The effects of ocean acidification on nitrogen cycling in permeable coastal sediments

M.Sc Thesis

Hannah Marchant

submitted to International Max Planck Research School of Marine Microbiology and the University of Bremen March 2010

1st reviewer: Marcel Kuypers, Max-Planck-Institute for Marine Microbiology, Bremen 2nd Reviewer: Marc Strous, Max-Planck-Institute for Marine Microbiology, Bremem

Summary

Summary Anthropogenic emissions of carbon dioxide to the atmosphere are changing the chemistry of the oceans; the 30% increase since pre-industrial times in H+ in the surface waters of the oceans has already resulted in a reduction of 0.1 pH. By the year 2300 further pH reductions could see decreases as large as 0.7 U. The effect of ocean acidification on biogeochemical cycles is therefore a vital research topic. Research into the responses of the nitrogen cycle is still in its infancy, changes in marine nitrogen fixation in the future ocean are perhaps the best characterized process so far, but work has focused on pure cultures in laboratory settings. There is very little experimental evidence to identify the response of nitrifiers and the role of denitrification has only been modeled. To date no studies have focused on the effect of ocean acidification in nitrogen cycling in marine sediments. Coastal permeable sediments contribute significantly to global nitrogen budgets, advective flushing within these sediments leads to daily fluctuations in oxygen, pH and nutrient dynamics. This project aimed to use a mesocosm approach to characterize the effects of midterm (4 weeks) ocean acidification on total denitrification, coupled denitrification-nitrification and nitrification within permeable sediments from the Wadden Sea. The study is split into two parts, a methods development section which details the set up of a seawater pCO2 manipulation system, the design and testing of flow through sediment columns and the development of a suitable method to measure denitrification and nitrification using isotope pairing techniques within the sediment columns. The main study focuses on a four week experiment in which replicate sediment columns were exposed to four different pCO2 treatments mimicking the last glacial maximum, ambient, year 2100 and year 2300 atmospheric carbon dioxide concentrations. pCO2 treatment had a marked effect on nitrate concentrations within the seawater supply reservoirs, in which a doubling of pCO2 was equivalent to a decrease in nitrate concentrations of 36%. Total denitrification did not change in the ambient and elevated pCO2 conditions, but the relative importance of coupled denitrification to the total denitrification increased with rising pCO2. Nitrification rates also increased across the treatments. Unexpectedly total denitrification rose in the last glacial maximum treatment. A model driven by the postulated change in nitrification in the seawater is proposed to explain future changes in sediment nitrogen cycling, concluding that ocean acidification may lead to greater N loss in coastal permeable sediments through tighter coupling of nitrification to denitrification. -2-

Acknowledgements

Acknowledgements I would like thank Marcel Kuypers, Tim Ferdelman, Dirk de Beer for their supervision on this project. Although not officially supervisors I would also like to thank Gaute Lavik and Marc Strous, whose advice and help was of great benefit. For many enlightening scientific discussions I would also like to thank Björn Rost, Jens Harder, Tim Kalvelage, Hang Gao, Felix Janssen and Judith Klatt I would like to thank Felix Raulf and Martin Glas, without whom the seawater pCO2 manipulation system would not have been possible. For technical help Gabi Klockgether, Gabi Schüßler, Kirsten Imhoff, Thomas Max, Andrea Schipper, Jan Fischer, Lubos Polerecky and Susanne Menger. And finally for giving up their free time to help me collect seawater in the rain, wind, and on once occasion -9ºC temperatures, Ivo Kostadinov, Jessica Fuessel, Elmar Pruesse and Emil Ruff.

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Introduction

Contents Part 1 – Main study............................................................................................................. - 6 Introduction ......................................................................................................................... - 6 Ocean acidification............................................................................................................ - 6 The carbon dioxide system in sea water............................................................................ - 7 Other acid-base equilibria in seawater .............................................................................. - 8 Ocean acidification and the carbon cycle.......................................................................... - 9 Ocean acidification and the nitrogen cycle ..................................................................... - 10 Nitrification ................................................................................................................. - 11 Nitrogen fixation ......................................................................................................... - 12 Denitrification ............................................................................................................. - 12 Denitrification in low pCO2 oceans ............................................................................ - 13 Ocean acidification and permeable coastal sediments .................................................... - 14 Current study ................................................................................................................... - 15 Methods .............................................................................................................................. - 16 Sampling Sites................................................................................................................. - 16 Sediment...................................................................................................................... - 16 Seawater ...................................................................................................................... - 17 Experimental set up......................................................................................................... - 17 Seawater acidification ................................................................................................. - 18 pH and DIC measurements ............................................................................................. - 19 TA and pCO2 determination............................................................................................ - 19 Nutrient analyses ............................................................................................................. - 19 Nitrate, nitrite and ammonia determination .................................................................... - 20 Denitrification and oxygen consumption incubations..................................................... - 20 Nitrification Incubations ................................................................................................. - 21 IPM Calculations............................................................................................................. - 22 Denitrification ............................................................................................................. - 22 Nitrification ................................................................................................................. - 22 Statistical analyses........................................................................................................... - 23 Results ................................................................................................................................ - 24 Seawater pCO2 manipulation........................................................................................... - 24 Carbonate chemistry of effluent pore water from sediment columns ............................. - 25 Nutrient concentrations ................................................................................................... - 29 Denitrification ................................................................................................................. - 30 Total denitrification..................................................................................................... - 31 Coupled nitrification – denitrification......................................................................... - 32 Nitrate depletion during denitrification incubations ................................................... - 33 Oxygen consumption rates .......................................................................................... - 33 Preliminary results of the nitrification incubations ......................................................... - 34 Background 29N production ............................................................................................ - 36 Discussion........................................................................................................................... - 37 Transformations in pore water carbonate chemistry ....................................................... - 38 O2 Consumption Rates (OCR) ........................................................................................ - 39 Nitrogen cycling.............................................................................................................. - 40 Reservoirs.................................................................................................................... - 40 Sediment...................................................................................................................... - 42 Nitrogen budgets in the Wadden Sea and beyond........................................................... - 46 -4-

Introduction Outlook............................................................................................................................... - 47 Part 2 – Method Development.......................................................................................... - 48 Method Development – Part 1.......................................................................................... - 49 Seawater Acidification System ........................................................................................... - 49 Method Development – Part 2.......................................................................................... - 55 Sediment Column Design.................................................................................................... - 55 Method development – Part 3 .......................................................................................... - 59 Nitrification and Denitrification Incubations ...................................................................... - 59 Summary and Conclusions............................................................................................... - 64 References .......................................................................................................................... - 65 -

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Introduction

Part 1 – Main study Introduction Ocean acidification Atmospheric carbon dioxide (CO2) has risen 31 % in relation to pre-industrial levels (Houghton et al., 2001); this is a direct response to anthropogenic emissions of CO2 from burning of fossil fuels, cement production and changes in land uses. Atmospheric CO2 concentrations are still rising at an unprecedented rate and by 2100 they are expected to exceed 1000 ppm, this is higher than anything experienced on Earth in the last 420,000 years and is at least an order of magnitude faster than in the past million yeas (Doney & Schimmel, 2007) (IPCC, 2001) (Fig.1).

Fig.1 CO2 fluctuations on an interglacial time scale (a) and within the last interglacial time period (b). Variations in deuterium (δD; black), and the atmospheric concentrations of the CO2 (red), CH4 (blue), and nitrous oxide (N2O; green). Shading indicates the last interglacial warm periods. The stars and labels indicate atmospheric concentrations at year 2000. Modified from the from the 4th IPCC Assessment report .

30 % of anthropogenically-produced CO2 has been absorbed by the surface layer of the oceans (Sabine et al., 2004), which is equivalent to an accumulation of 112 (±17)28 petagrams of carbon (Pg C) since the beginning of the industrial era. This has already resulted in a reduction of 0.1 pH units in surface waters (Haugan & Drange, 1996). If CO2 emissions continue to rise in an IS92a “business as usual” scenario then by 2100 models predict a decrease in pH of 0.3 - 0.5 units in the surface oceans (Caldeira & Wickett, 2003)(Fig.2). The -6-

Introduction effects of equilibration of CO2 with seawater must be understood before any discussion of the biological effects of this change.

Fig, 2. The projected change in pH of the oceans due to release of CO2 from human activities. Past and projected emissions of CO2 to the atmosphere are shown in the top bar in Gt C per year. The second bar shows historical atmospheric CO2 levels to 1975, observed atmospheric CO2 concentrations from 1975 to 2000 and predicted concentrations to year 3000. The bottom of the figure shows the projected average change in ocean pH with depth. Taken from Caldeira & Wickett, 2003,

The carbon dioxide system in sea water The projected 0.3–0.4 drop in surface ocean pH by the end of the 21st century is equivalent to approximately a 150% increase in H+ and 50% decrease in CO32− concentrations (Orr et al. 2005). This is because seawater carbonate chemistry is governed by a series of chemical reactions: CO2(atmos) ↔ CO2(aq) + H2O ↔ H2CO3 ↔ H+ + HCO3- ↔ 2H+ + CO32-

(1)

On a time scale of approximately one year air-sea gas exchange causes surface seawater to equilibrate with atmospheric levels. CO2 dissolves in seawater where almost all of it reacts with H2O to form carbonic acid, H2CO3. Further dissociation takes place and hydrogen ions are lost forming bicarbonate and carbonate ions (HCO3- and CO32- ). In surface seawater with

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Introduction pH of 8.1, nearly all inorganic carbon is in the form of bicarbonate ions (90%), carbonate ions make up most of the remainder of the pool (9%) and only 1% is dissolved CO2. The addition of CO2 causes increases in aqueous CO2, bicarbonate and hydrogen ions. These hydrogen ions are responsible for classical “ocean acidification”, pH decreases because it is a measure of –log10 [H+]. It is the increase in H+ that is responsible for the decline in carbonate ion concentrations. Over geological time scales the ocean has the capacity to buffer and restrict changes in pH that result from increases in atmospheric CO2. The carbonate buffer system in the oceans is described by the chemical equation:

H+ + CO32– ⇒ HCO3–

(2)

As CO2 dissolves in seawater the additional hydrogen ions react with carbonate ions and convert them bicarbonate, this reduces the concentration of hydrogen ions and as such the change in pH is lower than would be expected. This buffering capacity is not endless, as the process consumes carbonate ions, buffering capacity is diminished as CO2 increases. Over long term time scales of ocean mixing interactions with CaCO3- rich sediments buffers seawater chemistry further as acidic deep oceans lead to the dissolution of carbonate ion from the sediment (3). ← Mineral Formation CaCO3 ↔ Ca2+ + CO32– Dissolution→

(3)

Equation 3 highlights the most obvious consequence of ocean acidification on biological processes within the oceans. Many marine organisms build CaCO3 structures. These are dependent on the presence of bicarbonate and carbonate. As carbonate ion concentrations reduce, CaCO3 becomes more soluble, which may reduce shell formation.

Other acid-base equilibria in seawater An often neglected effect of ocean acidification is the broad alterations that are predicted in inorganic and organic seawater chemistry outside of the carbonate system. Natural sea water contains a number of acid-base species in significant amounts, the most common of these are -8-

Introduction minor nutrient species such as silicate, phosphate, iron and ammonia. The effect of pH on nutrient speciation can be modeled theoretically (Fig. 3)(Zeebe & Wolf-Gladrow, 2001). Few studies have been undertaken to date which quantify the effects of nutrient speciation on biological processes within the oceans. One example however is that the fraction NH-x available as NH3 will be reduced from 6% to 3% by 2100 (Bell et al. 2008, Bange, 2008.

Fig. 3. Theoretical changes in nutrient speciation of ammonia, silicate, phosphate and iron with pH (adapted from Zeebe & Wolf-Gladrow, 2001)

Ocean acidification and the carbon cycle Ocean acidification is likely to alter the biogeochemical dynamics of the carbon cycle. The first process by which this may occur is though decrease in CaCO3 saturation states and an increase in calcification rates. Andersson et al., (2005) observed that this could lead to a decrease in coral calcification rates in coastal regions by 40% by the end of the century, this could provide a weak negative feedback to rising anthropogenic CO2 and ocean acidification since dissolution of calcium carbonate minerals consumes CO2 and increases alkalinity, increasing the capacity of the ocean to absorb CO2 from the atmosphere. If surface ocean calcification were completely shut down then pCO2 in the surface oceans would decrease by about 10 to 20 atm (Gruber et al., 2004). However, anthropogenic inputs of CO2 are so high that these natural buffering systems are unlikely to provide significant negative feedbacks (Doney et al., 2009) The ratio of organic:inorganic carbon which is delivered to the sea floor will be effected by decreases in CaCO3 production. However, we do not yet know in which direction this will push the biological pump. It has been postulated that mineral particles of CaCO3 ballast organic carbon, increasing the sinking rates of fluxes (and therefore likelihood of burial) to the sea floor. CaCO3 dissolution may lead to less ballasting, increasing mineralization rates in -9-

Introduction shallow waters and thereby decreasing CO2 uptake efficiency (Armstrong et al., 2002, Passow et al., 2006). Alternatively if the ratio of organic: inorganic carbon reaching the deep sea is uncoupled, decreases in CaCO3 production would lead to dissolution of carbonate sediments – providing a negative feedback and raising ocean pH (Orr et al., 2005). The efficiency of the oceanic biological pump is inextricably linked to the nitrogen cycle. For example if exported material has higher C/N ratios then the pump is more efficient in exporting carbon (Doney et al., 2009). Therefore to understand how the oceans will respond to future acidification it is vital to know how the nitrogen cycle will be affected.

Ocean acidification and the nitrogen cycle Denitrification. Decrease in sediment NO3- uptake (Wood et al, 2009). Increase in sediment NO3- uptake (Widdicombe and Needham, 2007)

N2 fixation. Stimulated in Trichodesmium and Crocosphaera, inhibited in Nodularia. (Hutchins et al, 2007, Fu et al, 2008 and Czerny et al, 2009)

Nitrification/Ammonia oxidation. 40% reduction with a pH decrease of 0.5 units (Huesemann et al, 2002)

NH4+:NH3 ratio. Shift in chemical speciation with pH, decrease of pH may lead to halving of available NH3 by 2100 (Bange, 2008)

Fig 4. The Marine Nitrogen Cycle (redrawn from Hutchins et al., 2009) with a summary of the currently known impacts of ocean acidification

Nitrogen is a key limiting element in the oceans, whose availability is mediated by a number of microbial metabolisms and processes. Nitrogen cycling is a complex process owing to the eight-electron difference between the most oxidized and reduced compounds of N, which provides a large potential for redox cycling. Thus there are many possible processes within the nitrogen cycle which may be affected by ocean acidification, either directly or indirectly, the close linking of the nitrogen and carbon cycles mean that it is imperative to explore what these effects may be and to what extent they will alter the marine nitrogen budget. Research and awareness of the potential impacts of ocean acidification on the nitrogen cycle is growing - 10 -

Introduction steadily, however knowledge is still limited and frequently only pure cultures in laboratory settings have been investigated. The following section summarises the current knowledge and relates it to possible changes in nitrogen cycling that may occur in coastal marine environments (the focus of the current study).

Nitrification Nitrification, the oxidation of ammonium to nitrite and nitrate by autotrophic microbes may respond in two ways to ocean acidification. Theoretically nitrifiers are directly susceptible to changes in pCO2, Nitrosomonas and Nitrobacter are autotrophic bacteria which fix carbon using the Calvin cycle (Ward et al., 2008). This cycle relies on the RubisCO enzyme which is characterised by its low affinity for substrate CO2 (Rost et al., 2008) and therefore marine nitrifiers may potentially be CO2 limited or investing energy in carbon concentrating mechanisms (CCM’s). An increase in seawater CO2 may alleviate this limitation, or allow reallocation of resources previously required for CCM’s, thereby increasing nitrification rates. The study of the effects of ocean acidification on carbon acquisition is in its early stages in photosynthetic organisms and this hypothesis is a long way from being tested in marine nitrifiers. The second effect of ocean acidification on nitrification is indirect and related to the expected shift in the acid-base equilibrium of the ammonia/ammonium buffer system under different pH’s.

The enzymatic substrate during the first step of nitrification (carried out by

Nitrosomonas) may be NH3, rather than NH4+ (Ward et al., 2008). A reduction of the available NH3 available as substrate (3% in 2100 compared to 6% today, Bange et al., 2008) would inhibit nitrification rates. A single study has assessed the sensitivity of marine nitrification to ocean acidification, Huesemann et al., (2002) found that ammonium oxidation rates showed an almost linear decrease from pH 8 to pH 6.5 (with a 40% decrease between pH 8.0 and 7.5) in aphotic and euphotic seawater samples that had been treated with NaOH or bubbled with CO2 to reduce pH. From the basis of this evidence, shifts in chemical speciation appear to play a more important role in regulation of nitrification than any possible alleviation of carbon limitation. Further investigation is needed however, nitrification links ammonium and nitrate, the most reduced and oxidised forms of the nitrogen cycle together and therefore influences nitrogen dynamics considerably (Canfield et al., 2005). Nitrifiers are especially important in coastal environments, they generate a source of nitrate for denitrifying bacteria and couple an - 11 -

Introduction obligatory aerobic process with an anaerobic process, leading to nitrogen loss to the atmosphere (Herbert et al., 1999).

Nitrogen fixation The response of nitrogen fixation to ocean acidification has perhaps been the best studied part of the nitrogen cycle. A number of studies have been carried out on Trichodesmium (Levitan et al., 2007, Hutchins et al., 2007 and Ramos et al., 2007) In each it was shown that CO2 availability controls nitrogen and carbon fixation rates. Rates increased with pCO2 suggesting that this part of the nitrogen cycle is limited by present day CO2 levels. These results have significant implications for global nitrogen cycles as Trichodesmium is thought to provide up to 50% of new nitrogen in oligotrophic gyres (Karl et al. 2002). Hutchins et al., (2007) estimated that increased nitrogen fixation by this organism could account for an increase in fixed N of around 20 x 109 kg N yr-1 by 2100. These studies were all carried out with the same strain of Trichodesmium which may make this future estimate slightly ambitious, Fu et al., (2008) studied the important unicellular diazotroph Crocosphaera watsonii and observed enhanced N2 fixation rates under high pCO2. Which would support the estimate of increased N fixation.

However the trends in the

Crocosphaera study did not follow those of the Trichodesmium literature when limiting factors other than CO2 were introduced. Increased N fixation was no longer observable in Crocosphaera cultures which were iron limited, whereas in Trichodesmium cultures N fixation rates were still enhanced under high pCO2 and

phosphate limited conditions.

Therefore much more work is required before these results can be extrapolated to nitrogen fixation rates in future high CO2 oceans.

Cultures studies are often not indicative of

environmental responses. When considering shallow marine environments, where the availability of fixed nitrogen is considered to be a major factor regulating primary production, the microphytobenthos also play an important role. To date increases in the responses of this group of organisms to ocean acidification has not been determined.

Denitrification Denitrification occurs primarily in anaerobic zones and is responsible for the loss of fixed N from marine systems through a combination of heterotrophic N removal (classical denitrification) and anaerobic oxidation of ammonia (Anammox). There is no evidence that - 12 -

Introduction ocean acidification will directly effect the physiology of marine denitrifiers (Hutchins et al., 2009). However it is possible that denitrification will affected indirectly, changes in the other components of the nitrogen cycle - nitrification and nitrogen fixation may influence denitrification by altering the supply of reactive nitrogen compounds. Futhermore heterotrophic denitrification could be altered by changes in organic carbon supply. Only two studies have been carried out that may give an insight into changes in denitrification in response to ocean acidification. Widdicombe and Needham et al. (2007) and Wood et al., (2009) measured fluxes of ammonium, nitrate and nitrite in sediment cores which were exposed to elevated CO2 over 20 weeks. Wood et al., found that sediment uptake of nitrate decreased over time to such an extent that the sediment changed from being a sink to become a source of nitrate between pH 7.3 and pH 6.8. They could not determine whether this was due to a change in denitrification rates or due to changes in the microphytobenthos. Alternatively, using an almost identical experimental design Widdicombe and Needham found that nitrate flux into the sediment increased. A further study modelling the effects of increased C:N drawdown in response to ocean acidification had large implications for future denitrification in the oceans (Oschlies et al. 2008). In the model increased C:N drawdown led to a 50% increase in oxygen minimum zones. Oxygen minimum zones currently cover less than 1% of the oceans, but are responsible for 30 – 50 % of total oceanic N loss, which occurs 90 – 100 % through the process of anammox (Kuypers et al., 2005. Lam et al., 2007). Denitrification in sediments is considered as an important source of oceanic N loss, the most important zone of sediment denitrification has long been considered as organic rich continental margin sediments where 50–70% of marine denitrification occurs (Codispoti et al., 2001). Coastal permeable sediments have been identified as a major site of denitrification in a number of studies (Rao et al., 2008, Cook et al., 2006)

Denitrification in low pCO2 oceans Nitrogen isotope records from the last glacial maximum suggest that denitrification was greatly reduced and increased during the glacial-interglacial transition. Denitrification is highest in marine environments in sub-oxic zones and shelf sediments which are rich in organic material. As the last glacial period ended, continental warming increased, which increased the area of oxygen minimum zones, consequently increasing denitrification. The increased denitrification then provided a positive feedback to continental warming as it - 13 -

Introduction reduced ocean nitrate concentrations, decreasing the strength of the biological pump and producing N2O, a greenhouse gas (Altabet et al., 1995, Altabet et al., 2002). These records do not give us any indication of denitrification rates, just total denitrification.

Ocean acidification and permeable coastal sediments Until now the effect of ocean acidification on sediments has been largely neglected as sediments are assumed to be more impervious to changes in environmental conditions than pelagic realms as generally they already have elevated pCO2 and low pH. Furthermore most marine sediments are ventilated slowly, so changes in overlying water will affect sediment on a slower time scale, allowing buffering and adaptation. However near-term effects of increasing atmospheric pCO2 on the biology and chemistry of coastal permeable sediments may not be so limited, permeable sediments are flushed regularly with overlying water through advective processes and therefore changes in carbonate chemistry and the associated changes in nutrient concentrations of the overlying seawater could have a significant effect on sediment microbial biogeochemistry. The extremely limited number of studies on the affects of ocean acidification on marine sediment provide justification for further investigations. Widdicombe and Needham et al., (2007) and Wood et al. (2009) both showed changes in sediment nutrient fluxes in mesocosm studies in response to changes in overlying seawater pH. In Wood et al., small but significant changes in sediment pH were observed during environmentally relevant peturbations. Inagaki et al., (2006), investigated microbial communities and abundance in the sediment surrounding a deep sea natural CO2 lake, where strong natural pH gradients exist, they found that microbial communities and functioning may be affected by variations in CO2 concentrations and in situ pH, observing a strong decline in cell numbers toward the liquid CO2 interface of the lake on a scale of decimetres. Further evidence for the susceptibility of sediment environments to acidification can be taken from the terrestrial environment, in long term studies at the Hubbard Brook Environmental Forest system ecosystem acidification caused by acid rain has been monitored since the 1960’s. Analysis of the forest ecosystem suggests that is much more susceptible to the atmospheric input of acids than was originally expected on the basis of sulphur biogeochemistry (Likens, 1996). Soil nutrient environments in the system have been changed - 14 -

Introduction by anthropogenic nitrogen emissions and acidic deposition, causing nitrogen enrichment and cation depletion, which has influenced the growth dynamics of the forest (Naples and Fisk , 2010). Increased atmospheric carbon dioxide is known to affect the pH of soils, even though they already have acid pHs. A study which aimed to determine the effects of elevated CO2 on soil nitrogen cycling processes exposed soils to elevated atmospheric carbon dioxide (510 ppm) over seven years and found a drop in pH values of 5.77and 5.49 for ambient and elevated CO2, respectively (Zheng et al., 2008).

Current study The current study focuses on the effects of ocean acidification on nitrogen cycling within permeable coastal sediments. This is important because nitrogen cycling and primary production in coastal pelagic zones is strongly driven by the biological and chemical processes occurring in coastal sediments. It has been estimated that 30 to 80% of the phytoplankton nitrogen requirement in shallow coastal environments originates from the sediments (Dale and Prego, 2002). A change in export of fixed nitrogen from coastal zones to open ocean waters is likely to have important consequences for the global marine nitrogen budget and could drive changes in C:N ratios, in turn altering the efficiency of the biological pump. The specific aims of this study were to explore the changes in denitrification, nitrification and coupled nitrification-denitrification in permeable coastal sediments in response to ocean acidification. To gain further insights into the possible changes in these two elements of the nitrogen cycle a pCO2 condition was used which mimicked atmospheric conditions during the last glacial maximum (approximately 20,000 years ago) as well as two conditions in which pCO2 was increased.

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Results

Methods Sampling Sites Sediment

A

Sediment was sampled from the Janssand sand flat, which is located in the back barrier area of Spiekeroog Island in the East Frisian Wadden Sea, Germany (Fig. 5A). This site has been well described in previous studies (Billerbeck et al., 2006) and consists of three regions, the upper flat, the slope between the upper flat and the low water line and the low water line, (Fig 6)(Jansen et al., 2009). The sediment characteristics differ in these regions, however the entire flat is inundated with ca. 2m of seawater during high tide before exposure to the air for 6 – 8 h during low tide in a semi diurnal cycle. The sampling site (53.73515 ′N, 007.69913’E) was situated in the upper flat, which consists of silicate sands

B

with a mean porosity of 35%, mean grain size of 176 µm and mean permeability of 7.2–9.5 × 10−12 m2 in the upper 15 cm of sediment

Fig.5. Location of the sampling sites for (a) Sediment, located near the island of Spiekeroog and (b) Seawater located near Wilhemshaven, Germany. (Adapted from Billerbeck et al. 2006)

(Billerbeck et al., 2006). Sediment was collected on October 24 2009 at

low tide, sediment from the upper 5cm of the sand flat was pooled and homogenized and placed immediately into 12 sediment columns (see Method Development for description). These were closed and returned to the Max Planck Institute for Marine Microbiology, Bremen on October 25 2009, at which point they were supplied with unfiltered seawater from a single reservoir.

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Results

Seawater Sediment columns were supplied with unfiltered seawater. 300L was collected initially from the area of the Janssand sand flat parallel with sediment collection. Further collections took place on November 12, 2009, November 28, 2009, 13 December, 2009, January 3, 2010, January 15, 2010 and January 24, 2010. On these occasions between 200 and 300L of seawater was collected from the coastal region close to Wilhemshaven, Germany (53.61715’N, 8.1125’E)(Fig.5B). Seawater was stored in 10L containers and kept in the dark at 4°C until needed.

Fig. 6 Janssand tidal flat topography (from Billerbeck et al 2006) showing position of sampling location on the upper flat.

Experimental set up Prior to the start of the experiment, sediment columns were individually supplied with unfiltered seawater at 19°C. Inflow was drawn from a single reservoir which was continuously sparged with air. Peristaltic pumps pushed pore fluid upward through each reactor between inflow (bottom) and effluent (top) ports on a simulated tidal cycle (15 min pumping, 15 min no pumping for 6 hours, followed by six hours no pumping, 2.5ml/min flow rate). Flow rates were monitored and readjusted as needed. Tygon lab tubing (R-3603), which is designed for analytical applications and gas lines was used thoughout the experiment. On January 5, 2010 and January 8, 2010, control incubations in each sediment column were carried out to determine denitrification and nitrification rates respectively. On January 11, the 12 sediment columns were randomly assigned to one of four pCO2 conditions (nominally, 190, 380 [ambient seawater], 750 and 1200 µatm), leading to 3 replicates per condition. The experiment ran for 28 days until February 9, during which time during which time the - 17 -

Results reservoir water was monitored for pH, DIC, temperature, salinity, nitrate, nitrite and ammonium concentrations.

Seawater acidification Seawater carbonate chemistry in the 150, 750 and 1200 µatm reservoirs was adjusted by passing air with defined concentrations of carbon dioxide through unfiltered seawater contained within 40 L cylindrical reservoirs (Plexiglas), which were covered with aluminum foil to prevent light from entering (Fig. 7). In the ambient condition (380 µatm) the reservoir was aerated by sparging with air pumped from outside the laboratory. Defined concentrations of CO2 within the air supply were achieved by the following method; Carbon dioxide was removed from laboratory air by passing it through an adsorption dryer (K-MT 3-Lab, Domnick Hunter), the CO2-free air flow was controlled and monitored by mass flow controllers (Brock). Known amounts of CO2 were added from gas cylinders comprising of 5% CO2 in synthetic air or 2% CO2 depending on the final concentration required. The CO2 supply was controlled and monitored by rotameters (Purgemaster Durchflussmesser Series A6100, ABB Automation Products GmbH). The defined volumes of the two gases were mixed in Milli-Q water prior to being was passed through the water reservoirs as very fine bubbles, which enabled theCO2 gas to pass rapidly into solution. As the acidified water was taken from the reservoirs to supply the flow through reactors, it was replaced by unfiltered seawater from a separate tank held at the same temperature and aerated with air pumped from outside the laboratory. The replacement rate for each reservoir was 1.87 ml/min, this equated to a replacement of 9% of the CO2 equilibrated water each day, which was did not affect the reservoir pCO2. Lab Air

CO2 scrubber

CO2 free air

Mass flow controller Peristaltic pump

CO2 – 2 % or 5 %

Flow controller, rotameter

Mixing Flask

Sediment cores

Reservoir (40 L)

Fig. 7. Schematic diagram of seawater acidification system (see Method development for further details)

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Results

pH and DIC measurements The carbonate chemistry of the reservoirs was monitored by pH (3 times per week) and dissolved inorganic carbon (DIC) determination (once a week). The pH of the porewater at the effluent port of each column was measured at the end of the pumping cycle and at the beginning of the pumping cycle once a week. DIC from the column effluent was measured in parallel once a week. pH was measured immediately after sampling on the NBS scale with a commercial glass electrode (3-mm tip diameter, InLab 423, Mettler Toledo, Switzerland), the electrode was calibrated twice a day using a two point calibration, employing NBS buffers equilibrated to sample temperature (19°C). DIC was measured by coulometry, briefly, for each determination (within 2 hours of sampling) 10 ml of seawater was acidified to CO2 with 5ml of 20% phosphoric acid within an acidification module (UIC, Inc, Model 5012), this comprised of a potassium hydroxide scrubber to remove CO2 from the carrier gas and potassium iodide solution to remove other gas products which may effect the measurement. The CO2 gas stream was then passed into a glass cell within the coulometer (UIC, Inc, Model 5012 CO2 Coulometer) where it is quantitatively absorbed and reacts with monoethanolmine forming a titratable acid that causes a colorimetric pH indicator to fade. µg C per sample is then calculated by photodetermination of the colour change. The accuracy of the reaction was checked daily by titration of known amounts of calcium carbonate. The measurement range of the coulometer is between 1µg C and 10,000 µg C per sample.

TA and pCO2 determination pH, DIC, salinity and temperature were used to calculate total alkalinity and pCO2 using CO2Sys for excel (Pierrot et al., 2008).

Nutrient analyses

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Results Samples were collected for ammonium, nitrate and nitrite determination from the reservoirs twice a week. Immediately upon sampling seawater was transferred to 6 ml Exetainers (Labco, High Wycombe, UK) prefilled with 100 ml saturated HgCl2. Nutrient samples were collected from the porewater effluent of each column at the end of the pumping cycle and at the beginning of the pumping cycle twice a week.

Nitrate, nitrite and ammonia determination Combined nitrate and nitrite (NOx) was determined by a commercial chemiluminescence NOx analyzer after reduction to NO with acidic vanadium(II) chloride (Braman and Hendrix, 1989). Nitrite was determined after reduction to NO with potassium iodide. Ammonium was measured using a modified version of the indophenol blue method and was measured photometrically at 640 nm .

Denitrification and oxygen consumption incubations Denitrification rates were determined once a week at the end of a 6 hour pumping cycle using the isotope pairing technique (Nielsen, 1992), modified to simulate in situ pore water advection and so that destructive sampling was not necessary (see Method development for details). A sub sample of seawater from the reservoir supplying each column was amended to contain 50µm 15NO3- . 600 ml of amended seawater was then pumped into each core through the bottom inflow port at a rate of 22.5 ml/min, this was sufficient to entirely exchange the pore water within the column. Upon the completion of pumping the inflow port was closed using a 2-way valve. A sampling port consisting of 2 cm tygon tubing (I.D. 1mm) was connected to the 2 way valve and porewater was then collected by opening the valve and letting porewater flow directly into 6 ml exetainers prefilled with 100 ml saturated HgCl2 . Sampled porewater was replaced passively from a funnel containing unamended sea water placed at the outflow on the top of the column. Before each sample was collected, 3 ml of porewater was discarded to flush the tubing between the column and the sediment. 6ml samples were taken immediately after pumping had ended, then 10, 20, 35, 55 and 75 minutes after. Production of labeled N2 and O2 consumption within each sample was determined by measurement of isotopes of mass 28 (14N14N), 29 (14N15N), 30 (15N15N), 40 (Ar), 32 (O2), - 20 -

Results using a membrane inlet mass spectrometer (MIMS; GAM200, IPI). A standard two point calibration curve was constructed, based on isotope measurements of in air saturated and anoxic water. NOx and nitrite within each sample were measured as described previously and used to determine NO3- concentrations

Nitrification Incubations Nitrification rates were determined once a week at the end of a 6 hour pumping cycle. These incubations were carried out three days after denitrification incubations to ensure that all the labeled isotopes were removed from the cores. The incubation method was as described above, except the seawater was amended to a final concentration of 10µm

15

NH4+. 2.4 ml

seawater was replaced with a He headspace in each exetainer and samples were stored at room temperature until measurement. 14

N15N:14N14N ratios were determined by gas chromatography/isotope ratio mass

spectrometry (GC-IRMS)(Fissions VG optima), modified by the addition of a liquid nitrogen trap and a heater at 630°C before the column. . Each sample was measured twice by GC-IRMS , initially background 29N2 within the samples was measured. Two processes could be responsible for the production

14

N15N labeled

dinitrogen after addition of 15NH4+, the conversion of 15NH4+ label to dinitrogen by anaerobic ammonium oxidation (anammox) (4) and closely coupled nitrification-denitrification, in which the

15

NH4+ is oxidized to

NO3- which is converted to

15

29

N2 through dissimilatory

reduction . 15

NH4+ + NO2- → 29N2 + 2H2O

(4)

The second GC-IRMS measurement was carried out after removal of the background N2, followed by chemical conversion of nitrate and nitrite within the sample to N2. The excess of 29

N2 produced by this conversion was the basis for calculating nitrification rates from the

incubation series. Briefly, samples were transferred to 15 ml Sarstedt tubes and nitrate was reduced to nitrite by adding 0.3 to 0.5 g of spongy cadmium to each sample (McIllvin and Altabet, 2005). Spongy cadmium was produced from cadmium powder which was activated by rinsing in 6M HCl for one minute followed by ~10 washing steps in dH2O until the pH was neutralized. Reduction to nitrate is complete after 18 h, (pers. comm. J. Fuessel) and accordingly samples were placed on a horizontal shaker at 210 rpm for this period. Cadmium - 21 -

Results was removed by centrifugation (10 minutes, 4500 rpm) before 4.2 ml of each sample was decanted into 6 ml exetainers. Each sample was then sparged with helium for 8 minutes to remove N2. 200 µl of 6% sulfamic acid in 10% HCl was added via a gas tight syringe to reduce nitrite to dinitrogen and finally this reaction neutralized after 8 hours with 100µl NaOH. The isotope ratio of 29N2 was measured within 2 days.

IPM Calculations Denitrification Denitrification rates were calculated according to the isotope pairing procedures of Nielsen (1992). Areal production rates of 29N and 30N were calculated for 1m3 of sediment, assuming a mean sediment porosity of 35% and these rates were used to calculate (5) the denitrification rate of

15

NO3-, D15, (6) the denitrification rate of

denitrification in the

14

NO3-, D14 and (7) the total rate of

NO3- amended sediment, Dtot. Further calculations were then carried

15

out to determine (8) denitrification based on unlabelled NO3- from the porewater, Dw, (9) the rate of denitrification coupled to nitrification Dn,

Where and

15

14

D15 = p14N15N + 2 * p15N15N

(5)

D14 = D15 * p14N15N/(2 * p15N15N)

(6)

Dtot = D15 + D14

(7)

DW = D15* [14NO3-]/[15NO3-]

(8)

Dn = D14 - DW

(9)

[NO3-] is the nitrate concentration in the seawater before amendment with

[NO3-] is the concentration of 15NO3- added to the seawater.

Nitrification

- 22 -

15

NO3-

Results Production of 29N was determined for determined for 1m3 of sediment, assuming a mean sediment porosity of 35% at each time point and were corrected by the NOx measured in each sample. This allowed calculation of areal production rates of 29N2 for 1m3 of sediment, assuming a mean sediment porosity of 35%. The areal rates were used to calculate nitrification rates (10). { p14N15N /[NH4+], }* [NH4+]a

(10)

Where [NH4+], is the total concentration of ammonium in the seawater after addition of 15

NH4+ and [NH4+]a is the labeled ammonium in the seawater.

Statistical analyses All statistical analyses were carried out using PAST version 1.99

- 23 -

Results

Results Seawater pCO2 manipulation The experiment ran for 28 days, during which time the carbonate chemistry within the reservoirs was monitored by measuring pH, salinity and temperature three times per week and dissolved inorganic carbon (DIC) once a week. pCO2 and total alkalinity were calculated using CO2Sys (Pierrot et al., 2007). Calculated pCO2 values had a standard deviation of less than 40 µatm in all conditions and were close to the nominal values chosen at the start of the experiment (190, 380, 700 and 1100) (Fig. 8), total alkalinity was constant in all conditions (Table 1). 1400

pCO2 (µatm)

1200 1000 800 600 400 200 0 Last glacial Ambient pCO2 maximum pCO2

Year 2100 Year 2300 estimate pCO2 estimate pCO2

Fig. 8. pCO2 within the four supply reservoirs over 28 days (values represent means of 5 measurements, error bars are SD±1) Table 1. Parameters of the seawater carbonate system calculated from dissolved inorganic carbon (DIC), temperature, salinity, and pH using the CO2Sys program (Pierrot et al., 2007) (n=5; ±1SD Nominal pCO2 (µatm) Last glacial maximum pCO2

190

Ambient pCO2

380

Year 2100 estimate pCO2

750

Year 2300 estimate pCO2

1100

pCO2 (µatm) 208 (±18) 408 (±16) 671 (±50) 1131 (±80)

- 24 -

DIC

TA

(μmol/kgSW)

(μmol/kgSW)

2016 (±51)

2508 (±33)

2308 (±123)

2597 (±75)

2399 (±106)

2589 (±51)

2529 (±115)

2576 (±62)

pH (NBS) 8,42 (±0,02) 8,21 (±0,02) 8,02 (±0,02) 7,83 (±0,02)

Results

Carbonate chemistry of effluent pore water from sediment columns Transformations in the carbonate chemistry of the pore water at the effluent port of each sediment column were monitored each week by pH and dissolved inorganic carbon (DIC) measurements. Pore water for pH and DIC measurements was sampled at the end of six hour pumping cycle (Fig. 9). Each week a secondary pore water sample was collected at the start of a six hour pumping cycle (when no pumping had taken place for six hours). Absolute changes in the carbonate chemistry of effluent pore water were quantified and compared between the four pCO2 conditions, following this the change in TA, DIC and pH between the inflow and effluent pore water of each sediment column was compared, Two way ANOVA was used analyse whether there were significant differences between pCO2 treatments and over time by two way ANOVA . There was a significant variation in absolute pore water DIC, pH and total alkalinity (TA) (Table 2), pH differences were significant over time and between conditions, DIC and TA differences were only significant between pCO2 conditions. There were no interactions between time and treatment, so further analysis used 1 way ANOVA. Where variance was significant post-hoc pairwise comparisons, based on Tukey's HSD were noted and are reported in table 3a for variation over time (pH) and in table 3b for variations between pCO2 conditions (pH, DIC and TA). pH was significantly higher in the pore water of the last glacial maximum condition than in the two high pCO2 conditions, this trend was repeated in DIC concentrations and was more pronounced. Post hoc pairwise comparisons showed that the effluent porewater of the LGM condition had significantly lower amounts of dissolved inorganic carbon than all other pCO2 conditions. Furthermore in the elevated pCO2 conditions, the DIC of the year 2300 was significantly higher than the year 2100 estimate (mean values were 2948 and 3338 mmol/kgSW respectively). An increase in absolute DIC was expected in the higher pCO2 conditions and is an indicator of increased CO2 invasion. Total alkalinity differed significantly in the effluent pore water of the LGM condition and the high pCO2 condition; mean total alkalinity values were lower in the LGM condition and higher in the high pCO2 condition. When no pumping had taken place for 6 hours there was less variation between pCO2 treatments when pH values were compared. The only variable that showed a significant effect was the last glacial maximum condition, in which pH of pore water was higher than the other conditions

(Fig.

- 25 -

8).

Results

8,4 8,2 8,0

pH

7,8 7,6 7,4 7,2

Last Glacial Maximum Ambient Year 2100 estimate Year 2300 Estimate

7,0 6,8

Dissolved Inorganic Carbon (umol/kgSW)

6,6 4000

0

5

10

0

5

10

0

5

10

15

20

25

30

3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 4000

15

20

25

30

Total Alkalinity (umol/kgSW)

3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 15

20

25

30

Days Fig. 7. Measured values of pH and DIC and calculated values of TA in the porewater sampled from the effluent port of the sediment columns at the end of a pumping cycle. (n=3; error bars are SD±1)

- 26 -

Results

Last Glacial Maximum Ambient Year 2100 estimate Year 2300 Estimate

8,4 8,2 8,0

pH

7,8 7,6 7,4 7,2 7,0

0

5

10

15

20

25

30

Day Fig. 8. Measured values of pH in porewater sampled from the effluent port of the sediment columns at the start of a pumping cycle. (n=3; error bars are SD±1). Two way ANOVA showed a significant variation between the pCO2 conditions but not time. One way ANOVA showed that the last glacial maximum condition differed significantly from all other conditions, but no other data points differed significantly from each other

The change in pH, DIC and TA between the input seawater and the effluent pore water was calculated for each sediment column. Independent of condition pH was always lower after water had passed through the column and DIC and TA were always higher, which is concurrent with processes of organic matter remineralization occurring within the sediment. There were no significant differences in ΔDIC (inflow – effluent) between any of the pCO2 conditions. Neither were there any significant differences in ΔpH between pCO2 conditions, although there was a slight effect of time (p = 0.05). Significant differences were still apparent in the ΔTA between the pCO2 treatments (Fig. 9), the ΔTA of the high pCO2 condition was higher than in the LGM and ambient conditions (p = 0.02 and 0.03 respectively).

- 27 -

Results Table 2. Two way ANOVA , time and pCO2 condition were used as factors for analysis of variance of pH, DIC and TA values measured in the porewater effluent at the end of a pumping cycle. Factors show p values. n.s = not significant. Carbonate system parameter pH DIC TA

Total no. d.f 59 35 35

Factor 1: Time < 0.01 n.s. n.s.

Factor 2: pCO2 condition < 0.01 < 0.01 0.01

Interaction between factors n.s. n.s. n.s.

Table 3. One way ANOVA performed on carbonate system parameters measured in the porewater effluent of sediment columns after a 6 hour pumping cycle. ANOVA p values are reported followed by individual significant pairwise comparisons. A) ANOVA between time points disregarding the effect of pCO2 condition. B) ANOVA between pCO2 conditions disregarding and effect of time. pCO2 condition abbreviations, LGM = Last glacial maximum, Year 2100 = Year 2100 estimate, Year 2300 = Year 2300 estimate

a, Time

pH

One way Anova p