Control factors of the marine nitrogen cycle795493/... · 2015-03-31 · Control factors of the...
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Control factors of the marine nitrogen cycleThe role of meiofauna, macrofauna, oxygen and aggregatesStefano Bonaglia
Academic dissertation for the Degree of Doctor of Philosophy in Geochemistry at StockholmUniversity to be publicly defended on Wednesday 29 April 2015 at 13.00 in Nordenskiöldsalen,Geovetenskapens hus, Svante Arrhenius väg 12.
AbstractThe ocean is the most extended biome present on our planet. Recent decades have seen a dramatic increase in the number andgravity of threats impacting the ocean, including discharge of pollutants, cultural eutrophication and spread of alien species.It is essential therefore to understand how different impacts may affect the marine realm, its life forms and biogeochemicalcycles. The marine nitrogen cycle is of particular importance because nitrogen is the limiting factor in the ocean and abetter understanding of its reaction mechanisms and regulation is indispensable. Furthermore, new nitrogen pathways havecontinuously been described. The scope of this project was to better constrain cause-effect mechanisms of microbiallymediated nitrogen pathways, and how these can be affected by biotic and abiotic factors.
This thesis demonstrates that meiofauna, the most abundant animal group inhabiting the world’s seafloors, considerablyalters nitrogen cycling by enhancing nitrogen loss from the system. In contrast, larger fauna such as the polychaeteMarenzelleria spp. enhance nitrogen retention, when they invade eutrophic Baltic Sea sediments. Sediment anoxia,caused by nutrient excess, has negative consequences for ecosystem processes such as nitrogen removal because itstops nitrification, which in turn limits both denitrification and anammox. This was the case of Himmerfjärden andByfjord, two estuarine systems affected by anthropogenic activities, such as treated sewage discharges. When Byfjord wasartificially oxygenated, nitrate reduction mechanisms started just one month after pumping. However, the balance betweendenitrification and nitrate ammonification did not favor either nitrogen removal or its retention.
Anoxia is also present in aggregates of the filamentous cyanobacteria Nodularia spumigena. This thesis shows that evenin fully oxic waters, millimetric aggregates can host anaerobic nitrogen processes, with clear implications for the pelagiccompartment. While the thesis contributed to our knowledge on marine nitrogen cycling, more data need to be collectedand experiments performed in order to understand key processes and regulation mechanisms of element cycles in the ocean.In this way, stakeholders may follow and take decisions in order to limit the continuous flow of human metabolites andimpacts on the marine environment.
Keywords: Nitrogen cycle, denitrification, DNRA, anammox, anoxia, hypoxia, eutrophication, meiofauna, macrofauna,aggregates, cyanobacteria, Baltic Sea.
Stockholm 2015http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-115036
ISBN 978-91-7649-129-4
Department of Geological Sciences
Stockholm University, 106 91 Stockholm
Control factors of the marine
nitrogen cycle: the role of
meiofauna, macrofauna, oxygen
and aggregates
Stefano Bonaglia
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©Stefano Bonaglia, Stockholm 2015
ISBN 978-91-7649-129-4
Cover picture: Water meets sediments at Askö, southern Stockholm archipelago.
Printed in Sweden by Holmbergs, Malmö 2015
Distributor: Publit
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ABSTRACT
The ocean is the most extended biome present on our planet. Recent decades
have seen a dramatic increase in the number and gravity of threats impacting the
ocean, including discharge of pollutants, cultural eutrophication and spread of
alien species. It is essential therefore to understand how different impacts may
affect the marine realm, its life forms and biogeochemical cycles. The marine
nitrogen cycle is of particular importance because nitrogen is the limiting factor in
the ocean and a better understanding of its reaction mechanisms and regulation is
indispensable. Furthermore, new nitrogen pathways have continuously been
described. The scope of this project was to better constrain cause-effect
mechanisms of microbially mediated nitrogen pathways, and how these can be
affected by biotic and abiotic factors.
This thesis demonstrates that meiofauna, the most abundant animal group
inhabiting the world’s seafloors, considerably alters nitrogen cycling by enhancing
nitrogen loss from the system. In contrast, larger fauna such as the polychaete
Marenzelleria spp. enhance nitrogen retention, when they invade eutrophic Baltic
Sea sediments. Sediment anoxia, caused by nutrient excess, has negative
consequences for ecosystem processes such as nitrogen removal because it stops
nitrification, which in turn limits both denitrification and anammox. This was the
case of Himmerfjärden and Byfjord, two estuarine systems affected by
anthropogenic activities, such as treated sewage discharges. When Byfjord was
artificially oxygenated, nitrate reduction mechanisms started just one month after
pumping. However, the balance between denitrification and nitrate
ammonification did not favor either nitrogen removal or its retention.
Anoxia is also present in aggregates of the filamentous cyanobacteria Nodularia
spumigena. This thesis shows that even in fully oxic waters, millimetric aggregates
can host anaerobic nitrogen processes, with clear implications for the pelagic
compartment. While the thesis contributed to our knowledge on marine nitrogen
cycling, more data need to be collected and experiments performed in order to
understand key processes and regulation mechanisms of element cycles in the
ocean. In this way, stakeholders may follow and take decisions in order to limit
the continuous flow of human metabolites and impacts on the marine
environment.
Keywords: Nitrogen cycle; denitrification; DNRA; anammox; anoxia; hypoxia;
eutrophication; meiofauna; macrofauna; aggregates; cyanobacteria; Baltic Sea.
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TABLE OF CONTENTS:
LIST OF PAPERS ........................................................................................ 5
INTRODUCTION ....................................................................................... 6
NITROGEN, THE LIMITING FACTOR ................................................. 7
PROCESSES OF THE MARINE NITROGEN CYCLE ........................... 8
THE BALTIC SEA MARINE ENVIRONMENT .................................... 11
QUANTIFICATION OF SOLUTE FLUXES .......................................... 14
THE NITROGEN ISOTOPE PAIRING ................................................. 16
ENVIRONMENTAL FACTORS CONTROLLING NITROGEN
CYCLE ........................................................................................................ 18
1. Eutrophication and oxygen conditions ............................................. 18
2. Macrofauna and Meiofauna ............................................................... 21
3. The role of aggregates ....................................................................... 24
CONCLUSIONS AND FUTURE PERSPECTIVES ................................ 25
1. In situ studies for global N2 exchanges ............................................. 25
2. Role of infauna for methane and nitrous oxide emissions ................ 26
ACKNOWLEDGEMENTS ........................................................................ 27
REFERENCES........................................................................................... 28
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LIST OF PAPERS
This thesis is based on the following papers that are referred to in the text by
Roman numeral:
Paper I Bonaglia S, Deutsch B, Bartoli M, Marchant HK, Brüchert V
(2014) Seasonal oxygen, nitrogen and phosphorus benthic cycling
along an impacted Baltic Sea estuary: regulation and spatial
patterns. Biogeochemistry 119: 139-160
Paper II De Brabandere L, Bonaglia S, Kononets M, Viktorsson L,
Stigebrandt A, Thamdrup B, Hall POJ (2015) Oxygenation of an
anoxic fjord basin strongly stimulates benthic denitrification and
DNRA. To be submitted to Biogeochemistry
Paper III Bonaglia S, Bartoli M, Gunnarsson JS, Rahm L, Raymond C,
Svensson O, Shakeri Yekta S, Brüchert V (2013) Effect of
reoxygenation and Marenzelleria spp. bioturbation on Baltic Sea
sediment metabolism. Marine Ecology Progress Series 482: 43–55
Paper IV Bonaglia S, Nascimento FJA, Bartoli M, Klawonn I, Brüchert V
(2014) Meiofauna increases bacterial denitrification in marine
sediments. Nature Communications 5: 5133
Paper V Klawonn I, Bonaglia S, Brüchert V, Ploug H (2015) Aerobic and
anaerobic nitrogen transformation processes in N2-fixing
cyanobacterial aggregates. The ISME Journal
Paper I, III and IV: Stefano Bonaglia designed and conducted the research;
performed sampling at sea and carried out the experiments; performed stable
isotope analysis, analyzed data and wrote the paper. Paper II and V: Stefano
Bonaglia carried out sampling and conducted the experiments; was part of
analyzing nitrogen isotopes; contributed with data analysis and paper writing.
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INTRODUCTION
Nitrogen is among the seven most common elements in our galaxy and, alone, it
makes up approximately 78% of our planet’s atmosphere. Since its discovery in
1772 by Daniel Rutherford, research on this element has been constantly
performed, leading to a massive number of findings and applications. Nitrogen is
required by all living organisms because it takes part in the synthesis of the two
most abundant natural macromolecules, nucleic acids and proteins. Furthermore,
the atomic properties of nitrogen are quite unique: the oxidation states (OS) vary
from -3 in ammonium (NH4+) to +5 in nitrate (NO3
-) and, in this range, each
state is taken by at least one nitrogen molecule. Therefore, a large number of
microorganisms can take advantage of this extended redox range, and a vast
number of metabolic nitrogen pathways can take place in the microbial realm.
This Ph.D project was designed to gain understanding of how microbial nitrogen
pathways can be affected by various environmental stresses and biotic factors in
the marine environment. In particular it investigated the role of macrofauna,
eutrophication, oxygen conditions in sediments and algal aggregates on nitrogen
cycling processes. This project also aimed to describe and quantify the influence
of meiofauna (small metazoan living in the sediment) on microbial nitrogen
cycling.
This thesis introduction starts with two chapters that illustrate the nitrogen cycle
and review recent findings on the marine nitrogen cycle and its players. Then I
introduce the study area of the experiments carried out during my Ph.D. project,
in a wide sense the ocean, and more specifically the Baltic Sea marine
environment, which offered perfect empirical conditions for investigating the
biogeochemical transformations of nitrogen, such as lack of oxygen in the water
column, low biodiversity of benthic animals, presence of dense masses of
cyanobacteria, for example. I continue describing the main analytical
methodologies that were applied for determination of the different inorganic
nitrogen compounds encountered during the project. This thesis introduction
contains the main results and recapitulates the insights of the five papers
produced, and it ends with a presentation of the ongoing works including some
future perspectives.
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NITROGEN, THE LIMITING FACTOR
In an aquatic context, the nitrogen cycle is very likely the most intricate element
cycle, and thus among the most interesting biogeochemical cycles one could
decide to approach. Although the main pools of nitrogen are found in the
atmosphere and in the lithosphere, nitrogen in these compartments is nearly inert
(Canfield et al. 2010). In the oceans nitrogen is orders of magnitude less abundant
than in the Earth’s crust, mantle and atmosphere, but its importance is pivotal
because in primis this nutrient limits the global rates of net primary production
since it is needed by all forms of life, with no exceptions. Therefore the
availability of nitrogen controls both marine biogeochemical cycles and
ecosystem functioning.
In the marine environment, nitrogen fixation by plankton is the main provider of
bio-available, fixed nitrogen (Canfield et al. 2010; Codispoti et al. 2001), which
can be assimilated by different life forms, transformed by specialized
microorganisms, or lost from the aquatic system in different ways. Other major
sources of fixed nitrogen to the ocean are riverine inputs and atmospheric
deposition. In recent decades anthropogenic nitrogen loading has become a
substantial contributor of bio-available nitrogen to the sea, via land runoff and
discharge of treated sewage (Smith and Schindler 2009). Nitrogen carried by land
runoff, in particular, has massively increased following the dramatic increase in
the application of nitrogenous fertilizers in agriculture (Figure 1). Among the
main abiotic outputs, sedimentation and burial of organic material at the sea floor
play an important role, while as biotic sinks, denitrification and anammox are
bacterial processes that return fixed nitrogen to the atmosphere (Brandes et al.
2007).
Figure 1 – Global use of nitrogen and phosphorus fertilizers for all nations of the world except USSR, and global use of water for irrigation. Source: Tilman et al. (2001)
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It has recently been proposed that the marine nitrogen budget is very close to
being balanced (Devries et al. 2013). There is evidence, however, that even small
variations in the ratio between nitrogen fixation and denitrification would result
in change in climate over geological times (Falkowski 1997). In fact nitrogen is
the limiting nutrient in the ocean and abrupt changes in denitrification may affect
carbon sequestration through the biological pump (Codispoti et al. 2001).
Whether global marine nitrogen budget is in balance or not is still debated and
will be also matter of discussion in the near future (Gruber and Galloway 2008).
Interestingly, the supporters of the imbalanced scenario attribute the large
nitrogen deficit to high denitrification rates (Codispoti 2007; Codispoti et al.
2001). It results that adequate empirical measurements aimed at quantifying
microbial nitrogen cycle processes are of paramount importance when nutrient
budgets are made, especially in sensitive areas of the oceans where these
processes reach high rates.
PROCESSES OF THE MARINE NITROGEN CYCLE
Because of the steep gradient between oxic and anoxic conditions, both marine
sediments and water column chemoclines are characterized by a complex
nitrogen cycle, where a very diverse set of assimilatory and dissimilatory microbial
processes usually takes place. During this series of processes, nitrogen is either
oxidized or reduced, and intermediates are constantly produced and may
accumulate in the environment. This intricate set of transformations is
schematized in Figure 2.
Thanks to a very specialized community of N2-fixing microorganisms both from
the bacterial and archaeal domains, nitrogen enters the marine realm through the
reduction of N2 to NH4+. This process, called nitrogen fixation, is energy
demanding since the strong triple bond of the dinitrogen molecule needs to be
broken before nitrogen can be reduced. Once ammonium is fixed and present in
the environment it can be easily incorporated by prokaryotes and eukaryotes,
such as phytoplankton and macro-algae. Ammonium is the preferred form of
nitrogen for assimilation by living organisms that do not need to invest energy to
further reduce it, being NH4+ in its most reduced state (-3; Figure 2). When
nitrogen is incorporated by the organism it can be found in a number of different
molecules: amines, amino acids, acid amides, polyamides, polypeptides, etc. The
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breakdown of these molecules by hydrolysis generates NH4+ in a process named
ammonification. Ammonium can possibly continue its cycle by incorporation by
other organisms into other bio-molecules.
An alternative process dependent on ammonium is nitrification, which happens
in oxic conditions, where ammonium is oxidized to nitrite (OS +3), and then
nitrate (OS +5). This dissimilatory process can lead to production of nitrous
oxide (N2O; OS +1), a potent greenhouse gas, when oxygen concentrations
become lower and lower (Goreau et al. 1980). The second biotic source of
nitrous oxide is denitrification (Zumft 1997). This stepwise microbial process
takes place in anoxic or nearly anoxic conditions and reduces nitrate to gaseous
nitrogen (either N2O or N2), with nitric oxide (NO; OS +2) and nitrite as
intermediates. Ammonium oxidizing bacteria (AOB) and archaea (AOA) are
autotrophic because they synthesize bio-molecules from CO2 reduction, while
denitrifying bacteria are mainly heterotrophic since they preferentially use organic
substances as carbon source (Thamdrup 2012). As an alternative to organic
matter, autotrophic denitrifiers can oxidize reduced sulfur or reduced iron.
Figure 2 – A simplified representation of the marine nitrogen cycle and its microbial pathways. On top of the figure, numbers represent the oxidation state of nitrogen in the different compounds. N-fix=nitrogen fixation, Amm=ammonification, DNRA=dissimilatory nitrate reduction to ammonium.
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For a long time denitrification was considered the most important pathway for
reducing nitrate and nitrate (NOx) in the environment. Very recently, however,
the dissimilatory nitrate reduction to ammonium (DNRA) and the anaerobic
ammonium oxidation by nitrite (anammox) have been recognized as important,
alternative pathways to denitrification that can reduce NOx in nature (Burgin and
Hamilton 2007). DNRA is carried out by both autotrophic and heterotrophic
bacteria and instead of producing N2 as denitrification does, it yields NH4+, thus
increasing nitrogen retention in the ecosystem (Canfield et al. 2005). Anammox
bacteria, which are strictly autotrophic, were discovered based on their 16S rRNA
gene about 15 years ago (Strous et al. 1999). It was first thought that these
bacteria could only survive in artificial conditions such as sewage treatment
bioreactors, where they showed extremely slow growth conditions with average
doubling time of about two weeks (Kartal et al. 2013). However, a few years later,
the anammox reaction was proven to occur extensively also in nature, both in
marine sediments (Dalsgaard and Thamdrup 2002; Thamdrup and Dalsgaard
2002) and in the water column of oxygen minimum zones (OMZs) and anoxic
basins like the Baltic Sea (Dalsgaard et al. 2003; Hannig et al. 2007; Kuypers et al.
2005; Kuypers et al. 2003).
OMZs and sediments underneath shelf and coastal waters are recognized as the
most important sites for global nitrogen losses, accounting for about 60% of total
nitrogen removal from the system (Galloway et al. 2004; Seitzinger et al. 2006).
Between the two main biotic processes involved in nitrogen removal,
denitrification is globally more important (Devol 2015; Thamdrup 2012).
Anammox generally does not account for more than 20-30% of total benthic N2
production, and less than 10% in shallow coastal sediments (Thamdrup 2012).
The relative importance of anammox is even more variable in the OMZs and in
other anoxic water basins of the Earth, possibly due to the fact that these
environments present less stable conditions than sediments, and as a result they
are less suitable for slow growing bacteria than for facultative anaerobes like
denitrifiers.
Another recent finding that opens new views in the marine nitrogen cycle is
denitrification carried out by eukaryotes. Its discovery happened about 10 years
ago in a species of meiofaunal Foraminifera, and denitrification was suggested to
occur in its mitochondria (Risgaard-Petersen et al. 2006). Eukaryotic
denitrification has been found in ten other species of Rhizaria, which were shown
to be able to store nitrate in millimolar concentrations (Piña-Ochoa et al. 2010),
i.e. up to five orders of magnitude higher than the nitrate concentration in the
ocean. It may be that the bacterial mitochondrion carrying out denitrification was
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inherited by these eukaryotic cells, which suggests that also other eukaryotes
outside the taxon Rhizaria might have the same capability of reducing nitrate.
THE BALTIC SEA MARINE ENVIRONMENT
Approximately 70% of the Earth’s surface is covered by saline waters. Recent
decades have seen the oceans, and in particular the marine coastal areas, being
threatened by a series of environmental problems. Main impacts have arisen from
overfishing to spillages of toxic substances, from water acidification to global
warming and from discharge of radioactivity to decades of cultural
eutrophication. Certain aquatic areas of the world are more sensitive to these
threats because of their peculiar configuration, e.g. their morphology and
location.
The case of the Baltic Sea is emblematic. It is a semi-enclosed brackish basin
characterized by a high ratio between the area of the drainage basin and the area
covered by water (Leppäranta and Myrberg 2009). The water body is
approximately 400,000 km2 and is constituted by various sub-basins, separated by
sills and belts (Figure 3A). Its drainage basin is currently inhabited by more than
85 million people (Helcom 2009). Those factors plus the unique geomorphology
of the Baltic, which remains isolated from the North Sea by the shallow and
tangled Danish Straits, determine very long water residence time, strong
thermohaline stratification, and, as a consequence, high sensitivity for nutrient
enrichment both in the open sea and in the coastal areas.
Figure 3 – (A) Map of the Baltic Sea and its sub-basins, from Leppäranta and Myrberg (2009). (B) Bathymetric profile of the Baltic proper and average extension of anoxic, hypoxic and normoxic zones from a long-term dataset analysis (Conley et al. 2009b).
A B
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Eutrophication, the excessive nutrient enrichment that leads to accelerated
growth of primary producers in a water body, is particularly effective in enclosed
basins and coastal systems affected by poor water exchanges. Here another major
problem can develop, hypoxia, a condition of oxygen deficiency in the water
masses, which can be fatal for aquatic organisms dependent on oxygen for
respiration (Figure 3B). Generally, by definition, hypoxic zones of the oceans
have oxygen concentrations <60 µmol L-1 (Diaz and Rosenberg 2008; Rabalais et
al. 2010). When the situation is even more critical and benthic chemical and
biological reactions use up the little oxygen left, anoxia arises. The effects of
eutrophication and low oxygen concentrations on benthic metabolism and
nitrogen cycling processes were tested in papers I, II and III.
Hypoxia and anoxia were already present in the Baltic Sea during its formation,
more than 8000 years ago (Zillén and Conley 2010). However, eutrophication and
oxygen depletion have expanded further especially during the last 50 years and
now they affect not only the deeper basins but also a number of shallow coastal
zones (Conley et al. 2009a; Conley et al. 2011) with a negative impact on benthic
biota, which is generally dependent on oxygen for its metabolism (Karlson et al.
2002). While most of the macrofauna species decrease in abundance and in
biomass with declining oxygen levels (Diaz and Rosenberg 1995), marine
meiofauna is more resistant to oxygen and it shows unique adaptations to survive
hypoxia (Wetzel et al. 2001). Papers III and IV were conceived to describe the
effect of benthic macrofauna and meiofauna on nitrogen cycling and oxygen
dynamics.
In recent years a debate has been taking place about whether it would be more
effective to remediate the Baltic by reducing the nutrient inputs (Carstensen et al.
2014; Conley et al. 2009b), or using engineering solutions to increase the oxygen
level in the bottom waters (Stigebrandt and Gustafsson 2007; Stigebrandt et al.
2015). Paper II helped in gaining understanding of how large scale oxygenation
of a fjord in the west coast of the Baltic Sea affects benthic nitrogen removal and
recycling.
The Baltic Sea suffers from decades of diffuse and point nutrient loadings, e.g.
agricultural land run-off and sewage discharges, respectively. In the last century
nitrogen loads to the watershed, including atmospheric depositions, increased by
a factor of four (Schernewski and Neumann 2005). Although the nutrient trend
for the entire basin is clearly increasing, the surface layer of the Baltic Sea is
marked by a pronounced seasonality in nutrient abundances, with high
concentrations in winter and with values near the detection limits from early
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spring to late autumn (Nausch et al. 2008). This is obviously due to the broad
fluctuations in phytoplankton productivity during different times of the year.
Phytoplankton bloom is triggered when the upper mixed layer becomes shallower
than the light penetration depth and it causes indeed a rapid nutrient decrease
(Wasmund et al. 1998).
It is well known that marine phytoplankton takes up nutrients in a fixed
stoichiometry of carbon (C), nitrogen (N) and phosphorus (P), with the molar
ratio of C:N:P being 106:16:1 (Redfield et al. 1963). If one or more nutrients are
deficient, phytoplankton undergoes stress conditions due to the limiting element.
In the Baltic Proper nitrogen is the limiting factor, and the common reported
ratio of N:P is 7-9:1 (Nausch et al. 2008). This condition gives cyanobacteria an
advantage because they can carry out nitrogen fixation and can capitalize on the
mesohaline conditions prevailing in the central and eastern Baltic Sea (Stal et al.
1999; Wasmund 1997). Approximately 44% of the total primary production in
the Baltic Sea have been attributed to filamentous cyanobacteria (Ploug et al.
2010). Paper V investigated specifically how aggregates of filamentous
cyanobacteria could host a complete nitrogen cycling and therefore influence the
exchange of inorganic nitrogen in the pelagic ecosystem.
A substantial part of the primary production is assimilated or decomposed
already in the water column. Alternatively, dissolved and particulate organic
matter sink down to the sediment, leading to marked consumption of dissolved
oxygen by aerobic mineralization processes (Vahtera et al. 2007). When hypoxic
or anoxic conditions
establish, large amounts of
phosphorus are released from
the sediment to the water
column due to the reduction
of iron oxy-hydroxides bound
to phosphate (Rozan et al.
2002) (Figure 3B). In the
warm season cyanobacteria
are further stimulated when
the regenerated phosphate
reaches the upper water.
Beside this, oxygen depletion
tends to increase nitrogen
removal by denitrification
and anammox in the water
Figure 4 – The vicious circle leading to exacerbation of cyanobacteria blooms in the Baltic Sea. Source: Vahtera et al. (2007).
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column and in the sediment (Canfield et al. 2005; Hietanen et al. 2012) (Figure
3B), thus lowering the N:P stoichiometry even further.
Instead of “nutrients cycling” this series of mechanisms ongoing in the Baltic Sea
represents a particular case of “vicious circle” (Figure 4) (Tamminen and
Andersen 2007; Vahtera et al. 2007). Studies on Holocene sediments have given
evidence that cyanobacterial blooms have been a component of the Baltic Sea
ecosystem throughout its present brackish state (Bianchi et al. 2000). However, it
is important to emphasize that without the application of nutrient reduction
measures or an opportune, drastic change in the geochemical features of the
seafloor, these mechanisms will accentuate the eutrophic state of the Baltic Sea,
and its most discussed feature – the cyanobacterial blooms.
QUANTIFICATION OF SOLUTE FLUXES
Updated values for solute transport across marine interfaces (e.g. water-sediment
interface) and quantification of nitrogen processes are pivotal when nutrient
budgets are compiled in order to identify the correct remediation strategy and to
apply nutrient reduction measures to affected basins, like the Baltic Sea and other
endangered waterbodies around the world. One of the intents of this thesis was
to better understand benthic and pelagic biogeochemical processes that take place
in impacted Baltic Sea coastal areas. In particular, the central point I focused on
was to understand whether nitrogen removal (through denitrification and
anammox) outreached nitrogen regeneration (mainly through DNRA) or
viceversa.
The correct quantification of the solute exchange between different materials is
therefore essential. The experimental approaches developed to measure fluxes of
solutes can be categorized in two groups: (1) incubation of enclosed chambers (or
cores), where solutes fluxes are quantified by measuring the change of solute
concentrations in the water over time; (2) methods based on gradient description
and on the determination of differences in solute concentration in depth profiles
(both in the water column and in the sediment) (Zabel and Hensen 2006).
Both approaches were used extensively in the present thesis. Specifically,
incubation of sediment cores was applied in papers I, III and IV (Figure 5); in
situ chamber incubation was carried out in paper II; incubation of gas-tight vials
(Exetainers) with water and algal aggregates was employed in paper V. All these
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methods are based on the incubation of a medium (water, sediment, or both) in
enclosed experimental systems (e.g., plexiglas chambers and glass vials) in intact
or altered conditions (e.g., different oxygen levels, addition of benthic fauna) in
order to measure changes in solute
concentrations over time. In
particular, the core incubation
approach was preferred when
dealing with manipulated sediments
(Papers III and IV). The
manipulated sediment units, also
known as microcosms are extremely
useful when it is required to study
the effect of different treatments on
geochemical processes, and thus this
approach helps in the understanding
of cause-effect relationships
(Näslund et al. 2010).
The second approach was also
extensively used in the present
thesis (Papers I, III, IV and V).
Generally, concentration profiles obtained by extraction and subsequent analysis
of sediment porewater constituents have a poor vertical resolution (at most 3-4
mm). Unfortunately this does not allow the study of those oxidized compounds
that usually occur in the top few millimeters only, such as oxygen, nitrate and
nitrite. However, by means of microsensors a resolution of several micrometers
can be observed (Sweerts and De Beer 1989), and the fluxes can be calculated
from the concentration gradients by means of diffusive flux models (Berg et al.
1998) (Papers I and IV). Another great advantage of microelectrodes is their
versatility and applicability in different micro-environments, such as fecal pellets
or marine aggregates (Ploug et al. 1997) (Paper V).
When the gradient approach is used in sediments, however, it does not take into
account the convective flux caused by benthic fauna and, consequently, it might
underestimate the total solute exchange when biodiffusivity is an important factor
(Glud et al. 1994). The core incubation method has the strong advantage that it
also takes convective solute fluxes into account if the water overlying the
sediment is efficiently mixed (Figure 5), simulating the natural bottom water flow.
Another positive aspect of this latter method is that during a single incubation
several solute components can be investigated at the same time (e.g., gases,
Figure 5 – Example of sediment cores used in this thesis, i.e. plexiglas tubes filled half with sediment and half with water, with a stirring device placed in the water column.
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nutrients, metals). Lastly, the incubation method allows the artificial enrichment
with stable isotopes of nitrogen such as 15NO3- and 15NH4
+ in order to detect and
quantify rates of nitrogen processes, such as denitrification, anammox, DNRA,
nitrification and N2 fixation (Papers I to V).
THE NITROGEN ISOTOPE PAIRING
In recent decades several different methods have been developed in order to
detect and quantify rates of nitrogen cycle processes in marine and freshwater
environments. During the most recent years many improvements have been
achieved in this field, a consequence of the increased attention to sensitive issues
such as spreading of eutrophication and hypoxia (Groffman et al. 2006;
Steingruber et al. 2001). Among the different approaches to measure production
of nitrogenous gases, the most used ones are the acetylene inhibition technique
(Knowles 1990), the quantification of N2 production based on N2:Ar
measurements (Kana et al. 1994), and the 15N isotope pairing technique (Nielsen
1992).
Acetylene inhibition technique (AIT) is based on the inhibition of the last step of
denitrification, the reduction of N2O to N2 by acetylene. It is a simple method
based on the quantification of N2O by gas chromatography (Sørensen 1978).
Unfortunately nitrification is very sensitive to low concentration of acetylene and
is also inhibited when AIT is applied (Berg et al. 1982). Therefore this method
underestimates the total conversion to gaseous nitrogen in environments where
denitrification is based on nitrification. Moreover AIT does not allow estimation
of anammox, since the anammox reaction does not produce N2O.
The N2:Ar methodology obviates these underestimation problems because it
determines the total N2 being produced in the system through a membrane inlet
mass spectrometer (MIMS). This instrument allows rapid analysis and is highly
versatile, since it is not larger than a gas chromatograph and can be used on
shipboard (Kana et al. 1994). However, analysis with a MIMS requires very stable
temperature conditions since it measures the gas itself in the water samples, and
in experiments susceptible to low changes in temperature this might constitute a
serious problem (Eyre et al. 2002).
17
Compared to those two methodologies the isotope pairing technique (IPT), and
its revised version (Risgaard-Petersen et al. 2003), have two main advantages: the
very high analytical precision when an isotope ratio mass spectrometer (IRMS) is
employed as detector, and the fact that it makes it possible to dissect the sources
of N2 production. The combination of different incubation after amendment
with 15N isotope labeling of NO2-, NO3
- and NH4+ indeed allows discrimination
among the processes of nitrate reduction: denitrification of water nitrate (Dw),
denitrification coupled to nitrification (Dn), anammox and even DNRA, after
chemical oxidation of the ammonium pool to N2 with hypobromite solution
(Warembourg 1993), can be quantified.
The first task of this Ph.D. project was to establish the IPT and a method to
detect 15N-N2 and 15NH4+ at nanomolar concentrations with an IRMS as detector
for the first time in Sweden. A self-built gas separation line (Figure 6) was set up
and connected to an IRMS (Delta V Advantage, Thermo Scientific) through a
continuous flow interface (Conflo IV, Thermo Scientific). The construction of
the N2 line followed the scheme presented in Holtappels et al. (2011). Essentially
the line comprised a series of traps: (1) a liquid nitrogen trap to freeze water,
nitrous oxide, carbon dioxide and, partly, nitric oxide; (2) a copper column heated
to 650 °C to reduce oxygen; (3) a Porapak Q column to separate the gas species
before entering the Conflo (Figure 6).
The different masses of the N2 gas (masses 28, 29, and 30) were analyzed on a
triple collector of the Delta V Advantage and the areas (proportional to the
Figure 6 - Schematic representation of the gas separation line coupled to the isotope ratio mass spectrometer at Stable Isotope Lab, Stockholm University. Picture modified from Holtappels et al. (2011).
18
amounts) of the 14N14N, 14N15N, and 15N15N masses were recorded. After every
fifth sample, samples of air were injected as standard. The concentrations of 29N2
and 30N2 were calculated as excess over the natural abundances given by the air
standards following the rationale described in Holtappels et al. (2011).
ENVIRONMENTAL FACTORS CONTROLLING
NITROGEN CYCLE
1. Eutrophication and oxygen conditions
As previously discussed, coastal water quality is in decline and eutrophication is
spreading around the world as excessive nitrogen and phosphorus are finding
their way to the sea in increasing amounts
(Rabalais et al. 2009). Cultural
eutrophication may lead to hypoxia and
anoxia when harsh physical conditions
(e.g., scarce water circulation because of
wind absence, strong halocline formation)
encounter peculiar biological conditions
such as high oxygen consumption by the
microbial and faunal communities in the
water system. Paper I aims to investigate
the main pathways of sediment nitrogen
cycling along the axis of a Baltic Sea
estuary impacted by decades of heavy
nutrient discharges. Himmerfjärden is
affected by seasonal hypoxia and offers a
range of different sediment types and
physicochemical bottom water
characteristics that make it suitable for
understanding spatio-temporal dynamics
in nutrient cycling.
The inner part of Himmerfjärden (Figure
7) is usually stratified during summer and
experiences hypoxic bottom waters and
reduced sediments, especially in the deeper
areas of the estuary. In the outer basins hypoxia is only transient as attested by
Figure 7 – The study area of Paper I, Himmerfjärden. The four sampling stations, located along the eutrophication gradient (north-south), are H6, H4, H2 and B1. STP indicates the sewage treatment plant active in the area and serving about 300,000 people.
19
oxidized and macrofauna-inhabited superficial sediment, and it only occurs when
winds are weak and circulation is inhibited (Elmgren and Larsson 1997). In the
northern and innermost part of the estuary a sewage treatment plant (STP) has
been discharging treated sewage water since 1974 (Figure 7). In the late 80s the
STP used to discharge enormous quantities of nitrogen to the bay (~900 t yr-1),
and comparatively low quantities of phosphorus (~15 t yr-1). Since the late 90s
the nitrogen discharge was substantially reduced (~200 t yr-1) thanks to the
implementation of the tertiary treatment (Savage 2005). Treated sewage remains,
however, the main source of nitrogen to the estuary, which has been experiencing
eutrophication and oxygen paucity especially in its northernmost part.
Results from paper I suggest that the proportion of benthic nitrogen cycling
pathways differed in the basins of the estuary along the eutrophication gradient.
As expected, at the inner and most impacted station (H6, Figure 7), the heavy
loading and production of ammonium is partly decoupled from oxidation via
nitrification, reducing nitrogen loss via denitrification. Overall, most of the total
nitrogen is recycled to the pelagic zone by DNRA and ammonification.
Temperature is the main driver of nitrogen microbial activity at the central and
outer basins (stations B1, H2 and H4), where it correlated positively with
denitrification rates in agreement with previous studies (Rysgaard et al. 2004).
In the estuary bottom water oxygen concentrations also play an important
controlling role in nitrogen cycling. Oxygen depletion at H6 stimulated
ammonium effluxes and DNRA by a factor of ~4 and ~6, respectively, and
lowered rates of denitrification by 60%. In contrast, in January, fully oxygenated
bottom water at station H6 re-established nitrification, and this resulted in
denitrification rates comparable to those measured at the oxic stations, where an
efficient coupling between nitrification and denitrification outcompeted DNRA.
Long term anoxia, however, seems to affect the system in a different way. Paper
II investigated the effect of artificial oxygenation on nitrate reducing processes in
the By Fjord, a small fjord characterized by stagnant, permanently anoxic bottom
waters. The By Fjord is situated on the Swedish west coast and has a maximum
depth of about 50 m. It presents a strong oxycline (oxic-anoxic boundary)
between 13 and 20 m and it receives nutrient inputs both from the Bäve River
and the STP located close to the river’s mouth (Viktorsson et al. 2013). For more
than two years the fjord’s anoxic bottom waters received pumped oxygen-rich
water from the surface that interrupted the density stratification, generating
drastic changes in the chemistry and ecology of the seafloor (Stigebrandt et al.
2015).
20
NO3- (mol L-1)
10 12 14 16 18 20 22 24
DN
RA
tot /
(Dto
t + D
NR
Ato
t (
%)
40
45
50
55
60
65
70
75
Coefficients:b[0]13.3b[1]2.6r ²0.91
Contrary to the study in Himmerfjärden (Paper I), the By Fjord experiment
(Paper II) showed that oxygen presence stimulated both denitrification and
DNRA with rates of the two microbial processes being in the same range. It is
likely that in By Fjord nitrate reduction rates increased rapidly during the first six
months of oxygenation because
nitrate concentrations suddenly
increased in the bottom water.
However, the relative importance of
DNRA increased with time and
increasing concentrations of nitrate
(Figure 8). In this artificially
oxygenated sediment the large
sulfur bacteria Beggiatoa spp. might
have played an increasingly
important role in stimulating
DNRA and thus nitrogen recycling.
In both coastal systems N2
production by anammox was far
less important than production by
denitrification. If in Himmerfjärden it never accounted for more than 19% of the
total N2 yield, in By Fjord anammox contribution was estimated to be <4%.
Results from Paper I, in particular, showed that anammox rates had a striking
negative correlation with both organic carbon and nitrogen content in the
sediments. It is likely that at the more eutrophic sites anammox suffered from
substrate (i.e. nitrite) competition with heterotrophic denitrifiers and DNRA
bacteria. In any case, this limited importance of anammox seems to agree well
with previous studies that described shallow coastal environment as a minor
contributor of nitrogen losses (Dalsgaard et al. 2005; Thamdrup 2012).
Results from Papers I and II were important to demonstrate that impacted
coastal sediments are still important sinks of fixed nitrogen. For example, on an
annual scale 221 t of nitrogen are removed from Himmerfjärden sediment, which
is approximately 96% of the nitrogen discharged annually by the STP (Paper I).
It is evident that denitrification acts as the major nitrogen loss process for
external nitrogen inputs, as previously described for other coastal and shelf
ecosystems (Seitzinger 1988). However, it is appropriate to say that if
hypoxia/anoxia will expand further or become permanent, marine areas that
receive high organic loading such as estuaries and bays could undergo irreversible
Figure 8 – Influence of deep water nitrate concentrations on the relative importance of DNRA (DNRAtot) to total nitrate reduction (Dtot + DNRAtot).
21
shifts to nitrogen recycling to the pelagic zone, which in turn exacerbates
eutrophication and affects ecosystem functioning.
2. Macrofauna and Meiofauna
Benthic fauna describes those animals that live in the sediment (infauna) or on
top of the sediment (epifauna). Benthic fauna can also be differentiated upon the
body size: macrofauna represents animals with dimensions >1 mm while
meiofauna represents those with dimensions between 40 µm and 1 mm.
Burrowing macrofaunal organisms, such as polychaetes, bivalves, crustaceans,
insect larvae, are very important for regulation of benthic nutrient cycling and
microbial reaction rates (Aller 1982; Aller and Yingst 1985; Kristensen and
Kostka 2005). Meiofauna’s role on biogeochemical dynamics, however, is much
less known, with the exception of a few seminal papers (Aller and Aller 1992).
Macrofauna generally plays a positive role in nutrient dynamics and organic
matter mineralization through particle reworking and burrow ventilation (Aller
and Aller 1998; Kristensen et al. 2012). In particular, macrofauna commonly
helps the creation of oxic zones in the sediment, which stimulate nitrification. It
might also facilitate solute transport as a result of burrow ventilation, and
secretion of labile organic substances on burrow linings. These series of factors
may result in an increase in nitrogen removal as they all potentially enhance
microbial denitrification (Karlson et al. 2007; Kristensen and Kostka 2005; Stief
2013). However, this enhancement is not always observed since it is strictly
dependent on the bioturbation mode, which in turn depends on the morphology,
physiology and ethology of macrofauna.
Eutrophication and oxygen paucity might lead to abrupt changes to the benthic
biota. The ongoing shift in the macrofauna communities, for example, from an
amphipod-dominated to a small polychaete-dominated community as it is
happening in the Baltic Sea (Kotta and Ólafsson 2003; Perus and Bonsdorff
2004), might therefore lead to strong changes in the sediment biogeochemistry
and ecosystem functioning. In fact amphipods have been shown to stimulate
denitrification as a result of their reworking and bioturbation in the very top
centimeters of the sediment, where compounds are mobilized and easily oxidized,
serving as electron acceptors for different microbial metabolisms (Pelegri et al.
1994; Tuominen et al. 1999). In contrast, small polychaetes of the genus
Marenzelleria spp. (Figure 9) have the opposite effect and negatively affect
22
denitrification because they stimulate strictly anaerobic metabolism more than
aerobic metabolism (Paper III).
Paper III also demonstrates that
Marenzelleria spp. enhance significantly
nitrogen recycling, as a result of
DNRA rates being three times higher
in the oxidized sediment in presence
of the polychaete compared to the
same sediment without polychaetes.
Microelectrode profiling showed that
the sulfide front was closer to the
sediment surface in the Marenzelleria
cores, suggesting that sulfate
reduction was stimulated by the
polychaete, according to a previous
study (Kristensen et al. 2011). It is
therefore likely that high
concentrations of free sulfides
resulted to be toxic to heterotrophic
denitrifiers (Sørensen et al. 1980), and
might represent a competitive
advantage for DNRA bacteria (Paper
III).
Whereas an enormous body of literature has been dealing with macrofauna and
its effect on sediment biogeochemistry only a few papers have explored the role
of smaller benthic organisms such as meiofauna on benthic ecosystem services
(Nascimento et al. 2012; Näslund et al. 2010). The role of meiofauna needs to be
investigated more because benthic environments characterized by low inputs of
organic matter usually cannot sustain a structured and abundant endobenthic
macrofaunal community. On the contrary, meiofauna is often not only the most
abundant but also the faunal group with the highest biomass in this sort of
environments (Levin 2003; Rysgaard et al. 2000). Moreover, meiofauna is the
most diverse metazoan group in aquatic sediments and makes up approximately
60% of total metazoan abundance on our planet (Danovaro et al. 2010).
Paper IV investigated for the first time the role of meiofauna on benthic nitrate
reduction pathways. A density extraction method was used to isolate living
metazoans and to test the effect of low meiofauna and high meiofauna
Figure 9 – Specimens of Marenzelleria spp. digging into an artificial substrate (glass beads of 1 mm diameter).
23
abundances in the presence and absence of macrofauna for microbial nitrogen
cycling processes. As a major result, high bioturbation by meiofauna (Figure 10)
significantly enhanced the production of N2 by the denitrifying bacteria present in
the sediment. Meiofaunal denitrification (Risgaard-Petersen et al. 2006) was
excluded because incubation with nematode specimen and anoxic water amended
with 15NO3- did not produce any labeled N2 (Paper IV).
Bacterial denitrification was stimulated because it was tightly associated with
nitrification, and this latter process was in turn enhanced as a result of: (1)
increased solute transport and reaction rates associated with dense burrowing by
meiofauna in the oxic sediment strata (Figure 10) (Aller and Aller 1992); (2) large
excretion of ammonium by nematodes (Ferris et al. 1998), which usually
constitute the most abundant faunal taxon in marine sediments (Nascimento et
al. 2012). Marine meiofauna can be considered an important group of ecosystem
engineers since it has an effect on microorganisms (Näslund et al. 2010), related
processes such as microbial denitrification (Paper IV), and creation and
maintenance of idiosyncratic microhabitats (Badano and Cavieres 2006).
Conversely, macrofaunal groups like polychaetes and bivalves were demonstrated
to have a significant stimulatory effect on DNRA rates in the sediment (Paper
III, IV). Assuming that these macroorganisms would have a very small impact or
even no impact on phosphorus releases (Paper III), enhanced nitrogen recycling
to the water column might increase the N:P ratio in the pelagic realm, limiting
cyanobacterial blooms. However, this is just a hypothesis and more systematic,
seasonal, long-term studies are needed to demonstrate that the concept may be
valid in eutrophic ecosystems such the Baltic Sea.
Figure 10 – Optical photographs showing differences in the burrow patterns and distribution in sediment containing low meiofauna abundances and biomasses (left) and high meiofauna abundances and biomasses (right). Scale = 0.5 mm.
24
3. The role of aggregates
Aggregates, or marine snow, are formations of different particulate material such
as detritus, fecal pellets, algal filaments, inorganic material that can often be
found in the pelagic zone. Increasing eutrophic conditions, calm and warm
weather result in the formation of macroscopic aggregates (>0.5 mm) as the
surface waters of the Adriatic Sea and the Baltic Sea have testified (Degobbis
1989; Ploug et al. 2011). Because of their organic, often labile nature, they are
important in the marine food web, in the nutrient cycling and as hot-spots of
microbial processes such as carbon mineralization (Simon et al. 2002).
Although the last two decades have seen a considerable improvement in
understanding the role of aggregates for food webs and carbon turnover, their
contribution to nitrogen cycling process is still pretty much unknown. Paper V
aims to detect and quantify rates of both aerobic and anaerobic nitrogen
pathways inside and surrounding macroscopic aggregates. Aggregates of the
noxious algae Nodularia spumigena were collected in a Baltic Sea coastal area after
some days of calm and warm weather, which allowed them to float to the water
surface. These fresh aggregates were subject to microsensor (O2 and N2O)
measurements and incubation with the addition of different 15N inorganic
substrates.
Aggregates of Nodularia spumigena were relatively big (>3 mm), compact and
characterized by high oxygen uptake, conditions that made the aggregates’ core
anoxic (Paper V) (Ploug et al. 2011). High abundances of bacteria in the
aggregates (Kiørboe et al. 2002), anoxic conditions, and presence of nitrogenous
oxidized substrates made it possible to trigger the complete spectrum of nitrate
reduction mechanisms. However, the proportion between the final products of
these reductions revealed that DNRA was far more important than
denitrification, with anammox playing the less important role (Figure 11). If, on
one hand, unstable physico-chemical gradients hampered the anammox reaction,
it is still not clear why DNRA outcompeted denitrification. A hypothesis is that
high organic C:N ratios present in the aggregates’ anoxic cores would have
promoted DNRA bacteria, which yield more energy per mole of nitrate reduced
compared to denitrifiers (Giblin et al. 2013; Kraft et al. 2014).
25
Nitrification and N2 fixation were
also documented to happen in
association with N. spumigena
aggregates so Paper V showed that a
complete nitrogen cycling is present
and active in algal aggregates, even
when they are floating in fully aerated
waters. Albeit process rates could
always be detected and quantified, the
processes were considered potential
since the incubations were carried out
with excess of substrates (~30 µM).
However, in situ process rates of
DNRA and denitrification associated
to marine snow in oxygen minimum
zones (OMZs) might be even higher
than the potential rates reported in
Paper V because OMZs waters are
characterized by: (1) anoxia or
oxygen concentrations close to zero,
which expand the dimension of the
aggregate’s anoxic core; (2) abundant
dissolved nitrate and/or nitrite (De
Brabandere et al. 2014; Kuypers et al. 2005).
CONCLUSIONS AND FUTURE PERSPECTIVES
1. In situ studies for global N2 exchanges
The experimental work conducted for this thesis contributed to a better
understanding of the following: the regulative mechanisms of nitrogen
metabolism by meiofauna and peculiar macrofaunal groups, role of algal
aggregates in the marine environment by the point of view of the associated
microbial nitrogen pathways, and nitrogenous species dynamics at the
sediment/water interface for some anthropogenically affected coastal areas.
With the exception of Paper II, the studies were conducted under ex situ
conditions by means of sediment/water sampling and subsequent incubations in
Figure 11 – The entire spectrum of nitrogen cycling processes was observed in association with aggregates of Nodularia spumigena. The arrow width outlines the relative importance of each process.
26
the laboratory, trying to imitate in situ conditions. The main advantages of using
laboratory incubations are the limited cost and the fact that the effect of many
different conditions can be tested simultaneously by comparing different
treatments. On the other hand in situ studies, carried out by means of benthic
landers deployment, are absolutely necessary for studying the deep-sea conditions
(Tengberg et al. 1995). However, also in shallow coastal and shelf areas landers
might be preferred as they generally avoid artifacts and corrections introduced by
on land operations (i.e., strong changes in pressure, surface disturbance while
sampling and sub-coring, adjustment of extrinsic parameters such as temperature
or oxygen during incubation).
Autonomous free-fall benthic landers are perfectly suited to investigate in situ
nutrient benthic exchange and reaction processes (Tengberg et al. 1995). By
either extracting samples from or injecting solutions to the benthic incubation
chambers it is also possible to conduct in situ studies with 15N tracers, to
determine denitrification, DNRA and anammox (Paper II). Recent studies have
shown that coastal marine sediments might be a net source of nitrogen, as N2
fixation might be more important than denitrification (Fulweiler et al. 2007). In
situ incubations with benthic landers coupled to sensitive N2:Ar analysis would
allow the study of the total N2 exchange in areas of the sea where the proportion
between N2 fixation and N2 loss still need to be depicted, and where in situ
conditions are difficult to reproduce in the lab. To this end, the seafloor where
the oxycline intersects the sediment surface and large sulfur bacteria often
proliferate, might have a huge potential as N2 sink (Bonaglia, unpublished data),
and should be investigated further.
2. Role of infauna for methane and nitrous oxide emissions
Methane (CH4) and nitrous oxide (N2O) emissions from productive aquatic
ecosystems contribute globally to the greenhouse effect and as a consequence
they influence climate warming. In recent decades greenhouse gases emissions
have substantially increased mainly because human impact on ecosystems has
intensified considerably (Vitousek et al. 1997). Benthic environments such as
lake- and sea-floors are very important ecosystems, where a substantial part of
the organic matter produced or transported is remineralized into nutrients and
elements.
Benthic infauna has an essential role in sediment reworking, therefore favoring
mineralization and oxidation of metabolites (Paper IV and references therein).
27
Ecosystem stresses such as eutrophication, hypoxia/anoxia, biological invasions
and climate warming have been shown to deeply alter benthic faunal
communities. Recent studies have provided direct evidence of considerable
emissions of N2O indirectly associated with the metabolism of several
macrofaunal species (Heisterkamp et al. 2010; Stief et al. 2009). However, it is still
unclear which feedbacks affected benthic communities could have on that long
list of stresses and, in particular, on methane emissions and related global
warming.
A systematic ongoing study is investigating the contribution of common Baltic
Sea infauna taxa and biological invaders that recently colonized the Baltic Sea
sediments to nitrogen and carbon cycling, in terms of nitrogen- and carbon-gas
emissions. Questions such as the following will be addressed. Are benthic animals
important contributors to emissions of methane and nitrous oxide? Are the Baltic
Sea sediments a net source of greenhouse gases to the water and the atmosphere?
Is the invasive polychaete Marenzelleria spp. further enhancing these emissions? It
is hoped that concrete answers to those questions would help managers and
stakeholders to evaluate the actual sustainability of benthic aquaculture and to
take measures against invasion of allochthonous species.
ACKNOWLEDGEMENTS
I warmly thank my supervisors Patrick Crill, Volker Brüchert, Per Hall and
Christoph Humborg for valuable help in carrying out the research in these last
years. Also, my co-authors are acknowledged for their precious comments and
ideas on the thesis’ papers. I would like to thank especially Marco Bartoli,
Francisco Nascimento, Barbara Deutsch, Loreto De Brabandere, Isabell
Klawonn and Helle Ploug for practical support, inspiring discussions and very
useful advices. What I have learnt from the people listed in this paragraph goes
far beyond this thesis.
The following scientists are thanked for extremely valuable discussions and
advices throughout this project: Ragnar Elmgren, Jonas Gunnarsson, Ernest Chi
Fru (Stockholm University), Maren Voss (Leibniz Institute for Baltic Sea
Research), Bo Thamdrup (University of Southern Denmark), Hannah Marchand,
Gaute Lavik and Moritz Holtappels (Max Planck Institute for Marine
Microbiology). I would also like to thank Hildred Crill (Stockholm University)
who helped me to improve the English of this thesis.
28
I am very thankful to: Heike Siegmund (Stable Isotope Laboratory) and Henry
Holmstrand (Delta Facility) for assistance with analysis at the mass spectrometer;
Duc Nguyen and Jayne Rattray for sharing their skills in gas and liquid
chromatography; Anders Sjösten and his lab, Per Hjelmqvist and Jörgen Ek for
practical help with nutrient analysis. Thanks also to the Askö laboratory staff, the
crew of R/V Skagerak and Misha Kononets (University of Gothenburg) for their
help and fruitful collaboration during time at sea. Nils Ekeroth, Caroline
Raymond, Ola Svensson, Silvia Fedrizzi, Joanna Sawicka, Livija Ginters and
Linnea Isaksson are thanked for providing practical help and a very nice and
friendly working environment during field work at the Askö laboratory.
Sincere and warm thanks to my officemates, to my colleagues from the
Department of Geological Sciences and Applied Environmental Sciences, to the
Lappis crew and all the other friends who contributed to a good time inside and
outside the university. Forever I will be indebted to those people (you know who
you are) for their affection and for unique situations lived together. So, gratitude!
Of course, famiglia e amici are also warmly thanked for their long-distance support.
Finally I gratefully acknowledge funding from: two FORMAS projects that
mainly sponsored this work “Baltic Sea Ecosystem Adaptive Management
(BEAM)” and “Managing Baltic nutrients in relation to cyanobacterial blooms”;
Stockholm University’s Baltic Sea Centre, which provided extra funding for field
work at the Askö laboratory; K&A Wallenbergs stiftelse and the Geochemistry
group (Stockholm University), which covered part of the expenses for Ph.D.
courses and travels to international conferences.
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