
44
555
66
77
88

4.2 Origin of microbiological data
4.2.1 Samples of opportunity
Samples of opportunity are those samples taken during
a specific cruise or trip organized for a purpose other
than research. Sometimes such samples are collected
for microbial analysis.
Samples of opportunity have been very important in
the past. They have often generated the most relevant
changes of paradigms because they tend to be used to
test the possibilities of new technologies and method-
ologies. In general, new equipment and techniques are
applied only to a limited number of samples because
they are often expensive and time-consuming. As an
example, the first metagenomics studies were done
with samples collected in an isolated way, without any
study of the surrounding physical, biological or ecologi-
cal environment. Samples of opportunity are sometimes
collected from a sailboat with a bucket with no meas-
urements of chlorophyll or temperature to accompany
the data. This lack of concurrent determination of envi-
ronmental parameters certainly limits the possibilities of
posterior interpretation of the data. It is therefore rec-
ommended that after the initial “test-of-concept” of the
methodologies, this type of sampling should be avoided.
4.2.2 Oceanographic cruises
Oceanographic cruises are funded and undertaken
specifically for scientific research. Unfortunately, few
oceanographic cruises are purely dedicated to micro-
bial research. Shiptime is expensive and often has to
be shared with several other research groups. As a re-
sult, most research goals have to be agreed upon by the
participant crew and samples are not always taken at
the times and sites preferred by microbial researchers.
Furthermore, cruises tend to be of “one occasion only”
(e.g. Mediterranean cruise BOUM 1
). Specific exceptions
to this trend are cruises such as the Atlantic Meridional
Transect (AMT) which runs through the central Atlantic
Ocean annually, or the series of cruises that have ex-
plored with similar methodologies the deep waters of
the central Atlantic (cruises TRANSAT, ARCHIMEDES,
etc. 2
). In all these cases, however, the spatial component
of microbial diversity tends to be studied in more detail
than the temporal component.
While there are documents that explicitly describe the
consensus of the oceanographic community in terms
of unit usage in chemical oceanography and in bioge-
ochemical research 3
, there is currently no agreement
1. http://www.com.univ-mrs.fr/BOUM/
2. See also http://www.microbial-oceanography.eu/expeditions.html
3. UNESCO (1985) The international system of units (SI) in
on how samples should be collected and treated, and
which ancillary information should be collected along-
side the samples that will undergo molecular analyses
for diversity studies. It is suggested that recommenda-
tions should be set for the collection and storage of
marine microbial samples, taking into account the ex-
isting recommendations in place for genome analysis
and characteristic determination. Similarly, homogeneity
in how ancillary data should be collected, processed
and stored would be desirable. In a recent effort the
Genomic Standards Consortium (www.gensc.org) de-
fined metadata checklists for genomic, metagenomic
and marker genes sequences for a set of environments
which can guide the collection and storage of ancillary
data.
oceanography, UNESCO Technical Papers No. 45, IAPSO Pub. Sci.
No. 32, Paris, France, and UNESCO (1994) Protocols for the Joint Global
Ocean Flux Study (JGOFS) core measurements. IOC Manuals and
Guides, n. 29. 170 pp.
(a)
(e)
(b)
Figure 4.2. Examples of oceanographic ships used in scientific
cruises: (a) Hespérides (Spain); (b) Polarstern (Germany);
(c) Oceania (Poland); (d) Sorcerer at Helgoland Roads (Germany);
(e) Pourquoi pas? (France); (f) Pelagia (The Netherlands).
(From upper left to down right: courtesy J.M. Gasol, AWI,
Euroceans, Frank Oliver Glöckner, Ifremer, NIOZ)
(c) (d)
(f)

Bay, MOLA and Villefranche-point B stations, see Figure
4.3). This is a serious limitation to their utility. If all the
stations were to use the same methodologies, analyze
the same ancillary variables, and coordinate their ef-
forts, we would gain a much better understanding of
the seasonal variability of microbial diversity and func-
tion in the oceans surrounding Europe. Such integration
activities have been funded for some key open ocean
observatories across Europe (e.g. EuroSITES, ESONET)
but these do not yet have strong microbial diversity
components.
4.2.4 Automatic data acquisition
technologies
The wide-spread use of automatic sampling for micro-
bial diversity would certainly be desirable, but we are
still far from having at hand the technology needed for
such an endeavour. While sampling is relatively time-
consuming when large amounts of water need to be
processed e.g. in serial filtrations to account for organ-
isms of different size classes, processing of the samples
is particularly time-consuming. Firstly, processing re-
quires several series of filtration, DNA/RNA extraction,
PCR amplification, etc., and secondly, it is difficult to
technically automate the protocol. Two possibilities that
are under development and worth mentioning are the in
situ flow cytometers and the Environmental Sampling
Processor (ESP) (see also Chapter 3).
In situ flow cytometers take samples at regular intervals,
sometimes staining or fixing the samples, and run them
through one or several UV, blue or red lasers to identify
microbes either by their size, their morphology or their
DNA content. Some machines can be submerged and
operate on their own for months, reporting back to the
main lab by radio or other type of signalling. They are
already in operation in various places in the US, and a
company in Europe is producing several types of such
machines 10
. Some instruments (e.g. FlowCytobot in Gulf
of Mexico waters), have been able to produce data that
has allowed early warning of the appearance of toxic
dinoflagellates. They can still be considered, however,
in the development phase of most of the technologies.
The ESP goes a step further. It is currently a proto-
type created by the MBARI (Monterey Bay Aquarium
Research Institute) that provides on-site (in situ) collec-
tion and analysis of water samples from the subsurface
ocean. The instrument is an electromechanical/fluidic
system designed to collect discrete water samples,
concentrate microorganisms or particles, and automate
application of molecular probes which identify microor-
ganisms and their gene products. The ESP also archives
10. www.cytobuoy.com
4.2.3 Time-series and microbial
observatories
Fixed time-series stations sample the marine environ-
ment regularly in time. Fixed time-series observatories
for monitoring microbial diversity are predominantly
coastal, although some important open ocean stations
exist. For instance, two time-series stations initially
focused on marine biogeochemistry through the inter-
national JGOFS 4
project, the HOT and BATS stations 5
in the North Pacific and North Atlantic respectively, have
been fundamental in our furthering knowledge of the
magnitude and dynamics of microbes and microbial
processes in the ocean. However, other biogeochem-
ically-focused open-ocean time-series stations do
not have a strong biological microbial diversity com-
ponent (e.g. station ESTOC 6
near the Canary Islands
or VACLAN 7
in the deep North Atlantic waters). Except
for a Mediterranean station (DYFAMED 8
), the rest of
European stations with a component of microbial diver-
sity are coastal, such as stations Blanes Bay, MOLA and
SOLA in the NW Mediterranean region, Gulf of Naples,
Helgoland roads and L4 in the North Sea and English
Channel, and the RADIALES 9
in NE Iberian peninsula,
etc. These stations typically run their programs in a
largely uncoordinated way with the exception of EU-
funded projects (e.g. project BASICS and the Blanes
4. http://www1.whoi.edu/
5. http://hahana.soest.hawaii.edu/hot/
6. http://www.estoc.es/en/
7. http://www.vaclan-ieo.es/index_en.html.
8. http://outreach.eurosites.info/outreach/DeepOceans/station.php?id=2
9. http://www.seriestemporales-ieo.com/en/index.htm
Figure 4.3. Synchronicity in the development of populations of a
group of microbes (Cyanobacteria of the genus Synechococcus)
during 2003-2004 at three microbial observatories in the NW
Mediterranean separated by a total of ca 400 km: Blanes Bay,
station MOLA at Banyuls-sur-mer, and Point B at Villefranche-
sur-mer. Note the strong differences in spring and the similarities
in summer and autumn (unpublished data of P. Lebaron, M.
Weinbauer and J.M. Gasol).
0
2 10 4
4 10 4
6 10 4
8 10 4
1 10 5
1.2 10 5
1.4 10 5
1.6 10 5
0 100 200 300 400 500
Blanes (BBMO)
Banyuls (SOLA)
Villefranche (point B)
Synechococcuscellabundance(ml
-1
)
Days since Jan 1st, 2003

samples so that further analyses may be done after
the instrument is recovered. The machine has found
success in the detection of dinoflagellate harmful algal
bloom species, and in the detection of some specific
bacteria and other organisms by means of probe mi-
croarrays. Microfluidics systems that would allow a PCR
reaction followed by capillary electrophoresis are being
designed. No such technology is being produced or as-
sayed in Europe for marine research.
Relevant to instruments such as the one above, the term
“ecogenomic sensing” has been coined with the idea
of detecting molecular markers indicative of specific
organisms, genes or other biomarkers within an environ-
mental context. A field-portable device applied towards
oceanographic research or water quality monitoring
using wet chemistry molecular analytical techniques
to assess the presence and abundance of a specified
set of organisms, their genes and/or metabolites in near
real-time is the objective behind the ESP (Figure 4.4).
4.2.5 Experimental manipulations
Data often originate from controlled manipulation ex-
periments investigating marine microbial functioning.
Examples include mesoscosm experiments that have
tested the effect of nutrients on microbial dynamics, or
those that test for the effects of global change (tempera-
ture increases, acidification), or the addition of pollutants
(e.g. black carbon and oil, etc.) or natural products (aer-
osols, Sahara dust, etc.). Some of these experiments
have been performed in large-scale tanks in the open
ocean, extreme cases being the iron addition experi-
ments (e.g. SEEDS, EiFEX, SOIREE, SOFEX, SAGE…)
and the phosphorus in situ addition experiment (Eastern
Mediterranean Sea, CYCLOPS project). Manipulations
might also include the removal of predators (cascading
experiments) or many other biological forcings. In these
cases it is likely that the sample origin is the same for all
treatments, but that the main factor differing between
samples is the treatment. It is extremely important that
the data that come from these types of experiments are
correctly labelled as coming from an experiment, and
with labels that indicate the treatment. In that way, the
development of e.g. Alcanivorax and Cycloclasticus can
be associated to the presence of added hydrocarbons.
4.3 Current databases
and data handling practices
Whether the samples are obtained as part of the regular
sampling effort of an observatory, or as part of a more
sporadic cruise series, there are some conditions that
should be fulfilled in order to maximize utility of the mi-
crobial deliverables gathered. There is a need for:
(i) Well organized repositories of microbial cultures and
microbial genetic material, accessible to scientists
with ideas on how to analyze the material;
(ii) Samples must be ecologically-referenced, with as
much ancillary ecological and biogeochemical data
(metadata) collected alongside with the microbial
diversity data as possible; and
(iii) Sequence repositories which are geographically and
ecologically-referenced.
Figure 4.4. Illustration of an
economic sensors embedded
within an array of airborne and
in situ sensors to be used for
surveying microbial communities
(courtesy U.S. Department
of Energy Genomic Science
program – http://genomicscience.
energy.gov)

4.3.1 Reference culture storage
(#4 in Figure 4.1)
Classical marine microbiology still relies heavily on
the culturing of microorganisms for identification
and description of new species, and the biochemical
characterisation of novel gene products. The isolated
organisms are also useful as model organisms for
ecological or physiological experiments. The cultures
obtained during this type of research are valuable, but
need constant care, and as the number of cultures grow,
individual researchers cannot maintain these collections
as part of their work. A solution needs to be found to
store and maintain these culture collections.
This is currently provided by reference culture collec-
tions, such as the American Type Culture Collection
(ATCC), the German Collection of Microorganisms
and Cell Cultures (DSMZ), the Spanish Type Culture
Collection, or the Institute Pasteur Collection 11
. These
are institutions in which dedicated technicians take care
of the samples of type strains and produce copies for
the interested scientists. Nowadays, researchers that
keep in-house culture collections might send materi-
als to those who apply for samples depending on their
availability of time, but they are not formally in charge of
doing so. The type of repository that is needed is more
similar to some algal culture collections (such as the
Culture Collection Yerseke (CCY) at NIOZ or the Roscoff
Culture Collection (RCC) 12
) that maintain a diverse array
of ecologically relevant strains. Microdiversity and diver-
sification studies require that a large number of similar
microorganisms are being cultured and compared at the
biogeochemical, ecological and phylogenetic level. This
is not possible with standard culture collections.
Scientists should also be strict in that cultures and in-
formation about culturing requirements are sent to the
repositories when a paper describing some aspect of
an organism is published. Currently, we are faced with
the paradox that some organisms have their full genome
sequenced (and it can be downloaded from databases)
but the organism has never been deposited in a culture
collection in spite of the fact that several Nature papers
were published using this non-deposited model organ-
ism.
What is needed is (i) central collections, such as
specimen collections in museums with a rich set of elec-
tronically available metadata about the isolate; and (ii) a
descriptive catalogue describing what is available and
where. Both issues are currently targeted by the ESFRI
project MIRRI 13
.
11. http://www.lgcstandards-atcc.org/; http://www.dsmz.de/;
http://www.cect.org/; http://www.pasteur.fr/ip/easysite/pasteur
12. http://www.sb-roscoff.fr/Phyto/RCC/index.php?option=com_
frontpageItemid=1
13. http://www.mirri.org/
4.3.2 Genetic material repositories
(#5 in Figure 4.1)
General and well-organized repositories of microbial
genetic material do not exist. They are at the home in-
stitutions of the responsible scientists, in various states,
and there is no public access to them. However, the
scientists in charge should be able to obtain financial
support for these repositories from their institutions,
their governments, or the EU. The rationale for this is
firstly marine microbial genetic material is costly to ob-
tain, requiring sampling on expensive research cruises.
Secondly, repositories of historical samples could gen-
erate research by different groups or at different times,
as techniques are continuously being developed and
material collected years ago can be useful later on. We
are convinced that the final model should promote re-
positories of cultures (culture collections) as cited above,
in which a double funding model is used: some part of
the financing is obtained from the users, but the basic
expenses are paid by institution or government grants.
The EU could use some of their research funds for the
maintenance of these repositories.
An issue to discuss and study is the specific storage
conditions in which the genetic material should be main-
tained. This is a tricky question as ongoing research
tries to understand what makes DNA and RNA more
or less stable and what types of fixatives can be used.
Currently, samples tend to be stored in liquid N2 or in
-80°C freezers, but little is known about long-term DNA/
RNA/protein stability under the most common storage
protocols.
4.3.3 Sequence databases
(#6 in Figure 4.1)
The GenBank (NCBI) and EBI/ENA (EMBL) 14
sequence
databases (International Nucleotide Sequence Database
Collaboration (INSDC)) can be considered as data ar-
chives. Authors are obliged by journals to submit the
sequences described in their papers to the sequence
databases maintained by INSDC. Each sequence
submitted receives an accession number, and these
accession numbers are mandatory references in the
publication. As the amount of information in INSDC
increases with time, INSDC is less and less useful,
because it contains increasing low quality (short) envi-
ronmental sequences. Authors can then use the RDP10 15
database (which maintains a “classifier” function), or use
the curated SILVA 16
database and Greengenes 17
reposi-
tories. These databases include only the fraction of rRNA
gene sequences from INSDC which are of high quality
14. http://www.ncbi.nlm.nih.gov/genbank/ and
http://www.ebi.ac.uk/embl/
15. http://rdp.cme.msu.edu/
16. http://www.arb-silva.de/
17. http://greengenes.lbl.gov/

according to the criteria set by the curators. A database
which is specific for short-read sequences is the VAMPS
database system. 18
Metagenomic data are stored in specialized databases,
such as CAMERA or MG-RAST 19
. MEGAN 20
is down-
loadable software that allows interrogation of the SEED
and KEGG 21
genes databases. But it does not store the
data. MEGX 22
is an ecologically oriented web portal that
collects genome and metagenome data, displays it in
a geo-referenced global system that combines the ge-
netic data with ecological data obtained from the World
Ocean Atlas and World Ocean Database 23
. This system
integrates the SILVA database for rRNA gene-based di-
versity investigations.
Note that these systems are not a way for storing the
data. Instead, they are portals to integrate, access, and
compare the data.
4.3.4 Fingerprints and FISH/ARRAY samples
(#7 and #8 in Figure 4.1)
There is no centralized organized way of maintaining fin-
gerprinting images (e.g. DGGE gels) or data (e.g. ARISA
profiles). Similarly, the maintenance of array data and fil-
ters to perform FISH is done in house in each laboratory.
4.3.5 Ancillary data (aka contextual
or metadata) (#1, #2 and #3 in Figure 4.1)
Geographical and ecological accompanying data should
also be stored using a standardized scheme. Basic data
(geographical location, time) should always be accompa-
nied by basic oceanographic data (temperature, salinity,
chlorophyll or fluorescence). Nutrient concentrations,
particularly inorganic nutrients, should be mandatory.
Organic nutrients are also highly desired. Microbial
abundances should be reported alongside the method-
ology used to obtain them.
Initiatives exist that can be used as role models: the
Genomic Standards Consortium, an open-membership,
international working body founded in 2005 provides
mechanisms that standardize the description of ge-
nomes and the exchange and integration of genomic
data. Marine data initiatives and databases (EMODNet,
PANGAEA, SeaDataNet, Group on Earth Observations
(GEOSS), Global Monitoring for Environment and
Security GMES) 24
exist and new standards and mecha-
18. http://vamps.mbl.edu/
19. http://camera.calit2.net/ and http://metagenomics.anl.gov/
20. http://ab.inf.uni-tuebingen.de/software/megan/
21. http://www.theseed.org/wiki/Home_of_the_SEED and
http://www.genome.jp/kegg/
22. http://www.megx.net
23. http://www.nodc.noaa.gov/OC5/WOA05/pr_woa05.html
24. http://www.emodnet-chemistry.eu/; http://www.pangaea.de/; http://
www.seadatanet.org/; http://www.earthobservations.org/geoss.shtml;
nisms for seamless data transfer are being developed
and implemented.
4.3.6 The flow of data
The diagram in Figure 4.5 sketches, in a simplified way,
how data is generated from the successive research
activities and projects in research institutes, how data
are managed, and how these data are finally made avail-
able to the research community. In this scenario we will
assume the research activities start with the samples
taken, and measurements carried out on a research ves-
sel during a scientific cruise.
4.3.6.1 Scientific cruises, projects and funding
Marine scientists need to reserve/book ship time on
a scientific cruise organized by a ship operator. The
operator will record this booking in an administrative
database also called a cruise planning database. This
information is not always public, mostly only available
for the research operator, but some international initia-
tives share this kind of information. POGO and UNOLS
share the cruise schedules of their research vessels in
an attempt to optimize ship time. After each scientific
cruise, but with some lag, the principal investigator of
the cruise writes a cruise report describing what sta-
tions were visited, what kind of samples were taken,
and what scientific work was carried out by the partici-
pants of the cruise/campaign. Some public databases
have been built to capture this information. The ICES
ROSCOP forms and the SEADATANET cruise summa-
ry reports capture the information from these cruise
reports and make them available online. For research
organized within a publicly funded project (national,
European funds) the different progress reports will
also show a trace of the activities undertaken, and
the results so far. Sometimes administrative project
information can be found in different data systems of
science policy agencies and the EU funding adminis-
trations.
National marine data centres document these pro-
jects in a more scientific way, and some international
networks have organized shared project information re-
positories. The global change master directory (GCMD)
of NASA, and the European directory of marine en-
vironmental projects (EDMERP) started under the
Sea-search project, and continued under SEADATANET
to collect, standardize and make these project de-
scriptions publicly available. This documenting activity
generates what is recognized as “discovery metadata”
allowing scientists to discover ongoing marine research
projects, and contact their colleagues.
Publicly available cruise schedules, cruise summary re-
ports, and project information is important to optimize

expensive ship time and to enhance collaboration be-
tween scientists. The information collected is general,
but is made available a long time before the actual data
is released.
4.3.6.2 Data capture and documentation
The sampling history and analysis results are collected
in laboratory notebooks (paper or electronic), often with-
out clear guidelines. If the lab is better organized, these
data are sometimes entered into a research database
maintained by the most ‘IT-minded’ scientist in the lab
or department. It is not accessible to the wider com-
munity, and unless it is documented by a data centre,
its existence is not even known. Some national or in-
ternational projects organize entire work packages to
collect the data from the participating research groups.
Unfortunately, the data collected in such projects do not
always find their way to a professional data centre, and
risk getting lost after the project ends. In data manage-
ment terminology this collection of data is known as
a ‘dataset’, and the description of a dataset is called
‘metadata’. (Depending on the rules used to group data,
a dataset can be everything collected during a thesis,
a project, a specific campaign, or even all the data col-
lected by a research group during several decades).
Data centres describe datasets according to several
standards (ISO19115, Dublin core) and at different
levels of detail. The most general level is called ‘discov-
ery metadata’ and its purpose is to make the dataset
‘searchable’, ‘discoverable’. The metadata describes the
content of the dataset (which parameters it includes), the
period and region where it was collected, who collected
the data, where it is stored and under what conditions it
can be shared (a summary of the data policy).
Some international metadata directories include: The
register of resources started under the MARBEF NoE,
to describe all known marine biological data sets and
forms the basis for EUROBIS; the European Directory
of marine environmental data (EDMED) which was initi-
Figure 4.5. Data generation scheme providing a simplified overview of how data are generated from the successive research activities and
projects in research institutes, how these data are managed and finally made available to the research community. The red arrow represents
time. The dashes represent time gaps. The upper dark blue rectangle represents activities at the research institute, the lower blue rectangle
activities at a data centre. The middle orange rectangle represents the different data products that are generated. (Developed by F. Hernandez)

ated by BODC in 1991 under the EU MAST framework,
and collects discovery metadata of European datasets
described by the National Oceanographic Data Centres
in Europe, and is used by SEADATANET. The global
change master directory (GCMD) maintained by NASA
is a global environmental (meta)database.
4.3.6.3 Publications, data
and information databases
Many scientific results described in publications never
find their way to data centres or archives. And in many
cases, even if the publication is electronically available,
the information within is not sufficiently structured to be
re-used. This is mainly due to the fact that no common
rules exist for electronic data publishing. Consequently
the user is faced by a mixture of different data formats,
access policies and submission procedures if there is
an appropriate database at all.
When species are described in publications, a pletho-
ra of methods for naming, classifying and referencing
taxonomic entities has been used, but until recently, no
structured lists of species names were available, which
resulted in numerous multiple first descriptions of the
same species. Taxonomic databases, like ALGAEBASE
maintained at the University of Galway, the world reg-
ister of marine species (WORMS) maintained by VLIZ,
the catalogue of life (CoL) maintained by SPECIES2000,
the pan European species infrastructure (PESI), an FP7
project, are far from complete in terms of the number of
species names, let alone in terms of species information.
Particularly for the microbial taxa, databases are incom-
plete, which is partially due to the lack of a practical
“species concept” for microorganisms. The rules for the
formal description of a new microorganism in the litera-
ture require culturing and biochemical characterization
of the strain, which is not only time consuming, but of-
ten technically impossible. The commonly used species
registers lack the possibility to deal with non-taxonomic
entities like operational taxonomic units (OTU), which
poses problems in the standardization steps needed for
data integration of sequence and meta-omics observa-
tion data collected by microbiologists.
4.3.6.4 Archiving Oceanographic datasets
The purpose of data archiving is to make sure the data
is preserved for future use. Scientific data sets will
eventually get lost if not properly archived, because the
originating scientist left the institute, or because the me-
dia on which it was stored becomes unreadable. Data
archiving implies that the data set is documented, and
stored in a safe location. The data are often stored as
files, without need for further standardization, though
some data centres have developed ways of capturing
this heterogenic data in structured databases.
National Data Oceanographic Centres within the IODE
networks are mandated to collect and archive datasets.
The European data centres have organized themselves
in a real network thanks to the Sea-search project, and
subsequent SEADATANET projects.
The publishing network for geo-scientific and environ-
mental data (PANGAEA), is hosted by AWI and MARUM,
and used by the world data centre for marine environ-
mental sciences (WDC-MARE). It is one of the biggest
European marine data archives. Besides dealing with
project data, it also developed a publishing system for
archived data using references based on DOI’s.
Several years can pass before the data flows from the
researcher to a public data centre. This is often dic-
tated by the project funder, sometimes by the publisher
of a journal. The National Science Foundation requires
that any funded project entrusts the generated data to a
permanent data archive. The datasets in a data archive
are not publically accessible immediately. Access can
be restricted to a research group, or project partners.
Public archives also offer a moratorium period, during
which the submitted dataset is not publicly available.
This period is normally linked to the date of publication.
The dataset is then made available for download from
the data archive, or from the integrated data system.
4.4 From data to integration
and interpretation
The next stage in data management involves several
actions (standardization, quality control, biocuration) to
combine datasets from different sources (data provid-
ers) in one data system. This succession of activities is
also known as ‘integration of datasets’. This integration
gives higher value to the individual datasets; higher val-
ues depending on the actions taken for each specific
data system.
The data are at least transformed to a common format
and quality controlled. Standardization of the param-
eters measured, the units used, the time and location
and depth of the observation, the taxonomic entity, the
chemical compound are also essential for combining
data from different sources.
The deliverables collected during field work are finally
analyzed and interpreted and the final product (new
knowledge, whether published in manuscripts or not) is
created from the genomic (sensu lato) and environmen-
tal data, originating from field studies, lab analyses or
after computer work.
Again, the linkage between the final product and the
original data is often lost or, at least, cannot easily be

traced. Raw data, except for sequences deposited in
INSDC or sites like CAMERA, are very often absent from
the published manuscripts, and while at times raw data
are deposited in the Institutes’ websites, with time they
are likely to be lost.
Microbial biodiversity data obtained from samples of
opportunity or from samples taken on multipurpose
oceanographic cruises are too often collected in a non–
standardized way, less than optimally documented, and
fail to capture the temporal variation in microbial com-
1. Develop a Pan EU long-term programme for mul-
tidisciplinary sampling of each European sea for
at least 10 years. This should cover at least one
microbial observatory from each of the European
coastal and deep oceans and also deep-sea mi-
crobial observatories.
2. Stimulate the development of durable partnerships
between the marine microbial research commu-
nity and industry to develop and apply novel data
acquisition technologies involving microbial diver-
sity and function data. Automatic data acquisition
technologies will become important tools in the fu-
ture. It should be the aim of the European scientific
and industrial partners to lead the development
and application of this innovative technology in the
future.
3. Develop new standards and ontologies, and de-
velop or enhance existing data systems in order
to capture and interlink marine microbial informa-
tion with environment, habitat, genomic and spe-
cies information registers. New models need to be
developed to generate the missing data products
(derived data).
4. Enhance existing data and metadata repositories
to better fulfil the requirements for marine microbi-
ological applications. Many sequence data entries
available in public data repositories (INSDC) lack
the proper geo-referencing and environmental an-
notation needed for ecological studies.
5. Increase efforts to raise awareness and develop
strategies for the scientific marine microbiology
community to improve the (i) collection and report-
ing of ancillary data, (ii) storage of DNA, cultures,
microbial biomass, etc. in a well-organized re-
pository, (iii) linking samples to the ancillary (geo-
graphic, ecological, oceanographic, experimental)
data, and (iv) respect for international standards
for data reporting, and assure the maximal linkage
between all products of research (i.e. papers, cul-
tures, data, stored DNA, sequences, etc.). Training
efforts should be directed at the PhD level.
6. Organise repositories for all samples, cultures and
genetic materials collected during a project, and
find permanent facilities to host them. Samples,
cultures and genetic materials are valuable as ref-
erence for future research, but need constant care
and the individual scientists cannot maintain them
as part of their work.
7. Funding agencies need to contribute to address
the loss of data collected by public funds and
specify requirements for each project to perma-
nently archive and publish data generated by the
project in public data systems. Much of marine
biological, microbiological and environmental data
are still lost because they are stored on perishable
media, or buried in local archives which never be-
come public.
8. Build and run permanent (long-term funded) dedi-
cated data infrastructure nodes for microbial ma-
rine data analysis linked to existing data initiatives
like EBI, EMODNET, WORMS, LIFEWATCH. With
the explosion of Omics data, we will need faster,
more automatable data exchange systems. The
data delivery systems based on ‘discover, request
copy and download’ are fine to provide access to
some datasets, but are not suitable for the ma-
chine to machine interfaces that are needed to au-
tomate the data processing and analysis of these
huge data streams.
munities or their relation to environmental parameters.
Scientists research agendas and goals should take this
into account and adapt accordingly. It is not that the cur-
rent studies are not valid, but using organized sampling
systems would add considerable value to them. A few
observatories collect time-series data from fixed sta-
tions, but their monitoring programs often lack microbial
diversity data, and, when they do, their approaches are
not coordinated. For many research applications the
environmental (geographic, oceanographic, biogeo-
chemical, biodiversity, experimental manipulation) data
need to be collected at the same spot and time of the
microbial sample. In such cases, multidisciplinary sam-
pling is essential.

5.1 Introduction
The importance of marine microorganisms, their diver-
sity and impact, as well as advances in research toolkits
and infrastructures have all been addressed in this
report. Previous chapters have revealed important pro-
gress in our ability to address the societal and research
questions introduced in chapter 1 (see Information
Box 2). Important societal questions include how ma-
rine microbial research can contribute to addressing
some of the most important challenges of our time such
as human and environmental health, global change,
sustainable supply of healthy food and energy and
supporting a thriving European bioeconomy. However,
major gaps remain in our understanding of both the di-
versity and functioning of marine microorganisms which
hampers our ability to exploit the full range of opportuni-
ties that marine microbial research can provide for our
societies. Research and technology development are
the key to unlocking this societal potential. Before mak-
ing recommendations for future research priorities and
enabling actions, it is necessary to address the following
key scientific questions:
1. What is the nature of microbial diversity in the ocean
and seas?
2. Which taxa are most relevant in terms of function
across different ocean ecosystems?
3. Which metabolic pathways exist?
4. Which regulatory and signalling networks exist within
and between microbial communities?
5. How do environmental factors influence metabolism
and regulation within and between taxa?
6. How do microbial interactions influence ecology and
Because these scientific questions are central to our
ability to gain a deeper understanding of marine micro-
bial diversity and to reap the societal benefits associated
with that knowledge, this chapter uses them to struc-
ture a review of the European marine research status,
existing gaps and recommendations for future marine
research programmes.
Developments in the area of marine microbiological re-
search in Europe, and by extension marine research in
general, cannot be addressed in isolation from the in-
stitutional, funding and science policy landscape which
has evolved in the last decades, both in Europe and
beyond. Therefore, this chapter begins with a reflection
on European marine microbial research with respect to
the broader European science policy context and global
developments.
5.2 The status of European
marine microbial research
5.2.1 Science policy context
As pointed out in the Galway Declaration (2004) and
reinforced by the Aberdeen (2007) and Ostend (2010)
Declarations, marine science and technology plays an
essential role in generating the knowledge needed to
support the prosperity and well-being of Europeans,
especially in times of global economic, energy and en-
vironmental crisis.
Influenced by these initiatives and advances in science
and technology, the marine science policy landscape
has developed significantly during the last 10 years. The
Maritime Green Paper (2006) which developed the vision
for the Integrated Maritime Policy (IMP, 2007) highlighted
that marine research, and in particular marine biotech-
nological research, is a driver of economic activity as
it generates new knowledge in many scientific and
technological disciplines. Therefore, the impact of the
outputs extends much further than the marine field: it
stimulates activity in areas such as health, food, energy,
pharmaceuticals, environment and transport.
In September 2008 the EC delivered a European
Strategy for Marine and Maritime Research as the re-
search pillar of the European Integrated Maritime Policy
(IMP). It again prioritized marine biodiversity and bio-
technology research and recognised their potential to
contribute new knowledge, on which to base high value-
added products and processes supported by excellent
scientific research.
Marine microbiology research has grown consider-
ably during the past 15 years, partly as a result of the
wider recognition of the enormous potential of marine
biotechnology for the development of new applica-
tions and products. This has been reflected by many
science policy initiatives and events. For example, as a
result of the Bremen Meeting on Marine Biotechnology
Research (2007), the European Commission set-up a
Collaborative Working Group on Marine Biotechnology
(CWG-MB) which highlighted the potential of the sec-
tor and provided clear recommendations to support
its development in Europe. The EC-CWG culminated
in the FP7 Coordination and Support Action (CSA)
MARINEBIOTECH (www.marinebiotech.eu) which is pre-
paring (2011-2012) for an ERA-NET in the field of marine
biotechnology research.
In 2010, the Marine Board organised an ESF-COST
High-Level Conference on Marine Biotechnology and
published a position paper resulting from the work of
an expert working group on marine biotechnology. In
5. European status, research gaps and recommendations

2008 and 2010, EC-US Task Force on Biotechnology
Research workshops in Monaco and Washington re-
spectively, emphasized that microbes play a critical role
in the marine and global ecosystem and are considered
responsible for a significant proportion of biological ac-
tivity. In addition to highlighting research challenges and
opportunities, the workshops also stressed the need to
strengthen Europe’s bioinformatics research capability.
In the last decade, the European Commission has
recognised the need for European coordination and
collaboration in marine microbial and biotechnologi-
cal research and has funded, through its Framework
Programmes, several networks and projects designed to
support and stimulate research on marine diversity and
genomics. Two Networks of Excellence (NoEs), Marine
Biodiversity and Ecosystem Functioning (MarBef) and
Marine Genomics Europe (MGE), as well as a range of
integrated research projects such as HERMES (Hotspot
Ecosystems Research on the Margins of European
Seas) were able to bring together more than 150 institu-
tions, over 1000 researchers in over 30 countries (within
and outside Europe). Also at the pan-European level,
bringing together funding from national research fund-
ing agencies, the European Science Foundation (ESF)
managed a multi-national European investment in the
EuroDEEP initiative which aimed to describe, explain
and predict variations of biodiversity within and between
deep-sea habitats, with respect to deep-sea ecosystem
functioning and the global biosphere.
By funding the ASSEMBLE (2009-) research infra-
structure initiative and the EUROFLEETS (2009-)
infrastructure project, the integrated projects MAMBA
(Marine Metagenomics for new Biotechnological
Applications, 2009-), EuroMarine (2010-), MicroB3
(2012- ) and MaCuMBA (2012-), the EC has taken action
to specifically support marine biology and biotech-
nology in the 7th
framework programme. In particular
MicroB3 (2012-) and MaCuMBA (2012-) are specifically
addressing challenges associated with marine micro-
bial research. These major initiatives and several other
European projects have contributed to the initial devel-
opment of a European Research Area (ERA) for marine
sciences. The concept of the ERA, first coined at the
Lisbon European Council in March 2000 and given new
impetus in 2007 with the European Commission’s Green
Paper on ERA, aims to (i) improve dynamism and inno-
vation in all sectors of industry and services, resulting
in more and better jobs; (ii) address important issues of
a European and even global dimension, such as health,
energy supply and climate change; and (iii) create a so-
ciety in which knowledge is shared, taught and valued
as an essential source of personal and collective de-
velopment.
Interdisciplinary approaches, ranging from sampling and
lab work to bioinformatics is a prerequisite to be able to
address the complexity of the marine system. Integrating
marine sciences and bringing it from an institutional to a
European level has significantly improved the network-
ing between marine researchers and institutes. The
policies and programmes which have successfully been
implemented over the last years have laid a cornerstone
in our capacity meet the challenges of the 21st
century
in marine biology. The Tara and Malaspina cruises (see
Information Box 7) can be seen as a first spin-off of this
new kind of thinking in marine sciences.
5.2.2 Global developments
in marine microbial research
Globally, marine microbial research has gained signifi-
cantly more attention in recent years. One of the most
prominent projects of the last decade was the Global
Ocean Sampling Expedition (GOS) by J. Craig Venter,
an ocean exploration genome project with the goal
of assessing the genetic diversity in marine microbial
communities and to understand their role in nature’s fun-
damental processes. The project started as a Sargasso
Sea pilot sampling project in August 2003 and in 2004
extended to a two-year global circumnavigation us-
ing Craig Venter’s personal yacht, the Sorcerer II. The
first part of GOS, which sampled the North Atlantic,
Caribbean and a small part of the Pacific Ocean,
added DNA sequence information equivalent to 50%
of all protein-encoding sequences that had previously
been deposited in the INSDC (International Nucleotide
Sequence Database Collaboration). GOS confirmed
that marine microbes are diverse, revealing how little
is known about the genetic information of natural as-
semblages.
In terms of sequencing capacities, Europe currently
lags well behind the US and Asia. The Joint Genome
Institute (http://www.jgi.doe.gov/) as well as the Broad
institute (http://www.broadinstitute.org/) in the US are
the most prominent in the delivery of large scale envi-
ronmental sequencing projects and since 2008 the BGI
(formerly Beijing Genomics Institute, http://www.genom-
ics.cn) offers a sequencing service for nearly anything
that contains DNA. In Europe only two main sequenc-
ing centres are available: Genoscope in France (http://
www.genoscope.cns.fr) and the Sanger institute in the
UK (http://www.sanger.ac.uk/). With Sanger exclusively
focusing on medical applications, Genoscope is the only
resource for mid- to large-scale environmental sequenc-
ing approaches.
The requirements for data management and analysis
are increasing rapidly, mainly as a result of the grow-
ing and more diverse application of omics technologies.

Figure 5.1. Overview of the TaraOceans and Malaspina circumnavigation cruises (image by B. Garriz, E. Broglio and J.M. Gasol)
TaraOceans (2009-2012)
Malaspina(2010-2011)
Stimulated by the success of the GOS and Sorcerer
cruises, Europe recently launched two exciting cir-
cumnavigations with the Tara- and Malaspina cruises.
Tara-Oceans is a three year (2009-2012) circumglobal
expedition being carried out on the 36 metre research
schooner Tara (www.oceans.taraexpeditions.org).
The project is using satellite-derived information to
target mesoscale and sub-mesoscale structures of
scientific interest throughout the Earth’s ocean. The
scientists involved in the project have established a
standardized sampling protocol designed to sample
plankton communities from end-to-end, including vi-
ruses, Bacteria, Archaea, protists, and zooplankton.
The project’s aim is the reconstruction of ecosys-
tem community composition and understanding its
functional potential within the physico-chemical con-
straints of each sampling site.
The Malaspina circumnavigation expedition is an
interdisciplinary research project designed to as-
sess the impact of global change on the oceans
and explore their biodiversity (http://en.wikipedia.
org/wiki/Malaspina_Expedition_2010). The 250 sci-
entists on board the Hespérides and Sarmiento de
Gamboa have embarked on a nine-month expedition
(December 2010 to July 2011) combining pioneering
scientific research with training for young research-
ers, while advancing marine science and fostering
the public understanding of science. The project
is under the umbrella of the Spanish Ministry of
Science and Innovation’s Consolider – Ingenio 2010
program and is led by the Spanish National Research
Council (CSIC) with the support of the Spanish Navy.
Malaspina’s main objectives are: (i) to assess the im-
pact of global change on the oceans, (ii) explore the
biodiversity of the deep ocean, (iii) assess the impact
of the original Malaspina expedition, (iv) promote
marine science in Spain and public understanding of
issues in marine sciences and (v) to raise the inter-
est for marine sciences among the youth and training
of young scientists in a global perspective to ocean
sciences.
Information Box 7. European circumnavigation cruises

These technologies will result in a huge increase in the
amount of sequence and environmental data in the near
future which will need to be properly managed to al-
low analysis and meaningful research results. Currently,
the GOS circumnavigation data represents the largest
metagenomic dataset ever placed in the public domain,
with more than 13 million sequences and over 12 billion
base pairs of DNA. It is clear that the data of just the
European cruises can easily extend into the Petabyte
range (i.e. 1,000,000,000,000,000 bytes).
To cope with the size and complexity of such data-
sets the Community Cyberinfrastructure for Advanced
Marine Microbial Ecology Research and Analysis
(CAMERA) has been created in the US with funding from
the Gordon and Betty Moore Foundation. Also in the
US, the Integrated Microbial (Meta)genomes (IMG/M)
and MG-RAST (Metagenome Rapid Annotation using
Subsystem Technology) platforms hosted by the Joint
Genome Institute and the Argonne National Lab have
proven extremely useful for data analysis and annota-
tions, although not specifically created for the marine
sector.
In Europe, only EBI-ENA (EMBL) has the capacity to
store and make available at least parts of the data.
For bioinformatic data analysis, no dedicated marine
bioinformatics infrastructure like CAMERA exists in
Europe and data analysis is mostly carried out by the
biologists themselves. This situation has advantages in
terms of flexibility and the intimate connection between
the environment, samples, analysis and interpretation.
Unfortunately it also leads to heterogeneous approach-
es and “island solutions” which hamper large-scale data
integration and interpretation. Furthermore, it is often
difficult for individual research groups or even marine
institutes to reach a critical mass in bioinformatics that
can allow them to cope with the flood of data produced
by omics technologies.
5.3 Major research gaps and
challenges for future research
Although unprecedented technological improvements in
the last years have provided us with a wealth of knowl-
edge about the dominant types of microorganisms in the
oceans and their activities, there are still many research
questions that have not been solved, either because the
appropriate methodologies have not yet been developed
or applied or, more often, because answering these
questions implies an amount of work and resources that
is out of the reach of most laboratories.
The following key research priorities and needs are
proposed with respect to the main high-level scientific
questions:
(1) What is the nature of microbial diversity
in the oceans and seas?
Marine microbial biologists are often confronted with
the question: “How many microbial “species” exist in a
given environment?” In microbiology the real biodiver-
sity can, at best, only be estimated using an operational
approach to approximate a microbial “species”. This is
usually done by cultivation-independent techniques,
mostly based on the analysis of the ribosomal RNA
genes. Classical clone or single-cell based approach-
es cannot give a comprehensive picture of microbial
diversity due to the low statistical significance of the
results. Nevertheless, first usage of next generation se-
quencing (NGS) technologies suggests that any given
ecosystem is harbouring on the order of 104
microbial
“species”, which means that most of the species show
only very low abundances and comprise the so called
“rare biosphere”. This concept has important ecologi-
cal implications, as it would indicate that most bacterial
types exist everywhere and that most ecosystems have
the potential for responding to changes in environmental
conditions. For example, in the case of an oil spill, the
rare biosphere may contain organisms adapted to the
use of hydrocarbons which can become more abun-
dant. Recent reconsideration of the errors involved in
pyrosequencing have decreased the initial estimates of
how many different “species” an ecosystem could have,
but the numbers are still impressive and in the order of
thousands per sample.
The range of uncertainty expressed above indicates the
clear need for increased research in marine biodiversity.
Next Generation Sequencing now allows screening for
biodiversity for any “marker gene” at unprecedented
temporal and spatial scales. This could be used as the
starting point to determine the dynamics of microbial
communities as well as detecting recurring patterns of
microbial association and potential interactions. This
might provide an answer to the decades-old question
of whether everything is really everywhere. After a base-
line of biodiversity in the oceans has been determined,
anthropogenic influences that change biodiversity
could be distinguished from natural fluctuations. This
would also allow for monitoring the spread of potentially
pathogenic bacteria triggered by warming of the ocean
surface.
(2) Which taxa are most relevant
in terms of function across different ocean
ecosystems?
Recent research indicates that marine systems harbour
groups of organisms that are distinct from the ones
found in freshwater and terrestrial habitats. At least one
group, the alpha subclass of Proteobacteria, shows
a preference for the marine environment. The SAR11

clade, which can be found in nearly every marine sam-
ple, is a prominent example of this group. Although a
rather broad clade, even Euryarchaeota have turned out
to be a ubiquitous group preferentially found in marine
systems.
Although there are clear indications that core taxa ex-
ist, there is also evidence that changing environmental
conditions with water depth provide a stronger vertical
than horizontal determinant of microbial biodiversity
and distribution and that certan microbes are confined
to certain water masses. However, it appears that mi-
crobes are seasonal and that each year some appear in
winter while others occur in summer. What drives this
succession? What are the differences in the microbial
genomes that make a given SAR11 representative be a
winter “species”, while another one prefers the spring
temperatures?
We also need to understand how boundaries in the
ocean are formed which contain microbial communities
of similar structure. Like terrestrial ecologists we should
be able to define the biome and characterize the ecolog-
ical traits of the species that thrive in each biome. What
are the boundaries of the “ocean biomes”? This can
only be answered by a combined extensive effort with
an intensive effort at many sites: the microbial observa-
tories. Several such observatories are already operating
in Europe including the L4 Plymouth site, the Roscoff
station, Helgoland Roads in northern Europe, and the
Blanes Bay, Mola and Sola and Dyfamed stations in the
NW Mediterranean. However, this does not yet represent
a sufficiently comprehensive or coordinated network
and the maintenance of these sampling sites currently
relies too heavily on national funding which makes them
more exposed to the economic crisis.
The current lack of detailed data on “ocean biomes”
hampers our ability to determine which functions and
taxa are most relevant in the different ecosystems.
With the Earth Microbiome Project (EMP) the Argonne
National Lab in the US has recently begun to investi-
gate the diversity and function of the microbial world
more systematically and from the viewpoint of a microbe
(www.earthmicrobiome.org). EMP plans to sequence
200,000 samples resulting in a total of 2.4 petabases of
sequence data comprising of DNA, mRNA and rRNA.
While only a subset will be dedicated to the marine sys-
tem, this could nonetheless deliver first indications of
whether “ocean biomes” exist. An important advantage
of the EMP project is that sample preparation and ac-
quisition of contextual (meta)data will be centralized and
standardized, so that a direct comparison of the results
will be possible.
(3) What metabolic pathways, regulatory
and signalling networks exist?
Omics technologies open the possibility to provide an
inventory of enzymes, pathways and short regulatory
nucleotides of the marine system. With metatranscrip-
tomics and metaproteomics it is even possible to get
insights into the activity of genes at a certain point in
space or time. Nevertheless, because the reconstruction
of enzymatic functions and metabolic pathways using
omics technologies is mostly based on bioinformatic
predictions, the determination of the exact specificity
of the enzymes is still impossible. Additionally, in each
genome or metagenome investigated so far, up to 50%
of the predicted genes could not be assigned with a
function. These hypothetical or conserved hypothetical
genes most likely harbour the information of the specific
adaptations of the respective organism or community.
Single cell technologies can help to assign these genes
to specific taxa, but to elucidate their functions more
cultivates are a prerequisite.
If the quantity and quality of data increases substan-
tially, it may be possible to investigate the extent and
spreading of microbe-driven ecological functions in
microbial genomes. This relates to the resilience of a
microbial community to external forcings. Are functions
duplicated or multiplicated, or are there only a few or-
ganisms performing each function? Research to date
suggests that this depends on which function is consid-
ered: organic carbon respiration is probably widespread
and ecosystems are resilient in terms of performing this
function, while it is likely that the utilization of a specific
wavelength for phototrophic growth, for example, is less
common.
A dense network of environmental and genomic data
could also provide clues about the functions of hypo-
thetical genes by correlating environmental “biome”
features with recurring gene patterns of unknown func-
tions. Such “guilt by association” inferences are already
known to e.g. determine potential functions for genes as
part of transcriptional units (operons), but the approach
of using environmental parameters to predict functions
is in its infancy.
(4) How do environmental factors influence
metabolism and regulation within
and between taxa? How do microbial
interactions influence ecology and
The overwhelming and fascinating complexity uncov-
ered by omics techniques has become a major focus in
contemporary marine microbial ecology. The obvious
challenge to marine microbial ecologists is to trans-
form this avalanche of data at the molecular scale into
“knowledge and understanding” about the complex

interactions of marine organisms, their metabolic and
regulatory mechanisms, and ecosystem functioning in
general.
Following a traditional hypothetical-deductive per-
spective, science can be seen as a spiralling process
whereby observations lead to models (insights, hy-
potheses, etc.) which can be used to construct new
experiments that generate new data, and where each
stage brings us a further step towards better under-
standing. Looking back at the last decades of marine
microbial ecology, however, an important refinement to
this picture becomes obvious: journeys into new and
unknown territories require open-minded (i.e. model-in-
dependent) observations to allow an unbiased observer
to be able to see the unexpected and thus create a new
entrance into the hypothetical-deductive spiral. For ex-
ample, you need to know that there are ~109
bacteria
and ~1010
virus per litre of seawater before you can start
asking for mechanisms behind and relationships be-
tween such data.
A characteristic trait of a mature field of science is a
common theoretical framework within which observa-
tions can be organized and related, new observations
predicted, and experiments thus planned to challenge
the theory. Despite its progress, one can ask whether
marine microbiology scores particularly well according
to such a criterion. To a large extent the field is probably
still in the initial descriptive phase, gathering increasingly
detailed descriptions of the unknown. Significant efforts
are being invested to measure microbial diversity, but
to what extent is this still the initial hunt for a correct
number, more than a hypothesis-driven search for the
underlying mechanisms and relationships producing a
particular diversity? We know quite well the abundance
of viruses, but do we even have a satisfactory theory
explaining the order of magnitude of this number? We
can do meta-analyses generating correlation plots be-
tween bacterial production and primary production, but
to what extent can we match these correlations with
mechanistic explanations for how the system works? Is
the correct description of the state-of-the-art that we
have a lot of observations and data on how the marine
microbial ecosystem looks in terms of numbers, fluxes
and even species, strains and genes, but still a relatively
vague idea of the relationships between these numbers?
From this perspective, the new wave of molecular data is
flooding into a field that is immature in the sense of not
having an advanced theoretical framework within which
these data can be arranged, analyzed and interpreted.
In short, even with the vast amounts of data already
available, our hypothetical approaches are not suffi-
ciently advanced to permit large-scale predictions about
the influence of environmental factors on the diversity
and function of microbes and the influence of microbes
Summary Box 5.1.
Strategic research priorities and needs
1. Ensure appropriate and accurate data acquisition
to obtain more and better data;
2. Build a registry for samples and genetic materi-
als;
3. Develop innovative cultivation approaches and,
in turn, establish model organism databases and
genetic systems for marine organisms;
4. Improve classical methods/approaches, develop
novel techniques and combine their results with
abiotic and biotic information. By applying such
an approach it will become possible to generate
a more precise picture of the function, interaction
and diversity within marine microbial food-webs;
5. Generate a dense network of data on the marine
ecosystem by increasing both the number and
the technological capacities of marine observa-
tories;
6. Contextualise sequence data with a set of stand-
ardized parameters (metadata) of habitats
7. Improve data management to be able to coordi-
nate sampling, sample analyses and processing
of data and make this data openly available for
users;
8. Ensure interoperability of multidisciplinary data
repositories;
9. Implement well-curated and integrated ecologi-
cal/diversity/and genetic databases;
10. Fully implement a systems approach to microbial
ecology to understand ecosystem functioning;
11. Prepare European researchers to be able to par-
ticipate in, and benefit from, data delivered by in-
ternational projects and programmes.
on ecosystem functioning. However, when we lower our
expectations from a “universal model” towards the more
practical construction of tools with predictive power for
applied use in e.g. coastal zone management or climate
change prediction, a detailed knowledge about the mi-
crobial biodiversity and its functions in conjunction with
environmental parameters and anthropogenic forcing is
becoming possible.

As an overarching task, specialized and reliable da-
tabases for marker genes as well as functional genes
should be established and maintained by a set of highly
qualified bio-curators. The databases would help to
establish a clear baseline of biodiversity at the sam-
pling stations based on the analysis of marker genes.
Specialized databases for e.g. commercially useful
enzymes (food, energy, medicine) as well as specific
metabolic, functional or resistance genes could be
useful both in providing opportunities to develop com-
mercial products and processes and in monitoring the
status of the environment in times of global change.
The programme should also involve appropriate legal
and policy expertise to address issues concerning ac-
cess and benefit sharing, intellectual property, and data
as well as for the exploitation of high-potential commer-
cial applications.
The outcome of such a programme is expected to
be the following:
• A set of community-agreed standards and technolo-
gies for (automatic) sampling and data acquisition (lab
protocols), storage and exchange of data to reach
a new level of interoperability and data integration
across disciplines;
• Innovative software approaches for quality manage-
ment, data processing, data integration, accessibility
and visualisation;
• 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 in climate change and the ef-
fect of climate change on microbial communities;
• Delivery of new biocatalytic processes, enzymes, bio-
synthetic pathways and bioactive compounds for use
in biotechnological applications.
• An innovative legal framework and model contracts
for the protection and sustainable use of marine ge-
netic resources;
• A series of workshops, outreach and training ini-
tiatives to make the project results accessible for
researchers, industry, policy makers and the public
at large;
• A new generation of marine microbial researchers well
trained in oceanography, data management, omics
technologies and biodiversity policy;
• A new communication culture with bilateral under-
standing between computer scientists, biologists,
biochemists, bioinformaticians, oceanographers as
well as philosophy and legal experts.
The integrated programme will not only provide answers
to the high level societal questions and ecosystem
management; its interdisciplinary character will cross
5.4 Recommendations
Recommendation 1.
Establish a coordinated pan-European
research programme on marine microbiology
To address the societal and research questions raised
for marine microbial research on biodiversity and
ecosystem functioning at a comprehensive scale, a
long-term, pan-European research programme is ur-
gently needed. Now, at the onset of the data deluge,
this would prevent fragmentation and redundancies in
sampling and analysis, promote unified standards and
enhance interoperability and data integration. Advanced
research efforts must cross boundaries and involve a
range of disciplines including, for example, chemistry,
oceanography, ecology, genomics, information technol-
ogy as well as social sciences and even legal experts
to address issues related to intellectual property rights.
Such a large-scale programme should not displace the
funding of excellent individual investigators which has
been central to many scientific advances in marine mi-
crobiology, and to science in general. It should rather be
taken as an opportunity to promote marine microbiology
and embrace the opportunity to recruit and train the
very best students for a stimulating career in marine
microbiology research in line with Recommendation 1
above.
The proposed programme should:
• Have a duration of 10 years;
• Involve regular in-depth sampling and analysis at up
to 10 sampling sites selected at the onset of the pro-
gramme. These could be part of one major project
or a coordinated cluster of smaller sub-projects. The
selection should follow a screening procedure to find
a representative set of natural “ocean biomes”. Some
of the sampling sites should have a history of data
collection with respect to biodiversity, microbial and
environmental parameters;
• Sample selected sites at least once per week to moni-
tor changes in biodiversity and, at times of identified
fluctuation, apply the full range of omics technologies;
• Develop a set of standard operating procedures for
sampling, contextual (meta)data acquisition, data
management and processing;
• All data should be made publicly available in a “one-
stop-shop”, using the EBI-ELIXIR infrastructure, as
soon as they appear in a solid state;
• From the outset, biological data should be backed
up with oceanographic data from profile and remote
sensing systems. Moreover, automatic sampling and
data processing procedures should be developed in
parallel, integrating relevant industrial partners.

boundaries and support the development of a new cul-
ture of science communication. A better understanding
of the problems and needs of the different disciplines
and attitudes represented in a pan-European pro-
gramme will help in preventing fragmentation in the
European marine research area by substantially increas-
ing technological and knowledge levels.
Recommendation 2.
Create a European repository for cultivated
collections and a reference library
Even when we are capable of obtaining the full genome
sequence of uncultivated microorganisms through next
generation high-throughput metagenomic sequencing,
and can monitor their activities through transcriptom-
ics, proteomics and metabolomics, and have the
techniques to visualize these uncultivated microorgan-
isms in their natural environment, we will realize that
“cultivation-independent” strategies will only answer
part of our questions. To fully understand ecology and
life-cycle of a microorganism and its full biochemical
and acclimation potential, isolation and cultivation are
essential.
Unfortunately, there are currently no requirements for
scientists who publish results using not yet available
microorganisms to make these microorganisms avail-
able to the scientific community. In order to reproduce
the published results or to build on those results and
continue research, scientists depend on the willingness
of the owner of the strains to make them available which
can create problems. For example, the owner may plan
to continue research on that organism, or the strain has
been lost, or the owner is simply unwilling to cooperate
or respond.
Furthermore, the isolation, purification and cultivation
of microorganisms in the laboratory is often very dif-
ficult, tedious and laborious. Too often, at the end of
a project, the laboratory does not have the capacity
or willingness to maintain such cultures and they are
lost. Moreover, existing culture collections are often
only interested in taking a small selection of isolates,
such as novel microorganisms with unusual properties
or of proven biotechnological interest, because they
are limited in their financial and personnel capacities.
As a result, several laboratories have started their own
culture collections, often specializing in specific groups
of organisms. Where they exist, these collections are
frequently financed by short-term research funds and
such collections usually disappear when the scientist in
charge leaves or retires. More importantly, these culture
collections usually lack the capacity to supply cultures
to the broader scientific community.
Hence the following key steps are recommended:
• Create a virtual European repository and reference
library for cultivated marine microorganisms with a
single on-line portal and up-to-date catalogue (con-
taining all the information of the strains, their origin,
their growth media and cultivation conditions, (par-
tial) genome sequences, images, etc.) and access
procedure. Such a virtual repository could provide
information and access to all significant European
marine microbial culture collections and would re-
quire long-term financial support in cooperation with
the home institutes. The virtual repository will ensure
the quality control of the collections and control the
prompt execution of orders for strains by clients;
• Strengthen local culture collections to provide expert
knowledge and technology to:
– Investigate the taxonomic position of the individual
strains in the collection;
– Develop growth media and optimize cultivation con-
ditions;
– Develop long-term storage protocols for all strains
(e.g. by cryo-preservation or lyophilization) in or-
der to minimize the labour required for maintaining
growing cultures;
– Maintain a DNA library of the collection to supply
research groups who only need DNA of a particular
strain.
• Provide training for culture collection curators and
others involved in maintaining cultures of microor-
ganisms as well as for young scientists and research
assistants in order to educate the next generation of
dedicated culture collection curators.
Recommendation 3.
Create an integrated, multidisciplinary
European Centre for marine data management
and analysis
To address the research questions 1-4 (see Information
Box 2, page 15) and to prevent that the large-scale im-
plementation of omics technologies becomes disruptive
(where the data production is faster than the speed us-
ers are able to make use of it) it is recommended to
urgently establish a dedicated bioinformatic and data
management centre specialized in marine microbial
research. The centre should be designed as a special-
ised node of the EBI-ELIXIR infrastructure, to take full
advantage of the existing capacities and competences
in storage, processing and access to data.
The main tasks of the centre would be to:
• Process, analyse and integrate molecular data on
the diversity and function of marine organisms with
respect to the biotic and abiotic factors surrounding

general public as well as industry, funders and policy
makers.
The following key enabling actions should be supported
as a matter of urgency:
• Support and promote open days, movies and lectures
in public places and science educational facilities
such as aquaria and museums. This should include
integrated education material in schoolwork, and
more targeted publications in specialized media and
media campaigns towards policy makers;
• Develop specialized mobile exhibitions that evoke the
imagination and interest of the public at large. These
could include:
– An exhibition ship that travels along the coast of
Europe providing talks and lectures as well as
hands-on experience to discover the microbial
world in the marine environment;
– An exhibition/education/training centre on marine
microbial life made up from one or more containers
with installed microscopes and display materials
which can be transported by ship and by trucks
from city to city.
• Create programmes at national, regional and pan-
European level to improve graduate education. The
future of life sciences in the 21st century is closely
linked to the ability of scientists to develop and par-
ticipate in interdisciplinary projects embracing skills
and concepts from other disciplines. Hence, training
the next generation of marine microbiologists must
focus on the use of interdisciplinary and holistic ap-
proaches to solve a wide range of challenges specific
to dealing with marine microorganisms and the ma-
rine environment. Desirable actions include:
– Ensure that appropriate microbiology modules are
included in all bio-science undergraduate educa-
tional programmes;
– Initiate actions to ensure the participation of re-
searchers from non-marine backgrounds in marine
science and marine microbiology in particular, thus
providing a growing pool of exceptional research
talent for academic and industry needs;
– Organize regular trainings or summer schools on
marine microbiology subjects supported, for exam-
ple, by the EU Framework Programme.
them. Interoperability with oceanographic data and a
high-level of standardization will guarantee a seam-
less data integration between researchers, national
and European marine stations and EBI-ELIXIR;
• Build up specialist databases for marker genes and
omics to support the analysis of biodiversity and func-
tion. All data should be curated by domain experts
with respect to quality, consistency, interoperability
and compliance to standards. The respective knowl-
edge-enhanced databases will be a cornerstone for
the sustainable analysis of marine molecular data;
• Develop controlled vocabularies, ontologies and data
exchange formats in collaboration with experimental
scientists, data providers and EBI-ELIXIR. This will
enhance data annotation, electronic interoperability
among databases and data exchange among us-
ers. Emphasis should be placed on data and quality
management to allow sustainable data integration for
marine ecosystems biology and biotechnology;
• In support of the above, offer training facilities and op-
portunities in bioinformatics and data management.
The proposed Marine Bioinformatics Centre will enable
a new level of integrative and interdisciplinary marine
research, allowing unprecedented insights into the com-
plex interplay of marine organisms and their interaction
with the environment. The centre should act as a me-
diator between marine researchers and the EBI-ELIXIR
e-infrastructure to facilitate sustainable data manage-
ment and knowledge generation in Europe.
Recommendation 4.
Promote interest in marine microbial research
and improve training and education
Earth is a blue planet. With 70% of its surface covered
by the ocean and 40% of the world’s population liv-
ing within 50 km of a coastline, our heritage, economy
and well-being are inextricably linked with the marine
environment. Marine microbes are the “gatekeepers”
for the Earth System with an estimated contribution to
global primary productivity of between 50% and 90%.
It has become clear that marine microbes contribute
fundamentally to all global cycles of energy and matter
and it is evident that without their enormous power and
potential, Earth would not be a living planet. Although
these impressive facts are established, the propor-
tion of research funding allocated to marine research
and, in particular to marine microbial research, remains
incredibly low. It is imperative that the distinct role of
marine microbial communities in global change, sustain-
able supply of food and energy as well as human and
environmental health is brought to the attention of all
stakeholders from school children and students to the

sary to prevent the large scale implementation of omics
technologies turning into a disruptive process (where
the data production is faster than the speed users are
able to make use of it). It will provide critical support
and capacity to deal with the wealth of samples and
genetic information that will come on-stream through
the research in the framework of the pan-European re-
search programme on marine microbiology. Likewise,
material gathered during sampling campaigns would be
preserved and made available to a larger community if
the proposed European repository for cultivated collec-
tions and a reference library were to become a reality.
The combined set of recommendations, when realized,
will provide an enormous positive impact on European
marine microbial research and provide the necessary
push to improve Europe’s competitiveness in this im-
portant research area.
A key consideration is the urgency with which action
is required. By acting now to develop a coordinated,
planned and properly resourced programme, as de-
5.5 Implementing
the recommendations
The four high-level recommendations of this position
paper are designed in such a way that they are inter-
dependent, complimentary and positively reinforcing.
Creating better visibility and recognition for the im-
portance of marine microbial diversity and associated
research will assist in creating the necessary leverage
for appropriate science policy measures and funding of
relevant capacities and research activities. At the same
time, the realization of a major pan-European research
programme on marine microbiology will create a wealth
of material and opportunities for outreach and educa-
tion which will undoubtedly raise the profile of marine
microbial research in Europe. It will also help to increase
the appeal of this research field and attract the best
students for a career in this fascinating domain.
The creation of a multidisciplinary European Centre for
marine data management and analysis will be neces-
Figure 5.2. Schematic overview of main recommendations and their expected impacts in relation to addressing the key societal and
scientific challenges
Training
outreach
Freely available
data from
European sites
Tools for
sampling,
analysis and
storing of data,
cultures and
samples
Understand and
manage marine
microbial
communities in
relation to major
societal challenges
•Global change
•Sustainable food
and energy supply
•Human health
•Environmental
health
Fill gaps in scientiﬁc
knowledge
Communication outreach
Innovative solutions
in support of the
European
bioeconomy
Identity proﬁle
of European Marine
Microbiology
Research
European repository for
cultivated collections
reference library
Long term
pan-European
research
programme on
marine
microbiology
Integrated centre for marine
data management
analysis
Sampling
and
analysis
of key
European
sites
Develop
standard
operating
procedures
Develop and
populate
appropriate
databases
Create a
virtual
repository
Strengthen
local
culture
collections
Develop
standards

scribed above, we avert the damaging prospect of
further disintegration and fragmentation of the European
research effort which is inevitable under the “do noth-
ing” scenario. Without this integrating framework and its
key bioinformatics e-infrastructure, Europe will continue
to slide in its position against other advanced research
nations and, more importantly, opportunities to address
key economic, social and environmental challenges will
be lost. The European scientific community stands
ready to work in harmony to advance European marine
microbial research, knowledge and understanding. Let
us all, as stakeholders in this knowledge, work together
to deliver a true European Research Area in marine sci-
ence and microbiology.

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AOB: Ammonia oxidizing bacteria
ARISA: Automated ribosomal
intergenic spacer analysis
ASSEMBLE: Association of
European Marine Biological
Laboratories (EC FP7 research
infrastructure initiative)
ATCC: American Type Culture
Collection
ATP: Adenosine triphosphate
AWI: Alfred Wegener Institute for
Polar and Marine Research
BGI: Beijing Genomics Institute
BODC: British Oceanographic Data
Centre
CAMERA: Community
Cyberinfrastructure for Advanced
Microbial Ecology Research and
Analysis
CCN: Cloud condensation nuclei
CCY-NIOZ: Culture Collection
Yerseke – Royal Netherlands
Institute of Sea Research
cDNA: Copy- or Complementary
DNA; refers to DNA synthesized
from a messenger RNA (mRNA)
template in a reaction catalyzed by
the enzyme reverse transcriptase
and the enzyme DNA polymerase
CLAMER: Climate Change and
Marine Ecosystem Research
(EC FP7 Project)
CoML: Census of Marine Life
CSA: Coordination and Support
Action
CSIC: Spanish National Research
Council
CTD: Conductivity-temperature-
depth are standard oceanographic
parameters measured by, among
others, most research vessels,
floats and automated underwater
vehicles.
CWG-MB: Collaborative Working
Group on Marine Biotechnology
DGGE: Denaturing Gradient Gel
Electrophoresis
DMS: Dimethylsulfide
DMSP: Dimethyl sulfoniopropionate
DNA: Deoxyribonucleic acid;
contains the genetic instructions
used in the development and
functioning of all known living
organisms (with the exception of
RNA viruses).
DNRA: Dissimilatory nitrite or nitrate
reduction to ammonia
DOC: Dissolved organic carbon
DON: Dissolved organic nitrogen
DOP: Dissolved organic phosphorus
DSMZ: German Collection of
Microorganisms and Cell Cultures
EBI: European Bioinformatics
Institute (EBI)
EC: European Commission
EDMED: European Directory of
marine environmental data
EDMERP: European directory of
marine environmental projects
EiFEX: European Iron Fertilization
Experiment
ELIXIR: European Life-Science
Infrastructure for Biological
Information
EMBL: European Molecular Biology
Laboratory (EMBL)
EMBRC: European Marine Biological
Resources Centre (EMBRC)
EMODNet: European Marine
Observation and Data Network
EMP: Earth Microbiome Project
ENA: European Nucleotide Archive
EPS: Extracellular polymeric
substances
ERA: European Research Area
ESF: European Science Foundation
ESP: Environmental Sampling
Processor
ESTOC: European Station for Time
series in the OCean
EU: European Union
EUROBIS: European Ocean
Biogeographic Information System
EUROCORES: European
Collaborative Research Scheme
(developed and operated by the
European Science Foundation)
EUROFLEETS: Towards an Alliance
of European Research Fleets (EC
FP7 Research infrastructures
project)
EuroMarine: Integration of European
Marine Research Networks of
Excellence
FISH: Fluorescent In Situ
Hybridisation
FP: Framework Programme
GCMD: Global change master
directory
GEOSS: Group on Earth
Observations
GMES: Global Monitoring for
Environment and Security
GOS: Global Ocean Sampling
GPS: Global Positioning System
HABs: Harmful Algal Blooms
HERMES: Hotspot Ecosystems
Research on the Margins of
European Seas (EC FP6 Project)
HSV: Herpes Simplex Virus
ICES: International Council for the
Exploration of the Sea
ICoMM: International Census
of Marine Microbes
IMP: Integrated Maritime Policy
IMTA: Integrated Multi-Trophic
Aquaculture
INSDC: International Nucleotide
Sequence Database Collaboration
ISIS: In situ Iron Studies (consortium)
KEGG: Kyoto Encyclopedia of Genes
and Genomes
MaCuMBA: Marine Microorganisms:
Cultivation Methods for Improving
their Biotechnological Applications
(EC FP7 Project)
MAMBA: Marine Metagenomics for
new Biotechnological Applications
(EC FP7 Project)
Marbef: Marine Biodiversity and
Ecosystem Functioning (EC FP6
Network of Excellence)
MAR-FISH: Microautoradiography-
FISH
MBARI: Monterey Bay Aquarium
Research Institute
MDs: Microbial Deliverables. The
results of marine microbial
research (data, information
products, publications), and,
in particular, marine microbial
research products (samples,
cultures and sequences) which
are collectively called Microbial
Deliverables (MDs) for the purpose
of this position paper.
MEGAN: MEtaGenome Analyzer; a
software package developed and
used to analyze metagenomes
MEGX: is an ecologically oriented
web portal that collects genome
and metagenome data, displays it
in a geo-referenced global system
that combines the genetic data
with ecological data obtained from
the World Ocean Atlas and World
Ocean Database.
MEOR: Microbial Enhance Oil
Recovery
MGE: Marine Genomics Europe (EC
FP6 Network of Excellence)
MG-RAST: Metagenomics RAST
server; an automated analysis
platform for metagenomes
providing quantitative insights into
microbial populations based on
sequence data.
List of abbreviations and acronyms

MicroB3: Biodiversity, Bioinformatics,
Biotechnology (EC FP7 Project);
aims to develop innovative
bioinformatic approaches and
a legal framework to make
large-scale data on marine viral,
bacterial, archaeal and protists
genomes and metagenomes
accessible for marine ecosystems
biology and to define new targets
for biotechnological applications.
mRNA: messenger RNA
MRSA: Methicillin-resistant
Staphylococcus aureus
NASA: National Aeronautics and
Space Administration
NCBI: National Center for
Biotechnology Information
OIF: Ocean Iron Fertilisation
OMZ: Oxygen minimum zones
ORFs: Open reading frames
OTU: Operational taxonomic units
PANGEA Publishing network for geo-
scientific and environmental data
PCR: Polymerase Chain Reaction
PESI: Pan-European species
infrastructure
POC: Particulate organic carbon
POGO: Partnership for Observation
of the Global Oceans
RADIALES: Oceanographic time
series in northern Spain
RAS: Recirculating Aquaculture
Systems
RAST: Rapid Annotation using
Subsystem Technology
RCC: Roscoff Culture Collection
RFO: Research Funding Organisation
RNA: Ribonucleic acid
ROSCOP: Report of Observations/
Samples collected by
Oceanographic Programmes
RPO: Research Performing
Organisations
rRNA: ribosomal RNA
SAGE: Serial Analysis of Gene
Expression
SeaDataNet: Pan-European
Infrastructure for Ocean Marine
Data Management
SILVA: Comprehensive ribosomal
RNA databases
SIP: Stable-isotope probing
SOFEX: Southern Ocean Iron
Experiment
SOIREE: Southern Ocean Iron
Enrichment Experiment
sRNA: small RNA
SST: Sea surface temperature
T-RFLP: Terminal Restriction
Fragment Length Polymorphism
UNOLS: University-National
Oceanographic Laboratory System
UV: Ultra Violet
VMDC: Flanders Marine Data Centre
WDC-MARE: World Data Centre for
MARine Environmental sciences
WG MICROCEAN: Marine Board
Working Group on Marine
Microbial Diversity
WGA: Whole genome amplification
WORMS: World Register of Marine
Species

Contributing authors
Frank Oliver Glöckner (Chair)
Max Planck Institute for Marine
Microbiology Bremen and Jacobs
University Bremen, Germany
e-mail: fog@mpi-bremen.de
Adriano Bordalo
Institute of Biomedical Sciences,
University of Porto, Portugal
e-mail: bordalo@icbas.up.pt
Josep M. Gasol
Institute of Marine Sciences,
CSIC, Catalunya, Spain
e-mail: pepgasol@icm.csic.es
Francisco Hernandez
Flanders Marine Data Centre
(VMDC), Belgium
e-mail: tjess@vliz.be
Matthias Labrenz
Leibniz Institute for Baltic Sea
Research Warnemünde, Germany
e-mail: matthias.labrenz@io-
warnemuende.de
Fergal O’Gara
BIOMERIT Research Centre,
National University of Ireland Cork
(UCC), Ireland
e-mail: f.ogara@ucc.ie
Paraskevi Pitta
Hellenic Centre for Marine
Research, Institute of
Oceanography, Greece
e-mail: vpitta@hcmr.gr
Ruth-Anne Sandaa
University of Bergen, Norway
e-mail: Ruth.Sandaa@bio.uib.no
Lucas J. Stal
Netherlands Institute of Ecology
NIOO-KNAW, Netherlands
e-mail: L.Stal@nioo.knaw.nl
Elena Stoica
National Institute for Marine
Research and Development
“Grigore Antipa”, Romania
e-mail: estoica@alpha.rmri.ro
Marta M. Varela Rozados
Spanish Institute of Oceanography
(IEO) – Centro Oceanográfico da
Coruña, SPAIN
e-mail: marta.varela@co.ieo.es
Annex I. Participating authors

Printing: Drukkerij De Windroos NV (Beernem, Belgium)
May 2012
ISBN: 978-2-918428-71-8
The European Science Foundation (ESF) is an inde-
pendent, non-governmental organisation, the members
of which are 72 national funding agencies, research
performing agencies and academies from 30 countries.
Since its establishment in 1974, ESF, which has its head-
quarters in Strasbourg with offices in Brussels and Os-
tend, has assembled a host of organisations that span
all disciplines of science, to create a common platform
for cross-border cooperation in Europe.
ESF is dedicated to promoting collaboration in scientific
research and in funding of research and science policy
across Europe. Through its activities and instruments,
ESF has made major contributions to science in a global
context. ESF covers the following scientific domains:
• Humanities
• Life, Earth and Environmental Sciences
• Medical Sciences
• Physical and Engineering Sciences
• Social Sciences
• Marine Sciences
• Materials Science and Engineering
• Nuclear Physics
• Polar Sciences
• Radio Astronomy
• Space Sciences
www.esf.org
European Science Foundation

Marine Board
Wandelaarkaai 7
8400 Ostend • Belgium
Tel: +32 59 34 01 63
Fax: +32 59 34 01 65
info@marineboard.eu
www.marineboard.eu
May 2012 – Print run: 1000

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