Eutrophication Accelerates Carbonate Dissolution under High PCO2 Condition in Coral Reef Ecosystem

Incubation experiments were carried out to determine the effect of eutrophication on carbonate dissolution under high pCO2 (partial pressure of carbon dioxide) condition in coral reef ecosystem at Sesoko Island, Okinawa, Japan. Short incubation (24 h under natural illumination) and long incubations (4 days under dark condition) were carried out using white coral skeleton (without attachment of living organism, control); natural rubble (with associated epilithic and endolithic communities) and natural rubble with addition of dissolve organic matter (glucose and coral mucus). Addition of DOM significantly enhanced bacterial abundance (t-test; p=0.01) and net respiration (t-test; p=0.0001) with increasing pCO2 levels (p < 0.05) under natural illumination. Consistent with increase in respiration, dissolution rates also increased from 136.22±2.04 to 652.38±4.51 μmolmd. Under dark condition, where photosynthesis was inhibited, dissolution of calcium carbonate further increased with addition of different level of DOM. In addition of DOM incubation bottles, bacterial abundance increased by 3~4 orders of magnitude and the dissolution rates increased by 2.5~10 times more than the control. The results suggest that availability of organic matter in the reefs will enhance metabolic activities (respiration) of microbial communities associated with coral rubble which ultimately increase dissolution of calcium carbonate.


Introduction
Eutrophication is the enrichment of water bodies and associated sediments by inorganic plant nutrients (e.g.nitrate, phosphate) that occurs naturally or human activity (cultural eutrophication from fertilizer runoff and sewage discharge) and is particularly evident in slow-moving rivers and shallow lakes where high nutrient concentrations stimulate blooms of algae [1].On the other hand, organic matter in aquatic environment occurs in form of living organisms, organic detritus and dissolved substances.In case of coral reef ecosystems, habitually coral colonies release organic matter to the seawater as dissolved (DOM) and particulate organic matter (POM) [2].The coral-derived organic matter (DOC) is often collectively referred to as mucus, and that is rapidly utilized by bacteria for their enhanced growth and abundance.The coral mucus has been regarded as ecologically important, bacterial aggregation was found in coral mucus [3] and coral exudates actually enhanced the growth of pico-and nanoplankton [4].Some part of DOC in coral mucus was rapidly mineralized by bacteria into CO 2 in the reef sediment [5] and conversely, remain labile DOC contributes to long term C fixation as refractory organic matter [2].
Glucose, the most common monosaccharide (MCHO) in the seawater [6], was used as the MCHO supplement because seawater concentrations remain consistently low [7,8], possibly indicating rapid uptake to a threshold level by the indigenous bacterial populations.Nevertheless, the marine environment is extremely oligotrophic [9][10][11], which implies that microorganisms must face significant periods of shortage of carbon and energy [12,13].
The purpose of the study is to determine the effect of eutrophication accelerates carbonate dissolution by examining the response of coral rubble associated microbial community under high pCO 2 condition in coral reef ecosystem.We wanted to determine the level of DOM and pCO 2 has really enhanced carbonate dissolution by influencing bacterial activity.

Study area and collection of samples
The study area is located in a shallow fringing coral reef at Sesoko Island, Ryukyu Archipelago, Okinawa, Japan between 26° 38′ N and 127° 51′ E (Fig. 1).Coral rubble samples were collected in the middle of the lagoon at about 1~2 m depth, and coral mucus was collected from Acropora digitifera species using air exposure method [5] (Fig. 2) into cleaned syringe and transferred into washed clean beaker.Seawater was collected by using 10 L Nalgene bottle.After collection, seawater was filtered using a cartridge filter (0.2 μm isopore membrane filter) and dispensed into 1 L Nalgene bottles for incubation.All the bottles were washed using neutral Extran (MA02; MERCK) detergent and rinsed with Milli-Q water before use.

Experimental design
Incubation experiment were carried out in natural illumination (24 h short) and dark (4 day's long) under different level of pCO 2 conditions using natural rubble (NR: with associated epilithic and endolithic communities); natural rubble with addition of different level of glucose (NR+G) and natural rubble with coral mucus (NR+M) as source of organic matter to assess the role of organic maters on carbonate dissolution.As control, white coral skeleton (WCSk) was incubated in the same conditions as mentioned previously (Table 1).NR+G4 NR+G4 NR+G4 WCSk: White coral skeleton; TR: Treated rubble keeping only their endolithic communities; NR: Natural rubbles with associated epilithic and endolithic communities; NR+G: Natural rubbles with addition of organic matter (10 µM glucose); NR+M: Natural rubbles with addition of organic matter (10% coral mucus); G1, G2 and G4: Level of organic matter addition (1 µM, 2 µM and 4 µM of glucose); Natural illumination: Out door

Preparation of incubation experiment
Two small branches of natural coral rubbles of similar sizes were placed into 1 L Nalgene bottles which were filled with filtered seawater.Three (03) replicates were used for each incubation experiment.The different levels of pCO 2 in the incubation bottles were adjusted by injecting CO 2 saturated seawater into the bottles until pH values equivalent to the desired pCO 2 levels were obtained.CO 2 saturated seawater was prepared by bubbling pure CO 2 gas into natural seawater until pH was stable.Glucose stock solution was prepared as 1.8 g L -1 , and 100 µL, 200 µL, 400 µL and 1000 µL of this solution were added to the incubation bottles (1 L) to make final concentration of 1 µM, 2 µM, 4 µM and 10 µM.In case of coral mucus, 10% mucus solution was added to the incubation bottles.Incubations were conducted in natural temperature varied at 25C to 33C.Temperature and light intensity were monitored during the experiments using in situ sensor (MDS-MkV/T and MDS-MkV/L, Alec electronics).

Laboratory measurement and analysis
Measurement of short incubation experiment was done 24 times per day (1 h interval) and 4 times per day (6 h interval) for long incubation experiments.pH and DO were measured using a pH meter (with 3 sensors; temperature, pH and DO) ORION 4 STAR calibrated with NIST (NBS) scaled buffer solutions (Mettler pH 9.228 and 6.880 buffers at 20C).Alkalinity (A T ) was measured by the total alkalinity titrator (KIMOTO ATT-05).Reproducibility of the A T measurement was ± 2 µmol kg -1 (1σ; n = 10).
Heterotrophic bacteria were collected on 0.2 µm black polycarbonate filters by filtering 10 to 15 mL aliquots that were previously stained with DAPI (4',6-diamidino-2phenylindole) at 1 µg mL -1 concentration.Abundance was assessed by counting bacteria cells under an epifluorescence microscope (Nikon; ECLIPSE/E600), using a UV-filter.Net respiration of the rubble associated communities was calculated from changes in dissolve oxygen concentration and converted to carbon (C) using "Redfield-Richards Ratio" empirical formula [14].Statistical data were analyzed and the variances with 0.05 level of significance by using Microsoft Office Excel 2007.
Different levels of pCO 2 (ambient, 520, 720 and 1120 ppm) was calculated using pH and A T according to the carbonate equilibrium in seawater described by Millero and Fujimura et al. [15,16].Carbonate dissolution rates (µmol L -1 d -1 ) were analyzed using the alkalinity anomaly technique [16][17][18] following the equation: Dissolution rate = △A T /2, where △A T is the variation of A T (A T final -A T initial ) during the incubation period at the start and end of each incubation.The alkalinity anomaly method actually yields an estimate of the net value of the CaCO 3 precipitation/dissolution balance [19,20].
The carbonate system parameters in seawater and saturation state of aragonite (Ω a ) were calculated with the program CO2SYS [21] using the apparent equilibrium constants K′ 0 from Weiss [22], K′ 1 and K′ 2 from Mehrbach et al. [23] as described by Dickson and Millero [24] and the HSO 4 -constant according to Dickson [25].Saturation degree of aragonite (Ω a ) was calculated from pH, alkalinity, salinity and temperature data sets.The degree of saturation is defined as: Ω a = [Ca 2+ ] [CO 3 2-] / K′ sp , where K′ sp is the stoichiometric solubility product of aragonite derived from a function of salinity and temperature [26].

Addition of bioavailable organic matter
Bioavailable organic matter (glucose and coral mucus), was added to the incubation bottles to revitalize bacterial activity and their physiological activities accelerates carbonate dissolution.Addition of bioavailable organic matter enhanced bacterial abundance and dissolution rates were observed under natural rubble (NR), natural rubble with addition of glucose (NR+G) and with addition of coral mucus (NR+M) in ambient and high pCO 2 condition (Fig. 3 and Table 2).During 24 h natural illumination experiment, the bacterial abundance (t-test; p=0.01) and net respiration (t-test; p=0.0001) increased significantly with addition of bioavailable organic matter and also with increasing pCO 2 levels (p < 0.05).Consistent with increase in respiration, dissolution rates also increased.In the organic matter addition sample, bacterial abundance and dissolution rates both increased 5~6 times more than the natural rubble (NR) (Table 2).

Level of organic matter addition
During 4 day's long incubation experiment under dark condition, where photosynthesis was inhibited, dissolution of calcium carbonate increased with increasing pCO 2 levels and different levels (1 µM, 2 µM and 4 µM) of organic matter addition (glucose).After addition of different level of organic matter, the bacterial abundance and net respiration as well as dissolution rates were increased significantly (p < 0.05).During incubation experiment of without addition of organic matter, the bacterial abundance were 1.82±0.001×10 6cells mL -1 of sample water and dissolution rate were 70.67±1.22µmol m - 2 d -1 in the ambient condition, whereas in high pCO 2 (1120 ppm) condition were 2.39±0.001×10 6cells mL -1 and 84.32±0.97µmol m -2 d -1 respectively.But in case of different levels of organic matter addition, the bacterial abundance was increased 8.24±0.002×10 6~ 14.62±0.002×10 6cells mL -1 and dissolution rates were increased 152.22±1.11~ 318.93±1.13µmol m -2 d -1 with increasing both of glucose levels and pCO 2 levels (p < 0.05) (Table 3).There was strong positive correlation between different level of organic matter addition and calcium carbonate dissolution (r=+0.99;p=0.0001).

Carbonate dissolution vs. Saturation state
During 24 h natural illumination experiment, with increasing carbonate dissolution rates (from 136.22±2.04 to 652.38±4.51µmol m -2 d -1 ), aragonite saturation state (Ω arg ) were decreased (from 2.87 to 1.13) in ambient and high pCO 2 condition respectively.Coral mucus addition had more significant effect than glucose addition for carbonate dissolution as well as CO 3 2-saturation state.In case of 4 day's long incubation experiment (under dark condition), carbonate dissolution rates were increased from 70.67±1.22to 318.93±1.13µmol m -2 d -1 and aragonite saturation state (Ω arg ) were decreased from 1.82 to 0.96 under different level of organic matter (glucose) addition and different pCO 2 levels.There were strong negative correlation between carbonate dissolution rate and saturation state (r=-0.99;p=0.0002) (Fig. 4).
Table 3. Summary results of 4 day's long incubation under dark condition with different level of organic matter addition.

Addition of bioavailable organic matter
Natural seawater normally contains 10 5 to 10 6 bacterial cells mL -1 [27].The bacterial composition of natural seawater is regulated by such factors as grazing pressure [28,29], nutrient limitation or starvation [12,30], and various other physicochemical stresses (e.g., temperature variation, oxidative stress, etc.).Addition of bioavailable organic matter (glucose and coral mucus) to the samples was influencing bacterial activity [31,32], growth and their abundance (3~4 orders of magnitude); and caused subsequent increase of calcium carbonate dissolution (2.5~10 times more than in control) in the dissolution experiments (Table 2).In coral reef ecosystem, endolithic communities of coral rubble play a crucial role as primary producers and control calcification and dissolution of calcium carbonate by "Bio-Chemical Dissolution Processes (BCDP)" [31].The results suggest that availability of organic matter (eutrophication) accelerates carbonate Bacterial abundance [Cells ×10

Level of organic matter addition
Natural rubble (NR) is colonized by epilithic and endolithic communities with other organisms of Heterotrophs as foraminifera, nematodes and copepods.When photosynthesis is inhibited under dark condition, calcium carbonate dissolution was increased with increasing pCO 2 and addition of glucose levels.On the other hand, without any attachment of living organism, very small amount (00.02~30.38±0.84µmol m -2 d -1 ) of dissolution were observed from white coral skeleton (control) by the pressure of CO 2 only.This suggests that dissolution increased with enhance bacterial abundance and their physiological activities, and availability of organic matter also accelerates carbonate dissolution.Different level of organic matter (glucose) addition regulates bacterial activity as well as dissolution rates.The effect of adding glucose (3 mM) was examined by Eguchi et al. [33]; bacterial cells grow instantaneously and increased cell density after glucose addition, probably because of rapid initial intracellular poly glucose accumulation [34].Church et al. [35] found that biomass production of heterotrophic bacteria in the Southern Ocean was stimulated by addition of organic carbon.Organic carbon additions also consistently stimulated bacterial growth rates in the subarctic Pacific [12] and the equatorial Pacific [36].Moulin et al. [37] mentioned the bacterial respiration of organic matter can induce rapid dissolution of a significant amount of carbonate in the sediments.However, during 4 day's long incubation experiment under dark condition, we found 14 times higher bacterial abundance than the normal seawater condition with addition of different level of organic matter (1 µM, 2 µM and 4 µM glucose), and also found highly significant effect (r=+0.999; p=0.0005) on different level of organic matter addition.Consequently, we suggest that level of organic matter addition increased bacterial abundance and their physiological activity (especially respiration) regulates carbonate dissolution in seawater.

Carbonate dissolution vs. Saturation state
Calcium carbonate dissolution occurs due to reduction in CO 3 2-saturation state at elevated pCO 2 [38][39] and the value of Ω arg < 1 promotes dissolution [40][41][42][43][44].However, in coral reefs shows large variation in CO 2 concentration and Ω occur due to their high diversity and productivity.Therefore, below the super saturation threshold value 3~4 for aragonite [40][41] dissolution was observed.Previous studies reported that calcium carbonate dissolution occurred when Ω arg ranged from 4.38 to 2.84 [45]; 3.06 to 1.83 [46] and 3.7 to 1.3 [47].In our experiment dissolution of calcium carbonate occurred when the saturation state (Ω arg ) ranged from 2.87 to 1.13 in natural illumination experiment and from 1.82 to 0.96 in dark incubation experiment under different level of organic matter addition with increasing pCO 2 levels.We found strong negative correlation between carbonate dissolution rate and saturation state (r=-0.99;p=0.0002) (Fig. 4).

Conclusions
Eutrophication has a potentially important role for calcium carbonate dissolution.In coral reef ecosystems, coral reef itself release mucus as organic matter (DOM, POM) that enhancing growth of bacteria and their abundance.Thus, the levels of organic matter in the coral reefs also accelerate carbonate dissolution by enhancing bacterial activities (especially respiration) under different levels of pCO 2 .

Fig. 1 .
Fig. 1.Map showing the study area and the location of sample collection (○) at Sesoko Island, Okinawa, Japan (Fig. developed from Google map).

Table 1 .
Experimental design for short and long incubations.

Table 2 .
Summary result of 24 h natural illumination short incubation experiment.