Benthic metabolism and sediment nitrogen cycling
in Baltic Sea coastal areas:
the role of eutrophication, hypoxia and bioturbation
Stefano Bonaglia
ABSTRACT
Eutrophication is one of the greatest threats for the Baltic Sea, and one of its more critical consequences is bottom water hypoxia. Nutrient enrichment and oxygen-depletion affect both the deep central basins and a number of coastal areas, even though strategies for nutrient reduction have lately been implemented. In order to better understand why those threats are expanding and formulate more effective remediation strategies two main achievements are needed: (1) new data on benthic nutrient dynamics should be available in order to develop updated budgets for sensitive Baltic areas; (2) the main transformation processes and their regulation mechanisms (i.e. oxygen availability, presence of macrofauna, different organic loading scenarios) should be better constrained.
The aim of this licentiate thesis was to present a set of measurements of benthic biogeochemical processes under the conditions of eutrophication and advanced hypoxia. Thanks to a long-term experimental ex-situ approach, it was possible to test the effects of hypoxia, of re-oxygenation and bioturbation on benthic metabolism. Moreover, an annual nitrogen budget was proposed, based on measured data, for a number of stations along a nutrient-enriched estuarine system, where those processes were totally unexplored before.
This thesis was able to demonstrate that re-oxygenation of previously anoxic sediment has a positive effect on the ecosystem because of better retention of nutrients and efficient conversion of fixed nitrogen to nitrogen gas. Sediment colonization by the invasive genus
Marenzelleria counteracts some of the positive aspects provided by benthic oxygenation (in
particular, nutrient retention, N2 loss). A possible explanation for this reversal can be that
Marenzelleria does stimulate anaerobic more that aerobic metabolism.
Results from the seasonal study in the estuarine system suggest that at the outermost stations denitrification follows a pronounced seasonal pattern, primarily regulated by bottom water temperatures. At the innermost and impacted site oxygen level in the bottom water varies considerably during the year and causes denitrification/DNRA predominance to be the main nitrate reduction pathway. On an annual scale, the net amount of lost N2 is comparable at the four sampling sites and accounts for 96% of the total DIN discharged from the sewage treatment plant, suggesting that denitrification in the estuarine sediment acts as a major nitrogen sink for external N inputs.
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List of Contents:
1. Introduction: Baltic Sea nutrient cycles and aim of this thesis ... 3
2. Methods ... 6
2.1 Quantification of Benthic Solute Exchanges ... 6
2.2 The Nitrogen Isotope Pairing Approach ... 7
2.3 Studied Areas ... 9
3. Discussion of the Main Results ... 11
3.1 Paper I ... 11
3.2 Paper II ... 13
4. Conclusion and Future Perspectives ... 15
5. Acknowledgments... 18
6. References ... 20
Paper I ... 25
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1. Introduction: Baltic Sea nutrient cycles and aim of this thesis
In the last few decades marine and coastal areas have been threatened by several environmental issues. Main impacts have arisen from overfishing to spillages of toxic substances; from water acidification to global warming and cultural eutrophication. Some areas of the world are more sensitive to those threats because of their peculiar configuration, i.e. their morphology and location. This is the case of the Baltic Sea, a semi-enclosed brackish basin characterized by a high ratio between the areas of the drainage basin and its surface (Leppäranta and Myrberg 2009). The area of the Baltic Sea is greater than 400,000 km2 and has six sub-basins, separated by sills and belts, and the drainage area is inhabited by approximately 85 million people (Helcom 2009). Those factors plus the unique geomorphology of the Baltic, separated from the North Sea by the shallow and tangled Danish Straits, determine very long water residence time, strong thermohaline stratification (Fig. 1), and, as an obvious consequence, high sensitivity for nutrient enrichment both in the open sea and in the coastal areas.
Figure 1 – Water column profiles (n≈100) of salinity (a) and temperature (b) selected randomly from stations deeper than 100 m from the Baltic Proper. The profiles were collected between 1997 and 2005 (from May to September). SW indicates surface-water, MW middle-water, and DW deep-water. Modified from Håkanson & Bryhn (2008).
Once eutrophication is established in deep coastal environments, enclosed basins, and coastal systems affected by poor exchange of waters, another major problem arises: hypoxia, the oxygen deficiency in the water masses, usually defined as <60 µmol O2 L-1.
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Hypoxia was already present in the Baltic Sea during its formation, more than 8000 years ago but it has expanded further since 1960, and now it is affecting not only the deeper basins but also the coastal zones (Conley et al. 2009a; Conley et al. 2011). 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 or using engineering solutions to increase the oxygen level in the bottom waters (Conley et al. 2009b; Stigebrandt and Gustafsson 2007).
In fact, in the Baltic Sea diffuse and point sources from the land (e.g. agriculture, sewage treatment plants) are the main input of nutrient load. Recent studies have reported that in the last century nutrient loads to the watershed, including atmospheric depositions, increased by a factor of four (Schernewski and Neumann 2005). The Baltic Sea water is characterized by higher nutrient concentrations in winter and by values near the detection limit from early spring to late autumn (Nausch et al. 2008). This is obviously due to varying abundances of biological productivity during the seasons. Phytoplankton blooms cause a rapid nutrient depletion and generally start when the upper mixed layer becomes shallower than the light penetration depth (Wasmund et al. 1998).
It is well known that marine phytoplankton takes up nutrients in a precise molar ratio of C:N:P=106:16:1 (Redfield et al. 1963). If one or more nutrients are not present in the right proportion, phytoplankton undergoes stress conditions due to the limiting element. In the Baltic Proper N is the limiting factor, and the common reported ratio of N:P is 7-9:1 (Nausch et al. 2008). This condition gives the N2-fixing filamentous cyanobacteria an advantage because they are not limited by nitrogen and can capitalize on the mesohaline conditions prevailing in the central and eastern Baltic Sea (Stal et al. 1999; Wasmund 1997). A large number of the primary producers start to be decomposed already in the water column or, alternatively, they sink down to the sediment, in both cases leading to a marked consumption of dissolved oxygen (Vahtera et al. 2007).
Under hypoxic or anoxic conditions phosphate is released from the sediment to the upper mixed water layer because of the reduction of iron oxy-hydroxides (Rozan et al. 2002). In turn, in the warm season in the nutrient-depleted euphotic zone cyanobacteria blooms are highly stimulated when the regenerated phosphate reaches the upper water. Furthermore hypoxia and anoxia, besides increasing internal loading of phosphate, tends to increase nitrogen removal by denitrification and anammox both in the water column and in
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the sediment (Canfield et al. 2005; Hietanen et al. 2012), thus lowering the N:P stoichiometry even further (Vahtera et al. 2007).
Studies on Holocene sediments give evidence that such blooms have been a component of the Baltic Sea ecosystem throughout its present brackish state (Bianchi et al. 2000). Although widespread in lakes and coastal areas, they are generally not present in marine regions. It is important to emphasize that these conditions accentuate the eutrophic state of the Baltic Sea and its most discussed feature – the cyanobacteria blooms. This series of mechanisms acting in the Baltic Sea have been described as a “vicious circle” (Fig. 2) (Tamminen and Andersen 2007; Vahtera et al. 2007).
Figure 2 – The scheme reveals the main processes that involve nutrient transformations in the Baltic Sea environment. This complex set of interactions can be seen as a “vicious circle” (Vahtera et al. 2007).
Updated values for solute transport across the sediment-water interface and the description of the benthic processes of nitrogen and phosphorus removal are pivotal when nutrient budgets are compiled in order to identify the correct remediation strategy for the Baltic Sea and to apply the nutrient reduction measures to other similar basins around the world. The main intent of this thesis was to better understand benthic biogeochemical processes that take place in impacted Baltic Sea shallow and deep coastal areas. In particular the thesis focused on understanding whether nitrogen removal pathways (i.e., denitrification, anammox) or pathways of nitrogen regeneration (i.e., ammonification, DNRA) were quantitatively more important in those studied areas.
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Paper I had the main objective to test the effects of re-oxygenation and Marenzelleria spp. bioturbation on previously hypoxic sediment by means of a long term mesocosm experiment. Marenzelleria is a deep-burrowing invasive polychaete, which can cope with sulfidic environments. The main aim of the study was to investigate changes in solute exchange and nutrient cycling by means of core incubations technique.
In paper II we measured the main pathways of sediment N cycling and oxygen micro- and macro-dynamics along the axis of an impacted Baltic Sea estuary by means of extensive sampling and sediment cores incubations. The estuary is impacted by seasonal hypoxia and offers a range of different sediment types and physicochemical bottom water characteristics that is suitable for a seasonal study.
2. Methods
2.1 Quantification of Benthic Solute Exchanges
Measuring the exchange of solutes between the sediment and the water column is central to understand marine nutrient dynamics, their trends and their relation to eutrophication. Many approaches have been used to quantify the transport of solutes, but they can be all categorized in two main groups: (1) study of gradients based on the determination of differences in solute concentration in depth profiles (both in the water column and in the sediment); (2) sediment chambers (or cores) incubation, where solutes fluxes are quantified by measuring the change of solute concentrations in the overlying water over time (Zabel and Hensen 2006).
Both methods were applied in the present thesis, specifically in paper II. Unfortunately profiles obtained by porewater analysis have a poor vertical resolution (at most 3-4 mm), and this did not allow the study of nitrate and nitrite dynamics that usually occur in the top few millimeters. However, by means of oxygen microsensors a resolution of several micrometers can be observed and the fluxes can be calculated from the concentration gradients by means of diffusive flux models (Berg et al. 1998). This gradient approach does not take into account the convective flux caused by macrofauna and consequently
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underestimates the total solute exchange when bioturbation 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, simulating the natural bottom water flow. Another positive aspect of this method is that during a single incubation several solute components can be sampled at the same time (e.g., gases, nutrients, metals). For those reasons we preferred the latter method that was extensively used both for quantifying fluxes of nutrients and for measuring N-process rates, by means of 15
N additions.
2.2 The Nitrogen Isotope Pairing Approach
Different methods have been tested and used for detecting and quantifying N2 production in marine and freshwater sediment. During the last two decades many improvements have been achieved in this field, a consequence of the increased attention to sensitive issues such as the increase in coastal nitrogen concentrations, eutrophication and hypoxia (Groffman et al. 2006; Steingruber et al. 2001). Among the different approaches the most used are certainly the acetylene inhibition technique, the quantification of N2 production based on N2:Ar measurements, and the 15N isotope pairing technique. The last method has two main advantages: the very high analytical precision when an isotope ratio mass spectrometer is employed as detector, and the fact that it makes it possible to dissect the sources of N2 production. This is a very big achievement since the combination of different incubation techniques with 15N isotope labeling of NO2-, NO3- and NH4+ molecules allows discrimination among the processes of nitrate removal: denitrification of water nitrate (Dw), denitrification coupled to nitrification (Dn), anammox (An), and dissimilatory nitrate reduction to ammonium (DNRA).
Specifically, the first task of this thesis project was to set up a method for measuring labeled-N2 from gas samples (headspace technique). A self-built gas chromatographic line (Fig. 3) was connected to a continuous-flow isotope ratio mass spectrometer (IRMS). Its construction followed the scheme presented in Holtappels et al. (2011). Briefly, after incubating intact sediment cores with different amounts of 15NO3- samples of water and superficial sediment were withdrawn and stored in gastight glass vials (Exetainers, Labco
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Scientific, UK) for N isotope analysis. First, a headspace volume of helium (5.0 purity) was introduced to the Exetainers and the vials were left standing for 12 to 24 hours before the analysis. The isotopic composition of the N2 pool was determined by injecting manually 100 µl headspace with a gastight glass syringe (SGE Analytical Science, AU) to the GC line. The injected sample was carried by a helium stream (30 ml min-1) and passed through a series of traps: (1) a water freeze trap (liquid N2), (2) a copper reduction column heated to 650 °C, and (3) a GC column (Supelco Porapak Q, 1/8 in, 2 m) before entering the open split at the Conflo IV interface (Thermo Scientific, DE). The different masses of the N2 gas (masses 28, 29, and 30) were analyzed on a triple collector of a Delta V Advantage (Thermo Scientific) and the areas (amounts) of the 14N14N, 14N15N, and 15N15N masses were recorded. After every fifth sample, 10 µl 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).
Figure 3 – Schematic presentation of the headspace sampling and the gas chromatographic line used to prepare the sample before mass spectrometric analysis. Modified from Holtappels et al. (2011).
Moreover, this method allowed me to analyze the labeled fraction of the dissolved ammonium pool, in order to calculate DNRA rates. For the 15N-fraction of ammonium, water samples were transferred to small Exetainers, degassed with helium (5.0 purity) in order to remove all the background nitrogen gas, and finally treated with 200 µl hypobromite-iodine solution to oxidize NH4+ to N2. The sample was then treated as described above. The
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conversion was tested with standard solutions of 15NH4+ and 14NH4+ and was generally better than 92%. Finally in situ DNRA rates were calculated according to Risgaard-Petersen & Rysgaard (1995).
2.3 Studied Areas
The study presented in paper I was carried out with sediment from Kanholmsfjärden, a 100 m deep enclosed bay situated in the eastern part of Stockholm archipelago (Fig. 4).
Figure 4 – Location of the two study areas in the Stockholm archipelago. The surface associated with number 1 shows Kanholmsfjärden, the study area of paper I, and number 2 represents Himmerfjärden, the study site of paper II.
This bay has been monitored since 1982 and from that time on it has been documented that the bottom waters were cyclically anoxic/hypoxic (Stockholm Vatten AB, 2011). A pilot study of applied environmental engineering started in 2009 in order to artificially oxygenate the hypoxic deep water. A pump that distributed superficial water down to the anoxic bottom water was placed in the middle of the bay with the aim to introduce ~100 kg O2 s-1, a minimum condition to keep water well oxygenated (Stigebrandt and Gustafsson 2007).
The aim of the study presented in paper I was to test the effect of re-oxygenation and bioturbation of Kanholmsfjärden sediment by means of an ex situ experiment. In brief
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several sediment boxcore mesocosms were collected, brought to an experimental hall and re-oxygenated under controlled conditions. Four mesocosms were kept under hypoxic conditions and eight were supplied with oxygenated seawater. Specimens of Marenzelleria spp. were added to four of the oxygenated mesocosms. After two months of conditioning solute exchanges between the sediment and the water column were measured. The three different treatments (hypoxic sediment, oxic sediment and oxic plus macrofauna) were kept under the same conditions during the flux measurements, with the only difference that the mesocosms were sub-sampled in smaller sediment cores, which were easier to handle and necessary in destructive sampling as in the case for denitrification incubation. For a more detailed description of the methods used in this study, please see Materials and Methods in paper I.
In paper II the investigated area was Himmerfjärden, a N-S oriented estuary located in the southern Stockholm archipelago (Fig. 4). The estuary is divided into four sub-basins separated by W-E oriented sills. The drainage basin is quite large (1286 km2) compared to the embayment area (174 km2), but it is mainly covered by forest and does not supply heavy nutrient loading to the estuary. Himmerfjärden has an average depth of 17 m with deeper zones of almost 50 m and an average salinity of 6 ‰. The inner part of the estuary is usually stratified during summer and can experience hypoxic bottom waters and reduced sediments, especially in the deeper and more stagnant areas of the bay (Elmgren and Larsson 1997). In the outer basins hypoxia is only transient as attested by oxidized and macrofauna-inhabited superficial sediment, and it only occurs when winds are weak and circulation is inhibited.
In the northern and innermost part of the estuary a sewage treatment plant (STP) has been discharging treated sewage water since 1974. In the late 80s the STP discharged an enormous quantity of total nitrogen to the bay (~900 t yr-1) and a comparatively low quantity of total phosphorus (~15 t yr-1). Since 2000 the N discharge was substantially reduced thanks to more advanced technology and the introduction of tertiary water treatment. Except for 2007-2008 the bay has received 300-350 t N yr-1 but there is still ~15 t P yr-1. Considering that the STP discharge and the water from northern Lake Mälaren are the main sources of nutrients to the inner bay, it is evident that the innermost part of the estuary is under eutrophic conditions and the outer areas present more pristine conditions.
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For the Himmerfjärden study three stations were chosen along the eutrophication gradient of the estuary plus one station outside the bay serving as a control station. The main aim was to explore the differences in sediment N cycling and the dynamics of oxygen consumption along this impacted estuary. An extensive annual study was carried out and the same methodologies of sampling and sediment incubation were repeated from May 2011 to January 2012. For a full description of the techniques used in this study, please refer to paper II.
3. Discussion of the Main Results 3.1 Paper I
The reversal from hypoxic to normoxic conditions in the mesocosm experiment led to major changes in the benthic metabolism of Kanholmsfärden sediment. Oxygen uptake was stimulated by a factor of greater than three, ammonium efflux stopped, and regeneration of manganese and silica was substantially reduced (Fig. 5a,b,c,d). On the other hand phosphate flux was not altered significantly as for the other investigated solutes (dissolved CH4 and Fe). Denitrification was stimulated by a factor of six and DNRA almost completely stopped (Fig. 5e,f). All those alterations in the benthic processes induce a positive effect at the ecosystem level because better retention of silica and greater conversion of fixed nitrogen to N2 gas would decrease the amount of recycled nutrients that are normally available to primary production. In turn, this would decrease eutrophication.
In the Baltic Sea, colonization by macrofauna, particularly of the invasive genus
Marenzelleria, is expected once normoxia is established (Karlson et al. 2011). Sediment cores
with these polychaetes generated significantly higher effluxes of manganese, dissolved inorganic nitrogen and dissolved silica compared to normoxic non-bioturbated sediment (Fig. 5b,c,d). These results suggest that the doubled O2 uptake in the bioturbated treatment compared to the oxic non-bioturbated one was mainly due to respiration by Marenzelleria (67%) and secondarily due to additional chemical and biological oxidation in the burrow walls (33%). Surprisingly, the polychaete presence and activity stimulated neither nitrification nor denitrification. In contrast, denitrification was even lower and DNRA rates
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were higher in the bioturbated treatment compared to the oxic non-bioturbated one (Fig. 5e,f). This was possibly due to the more sulfidic environment established in the presence of
Marenzelleria, a hypothesis first suggested by Kristensen et al. (2011) and confirmed by the
O2 and H2S microprofiles. HY NO NOB 0 20 40 60 80 H4SiO4 flux d HY NO NOB 0 2 4 6 8 10 12 Dw Dn e HY NO NOB -20 0 20 40 60 c HY NO NOB 0 1 2 3 4 5 6 DNRAw DNRAn f HY NO NOB 0 30 60 90 120 150 180 Mn flux b HY NO NOB µm ol m -2 h -1 -600 -500 -400 -300 -200 -100 0 O2 flux a µm ol m -2 h -1 NO3 flux NH4+ flux
Figure 5 – Net fluxes of a) oxygen, b) manganese, c) nitrate and ammonium, d) phosphate; e) rates of nitrification-coupled (Dn) and of water column nitrate denitrification (Dw); f) rates of dissimilatory nitrate reduction to ammonium coupled to nitrification (DNRAn) and fueled by water column nitrate (DNRAw) measured in intact sediment cores for the hypoxic (HY), normoxic (NO) and normoxic-bioturbated (NOB) treatments. Averages ± standard error are reported; positive fluxes are from the sediment to the water column.
Therefore, as one of the main findings of this work, it was demonstrated that repopulation of previously anoxic bottoms of the Baltic Sea with invasive polychaetes partially counteracts the positive nutrient retention effects of benthic oxygenation. Paper I has general implications for predicting the response of anoxic seafloors following reoxygenation. One of the aspects introduced by the approach described here compared to other papers dealing with oxic-anoxic shift and bioturbation was that intact sediment was used instead of sieved one, resulting in unaltered pools and profiles. Moreover, the sediment was conditioned and manipulated for more than seven weeks, which makes it a much better representation of what really happens in situ.
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3.2 Paper II
Results from paper II suggested that the proportion of benthic nitrogen cycling pathways differed in the basins of the estuary along the eutrophication gradient. At station H6, the inner and most impacted one, the production of ammonium was decoupled from oxidation via nitrification, preventing nitrogen loss via denitrification. Overall, at station H6, the ratio between N2 fluxes and the sum of inorganic nitrogen species efflux was much lower than at the less impacted sites (H4, H2 and B1), resulting in higher nitrogen retention and recycling to the pelagic zone (Fig. 6).
Figure 6 - Summary of the benthic nitrogen pathways for the four sampling. All rates are reported as µmol N m-2 h-1 and are annual averages ± standard deviation. The production of ammonium was not measured in this study and has to be considered a potential ammonification rate, sensu Dalsgaard (2003). Dn represents rates of nitrification-coupled denitrification; Dw is water column nitrate denitrification; DNRAn represents rates of dissimilatory nitrate reduction to ammonium coupled to nitrification; DNRAw is DNRA fueled by water column nitrate; DE is denitrification efficiency, sensu Eyre & Ferguson (2009).
At the central and outer basins temperature was the main driver of microbial activity and correlated positively with denitrification rates. Rates in summer were up to eight fold
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higher in summer compared to winter and spring. Since organic matter content and oxygen concentrations did not correlate with denitrification, it is likely that the higher rates were mainly due to temperature increase. Organic enrichment inhibited N2 production via anammox both at the impacted site H6 and at the more pristine ones (H4, H2, B1). The level of oxygen in bottom water proved to be another important controlling factor for nitrogen cycling. In October, oxygen depletion at H6 exacerbated the above mentioned processes, stimulating ammonium effluxes and dissimilatory nitrate reduction to ammonium (DNRA) by a factor of ~4 and ~6, respectively, and lowering 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 the ones measured at the oxic stations in summer.
A major result from this study was that, surprisingly, on an annual scale the net amount of N2 was comparable at the four stations and it accounted for ~90 mmol N m-2 yr-1. This result has implications for nutrient budget estimates because it shows that under transient and seasonal hypoxia a system can quickly recover from reducing conditions and counteract ammonium regeneration processes with nitrogen removal processes. However, it seems evident that if hypoxic periods will expand further or become permanent, coastal areas that receive high organic loading such as estuaries and bays could undergo irreversible shifts to N recycling to the pelagic zone, in turn exacerbating eutrophication.
Moreover this study was important to demonstrate that sediment from Baltic Sea coastal areas are important sinks of fixed nitrogen, since there is a lack of systematic studies focusing on rates measurement, except from the studies conducted in the Gulf of Finland (Hietanen 2007; Hietanen and Kuparinen 2008; Jäntti et al. 2011). On an annual scale 221 t N are removed from Himmerfjärden sediment, which is approximately 96% of the DIN discharged annually by the sewage treatment plant. It is evident that denitrification in the estuarine sediment acts as the major nitrogen loss process for external N inputs, as previously described for a number of other coastal and shelf ecosystems (Seitzinger 1988).
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4. Conclusion and Future Perspectives
In situ versus ex situ studies
The experimental work conducted for this thesis contributed to a better understanding of nutrient dynamics and rates of microbial processes at the sediment/water interface in some impacted coastal Baltic Sea ecosystems. Both studies were conducted under ex-situ conditions by means of sediment sampling and subsequent micro- and mesocosm incubations. 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, are necessary for studying the deep-sea but they may even be preferable for shallow shelf seas (Tengberg et al. 1995). Benthic landers in particular are a well-suited technology for upscaling measured fluxes and rates to calculate areal budgets, since in situ work generally avoids artifacts and corrections introduced by on land operations (i.e. sampling, sub-coring, adjustment of extrinsic parameters such as temperature of oxygen during incubation).
Thanks to a cooperation that was recently established with the Benthic Biogeochemistry Group, Department of Chemistry, University of Gothenburg, we will conduct a study focusing on comparing ex situ and in situ techniques to investigate benthic processes in the northern Baltic in the spring and autumn 2013. The Göteborg Big Lander is an autonomous free-fall benthic lander that can either extract samples from or inject solutions to the benthic incubation chambers for in-situ incubations. This feature allows use of the chambers for in situ studies with 15N tracers, and measurement of in situ denitrification and DNRA. The study areas will be the Gulf of Bothnia and the Bothnian Bay, two areas where very few recent nitrogen cycling experiments have been conducted (Stockenberg and Johnstone 1997; Tuominen et al. 1998).
Meiobenthic bioturbation: the importance of meiofauna in marine nitrogen cycling
It is well established that macrofauna has a large large impact on sediment biogeochemistry because it alters and regulates solute fluxes and changes microbial dynamics (Kristensen and Kostka 2005). However, marine sediments, especially in the near-surface, are inhabited by another important group of animals, the meiofauna. Meiofauna is
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orders of magnitude more abundant and presents a much more diverse community structure than macrofauna, and its importance for sediment microbial pathways is very poorly understood. One of the main reasons why the meiobenthos has so far been scarcely investigated is due to the fact that these metazoans are extremely difficult to handle, since they are small (40µm to 1mm) and therefore very fragile. The first authors who recognized the importance of meiofauna on sediment nutrient cycling were Aller & Aller (1992). In their study they found that meiofauna enhanced solute exchanges by a factor of 2 compared to control sediment without meiofauna.
Meiobenthic bioturbation is very important especially in muddy sediment since it has been shown that smaller grain sizes imply more abundant meiofauna (Cullen 1973). Only very recently studies have addressed the role of meiofauna in benthic metabolism and its stimulation of microbial activity (Nascimento et al. 2012; Rysgaard et al. 2000). Furthermore it has been described that foraminifera, unicellular eukaryotes very common to the marine environment, are even capable of complete denitrification (Risgaard-Petersen et al. 2006). However, there is still a great lack of empirical studies that consider the role of meiofauna bioturbation and irrigation for carbon and nutrient cycles. In particular it is still unknown if and how meiofauna can regulate N release and transformations (e.g., denitrification and DNRA).
I have therefore initiated a pilot study in this direction. During summer 2012 a study was carried out and a density extraction method was used to isolate meiofauna specimens (Nascimento et al. 2012). The extraction was not harmful for metazoans and, by manipulating sieved sediment cores, the effect of low meiofauna and high meiofauna abundances were tested on benthic metabolism, in presence and in absence of macrofauna. In particular, (1) O2, DIC, CH4, and N2 net fluxes; (2) denitrification and DNRA rates; (3) nutrient exchanges were quantified. There are only preliminary results available so far, but based on these data it was already evident that meiofauna fosters both O2 uptake and N2 efflux significantly.
Coupling benthic N, S and P cycling in Baltic Sea oxygen-poor environments
Nitrogen and sulfur cycles are tightly coupled, especially in environments where sulfate is abundant (> 500 µM) and organic loading is high (Fossing et al. 1995). When
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bottom waters undergo severe hypoxic conditions reduced compounds such as ammonia and hydrogen sulfides accumulate in the sediment, and, if hypoxia persists, they can be detected even in the water column. However, a few electron acceptors might still be available, and organisms that can store those oxidized compounds have a metabolic advantage. This is the case with large filamentous sulfur bacteria, Beggiatoa spp. and
Thioploca spp., which can carry out sulfide oxidation with the nitrate stored in their
intracellular vacuoles (Jørgensen and Nelson 2004). Large sulfur bacteria are widespread and in particular the genus Beggiatoa can be found both in freshwater and marine sediments (Jørgensen and Nelson 2004). In the Baltic Sea Beggiatoa has been detected both in coastal areas and in the central basins (Jäntti and Hietanen 2012; Rosenberg and Diaz 1993).
Moreover, marine sediments are thought to be the main sink of phosphorus because of burial of inorganic phosphate and its conversion to phosphate minerals. It has been widely recognized that during hypoxia the reduction of iron oxyhydroxides cause the release of iron-bound P to the porewater and consequently to the water column (Rozan et al. 2002; Sundby et al. 1992). Only very recently it has been shown that under anoxic conditions large sulfur bacteria (Beggiatoa, Thioploca) not only store phosphorus in the form of poly-P granules as energy source, but they can also catalyze authigenic apatite precipitation (Goldhammer et al. 2010), and therefore enhance P sequestration under reducing conditions.
An ongoing study is addressing the relationship between Beggiatoa appearance and benthic nitrogen and phosphorus cycling in hypoxic Baltic Sea sediments. It has been shown that Beggiatoa performs dissimilatory nitrate reduction that leads to ammonium rather that nitrogen gas (Jørgensen and Nelson 2004), but it is still not clear how Beggiatoa affects N2O emissions, N2 fixation, ammonium oxidation via anammox, and ammonium oxidation carried out by archaea (AOA). Moreover, an important question is whether the presence of
Beggiatoa might lead to poly-P formation and P sequestration, as it was previously described
for the Namibian shelf (Goldhammer et al. 2010).
Therefore a series of measurements using stable (15N) and radioactive (33P) will be carried out with sediment from the open Baltic where the redox-cline intersects the sediment surface to dissect those questions. Analysis of compound specific lipids (ladderanes and crenarchaeols) would allow for the identification and confirmation of the presence of anammox bacteria and Crenarchaeota. This latter technique can be applied
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thanks to the recent installation of a new Gas Chromatography-Mass Spectrometer and a new High Performance Liquid Chromatography-Mass Spectrometer, and will be carried out in collaboration with Dr. Jayne Rattray, Department of Geosciences, Stockholm University. A preliminary study already showed that those sediments have a huge potential of N2 uptake and, by using 15NO3--tracer experiments, releases of N2O were found to be almost double than N2 efflux (Bonaglia, unpublished data).
5. Acknowledgments
I am very grateful to the main projects financing this work: “Baltic Sea Ecosystem Adaptive Management (BEAM)” and “Managing Baltic nutrients in relation to cyanobacterial blooms (CYANOS)”, both funded by FORMAS. I am very thankful to Stockholm University Marine Research Centre which provided extra funding for boat time and staying at the Askö Laboratory.
I would like to thank my supervisors (Volker Brüchert, Helle Ploug, Per Hall, Moritz Holtappels) and co-authors for valuable help in carrying out the research in those first two years. I would also like to thank Barbara Deutsch, Isabell Klawonn, Joanna Sawicka, Nils Ekeroth, Silvia Fedrizzi, and the Askö Laboratory staff for their help in sampling and providing such a nice and friendly working environment. I want to thank the following scientists for extremely valuable discussions and advice: Ragnar Elmgren and Patrick Crill (Stockholm University), Loreto De Brabandere (Vrije Universiteit Brussel), Hannah Marchand and Gaute Lavik (MPI Bremen). A special thanks goes to Jonas Gunnarsson, Lars Rahm, Caroline Raymond and Ola Svensson: without their work the ms on Marenzelleria would not exist.
I am very thankful to the SIL and Delta staff, in particular Heike Siegmund and Henry Holmstrand, for assistance within mass spectrometry, and Duc Nguyen for sharing his experience in gas chromatography. Thanks to Anders Sjösten and his lab, Per Hjelmqvist and Jörgen Ek for support with nutrient analysis. I would also like to thank Hildred Crill who helped me to improve the English of this thesis.
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A very special thanks goes to Marco Bartoli (University of Parma) who initiated me into this job and supported me so kindly during those two years. Without his help, I would not have been able to make it.
I would also like to thank my officemates, colleagues and friends from IGV, ITM, ECO and Göteborg Universitet for good discussions and their friendship.
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