Production of Dissolved and Particulate Organic Matter by the Reef ...

BULLETIN OF MARINE SCIENCE, 82(2): 237–245, 2008

CORAL REEF PAPER

Production of dissolved and particulate organic matter by the reef-building corals Porites cylindrica and Acropora pulchra Yasuaki Tanaka, Toshihiro Miyajima, Isao Koike, Takeshi Hayashibara, and Hiroshi Ogawa Abstract

Coral colonies are one of the major producers of dissolved and particulate organic matter (DOM and POM, respectively) in coral reefs. To investigate the net release rates of DOM and POM and the production ratios (DOM:POM), the reef-building corals Porites cylindrica (Dana, 1846) and Acropora pulchra (Brook, 1891) were incubated separately and accumulation rates of DOM and POM in the overlaying seawater were simultaneously measured over 4 d. Release rates of the newly synthesized organic matter by symbiotic algal photosynthesis were also measured using a 13C-labeling technique. Dissolved organic carbon (DOC) was produced at rates of 340 and 380 nmol C cm−2 d−1 for P. cylindrica and A. pulchra, respectively, and particulate organic carbon (POC) was produced at 590 and 740 nmol C cm−2 d−1, respectively. POC was produced at a significantly higher rate than DOC for both coral species: the average ratios were 0.6 and 0.5 for P. cylindrica and A. pulchra, respectively. Particulate organic nitrogen was also produced at a significantly higher rate than dissolved organic nitrogen. These results indicate that reef-building corals produce more POM than DOM at least over the time scale of a few days. Newlysynthesized organic carbon accounted for < 10% of the accumulated bulk DOC, suggesting that most of the released DOC was derived from previously-synthesized organic carbon.

Coral reefs are regions of high gross primary productivity in spite of low nutrient concentration in the water column (Odum and Odum, 1955). This high productivity has been explained by the tight recycling of inorganic nutrients within the reef (van Duyl and Gast, 2001), with the net production of organic matter typically being small due to respiratory consumption (Kinsey, 1985). On the other hand, some direct measurements of organic matter concentration have suggested that both dissolved (DOM) and particulate organic matter (POM) are exported from reefs to the offshore (Charpy and Charpy-Roubaud, 1991; Delesalle et al., 1998; Hata et al., 1998, 2002). Sources for DOM and POM in reef waters are varied (van Duyl and Gast, 2001). In particular, corals release 6%−40% of the photosynthetically fixed carbon (C) by the symbiotic algae (zooxanthellae) as mucus and DOM to the ambient seawater (Crossland et al., 1980a; Muscatine et al., 1984; Crossland, 1987; Ferrier-Pagès et al., 1998). High contribution of coral mucus to the bulk POM is also suggested from the observation of low C to nitrogen (N) ratios of the POM (Hata et al., 2002). Although a number of previous studies have focused on the chemical composition (i.e., proteins, carbohydrates, and lipids) of the released organic matter (Krupp, 1984; Meikle et al., 1988; Vacelet and Thomassin, 1991), sizes of the organic matter have rarely been investigated, thus there are few comparisons of the production rates between DOM and POM. The size of organic matter strongly affects its subsequent pathways. For example, DOM in reef waters is mainly taken up by heterotrophic bacteria (van Duyl and Gast, Bulletin of Marine Science

© 2008 Rosenstiel School of Marine and Atmospheric Science of the University of Miami

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2001) and incorporated into the microbial food web, while the larger POM is utilized by reef fishes (Benson and Muscatine, 1974), soft corals (Fabricius and Dommisse, 2000), and partially sinks down and is mineralized in the reef sediment (Wild et al., 2005). To understand the biological availability and subsequent C and N pathways, information concerning the sizes of organic matter produced by reef-building corals is necessary. With regard to carbon cycling by the coral colony, it is also important to measure the rates at which newly synthesized organic matter in the coral colony is released into the ambient seawater. It has been shown using 14C that more than half of photosynthetically-fixed C can be lost from the coral colony within a few days (Cooksey and Cooksey, 1972; Crossland et al., 1980b), which was ascribed to the host and algal respiration and organic matter release. To clarify whether the loss of the new photosynthetic products is due to respiration or organic matter release, the release rate of the newly synthesized organic C into the seawater needs to be measured directly. The purpose of the present study was to investigate the net production rates of DOM and POM by reef-building corals and the production ratio (DOM:POM) for both C and N, and to ascertain the rate of release of newly synthesized organic C by the symbiotic algae as DOC. Two species of reef-building corals were incubated separately in a closed system with 13C-labeled dissolved inorganic C (DIC), and the accumulation rates of DOM, POM, and 13C-labeled DOC in the incubated seawater were simultaneously measured over 4 d. Materials and Methods Collection and Preparation of Corals.—The experiment was performed using the zooxanthellate corals Porites cylindrica (Dana, 1846) and Acropora pulchra (Brook, 1891), which were collected on a reef flat of Shiraho Reef at Ishigaki Island (24°21´−31´N, 124°4´−16´E), Japan in August 2004. Both species were collected at a water depth of about 1 m at low tide. Branches of approximately 8 cm were cut and maintained for several days before each experiment in an outdoor aquarium, which was continuously supplied with seawater pumped from the field. In total, six bottles were prepared for the two coral species, i.e., three coral branches and three transparent polycarbonate bottles (inner volume 10 l) per species. Each bottle had 8.0 L of 0.2 µm-filtered natural seawater that contained 0.06−0.07 µmol L−1 NO3−, 0.01−0.03 µmol L−1 NO2−, 0.7−1.1 µmol L−1 NH4+, and 0.005−0.02 µmol L−1 PO43−. One coral branch having a surface area of 103−128 cm2 was suspended in each bottle using a nylon line, taking care not to hang the coral upside down, and the seawater in the bottle was agitated by a magnetic stirrer positioned at the bottom. At the beginning of the experiment, NaH13CO3 was added to the seawater to follow newly synthesized organic C. The final concentration of the added 13 C was 0.2 mmol L−1, representing 10 atom % of 13C. Incubation was carried out for 4 d under natural sunlight conditions at a temperature of 28.7 ± 0.8 °C (mean ± SD, range 27.2−30.6 °C), which was maintained by running seawater outside the bottles. Subsamples (350 ml) of the incubation seawater were collected once a day at 1400 hrs. Each sample was immediately filtered by passage through a pre-combusted Whatman GF/F filter (pore size 0.7 µm) using a hand-operated vacuum pump (pressure < 0.02 MPa). A part of the filtrate was transferred into pre-combusted glass ampoules (20 ml), which were quickly sealed and frozen at −20 °C until analysis of DOM and inorganic nutrients. The remaining filtrate was also frozen in an acid-washed polypropylene bottle for determining 13C isotope enrichment of DOC. Enough POM to detect the amount was not collected on the GF/F filter by sampling

TANAKA ET AL.: ORGANIC MATTER RELEASE FROM CORALS

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seawater of 350 ml. Instead, subsamples for determining POM release rates from the corals were collected at the end of the 4 d experiment by filtering all of the remaining incubation seawater using Whatman GF/F filters. The filters were frozen at −20 °C until analysis. Subsamples for 13C isotope enrichment of DIC were taken everyday in glass vials and im�� mediately fixed with saturated HgCl2 (final concentration 0.4%, w/v). Subsamples for bacterial abundance were fixed with pre-filtered formalin in acrylic tubes (final concentration 2%, v/v). After incubation, the coral skeletons were stored at −20 °C for determining the coral surface area using the aluminum foil method (Marsh, 1970). Analyses.—The concentrations of DOC and total dissolved N (TDN) were measured by the high temperature catalytic oxidation method (HTCO), using TOC 5000 (Shimadzu) and ECL-880 US (Yanaco) (Ogawa et al., 1999). DON concentrations were calculated by subtraction of total dissolved inorganic N (DIN) from TDN. DIN (NO3−, NO2−, NH4+) was quantified with a nutrient analyzer AACS-III (BRAN+LUEBBE). 13 C isotope enrichment of DIC was determined by gas chromatography/isotope ratio mass spectrometer (GC-IRMS) as previously described (Miyajima et al., 1995) using GC-6890 (Agilent Technology) and DELTA plus XP (Finnigan). 13C isotope enrichment of DOC was measured by combination of wet oxidation and GC-IRMS. Briefly, 3 ml of 3.3% K2S2O8 (w/w) and 200 µl of 30% H3PO4 (v/v) were added to a 20 ml glass ampoule containing 15 ml of sample seawater. Then, DIC in the seawater was removed by bubbling N2 gas at 200 ml min−1 for 10 min. After immediately sealing the ampoule under N2 purge, it was autoclaved at 121 °C for 60 min. DOC in the seawater was oxidized to CO2 with this procedure. CO2 in the headspace of the ampoule was injected with a gas-tight syringe into the GC-IRMS. The analytical precision of this method is < 1‰ δ13C (relative to PDB), which is sufficiently precise to measure the isotope ratios of 13C-tracer samples. However, this technique causes some chloride ions to be oxidized into gaseous chlorine, which may be harmful to the analytical instruments. The removal of chlorine should be resolved in the future. GF/F filters for POM analysis were dried in an oven at 50 °C for 3 hrs, and then treated with the vapor of 12N HCl for 12 hrs to remove inorganic C. After evaporating extra HCl on a hot plate (80 °C) under vacuum, the filters were dried again for 2 hrs in the oven at 50 °C. Quantification of POM was determined by a CHN elemental analyzer (Fisons; NA-1500). Bacterial abundance was directly counted by epifluorescence microscopy after staining with 4',6-diamidino-2-phynylindole (DAPI) as previously described (Porter and Feig, 1980). At least 400 cells were counted per one sample. Because DAPI-stains include all DNAcontaining cells, irrespective of whether they are alive or dead (Zweifel and Hagström, 1995), the obtained data would be maximum estimation of the bacterial abundance. Moreover, the counts of 400 cells theoretically involve a standard deviation of 5% for the obtained data. Thus, further detailed studies could be suggested for more accurate bacterial response to the organic matter released from the corals. Calculations and Statistical Analyses.—Release rates (R: µmol cm−2 d−1) of bulk DOM and POM were calculated as follows: R = ∆OM × V S−1 T−1

(1)

where ∆OM is an accumulated DOM (or POM) concentration (µmol L−1) during the sampling interval (T: day), and V and S are water volume (L) and coral surface area (cm 2), respectively. Average release rates of DOM during the 4 d experiment were determined for each incubation bottle. Newly synthesized and subsequently released DOC (DOCnew) during the 4 d experiment was calculated using 13C isotope ratios of DOC and DIC as described by the following equation: DOCnew (µmol L−1) = (excess 13C amount in bulk DOC) (average APE 13C of DIC in seawater × 0.01)−1 (2)

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where APE 13C is atom % of excess 13C and was calculated as APE 13C (%) = (atom % 13C of sample) − (atom % 13C of natural seawater)

(3)

The atom % 13C of DIC in the natural seawater, i.e., in the beginning of the coral incubation, was 1.11%. Excess 13C amount in bulk DOC (µmol L−1) = bulk DOC (µmol L−1) × (APE 13C of bulk DOC) × 0.01 (4) The atom % 13C of bulk DOC in the natural seawater was 1.08%. Release rates of DOCnew from the incubated corals were calculated according to equation (1) and the average release rates for the 4 d experiment were determined for each incubation bottle. Release rates of DOM, POM and DOCnew are expressed as mean ± standard error (SE) of three incubations per species. DOM and POM release rates were compared using a Student’s t-test (n = 3 per species). Linear regressions on incubation period to APE13C of DOC were conducted with Sigma Plot 8.02 software (SPSS Inc.)

Results DOC and DON concentrations in the incubation seawater were initially 63 µmol C L−1 and 4.9 µmol N L−1, and increased by 21−30 µmol C L−1 and 0.8−1.8 µmol N L−1 after the 4 d experimental period (Fig. 1). The average DOC accumulation rates normalized to the unit surface area of the coral were 344 ± 9 and 379 ± 41 nmol cm−2 d−1 for P. cylindrica and A. pulchra, respectively (mean ± SE; Table 1). The DON accumulation rates were 18 ± 2 and 14 ± 2 nmol cm−2 d−1 for P. cylindrica and A. pulchra, respectively (mean ± SE; Table 1). APE 13C of bulk DOC, which was normalized to the coral surface area of 100 cm2, increased by 0.14%−0.18% during the 4 d coral incubation (Fig. 2). The average APE 13 C of DIC was within the range of 8.3%−8.5% for all the incubations. Using the

Figure 1. An example of the increased concentrations of DOC and DON (∆DOC and ∆DON), bacterial abundance, and estimated bacterial C biomass in one of the incubation bottles for Porites cylindrica. Bacterial C biomass was estimated using the factor of 30 fg C cell−1 (Fukuda et al., 1998). Error bars represent standard deviation (SD) of analysis for DOM concentrations, and standard error (SE) for bacterial abundance and estimated bacterial C.


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Table 1. DOM and POM accumulation rates (nmol cm−2 d−1) in coral-incubated seawater. Rates were normalized to unit surface area of the coral. DOCnew (nmol cm−2 d−1) is newly synthesized and subsequently released organic C in the form of DOM. Data are represented as mean ± SE (n = 3 per species). Bulk DOM Bulk POM DOC DON C:N DOCnew POC PON C:N DOC:POC DON:PON P. cylindrica 344 ± 9 18 ± 2 19 ± 2 24 ± 1 592 ± 55 31 ± 2 19 ± 3 0.60 ± 0.07 0.57 ± 0.04 A. pulchra 379 ± 41 14 ± 2 27 ± 6 33 ± 2 744 ± 94 48 ± 1 16 ± 2 0.51 ± 0.03 0.30 ± 0.04

values, average accumulation rates of DOCnew were calculated to be 24 ± 1 and 33 ± 2 nmol cm−2 d−1 for P. cylindrica and A. pulchra, respectively (mean ± SE; Table 1). Bulk POC and PON accumulated by 49−78 µmol C L−1 and 2.7−4.6 µmol N L−1 during the 4 d incubation. The average POC accumulation rates per unit surface area of the coral were 592 ± 55 and 744 ± 94 nmol cm−2 d−1 for P. cylindrica and A. pulchra, respectively, and the PON accumulation rates were 31 ± 2 and 48 ± 1 nmol cm−2 d−1, respectively (mean ± SE; Table 1). DOC:POC ratios of the accumulated organic matter were significantly lower than one for P. cylindrica (t-test: t = 3.86, P < 0.05) and A. pulchra (t-test: t = 6.34, P = 0.01). The actual DOC:POC ratios were 0.60 ± 0.07 for P. cylindrica (mean ± SE, n = 3) and 0.51 ± 0.03 for A. pulchra (mean ± SE, n = 3), respectively. DON:PON ratios were also significantly lower than one for P. cylindrica (t-test: t = 19.9, P < 0.01) and A. pulchra (t-test: t = 25.0, P < 0.001). The average DON:PON ratios were 0.57 ± 0.04 for P. cylindrica (mean ± SE, n = 3) and 0.30 ± 0.04 for A. pulchra (mean ± SE, n = 3). Total bacterial numbers were 5.0 × 105 and 2.0 × 105 cells ml−1 before the incubation of P. cylindrica and A. pulchra, respectively. During the coral incubation, the abundance increased to 0.96−1.4 × 106 cells ml−1 for P. cylindrica, depending on the bottles, and to 5.2−7.4 × 105 cells ml−1 for A. pulchra. The fastest increase rate (5.3 × 105 cells ml−1 d−1) was observed during the first day in one of the three bottles for P. cylindrica (Fig. 1).

Figure 2. Atom % excess 13C (APE 13C) of bulk DOC in the incubation seawater. Data are normalized to the coral surface area of 100 cm2, and are shown as mean ± SD of three incubations per species.

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Discussion The present study is the first reported simultaneous measurement of the net release rates of DOM and POM from coral-algal symbiotic colonies. This approach has shown that the reef-building corals produce both DOM and POM simultaneously but POM accumulates more than DOM, with the DOM:POM ratios reaching 0.3−0.6. The observed release rates of DOC (344−379 nmol cm−2 d−1) and POC (592−744 nmol cm−2 d−1) are comparable with �� previously �� reported values of the continuous �� daytime release of 150 nmol C cm−2 hr−1 as TOC from Acropora palmata (Lamarck, 1816) (Means and Sigleo, 1986), 1.8 and 2.3 µmol C cm−2 d−1 as mucus plus DOClipid from Acropora variabilis (Klunzinger, 1879) and Stylophora pistillata (Esper, 1797), respectively (Crossland, 1987), and 58 and 83 nmol C cm−2 hr−1 as POC during the daytime from Acropora millepora (Ehrenberg, 1834) and Acropora aspera (Dana, 1846), respectively (Wild et al., 2005). Presently, subsamples of the incubation seawater were taken once a day for DOM and at the end of the 4 d incubation for POM, and the net release rates of DOM and POM were calculated. A part of the released DOM might have been rapidly incorporated by bacteria (van Duyl and Gast, 2001), and it was also likely that the socalled “bottle effect” might have occurred, enhancing bacterial growth. Ferrier-Pagès et al. (1998) suggested the importance of the microbial community in the mucus layer of the coral colony for rapid decomposition of the released DOM. Bacterial consumption would result in the underestimation of the gross DOM release rates. Assuming that each bacterial cell contains 30 fg C (Fukuda et al., 1998) and growth efficiency was 20%, the observed increases of bacterial abundance during the 4 d incubation (4.6−9.0 × 105 cells ml−1 and 3.2−5.4 × 105 cells ml−1 for P. cylindrica and A. pulchra, respectively) corresponded to 3.9−11 µmol C L−1. Calculating for each incubation bottle, the bacterial consumption was equivalent to 14%−44% of the net accumulated DOC at the end of the experiment (21−30 µmol L−1). This suggests that gross DOM release from the corals might have been 1.1−1.4 times higher than the obtained net release rates. Moreover, the gross release rates would increase still further, considering the contribution of the microbial community in the mucus layer of the coral. For both coral species, the accumulation rates of POM were significantly higher than that of DOM for both C and N. The accumulated DOC:POC ratios were 0.60 and 0.51 for P. cylindrica and A. pulchra, respectively, and DON:PON ratios were 0.57 and 0.30. The bulk POM probably includes components such as bacteria, zooxanthellae released from the host (Hoegh-Guldberg et al., 1987), and non-living coral mucus. C:N ratios of the POM (19 and 16 for P. cylindrica and A. pulchra, respectively) suggest that the corals released C-rich organic matter as POM into the ambient seawater. Previous studies have reported that coral mucus includes organic matter of high C:N ratios, such as wax esters (Benson and Muscatine, 1974; Vacelet and Thomassin, 1991) and carbohydrates (Meikle et al., 1988; Coffroth, 1990), which is consistent with our observations. There are very little data concerning the size distribution of organic matter in coral reef waters (Hata et al., 2002). These authors measured fluxes of DOM and POM in Shiraho Reef in Ishigaki Island, and reported that the DOC:POC ratio was approximately 6. This ratio is markedly higher than that reported here (0.30−0.6) and suggests that reef communities other than corals (i.e., sediments, algae, and


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seagrasses) release organic matter to the ambient seawater at a higher DOC:POC ratio than that from coral colonies. DOM:POM ratios in actual reef waters would be affected by the particular biota predominant in the area. The present study suggests that coral colonies themselves accumulate more POM than DOM, at least on the time scale of a few days. The total accumulation rates of organic C (DOC + POC) were 0.9 and 1.1 µmol C cm−2 d−1 for P. cylindrica and A. pulchra, respectively. Using rates of tissue production by algal photosynthesis of 10 µmol C cm−2 d−1 for A. pulchra (Tanaka et al., 2007), the total organic C release accounts for approximately 10% of the net tissue production for both coral species. The ratio is within the range of previously reported values of 6%−40% (Crossland et al., 1980a; Muscatine et al., 1984; Crossland, 1987; FerrierPagès et al., 1998). The present study has also shown the effectiveness of a new method for determining 13 C enrichment of DOC. Average DOCnew accumulation rates were 24 and 33 nmol cm−2 d−1 for P. cylindrica and A. pulchra, respectively. The DOCnew accounted for 7.0% and 8.7% of the bulk DOC, suggesting that > 90% of the released DOC was derived from not newly synthesized C, but previously stored organic C pool. As considered earlier, the ratio of DOCnew release to the net tissue production could be < 1% for both coral species. This suggests that > 99% of the newly synthesized organic C by the symbiotic algae was not released as DOC within a few days. In previous studies, almost half of photosynthetically fixed C was lost from the coralalgal symbiotic colony within a few days, and was ascribed to host and algal respiration and/or organic matter release (Cooksey and Cooksey, 1972; Crossland et al., 1980b). The present study has shown that the rapid loss of the newly fixed C might not be caused by DOC release but, presumably, by respiratory use. Total accumulation rates of organic N (DON + PON) were 49 and 62 nmol N cm−2 d−1 for P. cylindrica and A. pulchra, respectively (Table 1). The N release was very low compared to the whole tissue biomass (50 µmol N cm−2 for A. pulchra; Tanaka et al., 2007). Coral mucus has low C:N ratios of 6−13 (reviewed by Brown and Bythell, 2005), and thus copious mucus release due to stressful conditions, such as sedimentation and aerial exposure, will result in severe N loss for the coral colonies. However, present observations imply that DON and PON release into the ambient seawater is not a severe N loss for the coral tissue at least in the condition of the present experiment, where the corals were incubated in gently stirred seawater without air exposure. In summary, this study investigated net production rates of DOM and POM from two reef-building corals, and concluded that more POM is produced than DOM. The ratios were not determined from the gross release rates from the corals, but rather from the net accumulation rates over a time scale of a few days. The produced DOM and POM would be gradually utilized by microorganisms, depending on the sizes of the organic matter. Our use of a new 13C labeling technique suggested that net production of organic C by algal photosynthesis is mostly (> 99%) stored in the colony and not used for the release of DOC within a few days. Less than 10% of the released bulk DOC was derived from the newly synthesized organic C, implying that > 90% of the released DOC is likely derived from previously stored organic C pools in the coral colony.

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Acknowledgments We are grateful to H. Fukuda and Y. Umezawa (Ocean Research Institute) for assistance of the experiment. This study was financially supported by CREST, R&D of Hydrological Modeling and Water Resources System (Japan Science and Technology Agency) and The 21st Century COE Program, Biodiversity and Ecosystem Restoration Research Project (Japan Ministry of Education, Culture, Science and Technology).

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Date Accepted: 28 December, 2007.

Addresses: (Y.T., T.M., I.K., H.O.) Marine Biogeochemistry Laboratory, Ocean Research Institute, The University of Tokyo, Minamidai 1-15-1, Nakano, Tokyo, 164-0014, Japan. (T.H.) Seikai National Fisheries Research Institute, Fukai-Ota 148-446, Ishigaki, Okinawa, 907-0451, Japan. Corresponding Author: (Y.T.) Telephone: (81)-3-5351-6525, Fax: (81)-3-5351-6461, E-mail: .