Control Factors of the Marine Nitrogen Cycle

Home , Denitrification, Lithotroph, Nitrification, Nitrogen cycle, Nitrogen fixation

Control factors of the marine nitrogen cycle The role of meiofauna, macrofauna, oxygen and aggregates Stefano Bonaglia Academic dissertation for the Degree of Doctor of Philosophy in Geochemistry at Stockholm University to be publicly defended on Wednesday 29 April 2015 at 13.00 in Nordenskiöldsalen, Geovetenskapens hus, Svante Arrhenius väg 12.

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.

Stockholm 2015 http://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

©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

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.

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

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.

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.

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)

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

+ 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.

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

10 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.

A B

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).

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

12 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 Figure 4 – The vicious circle leading to exacerbation of cyanobacteria blooms in the tends to increase nitrogen Baltic Sea. Source: Vahtera et al. (2007). removal by denitrification and anammox in the water

13 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

14 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 Figure 5 – Example of sediment cores (Näslund et al. 2010). used in this thesis, i.e. plexiglas tubes filled half with sediment and half with The second approach was also water, with a stirring device placed in the water column. 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,

15 nutrients, metals). Lastly, the incubation method allows the artificial enrichment 15 - 15 + with stable isotopes of nitrogen such as NO3 and NH4 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).

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 15 - - + with N 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.

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).

The first task of this Ph.D. project was to establish the IPT and a method to 15 15 + detect N-N2 and NH4 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

17 amounts) of the 14N14N, 14N15N, and 15N15N masses were recorded. After every 29 fifth sample, samples of air were injected as standard. The concentrations of N2 30 and N2 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 Figure 7 – The study area of Paper I, understanding spatio-temporal dynamics Himmerfjärden. The four sampling in nutrient cycling. stations, located along the eutrophication gradient (north- The inner part of Himmerfjärden (Figure south), are H6, H4, H2 and B1. STP 7) is usually stratified during summer and indicates the sewage treatment plant experiences hypoxic bottom waters and active in the area and serving about 300,000 people. reduced sediments, especially in the deeper areas of the estuary. In the outer basins hypoxia is only transient as attested by

18 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).

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

75 nitrate concentrations suddenly

70 (%) increased in the bottom water.

Coefficients: 

b[0] 13.3 tot However, the relative importance of b[1] 2.6 65 r ² 0.91 DNRA increased with time and 60

+ DNRA increasing concentrations of nitrate

55 tot (Figure 8). In this artificially 50 /(D tot oxygenated sediment the large 45 sulfur bacteria Beggiatoa spp. might 40 DNRA 10 12 14 16 18 20 22 24 have played an increasingly - -1 NO3 (mol L ) important role in stimulating Figure 8 – Influence of deep water nitrate DNRA and thus nitrogen recycling. concentrations on the relative importance In both coastal systems N2 of DNRA (DNRAtot) to total nitrate reduction (Dtot + DNRAtot). 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

20 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

21 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 Figure 9 – Specimens of Marenzelleria denitrifiers (Sørensen et al. 1980), and spp. digging into an artificial substrate might represent a competitive (glass beads of 1 mm diameter). 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

22 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 15 - with NO3 did not produce any labeled N2 (Paper IV).

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.

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.

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).

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 Figure 11 – The entire spectrum of characterized by: (1) anoxia or nitrogen cycling processes was observed oxygen concentrations close to zero, in association with aggregates of which expand the dimension of the Nodularia spumigena. The arrow width aggregate’s anoxic core; (2) abundant outlines the relative importance of each process. 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

25 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).

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.

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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