Bacterial production and transformation of dissolved neutral sugars and amino acids in seawater

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Introduction
Approximately 700 Pg (10 15 g) of carbon in the ocean is in the form of dissolved organic matter (DOM) and consist of a broad range of different chemical compounds spanning a continuum of sizes and reactivities (Hansell, 2013;Siegenthaler and Sarimento, 1993).Although all organic matter originates from organisms, only about 6.6 % of surface DOM and 2 % of deep ocean DOM is identified as specific biomolecules such as neutral sugars and amino acids (Benner, 2002).Despite their low concentrations, the rapid turnover of simple biomolecules suggests that they play an important role in the cycling of carbon and nitrogen in the ocean (Rich et al., 1997(Rich et al., , 1996;;Skoog et al., 1999).However, several studies have also indicated that some biomolecules can resist bacterial degradation over year long timescales (Kirchman et al., 2001;Ogawa et al., 2001).Heterotrophic bacteria are well known sources of semi-labile and refractory DOM (Kaiser and Benner, 2008;McCarthy et al., 1998;Ogawa et al., 2001), and ∼ 23 % of the entire DOC pool is estimated to derive from bacteria via the microbial carbon pump (microbial transformation of bioavailable DOM to refractory DOM) (Benner and Herndl, 2011;Jiao et al., 2010).However, still only little is known about microbial production of specific semi-labile and refractory biomolecules.
The most common biomolecules in the oceanic DOM pool are carbohydrates (Benner et al., 1992), with neutral sugars constituting up to half of the total carbohydrate pool (Biersmith and Benner, 1998).Seven different neutral sugars are commonly detected (fucose, rhamnose, arabinose, galactose, glucose, mannose and xylose) and the amount of each neutral sugar relative to the total amount of neutral sugars (mol %) differs among DOM released by different groups of organisms (Lazareva and Romankevich, 2012).Freshly released neutral sugars from different algal species exhibit great variation in mol % but generally have high contributions of glucose and galactose, each sometimes reaching values above 50 mol % (Amon and Benner, 2003;Biersmith and Benner, 1998;Hama and Yanagi, 2001).Surface ocean DOM is also rich in glucose and galactose with many studies reporting ∼ 20 mol % for each of the two compounds Figures  (Goldberg et al., 2009;Kaiser and Benner, 2009;Skoog and Benner, 1997).With depth, glucose and fucose increase in relative abundance, accounting for about 20-40 % and 16-19 % in the deep ocean, respectively (Kaiser and Benner, 2009;McCarthy et al., 1996).The composition found in the ocean reflects both the neutral sugars released by different organisms and the neutral sugars persisting in organic matter after long-term bacterial degradation.Knowledge of the composition of neutral sugars produced by heterotrophic bacteria and their contribution to organic matter cycling is therefore key to understanding the origin and fate of these specific DOM components in the ocean.D-enantiomers of amino acids are useful as bacterial biomarkers, since bacteria are the predominant source of D-amino acids in seawater (Kaiser and Benner, 2008).Four different pairs of L-and D-amino acids are important in DOM (aspartic acid, glutamic acid, serine and alanine) and the amount of D relative to L (the D/L ratio) has been used as an indicator of amino acid bioavailability (Jørgensen et al., 1999).Freshly released DOM from phytoplankton has a low D/L ratio, but the ratio increases during bacterial degradation of DOM (Amon et al., 2001) due to direct release of D-amino acids by bacteria during growth (Kawasaki and Benner, 2006); viral lysis of cells and the subsequent release of cell wall D-amino acids (Middelboe and Jørgensen, 2006); and a presumably higher bioavailability of L-amino acids (Amon et al., 2001;Hopkins et al., 1994;Pérez et al., 2003).The presence of grazers such as flagellates can also increase the bacterial uptake of D-amino acids, possibly due to a higher release from bacterivory and subsequent microbial uptake (Pérez et al., 2003).The D/L ratio does not follow a certain pattern with depth in the ocean.Both a decrease, an increase and no change has been observed with depth in different studies (Jørgensen et al., 1999;Kaiser and Benner, 2008;McCarthy et al., 1998;Pérez et al., 2003), making it difficult to distinguish between different sources and sinks.
In the present study, we investigated the concentration, composition and bioavailability of neutral sugars and amino acids in Arctic and Atlantic seawater.and amino acids were investigated.The aim was to compare the molecular composition of biomolecules in DOM produced by bacteria and in DOM remaining after long-term bacterial degradation and to use these molecular signatures of bacterial activity to further understand the origins of neutral sugars and amino acids in the ocean.

Sampling site and incubation experiments
Seawater for long-term incubations was collected from two different locations in the Denmark Strait: at 10 m depth in the warm (9.8 • C) north going North Icelandic Irminger Current (the Atlantic sample) and at 80 m depth in the cold (−1.6 • C) south going East Greenland Current (the Arctic sample, Fig. 1).The Arctic Ocean receives a large amount of freshwater, supplying 25-36 Tg (10 12 g) terrigenous dissolved organic carbon (DOC) per year (Raymond et al., 2007), and up to 41 % of this DOC is exported through the Fram Strait (Opsahl et al., 1999).The East Greenland Current transports cold Arctic water from the Fram Strait south along the Greenland shelf where it mixes with water from the North Atlantic Ocean (Stein, 1988).The seawater collected for bioassay incubations included terrigenous DOM from the Arctic Ocean as well as marine DOM from the Atlantic Ocean.Three treatments of Arctic and Atlantic samples were incubated for 32 days in the dark at 18 filtered through a GF/C filter (Whatman, ∼ 1.2 µm).Both filters were cleaned with amble amounts of seawater before the filtrates were collected.The filtrates were acclimated to 18 • C in the dark for approximately 24 h and the incubations were initiated by adding a 10 % bacterial inoculum to the 0.2 µm filtered water in acid washed amber glass bottles.Each of the six treatments consisted of 9 amber glass bottles (200 mL) of seawater and three bottles were sacrificed during each subsampling.The artificial seawater for ASW glu samples was prepared as described by Kester et al. (1967) and adjusted to the salinities of the NSW samples (35.1 and 33.5 for Atlantic and Arctic seawater, respectively) by further addition of MilliQ water.Subsamples for bacterial abundance (all heterotrophic prokaryotes), DOC concentration, dissolved combined neutral sugars and amino acids were taken on days 0, 6 and 32.Bacterial abundance and DOC were measured from all triplicate bottles while neutral sugars and amino acids were measured from a single bottle.All samples from day 0 were taken approximately 12 h after the addition of inoculum, glucose and nutrients.

Sample analysis
Subsamples for determination of bacterial abundance were fixed with glutaraldehyde (1.3 % final concentration) and stored frozen (−80 • C) until measurement.Bacteria were counted by flow cytometry on a BD FACS Canto II flow cytometer using the nucleic acid stain SYBR Green to stain the fixed cells (Marie et al., 1997).Subsamples for measurement of DOC concentration were 0.2 µm filtered (Acrodisc) and collected in acid cleaned high-density polyethylene (HDPE) bottles.The samples were acidified with 2 M HCl to a pH of 2 and stored cold (5 • C) until analysis on a Shimadzu TOC-V CPH analyzer.The instrument was calibrated using a standard series made from acetoanilide and performance was evaluated Neutral sugars and amino acids were measured from 0.2 µm filtered subsamples collected in acid cleaned HDPE bottles that were stored frozen (−20 • C) until analysis.The concentration of free and combined hydrolyzable neutral sugars (fucose, rhamnose, arabinose, galactose, glucose, mannose and xylose) was measured on a Dionex 500 ion chromatography system with pulsed amperometric detection (PAD) as described by Skoog and Benner (1997) and Kaiser and Benner (2009).The relative deviation from the mean of replicate hydrolysis (excluding replicate experiments) was 29-40 % for concentrations < 20 nM and 10-33 % for concentrations > 20 nM of individual neutral sugars.For total hydrolyzable neutral sugars the relative deviation from the mean was 15 %.Free and combined hydrolyzable amino acids were analyzed according to Kaiser and Benner (2005) on an Agilent 1260 UPLC using a fluorescence detector and corrected for hydrolysis induced racemization Kaiser and Benner 2005).The concentration of the following amino acids was measured: L-and D-asparagine (asparagine and aspartic acid), L-and D-glutamine (glutamine and glutamic acid), L-and D-serine, Lhistidine, L-threonine, glycine, L-arginine, β-alanine, L-and D-alanine, γ-aminobutyric acid, L-tyrosine, L-valine, L-methionine, L-phenylalanine, L-isoleucine, L-leucine and L-lysine.The relative deviation from the mean of duplicate hydrolysis (excluding replicate experiments) was 4-23 % for concentrations < 10 nM and 3-8 % for concentrations > 10 nM of individual amino acids.For total hydrolyzable amino acids the relative deviation from the mean was 8 %.

Terminology and calculations
The sum of all neutral sugars and the sum of all amino acids measured are termed total hydrolyzable neutral sugars (THNS) and total hydrolyzable amino acids (THAA), respectively.The contributions of THNS and THAA to total DOM (the yields) were calculated as the ratio of carbon bound in either THNS or THAA to the total concentration of DOC.For THAA, the calculations excluded the non-protein amino acids β-alanine and γ-aminobutyric acid.The mol % of a specific compound was calculated as the ratio Figures of the compound (given in moles) to the total concentration of compounds, THNS or THAA (given in moles).
To investigate the neutral sugars produced by bacteria after utilization of the added glucose, the concentration and composition of neutral sugars on day 6 in glucose enriched treatments were compared to the corresponding values of NSW samples.For NSW glu samples, it was necessary to take into account that a fraction of neutral sugars were bound in natural oceanic DOM.Assuming that bacterial transformation of natural oceanic DOM was similar in NSW and NSW glu samples, a subtraction of the concentration of individual sugars (NS) in NSW samples from the corresponding concentrations in the NSW glu samples allowed an estimation of neutral sugars produced by bacteria in NSW glu samples: For ASW glu samples, only the neutral sugars bound in the 10 % inoculum had to be subtracted, assuming similar concentrations and compositions in the inoculum and the NSW samples: NS bacterially produced = NS ASW glu − 0.1 • NS NSW .

Bacterial abundance and dissolved organic carbon
The abundance of bacteria was low at the beginning of the experiments, with less than 0.4×10 6 cells mL −1 in Atlantic samples and less than 0.2×10 6 cells mL −1 in Arctic samples (Fig. 2a-c).During the first 6 days of the experiment, the abundance increased considerably in all treatments, reaching 1.3 × 106 and 0.4 × 10 6 cells mL −1 in Atlantic and Arctic NSW samples and between 3.7 × 10 6 and 5.2 × 10 6 cells mL −1 in glucose enriched treatments.From day 6 to day 32, the bacterial abundance stayed constant or Figures

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Full decreased slightly in all samples.The initial DOC concentration in Atlantic and Arctic samples was approximately 60, 115 and 70 µM in NSW, NSW glu and ASW glu samples, respectively (Fig. 2d-f).DOC concentrations decreased in all treatments during the first 6 days and stayed approximately constant from day 6 to day 32.The net DOC consumption in the Atlantic samples was 4±3.6 , 52±3.6 and 58±1.4 µM in NSW, NSW glu , ASW glu samples, respectively.The corresponding values for the Arctic samples were 2±1.5, 52±0 and 56±2.1 µM DOC.The DOC consumption after 32 days corresponded to 7 and 1 % of total DOC in the Atlantic and Arctic NSW samples, respectively.

Neutral sugars
The initial concentrations of THNS ranged from 210 to 339 nM in NSW samples and from 7041 to 7850 nM in the glucose enriched samples.THNS decreased in all treatments over time, particularly during the first 6 days of the incubations (Fig. 3a-c).After 32 days, the net consumption of THNS in the Atlantic samples was 130, 7585 and 6992 nM in NSW, NSW glu , ASW glu , respectively.The corresponding values for Arctic samples were 84, 6990 and 6943 nM.Initially, THNS constituted 3.5 and 2.4 % of total DOC in Atlantic and Arctic NSW samples, respectively.The yields decreased to 2.5 and 1.7 % on day 6 and continued to decrease at a slower pace during the remaining of the incubations (Fig. 3d).In the glucose enriched treatments, the yields were approximately 40 and 63 % in NSW glu and ASW glu samples, respectively, decreasing to ∼ 3 and 9 % after 6 days (Fig. 3e and f).The molecular composition (mol %) of neutral sugars changed over time but varied little between treatments (Table 1).Galactose and glucose were the most abundant neutral sugars in all samples, together comprising between 38 and 74 mol %.The glucose mol % increased from day 0 to day 6 in all samples (disregarding glucose treatments, day 0) followed by a decrease from day 6 to day 32.The galactose mol % followed the opposite pattern: decreased during the first Introduction

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Full Due to the high concentration of glucose on day 0 in the glucose enriched treatments, these samples were diluted 100 times prior to analysis.Consequently, no other neutral sugars were determined on day 0. The molecular composition of neutral sugars produced by bacteria in the glucose enriched treatments during the first 6 days was calculated as described in Sect. 2. It was characterized by a high glucose mol % (47 ± 12) and a relatively low mol % of the remaining neutral sugars, ranging from 3 ± 2 to 14 ± 9 (Table 1).After 32 days, the molecular composition in all samples was characterized by a high galactose mol % (33 ± 11), a lower glucose mol % (22 ± 8) and an even lower mol % of the remaining neutral sugars, ranging from 7 ± 5 to 11± 3.

Amino acids
The initial concentration of THAA ranged from 170 to 244 nM in all treatments (Fig. 4ac).During the incubations, the concentration of THAA decreased in the Arctic and Atlantic NSW samples.The glucose enriched samples showed an increased concentration of THAA on day 6 followed by a decrease.The only exception was the Arctic ASW glu sample, which like NSW samples showed a decreasing THAA concentration throughout the incubation.The yield of THAA in NSW samples decreased from 1.4 to 1.1 % and from 1 to 0.8 % for Atlantic and Arctic treatments, respectively (Fig. 4d).The yield in NSW glu samples increased from 0.7 and 0.6 % on day 0 to 1.5 and 1.8 % on day 6 and ended at 1.5 and 0.8 % on day 32 in Atlantic and Arctic samples, respectively (Fig. 4e).The yield in ASW glu samples increased from 1.1 and 1.0 % on day 0 to 7.3 and 2.1 % on day 6 and decreased to 2.3 and 0.7 % on day 32 in Atlantic and Arctic samples, respectively (Fig. 4f).
The molecular composition (mol %) of amino acids varied little from sample to sample and did also not show any notable changes over time (Table 2).Glycine was the most abundant amino acid comprising between 26 and 40 mol %.The four amino acids from which both the L-and the D-enantiomers have been measured (asparagine, glutamine, serine and alanine) were the second-most abundant amino acids comprising between 6 and 18 mol %.The concentration of D-amino acids only changed slightly while L-amino Introduction

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Concentration and bioavailability
In NSW samples, the initial DOC concentrations (61-63 µM) were similar to previous measurements from the region (e.g.Amon et al., 2003;Benner et al., 2005).After 32 days, 7 and 1 % of the DOC was consumed in Atlantic and Arctic NSW samples, respectively.These values are comparable to estimates by Amon and Benner (2003) who estimated around 10 % of Atlantic and 2 % of Arctic DOC to be labile.The initial concentrations of THNS in NSW samples (210 and 339 nM) were within the range of concentrations (60-409 nM) found in ultrafiltered surface samples (< 0.1 µm, < 100 m) from the region (Amon and Benner, 2003), and GF/F filtered surface samples from the Sargasso Sea which ranged from ∼ 180 to 450 nM (Goldberg et al., 2009).A considerable fraction of the neutral sugars in NSW samples were labile, as indicated by the preferential removal of neutral sugars (Fig. 3d).This trend has also been observed in previous studies (Amon and Benner, 2003;Amon et al., 2001).The initial concentrations of THAA in NSW samples (189 and 244 nM) corresponded well with values from surface waters of the Sargasso Sea (∼ 150-200 nM, Lee and Bada 1977;Kaiser andBenner 2008, 2009) and the Arctic Ocean (∼ 150-500 nM, Dittmar et al., 2001;Shen et al., 2012).The preferential removal of amino acids in NSW samples indicated that a fraction of amino acids was labile (Fig. 4d).Our study supports the general notion that neutral sugar and amino acid yields can be used as biochemical indicators of DOC bioavailability (Amon et al., 2001), since the highest DOC consumption was observed in the Atlantic NSW sample, which was also associated with the highest yields of THNS and THAA.Introduction

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Full In the glucose enriched samples, 49-56 µM DOC was consumed during the experiment.The DOC samples on day 0 were collected 12 h after the addition of glucose, and the actual DOC consumption was therefore likely up to 12-17 µM higher (estimated from the missing free glucose).Taking this into account, the DOC consumptions agreed well with the amount of added glucose and possibly a small contribution from the original DOM pool.The DOC consumption estimates were calculated from the missing free glucose: glucose was added to a final concentration of 10 000 nM before addition of the inoculum.However, the concentration measured at the beginning of the experiment ranged from 7041 to 7850 nM.Sample hydrolysis possibly also altered some of the added glucose, resulting in lower measured concentrations (Skoog and Benner, 1997).The DOC consumption in NSW glu samples (52 µM) was lower than in the ASW glu samples (56-58 µM), despite of a higher DOC concentration and an equal amount of glucose.Although this discrepancy is small, it may represent a minor contribution of labile DOC associated with the inorganic salts used to prepare the ASW glu samples or an inhibition of degradation of natural DOM in the presence of a simple labile substrate (Gontikaki et al., 2013).However, further studies are warranted to resolve the importance of the latter process.The significant drop in neutral sugar yields clearly reflects the labile nature of added free glucose.The amino acid yields increased at the beginning of the incubations and decreased from day 6 to day 32 -a trend also observed in previous studies (Kawasaki and Benner, 2006;Ogawa et al., 2001).The initial increase in amino acid yields is due to the significant increase in bacterial abundance and subsequent release of amino acids.

Bacterial production of neutral sugars during the first 6 days
Bacterial production and subsequent release of neutral sugars was calculated from the glucose enriched treatments as described in Sect. a relatively low mol % of the remaining neutral sugars (Fig. 5), and are strikingly similar to the composition of bacterial DOM found in a study by Ogawa et al. (2001) after 7 days of incubation.Similar patterns have also been observed in algal-derived DOM (Hama and Yanagi, 2001;Lazareva and Romankevich, 2012), e.g. in DOM from a fresh diatom culture (Biersmith and Benner, 1998) and from sea ice algae (Amon and Benner, 2003).However, DOM released by other algal cultures exhibit different molecular compositions of neutral sugars with galactose or xylose being most abundant (Biersmith and Benner, 1998).In the ocean, glucose is generally the most abundant neutral sugar, however, values above 30 mol % are only observed occasionally (Table 3).Amon and Benner (2003) suggested that degradation processes rather than production processes determine the neutral sugar composition in the ocean, based on the similarities observed in samples of different origin (terrestrial and marine) and from different locations (oceanic regions and water masses).This can possibly explain the difference in molecular composition of DOM in the ocean and the bacterially-produced DOM after 6 days in the present study.Only small differences in neutral sugar composition between Atlantic and Arctic samples were observed after 6 days of incubation despite the different bacterial inocula.Moreover, there was a striking similarity in the bacteria-derived neutral sugar composition observed in the present study and in the study by Ogawa et al. (2001).Together, these results suggest that bacterially-produced neutral sugars, independent of bacterial community structure, are important in shaping the molecular composition of neutral sugars in seawater.

Bacterial degradation of neutral sugars
After 32 days, a clear degradation signature had emerged with glucose being less important and galactose being more important (Fig. 6).This trend has also been seen in studies of marine sediments (Oakes et al., 2010).The majority of the remaining DOM after 32 days was assumed to be semi-labile or refractory, and the striking similarity between treatments indicates that the molecular composition of semi-labile or refractory neutral sugars (i.e.material persisting longer that 32 days) attains a fairly Figures

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Full specific molecular composition, irrespective of initial DOM composition.Furthermore, the similarity between treatments suggests that bacterial degradation processes shape the composition of semi-labile or refractory neutral sugars.After 32 days, about 85 to 87 % of the neutral sugars present in ASW glu samples are of bacterial origin (i.e.produced by bacteria during glucose assimilation), while only 13 to 15 % originate from the DOM added with the inoculum (excluding glucose), assuming that neutral sugars in the inoculum follows the same degradation pattern as in NSW samples and that no refractory neutral sugars were added with the inorganic salts while preparing the artificial seawater.Hence, the molecular composition observed in the ASW glu treatments is primarily the result of bacterially-produced and bacterially-altered molecules containing neutral sugars.In NSW samples, however, the molecular composition after 32 days primarily reflects the natural background level of refractory and semi-labile neutral sugars present in the seawater when collected.Since this background signature is approaching the molecular composition of bacterially-produced neutral sugars in ASW glu samples remaining after 32 days, we hypothesize that semi-labile and refractory neutral sugars primarily originate from bacterial processing of DOM and bacterial remains.Mesocosm studies have shown that 91-94 % of dissolved neutral sugars accumulated during an algal bloom were degraded within 15-20 days (Kragh and Søndergaard, 2009;Meon and Kirchman, 2001), and here we find that only 32-49 % of the neutral sugars produced by bacteria in ASW glu samples were degraded within a period of 26 days, from day 6 to day 32.Neutral sugar containing molecules produced by bacteria appear to be less bioavailable than those produced by algae.
The molecular composition of neutral sugars in the deep ocean is significantly different from that observed at the end of our incubation experiments, although both reflect a high degree of bacterial degradation and transformation.Our results indicate that bacterially degraded neutral sugars have a high galactose mol % (Table 1, Fig. 6), but measurements in the deep ocean reveal a high glucose mol % (Table 3).This discrepancy has been seen in other studies as well (e.g.Amon et al., 2001) and can be due to fundamental environmental differences between the ocean interior and bioassay in-Introduction

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Full cubations and the different time scales of carbon cycling.However, the difference can possibly also be due to differences in diagenetic state of the neutral sugars in the deep ocean and in the surface waters sampled for this study.In addition, it is possible that bacterial degradation of particulate organic material (POM) plays a major role in shaping the molecular composition in the deep ocean.POM supports a major fraction of the respiratory carbon demand below the photic layer (Arístegui et al., 2002) and POM is known to have a high glucose content (Hernes et al., 1996;Panagiotopoulos and Sempéré, 2007).In our incubations, only the 10 % inoculum contained POM, and it is likely that our results mainly reflect bacterial degradation of DOM, either DOM produced by bacteria from glucose or DOM initially present in the samples, while deep ocean observations mainly reflect bacterial degradation of POM.Finally, the high glucose mol % in the deep ocean possibly reflects bacterially-produced neutral sugars with a high glucose content (Fig. 5), or other neutral sugar sources also having a high glucose mol %, e.g.submarine vent microbes (Skoog et al., 2007).In the Pacific Ocean, the glucose mol % is significantly higher than in the Atlantic Ocean (Kaiser and Benner, 2009), supporting the hypothesis that the bacterially-produced neutral sugars become more important with time in the deep ocean.However, further studies are needed to fully understand bacterial degradation of neutral sugars bound in DOM and POM and the connection between in situ measurements and incubation studies.

Bacterial degradation of amino acids
The molecular composition of amino acids did not vary considerably among samples nor change over the time course of the experiments (Table 2).The most abundant amino acid was glycine with a mol % between 26 and 40 followed by aspartic acid, glutamic acid, serine and alanine, which all typically ranged from 10 to 15 mol %.The same trends are seen in the ocean, except that the mol % of glycine is somewhat lower (15-32) and the mol % of γ-aminobutyric acid increases with depth (Table 4).A high glycine content is generally associated with highly degraded organic matter (Dauwe et al., 1999), however, the high mol % observed in our incubations are rarely seen in Introduction

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Full the ocean (Table 4).The amino acid γ-aminobutyric acid is known to increase with depth in the ocean and has therefore been used as an indicator of organic matter diagenesis (Dauwe and Middelburg, 1998;Davis et al., 2009).No consistent increase in γ-aminobutyric acid is seen during our incubations, suggesting that the increase observed in the ocean could result from long-term degradation.This hypothesis is consistent with results from a study by Davis et al. (2009) who found an increase in γ-aminobutyric acid of only 0-7 mol % during 20-33 days incubation experiments.The four measured amino acids with L-and D-enantiomers exhibited a clear degradation pattern (Fig. 7).Despite of very different D/L ratios at the beginning of the experiment, all treatments ended up having almost identical ratios after only 32 days (Table 2).The D/L ratios of amino acids in NSW samples generally remained constant or increased slightly during the incubation.In the glucose enriched samples, however, the ratio increased significantly and after 32 days all treatments had almost similar D/L ratios.This trend was especially clear for aspartic acid (except the Arctic ASW glu sample indicated on the figure) and serine, where the variability after 32 days was minimal.As an exception, Atlantic and Arctic samples seemed to have slightly different D/L ratios of glutamic acid after 32 days, with Atlantic samples being below 0.27 and Arctic samples above 0.35.Hence, the amount of D-amino acids produced relative to L-amino acids utilized was higher in Arctic samples than in Atlantic samples.Previous studies have also reported increased D/L ratios with time in degradation experiments (Amon et al., 2001;Jørgensen et al., 1999) and with depth in the ocean (Dittmar et al., 2001;Kaiser and Benner, 2008).The common endpoints in D/L ratios observed here have not previously been observed and suggest that a degradation signature exists.It appears that DOM source (Arctic DOM, Atlantic DOM and glucose) and bacterial community (Arctic vs. Atlantic) have a minor influence on D/L amino acid ratios, except for glutamic acid where bacterial community structure to some extent might influence the ratio.The fact that D/L amino acid ratios in NSW samples equals bacterially-produced D/L ratios in ASW glu samples after 32 days indicates that bacteria are the dominant source of amino acids in semi-labile and refractory DOM.Introduction

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Full The D/L ratios observed at the end of the incubation experiments were considerably higher than D/L ratios in the deep ocean (Kaiser and Benner, 2008;McCarthy et al., 1998;Pérez et al., 2003).In our experiments as well as in the ocean, the highest D/L ratio is that of aspartic acid, followed by the D/L ratio of alanine and the (almost equal) D/L ratios of glutamic acid and serine.However, the D/L ratios observed after 32 days in our incubations were about 0.1 to 0.3 units higher than in the deep ocean (Fig. 7, McCarthy et al., 1998).The time scale of the incubation experiments was much shorter than the time scale of ocean circulation, and it is possible that deep ocean amino acids exhibit a different D/L ratio due to a higher degree of diagenesis.The observed difference could also reflect the fact that the incubations included a larger fraction of DOM of bacterial origin and therefore had a higher D-amino acid content than DOM and POM in the ocean.The main sources of D-amino acids are bacterial cell wall-membrane components including peptidoglycans, lipopolysaccharides and lipopeptides (Kaiser and Benner, 2008).The ASW glu samples demonstrated that during bacterial utilization of glucose and subsequent degradation of bacterial remnants, DOM with high D/L amino acid ratios is produced.The DOM and POM available for bacterial degradation in the ocean, however, is probably derived from many different sources (Kaiser and Benner, 2008).

Implications
Bacteria play an important role in shaping the concentration and composition of neutral sugars and amino acids in the ocean.Our results show that bacteria are capable of changing the composition of biomolecules bound in DOM significantly within short time scales and that biomolecule yields can be used as indicators of DOC bioavailability.Bacterial transformation of labile DOM to refractory DOM via the microbial carbon pump has been suggested as an important production pathway of refractory DOM (Jiao et al., 2010;Ogawa et al., 2001).Results from the present incubations indicate that the microbial carbon pump also applies for neutral sugars and amino acids: the molecular Figures composition of biomolecules produced by bacteria and biomolecules remaining after degradation of bacterial remnants in ASW glu samples was strikingly similar to the composition of semi-labile or refractory biomolecules remaining in NSW samples after 32 days, suggesting that bacterially-produced biomolecules persist for long periods in the ocean.Introduction

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Full  1.Molecular composition of neutral sugars in the six different treatments during the incubations.The neutral sugars produced by bacteria in the glucose enriched samples during the first 6 days (n = 4, see Methods for explanation of the calculations) and the neutral sugars remaining after 32 days (n = 6) are given as means and standard deviations.Nd = not determined.

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Full  Full  Full Discussion Paper | Discussion Paper | Discussion Paper | During bioassay incubations (32 days) of seawater samples and parallel glucose enriched artificial seawater samples, the bacterial production and decomposition of individual neutral sugars Discussion Paper | Discussion Paper | Discussion Paper | • C: a natural seawater sample (NSW), an artificial seawater sample with 60 µM glucose C as the only carbon source (ASW glu ) and a natural seawater sample spiked with 60 µM glucose C (NSW glu ).Hence, the carbon pool consisted of either natural DOM, glucose or both.All samples were amended with inorganic N and P to have 10 µM KNO 3 and 2 µM Na 2 HPO 4 at the beginning of the incubations.N and P were added in excess to reduce the risk of nutrient limitation during the incubations.Onboard the ship, the majority of the seawater was filtered through a 0.2 µm cartridge filter (Millipore Opticap) to obtain the DOM fraction, and a small bacterial inoculum was Discussion Paper | Discussion Paper | Discussion Paper | using deep-sea water reference material made available by the Hansell CRM Program.The measured concentration of DOC in the deep-sea reference (41-43 µM) corresponded well with values from the Hansell CRM Program (41-44 µM).Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | acids generally decreased in concentration during the time course of the experiments, leading to an increased D/L ratio with time (Table Discussion Paper | Discussion Paper | Discussion Paper | 2. The amount of neutral sugars produced during the first 6 days of our incubation (104-208 nM) was within the range of concentrations of neutral sugars observed in the ocean (20-800 nM, Benner, 2002).The molecular composition was characterized by a high glucose mol % (47 ± 12) and Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Skoog, A., Vlahos, P., Rogers, K. L., and Amend, J. P.: Concentrations, distributions, and energy yields of dissolved neutral aldoses in a shallow hydrothermal vent system of Vulcano, Italy, Org.Geochem., 38, 1416-1430, doi:10.1016/j.orggeochem.2007.03.005, 2007.Stein, M.: Variability of water masses, currents and ice in Denmark Strait, Sci.Counc.Stud., 12, 71-84, 1988.Yamashita, Y. and Tanoue, E.: Distribution and alteration of amino acids in bulk DOM along a transect from bay to oceanic waters, Mar.Chem., 82, 145-160, doi:10.1016DiscussionPaper | Discussion Paper | Discussion Paper | Table Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper |

Fig. 7 .
Fig. 7. Mean amino acid D/L ratios on day 32 of the incubations.Means and standard deviations were calculated from all samples on day 32 (n = 6).Asx did not include the artificial Arctic sample which had a considerably higher D/L ratio than the rest of the samples (indicated with a star).

Table 2 .
Molecular composition (mol %) of amino acids and D/L ratios in the six different treatments during the incubations.The amino acids His, Thr, Tyr, Val, Met, Phe, Ile and Lys all have mol % below 5 and are therefore not included in this table.The amino acids provided in the table accounted for 91-98 mol %.The molecular composition and D/L ratios after 32 days are given as means and standard deviations calculated from all six treatments.The only exception is the D/L ratio of Asx which did not include ASW glu Arctic.
a Mannose and xylose were stated as one value, and for simplicity, this value has been split up into two identical mol %.Introduction
a Most sampling sites were shallower than 200 m.b Samples collected < 300 m were GF/F filtered and samples collected > 300 m were unfiltered.Introduction