Insights into estuarine benthic dissolved organic carbon (DOC) dynamics using δ13C-DOC values, phospholipid fatty acids and dissolved organic nutrient fluxes

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Geochimica et Cosmochimica Acta 75 (2011) 1889–1902 www.elsevier.com/locate/gca

Insights into estuarine benthic dissolved organic carbon (DOC) dynamics using d13C-DOC values, phospholipid fatty acids and dissolved organic nutrient fluxes Damien Maher ⇑, Bradley D. Eyre Centre for Coastal Biogeochemistry, Southern Cross University, P.O. Box 157, Lismore, NSW 2480, Australia Received 19 July 2010; accepted in revised form 4 January 2011; available online 13 January 2011

Abstract Benthic dissolved organic carbon (DOC) flux rates and changes in DOC isotope ratios, along with nutrient fluxes, phospholipid fatty acids concentration and carbon isotope ratios were measured in productive estuarine sediments over a diel cycle to determine the mechanisms driving benthic–pelagic coupling of DOC. There was uptake of DOC during the dark and efflux during the light at all sites. DOC uptake rates were related to benthic respiration (dark O2 uptake) and effluxes were coupled to the trophic status (ratio of production to respiration) of the sediments. Highest uptake and efflux rates were observed at two high nutrient concentration sites. The DOC:DON ratio of water column dissolved organic matter (DOM) decreased during the dark and increased during the light indicating preferential uptake and release of carbon rich dissolved organic matter. The calculated carbon isotope ratio of the DOC taken up by the benthos was significantly more depleted than the bulk water column DOC pool, suggesting preferential uptake of selected components of the water column DOC pool. Generally the isotope ratio of the DOC released during the light was more enriched than that taken up during the dark, which suggests that the benthos has the potential to significantly alter the estuarine DOC pool. Uptake and efflux were coupled to respiration and algal grazing/mineralization, therefore increased nutrient loading may shift the composition of the estuarine DOC pool through changes in the magnitude of benthic DOC fluxes. A combination of biological (diel shifts in DOC production and consumption) and abiotic processes (flocculation) appear to be driving the observed benthic DOC dynamics at the study sites. This study was the first to measure carbon isotopic changes in the water column DOC pool due to benthic processes, and shows that the benthos can alter the estuarine DOC pool through diel differences in DOC uptake and efflux. Ó 2011 Elsevier Ltd. All rights reserved.

1. INTRODUCTION Dissolved organic matter (DOM) is the largest reservoir of organic matter in the ocean, and is one of the largest reactive pools of carbon on earth (Schlesinger, 1991). The oceanic DOM pool is comprised of autochthonous and allochthonous material. The autochthonous pool is ultimately sourced from in situ primary production, and the allochthonous pool is derived from atmospheric and

⇑ Corresponding author. Tel.: +61 266 203577; fax: +61 266 212669. E-mail address: [email protected] (D. Maher).

0016-7037/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.gca.2011.01.007

riverine sources (Hansell and Carlson, 2002). Despite the obvious importance of the oceanic DOM reservoir, it is still largely unconstrained (del Giorgio and Duarte, 2002). As river inputs to the ocean generally pass through estuaries, these ecosystems may exert a strong control over the quality and quantity of the river DOM load delivered to the ocean. During high flows estuaries typically act purely as a conduit of DOM from the catchment to the coastal ocean (Eyre and Twigg, 1997; McKenna, 2004; Kaldy et al., 2005), however, estuaries can also modify the DOM load through removal and production processes. Quantification of these processes can be complicated due to short water residence times and the complex interactions between organic matter sources and sinks. Integration of stable

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and radio-isotope values of dissolved organic carbon (DOC), concentration data, and molecular composition of the DOC pool along estuarine gradients has shown that the estuarine DOC load can be significantly altered in terms of both concentration and composition compared to what would be expected under conservative mixing (Raymond and Bauer, 2001; Otero et al., 2003; Wang et al., 2004b). The DOC load can be changed along an estuarine gradient by various biological, physical and chemical processes. Biological sources of DOC include virus-mediated cell lysis (Fuhrman, 1999; Middelboe et al., 2003), bacterial excretion of capsular material (Stoderegger and Herndl, 1998), autotrophic excretion of DOC exudates (Staats et al., 2000; Underwood and Paterson, 2003) and grazer-mediated release (“sloppy feeding”) (Strom et al., 1997). Biological sinks include bacterial utilization (Moran and Hodson, 1990) and autotrophic uptake (Admiraal et al., 1984; Vonk et al., 2008). The DOC load can also be modified through physical and chemical processes including flocculation (Sholkovitz, 1976), photodegradation (Wetzel et al., 1995; Obernosterer and Benner, 2004), sediment resuspension (Guo and Santschi, 1997) and groundwater exchange (Santos et al., 2009) The use of stable isotopes to study estuarine DOC dynamics has lead to a paradigm shift from estuaries being seen strictly as conduits of DOC from terrestrial to marine environments, to a more recent view that these ecosystems act as reactors, transforming DOC through complex addition and removal processes. Although it is generally accepted that a portion of the riverine DOC is removed during estuarine transition, and replaced with estuarine-derived DOC, the formation and cycling of DOC in estuaries is still poorly understood (Wang et al., 2004a). In estuaries the processes linked to DOC transformations may be concentrated at the sediment surface due to high rates of autotrophic and heterotrophic production, and high rates of organic matter remineralization (Wollast, 1991). Benthic DOC fluxes have been shown to oscillate between uptake and effluxes over diel cycles (Velimirov, 1986; Ziegler and Benner, 1999; Porubsky et al., 2008), however the composition of the DOC pool exchanged between the benthos and the water column has not been studied. Under light conditions the water column DOC pool may be supplemented with autotrophic-derived DOC and during the dark, there may be uptake of DOC through bacterial consumption and/or autotrophic uptake. Autotrophic exudates typically have a high C:N ratio and are predominantly comprised of carbohydrates (Hoagland et al., 1993; Underwood and Paterson, 2003; Oakes et al., 2010a), although the C:N ratio can vary between the high molecular weight (HMW) and low molecular weight (LMW) fractions (Biddanda and Benner, 1997). Further, the C:N ratio of exuded DOM can vary significantly seasonally, for example Barro´n and Duarte (2009), found that the C:N ratio of DOM released from a Posidonia oceanica bed varied from 3 to 205 annually. Autotrophs and heterotrophs may preferentially take up nitrogen rich DOC such as amino acids particularly under nutrient limiting conditions (Thingstad et al., 1997; Veuger et al., 2007; Eyre et al., 2008). This suggests that the composition of the

estuarine DOC load may change over a diel cycle through differences in the DOC consumed and produced, even if these processes are balanced in terms of bulk flux rates. Complicating these biological pathways are physico-chemical DOC transformations (e.g. photodegradation and flocculation) which may also alter the composition of the DOC pool. We hypothesize that productive sediments in estuarine systems will alter the bulk DOC load through differences in the DOC produced and consumed, and that the magnitude of the differences will be related to primary and secondary production, and nutrient availability. To test this hypothesis we undertook a series of six benthic chamber incubations in productive sites (seagrass meadows, subtidal and intertidal shoals) in a south-east Australian warm-temperate estuary. We measured changes in concentration and carbon stable isotope ratios of DOC (d13C-DOC) and nutrient concentrations over a diel cycle. We also measured the carbon isotope ratios of seagrass, microphytobenthos (MPB), bacteria, and bulk sediment to try to determine the pathways of DOC production and consumption. 2. METHODS 2.1. Study area The study sites are located in the Camden Haven estuary, a warm temperate south-east Australian barrier micro-tidal (1 m tidal range) estuary (Fig. 1). The estuary contains significant seagrass meadows of Zostera capricorni, Halophila ovalis and Ruppia megacarpa, covering 40% of the estuary area (Eyre and Maher, 2010). Annual average rainfall in the study area is 1540 mm most of which falls during the summer, and the annual mean maximal daily temperature ranges from 17 to 26 °C (climate data available from www.bom.gov.au). A total of six sites were selected with four seagrass sites, one intertidal site and one subtidal site (Table 1). These sites were selected as they likely represent areas of high production, respiration and benthic DOC flux rates and therefore are areas most likely to influence to the bulk estuarine DOC pool. The estuary is oligotrophic with annual dissolved inorganic nitrogen (DIN) and dissolved inorganic phosphorus (DIP) concentrations of 2 and 0.6 lM respectively, and pelagic chlorophyll a concentrations generally lower than 5 mg m3. However within the estuary there are small areas with generally elevated nutrients associated with point source discharges, i.e. sites CHZ1 and CHY which received runoff from a caravan park and adjacent farm land. 2.2. Benthic flux incubations Benthic flux incubations were carried out in situ during autumn (April) 2007 using acrylic benthic chambers similar in design to those of Webb and Eyre (2004), with laminar flow provided by a diffuser system. Prior to the field trip all chambers were cleaned with weak HCl acid and soaked in Milli-Q water for a week. Triplicate chambers were placed in the sediment at each site, and left open for 24 h prior to commencement of incubations. Water within the chambers was continuously being exchanged with the

Benthic d13C-DOC fluxes

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Fig. 1. Location of the study area and sites.

water column via a submersible pump during the 24 h equilibration period. Chambers were then sealed 1 h prior to dusk. Water samples (40 mL) were collected at dusk, midnight, dawn and midday from inline sample ports using a sample rinsed polypropylene syringe, with water replaced by an inline reservoir containing site collected water. Dissolved oxygen concentrations were measured at each sample time using a Hach LDO DO meter (±0.01 mg L1) to determine community production and respiration rates.

Over the course of the incubation less than 1% of the volume was removed by sampling (chamber volume 50 L). Water samples were immediately filtered through pre-combusted (6 h, 600 °C) Whatman GFF filters into pre-combusted (6 h, 600 °C) borosilicate vials, and placed on ice until frozen (within 30 min) and kept at 20 °C until analysis. Upon completion of the incubations, sediment samples (top 1 cm) from within each chamber were collected with acrylic cores (95 mm ID) for determination of chlorophyll a and

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Table 1 Study site locations and properties. Site

Lat/long

Habitat type

Depth (m)

Sediment type

Salinity (&)

Temperature (°C)

DIN (lM)

DIP (lM)

CHZ1

31°310 2100 S, 152°480 2400 E 31°310 2000 S, 152°480 2300 E

Zostera capricorni meadow – impacted, elevated DIN Intertidal sand– impacted, elevated DIN. Heavily bioturbated by burrowing thalassinidean shrimp Trypaea australiensis Subtidal mud

0.5

33

22

7

0.6

Intertidal

Muddy sand Sand

33

22

7

0.8

1

Sandy mud

31

21

0.4

0.5

Ruppia megacarpa meadow

0.5

Mud

31

21

0.6

0.5

Zostera capricorni meadow

2

Mud

32

20

0.3

0.4

Halophila ovalis meadow

2

Mud

32

20

0.3

0.4

CHY

CHA CHR CHZ2 CHH

31°360 3900 S, 152°450 2600 E 31°360 3800 S, 152°450 2700 E 31°370 300 S, 152°450 30’’E 31°37’2’’S, 152°450 2900 E

total organic carbon (TOC) and phospholipid fatty acid (PLFA) concentrations and carbon isotope ratios. Seagrass blades were also collected for d13C analysis. 2.3. d13C-DOC analysis The d13C-DOC was analyzed using the wet oxidation method of St-Jean (2003), using a OI TOC1030W analyzer (Oakes et al., 2010b). Briefly, 5 mL of sample was introduced to a closed reaction chamber on the TOC analyzer via an autosampler. DIC was removed by acidification (5% H3PO4), and the CO2 gas was purged from the reaction chamber with helium carrier gas. 10 mL of sodium persulphate (20% w/w) was added to the reaction chamber which was then heated to 98 °C, oxidizing the DOC to CO2. The reaction time was set to 10 min. Upon completion of the oxidation reaction the evolved CO2 gas was purged with helium, and water vapor and halogens were removed by a series of inline traps and scrubbers. The gas stream was passed through a nondispersive infrared detector to obtain CO2 concentrations, and introduced to the IRMS (Thermo Delta V+) through a continuous flow interface (Conflo III). The carbon isotope ratios were calculated relative to the international standard (PDB). DOC concentrations were calibrated using a five point calibration curve with 0, 1, 2, 5 and 10 ppm C glucose solutions. All standards and reagents were made using Milli-Q water. Isotope ratios of the glucose were compared between conventional EAIRMS and the d13DOC method and were within 0.8& (n = 3). Isotope and concentration accuracy during analysis was maintained by inserting standards of known concentration and isotope value every fifth sample. 2.4. Nutrient analysis Nutrient concentrations were determined colourmetrically using a Lachate flow injection analyzer. Total dissolved nitrogen (TDN) and total dissolved phosphorus (TDP) concentrations were determined after persulphate

digestion (Valderrama, 1981). Detection limits for TDN and TDP were 0.7 and 0.16 lM, and errors (CV%) are 4.1% and 3.1%, respectively (Eyre and Ferguson, 2002). Inorganic nutrients were determined using the standard Lachat methods (Lachat, 1994) with detection limits of 0.03, 0.07 and 0.35 lM for PO4, NOx and NH4, respectively, and associated errors of 2.3%, 3.6% and 5.1%, respectively (Eyre and Ferguson, 2002). DON was determined by difference between TDN and dissolved inorganic nitrogen (DIN = NH4 + NO3 + NO2). DOP was determined by difference between TDP and PO4. Certified reference materials and check standards were run in conjunction with samples to maintain analytical accuracy. The limits of DOC, DON and DOP flux measurements were 0.7, 2.9 and 0.7 lmol m2 h1 respectively based on detection limits, sampling intervals and CV% errors. 2.5. Sediment organic carbon (OC), seagrass, bacteria and MPB isotope analysis Samples of the top 1 cm of sediment and seagrass blades were lyophilized, finely ground, and were analyzed by standard methods using a Flash EA coupled to a Thermo Delta V+ IRMS via a continuous flow interface. Linearity of instrument response was checked by inclusion of standards every fifth sample. Precision for d13C and TOC concentration were 0.1& and 0.1%, respectively. The d13C value of bacteria and MPB at each site was determined by analysis of the bacteria (i14:0, i15:0, a15:0, 10Me16:0, i17:0 and a17:0; Ratledge and Wilkinson, 1988) and algae (16:1x7, 20:5x3 and 22:6x3; Volkman et al., 1989) specific phospholipid fatty acids (PLFA) by GC-C-IRMS (Boschker et al., 1999). The d13C values of individual biomarker PLFA were averaged to estimate bacterial and algal community d13C. It should be noted that the algal PLFA pool consists of a mix of both benthic and freshly deposited phytoplankton. The average d13C value of long chain fatty acids (LCFA) (even numbered saturated fatty acids chain length C24  C30) was used as a proxy for the isotope ratio of

Benthic d13C-DOC fluxes

terrestrial plant inputs (Canuel et al., 1995; Canuel et al., 1997). Both the d13C and relative abundance (i.e. % of total identified PLFA) for individual, algal, bacteria and LCFA PLFA are presented. The CV (%) of d13C on replicate standards was 0.23& (n = 4). Benthic chlorophyll a and pheophytin concentrations were determined spectrophotometrically after extraction with 90% acetone, using the equations of Jeffrey and Welschmeyer (1997). We also collected water samples from the riverine end member for nutrient and d13C-DOC analysis three times during the field campaign, which were sampled and analyzed as above. 2.6. Flux calculations Flux calculations of DOC and O2 across the sediment water interface were determined using the formula:

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F ¼ ½ðCt1  Ct0   V =A=T where F = flux rate (lmol m2 h1), Ct1 and Ct0 are the final and initial concentrations, respectively (lM), V is the volume of the chamber, A is the surface area of sediment covered by the chamber (m2) and T is the incubation time (h). Respiration, net primary productivity, gross primary productivity and productivity:respiration ratio were determined as: Respiration ðRÞ ¼ Dark O2 flux rate Net primary production ðNPPÞ ¼ Light O2 flux rate Gross primary productivity ðGPPÞ ¼ Light O2 flux rate  Dark O2 flux rate Productivity : respiration ratio ðP=RÞ ¼ GPP  daylight hours=R  24:

Fig. 2. Diel changes in DOC concentration and d13C-DOC in the benthic chambers at each site (mean ± SE, n = 3).

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The dark period is defined as the period between dusk and dawn, and the light period is defined as the period between dawn and midday. DOC:DON and DOC:DOP ratios were calculated using the molar ratios DOC, DON and DOP of samples collected from the chambers at dusk, dawn and midday. An isotope mass balance was used to determine the d13C value of the DOC taken up or effluxed by the sediments:

d13 C  DOCflux ¼ ½ðDOCm1  d13 C  DOC1 Þ  ðDOCm2  d13 C  DOC2 Þ=ðDOCm1  DOCm2 Þ where DOCm1 and DOCm2 are the initial and final DOC concentrations respectively and d13C-DOC1 and d13CDOC2 are the initial and final d13C value of the DOC, respectively.

Fig. 3. (A) Benthic DOC flux rates. B. Benthic O2 flux rates C. Calculated benthic d13C-DOCflux values for dark and light incubations. ** represents sites with statistically significant differences between dark and light values (t-test, p < 0.05) (mean ± SE, n = 3).

Benthic d13C-DOC fluxes

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2.7. Statistical analyses A paired t-test (a = 0.05) was used to test for significant differences in bulk flux rates and d13C-DOCflux between dark and light treatments at each site. One-way ANOVA analyses was used to test for differences in fluxes between seagrass and unvegetated sediments. Linear regression was used to test for relationships between parameters. All analyses were undertaken using SPSS v 17.0. 3. RESULTS 3.1. Diel DOM dynamics All sites showed DOC uptake during the dark and DOC release during the light (Fig. 2). d13C-DOC also followed a distinct diel pattern with values becoming more enriched during the dark, and more depleted during the light. d13C-DOC values tracked changes in concentration at all sites, indicating that compositional changes in the bulk DOC pool occurred in concert with concentration changes. DOC uptake rates ranged from 800 to 3000 lmolC m2 h1, and efflux rates from 1400 to 4500 lmolC m2 h1 (Fig. 3A). DOC efflux rates during the light were higher than uptake during the dark, (paired t test, p < 0.01), and net flux rates ranged from 113 to 1632 lmol C m2 h1, indicating the benthos was a net source of DOC over a diurnal cycle. There was no significant difference between seagrass and non seagrass dark (one-way ANOVA F1,16 = 3.189, p = 0.093) and light (one-way ANOVA F1,16 = 0.439, p = 0.517) DOC fluxes indicating that fluxes were not influenced by the presence of seagrasses. Respiration (R) rates ranged from 3000 to 12000 lmol O2 m2 h1 across all sites, and net primary productivity (NPP) ranged from an uptake of O2 at sites CHZ1 and CHY sites which were net heterotrophic during the day (4000 and 6000 lmol O2 m2 h1, respectively), to an O2 release of 10000 lmol O2 m2 h1 at site CHZ2 (Fig. 3B). Net O2 fluxes indicated that all sites with the exception of CHZ2 were heterotrophic. DON and DOP fluxes were more variable within and between sites (Fig. 4A and B), and the diel pattern observed for DOC fluxes was not observed across all sites. Dark fluxes of DON ranged from an uptake of 100 lmol N m2 h1 (sites CHA, CHH) to an efflux of 190 lmol N m2 h1 (CHZ2). Light DON fluxes ranged from an uptake of 360 lmol N m2 h1 at the two Z. capricorni meadow sites to an efflux of 300 lmol N m2 h1 at the two non-seagrass sites (CHY, CHA). DON fluxes at site CHR were not significantly different from zero. Dark DOP fluxes were either not significantly different from zero (CHY, CHR and CHZ2) or uptakes. Light DOP fluxes from not significantly different to zero (CHY, CHA, CHR) to an efflux of 10 lmol P m2 h1 (CHH). With the exception of site CHA, all sites exhibited a decrease in the DOC:DON ratio of the water column DOM over the dark period (Fig. 4A). The largest decreases were observed at the high nutrient sites (CHZ1 and CHY). During the light period, DOC:DON ratios increased at all sites except CHY and CHA. Midday and dusk DOC:DON

Fig. 4. (A) DON fluxes; (B) DOP fluxes and (C) Diel changes in water column DOC:DON ratios. D. Diel changes in water column DOC:DOP ratios (mean ± SE, n = 3). ns, Flux not significantly different from zero, **, significant difference between dark and light fluxes, a, significant different between dusk and dawn, b, significant difference between dawn and midday (t-test p < 0.05).

ratios were similar at all sites except CHY. DOC:DOP ratios exhibited a similar diel pattern as DOC:DON ratios, with a decrease during the dark, and increase during the light (Fig. 4B).

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3.2. PLFA, d13C-DOCflux and pigments A total of 25 individual PLFAs were identified, ranging in length from C12:0 to C30:0 (Table 2). C16:0 was the dominant PLFA in all samples contributing 24–30% of the total identified PLFA abundance. Bacterial PLFAs contributed 15–23% of the total identified PLFA, and algal and LCFA contributed 3–10% and 2–6%, respectively. Algal PLFAs were generally more enriched in d13C than LCFA although due to the high variation between the values for individual PLFAs the difference was only statistically significant at the

two non-seagrass sites CHA and CHY (t test, p < 0.01). Bacterial PLFA and sediment OC d13C values were more enriched than the algal PLFA d13C values at each site and LCFA were the most depleted organic matter source with values from 26& to 31& (Table 3). Water column d13C-DOC was more depleted at the two high nutrient sites (CHZ1 and CHY), as were sediment OC and seagrass d13C values. LCFA d13C values were generally depleted by 10& compared to sediment OC values. Highest chlorophyll a (30 mg m2) and pheophytin (20 mg m2) concentrations were found at the high nutrient sites CHZ1

Table 2 d13C values of DOC flux, organic matter sources and microbial community (mean ± SE). Note PLFA samples represent pooled samples from triplicate chambers. Site

Dark d13C-DOCflux

Light d13C-DOCflux

Water Column d13C-DOC

Seagrass d13C

Sediment OC d13C

Sediment OC (%)

Chlorophyll a (mg m2)

Pheophytin (mg m2)

CHZ1 CHY CHA CHR CHZ2 CHH

27.38 27.26 29.17 25.03 32.12 19.70

22.16 27.78 19.55 24.80 24.26 18.00

20.78 19.18 13.35 13.42 15.54 14.51

19.12 (0.31)

20.46 21.42 15.81 16.00 16.40 15.55

1.82 1.96 2.35 2.05 3.11 4.33

32.30 (5.52) 29.17 (2.98) 15.34 (0.81) 14.72 (0.07) 7.79 (1.48) 14.93 (3.08)

22.41 (3.12) 15.06 (2.01) 8.12 (0.68) 10.47 (0.76) 8.88 (1.71) 13.72 (3.16)

(0.78) (0.34) (0.14) (0.18) (1.04) (1.26)

(0.95) (0.66) (0.39) (0.58) (0.73) (0.81)

(0.08) (0.34) (0.25) (0.18) (0.53) (0.34)

15.68 (0.51) 12.98 (0.13) 13.20 (0.59)

(0.27) (0.11) (0.10) (0.05) (0.34) (0.09)

(0.14) (0.23) (0.23) (0.07) (0.55) (0.29)

Table 3 Relative abundance (% of total) and d13C values for individual PLFA. bd – below detection limits. d13C values for RAlgal and RLCFA PLFA are mean ± SD. PLFA

CHZ1 13

12:0 i14:0 14:0 i&a15:0 15:0 i16:0 16:3 16:1w7 16:1w5 16:0 10Me16:0 i&a17:0 17:1 17:0 18:2/18:1 18:0 20:5w3 20:0 22:6w3 22:0 24:0 26:0 28:0 30:0 RBacteria PLFA (i14:0, i15:0, a15:0, i16:0, 10Me16:0, i17:0, a17:0) RAlgal PLFA (16:1w7, 20:5w3, 22:6w3) RLCFA PLFA (P24:0)

CHY 13

CHA 13

CHR 13

CHZ2 13

CHH

% (d C)

% (d C)

% (d C)

% (d C)

% (d C)

% (d13C)

0.1 (bd) 1.4 (20.4) 4.1 (23.1) 13.9 (21.5) 3.1 (21.0) 0.9 (21.8) 0.9 (19.9) 2.7 (24.6) 10.8 (24.1) 23.7 (23.8) 3.8 (25.9) 3.2 (23.5) 1.9 (24.0) 2.1 (22.1) 16.1 (24.7) 4.9 (27.5) 0.5 (30.5) bd 1.6 (30.3) 0.9 (28.2) bd 1.3 (30.3) 1.5 (31.9) 0.7 (31.3) 23.2 (22.6 ± 2.1) 4.8 (28.4 ± 3.4) 3.5 (31.2 ± 0.8)

0.2 (bd) 0.8 (24.8) 7.9 (23.5) 7.1 (21.3) 9.8 (16.4) 1.1 (27.5) 1.0 (23.2) bd 14.0 (23.1) 30.7 (21.6) 2.4 (26.5) 3.5 (20.4) 1.4 (27.2) 1.8 (21.8) 10.5 (25.6) 3.0 (27.8) 0.9 (22.5) bd 1.6 (22.7) 0.6 (33.8) bd 1.1 (31.3) 0.4 (29.9) 0.4 (31.7) 14.8 (25.4 ± 3.3) 2.9 (22.6 ± 0.1) 1.9 (31.0 ± 0.9)

0.2 (bd) 1.0 (20.2) 4.1 (22.0) 8.8 (20.3) 2.4 (19.6) 0.7 (20.7) 0.6 (19.5) 2.0 (20.2) 8.2 (23.8) 24.7 (21.5) 3.9 (25.4) 3.9 (22.5) 1.4 (23.3) 2.7 (22.2) 16.5 (23.2) 4.8 (24.1) 5.3 (22.8) 1.4 (25.0) 3.2 (21.2) 1.1 (28.7) bd 2.5 (27.2) bd 1.7 (28.7) 18.2 (21.6 ± 2.5) 10.5 (21.4 ± 1.3) 3.4 (27.9 ± 1.0)

0.2 (bd) 1.1 (20.1) 4.8 (22.2) 9.4 (21.3) 3.3 (18.9) 0.7 (22.7) 0.7 (20.6) 3.0 (24.4) 10.7 (22.8) 26.2 (21.7) 7.2 (26.1) 2.3 (23.0) 1.3 (24.0) 2.6 (23.4) 12.9 (24.5) 4.0 (25.7) 3.4 (26.2) 0.5 (26.3) 0.5 (22.4) 0.3 (29.1) 0.6 (30.5) 1.9 (29.5) bd 2.6 (30.4) 20.6 (22.2 ± 2.7) 6.9 (24.3 ± 1.9) 5.0 (30.1 ± 0.6)

0.2 (bd) 1.3 (19.5) 4.2 (22.6) 11.2 21.1) 2.4 (21.9) bd 0.7 (21.4) 3.2 (24.2) 9.0 (25.0) 27.6 (24.6) 3.6 (24.1) 2.0 (21.1) 0.9 (18.5) 1.4 (18.2) 14.1 (24.2) 5.2 (26.5) 4.1 (29.2) 0.6 (28.3) 1.8 (22.6) 0.4 (26.7) 0.5 (32.8) 0.6 (29.6) 3.1 (29.5) 2.0 (27.5) 18.2 (21.2 ± 2.1) 9.1 (25.3 ± 3.4) 6.1 (29.9 ± 2.1)

bd 1.6 (18.2) 4.4 (21.1) 12.9 (21.0) 3.0 (20.7) 0.8 (23.9) 0.4 (19.7) 3.1 (19.9) 7.2 (22.9) 25.1 (22.0) 4.2 (25.9) 2.7 (23.2) 1.2 (25.1) 2.5 (23.9) 12.9 (23.3) 4.6 (25.1) 4.2 (25.6) 1.5 (24.6) 2.5 (23.7) 1.0 (26.1) bd 0.9 (26.0) bd 3.3 (26.7) 22.2 (22.0 ± 3.4) 9.8 (23.1 ± 2.9) 4.2 (26.3 ± 0.5)

Benthic d13C-DOC fluxes

and CHY which were generally twice as high as the other sites (Table 3). The d13C-DOC value of the riverine end member during the study was 29.39 ± 1.9& (n = 3). d13C-DOCflux values differed between the dark and light incubations at most sites (Fig. 3C). The value of the d13CDOCflux during the dark was similar to, or more depleted than light d13C-DOC flux values. With the exception of site CHH, dark d13C-DOCflux values ranged from 25& to 32&. Site CHH had a more enriched dark d13C-DOCflux than the other sites of 19&. These values are considerably more depleted than the bulk water-column DOC pool which ranges from 20& to 13& (Table 2). Light d13CDOCflux values ranged from 18& at site CHH to 27& at site CHY. Significant differences (t test, p < 0.001) between light and dark d13C-DOCflux values were found at sites CHZ1, CHA and CHZ2. 4. DISCUSSION 4.1. Experimental design Benthic chambers have been used extensively to measure DOC fluxes (e.g. Burdige and Homstead, 1994; Ziegler and Benner, 1999; Barro´n et al., 2004; Maher and Eyre, 2010), however this is the first study to measure changes in d13CDOC associated with benthic DOC fluxes. Therefore, it is appropriate to discuss the caveats associated with the experimental design, and how these may influence the interpretation of the results. Firstly, we used limited sampling intervals to estimate the fluxes of oxygen, DOM and d13C-DOC and assumed that these rates were representative of the dark and light periods. Our measured rates during the dark encompass the full dark cycle (i.e. from dusk to dawn) and therefore should represent the net change during this period. In contrast, the light samples were only collected from dawn and midday and our assumption is that the first half of the light cycle is representative of the full light cycle. This methodology was employed to minimize the potential errors that may be introduced by nutrient limitation during the light and potential underestimation of metabolic rates due to bubble formation within the chambers. We assume that the changes in DOC concentration and d13C value are associated with benthic fluxes of DOC which have a distinct d13C value. There may however be preferential consumption/release of isotopically distinct DOC components within the benthic boundary layer. The magnitude of these changes is likely to be linked to phytoplankton and bacterioplankton production rates which annually are only 30% of benthic production and respiration in the Camden Haven system (Eyre and Maher, 2010), and would be significantly lower within the benthic chambers due to lower light availability at the benthos. Alteration of the DOC pool can also occur through photochemical reactions which mineralize DOC to DIC, and also fractionate the d13C value of the remaining DOC (Va¨ha¨talo and Wetzel, 2008). Algal and terrestrial derived DOC has been found to be resistant to photochemical alteration over the short time scales relevant to the light incubation period during this study (Thomas and Lara, 1995; Obernosterer and Benner, 2004), further,

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the DOC concentration increased during the light indicating that DOC production, rather than mineralization was the dominant process. Therefore we have assumed that photochemical transformations of the DOC pool were negligible. 4.2. DOM fluxes This study presents the first attempt at quantifying the changes in the bulk DOC pool due to benthic processes using changes in the natural abundance of d13DOC during benthic flux incubations. DOC was taken up by the benthic communities during the dark, and released during the light, at all sites. This diel pattern has been reported previously for seagrass meadows (Velimirov, 1986; Ziegler and Benner, 1999; Maher and Eyre, 2010) and intertidal shoals (Porubsky et al., 2008; Maher and Eyre, 2010) and indicates that DOC is produced in excess of demand within the sediments during the light and that during the dark there is a benthic DOC deficit, and some of the benthic carbon demand is supplied by the water column. Complicating this supply versus demand view of benthic DOC cycling are physico-chemical processes such as flocculation, along with the composition and lability of the DOC pool. The light DOC efflux may be due to DOC exuded from autotrophs, DOC leached from deposited particulate matter or DOC release through grazing of particulate organic matter. Daytime DOC effluxes have been found to be positively correlated with NPP in seagrass beds (Ziegler and Benner, 1999) and algal biofilms (Ziegler et al., 2009) due to exudation of DOC by autotrophs. However in this study we found a significant (R2 = 0.410, n = 18, p < 0.01) negative correlation between NPP and light DOC fluxes (Fig. 5A). Light DOC fluxes were also negatively correlated to the ratio of production to respiration (P/R ratio) of the benthic community (R2 = 0.423, n = 18, p < 0.01, Fig. 5B). Therefore, heterotrophic, rather than autotrophic processes appear to be driving the light DOC flux during this study. DOC exudation by autotrophs under light conditions may have been rapidly utilized by the bacterial community (Cook et al., 2007; Ziegler et al., 2009), satisfying the benthic microbial C demand. Light-enhanced increases in respiration rates have been linked to increases in oxygen penetration depths in benthic phototrophic communities (Epping and Jørgensen, 1996), therefore increasing DOC release as the sediments become more heterotrophic may be driven by aerobic degradation of sediment organic matter to DOC. Barro´n et al. (2004) found that DOC fluxes from seagrass meadows increased with community respiration, and hypothesized that this was due to allochthonous organic matter inputs. Light DOC efflux during our study may have been due to hydrolysis of a combination of deposited estuarine particulate organic matter, phytodetritus and benthic microalgae. This is evident from the positive relationship between light DOC flux and benthic chlorophyll a (Fig. 5C) and pheophytin (Fig. 5D) concentrations, as well as from the relationship between the light d13DOCflux and d13C of the LCFA (see later discussion). Chlorophyll a and pheophytin concentrations represent fresh and degraded algal material respectively, and the

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Fig. 5. (A) Light DOC flux versus light O2 flux. (B) Light DOC flux versus P/R ratio. (C) Light DOC flux versus benthic chlorophyll a. (D) Dark DOC flux versus dark O2 flux for individual chamber incubations. (E) Dark DOC flux versus benthic pheophytin concentration. (F). Light d13C-DOCflux values versus LCFA d13C values (mean ± SE, n = 3).

LCFA is a measure of terrestrial organic matter. During the dark there may be a benthic C deficit due to reduced exudation by autotrophs, leading to DOC uptake (see below). Unraveling the mechanisms driving dark DOC uptake is more complicated. The decrease in DOC:DON and DOC:DOP ratios during the dark indicate a preferential uptake of carbon rich organic matter. DOC:DIN and DOC:DIP ratios also decreased during the dark (data not shown). Bacteria (Bauerfeind, 1985; Veuger et al., 2007), seagrass

(Vonk et al., 2008) and microalgae (Admiraal et al., 1984; Linares and Sundba¨ck, 2006; Cook et al., 2009) have been shown to actively take up DON substrates. The uptake of carbon rich organic matter, particularly in an oligotrophic system, seems somewhat counterintuitive. The preferential uptake of carbon-rich dissolved organic matter during the night could be associated with a biological uptake of carbohydrates. Carbohydrates not only constitute a large proportion of the estuarine DOC pool (Wang et al., 2009), but can

Benthic d13C-DOC fluxes

be a dominant substrate for benthic bacterial metabolism (Fischer et al., 2002). Benthic N and P demand during the dark may have been met by DIN and DIP produced by remineralization of organic matter and/or by diffusive supply from the pore waters, which typically have highly elevated concentrations of DIN and DON, particularly in seagrass sediments (Lee and Dunton, 1999; McGlathery et al., 2001). There was a significant positive relationship between dark O2 and DOC uptake (with two outliers excluded in Fig. 5E). This suggests a coupling between R and DOC uptake. Assuming all DOC uptake was associated with biological demand, and all oxygen uptake was associated with community R, and that there was a respiratory quotient (RQ) of 1, the water column supplied 20% of the total benthic carbon demand through DOC uptake during the dark (i.e. slope of regression; Fig. 5E). The remaining carbon demands of the benthic community would be met by sediment organic matter. The efflux of DOC from the sediment to the water column during the light, and uptake during the dark, highlights the importance of benthic–pelagic coupling in the estuarine DOC cycle. The few studies on benthic DOC fluxes have focused on either autotrophic production during the light, and its control over bacterioplankton production (e.g. Ziegler and Benner, 1999), or the role of the benthos as a DOC source to the water column (e.g. Burdige and Homstead, 1994; Alperin et al., 1999; Burdige et al., 1999), as such, the mechanisms driving dark DOC uptake require further study, as this may be a potential sink for organic carbon within aquatic systems. 4.3. d13C-DOC fluxes The d13C-DOC value of the water column represents the mixture of various autochthonous and allochthonous sources of DOC to the estuary. Terrestrial derived organic matter sourced from C3 plants range from 30& to 26& (Peterson and Fry, 1987). Salt marshes (which have a more enriched d13C value of 13 to 16& due to C4 metabolism; Smith and Epstein, 1971) may export DOC (e.g. Peterson et al., 1994) or remineralize DOC within the marsh complex itself (Dame et al., 1991), however these habitats only cover a very small percentage (0.1%) of the catchment area. Therefore terrestrial inputs of DOC would be expected to have a d13C value similar to that of the C3 plants, i.e. 30&. This is similar to the value of the freshwater d13C-DOC value measured during the study (i.e. 29.39 ± 1.9&). This value is significantly more depleted than the water column d13DOC values which are similar to the seagrass and algal d13C values (Table 3), indicating that the bulk estuarine DOC pool is composed of predominantly autochthonous DOC. Differences between the dark and light d13C-DOCflux values (Fig. 3C) and diel variation in flux direction (Fig. 3A), indicate that the pool of DOC consumed by the benthos differs from that produced. It also suggests preferential uptake of selected fractions of the bulk DOC pool during the dark. The benthic uptake of isotopically depleted DOC during the dark and release of isotopically enriched DOC during the light has not previously been

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documented. We expected to see little discrimination in the pool of DOC taken up by the sediments. The benthic DOC fluxes appear to be related to a combination of biological and/or abiotic processes. Two hypothesis are presented to explain the benthic DOC dynamics at the study site: (1) temporal variability in microbial substrate production and consumption, and (2) diel shift between biological production (during the light), and abiotic flocculation of DOC during the dark. It is likely that a combination of these factors drives the DOC fluxes at the study sites. The generally depleted value of the dark d13C-DOCflux relative to the bulk water column DOC, suggests that either there is preferential uptake of depleted compounds of estuarine origin, or, uptake of DOC derived from terrestrial sources. Despite the relationship between dark DOC flux rates and R (Fig. 5D), the isotope ratio of bacterial PLFA and the dark d13C-DOCflux values were not correlated (R2 = 0.0001, n = 6). Bacteria and algae d13C were significantly correlated (R2 = 0.7086, p = 0.036, n = 6), indicating close coupling between bacteria and algae. However, it is important to note that PLFA samples were collected at the completion of the light incubation, and therefore are likely to represent the carbon source of bacteria during the light. Bacteria may have utilized selected components of the water column DOC during the dark, and algal derived DOC during the day. DOC exudation by benthic algae has been found to be light dependant and driven by oxygenic photosynthesis (Staats et al., 2000), and benthic bacterial production within seagrass communities has been found to show diurnal variation, with production rates up to ten fold higher during the day (Moriarty and Pollard, 1982). Recent evidence indicates that coastal communities of bacteria are capable of metabolizing a wide range of substrates, which is believed to be associated with heterogeneity in DOC supply (Mou et al., 2008). This suggests that benthic bacteria should be able to efficiently switch DOC substrates depending upon supply. To our knowledge, no studies have looked at changes in benthic bacterial DOC substrate utilization between day and night. Uptake of 13C-depleted organic matter relative to the bulk organic matter pool has been reported for bacterioplankton in the Mississippi River plume (Kelly and Coffin, 1998). The primary carbon source for bacteria in this system may have been from depleted sources linked to methane oxidation, nitrification, hydrocarbon seeps or selective uptake of autochthonous lipids. Alternatively, uptake of 13 C-depleted DOC may be related to terrestrial-derived organic matter. The evidence supporting heterotrophic reliance on terrestrial derived DOC in aquatic systems is equivocal, and may be linked to trophic status. For example, McCallister and del Giorgio (2008) found that terrestrial derived organic carbon was the main substrate for bacterial respiration in oligotrophic systems, and autochthonous organic matter becomes more important under eutrophic conditions. Further, the diel dynamics of autochthonous DOC production may lead to increased reliance upon allochthonous DOC during the dark (see earlier discussion). There was no relationship between bacterial d13C values and dark d13C-DOCflux (p > 0.05). This may be associated

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with a diel shift in substrate metabolism (see earlier discussion) and/or a combination of abiotic and biotic processes driving dark uptake of DOC. Low pH has been found to favor aggregation of humic substances (Maignan, 1983), and pH dropped during the night at all sites due to respiration, and subsequent increases in dissolved CO2 concentration. While the positive relationship between dark DOC and O2 uptake (Fig. 5D) supports the hypothesis of biological uptake it may also reflect pH induced changes in flocculation rates associated with benthic R. Vascular plants have been found to be the dominant source of humic substances to the coastal ocean (Moran and Hodson, 1990), and the contribution of vascular plants to the humic substance pool is likely to be even greater in estuaries considering it has been found that up to 80% of humic acids are removed during estuarine mixing (Sholkovitz et al., 1978). Estuarine humic substances have been found to have a significantly more depleted d13C value than the bulk DOC pool (Otero et al., 2003), further indicating a terrestrial source. It would be expected that the humic substance pool in the Camden Haven estuary would have a typically terrestrial d13C value, similar to the calculated dark d13C-DOCflux values found in this study. Therefore, abiotic aggregation and flocculation of humic substances under decreasing pH would explain both the relatively depleted value of the dark d13C-DOCflux (compared to the bulk water column DOC pool) and the lack of correlation between the benthic microbial d13C values and the dark d13C-DOCflux value. There was a positive relationship between pH and DOC concentration (R2 = 0.305, p < 0.001, n = 72), however; pH was also positively correlated to O2 (R2 = 0.718, p < 0.01, n = 72) and DOC and O2 were also positively correlated (R2 = 0.449, p < 0.01, n = 72), making it difficult to determine if pH decreases were driving DOC flocculation, or whether pH just covaried with DOC concentration as a result of respiration and production. The mechanisms driving dark DOC uptake in estuarine sediments are clearly an area for further research. During the light, the value of the d13C-DOCflux was most closely related to the d13C value of LCFA isolated from the sediments (Fig. 5F). The fractionation factor between the isotope ratio of the light DOC flux and LCFA is 4–6& which is similar to reported values for fractionation associated with lipid synthesis (Boschker et al., 1999; Jones et al., 2003). This relationship suggests that the light DOC flux is coupled to the release of organic matter derived from early diagenesis of higher plants (terrestrial and seagrass) rather than MPB exudates. The light d13C-DOCflux values are also depleted by 2–8& compared to bulk sediment d13C (Table 3) which indicates that a depleted fraction of the sediment OM pool is released to the water column. 4.4. Influence of nutrient availability on DOC fluxes Light efflux rates of DOC were inversely related to NPP and P/R ratios (Fig. 5A and B), and positively correlated to chlorophyll a and pheophytin concentrations (Fig. 5C and D). Dark DOC fluxes where positively related to benthic respiration (Fig. 5E) and pheophytin concentrations (R2 = 0.558, p < 0.001, n = 18). Therefore, under increasing

nutrient supply the associated increase in algae grazing/ mineralization and benthic respiration will increase both dark uptake and light efflux rates. The increase in dark uptake balances the increased light efflux, leading to no significant increase in net flux rates with increasing respiration (R2 = 0.009, p > 0.05, n = 18). However, the d13C values of the DOC taken up and DOC effluxed suggest that there are differences in these pools of DOC. We would therefore expect that the composition of the estuarine DOC pool would change significantly between estuaries of differing trophic status. DOC composition may also vary with estuarine geomorphology, climatic conditions, freshwater input and seasonally. To our knowledge there has been no comparative work done on how the composition of the estuarine DOC pool changes with increasing eutrophication or across a range of estuarine conditions. River inputs of DOM to the ocean are a significant term in the global carbon budget, and is believed to support oceanic respiration in less productive regions of the ocean (Duarte and Regaudie-de-Gioux, 2009). Benthic processes may contribute to changes in the concentration and composition of DOC as it is transported through estuaries and therefore has implications for the global carbon budget, particularly if the reactivity of the DOC pool is altered. 5. SUMMARY This study provides evidence that productive estuarine habitats alter the riverine DOC pool through diel shifts in benthic uptake and efflux, which differ in source, and, most likely, composition. While the exact mechanisms driving the diel shifts in benthic flux direction and composition are not entirely clear, it appears that a combination of biological (diel shifts in DOC production and consumption) and abiotic processes (flocculation) are driving the observed benthic DOC dynamics at the study sites. The present study’s findings are similar to previous studies that have found that estuaries alter the DOC pool through simultaneous production and removal processes. Further research into both the composition and reactivity of the estuarine DOC pool, and its relationship to benthic–pelagic coupling are clearly required to further constrain the global carbon budget. ACKNOWLEDGEMENTS We thank P. Squire, M. Bautista, J. Oakes, T. Browne and D. Erler for assistance with fieldwork, and I. Alexander for nutrient analysis. B. Jones is gratefully acknowledged for his assistance with stable isotope and PLFA analysis. I. Santos provided useful comments on the manuscript. We thank the AE and three anonymous reviewers for their comments which improved this manuscript significantly. This study was funded by Port Macquarie Hastings Council and an Australian Postgraduate Award (APA) to DM, and ARC Discovery (DP0342956) and ARC Linkage (LP0212073) grants awarded to BE.

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