Download Microbial Ecosystem Functions Along the Steep Oxygen

Have you ever dreamed of going to an anoxic world? This thesis
takes you on a fascinating journey along the steep oxygen gradient at
the deepest point of the Baltic Sea, the Landsort Deep. On your trip
from the oxygenated surface water into the anoxic deep water and
sediment, you will meet many microorganisms whose importance
for sustaining ecosystem functions cannot be underestimated. Meet,
for example, the organic matter degraders, fermenters, sulphate
reducers, methane producers and methane oxidisers. Find out if they
are more susceptible to share genetic material compared to other
microorganisms. And finally, are the Cyanobacteria in the dark and
anoxic sediment dead or alive?
Microbial Ecosystem Functions Along the Steep
Oxygen Gradient of the Landsort Deep, Baltic Sea
Petter Thureborn carries out research in the field of microbial ecology with a special focus on the Baltic Sea. This is his PhD thesis.
Environmental Science, School of Natural Sciences, Technology and
Environmental Studies, Södertörn University.
PETTER THUREBORN
Distribution: Södertörn University, Library, SE-141 89 Huddinge. [email protected]
Microbial Ecosystem Functions Along
the Steep Oxygen Gradient of the
Landsort Deep, Baltic Sea
Petter Thureborn
Microbial ecosystem functions along
the steep oxygen gradient of the
Landsort Deep, Baltic Sea
Microbial ecosystem functions along
the steep oxygen gradient of the
Landsort Deep, Baltic Sea
Petter Thureborn
Environmental Science,
School of Natural Sciences, Technology and Environmental Studies,
Södertörn University
Södertörn University
The Library
SE-141 89 Huddinge
www.sh.se/publications
© Petter Thureborn
This research was funded by the Foundation for Baltic and East European
Studies 1169/42/2007:17 and the EU Metaexplore project (KBBE-222625).
Cover image: Tolypothrix (Cyanobacteria) by Matthew J. Parker
CC BY-SA 3.0
(http://creativecommons.org/licenses/by-sa/3.0), via Wikimedia Commons
Cover layout: Jonathan Robson
Graphic form: Per Lindblom & Jonathan Robson
Printed by Elanders, Stockholm 2016
Södertörn Doctoral Dissertations 125
ISSN 1652–7399
ISBN 978-91-87843-58-7 (print)
ISBN 978-91-87843-59-4 (digital)
Abstract
Through complex metabolic interactions aquatic microbial life is essential as a
driver of ecosystem functions and hence a prerequisite for sustaining plant and
animal life in the sea and on Earth. Despite its ecological importance, information on the complexity of microbial functions and how these are related to
environmental conditions is limited. Due to climate change and eutrophication,
marine areas facing oxygen depletion are increasing and predicted to continue to
do so in the future. Vertically steep oxygen gradients are particularly pronounced in the Baltic Sea. In this thesis, therefore, the ecosystem functions of microbial communities were investigated, using metagenomics, to understand how
they were distributed along the steep oxygen gradient at the Landsort Deep, the
deepest point of the Baltic Sea. Furthermore, microbial communities from the
Landsort Deep transect were compared to microbial communities of other
marine environments to establish whether the environment at this site resulted
in a characteristic community. To reveal what microbial community functions
and taxa were active in the anoxic sediment a metatranscriptomic approach was
used. Results showed a marked effect of the coupled environmental parameters
dissolved oxygen, salinity and temperature on distribution of taxa and particularly community functions. Microbial communities showed functional capacities consistent with a copiotrophic life-style dependent on organic material
sinking through the water column. The eutrophic condition with high organic
load was further reflected in the metatranscriptome of the anoxic sediment community, which indicated active carbon mineralisation through anaerobic heterotrophic-autotrophic community synergism. New putative linkages between
nitrogen and- sulphur metabolisms were identified at anoxic depths. Furthermore, viable Cyanobacteria in the anoxic sediment was evident from the transcript analyses as another reflection of marine snow. High abundance and
expression of integron integrases were identified as a characteristic feature of the
Landsort Deep communities, and may provide these communities with a mechanism for short-term-adaptation to environmental change. In summary, this
thesis clearly documents what impact eutrophication and oxygen depletion have
on microbial community functions. Furthermore, it specifically advances the
mechanistic insight into microbial processes in anoxic deep-water sediment at
both genomic and transcriptional level. Given the predicted progress of oxygen
depletion in marine and brackish environments, this work advances information
necessary to estimate effects on marine and in particular brackish ecosystem
functions where anoxic conditions prevail.
Keywords: microbial ecology, ecosystem functions, microbial community, bacteria, archaea, metagenomics, metatranscriptomics, oxygen gradient, hypoxia,
sediment, Baltic Sea.
Sammanfattning
Mikroorganismer är essentiella för fungerande ekosystemfunktioner i akvatiska
miljöer och därmed en förutsättning för övrigt växt- och djurliv på vår planet.
Trots deras ekologiska nyckelroll är kunskapen om mikroorganismernas funktion och komplexitet samt hur dessa är relaterade till miljön begränsad. På grund
av eutrofiering och klimatförändringar har marina områden som lider av
syrebrist ökat och en ytterligare utbredning av marina och bräckta områden med
syrebrist är predicerad i framtiden. Stora områden av Östersjön kännetecknas av
vertikala syregradienter med syresatt ytvatten och anoxiskt bottenvatten. I denna
avhandling undersöktes därför med metagenomik hur mikrobiella ekosystem
funktioner var utbredda längs den vertikala syregradienten i Östersjöns djupaste
del, Landsortsdjupet. Dessutom jämfördes de mikrobiella samhällena från Landsortsdjupet med mikrobiella samhällen från andra marina miljöer för att utröna
om den karakteristiska miljön i Landsortsdjupet återspeglade de mikrobiella
samhällen som lever där. För att undersöka vilka mikroorganismer samt vilka
mikrobiella ekosystemfunktioner som var aktiva i det anoxiska sedimentet i
Landsortsdjupet användes metatranskriptomik. Resultaten visade en stark korrelation mellan miljöparametrarna syrehalt, salinitet och temperatur och fördelningen av mikrobiell taxa och i synnerhet mikrobiell funktion längs Landsortsdjupets transekt. De mikrobiella samhällena uppvisade en funktionell kapacitet
förenlig med en livsstrategi beroende av organiskt material som sjunker genom
vattenkolonnen som en konsekvens av eutrofiering. Eutrofa förhållanden med
hög halt av organiskt material var även återspeglad i metatranskriptomet från
det anoxiska sedimentet, som indikerade aktiv mineralisering av organiskt kol
genom anaerob heterotrof-autotrof synergism. Nya möjliga kopplingar mellan
kväve- och svavelmetabolism identifierades i det anoxiska vattnet. Vidare visade
resultat från metatranskriptom-analys att livsdugliga cyanobakterier var abundanta i det mörka och anoxiska sedimentet, vilket även detta kan vara en konsekvens av sjunkande organiskt material. Hög abundans och hög transkribering av
integron gener kunde identifieras som ett karakteristiskt kännetecken hos
mikrobiella samhällena i Landsortsdjupet vilket skulle kunna förse dem med en
mekanism för anpassning till miljöförändringar. Sammanfattningsvis dokumenterar denna avhandling tydligt vilken påverkan eutrofiering och syrebrist har på
mikrobiella funktioner. Dessutom för den specifikt kunskapen om mikrobiella
processer i anoxiska djupvattensediment framåt på både genom- och transkriptionsnivå. Mot bakgrund av en predicerad ökning av syrebristen i marina miljöer, bidrar denna avhandling med information viktig för att kunna förutse vilka
effekter anoxiska förhållanden kan komma att få på ekosystemfunktioner i
marina miljöer och i brackvattenmiljöer i synnerhet.
Table of contents
List of Papers ........................................................................................................................11
Abbrevations ........................................................................................................................12
Introduction .........................................................................................................................13
Study system – the Landsort Deep, Baltic Sea .................................................................19
Aim and objectives of the thesis ........................................................................................23
Methodological design, approach and considerations...................................................25
Central results and Discussion ..........................................................................................31
Conclusions ..........................................................................................................................47
Acknowledgements .............................................................................................................49
References.............................................................................................................................51
Paper I ...................................................................................................................................67
Paper II..................................................................................................................................81
Paper III ..............................................................................................................................103
List of Papers
This thesis is based on the following papers:
I. Thureborn P, Lundin D, Plathan J, Poole AM, Sjöberg B-M, Sjöling S. (2013)
A Metagenomics Transect into the Deepest Point of the Baltic Sea Reveals Clear
Stratification of Microbial Functional Capacities. PLoS ONE 8(9): e74983.
doi:10.1371/journal.pone.0074983
II. Thureborn P, Franzetti A, Hu YOO, Sjöling S, Lundin D. A dark, anoxic
mausoleum for DNA – perceived and actual community structure in the Landsort
Deep sediment, the Baltic Sea. Submitted manuscript
III. Thureborn P, Franzetti A, Lundin D, Sjöling S. (2016) Reconstructing
ecosystem functions of the active microbial community of Baltic Sea oxygen
depleted sediments. PeerJ. 4:e1593; DOI 10.7717/peerj.1593
Papers are reprinted with permission from the respective publisher.
My contributions to the papers included in this thesis have been as follows:
Paper I – Planned the study, wrote the manuscript, performed sampling and lab
experiments, performed bioinformatics and statistical analyses, made the figures
and tables.
Paper II – Wrote the manuscript, performed sampling and lab experiments.
Paper III – Planned the study, wrote the manuscript, performed sampling and
lab experiments, performed bioinformatics and statistical analyses, made the
figures and tables.
11
Abbrevations
Anammox = Anaerobic ammonia oxidation
AMZ = Anoxic marine zone
ANME = Anaerobic methanotrophic archaea
AOM = Anaerobic oxidation of methane
CA = Correspondence analysis
CCA = Canonical correspondence analysis
DNA = Deoxyribonucleic acid
DOC = Dissolved organic carbon
eDNA = Extracellular DNA
iDNA = Intracellular DNA
LD = Landsort Deep
mRNA = Messenger RNA
OMZ = Oxygen minimum zone
OTU = Operational taxonomic unit
pMMO = Particulate methane monooxygenase
PSP = Protein synthesis potential
PSU = Practical salinity unit
RNA = Ribonucleic acid
rRNA = Ribosomal RNA
12
Introduction
Microbial ecosystem functions
Bacteria and Archaea are the most abundant, ubiquitous and diverse life forms
of the oceans [1,2,3] and bacterial and archaeal life hold a vast reservoir of genetic and metabolic potential [4]. Furthermore, Bacteria and Archaea are unique in
their capability to carry out respiration with molecules other than oxygen (e.g.
NO2-, NO3-, SO42-, Fe3+), which enables metabolism in the absence of oxygen (i.e.
anaerobic metabolism) [5]. This is important since it extends the window of
accessible habitats for Bacteria and Archaea into environments with no oxygen
(i.e. anoxic environments). In combination with their capacity for both heterotrophy (i.e. usage of organic carbon as carbon source) and autotrophy (i.e. inorganic carbon is used as carbon source) it makes Bacteria and Archaea essential
drivers of the Earth’s biogeochemical cycles (e.g. carbon, nitrogen, phosphorus
and sulphur) in both oxic and anoxic environments [5,6]. Furthermore, Bacteria
and Archaea perform other important functions such as degradation of pollutants and producers of bioproducts. Ultimately, processes mediated by Bacteria and Archaea are key in sustaining marine ecosystem functions like, for
example, nutrient transport and turnover, primary and secondary production
and climate feedback [7].
Bacteria and Archaea are – together with most protozoan species, some algae
and fungi and a few animals – commonly called microorganisms. This thesis is
focused on Bacteria and Archaea, which are below referred to as microorganisms
Microbial functions of importance for marine carbon transformations
The majority of the fixation of atmospheric carbon dioxide (CO2) occurs through
oxygenic photosynthesis in Cyanobacteria and eukaryotic phytoplankton (e.g.
diatoms and dinoflagellates) [8]. This is essential for marine life as it provides
energy and nutrients to the pelagic food web. Aerobic heterotrophic bacteria
perform an important function in retaining this energy, by uptake and assimilation of released dissolved organic carbon (DOC) from phytoplankton, in the
pelagic food web [9,10,11,12]. Energy that would otherwise be lost to the oceans’
interior thus becomes available to fish and top-predators via a grazing cascade of
ciliates, flagellates and zooplankton [10]. In hydrolysis of aggregates of sinking
organic matter (i.e. marine snow), microorganisms perform another important
function in releasing DOC and other nutrients in the pelagic, but also in the
oceans’ interior [12]. It is estimated that 90% of the marine primary production
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PETTER THUREBORN
is mineralised by aerobic heterotrophy in the surface water. This is for open sea,
however, and in coastal waters more of the primary production sinks to deep
water and the seafloor [13]. However, not all organic carbon is degraded and
mineralised by microorganisms on the seafloor. This leads to large sequestration
of both carbon and other nutrients in the marine environment that influences
atmospheric CO2 levels [14,15].
A major part of the microbial processes involved in biogeochemical cycling
are only performed under hypoxic and anoxic conditions [5]. The seafloor (i.e.
the sediment) holds the most substantial anoxic niche in the marine environment [16]. Anoxic water is typically restricted to oxygen minimum zones
(OMZs) or anoxic marine zones (AMZs) in areas of large upwelling or anoxic
basins [17].
In anaerobic habitats heterotrophic metabolism via respiration of oxygen is
replaced by fermentation and anaerobic respiration using alternative electron
acceptors (e.g. NO2-, NO3-, SO42-, Fe3+). The dominant process in which organic
carbon is mineralised under anoxic conditions is sulphate reduction [18]. Sulphate reduction accounts for 50% of the organic carbon mineralisation in coastal
marine sediments [19,20] and marine sediments are the most important sink for
sulphate in the sea water [21]. Hydrogen sulphide (H2S) produced from sulphate-reduction is commonly oxidised by sulphur-oxidising bacteria, which has
implications for dark carbon fixation [22] and nitrogen cycling [23], but at high
concentration also forms manganese sulphide precipitates [24,25]. Complete
mineralisation of organic matter is not possible without methanogenic archaea
that carry out the final step of the transformation of organic matter. Methanogens have the capacity to produce methane through reduction of different
substrates (e.g. CO2, acetate, methylamines, methanol) [26]. Mineralisation of
organic matter in anoxic environments is performed by close interaction of
fermenters, sulphur respirers, and methanogens. These interactions increase the
overall mineralisation efficiency since concentrations of metabolites stay low and
hence avoid the build up of metabolites in the immediate environment, which
would reduce the mineralisation rate [27]. Carbon mineralisation is directly
coupled to nitrogen metabolism through the denitrification process (NO3
NO2-  NO  N2O  N2). Denitrification is, together with anaerobic ammonia
oxidation (anammox) (NO2- + NH4+  N2), responsible for all the nitrogen loss
in the oceans. These processes are therefore important in balancing the global
nitrogen-budget [28]. Denitrification and anammox both occur in OMZ/AMZ
water and sediment [29], but seem to be separated in time and space
[30,31,32,33,34]. The separation of these processes has been proposed to be controlled by the composition and temporary supply of organic carbon [28].
The biogeochemical cycling of elements are coupled through microbial
processes in both oxic and anoxic environments. However, the complexity of
these microbial processes, particularly in environments with low or no oxygen, is
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MICROBIAL ECOSYSTEMS FUNCTIONS
not fully understood [17]. With predictions of increasing hypoxic and anoxic areas
in the future [35] and the impact it may have on marine ecosystem functions,
further investigations of microbial processes in environments with different
oxygen conditions, and particularly in anoxic environments, are much needed.
The unseen majority
The importance of marine microorganisms for marine biogeochemical cycles
has been known for a long time [36], although only a minority of the microbial
cells in seawater have been accessed by cultivation in the laboratory [37]. This
cultivation discrepancy, which is true for other environments as well, has been
named the “great plate anomaly” [38]. Although cultivation methods improved,
the advancements in molecular methods including direct DNA extraction,
cloning and sequencing of specific genes from environmental samples were
groundbreaking in the way they exposed the, up until that point, “black box” of
microbial taxonomic diversity [39,40]. Since the earlier studies of marine
microbial diversity, based on 16S ribosomal RNA (rRNA), for example in the
Sargasso Sea by Giovannoni and colleagues [41], the ubiquitousness and phylogenetic diversity of microorganisms has become apparent in habitats spanning
from marine surface water and ice to deep sea subsurface sediment [3,42]. Total
marine microbial diversity has been estimated to consist of up to two million
taxa [1] and the number of microbial species identified by 16S rRNA analysis on
Earth currently exceeds 4 million [43]. Typically, the composition of microbial
assemblages are dominated by a few populations whereas the largest diversity is
to be found in the large numbers of low-abundance populations, also called the
‘rare biosphere’ [44,45].
About 40 years of investigations, together with major progress in method
development, have exposed the unseen majority and highlighted the ubiquitousness of microorganisms in marine environments. Typically, these studies have
focused on taxonomic diversity and the abundance of microorganisms. However, information on what the microorganisms are doing and how their
metabolic and regulatory capacities relate to the environment is, in several
aspects, uncharted. Developing a more advanced knowledge of what functions
microorganisms perform in the environment is therefore necessary in order to
increase our understanding of their role in, and impact on, ecosystem functions.
How to identify microbial functions from genomic data
Through adaptation to environmental conditions, evolution shapes the genomic
functional content of microorganisms, which leads to functional divergence
among microorganisms. For taxonomy to be ecologically meaningful, it would
therefore be suitable if any microbial taxon was a reflection of its genomic
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PETTER THUREBORN
functional content. This is not the case, however, since microbial taxonomy is
typically based on phylogenetic 16S rRNA gene analysis ([46] and references
therein). This discrepancy between functional divergence and phylogenetic
divergence of taxonomic marker genes evokes the question; What is the most
suitable approach to investigate microbial functions from genomic data, infer
function from taxonomy based on phylogenetic 16S rRNA gene analysis or from
analysis of functional genes (i.e. protein-coding genes) directly? Studies suggest
that species-function relationships exist and that phylogenetically closely related
taxa have generally more similar functions compared to distantly related species
[47]. However, any general relationship between 16S rRNA phylogeny and
microbial function may be difficult to establish. As pointed out by Fenchel [48]
the microbial taxonomic diversity estimated from neutral mutations of the 16S
ribosomal gene does not necessarily reflect functional diversity in terms of
physiological and morphological properties that better define the ecological
functions of the organism. This functional divergence is caused by substantial
genetic variations enabled by genome plasticity between phylogenetically closely
related species [49] and makes functional interpretation from 16S rRNA sequence data problematic. However, by adopting the concept of functional traits used
in plant and animal ecology, attempts have been made to define organisms from
their ecological roles in microbial ecology too [50,51]. A functional trait has to
be a measurable feature – that is linked to the organism’s fitness or performance
– and how to establish and identify them is a big challenge [52,53]. Detailed
understanding of functional traits, as for microbial ecology in general, is to be
found among microorganisms that have been readily cultivable in the laboratory
[52]. For example, functional traits were successfully identified for different
Prochlorococcus ecotypes (i.e. one species occupying different niches) by comparing their respective abundance along environmental gradients [54]. The
evidence for function as ecological determinant of microbial species has grown
[52,55,56] and it has been shown that environmental conditions decide the
occurrence of functional traits in microbial communities [57]. Given that functional traits are linked to the microorganisms’ fitness, the environment will
select for these microorganisms (i.e. environmental filtering). This is in agreement with the organism-centric Baas-Becking theory ‘everything is everywhere,
but the environment selects’ [58] and led Raes and colleagues to postulate the
function-oriented theory ‘all functions are everywhere, but the environment
selects’ [57]. It should be noted, however, that through their metabolism and
interaction with the environment microorganisms affect the abiotic environment. Hence, the relationship between microorganisms and the surrounding
environment is bidirectional. Nonetheless, comparisons of microbial functional
data from different environments have huge potential to reveal what functions
are favourable for certain environmental conditions.
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MICROBIAL ECOSYSTEMS FUNCTIONS
An aspect to consider when investigating microbial functions is to choose at
what ecological level this should be done. The ecological levels of microorganisms within a habitat range from individual- and populations of cells to an
integrated community [59]. Different populations within the community occupy
different niches (i.e. have different functional roles) and both positive and
negative interactions occur [60]. Integrated coexistence of different populations
in a contiguous environment, with for example metabolic cooperation [61,62,63]),
may be one reason for the widespread unculturability among microorganisms
[64,65]. It is through the cooperation and interactions between different microorganisms that microbial processes sustain important ecosystem functions and
make the microbial community an ecologically meaningful unit to study.
To increase the knowledge about microbial functions further investigations
on protein-coding genes are preferential over taxonomic markers, especially as
the direct analyses of metabolic turnover rates at all community levels is less
feasible. Combining functional genetic data with environmental parameters has
the potential to identify microbial functions that are dominant in specific
environments. Furthermore, it is important to better understand the complexity
of how microorganisms interact and together perform important functions in
the marine ecosystem. To do this, a method by which all populations in a community can be analysed simultaneously for their genetic functional information
is suitable.
Environmental community genomics and transcriptomics
Investigations of total microbial communities of different environments can be
achieved by using a suite of molecular methods; metagenomics, metatranscriptomics, metaproteomics and metabolomics, commonly referred to as environmental community ‘omics’ or meta’omics’. Within this thesis meta’omics’ includes only metagenomics (i.e. environmental community genomics) and metatranscriptomics (i.e. environmental community transcriptomics) that are based
on analysis of the nucleic acids DNA and RNA, respectively. Direct sequencing
of nucleic acids (i.e. DNA and RNA) from environmental samples or single cells,
bypasses limiting cultivation, PCR and cloning steps and enables access to all
genomes within a sampled environmental microbial community, see e.g. [66,67].
This facilitates investigation of profiles of functional capacity (i.e. protein-coding
genes) and activity (i.e. gene transcripts) and taxonomic composition of a total
community in its natural environment. Hence microbial studies are not restrictted to artificial laboratory setups and to selected populations. However, this
approach can also be applied in controlled laboratory experiments and reap
benefits. Importantly, metagenomics and metatranscriptomics can provide data
on microbial community functions and how these relate to biogeochemical
processes and ecosystem functions [68]. For example, comparative metageno17
PETTER THUREBORN
mics of microbial communities from totally different habitats [69,70], or along
environmental gradients [71,72,73,74] have been performed in order to link
microbial functions to environmental conditions. Correlation of extensive metagenomic data with environmental data may help to identify the drivers of metabolic pathways [75]. Similarly, metagenomic investigations of community functions have been instrumental in the prediction of ecosystem functions (e.g. primary production and nitrogen fixation) [57,75].
Consequently, meta‘omics’ has proved to be a successful approach in providing insight into microbial processes and interactions within microbial communities and how microbial community functions relate to changing environmental conditions. Thus, meta’omics’ is a suitable tool to use when continuing
to investigate the underexplored complexity of microbial processes and how
these are related to the environment.
18
Study system – the Landsort Deep, Baltic Sea
The Baltic Sea is a brackish water body with large riverine input of freshwater
from the surrounding drainage area and irregular inflow of saltwater from the
North Sea [76]. Saline inflows are forced by differences in sea levels, caused by
wind and water pressure, or the salinity gradient between the Baltic Sea basins
([77] and references therein). However, only large inflows of oxygenated water
have the potential to oxygenate the deep water because of the permanent vertical
density gradient [77]. The two latest inflows of oxygenated saltwater from the
North Sea occurred in 2003 and 2014 [77]. The Baltic Sea is divided into six different basins separated by sills including the Baltic Proper, Bothnian Bay,
Bothnian Sea, Gulf of Finland, Gulf of Riga and Kattegatt (Figure 1) that further
limit water mixing. The specific water regime and bathymetry cause a horizontal
salinity gradient from south to north (30 to 0 PSU) in the Baltic Sea and in the
Baltic Proper, the central Baltic Sea (Figure 1), this also creates a strong vertical
salinity gradient from surface to bottom (i.e. a halocline) that prevents mixing of
surface- and deep water. In the northern Baltic Proper, salinity ranges from 6 to
8 PSU in the surface water and up to 11 to 14 PSU in the bottom water. In combination with high heterotrophic respiration during mineralization of deposited
organic matter this results in stagnant, oxygen-depleted water below the halocline [78,79,80]. In the past century, a ten-fold increase in oxygen-depleted areas
has occurred in the Baltic Sea, mainly due to eutrophication and the water
regime [81]. This has resulted in one of the largest hypoxic or anoxic and sulphidic areas in the world [82]. Oxygen depletion alters the biogeochemical cycles
of nutrients and ecosystem energy flows [83,84], in which microbial populations
have an essential function [5]. Further expansion of oxygen-depleted areas in the
Baltic Sea is predicted in the future [85].
19
PETTER THUREBORN
Figure 1. The Baltic Sea.
The Baltic Sea with its six major basins; Kattegatt, Baltic Proper, Gulf of Riga, Gulf of Finland,
Bothnian Sea and Bothnian Bay. Red circle indicates the position of the Landsort Deep (LD),
the study system used in this thesis. Dotted lines show the boundaries of the Baltic Proper.
Original map retrieved from d-maps.com,
http://www.d-maps.com/carte.php?num_car=2072&lang=en
Microbial research studies in the Baltic Sea have investigated transitions of both
taxonomic [86] and functional [73] composition along a salinity gradient and
over seasons e.g. [87,88] in surface water. Furthermore, a number of studies have
investigated microbial community structure [89] and specific microbial functions e.g. methane oxidation [90], acetate uptake [91], nitrogen fixation [92],
denitrification [93,94], dark carbon fixation [22], sulphur oxidation [95,96] and
ammonia oxidation [97] in pelagic redoxclines. Microbial community structure
20
MICROBIAL ECOSYSTEMS FUNCTIONS
and activity [98,99] and specific functions e.g. denitrification and DNRA [100]
of coastal sediment communities have also been explored. However, investigations of total microbial community functions of the Baltic Sea are still in their
infancy, which is specifically evident for anoxic deep water and sediment. Investigation of total microbial community functions of the Baltic Sea, using a
meta’omics’ approach, has the potential to reveal novel information on microbial metabolic interactions in a brackish and eutrophied environment. Specifically, investigation of total microbial communities along the strong salinity and
oxygen gradients in the Baltic Sea may reveal important information. Such information is key for an improved understanding of how microbial ecosystem
functions are affected by environmental change.
The chosen study site for this thesis was Landsort Deep (LD) (lat 583591 N,
long 01814.26 E), the deepest point in the Baltic Sea (459 m), which is situated in
the North West Baltic Proper (Figure 1) and is one of the environmental monitoring sites, BY31. Because of the permanent halocline, which prevents mixing of
surface and bottom water, and the specific bathymetry and large depth that
restrain the rare intrusions of oxygenated water to reach the deep water, the
ventilation of the deep water at this site is minimal. Consequently, the oxygen
and salinity gradient at LD is the steepest and most persistent in the Baltic Sea.
Lamination of the LD sediment indicates anoxic conditions at the sediment
surface for the past ca. 4500 14C years [24]. It is important to point out that LD
sediment, as an unusually deep site of the Baltic Sea, is more environmentally
stable than many other bottom sites. It is in that sense a specific environment
and not in all aspects representative for the rest of the Baltic Sea. Such stable
anoxic conditions have been shown to increase preservation of DNA from dead
cells [101]. Furthermore, remnants of phytoplankton blooms and other particulate organic carbon (POC) sink and accumulate on the sediment surface,
resulting in some of the highest POC values measured in the world at the Baltic
Proper deep [102]. For these reasons, extreme environmental stratification and
permanent anoxia, LD provides a suitable system to investigate how microbial
ecosystem functions relate to the environment (e.g. different oxygen conditions)
and what microbial taxa and functions are active in organic carbon mineralisation in a stable anoxic environment.
21
Aim and objectives of the thesis
Microbial community processes are important for marine ecosystem functioning
[5]. It is also known that environmental change has an impact on microbial
community functions but not to what extent [103,104,105]. The limited detailed
information on how microbial communities relate to both biotic and abiotic
factors in marine environments is a hindrance for successful ecosystem simulation models [106]. To fill this knowledge gap it is of great importance to investigate in detail relationships among microbial processes and how these are
affected under different environmental conditions. At a fundamental level this
will improve our understanding of microbial ecosystem functions and ultimately
increase the ability to predict how marine ecosystems will be affected by environmental change.
With the prospect of a progression of oxygen depletion of marine environments globally and in the brackish and eutrophied Baltic Sea in particular, the
main aim of this thesis was to investigate and understand how microbial community functions relate to differences in oxygen conditions in a brackish and
eutrophied water ecosystem. Furthermore, this thesis aimed to investigate what
taxa and functions are active in anoxic sediment with high organic load as a
consequence of eutrophication. The specific objectives of this thesis were to
investigate i) what functional capacities and activities are characteristic for
microbial communities of the anoxic water and sediment ii) whether the specific
environmental conditions of the Baltic Sea are reflected in microbial community
structure and functional capacity. To meet these objectives, the following research questions were addressed, using the Landsort Deep as a study system:
Q1. How are functional capacities and taxa of microbial communities
stratified along a steep oxygen gradient in a brackish and eutrophied
system?
Q2. Given the relative isolation and strong environmental stratification
at Landsort Deep, do its microbial communities hold any unique taxa
and functional capacities compared to other environments?
Q3. Given that anoxic sediments are hot spots of preserved DNA from
dead cells and that deposition of organic carbon at the sediment in
eutrophied systems is high, what taxa and microbial community
functions were active in the anoxic sediment of Landsort Deep?
23
Methodological design, approach and considerations
Material for this thesis work was retrieved on two sampling cruises to Landsort
Deep. The first cruise was carried out on the 15th of April 2009 and included
sampling of microbial communities from surface water, mixed layer, deep water
and sediment (Paper I). The second cruise was accomplished on the 21st of April
2010 and included sampling of sediment (Paper II & III). For detailed information on sampling and processing of samples see respective paper.
To address the research questions three different methodological approaches
were used, all three are based on nucleic acids from total microbial communities
in their natural environment. In principle, the results from all three approaches
were generated in a similar way. Nucleic acids were extracted from the environmental sample and sequenced with high-throughput sequencing technologies.
Subsequently, the generated sequence data was checked for quality and compared
to databases of previously functionally and taxonomically annotated genes,
which enables interpretation of microbial metabolic metabolisms (Figure 2).
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PETTER THUREBORN
Figure 2. Microbial community metabolism drives ecosystem functions.
This model shows important ecosystem functions and how organisms via ecological interactions
sustain these (right panel). The left panel shows a metagenomics approach with the flow of
information from microbial (i.e. bacterial and archaeal) genomes to microbial functions.
Analyses of microbial genomes of the total microbial community have the potential to provide
information on hypothetical linkages of metabolisms and regulatory pathways between
different populations in the community. Ultimately, this information could improve the understanding on how microbial processes affect ecosystem function. POC, particulate organic carbon;
DOC, dissolved organic carbon; DMS, dimethyl sulphide. Modified from DeLong 2009.
26
MICROBIAL ECOSYSTEMS FUNCTIONS
To investigate how functional capacity and taxa of microbial communities were
stratified along the steep oxygen gradient of the Landsort Deep and to compare
LD microbial communities to microbial communities from other marine environments in Paper I, a metagenomics approach was used. Water and sediment
with distinct differences in environmental conditions (surface water, mixed
layer, anoxic- water and sediment) were sampled (Figure 3). Total community
DNA (i.e. the metagenome) was extracted from the different samples and used
to determine functional capacity and taxonomic composition of the microbial
communities. In order to identify what taxa were potentially active in the anoxic
Landsort Deep sediment, 16S rRNA analysis of total community RNA together
with 16S rDNA amplicon analysis was used in Paper II (Figure 3). With the
assumption that rRNA indicates intact ribosomes that are necessary for protein
synthesis, a ratio of taxa-specific 16S rRNA to 16S DNA was calculated and
applied in this thesis as an estimate of protein synthesis potential (PSP) [107] of
different taxa. In order to investigate what specific microbial functions were
active in the anoxic LD sediment in Paper III, a metatranscriptomics approach
was used. Through this approach, active microbial functions were identified
from total community mRNA (i.e. the metatranscriptome) that was extracted
directly from the sediment (Figure 3).
27
PETTER THUREBORN
Figure 3. Environmental characteristics and sampling strategy at Landsort Deep.
The figure presents an overview of which nucleic acids, DNA, rRNA, mRNA, that were sampled and analysed in Paper I, II and III, respectively. It also shows at what depth the samples
were retrieved and the steep oxygen, salinity and hydrogen sulphide gradients along the
Landsort Deep trench.
Meta’omics’ methods have many advantages that are outlined above (see Environmental community ‘omics’). However, there are also some limitations with these
methods. Technical challenges include development of DNA and RNA extraction
and sequencing methods that do not introduce bias towards certain genomes as
this makes comparative investigations less reliable [108,109,110,111,112]. The
short half-life of RNA is another problem during sampling and extraction
procedures. This could however be overcome by using RNA stabilising reagents
as in Paper II and III. Furthermore, the process to get meaningful functional
28
MICROBIAL ECOSYSTEMS FUNCTIONS
information from sequence reads including gene prediction and functional
annotation is not always straightforward. Gene prediction and functional annotation of sequence data are constrained by short read lengths, sequence
quality and large datasets without gene-linkage context, but improvements on
sequence read length combined with increased computer capacity and development of bioinformatics tools promise to resolve some of these problems [7]. In
Paper III, where short 100 base-pair paired-end Illumina reads was used,
developing an assembly-based bioinformatics pipeline circumvented these problems by increasing sequence length, which improved gene prediction and functional annotation. Meta’omics’ is also heavily reliant on compiled databases of
previously functionally annotated genes against which sequence data are
searched for functional assignment. These databases are still skewed against cultivated microorganisms and many genes have yet to be assignned a function
[113,114]. As a consequence, only about 20% of the potential genes in metagenomic and metatranscriptomic studies are typically assigned to a function [72].
Most probably improvements in cultivation techniques will improve meta‘omics’ methodologies and therefore a parallel evolution of these approaches is
preferable. Another drawback in analysis of total communities using meta’omics’ is that the harvested DNA and RNA sequences that are assembled into
metabolic pathways do not necessarily originate from the same cell or even
population of cells, which may cause over estimation of the metabolic capacity of
the community. Importantly, meta’omics’ methods generate qualitative data on
functional capacity and activity, but can neither be used as complete evidence
that microbial processes actually occur, nor can the data be used quantitatively
to estimate process rates. Notwithstanding, meta’omics’ approaches are unique
in the way they provide access to all genes and transcripts in a microbial
community, which provides researchers with an excellent opportunity to study
how microbial functions may interact with its biotic and abiotic environment.
Differences between DNA, rRNA and mRNA make them more or less suitable for certain investigations. Due to the high stability of the DNA molecule,
harvested environmental DNA may include both extracellular DNA (eDNA)
and intracellular DNA (iDNA) from deceased cells [115]. This is especially
important to consider when performing metagenomic analyses of sediment
communities, which receive organic material sinking through the water column
[116]. Neither does DNA analysis provide any evidence that the genes it encodes
are expressed. Consequently, interpretations of functional activity based on
DNA data can be misleading. However, the relative stability of the DNA
molecule makes it a reliable identifier of what is there in an environment, which
is good for comparisons over longer time scales and to get insight into integrated
terrestrial and marine ecological processes [115].
An advantage of RNA-based approaches is that rRNA, and particularly
mRNA, has a shorter half-life compared to DNA [117]. The presence of gene
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PETTER THUREBORN
transcripts in a sample is therefore a better indicator of microbial activity since it
is more probable that it reflects recent or ongoing gene transcription, and hence
more commonly used e.g. [72,118,119]. Ribosomal RNA forms secondary structures and is as a consequence more stable than mRNA. Other concerns about
using rRNA as an indicator of activity are the contradictory relationships
between rRNA, cell growth and activity, further complicated by the differences
in lifestyle between taxa [107]. However, by the presence of rRNA indicating
intact ribosomes, rRNA data can provide evidence of the potential for protein
synthesis [107]. Comparisons of abundance in rRNA molecules are problematic
because of differences in gene copy numbers in different genomes [107]. In
Paper II, this was taken care of by calculating a ratio of RNA to DNA, thus
producing a more comparable estimate of protein synthesis potential in different
taxa, compared to using RNA only. Not that the use of mRNA as a proxy for
microbial activity is without caveats, as it does not necessarily prove active
microbial processes. To date, detailed information on regulation of gene expression and stability of gene transcripts in organisms in the natural environment is
scarce, which limits the use of this method as an indicator of activity. Future
efforts combining metatranscriptomics with proteomics have the potential to
provide insight into the relationship between gene transcripts and protein
expression, although the presence of a protein does not indicate protein activity
per se [120]. A more stringent interpretation is to view gene transcripts as indicators of upregulation and downregulation of genes, as pointed out, for example,
by Prosser [121], since the regulation is a reflection of the influence that the
environment and the community interactions have on the microbial community. Importantly, however, from the central dogma of molecular biology that
describes the information flow in biological organisms from DNA replication,
transcription of DNA and translation of gene transcripts into proteins [122], it is
clear that RNA is a step closer to “real” activity in the form of active protein and
enzyme activity compared to DNA. Ribosomal RNA and mRNA in particular
provide a measurable biomarker that is highly sensitive to environmental
changes [123]. Therefore, metatranscriptomics is a more suitable method than
metagenomics with which to investigate cell activity and potential metabolic
activity of a total community and to provide new information on potential
microbial processes and interactions. Protein-coding gene transcripts can also
give taxonomic information, but a combined approach with 16S RNA gene transcripts, as used in this thesis, has an advantage because of the suitable taxonomic
resolution of the ribosomal gene.
30
Central results and Discussion
Q1. How are functional capacities and taxa of microbial communities
stratified along a steep oxygen gradient in a brackish and eutrophied
system?
Environmental stratification reflected in functional capacities
Given the substantial differences in environmental conditions across the four
sampled environments (surface water, mixed layer, anoxic- water and sediment)
at LD (Figure 3), investigations in Paper I sought to establish how microbial
communities with their functional capacities were stratified. Furthermore, it
sought to identify what changes in environmental conditions the functional
capacities most strongly responded to. Results revealed clear stratification of
both functional capacities and taxonomic composition across the LD transect, as
shown by correspondence analysis (CA) (Figure 2, Paper I). Stratification was
most evident for functional capacities, with 76% of the total variation corresponding to the first CA axis (CA1) compared to 59% for taxonomic composition.
This indicates that environmental conditions are better at predicting functional
gene repertoires than taxonomic composition at LD. Furthermore, it suggests
that some taxa may hold a gene repertoire that enables survival under different
environmental conditions across the LD transect. Both functional and taxonomic composition showed strongest response to changes in dissolved oxygen,
salinity and temperature, as shown by canonical correspondence analysis (CCA)
(Table S4, Paper I). Although the differences in temperature along the depth
gradient were small at LD it seem to have an impact on the microbial distribution, which is in agreement with what other researchers have found in the
oceans globally [124]. Salinity and dissolved oxygen are strongly correlated at
LD and it could not be distinguished which of the two had the strongest impact
on the microbial communities. Access to oxygen or not dramatically alters what
genetic prerequisites are needed for microbial respiration [5]. Thus, the clear
stratification of functional capacities and taxa found in Paper I is certainly to a
large extent a reflection of the distinct differences in oxygen availability in the
sampled water and sediment. The Baltic Sea is unique with its salinity gradient in
both lateral and vertical directions and its influence on microbial communities
in surface water is documented [86,125,126]. Results presented here suggest that
salinity may have an impact on microbial community structure and function,
also at deeper depths. Together, results in this thesis indicate that Baltic Sea
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PETTER THUREBORN
microorganisms have adapted to the unique oxygen and salinity regimes of the
Baltic Sea.
The strong stratification of microbial genes at LD, which is shown in Paper I,
revealed what functions (and taxa) were associated with certain oxygen and
salinity conditions. For example, the anoxic 400 m water column and the sediment LD communities were both overrepresented in hydrogenase and anaerobic
reductase genes, which are important for anaerobic respiration and hence for life
in an anoxic habitat. Furthermore, typical anaerobic taxa such as sulphatereducing deltaproteobacteria and methanogenic archaea were more abundant in
the anoxic environment. Interestingly, results revealed a comparatively high
abundance of genes associated with chemotaxis, pilus formation, quorum
sensing, biofilm formation and degradation of polysaccharides and amino sugars
(e.g. chitin). These are all genetic prerequisites for attachment to and utilisation
of organic material at anoxic conditions. Together with a comparatively high
functional capacity in motility, regulation and cell signalling and defence
mechanisms and a comparatively low capacity for fatty acid and secondary
metabolism (Figure 5, Paper I), results of this thesis suggest that functional
capacities of the anoxic LD communities are consistent with a copiotrophic lifestyle [127] dependent on marine snow including for example phytoplankton and
chitin-rich exoskeleton remnants [128] (Figure 4).
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MICROBIAL ECOSYSTEMS FUNCTIONS
Figure 4. Functional capacities of the microbial communities of Landsort Deep.
Selected functional characteristics of the Landsort Deep communities revealed by comparative
metagenomics in Paper I. Detected functional capacities (in red colour), suggest linkages
between nitrogen and sulphur metabolisms at the anoxic depth. Moreover, functional capacities suggest that the microbial communities are adapted to a copiotrophic life-style at depth,
dependent on sinking organic matter. A high capacity for genetic exchange was evident, particularly in the anoxic water and sediment. Org, organic carbon; Org-N, fixed organic nitrogen.
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PETTER THUREBORN
The high representation of genetic prerequisites for a copiotrophic life-style is
most likely an effect of high deposition of organic material caused by eutrophication in the Baltic Sea. The results are further supported by results from a
comparative analysis of LD communities with other marine microbial communities (Figure 5, Paper I). They showed that these functional traits were
shared with microbial communities from similar environments (i.e. Tonya Seep
[129] and Marmara Sea [130] sediment) further linking them to environments
with high loading of organic material. Ultimately, the results show that LD
microorganisms hold a functional capacity to profit from the current eutrophication situation, by utilising the organic material at anoxic depths returning carbon dioxide and nutrients to the pelagic food web.
Stratification and inter-linkage between nitrogen and
sulphur metabolisms
Microbial mediated biogeochemical cycling of elements links aerobic and
anaerobic processes and a large portion of the microbial processes are performed
only in habitats without oxygen [5]. One of the functional capacities that showed
a clear stratification across the oxygen gradient was functional capacity involved
in nitrogen transformations (Figure 3, Paper I). According to metagenomic
analyses, the nitrogen removal process denitrification was overrepresented
under anoxic conditions, where nitrate/nitrite were absent, compared to the
mixed layer with hypoxic conditions. Nitrogen removal through heterotrophic
denitrification is understood to decrease with increased oxygen depletion
because of its dependency on NO2- and NO3- through nitrification at oxic conditions [100,131]. Taxonomic assignment of the denitrification genes revealed
that more than 30% were best matched to the epsilonproteobacterium Sulfurimonas gotlandica GD1 (1% of total community at species level) that was
abundant at anoxic depth (Figure S2, Paper I). This species of Epsilonproteobacteria has the genetic capacity for chemolithotrophic denitrification through
sulphur oxidation [132], which is consistent with the results in Paper I that 20%
of the sulphur oxidation genes were linked to Epsilonproteobacteria in the
anoxic water at LD. Sulphur-oxidising Epsilonproteobacteria have been found to
be an important catalyst of chemolithoautotrophic denitrification and dark CO2fixation in the pelagic redoxcline at Gotland Deep, Baltic Sea [22,95]. Thus, the
high denitrification capacity of this organism in the anoxic water of Landsort
Deep as revealed in Paper I suggests that its important role in nitrogen and
sulphur transformations could be extended beyond the redoxcline into the
anoxic water (Figure 4). The importance of autotrophic denitrification in AMZs
globally has been highlighted since the recognition of a cryptic sulphur cycle in
these zones [17,23]. In the majority of these zones, however, autotrophic denitrification is linked to the Gammaproteobacteria 'SUP05' [23,133]. Notably, when
34
MICROBIAL ECOSYSTEMS FUNCTIONS
compared to the communities of Chile OMZs [23], the LD community of anoxic
water showed a comparatively high abundance of Sulfurimonas-like Epsilonproteobacteria and a low abundance of the SUP05-cluster of Gammaproteobacteria (Figure S3, Paper I). Fully sulphidic systems such as the Baltic Proper
basins differ in chemistry to the majority of the world's AMZs [17]. Results in
this thesis suggest that sulphur-oxidising Epsilonproteobacteria have the necessary genetic requirements, compared to SUP05, to be the main denitrifier in the
euxinic parts of the Baltic Sea as well as in other euxinic environments such as
the Black Sea, Cariaco Basin, Saanich Inlet and Namibian Shelf.
Another important nitrogen removal process under anoxic conditions is
anaerobic ammonia oxidation (anammox) where ammonia is anaerobically
oxidised using NO2- as electron acceptor [134]. No genes for the anammox
process were detected in the water and sediment that were analysed in Paper I &
III. Possibly, the absence of functional anammox genes in the LD metagenomes
may be a consequence of finite sampling. However, these results are consistent
with another study at LD [135] where no anammox activity was detected at
similar depth (77 m) as the mixed layer (75 m) in Paper I. In addition, a high
abundance of denitrification genes detected at LD (Paper I) suggests that
anammox is relatively irrelevant for nitrogen removal at LD and possibly not in
the Baltic Sea open water.
The relative contribution of denitrification and anammox in marine nitrogen
removal has been proposed to be controlled by organic matter [28]. This suggests that the absence of anammox capacity could be a reflection of high carbon
loading [102] at LD. Studies of the relative contribution of denitrification and
anammox in the oceans, however, have been focused on heterotrophic denitrification and are most extensively studied in the oceans’ OMZs [28]. Results of
this thesis, Paper I, suggest that autotrophic denitrification is important for
nitrogen removal in sulphidic environments, such as LD, and that organic
matter is not the only factor that controls the relative contribution of the different nitrogen removal processes.
Notably, results in Paper I showed a high abundance of sulphide insensitive
and ammonia oxidising Thaumarchaeota (Nitrosopumilus maritimus) in the LD
anoxic water (7.1%) and mixed layer (6.2%) (Figure S2, Paper I). These
numbers were also found to be high when compared to other marine environments (Figure S3, Paper I). Results also revealed functional capacity for archaeal
ammonia oxidation (AOA) through the presence of archaeal amoABC genes (n
= 9) at these depths. These findings are in agreement with other studies of ammonia oxidation at suboxic depth [97,136,137] and suggest an important role for
archaeal ammonia oxidation at suboxic depths in the Baltic Proper. However,
the high abundance of Thaumarchaeota in the deep anoxic water at Landsort
Deep as revealed in Paper I was more unexpected, as completely anoxic waters
are typically occupied by anammox bacteria [138,139,140]. These, however, were
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PETTER THUREBORN
only detected at low numbers in anoxic LD water and for which no hydrazine
oxidoreductase genes (hzo) were detected (see above) (Paper I). On the other
hand, nitrification activity [141,142] and gene expression of aerobic ammonia
oxidation genes [143] in different AMZs suggest there is potential for archaeal
aerobic ammonia oxidation in these environments too [17]. Additionally, Baltic
Sea Thaumarchaeota has been shown to persist under high sulphide concentrations and actively oxidise ammonia at low sulphide concentrations [136].
Whether nitrification is active in the deep anoxic and sulphidic water at
Landsort Deep or a reflection of a latent nitrification capacity that becomes
activated by occasional oxygen intrusions remains an open question. As nitrification would fuel autotrophic denitrification, such latent capacity could be of
particular importance for removal of ammonia that has accumulated in-between
inflows of oxygenated water at Landsort Deep. Interestingly, as indicated by the
comparatively high abundance of Thaumarchaeota at Landsort Deep compared to
other environments (Paper I), this would possibly reflect an adaptation to the
irregular fluctuations of chemistries in Baltic Sea basins, which is a consequence of
the special bathymetry and water regimes of this environment.
Results in Paper I revealed further potential linkage of nitrogen and sulphur
metabolisms at LD through the identification of the minimum gene set (nifHDK
and nifENB) required for nitrogen fixation [144] in genomes of sulphatereducing deltaproteobacteria in the anoxic deep water and sediment. The
functional capacity for nitrogen fixation was clearly enriched in the anoxic water
and sediment compared to surface water and mixed layer at LD (Figure 3, Paper
I) and showed strong correlation to both distribution of sulphate reduction
capacity (Pearson’s r = 0.94) and Deltaproteobacteria (r = 0.95) across the LD
transect. The lack of functional capacity for nitrogen fixation in LD surface
water could probably be explained by the sampling time, i.e. during spring
bloom that consists of phytoplankton rather than diazotrophic cyanobacteria,
and removal of filamentous diazotrophs by filtering. High concentration of
ammonium, as it is below the halocline at LD, is typically considered to restrict
nitrogen fixation [145]. However, heterotrophic diazotrophs have been reported
in a set of marine OMZs [146,147,148] and not least in suboxic and anoxic water
at Gotland Deep and Bornholm Basin in the Baltic Sea [92]. This corroborates
the findings in Paper I, which suggest that sulphate-reducing deltaproteobacteria may be a key diazotroph in sulphidic environments of the Baltic Sea.
In all, this thesis showed how the characteristic salinity and oxygen stratification of the LD transect were reflected in functional capacities and taxonomic
composition of microbial communities. Metagenomic data revealed functional
traits associated with a life-style dependent on sinking organic matter at anoxic
depths, clearly indicating the effect of eutrophication on these communities.
Interestingly, results in this thesis also reveal new potential links between nitro-
36
MICROBIAL ECOSYSTEMS FUNCTIONS
gen and sulphur metabolisms that may be characteristic of the euxinic conditions that prevail below the halocline in the Baltic Sea.
Q2. Given the relative isolation and strong environmental stratification
at Landsort Deep, do its microbial communities hold any unique taxa
and functional capacities compared to other environments?
Similarities of LD communities to communities of other
marine environments
Extreme stability of the stratification at LD and the relative isolation of the LD
deep water and sediment make it very different from most other marine environments. This thesis work investigated whether LD microbial communities
show both taxonomic makeup and functional capacities similar to microbial
communities from similar environments, or whether extreme stability and
isolation of the environment at LD, particularly below the halocline, has resulted
in unique communities with more similar genetic repertoires than that of other
communities. To answer this question the functional capacity and taxonomic
profiles were compared through comparative metagenomic analysis of 11 different sites and 27 metagenomes from marine water columns and sediment, but
also from soil and compost (Paper I). Results showed that the anoxic water and
sediment LD communities were more similar to each other than to any other
community in terms of taxonomic composition. These communities showed
highest similarities to communities of other hypoxic and anoxic sediment. This
was apparent both for taxonomic makeup and functional capacities (Figure 4,
Paper I). Similarities between these communities included enrichment in
functional capacities of copiotrophic, anaerobic metabolisms and taxa such as
sulphate-reducing bacteria and methanogens. Possibly, these similarities are a
reflection of shared environmental characteristics such as anoxia and high nutrient and sedimentary load. Furthermore, results in Paper I revealed that the LD
surface water community was most similar to communities of surface water of
the Western English Channel. This possibly reflects the shared coastal environmental characteristics including the receipt of organic material and nutrients via
riverine input [149,150]. The similarity was most distinct for taxonomic composition with enrichment of taxa like Sphingobacteriaceae, unclassified Flavobacteria and Methylophilales (Figure S3, Paper I), which are associated with
high levels of organic particles [151]. The LD community from the mixed layer
was an outlier in terms of taxonomic composition, but showed most similarity in
functional profiles to two deep water communities, from the Marmara Sea (1
km)[130] and the Puerto Rico Trench (6 km) [152] (Figure 4, Paper I).
Speculatively, this is an effect of the eutrophication and limited water mixing
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PETTER THUREBORN
that results in stagnant conditions and hypoxia shifting high up in the water
column at LD.
Consequently, with the exception of taxonomic composition of the anoxic LD
water and sediment communities, results in Paper I show that LD communities
of different water depth are more similar to other communities of similar environments than to each other, emphasising the strong stratification of the communities in LD. Other studies similarly show that biogeochemical conditions are
a good predictor of microbial functional capacities [70,153]. In this thesis,
functional capacities associated with copiotrophic metabolism were successfully
shown to be associated with anoxic marine environments with high organic
load, by using comparative metagenomics. This underscores the strength of this
method to identify relationships between microbial community functions and
environment.
Characteristic taxa of Landsort Deep
As shown by the comparative metagenomics analyses, Paper I, and as described
above, similar environments contain microbial communities with similar
functional and taxonomic profiles. However, the comparative metagenomics
results also reveal taxa characteristic for the LD communities. In the surface
water community, Flavobacteria was significantly overrepresented compared to
communities from other habitats, including the Western English Channel – the
most similar waters in the analysis. Flavobacteria are common in coastal waters
and have the capacity to transform a variety of carbon sources [151,154]. Indeed,
a third of the functional genes involved in degradation of organic matter could
be matched to Flavobacteria, as shown in Paper I. These results support Flavobacteria as an important actor in transforming carbon from algal blooms [155]
and riverine inputs [156] in the brackish Baltic Sea surface waters. Other researchers have proposed a global brackish microbiome as a result of strong
selection pressure in brackish environments [126] and an autochthonous estuarine
community have been suggested to inhabit the brackish waters of the Baltic Sea
[157]. Interestingly, metagenomics results presented in this thesis suggest that
the Baltic Sea environment has uniquely shaped flavobacterial genomes.
A striking feature of the microbial community at LD was the five-fold higher
abundance of the actinobacterial genus Mycobacteria in the sediment metagenome compared to other metagenomes included in the analysis (Figure S3,
Paper I). Most of the Actinobacterial sequences were best matched to aerobic
pathogenic Mycobacteria and indicates that their presence in the LD sediment
could be a result of sedimentation from the oxygenated water column. However,
while the phylum Actinobacteria was relatively abundant across the whole LD
transect, Mycobacteria was significantly overrepresented in the sediment. Even
though Mycobacteria was low in numbers in the water column, the extremely
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MICROBIAL ECOSYSTEMS FUNCTIONS
high abundance in the sediment could be a result of sedimentation and accumulation of mycobacterial cells. However, the large difference in abundance of
Mycobacteria between water column and sediment opens for the possibility that
these bacteria are indeed autochthonous for the LD sediment. Results in Paper
II showed that Mycobacteriaceae had as high PSP as families of Deltaproteobacteria and Chloroflexi, which are typically expected to be active members of
the sediment community. Furthermore, findings in Paper III that Actinobacteria, including Mycobacteria, was the fourth most abundant taxa in the
sediment metatranscriptome strongly suggest that Mycobacteria is indeed a
member of a local and active sediment community at LD (Figure 5). In studies
by others, growth of strains of Mycobacteria have been shown to be stimulated
by fulvic and humic acids [158], which are both common constituents of Baltic
Sea sediments [159]. Results in Paper I & III furthermore indicate that mycobacterial functional capacity and activity are associated with the degradation of
organic compounds. Thus, results in this thesis clearly suggest that Mycobacteria
could play an active role in anaerobic degradation of organic compounds in the
Landsort Deep sediment.
Figure 5. Active community transformation of organic matter in the LD sediment.
This overview summarises the most important results from analysis of total RNA in Paper II
and III. Community transcripts strongly indicate active carbon transformation via enzymatic
hydrolysis, fermentation, dissimilatory sulphate reduction, methanogenesis and anaerobic
methane oxidation. A high abundance of cyanobacterial rRNA and mRNA was identified in
the sediment and incites questions about what, if any, functional role they may have in the
anoxic sediment. Furthermore, the overview illustrates potential genetic exchange through
integron recombination, as indicated by high integron integrase transcript abundance in the
LD sediment. VFA, volatile fatty acids.
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PETTER THUREBORN
Environmental adaptation
Short generation times, large population sizes and high dispersal rates make
microorganisms well equipped to adapt to different environmental conditions
[160,161,162]. Importantly, these organisms also undergo events of horizontal
gene transfer facilitated by different mobile genetic elements (MGEs) like
plasmids, transposons and integrons or via viral infection [163,164]. Horizontal
gene transfer provides a mechanism through which advantageous genes can be
spread rapidly within the microbial community and hence increase the potential
of adaptation to environmental change [165,166]. Against this backdrop it was
interesting that results in Paper I revealed a comparatively high abundance of
integrons in the LD communities (Figure S6, Paper I). Whereas the high integron abundance was most marked for sediment and deep sea habitats in the
comparative metagenomics analysis, which is in agreement with results of others
[71,139,167], it was also a characteristic feature of LD surface and mixed layer
when compared to metagenomes of other marine surface water. Integrons are
mobile genetic elements with a potential to facilitate dissemination of a large
pool of adaptive genes between microbial genomes [168]. Integron recombination events can be induced by environmental stress (i.e. a change in environmental conditions that is negative for a given microorganism) [169]. Thus an
increased frequency of integrons could be interpreted as a reflection of previous
disturbance but also a capacity for future or ongoing short-term environmental
adaptation. High abundance of integron genes has been found to correlate with
increased levels of environmental pollution [170,171,172] and the class 1 integron-integrase gene has been proposed as a proxy for anthropogenic pollution
[173]. High integron abundance in the LD communities may be a reflection of
the documented pollution at this site [174]. Interestingly, the LD sediment (same
sample as in this thesis) has been shown to contain a high abundance of IncP
alpha plasmids, which are also mobile genetic elements that may increase the
recipient’s fitness in polluted environments [175]. Moreover, findings in Paper I
point to a high abundance of resistance genes for cobalt, cadmium and zinc in
the anoxic 400m and sediment LD communities. Most likely this reflects the
high heavy metal concentrations at Landsort Deep [174,176]. Generally, this
may reflect the biogeochemistry of this environment with adsorption of the
metals to biogenic material, or the formation of sulphides, sedimentation and
accumulation of particles onto the sediment [177]. This was corroborated in the
comparative metagenomics analysis, which showed that also Marmara Sea and
Tonya Seep sediments and Puerto Rico Trench deep water likewise showed a high
abundance of cadmium-, cobalt- and zinc resistance genes (Figure S6, Paper I).
The high integron abundance in the LD sediment was further supported by
comparatively high expression of the integron integrases in the LD sediment,
which was evident from comparative metatranscriptomic analysis (Figure S2,
40
MICROBIAL ECOSYSTEMS FUNCTIONS
Paper III). As genetic recombination in bacteria can be induced by a change in
environmental conditions [169], results in this thesis thus suggest ongoing
genetic recombination in the LD sediment microbial community, which may
provide this community with a mechanism for short-term adaptation to environmental change (Figure 5). Together with other factors (e.g. short generation time and high mutation rate), integrons may be a reason that no difference
could be observed in species richness along the Baltic Sea salinity gradient [86],
which is in clear contrast to the decline in species richness of macroorganisms,
which lack the capability for genetic mobility, in brackish waters [178]. A
successful integron mediated response to environmental change would, however, need the transferred gene to have a function that increases the fitness of the
recipient microorganism. In the future, identification of the genes carried on
integrons in the LD communities thus has potential to reveal what adaptive
strategies are of importance for microbial communities in a brackish environment under anthropogenic pressure.
In this thesis, characteristic taxa and functions of LD communities were
revealed by comparative metagenomics and metatranscriptomics. Characteristic
taxa for the Baltic Sea were identified and may reflect novel functional adaptations to the specific environmental conditions that prevail in brackish and
eutrophied systems. As fine-tuned functional adaptations would not stand out in
functional annotation, in-depth analysis of the genomes of characteristic taxa
may be a way forward in identifying key functions of microorganisms that thrive
in the Baltic Sea. The identification of the presence of integrons that may go
through recombination suggests a possible mechanism for mobile genetic
exchange. The detection of high integron abundance and expression, which has
a potential to increase environmental fitness of microorganisms, merits further
studies on integrons as these may carry important information on functional
adaptations to the Baltic Sea environment.
Q3. Given that anoxic sediments are hot spots of preserved extracellular
DNA and that deposition of organic carbon at the sediment in
eutrophied systems is high, what taxa and microbial community functions were active in the anoxic sediment of Landsort Deep?
Pelagic – seafloor connectivity
In Paper I, metagenomic analyses exposed a diverse repertoire of bacterial and
archaeal genes in the Landsort Deep sediment. Results also revealed that a significant portion (85%) of the species detected in the LD surface water metagenome were also present in the LD sediment metagenome. That is a very high
overlap in taxonomic composition compared to the 10% overlap Zinger and
colleagues [154] found between pelagic and benthic realms globally, using 16S
41
PETTER THUREBORN
ribosomal DNA as phylogenetic marker. It should be noted, however, that the
large overlap in Paper I is probably overestimated, caused by the approach used
in this study (i.e. analysing protein-coding genes instead of 16S rRNA genes).
Still, the presence of phototrophic bacteria, e.g. Cyanobacteria spp., typically
aerobic bacteria Flavobacteria, and Mycobacterium spp. in the LD sediment,
raised the question of which portion of the diversity was attributed to active taxa
and which was just a signal of preserved eDNA or iDNA from dead cells.
In Paper II, results revealed large variations in protein synthesis potential
(PSP) (total RNA/rDNA ratios) among operational taxonomic units (OTUs)
(Figure 2, Paper II). More than 50% of the OTUs detected had a PSP < 0.5,
which suggests that many cells were in a phase of degradation when the sediment was sampled. This is in agreement with observations that most of the
microbial cells in the environment are inactive, dormant or dead [179] and that
marine anoxic sediments are hotspots of eDNA [101]. However, results also
revealed three taxonomic groups that had significantly higher totalRNA/rDNA
ratio distributions (sign test, p < 0.05) among their OTUs compared to the community median. Interestingly, these included not only Deltaproteobacteria that
are known to contain many anaerobic species [180], but also Actinobacteria,
including Mycobacteriaceae, and Cyanobacteria (Figure 2, Paper II).
Cyanobacteria in particular had a very high protein synthesis potential (i.e.
ten times higher compared to other taxa in the LD sediment) (Figure 2, Paper
II). These results were corroborated by the fact that the cyanobacterial mRNA
transcript to gene ratio was the highest identified in the metatranscriptomic
analysis (Figure 4, Paper III). Cyanobacteria of the LD sediment was dominated
by unicellular Synechococcus spp. that was three times more abundant compared to filamentous bloom-forming Anabaena spp. (Paper II). Both Synechococcus spp. and Anabaena spp. temporarily reach high numbers in Baltic Sea
surface water during summer [157,181] and ultimately sink through the water
column and provide nutrients to the benthic communities [182,183,184].
Nonetheless, the high PSP of Cyanobacteria detected in Paper II was unexpected
given the hypothesis that their presence signals a sediment graveyard as a result
of Baltic Proper marine snow. Since other common Baltic Sea surface water such
as Flavobacteria (Paper I) and Verrucomicrobia [185] had much lower PSP in
the sediment as shown in (Figure 2, Paper II), the questions arise: Why do the
Cyanobacteria show such high PSP? Could they contribute actively to the ecosystem functioning in the sediment?
The extremely high ratio of ribosomal RNA to DNA in Cyanobacteria may at
least partly be a reflection of their physiology. Large numbers of protein bound
in photosystems in thylakoid membranes that need frequent replacement due to
photo-induced damage would need large numbers of membrane-bound ribosomes to replenish photosystems.
42
MICROBIAL ECOSYSTEMS FUNCTIONS
Possibly the higher PSP in Cyanobacteria signals a physiological preparedness
for changing environmental conditions. Such a feature would be consistent with
the fact that filamentous Cyanobacteria which enters a dormant state forms
dormancy cells (akinetes) that contain higher numbers of both ribosomes and
genome copies compared to vegetative cells [186]. Furthermore, benthic akinetes
have been suggested to have an important role in overwintering and bloom
dynamics of Baltic Sea Anabaena species as they migrate to the surface water
[187,188]. However, even though LD sediment Cyanobacteria may have physiological preparedness for future migration to surface water, it seems less likely
that they would do so from 500 m depth at LD given the current knowledge of
buoyancy regulation in oceanic cyanobacteria [189]. Somewhat different observations have been made for Synechococcus, for which growth rates have been
shown to be positively correlated with RNA and DNA content in both freshwater and marine Synechococcus spp. [190]. Moreover, the same authors found
that the RNA/DNA ratio was positively correlated to growth rate in marine
Synechococcus sp. strain WH 8103. By using diurnal batch cultures Lepp and
Schmidt [190] also showed that nucleic acids (i.e. DNA, RNA, 16S rRNA)
increased during periods of darkness and decreased during periods of light in
Synechococcus sp. strain PCC 6301 and proposed it to be a reflection of a period
of de novo synthesis without concurrent cell division. Consequently, the high
PSP of Cyanobacteria revealed in this thesis could be either a reflection of
growing cells or cells arrested in a “dark” mode with no cell division in the dark
Landsort Deep sediment.
These results suggest that the Cyanobacteria have the potential to be active in
the deep sea sediment. Cyanobacterial species have previously been reported to
grow heterotrophically under aerobic [191,192] and anoxic [193] dark conditions and recently Melainabacteria, a new class within Cyanobacteria with
capacity for fermentative metabolism, was identified [194,195]. However, the
transcriptome investigated in Paper III did not reveal any cyanobacterial metabolic activity. Analysis of the cyanobacterial transcriptome revealed a high
abundance of transcripts encoding the cold-shock DEAD-box protein A. This
protein has been suggested to be involved in ribosome biogenesis, RNA turnover
and translation initiation and cyanobacterial stress response and adaptation to
low temperature [196,197], which could indicate a role in adaptation to low
temperature for the LD sediment Cyanobacteria.
From results presented in this thesis it seems that Cyanobacteria, probably
recruited from the water column, are indeed alive in the LD sediment (Figure 5).
The finding that sedimented pelagic Cyanobacteria could be viable members of
the anoxic sediment, with a high potential for protein synthesis, challenges the
current view of sedimenting cyanobacterial blooms as a carbon source for
heterotrophic growth only. Whether these Cyanobacteria are metabolically
active, growing, dormant or just waitin’ around to die in the LD sediment is not
43
PETTER THUREBORN
yet fully clear and merits further studies on cyanobacterial physiology in dark
and anoxic environments.
Active carbon transformations in the anoxic sediment
Microbial processes on the oceans’ seafloors are important for marine carbon
cycling. They determine how large portions of deposited organic matter are
stowed away over geological time and how much is mineralised and returned to
the ocean as CO2 and inorganic nutrients. As preservation of organic matter has
been shown to increase anoxic sediments [198] and the deposition of organic
material at the Baltic Proper seafloor is one of the highest in the world [102], this
thesis work investigated, by metatranscriptomics, whether the LD sediment
microbial community was active in carbon mineralisation.
Results in Paper III revealed that a third of the total functional capacity in the
LD sediment that was captured (i.e. the metagenome) was expressed and that a
metabolically active microbial community inhabits the anoxic deep sea Baltic
Proper sediment.
Gene transcripts with importance for enzymatic hydrolysis of organic matter
indicated active degradation of organic matter into smaller organic compounds
in the sediment. The abundance of these transcripts was comparatively high in
relation to sediments of other environments (Figure S2, Paper III), which may
be a reflection of the high deposition of organic carbon at LD [102]. Since suites
of gene transcripts associated with the anoxic metabolic processes fermentation,
dissimilatory sulphate reduction and methanogenesis could be identified, this
study strongly indicates active transformation of organic carbon at this site,
deposited as a consequence of eutrophication (Figure 6, Paper III).
Specifically, the LD sediment community displayed transcription of the complete methanogenic pathway. Gene transcripts were identified for all methanogenic pathways from CO2, acetate, methanol and methylamines, respectively
(Paper III). Combination of taxonomic information and transcript abundance
data suggests CO2 as the main electron acceptor for this process in LD sediment
(Table S6, Paper III). Moreover, transcripts encoding the methanogenic key
enzyme methyl coenzyme M reductase (mcrA) were clearly overrepresented in
the LD sediment compared to Peru Margin sub-seafloor [199] and Arctic Jan
Mayen vent field [200] sediments (Figure S2, Paper III). The identification of
methanogenesis as one of the major active ecosystem processes at this site corresponds well with both the metagenomic results (Paper I) and is corroborated by
the high methane concentrations in the anoxic water of Landsort Deep, shown
by other researchers [90,201]. Interpretation of transcripts of methanogenic
genes is however cryptic since the methanogenic pathway could be driven in
both directions, in reverse catalysing anaerobic oxidation of methane (AOM)
[202]. Indeed, both methanogenic archaea and anaerobic methanotrophic
44
MICROBIAL ECOSYSTEMS FUNCTIONS
archaea (ANME) were found to be active members of the LD sediment community, according to phylogenetic analysis of transcripts of the methanogenic
key gene mcrA (Figure S6, Paper III). Results also revealed transcripts for the
particulate methane monooxygenase (pMMO) from the methanotrophic
methylococcaceae. This enzyme converts methane to methanol in methanotrophic bacteria [203]. Methane has been shown to be oxidised aerobically by
bacteria in the LD redox zone at 70-115 m, but only proposed to be anaerobically oxidised by ANME at anoxic depth [90]. One mechanism through which
methane can be oxidised anaerobically is with nitrate as electron acceptor [203].
Concurrent presence of transcripts for nitrate and nitrite reductase with pMMO
transcripts, as shown in Paper III, thus opens for the suggestion that Methylococcaceae oxidises methane by nitrate reduction in the anoxic LD sediment.
Importantly, results in this thesis show active transformations of organic
carbon in the anoxic LD sediment (Figure 5). Mutual evidence of active fermentation, sulphate reduction and methanogenesis indicates efficient mineralisation
of organic carbon into methane by heterotrophic-autotrophic community
synergism, which is anaerobically oxidised by both Archaea and Bacteria. Active
microbial mineralisation in the Landsort Deep sediment shows that all organic
matter reaching the seafloor as a consequence of eutrophication is not buried in
the sediment and should not be considered as carbon sink only. Instead, through
microbial activities, the sediment functions as a regenerator of previously fixed
CO2 and nutrients, making it available again to the organisms in the ecosystem.
45
Conclusions
This thesis work documents for the first time how the extreme and vertical
oxygen gradient in the Baltic Proper is reflected in the functional capacity and
taxonomic composition of the microbial communities that inhabit these waters
and sediments. The unique oceanographic characteristics and eutrophication of
the Baltic Proper that results in accumulation of organic material, oxygen
depletion and microbial production of sulphide were reflected in functional
capacities of the communities. Functional capacities of deep anoxic water and
sediment communities clearly indicate a life-style adapted to sinking organic
material. In addition, they suggest new links between carbon, nitrogen and
sulphur cycling in the sulphidic water of the Baltic Sea. Furthermore, this thesis
provides new qualitative information on how organic matter is actively
mineralised in the anoxic sediment at Landsort Deep and shows that the deep
water sediment does not only function as a carbon sink. Results further suggest
that the unique environmental characteristics of the Baltic Sea have sculpted
some characteristic taxa, but also that the microbial communities make use of
mechanisms for short-term adaptation to a changing environment. Importantly,
this thesis work advances the understanding, at genomic and transcriptional
level, of microbial ecosystem functions in deep anoxic water and sediments.
Information retrieved here provides mechanistic insight into biogeochemical
processes valuable for biogeochemical modeling. Specifically, with the current
progression of oxygen depletion in the Baltic Sea and globally, information
generated in this thesis is important to include in modeling efforts to predict
how ecosystem functions are affected by increased anoxia.
47
Acknowledgements
Tack till alla er som bidragit, hjälpt, lyssnat och stöttat. Ni vet vilka ni är.
49
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Södertörn Doctoral Dissertations
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Jolanta Aidukaite, The Emergence of the Post-Socialist Welfare State: The case of the Baltic
States: Estonia, Latvia and Lithuania, 2004
Xavier Fraudet, Politique étrangère française en mer Baltique (1871–1914): de l’exclusion à
l’affirmation, 2005
Piotr Wawrzeniuk, Confessional Civilising in Ukraine: The Bishop Iosyf Shumliansky and the
Introduction of Reforms in the Diocese of Lviv 1668–1708, 2005
Andrej Kotljarchuk, In the Shadows of Poland and Russia: The Grand Duchy of Lithuania
and Sweden in the European Crisis of the mid-17th Century, 2006
Håkan Blomqvist, Nation, ras och civilisation i svensk arbetarrörelse före nazismen, 2006
Karin S Lindelöf, Om vi nu ska bli som Europa: Könsskapande och normalitet bland unga
kvinnor i transitionens Polen, 2006
Andrew Stickley. On Interpersonal Violence in Russia in the Present and the Past: A
Sociological Study, 2006
Arne Ek, Att konstruera en uppslutning kring den enda vägen: Om folkrörelsers modernisering i skuggan av det Östeuropeiska systemskiftet, 2006
Agnes Ers, I mänsklighetens namn: En etnologisk studie av ett svenskt biståndsprojekt i
Rumänien, 2006
Johnny Rodin, Rethinking Russian Federalism: The Politics of Intergovernmental Relations
and Federal Reforms at the Turn of the Millennium, 2006
Kristian Petrov, Tillbaka till framtiden: Modernitet, postmodernitet och generationsidentitet i
Gorbačevs glasnost’ och perestrojka, 2006
Sophie Söderholm Werkö, Patient patients?: Achieving Patient Empowerment through Active
Participation, Increased Knowledge and Organisation, 2008
Peter Bötker, Leviatan i arkipelagen: Staten, förvaltningen och samhället. Fallet Estland, 2007
Matilda Dahl, States under scrutiny: International organizations, transformation and the
construction of progress, 2007
Margrethe B. Søvik, Support, resistance and pragmatism: An examination of motivation in
language policy in Kharkiv, Ukraine, 2007
Yulia Gradskova, Soviet People with female Bodies: Performing beauty and maternity in
Soviet Russia in the mid 1930–1960s, 2007
Renata Ingbrant, From Her Point of View: Woman’s Anti-World in the Poetry of Anna
Świrszczyńska, 2007
Johan Eellend, Cultivating the Rural Citizen: Modernity, Agrarianism and Citizenship in Late
Tsarist Estonia, 2007
Petra Garberding, Musik och politik i skuggan av nazismen: Kurt Atterberg och de svensktyska musikrelationerna, 2007
Aleksei Semenenko, Hamlet the Sign: Russian Translations of Hamlet and Literary Canon
Formation, 2007
Vytautas Petronis, Constructing Lithuania: Ethnic Mapping in the Tsarist Russia, ca. 1800–
1914, 2007
Akvile Motiejunaite, Female employment, gender roles, and attitudes: the Baltic countries in a
broader context, 2008
Tove Lindén, Explaining Civil Society Core Activism in Post-Soviet Latvia, 2008
24. Pelle Åberg, Translating Popular Education: Civil Society Cooperation between Sweden and
Estonia, 2008
25. Anders Nordström, The Interactive Dynamics of Regulation: Exploring the Council of
Europe’s monitoring of Ukraine, 2008
26. Fredrik Doeser, In Search of Security After the Collapse of the Soviet Union: Foreign Policy
Change in Denmark, Finland and Sweden, 1988–1993, 2008
27. Zhanna Kravchenko. Family (versus) Policy: Combining Work and Care in Russia and
Sweden, 2008
28. Rein Jüriado, Learning within and between public-private partnerships, 2008
29. Elin Boalt, Ecology and evolution of tolerance in two cruciferous species, 2008
30. Lars Forsberg, Genetic Aspects of Sexual Selection and Mate Choice in Salmonids, 2008
31. Eglė Rindzevičiūtė, Constructing Soviet Cultural Policy: Cybernetics and Governance in
Lithuania after World War II, 2008
32. Joakim Philipson, The Purpose of Evolution: ‘struggle for existence’ in the Russian-Jewish press
1860–1900, 2008
33. Sofie Bedford, Islamic activism in Azerbaijan: Repression and mobilization in a post-Soviet
context, 2009
34. Tommy Larsson Segerlind, Team Entrepreneurship: A process analysis of the venture team
and the venture team roles in relation to the innovation process, 2009
35. Jenny Svensson, The Regulation of Rule-Following: Imitation and Soft Regulation in the
European Union, 2009
36. Stefan Hallgren, Brain Aromatase in the guppy, Poecilia reticulate: Distribution, control and
role in behavior, 2009
37. Karin Ellencrona, Functional characterization of interactions between the flavivirus NS5
protein and PDZ proteins of the mammalian host, 2009
38. Makiko Kanematsu, Saga och verklighet: Barnboksproduktion i det postsovjetiska Lettland,
2009
39. Daniel Lindvall, The Limits of the European Vision in Bosnia and Herzegovina: An Analysis
of the Police Reform Negotiations, 2009
40. Charlotta Hillerdal, People in Between – Ethnicity and Material Identity: A New Approach to
Deconstructed Concepts, 2009
41. Jonna Bornemark, Kunskapens gräns – gränsens vetande, 2009
42. Adolphine G. Kateka, Co-Management Challenges in the Lake Victoria Fisheries: A Context
Approach, 2010
43. René León Rosales, Vid framtidens hitersta gräns: Om pojkar och elevpositioner i en multietnisk skola, 2010
44. Simon Larsson, Intelligensaristokrater och arkivmartyrer: Normerna för vetenskaplig skicklighet i svensk historieforskning 1900–1945, 2010
45. Håkan Lättman, Studies on spatial and temporal distributions of epiphytic lichens, 2010
46. Alia Jaensson, Pheromonal mediated behaviour and endocrine response in salmonids: The
impact of cypermethrin, copper, and glyphosate, 2010
47. Michael Wigerius, Roles of mammalian Scribble in polarity signaling, virus offense and cellfate determination, 2010
48. Anna Hedtjärn Wester, Män i kostym: Prinsar, konstnärer och tegelbärare vid sekelskiftet
1900, 2010
49. Magnus Linnarsson, Postgång på växlande villkor: Det svenska postväsendets organisation
under stormaktstiden, 2010
50. Barbara Kunz, Kind words, cruise missiles and everything in between: A neoclassical realist
study of the use of power resources in U.S. policies towards Poland, Ukraine and Belarus
1989–2008, 2010
51. Anders Bartonek, Philosophie im Konjunktiv: Nichtidentität als Ort der Möglichkeit des
Utopischen in der negativen Dialektik Theodor W. Adornos, 2010
52. Carl Cederberg, Resaying the Human: Levinas Beyond Humanism and Antihumanism, 2010
53. Johanna Ringarp, Professionens problematik: Lärarkårens kommunalisering och välfärdsstatens förvandling, 2011
54. Sofi Gerber, Öst är Väst men Väst är bäst: Östtysk identitetsformering i det förenade
Tyskland, 2011
55. Susanna Sjödin Lindenskoug, Manlighetens bortre gräns: Tidelagsrättegångar i Livland åren
1685–1709, 2011
56. Dominika Polanska, The emergence of enclaves of wealth and poverty: A sociological study of
residential differentiation in post-communist Poland, 2011
57. Christina Douglas, Kärlek per korrespondens: Två förlovade par under andra hälften av 1800talet, 2011
58. Fred Saunders, The Politics of People – Not just Mangroves and Monkeys: A study of the
theory and practice of community-based management of natural resources in Zanzibar, 2011
59. Anna Rosengren, Åldrandet och språket: En språkhistorisk analys av hög ålder och åldrande i
Sverige cirka 1875–1975, 2011
60. Emelie Lilliefeldt, European Party Politics and Gender: Configuring Gender-Balanced
Parliamentary Presence, 2011
61. Ola Svenonius, Sensitising Urban Transport Security: Surveillance and Policing in Berlin,
Stockholm, and Warsaw, 2011
62. Andreas Johansson, Dissenting Democrats: Nation and Democracy in the Republic of
Moldova, 2011
63. Wessam Melik, Molecular characterization of the Tick-borne encephalitis virus: Environments and replication, 2012
64. Steffen Werther, SS-Vision und Grenzland-Realität: Vom Umgang dänischer und „volksdeutscher” Nationalsozialisten in Sønderjylland mit der „großgermanischen“ Ideologie der
SS, 2012
65. Peter Jakobsson, Öppenhetsindustrin, 2012
66. Kristin Ilves, Seaward Landward: Investigations on the archaeological source value of the
landing site category in the Baltic Sea region, 2012
67. Anne Kaun, Civic Experiences and Public Connection: Media and Young People in Estonia,
2012
68. Anna Tessmann, On the Good Faith: A Fourfold Discursive Construction of Zoroastripanism
in Contemporary Russia, 2012
69. Jonas Lindström, Drömmen om den nya staden: stadsförnyelse i det postsovjetisk Riga, 2012
70. Maria Wolrath Söderberg, Topos som meningsskapare: retorikens topiska perspektiv på
tänkande och lärande genom argumentation, 2012
71. Linus Andersson, Alternativ television: former av kritik i konstnärlig TV-produktion, 2012
72. Håkan Lättman, Studies on spatial and temporal distributions of epiphytic lichens, 2012
73. Fredrik Stiernstedt, Mediearbete i mediehuset: produktion i förändring på MTG-radio, 2013
74. Jessica Moberg, Piety, Intimacy and Mobility: A Case Study of Charismatic Christianity in
Present-day Stockholm, 2013
75. Elisabeth Hemby, Historiemåleri och bilder av vardag: Tatjana Nazarenkos konstnärskap i
1970-talets Sovjet, 2013
76. Tanya Jukkala, Suicide in Russia: A macro-sociological study, 2013
77. Maria Nyman, Resandets gränser: svenska resenärers skildringar av Ryssland under 1700talet, 2013
78. Beate Feldmann Eellend, Visionära planer och vardagliga praktiker: postmilitära landskap i
Östersjöområdet, 2013
79. Emma Lind, Genetic response to pollution in sticklebacks: natural selection in the wild, 2013
80. Anne Ross Solberg, The Mahdi wears Armani: An analysis of the Harun Yahya enterprise,
2013
81. Nikolay Zakharov, Attaining Whiteness: A Sociological Study of Race and Racialization in
Russia, 2013
82. Anna Kharkina, From Kinship to Global Brand: the Discourse on Culture in Nordic
Cooperation after World War II, 2013
83. Florence Fröhlig, A painful legacy of World War II: Nazi forced enlistment: Alsatian/
Mosellan Prisoners of war and the Soviet Prison Camp of Tambov, 2013
84. Oskar Henriksson, Genetic connectivity of fish in the Western Indian Ocean, 2013
85. Hans Geir Aasmundsen, Pentecostalism, Globalisation and Society in Contemporary Argentina, 2013
86. Anna McWilliams, An Archaeology of the Iron Curtain: Material and Metaphor, 2013
87. Anna Danielsson, On the power of informal economies and the informal economies of power:
rethinking informality, resilience and violence in Kosovo, 2014
88. Carina Guyard, Kommunikationsarbete på distans, 2014
89. Sofia Norling, Mot ”väst”: om vetenskap, politik och transformation i Polen 1989–2011, 2014
90. Markus Huss, Motståndets akustik: språk och (o)ljud hos Peter Weiss 1946–1960, 2014
91. Ann-Christin Randahl, Strategiska skribenter: skrivprocesser i fysik och svenska, 2014
92. Péter Balogh, Perpetual borders: German-Polish cross-border contacts in the Szczecin area,
2014
93. Erika Lundell, Förkroppsligad fiktion och fiktionaliserade kroppar: levande rollspel i
Östersjöregionen, 2014
94. Henriette Cederlöf, Alien Places in Late Soviet Science Fiction: The “Unexpected Encounters”
of Arkady and Boris Strugatsky as Novels and Films, 2014
95. Niklas Eriksson, Urbanism Under Sail: An archaeology of fluit ships in early modern everyday
life, 2014
96. Signe Opermann, Generational Use of News Media in Estonia: Media Access, Spatial Orientations and Discursive Characteristics of the News Media, 2014
97. Liudmila Voronova, Gendering in political journalism: A comparative study of Russia and
Sweden, 2014
98. Ekaterina Kalinina, Mediated Post-Soviet Nostalgia, 2014
99. Anders E. B. Blomqvist, Economic Natonalizing in the Ethnic Borderlands of Hungary and
Romania: Inclusion, Exclusion and Annihilation in Szatmár/Satu-Mare, 1867–1944, 2014
100. Ann-Judith Rabenschlag, Völkerfreundschaft nach Bedarf: Ausländische Arbeitskräfte in der
Wahrnehmung von Staat und Bevölkerung der DDR, 2014
101. Yuliya Yurchuck, Ukrainian Nationalists and the Ukrainian Insurgent Army in Post-Soviet
Ukraine, 2014
102. Hanna Sofia Rehnberg, Organisationer berättar: narrativitet som resurs i strategisk kommunikation, 2014
103. Jaakko Turunen, Semiotics of Politics: Dialogicality of Parliamentary Talk, 2015
104. Iveta Jurkane-Hobein, I Imagine You Here Now: Relationship Maintenance Strategies in
Long-Distance Intimate Relationships, 2015
105. Katharina Wesolowski, Maybe baby? Reproductive behaviour, fertility intentions, and family
policies in post-communist countries, with a special focus on Ukraine, 2015
106. Ann af Burén, Living Simultaneity: On religion among semi-secular Swedes, 2015
107. Larissa Mickwitz, En reformerad lärare: konstruktionen av en professionell och betygssättande
lärare i skolpolitik och skolpraktik, 2015
108. Daniel Wojahn, Språkaktivism: diskussioner om feministiska språkförändringar i Sverige från
1960-talet till 2015, 2015
109. Hélène Edberg, Kreativt skrivande för kritiskt tänkande: en fallstudie av studenters arbete
med kritisk metareflektion, 2015
110. Kristina Volkova, Fishy Behavior: Persistent effects of early-life exposure to 17α-ethinylestradiol, 2015
111. Björn Sjöstrand, Att tänka det tekniska: en studie i Derridas teknikfilosofi, 2015
112. Håkan Forsberg, Kampen om eleverna: gymnasiefältet och skolmarknadens framväxt i
Stockholm, 1987–2011, 2015
113. Johan Stake, Essays on quality evaluation and bidding behavior in public procurement auctions, 2015
114. Martin Gunnarson, Please Be Patient: A Cultural Phenomenological Study of Haemodialysis
and Kidney Transplantation Care, 2016
115. Nasim Reyhanian Caspillo, Studies of alterations in behavior and fertility in ethinyl estradiolexposed zebrafish and search for related biomarkers, 2016
116. Pernilla Andersson, The Responsible Business Person: Studies of Business Education for
Sustainability, 2016
117. Kim Silow Kallenberg, Gränsland: svensk ungdomsvård mellan vård och straff, 2016
118. Sari Vuorenpää, Literacitet genom interaction, 2016
119. Francesco Zavatti, Writing History in a Propaganda Institute: Political Power and Network
Dynamics in Communist Romania, 2016
120. Cecilia Annell, Begärets politiska potential: Feministiska motståndsstrategier i Elin Wägners
‘Pennskaftet’, Gabriele Reuters ‘Aus guter Familie’, Hilma Angered-Strandbergs ‘Lydia Vik’
och Grete Meisel-Hess ‘Die Intellektuellen’, 2016
121. Marco Nase, Academics and Politics: Northern European Area Studies at Greifswald University, 1917–1992, 2016
122. Jenni Rinne, Searching for Authentic Living Through Native Faith – The Maausk movement
in Estonia, 2016
123. Petra Werner, Ett medialt museum: lärandets estetik i svensk television 1956–1969, 2016
124. Ramona Rat, Un-common Sociality: Thinking sociality with Levinas, 2016
125. Petter Thureborn, Microbial ecosystem functions along the steep oxygen gradient of the
Landsort Deep, Baltic Sea, 2016
Have you ever dreamed of going to an anoxic world? This thesis
takes you on a fascinating journey along the steep oxygen gradient at
the deepest point of the Baltic Sea, the Landsort Deep. On your trip
from the oxygenated surface water into the anoxic deep water and
sediment, you will meet many microorganisms whose importance
for sustaining ecosystem functions cannot be underestimated. Meet,
for example, the organic matter degraders, fermenters, sulphate
reducers, methane producers and methane oxidisers. Find out if they
are more susceptible to share genetic material compared to other
microorganisms. And finally, are the Cyanobacteria in the dark and
anoxic sediment dead or alive?
Microbial Ecosystem Functions Along the Steep
Oxygen Gradient of the Landsort Deep, Baltic Sea
Petter Thureborn carries out research in the field of microbial ecology with a special focus on the Baltic Sea. This is his PhD thesis.
Environmental Science, School of Natural Sciences, Technology and
Environmental Studies, Södertörn University.
PETTER THUREBORN
Distribution: Södertörn University, Library, SE-141 89 Huddinge. [email protected]
Microbial Ecosystem Functions Along
the Steep Oxygen Gradient of the
Landsort Deep, Baltic Sea
Petter Thureborn