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

2

©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

3

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

5

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.

6

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

9

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.

10

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

11

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

13

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).

14

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

15

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.

16

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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