Anja Engel
email: anja.engel@awi.de

Nicole Händel

Julia Wohlers

Mirko Lunau

Hans-Peter Grossart

Ulrich Sommer

Ulf Riebesell

Ulrich Sommer³

à la diffusion de documents scientifiques de niveau

Keywords: DOM, phytoplankton, sea surface warming, mesocosms, polysaccharides, amino acids, microbial loop DOM, phytoplankton, sea surface warming, mesocosm, polysaccharides, amino acids, microbial loop

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Effects of sea surface warming on the production and composition of dissolved organic matter during phytoplankton blooms: Results from a mesocosm study

Introduction

The cycling of organic matter in the surface ocean is regulated by a complex interplay of production and loss processes. Loss of organic matter occurs primarily through heterotrophic activities, resulting in solubilisation and subsequent remineralisation of organic compounds, or through gravitational settling of particles to the deeper ocean. The balance between remineralisation and sinking processes thereby determines the efficiency of organic matter export to the deeper ocean. Particulate organic matter (POM) drives food web interactions at the higher trophic levels as well as export fluxes. The vast majority of organic matter in the ocean, however, is dissolved, comprising in general more than 90% of total organic carbon (TOC), equivalent to 662 Pg C [START_REF] Hansell | Dissolved organic matter in the ocean: New insights stimulated by a controversy[END_REF].

Marine dissolved organic matter (DOM) is produced primarily by extracellular release from phyto-and bacterioplankton cells (Fogg 1983[START_REF] Ogawa | Production of refractory dissolved organic matter by bacteria[END_REF], enzymatic solubilization of particles [START_REF] Cho | Major role of bacteria in biogeochemical fluxes in the ocean's interior[END_REF][START_REF] Smith | Intense hydrolytic enzyme activity on marine aggregates and implications for rapid particle dissolution[END_REF], viral lysis (Fuhrmann 1999), and zooplankton grazing [START_REF] Copping | Carbon budget of marine phytoplankton-herbivore system with carbon-14 as tracer[END_REF][START_REF] Nagata | Production mechanisms of dissolved matter[END_REF]. Major loss processes for DOM are microbial uptake and remineralisation [START_REF] Azam | Size distribution and activity of marine microheterotrophs[END_REF], and photochemical decomposition [START_REF] Mopper | Photochemical degradation of dissolved organic carbon and its impact on the oceanic carbon cycle[END_REF]. Another important loss of DOM is the aggregation of high-molecular weight components (HMW>1kDa) into gel particles, a physical process that transfers dissolved molecules into the particulate size spectrum [START_REF] Leppard | The characterization of algal and microbial mucilages and their aggregates in aquatic ecosystems[END_REF][START_REF] Kerner | Self-organization of dissolved organic matter to micelle-like microparticles in river water[END_REF], Engel et al. 2004[START_REF] Verdugo | The oceanic gel phase: a bridge in the DOM-POM continuum[END_REF]). Export of DOM from the ocean's surface to the deeper ocean contributes significantly to the biological pump [START_REF] Hopkinson | Efficient export of carbon to the deep ocean through dissolved organic matter[END_REF].

When biological production and loss processes are decoupled, either temporarily or spatially, DOM accumulates. This is most evident in oceanic regions that experience annual phytoplankton blooms [START_REF] Carlson | Production and Removal Processes[END_REF]. The percentage of organic matter that is released by (Myklestad et al. 1972, Biddanda and[START_REF] Biddanda | Carbon, nitrogen, and carbohydrate fluxes during the production of particulate and dissolved organic matter by marine phytoplankton[END_REF]. Especially towards the end of nutrientlimited phytoplankton blooms, enhanced leakage of low-molecular weight compounds, and exudation of larger molecules therefore generate high DOM concentrations (Søndergard et al. 2000, Engel et al. 2004). [START_REF] Hansell | Net community production of dissolved organic carbon[END_REF] estimated that 1.2 Gt C year -1 or 17% of global new production escapes rapid microbial utilization, accumulates in the surface waters and is available for export to the ocean's interior.

The reactivity of organic matter is often described by its turnover time. Especially for DOM, which to a large extent is chemically uncharacterized, the times-scale of cycling have been used to differentiate between labile (hours to days), semi-labile (weeks to months) and refractory (months to years) components [START_REF] Anderson | A one dimensional model of dissolved organic carbon cycling in the water column incorporating combined biologicalphotochemical decomposition[END_REF]. The turnover of DOM is not solely a function of its chemical quality, but also depends of the abundance, species composition and activity of microorganisms that consume DOM, and of other factors such as temperature, light and nutrients [START_REF] Hansell | Net community production of dissolved organic carbon[END_REF]. Therefore, turnover times of DOM vary greatly over time and space. A more direct indication of the reactivity and nutritious quality of organic matter can be obtained from molecular compound analysis (e.g., [START_REF] Lee | Particulate amino acids in the sea: Effects of primary productivity and biological decomposition[END_REF][START_REF] Cowie | Sources and reactivities of amino acids in a coastal marine environment[END_REF], Hernes et al., 1996). Carbohydrates (CHO) and amino acids (AA) are the major characterized components of organic matter in marine systems (e.g., [START_REF] Wakeham | Molecular indicators of diagenetic status in marine organic matter[END_REF][START_REF] Pakulski | Abundance and distribution of carbohydrates in the ocean[END_REF][START_REF] Benner | Chemical composition and reactivity[END_REF][START_REF] Carlson | Production and Removal Processes[END_REF]).

While dissolved monomeric, i.e. free, amino acids and carbohydrates are rapidly taken up by organisms, in particular by heterotrophic bacteria, combined carbohydrates of high molecular weight (>1kDa), i.e. polysaccharides, and combined amino acids, e.g. proteins, represent the major fractions of the accumulating DOM during phytoplankton blooms (Søndergard et al. 2000). Although knowledge on the temporal and spatial variability of polysaccharides and amino acids in the water column has greatly increased over the last decades, the mechanistic understanding of their environmental and biological control is still fragmentary. In general, the metabolic cycling of organic components is catalyzed by enzymes, whose specific requirements for activation energy determine the rate of the reaction. The optimum temperature and the sensitivity to changes in temperature vary between enzymes and so does the temperature effect on the various metabolic processes [START_REF] Brown | Toward a metabolic theory of ecology[END_REF]. As a consequence, the overall influence of rising temperature on a dynamic ecosystem and on the fluxes of organic matter therein is difficult to predict. Moreover, rates of metabolic processes are co-determined by many other factors, such as substrate quality, availability of co-enzymes and organism size.

Based on data from various oceanic provinces, [START_REF] Kirchman | Microbial growth in the polar oceans-role of temperature and potential impact of climate change[END_REF] recently reassessed the temperature effect on the ratio of bacterial to primary production. They suggested that the apparent temperature sensitivities of marine bacteria in waters <4°C are strongly co-determined by the availability of nutritious, labile DOC. Following this argument, the influence of global warming on microbial organic matter carbon cycling in cold seas may be rather small, if the amount of labile DOC was not to change.

During a mesocosm study, [START_REF] Wohlers | Changes in biogenic carbon flow in response to sea surface warming[END_REF] recently observed that increasing temperatures by +0, +2, +4, and +6°C resulted in a higher partitioning of carbon to the DOM pool, and in an overall decrease of the net biological draw-down of dissolved inorganic carbon (DIC). Here, we give detailed information on the compositional and quantitative changes of DOM produced during that mesocosm study. We focuss particularly on the question how polysaccharides and amino acids, representing the major nutritious components of DOM, may respond to sea surface warming in the course of phytoplankton blooms in cold seas.

Mesocosm set-up

An indoor mesocosm study was conducted between 6 th January and 5 th February 2006.

Eight mesocosms with an approx. volume of 1400 L each were set up in four temperaturecontrolled climate chambers [START_REF] Sommer | An indoor mesocosm system to study the effect of climate change on the late winter and spring succession of Baltic Sea phyto-and zooplankton[END_REF]. Unfiltered seawater containing a natural winter/spring plankton community was pumped from 6 m depth in Kiel Fjord, Baltic Sea, and was simultaneously filled into the mesocosms. Mesozooplankton from net catches in Kiel Bight was added in natural densities of approximately 10 individuals L -1 .

Temperature of the four climate chambers was adjusted to 2.5, 4.5, 6.5 and 8.5°C, respectively. The lowest temperature of 2.5°C (in situ treatment +0°C; mesocosms M7 and M8) was determined based on a 10-year meteorological database for Kiel Bight (1993Bight ( -2002) ) for January and February. The elevated temperature regimes, hereafter named +2°C (M5 and M6), +4°C (M3 and M4), and +6°C (M1 and M2), were chosen according to the projected increase in global mean sea surface temperature of up to 6.4°C (high scenario A1FI) until the end of the 21 st century (summarised by [START_REF] Solomon | Climate Change 2007: The Physical Science Basis. Contribution of Working Group I to the Fourth Assessment Report of the Intergovernmental Panel on Climate Change[END_REF]. Light was supplied by light benches above each mesocosm, containing different types of light tubes in order to supply close-to-natural light conditions (T5, 10x JBL Solar Tropic [4,000 K], 2x JBL Solar Natur [9,000 K]). The light benches were controlled by separate computer units (ProfiLuxII). With these, a triangular light curve was created with a light: dark-cycle of 12h: 12h. At sunrise, the initial light intensity was 40 % of the total capacity, gradually increased up to 100 % at midday and then gradually decreased again to 40 % at sunset. The maximum light intensity at After the addition of inorganic nitrate, we followed the build-up and decline of phytoplankton blooms in each mesocosm over a course of 30 days. During the experiment, the water body was gently mixed with a propeller attached to the side of the mesocosm. With this, cells and smaller particles were kept in suspension, whereas bigger particles and aggregates, forming at the end of the bloom, sank out of the water column.

Sampling

Water samples were taken daily by lowering a silicon tube into the middle of the mesocosm, closing it at the top, pulling the upper end out and filling a clean canister with the water by means of gravitation.

Bulk chemical analysis

A variety of chemical and biological analyses were performed throughout the experiment. Dissolved inorganic nutrients were measured with an autoanalyzer (AA II) on water samples filtered through 5.0 µm cellulose acetate following the protocol by Hansen and [START_REF] Koroleff | Determination of nutrients[END_REF]

Combined carbohydrates

Hydrolysable combined carbohydrates in the molecular fraction >1kDa, i.e.

polysaccharides, were collected daily from each mesocosm. For total polysaccharides (TCCHO) including particulate and dissolved components, two replicate samples of 20 ml were filled into combusted glass vials using 50 ml disposable syringes. For dissolved polysaccharides (DCCHO), two replicate samples were filtered through 0.45 µm syringe filters (GHP membrane, Acrodisk, Pall) before filling the sample into combusted glass vials.

All samples were immediately frozen and stored at -20°C. Prior to filtration, all syringes and syringe filters were rinsed with several ml of Milli-Q water first and seawater sample thereafter. The samples were thawed immediately before anlaysis. Desalination of the samples was conducted by membrane dialysis (1kDa MWCO, Spectra Por) for 6h at 0°C. Monomeric carbohydrates were yielded after acid hydrolysis of desalinated samples using 0.8M HCl final concentration for 20h at 100°C, and neutralisation through acid evaporation (N 2 ).

Carbohydrate monomers were determined by ion chromatography on a Dionex ICS 3000 system using high performance anion exchange chromatography (HPAEC) coupled with pulsed amperometric detection (PAD) following the protocol of [START_REF] Piontek | Acidification increases microbial polysaccharide degradation in the ocean[END_REF].

Dissolved amino acids

Dissolved free amino acids (DFAA) and combined amino acids (DCAA) were determined by HPLC after ortho-phthaldialdehyde derivatization [START_REF] Lindroth | High performance liquid chromatography determination of subpicomole amounts of amino acids by precolumn fluorescence derivatisation with OPA[END_REF].

Therefore, 20-mL samples were filtered through 0.45 µm syringe filters with low protein binding affinity (GHP membrane, Acrodisk, Pall Corporation) and stored at -20°C. Samples were thawed immediately before analysis. Dissolved combined amino acids (DCAA) were hydrolyzed with 6 N HCl at 155°C for 1 h before analysis. The following standard amino acids were used: aspartic acid (Asp), glutamic acid (Glu), histidine (His), serine (Ser), arginine (Arg), glycine (Gly), threonine (Thre), alanine (Ala), tyrosine (Tyr), valine (Val),

Statistical treatment of data

Mean values were compared by means of a t-test. Significance of the correlation coefficient (r 2 ) against H o : ρ=0, was tested by a Student-test according to [START_REF] Sachs | Angewandte Statistik[END_REF], with a degree of freedom, df=n-2. The influence of the temperature treatment on biological or chemical variables was determined by means of the analysis of variance (ANOVA).

Significance level of each test was p<0.05.

Results

The general development of the phytoplankton bloom is described in more detail in [START_REF] Wohlers | Changes in biogenic carbon flow in response to sea surface warming[END_REF]. Briefly, a bloom developed in each of the mesocosms after nutrient addition and was dominated by diatoms (in particular Skeletonema costatum, Chaetoceros spp. and Thalassiosira spp.). Bloom development was reflected after day 5 by an increase in Chl a concentration, and a rapid decline in dissolved inorganic nutrients. Rising water temperature led to an earlier onset of the bloom with an acceleration of 1 day/°C. Maximum and average Chl a concentrations, and primary production rates did not differ significantly between treatments. Accumulation of POM during the experiment ranged between 21 and 36 µmol L -1 PN, 0.67-0.79 µmol L -1 POP, and 220-393 µmol L -1 POC with no significant effect of temperature. In addition, no temperature effect was observed for rates of nutrient drawdown. In contrast, the net drawdown of DIC decreased significantly with increasing respectively. After the onset of nutrient depletion, average DOC concentration in the mesocosms significantly increased with temperature (p<0.005). At this time, average DON concentration in all mesocosms was 8.39±1.64 µmol L -1 and did not respond to the temperature treatment. A higher net respiratory release of carbon was observed during the post-bloom phase in the mesocosms at +4°C and +6°C. Maximum rates of secondary production of free-living bacteria were higher in the warm mesocosms.

Concentration and composition of polysaccharides

Initial total polysaccharide (TCCHO) concentration was 1.60±0.19µmol L -1 , with 1.23±0.19 µmol L -1 being comprised of dissolved polysaccharides (DCCHO). Following biological production, polysaccharide concentration increased in all mesocosms to maximum values of 51µmol L -1 for TCCHO (+2°C M6) and 24µmol L -1 for DCCHO (+4°C M4). In all mesocosms, TCCHO increased before DCCHO, indicating a general direction of carbohydrate partitioning from the particulate into the dissolved phase (Fig. 1).

Temperature affected the temporal development of polysaccharide concentration significantly, leading to an earlier exponential increase of TCCHO (p<0.05), and of DCCHO (p<0.01), and to earlier peak concentrations in DCCHO (p<0.001). For DCCHO, the timing of increase in concentration was accelerated by 1.6 days/°C (Fig. 2a). The time span between the increase in TCCHO and in DCCHO narrowed with increasing temperature by 0.87 days/°C (p<0.05), indicating a faster partitioning of carbohydrates from the particulate into the dissolved pool at higher temperatures (Fig. 2b). Concentration of DCCHO declined after 3 Temperature also affected the absolute amount of total and of dissolved polysaccharides (ANOVA, p<0.005). The maximum yield, defined as the difference between the maximum and the initial concentration of a component:

[Yield max ]=[C max ]-[C initial ],
significantly increased with temperature for DCCHO (p<0.005) (Fig. 2c). In contrast, a maximum yield of TCCHO was observed in the coldest mesocosms, and decreased with increasing temperature (p<0.005)(Fig. 2d). This points towards a decline in the yield of particulate polysaccharides with rising temperature. Over the course of the experiment, the average ratio of [DCCHO]: [TCCHO] was 35, 40, 49 and 59% in the +0, +2, +4, and +6°C treatments, respectively. During the post-bloom phase, polysaccharides in the warmer mesocosm (+6°C and +4°C) were almost completely contained in the dissolved phase (Fig. 1).

On few individual days, concentrations of DCCHO slightly exceeded those of TCCHO, which can be explained by analytical and sample variability.

In all mesocosms, molar composition of total polysaccharides showed a clear dominance of Glucose (Glc) (Table 2), which is derived as a primary product from algal photosynthesis. Reflecting algal productivity, mole percentages of Glc in polysaccharides increased sharply around the Chl a peak, and dominated carbohydrate composition by >80%

during the bloom and post-bloom phase. Percentages of Glc were generally higher in TCCHO than in DCCHO (Fig. 3, Table 2), reflecting the production of Glc by algal photosynthesis and its preferential removal from the dissolved phase. The increase of mole percentages of Glc in DCCHO followed the increase in TCCHO much sooner in the warmer treatments than in the colder, illustrating again the more rapid partitioning of polysaccharides into DOM (Fig. 3).

Dissolved amino acids (DAA)

In all mesocosms, total dissolved amino acids (DAA), including dissolved free amino acids (DFAA) and dissolved combined amino acids (DCAA), started with an average concentration of 1.72±0.67 µmol L -1 , and increased continuously at an average rate of about 0.79±0.20 nmol L -1 h -1 (n=99, r²=0.15, p<0.001) (Fig. 5) until the end of the study. The In general, the molecular composition of DAA was quite similar in all mesocosms and not significantly related to the temperature treatment (ANOVA, p>0.05). DAA mole composition in all mesocosms was dominated by neutral compounds, with Gly and Ala having the highest contributions of 15% and 14%, respectively, followed by the hydroxilic amino acid Ser (~13%) and the acidic amino acid Glu (~10%). The lowest contribution was due to the aromatic amino acid Phe.

The mole composition of amino acids reflects the diagenetic state of organic matter, which can be estimated by the degradation index (DI) of Dauwe and Middelburg (1998, see also [START_REF] Dauwe | Linking diagenetic alteration of amino acids and bulk organic matter reactivity[END_REF]. For the calculation of DI from DAA in this study, mole percentages of amino acid were standardized using averages, and standard deviations as given in [START_REF] Dauwe | Linking diagenetic alteration of amino acids and bulk organic matter reactivity[END_REF] and multiplied with factor coefficients as determined by these authors after Principal Component Analysis (PCA) of 28 marine samples of different diagenetic state. The DI represents the cumulative variation of amino acids relative to the average composition of the 28-sample dataset weighed by the factor coefficient of the first axis. Lower values indicate more degraded, higher values rather fresh organic material. DI values typically range between +2 and -2. During this study, DI values were generally >0, reflecting the freshness of organic matter produced during the phytoplankton bloom (Fig. 6). A significant change in DI over the duration of the experiment was observed for the two mesocosms of the +6°C treatment solely.

Here, a linear decline of DI indicated the proceeding degradation of organic matter (p<0.001).

During the post-bloom phase, i.e. after day 17, DI values of the +6°C and +4°C treatments were significantly lower than those of the +0°C treatment (p<0.001), indicating that DAA were more degraded at higher temperatures. Therefore, DFCHO are generally turned over rapidly in the presence of heterotrophic microbes, and concentrations of DFCHO are often <100nM [START_REF] Rich | Concentrations and uptake of neutral monosaccharides along 140 degrees W in the equatorial Pacific: Contribution of glucose to heterotrophic bacterial activity and the DOM flux[END_REF][START_REF] Skoog | Microbial glucose uptake and growth along a horizontal nutrient gradient in the North Pacific[END_REF].

Contribution of CHO and AA to carbon and nitrogen cycling

Before the onset of the phytoplankton bloom, the average molar C:N ratio of DCCHO was 24±7 in all mesocosms. Due to the high amount of Glc released into DOM, the C:N ratio of DCCHO clearly increased during and after the bloom, yielding maximum ratios of 450-500 on individual days in the +6°C and +4°C mesocosms. The overall average C:N ratio of DCCHO was 122±125 (n=194).Temperature significantly affected the C:N ratio of DCCHO (ANOVA p<0.001), leading to higher values in the warmer mesocosms (+4°C and +6°C).

For TCCHO, the C:N ratio was generally lower than for DCCHO, and yielded an overall average value of 91±77 (n=194). This can be explained by the higher mole percentages of amino sugars in TCCHO than in DCCHO (Table 2). The initial C:N ratio of Amino acids contributed on average 5.4±2.4% to DOC and 35±15% to DON. The average C:N ratio of DAA during this study was 3.2±0.26 (n=186). This ratio did not change significantly over time and was not affected by the temperature treatment (ANOVA, p>0.05).

During this mesocosm study, DCCHO and DAA were determined as major components of freshly produced, labile to semi-labile DOM. In order to identify the role of polysaccharides and amino acids in DOM dynamics, we calculated the total sum of carbon and nitrogen in the identified molecular components of DCCHO and DAA, and refer to this as iDOC and iDON.

In all mesocosms, new DOM was produced shortly after the depletion of nitrate (Fig. 7). Initially, iDOC contributed on average 11.7±3.6% to DOC, and increased to maximum values at the onset of the post-bloom phase. Maximum percentages of iDOC of about 90% DOC were observed in the +4°C treatments. The average concentration of iDOC was significantly different between the temperature treatments (ANOVA, p<0.001), yielding 61, 122, 175, and 132 µmol C L -1 in the +0, +2, +4, and +6°C treatment, respectively. Due to the much higher accumulation of DCCHO than of DAA, molar C:N ratios of freshly produced DOM* increased strongly during the experiment and yielded maximum ratios of 39, 65, 87, and 74 for the +0, +2, +4 and +6°C treatments, respectively. This induced an increase in C:N ratios of total DOC from an average initial value of 6.3±1.0 to maximum values of 28, 32, 41, and 29 for the +0, +2, +4 and +6°C treatments, respectively. chosen for this experimental study can be described as a high light, high nutrient, new production system, such as is typical for phytoplankton spring blooms at higher latitudes.

Temperature effects on polysaccharide production and release

Phytoplankton growth and productivity are co-determined by light, nutrients and temperature [START_REF] Verity | Effects of temperature, irradiance, and daylength on the marine diatom Leptocylindrus danicus cleve. I. Photosynthesis and cellular composition[END_REF][START_REF] Geider | Light and temperature dependence of the carbon to chlorophyll a ratio in microalgae and cyanobacteria: implications for physiology and growth of phytoplankton[END_REF][START_REF] Schofield | Impact of temperature on photosynthesis in the toxic red-tide dinoflagellate Alexandrium fundyense[END_REF]. Temperature is considered to affect the synergy between electron transport and carbon fixation in algal photosynthesis [START_REF] Verity | Effects of temperature, irradiance, and daylength on the marine diatom Leptocylindrus danicus cleve. I. Photosynthesis and cellular composition[END_REF].The photosynthetic response to temperature depends on the amount of PAR, and is different between sub-saturating and saturating light levels (Davison, 1991).

Carbohydrates are the primary product of photosynthesis and play a central role in cell growth and metabolism. Investigations of temperature effects on the release of organic carbon or carbohydrates from the cell are scarce, and limited to phytoplankton culture studies conducted under nutrient replete conditions [START_REF] Verity | Effects of temperature, irradiance, and daylength on the marine diatom Leptocylindrus danicus cleve. I. Photosynthesis and cellular composition[END_REF], Wolfstein and Staal 2002, Claquin et al. 2008).

Carbohydrates are often exuded from phytoplankton cells in form of high molecular weight polysaccharides (>1kDa) (Myklestad et al. 1972, Biddanda and[START_REF] Pakulski | Abundance and distribution of carbohydrates in the ocean[END_REF] supposedly due

to an imbalance of nutrient acquisition and primary production [START_REF] Wood | Paradox lost? On the release of energy-rich compounds by phytoplankton[END_REF]Van Walen 1990, Engel et al. 2004). Dissolved polysaccharides can aggregate in the extracellular medium to form TEP. Claquin et al. (2008) reported that TEP formation in nutrient replete phytoplankton cultures increased with temperature until an optimum of about 22 -25°C.

During this study, we observed that total combined Glc (TGlc) sharply increased together with Chl a concentration in all temperature treatments. Higher temperatures led to an Chl a specific maximum primary production rates increased with temperature under high light condition [START_REF] Verity | Effects of temperature, irradiance, and daylength on the marine diatom Leptocylindrus danicus cleve. I. Photosynthesis and cellular composition[END_REF][START_REF] Schofield | Impact of temperature on photosynthesis in the toxic red-tide dinoflagellate Alexandrium fundyense[END_REF]. [START_REF] Schofield | Impact of temperature on photosynthesis in the toxic red-tide dinoflagellate Alexandrium fundyense[END_REF] suggested that a more efficient photosynthesis at higher temperatures could be attributed to the higher activity of enzymes involved in the dark reactions, yielding a higher production of carbohydrates per unit Chl a. On the other hand, carbohydrates are the main substrates for cellular respiration, which has been shown to increase with temperature in both autotrophic [START_REF] Verity | Effects of temperature, irradiance, and daylength on the marine diatom Leptocylindrus danicus Cleve. III. Dark respiration[END_REF], and heterotrophic plankton [START_REF] Hoppe | Climate warming during winter affects the coupling between phytoplankton and bacteria during the spring bloom: Results from a mesocosm study[END_REF]. [START_REF] Wohlers | Changes in biogenic carbon flow in response to sea surface warming[END_REF] reported that the community respiration rate during this mesocosm study also increased with temperatures. Higher respiration of carbohydrates by the microbial community was likely the major loss for carbohydrates in this study and may explain the lower yield of TCCHO in the warmer mesoscosms.

The faster and higher partitioning of carbohydrates into DOM during our study suggests that temperature not only affected the rate of processes involved in carbohydrate production and respiration, but also those of organic matter release. A potential explanation may be given in line with the 'overflow-hypothesis' that considers exudation as a release of excess photosynthetic products [START_REF] Wood | Paradox lost? On the release of energy-rich compounds by phytoplankton[END_REF]VanValen 1990, Schartau et al. 2007). Because the rate of nutrient draw down was similar in all mesocosms, a faster accumulation of carbohydrates at higher temperatures could increase the intracellular amount of excess organic carbon, and therefore the exudation rates of polysaccharides. In general, the percentage extracellular release (PER) of polysaccharides from phytoplankton in marine systems varies strongly between phytoplankton species and environmental conditions (see Carlson, 2002 for review). Up to 75% of primary production can be released by the phytoplankton cell as DOC [START_REF] Baines | The production of dissolved organic matter by phytoplankton & its importance to bacteria, patterns across marine & freshwater systems[END_REF]Pace 1991, Wolfstein et al. 2002), with the largest fraction in form of polysaccharides. Studying the extracellular carbon release by phytoplankton in the Southern Ocean, [START_REF] Moran | Response of Southern Ocean phytoplankton and bacterioplankton to short-term experimental warming[END_REF] showed that a warming by 2°C resulted in a ~54% increase in PER during short-term incubations. Our results strongly support the assumption that phytoplankton exudation is temperature sensitive. Here, a temperature rise from 2°C to 8°C clearly accelerated and increased the release of polysaccharides into DOM, and enabled an earlier, faster and higher supply of labile DOM to the heterotrophic microbial community.

Temperature effect on amino acid production and release

Amino acids represent the largest identifiable pool of DON and typically range between 0.15 to 4.20 µmol L -1 in marine systems (Bronk 2002, Kaiser andBenner 2009).

DAA are the major resource for the growth of heterotrophic microbes [START_REF] Rosenstock | Use of dissolved combined and free amino acids by planktonic bacteria in Lake Constance[END_REF][START_REF] Carlson | Production and Removal Processes[END_REF][START_REF] Bronk | Dynamics of DON[END_REF][START_REF] Simon | Different coupling of dissolved amino acid, protein, and carbohydrate turnover to heterotrophic picoplankton production in the Southern Ocean in austral summer and fall[END_REF] and have been shown to supply ~50% of the bacterial nitrogen demand in coastal and estuarine systems (Keil andKirchman 1993, Middelboe et al. 1995). In addition, some phytoplankton species have been shown to possess ectoenzymes that cleave NH 4 + from amino acids for subsequent uptake by the autotrophic cell [START_REF] Palenik | Cell-surface oxidation of amino acids in seawater[END_REF].

DFAA are released from organisms by diffusive leakage, cell break-up by grazing or result from enzymatic cleavage of DCAA [START_REF] Smith | Intense hydrolytic enzyme activity on marine aggregates and implications for rapid particle dissolution[END_REF]. During this study no significant differences were observed in DFAA concentration over time or between temperature treatments. This suggests that the overall balance of production and loss processes for DFAA was similar in all mesocosms, and may rather be related to inorganic and particulate organic nitrogen cycling, which did not display temperature sensitivities during this study either.

However, DFAA are particularly labile components of marine DOM. Because DFAA are small enough to be directly taken up by microbes, their turn-over times range within a few hours, which is smaller than the sampling interval applied during this study. Therefore, differences between mesocosms with respect to DFAA production and consumption rates could not be resolved here.

During this study the identifiable DAA represented on average 35±15% of DON. This value is higher than observed in field studies, where DAA percentages of DON were reported to vary between 1 and 13% [START_REF] Bronk | Dynamics of DON[END_REF], and emphasizes the fresh nature of DOM in our mesocosms. Concentrations of DAA increased continuously during the bloom, due to the accumulation of DCAA. Processes of DAA production were not determined directly during this study. However, we assume that active exudation from phytoplankton was likely not the major source for DCAA, because this would constitute the loss of a limiting resource as nitrogen depletion was observed in the course of the study. Grossart et al. (2007) did not observe an accumulation of DCAA during mesocosm experiments with phytoplankton cultures and bacterial assemblages, despite an increase in DFAA. Another source of DCAA is the release from microbes during zooplankton grazing, and viral lysis [START_REF] Bronk | Dynamics of DON[END_REF]. Because the accumulation of DCAA during this study did not differ significantly between the treatments, processes responsible for DCAA production either did not respond to rising temperature, or loss processes, e.g. bacterial consumption of DCAA, responded likewise. A close coupling between DCCA production and subsequent consumption by phyto-and bacterioplankton has been observed previously in other studies [START_REF] Kawasaki | Bacterial release of dissolved organic matter during cell growth and decline: Molecular origin and composition[END_REF]Benner 2006, Grossart andSimon 2007). We did notice a significant diagenetic change in amino acid composition over time in the mesocosms with the warmest treatment (+6°C), which points to an intense microbial processing of DAA. This may partly be due to the longer duration of the post-bloom period in the +6°C treatment, giving the heterotrophic community more time to evolve. [START_REF] Thingstad | Counterintuitive carbon-tonutrient coupling in an Arctic pelagic ecosystem[END_REF] suggested that more nutrients get recycled in mature microbial food-webs, having a higher abundance of microzooplankton. As a consequence of a higher availability of regenerated nutrients, bacteria are then better able to degrade freshly produced DOM. The decline of DCCHO concentration in the +6°C mesocosms towards the end of the study likely reflected such an intensified microbial degradation of fresh DOM.

Consequences for carbon and nitrogen cycling

Marine microbes, and heterotrophic bacteria in particular, are the major sink of fresh DOM in the ocean. It has been suggested that marine bacteria are sensitive to global warming, but that individual processes such as bacterial respiration (BR) and bacterial biomass production (BBP) may respond differently to temperature [START_REF] Vazquez-Dominguez | Ocean warming enhances respiration and carbon demand of coastal microbial plankton[END_REF][START_REF] Kritzberg | Temperature and phosphorus regulating carbon flux through bacteria in a coastal marine system[END_REF]. The response of bacterial growth efficiencies (BGE) to temperature, with BGE= BBP/ (BBP+BR), is co-determined by the quality of the organic resources (delGiorgio and Cole, 1998). Consumption of carbon-rich substrates, such as carbohydrates, may enhance bacterial respiration and thus lower BGE, while nitrogen-rich compounds, such as amino acids, stimulate cell growth and increase BGE [START_REF] Kroer | Bacterial growth efficiencies on natural dissolved organic matter[END_REF].

During this study, we observed that rising seawater temperature in a range of 2°-8°C selectively enhanced the release of polysaccharides during phytoplankton blooms that are terminated by nutrient depletion. The higher release of polysaccharides induced a rapid increase in the C:N ratio of fresh DOM. Warming also increased community respiration, 2009). The stimulation of bacterial respiration has also been observed earlier in other experimental studies that simulated effects of sea surface warming on bacterioplankton communities [START_REF] Vazquez-Dominguez | Ocean warming enhances respiration and carbon demand of coastal microbial plankton[END_REF][START_REF] Hoppe | Climate warming during winter affects the coupling between phytoplankton and bacteria during the spring bloom: Results from a mesocosm study[END_REF][START_REF] Kritzberg | Temperature and phosphorus regulating carbon flux through bacteria in a coastal marine system[END_REF].

We therefore conclude that warming leads to a selective accumulation of carbon rich components of DOM, as a result of a faster extracellular release of carbohydrates by phytoplankton. This, in turn, could result in accelerated microbial respiration and hence carbon dioxide liberation. This scenario may be specifically applicable to boreal areas with present day temperatures of <4°C and at times when phytoplankton blooms are terminated by nutrient limitation. Along with the enhanced release of DOC from phytoplankton cells, one may expect a reduction in the C:N ratio of sinking particles, as was indeed observed during this mesocosm experiment [START_REF] Piontek | Effects of rising temperature on the formation and microbial degradation of marine diatom aggregates[END_REF].

In response to an increased availability of labile carbon, and hence of energy, marine bacteria are better capable to utilizing inorganic nitrogen resources [START_REF] Kirchman | Carbon limitation of ammonium uptake by heterotrophic bacteria in the sub-arctic pacific[END_REF]Goldman and Dennett, 1991[START_REF] Kirchman | Uptake and regeneration of inorganic nutrients by marine heterotrophic bacteria[END_REF][START_REF] Thingstad | Counterintuitive carbon-tonutrient coupling in an Arctic pelagic ecosystem[END_REF]. A higher supply with fresh DOC during warming may thus enhance the resource competition between heterotrophic bacteria and phytoplankton, and may even exacerbate nutrient limitation of phytoplankton.

A presently inadequate supply of labile DOC has been suggested to co-limit marine bacteria in polar seas, in addition to temperature [START_REF] Kirchman | Microbial growth in the polar oceans-role of temperature and potential impact of climate change[END_REF]). If our experimental findings are applicable to polar microbial systems, the predicted increase in sea surface temperature by 2-6°C (IPCC 2007) may alleviate carbon limitation of polar bacteria through an enhanced availability of dissolved carbohydrates. This process may become particularly important as the bacterial carbon demand also increases with temperature [START_REF] Pomeroy | Temperature and substrates as interactive limiting factors for marine heterotrophic bacteria[END_REF]. Applying a pelagic food-web model, [START_REF] Laws | Temperature effects on export production in the open ocean[END_REF] identified temperature as a major control of the ratio of new, respectively export, production to regenerated production (e/f-ratio) in various oceanic provinces; in warmers seas, a larger fraction of primary production is supported by regenerated nutrients and does not contribute to export fluxes. The regeneration of nutrients in pelagic food-webs is primarily controlled by the microbial loop, i.e. the cycling of elements and energy through processes of extracellular release of primary production, subsequent bacterial utilization of DOM, and grazing by heterotrophic nanoflagellates [START_REF] Azam | The ecological role of water column microbes in the sea[END_REF]). If the bottom-up control of the microbial loop in the ocean changes in response to global warming, such as suggested from our mesocosm study, nutrient regeneration in the future surface ocean may increase, potentially reducing e/f ratios.

However, warming is only one factor in the complex of environmental changes in the ocean that are expected to proceed rapidly in the wake of climate change. Differences in stratification depth and intensity may occur and will alter the supply of nutrients to the surface ocean, as well as the light regime experienced by the phytoplankton communities. Uptake of anthropogenic CO 2 by the surface ocean has induced the carbonation as well as the acidification of seawater. In a recent study, [START_REF] Piontek | Acidification increases microbial polysaccharide degradation in the ocean[END_REF]

  dissolved pool often increases when nutrient concentration declines

  Abbreviations and acronyms are defined inTtable 1.

  five times a week with a submersible 4π PAR sensor (LiCOR Inc, US) at a depth of approx. 10 cm and was on average 179 ± 24 µmol photons m -2 s -1 .Temperature, salinity and pH were measured three times a week with a WTW conductivity and pH probe. Initial concentrations of dissolved inorganic phosphate (OH) 4 ) were approximately 0.9 µM, 8 µM, and 20.4 µM, respectively. Due to the unusually low NO 3 -concentration in that year, we added additional NO 3 -to a final concentration of 21.1 µM to ensure the development of a phytoplankton bloom.

  ), isoleucine (Ileu), leucine (Leu). Alpha amino butric acid was used as an internal standard to account for losses during handling. For one mesocosm per temperature treatment, dissolved combined amino acids (DCAA) and dissolved free amino acids (DFAA) were determined separately. Concentrations of DCAA after hydrolysis are given as monomer equivalents.

  503±50, 485±58, 435±73, and 350±34 µmol C L -1 at +0, +2, +4, and +6°C, respectively. DOC accumulated in the course of the experiment, particularly after the onset of nutrient depletion. DOC accumulation ranged between 45 and 147 µmol L -1 , with the lowest values of 45 and 74 µmol L -1 being observed in the +0°C mesocosms M7 and M8,

  warmest treatment (+6°C), but continued to increase in the colder mesocosms until the end of the experiment.

  efficiency in Glc production between the temperature treatments, Glc concentration was related to Chl a. Maximum Chl a concentration varied between 40 and 68 µg L -1 , but Chl a variability was not related to the temperature treatment[START_REF] Wohlers | Changes in biogenic carbon flow in response to sea surface warming[END_REF]. Specifically, Chl a concentrations in the +4°C treatment were similar to those in the +0°C treatment. The onset of Chl a increase, however, was earlier at higher temperatures with an acceleration of 1 day/°C[START_REF] Wohlers | Changes in biogenic carbon flow in response to sea surface warming[END_REF]). The ratio of total Glc to Chl a ([TGlc]:[Chl a]; µmol:µg) differed significantly between the temperature treatments (ANOVA, p<0.005), but was more similar among the warmer treatments (+4°C and +6°C). During the first 10 days, [TGlc]:[Chl a] decreased in all mesocosms (Fig.4), as a consequence of Chl a synthesis preceding new Glc production. After the onset of the bloom, [TGlc]:[Chl a] increased steadily in all mesocosms, but earlier in the warmer mesocosms. The temporal rate of change, i.e.∆([TGlc]:[Chl a]):∆(t), was determined by linear regression for five days of exponential increase (with p<0.005 for each regression), and yielded values of 0.10±0.032,0.16±0.002, 0.22±0.005, 0.20±0.32 µmol TGlc µg -1 Chl a d -1 for the +0, +2, +4, and +6°C treatment, respectively. Significant differences between the rates of change in Chl a specific TGlc, however, were only determined between the +0°C and the +4°C treatment (t-test, p=0.05, n=10). During the post-bloom phase, phytoplankton cells settled to the bottom of the mesocosms, and Chl a concentration decreased. At this time, ratios of [TGlc]:[Chl a] were more than five times higher in the warmer treatments (+4°C and +6°C) compared to the +0°C treatment, primarily due to the higher abundance of dissolved Glc.Percentages of other carbohydrates decreased relative to Glc in the course of the bloom. Quantitatively, the acidic sugar Glc-URA was the second most important carbohydrate with yields of 1-18%. The individual molar percentages of the neutral sugars Fuc, Rha, Gal, and Man/Xyl ranged between 1-12%. The deoxysugars Fuc and Rha were generally more enriched in DCCHO than in TCCHO, underlining the more refractory when compared to POM. Percentages of deoxysugars were ~30% of DCCHO, initially, and increased in each mesocosm before the peak of the bloom, to maximum values of 38-44%, and decreased thereafter to minimum values of 1.8-9.5%. The amino sugar Glc-AM comprised about 1.5-3.6% of carbohydrates. Glc-AM had generally higher percentages in TCCHO than in DCCHO, indicating that particulate Glc-AM was either less solubilised than the other carbohydrates, or dissolved Glc-AM more rapidly incorporated into particles. The acidic sugar Gal-URA was below the detection limit in most samples and contributed < 2% to total carbohydrates. Acidic polysaccharides represent a characteristic fraction of transparent exopolymer particles (TEP), which are determined by staining with the acidic-sugars-specific dye Alcian Blue[START_REF] Alldredge | The abundance and significance of a class of large, transparent organic particles in the ocean[END_REF]. TEP production during this mesocosm study was relatively low, yielding an overall average concentration of 193±131 µg Xeq. L -1[START_REF] Wohlers | Changes in biogenic carbon flow in response to sea surface warming[END_REF]. TEP concentration sharply increased in the +6°C mesocosm (M1 and M2) after day 20. In these mesocosms, TEP concentration was significantly correlated to the mole percentage of Glc-URA (p<0.001, data not shown).

  overall average concentration of DAA was 2.23±0.79 µmol L -1 (n=99). DFAA were determined separately for one mesocosm (+6°C M2, +4°C M4, +2°C M5, +0°C M7) per temperature treatment. There, DFAA concentrations ranged between 80 and 360 nmol L -1 (n=81) and comprised between 5 and 20% of DAA. Concentrations of DFAA in the time, but did not exhibit significant differences between the temperature treatments (ANOVA, p=0.30).

  observations, TCCHO contributed 39±28% to TOC (calculated as sum of POC and DOC)(n=194), DCCHO 24±23% (n=194) to DOC, and particulate polysaccharides (PCCHO, calculated by subtracting DCCHO from TCCHO), comprised 57±44 % of POC (n=181). Polysaccharide production contributed largely to the build-up of DOC during the course of the bloom, yielding a non-DCCHO background DOC concentration of about 120 µmol L -1 (data not shown). Besides polysaccharides, dissolved free carbohydrates (DFCHO) are an important carbohydrate source for heterotrophic organisms.DFCHO were not determined during this study, and are thus not included in the estimation of fresh DOC and DON. However, we can assume that DFCHO were quantitatively negligible for DOC accumulation compared to the observed high concentrations of DCCHO. In contrast to DCCHO, DFCHO are low molecular weight compounds, consisting of mono-and dimeric carbohydrates that can be taken up by the heterotrophic cell without prior enzymatic cleavage.

  increased in the course of the phytoplankton bloom to maximum values of 300-360.

  how an increase in sea surface water temperature of boreal seas, such as those projected in the context of global warming until the year 2100, may affect the production and composition of labile DOM during phytoplankton blooms. The scenario

  the bloom development and of polysaccharides production, and to a faster and more intense partitioning of polysaccharides into DOM. During the rise of the bloom, the rate increase of Chl a-normalised TGlc concentration was higher in the warmer treatments, suggesting a more efficient photosynthesis of the phytoplankton community. This interpretation agrees with earlier observation from phytoplankton cultures showing that the

  reduced net draw-down of DIC over the course of the bloom(Wohlers et al.

Figure 1 :

 1 Figure 1: Concentrations of dissolved (grey bars) and total (black bars) combined

Figure 2a- d :

 d Figure 2a-d: Temperature effects on the onset of DCCHO production (a), the time-lag

Figure 3 :

 3 Figure 3: Molar percentages of Glucose (Glc, %) in TCCHO (open circles) and DCCHO

Figure 4 :

 4 Figure 4: Total glucose (TGlc) normalised to Chl a in the course of the phytoplankton

Figure 5 :

 5 Figure 5: Increase of dissolved amino acids (DAA) concentrations, including dissolved free

Figure 6 :

 6 Figure 6: Decline in the degradation index (DI) of dissolved amino acids (DAA) in the +6°C

Figure

  Figure 7a-d: Changes in the ratios of freshly produced and identified DOC (iDOC) to total DOC (iDOC:DOC, open circles), and of nitrate concentrations (µmol L -1 ) (dotted line,

  Figure2a-d

Table 1 :

 1 demonstrated that ocean acidification accelerates the decomposition of polysaccharides by bacterial extracellular enzymes. Thus, future changes in the ocean may not only enhance the supply of labile DOM, in case of warming, but also accelerate its microbial metabolism, in the case of acidification, with the overall result of higher CO 2 remineralisation. Abbreviations and acronyms.

	F o r
	P
	e
	e r
	R
	e
	v i e
	w

Table 1 :

 1 Abbreviations and acronyms.

	AAS	Amino acids	Glc-AM	Glucosamine
	Asp	Aspartic acid	His	Histidine
	Arg	Arginine	Leu	Leucine
	Ala	Alanine	ILeu	Isoleucine
	Ara	Arabinose	Gly	Glycine
	Chl a	Chlorophyll a	HMW	high-molecular weight

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Acknowledgements

This work was funded by the DFG (German Research Foundation) as part of the priority program 1162 AQUASHIFT and by the Helmholtz Association (HZ-NG-102). P.

Fritsche, A. Ludwig, J. Piontek, G. Langer, H. Johannsen and K. Schulz are gratefully acknowledged for technical assistance and help during sampling.