Unexpected results from direct measurement, with a torsion microbalance in a closed system, of calcification rates of the coral Agaricia agaricites (Scleractinia:Agariicidae) and concomitant changes in seawater pH
Resultados inesperados de medición directa, con una microbalanza de torsión en un sistema cerrado, de las tasas de clacificación de los corales Agaricia agaricites (Scleractinia:Agariicidae) y concomitantes cambios de pH en el medio del mar
Ocean acidification is impacting the calcification of corals, but the mechanisms of calcification are still unclear. To explore the relationship between calcification and pH, small pieces of coral were suspended from a torsion microbalance in gently stirred, temperature controlled, seawater in a closed chamber. Net calcification rate and pH were continuously monitored while light, temperature or pH could be manipulated. The coral pieces were from the edges of thin plates of Agaricia agaricites and were studied alive and freshly collected. Unexpectedly, when calcification was taking place (n=9, 0.082 mg.hr-1.cm-2), as determined by weight increase, the pH of the surrounding seawater medium changed little (n=10, -0.0047 pH units.hr-1.cm-2). When calcification was not taking place the decrease of seawater pH was an order of magnitude higher, -0.013 pH units.hr-1.cm-2. This is the opposite of what is expected when calcium carbonate (CaCO3) forms. Similarly, fresh skeleton initially showed no change of pH in the seawater medium although the rates of weight gain were high (upto 1.0 mg hr-1.cm-2). After 10 hours, as the rate of deposition decreased following a generalized Michaelis-Menten growth curve, the pH began to decrease dramatically indicating an increase of CO2 in the seawater. These unexpected results can be explained if unstable calcium bicarbonate (Ca(HCO3)2) is formed in the organic matrix/carbonic anhydrase surface and slowly transforms later to CaCO3. Pieces of living coral monitored in the chamber for 30 hours gained weight during the day and loss it at night. The loss would be consistent with the transformation of Ca(HCO3)2 to CaCO3 with the release of CO2. The mean calcification rate of live coral was greater (n=8, p=0.0027) in high light (120 μmol.s-1.m-2) at 0.098 mg.hr-1.cm-2, compared to 0.063 mg.hr-1.cm-2 in low light (12 μmol.s-1.m-2). However, at the same time the mean rate of pH change was -0.0076 under low light compared to -0.0030 under high light (n=8, p=0.0001). The difference can be explained by CO2 being used for photosynthesis by zooxanthellae. The deposition rate of live coral was not affected by the addition of phosphate but the rate of weight gain by the freshly collected skeleton was strongly enhanced by phosphate. These results indicate that care should be applied in the application of the alkalinity anomaly technique for the measurement of calcification in corals.
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La acidificación del océano está impactando la calcificación de los corales, pero los mecanismos de la calcificación son aún inciertos. Para explorar la relación entre la calcificación y pH, pequeños trozos de coral fueron suspendidos en una microbalanza de torsión en agitado suave, temperatura controlada, y agua de mar en una cámara cerrada. La tasa de calcificación neta ]]>
Agaricia agaricites y se estudiaron vivos y recién colectados. Inesperadamente, cuando la calcificación (n= 9, 0.082 mg.hr-1.cm-2) se estaba dando, según lo determinado por el aumento de peso, el pH del agua de mar circundante cambió poco (n = 10,-0.0047 pH units.hr-1.cm-2). Durante los períodos cuando la calcificación no se estaba dando la disminución del pH del agua de mar era un orden de ]]>
hr-1.cm-2. Esto es exactamente lo contrario de lo que se espera cuando se forma carbonato de calcio (CaCO3). Del mismo modo un esqueleto recién colectado al inicio no mostró cambios de pH en el agua de mar aunque eran muy altas ]]>
hr-1.cm-2). Después de 10 horas, la tasa de deposición disminuyó hasta seguir una curva de crecimiento generalizada de Michaelis-Menten, el pH comenzó a disminuir drásticamente, lo que indica un aumento de CO2 en el agua de mar. Estos resultados inesperados pueden explicarse si el bicarbonato de calcio inestable (Ca(HCO3)2) se forma en la superficie de la anhidrasa carbónica/matriz orgánica y lentamente se transforma más tarde a CaCO3. Piezas de coral vivo vigiladas en la cámara durante 30 horas ]]>
CaCO3 con el lanzamiento de CO2. La tasa de calcificación media de coral vivo fue mayor (n= 8, p= 0.0027) en luz alta (120 μmol.s-1.m-2) a 0.098 ]]>
hr-1.cm-2, en comparación con 0.063 mg.hr-1.cm-2 en condiciones de poca luz (12 μmol.s-1.m-2). Sin embargo, al mismo tiempo ]]>
CO2 está siendo utilizado para la fotosíntesis por zooxantelas. La tasa de deposición de coral vivo no fue afectada por la adición de fosfato pero la tasa de ganancia de peso de los esqueletos recién colectados era fuertemente reforzada por fosfato. Estos ]]>
The processes involved in the production of an aragonite (CaCO3) skeleton in corals are ]]>
-1 of calcium ions (Ca2+), dissolved inorganic carbon is present as carbon dioxide (CO2), carbonic acid (H2CO3), bicarbonate (HCO3-) and carbonate (CO32-), with the equilibria ]]>
Ca2+ and CO32- . However, Lee, Park, Kwak & Cho (2010) have reported crystalline aragonite deposition from a solution of CaCl2 containing carbonic anhydrase using ]]>
CO2 directly from the atmosphere. The sources of calcium and carbon and how they reach the calcification site have been reviewed by Cohen and McConnaughey (2003), Furla, Galgani, Durand and Allemand (2000) and Allemand et al. (2004; 2011). Carbon dioxide which can move freely through cell membranes was proposed by McConnaughey (1989) as a substrate for calcification to account for 18O and 13C deficiencies in coral skeletons (McConnaughey, 2000). The response of corals to changes ]]>
CO2 partial pressures and temperature was reviewed by Reynaud et al. (2003). While calcification and photosynthesis may compete for the same DIC pool they are sometimes regarded as complementary (Gattuso, Allemand & Frankignoulle, 1999) rather than in competition (Langdon & Atkinson, 2005). With a focus on ocean acidification as the result of increased anthropogenic carbon dioxide in the atmosphere, calcification has been increasingly linked to the calcium-carbonate saturation state (ω) which is the ratio of the ion concentration ]]>
Ca2+] x [CO32-]) to the solubility product of the mineral deposited, in this case aragonite (Allemande et al. 2004). As acidity increases the relative concentration of carbonate in seawater is reduced and has been used to predict decreased calcification at the organism and community levels (Kleypas et al., 1999; Gattuso et al., ]]>
The mechanisms of calcification have been fully reviewed by Allemand et al. (2011). Active calcification by the coral involves secretion of a layer of organic matrix (reviewed by Allemand, Tambutté, Girard & Jaubert, 1998 and Allemand et al., 2011). Sandeman (2012) presented evidence ]]>
Ca2+, HCO3- and CO32- cannot move passively through cell membranes so either active transport via a transcellular route or passive diffusion of ions or seawater using a paracellular route at the boundaries of cells or some ]]>
CO2 for photosynthesis and CO2 is released as the result of respiration by coral tissue and their symbionts. Sandeman (2012) presented evidence that CO2 is the substrate for calcification by living and freshly waterpiked coral skeleton and that the enzyme carbonic anhydrase appears to be incorporated in the organic matrix secreted by the calicoblastic layers. It was also shown that for dead coral skeleton in sea water
CO32- is the substrate and at pH> 7.4 the skeleton gained weight and at ph<7.4 dissolved.
Calcification rates of corals are commonly derived by buoyant weighing techniques (Davies, 1989), uptake of the radioactive isotope 45Ca (e.g. Moya et al., 2006; Al-Horani, ]]>
Porites porites has been used to measure calcification rates over time periods of less than a day but a disadvantage in physiological studies is that the coral or a coral ‘nubbins’ has to be transferred to and from a balance for weighing. For ]]>
Agaricia could be ignored.
In an earlier paper Sandeman (2012) reported on a torsion balance developed to investigate calcification in small pieces of the coral Agaricia agaricites. A major disadvantage of the technique was that in an open system, the seawater medium could ]]>
CO2 with the air, changing the pH and bringing uncertainty to the carbon chemistry. In this study the torsion balance has been completely enclosed in a sealed chamber to avoid the exchange of CO2 with the air. The pH electrodes and their associated temperature sensors have been incorporated so that pH can be continuously monitored ]]>
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Seawater contains about 10.3mmol.Kg-1 of calcium ions (Ca2+), dissolved inorganic carbon is present in seawater as carbon dioxide (
CO2), carbonic acid (H2CO3), bicarbonate (HCO3-) and carbonate (CO32-), the equilibria between the ]]>
CO2(Aq) ↔ HCO3- ↔ CO32- (Eq. 1.)
+H2O +H+ +H+
The equilibria between the species of carbon are governed by their equilibrium constants and their ]]>
Fig. 1 which is similar to Fig. 1. 1. 3 in Zeebe and Wolf-Gladrow (2000) but is for seawater (S= 35.5 and T= 28°C) used in this study. In general, at normal seawater pH (8-8.2), the bulk of the carbon is present as HCO3- with a small quantity as CO32- and even smaller quantity as CO2. If, as in the acidification of sea as the result of increased atmospheric carbon dioxide, there is an increase in dissolved CO2, the total dissolved organic carbon (DIC) increases but ]]>
Fig. 1). If CO2 in the system decreases the opposite happens, DIC decreases and TA remains the same and there is a consequent increase in pH. If calcification takes place with the formation of calcium carbonate as a result of the following reaction (Zeebe & Wolf-Gladrow, 2000):
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Ca2+ + CO32- → CaCO3 (Eq. 2.)
DIC decreases by 1 ]]>
Fig. 1, results in a lower pH. If CaCO3 dissolves the direction of the arrow is reversed and there is an increase of 1 unit in DIC and two units of TA and the pH ]]>
CaCO3 formation is the same whether the source of carbon is CO32-, HCO3- or CO2 (Zeebe & ]]>
The aim in this study was use the improved torsion balance to follow up on the rather extraordinary findings related to freshly waterpiked Agaricia skeletons (Sandeman, 2012) and to further explore the indications that calcification in the living coral might involve
CO2 rather than CO32-. Generally two of DIC, TA, pH and [CO2] are required to calculate the components of the carbonate system in seawater. The experimental set-up used here permits continuous monitoring of pH change and weight change of the coral. If ]]>
Materials and Methods
Small pieces of Agaricia agaricites were snipped from the edge of thin plates of young colonies growing near the reef crest opposite the Discovery Bay Marine Laboratory. The ]]>
2) then suspended, in a horizontal orientation, by loops of thin (0.025mm diam.) polyester monofilament in gently flowing seawater in a seawater table. The pieces were used in experiments within an hour of collection. There is some indication that orthophosphate in low concentrations inhibits the deposition and dissolution of calcium carbonate (Pytkowicz, 1973; Morse, 1974; Burton & Walter, 1990) and for live corals and freshly waterpiked coral skeleton the seawater used in the experimental ]]>
CO2], [CO32-] and ω for each batch of seawater (Table 1) were estimated using the CO@SYS program. From the initial salinity, temperature, total alkalinity (TA) and pH for each batch of seawater the total dissolved carbon (DIC) was obtained. The experimentally measured change of weight of the skeleton then permits calculation of the carbonate withdrawn from the seawater medium and estimation of DIC at the end of each experiment.
The balance (Fig. 2) chambers (volume ≈ 1lL) were filled with millepore filtered seawater brought to the temperature at which the experiment was to be conducted (27°C). Temperature sensing thermistors, control circuits, and insulated heater coils maintained a constant temperature within 0.2 °C. Small magnetic stirrers (1.0x0. cm) gently and continuously circulated the ]]>
CO2 content using a syringe through the hole in the centre of the access window. For higher pH levels water in the vessel was exchanged with seawater that had been bubbled with CO2 free air. Two pH meters (Omega, ]]>
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Pieces of coral were carefully inspected before each experiment and were rejected if there were signs of damage to the living tissue on the upper or lower surfaces or if mucus was present. A piece of coral was suspended by its monofilament loop from a hook at the end of the carbon fibre beam of the torsion balance (Fig. 2). The beam was balanced by sliding the counterweights along the other arm of the beam so that the reflected spot from the ]]>
Fig. 2). The lid with the pH and temperature probe was placed on the chamber and pressed into place so that its o-ring sealed the system, air bubbles were removed and sea water added through the access window. Finally the access window closure was put in place and its centre hole covered with a glass cover-slip and silicone grease seal. Change in weight as aragonite skeleton is deposited result in movement of the laser beam spot on the scale and readings were taken at ]]>
A series of small solid aragonite cubes (1-3mg ) were prepared beforehand, these were cut from Agaricia skeletons, and their dry weights were accurately known. Each cube was attached to a 20 cm length of extremely fine monofilament consisting of ]]>
Results
Waterpiked coral: Freshly waterpiked corals showed the pattern of weight gain reported previously ]]>
Fig. 3A) the initial rate (first hour) of weight gain of 0.97 mg.h-1 decreased following the generalized Michaelis Menten growth curve (Lopez et al., 2000; Sandeman, 2012) for the first 15 hours then maintained a steady rate of increase of 0.072mg.hr-2.cm-2 until the termination of the experiment ]]>
hr-1 to a constant rate of 0.072mg hr-1. However, as the changeover point was reached the seawater pH started to drop and eventually reached a steady rate of change of 0.0047 pH units.hr-1.cm-2. The mean initial calcification rate (n=4), over the first hour, was 0.84mg.hr-1.cm-2 with the pH changing at -0.00041units.
hr-1.cm-2. After reaching a steady rate of increase the mean calcification rate was 0.036mg.hr-1.cm-2 and the pH rate of ]]>
hr-1.cm-2.
Over the pH range of 6.9-8.6 ]]>
43- (Burton &Walton, 1990). Experiments to see how freshly waterpiked coral responded to 50μmol of orthophosphate gave the surprising result that rather than being inhibited the deposition rate on waterpiked coral (Fig. 3B) increased by over an order of magnitude from 0.04mg.h-1 to 0.88mg.h-1. The experiment was repeated four times with similar results.
Live coral: When placed in the sealed chamber live corals commonly showed no weight increase for the first few hours then started calcifying at a steady rate. During the period when no calcification was taking place the pH of the chamber decreased steadily. After calcification started, as indicated by weight ]]>
Fig. 4A) during the initial period with no calcification taking place the pH decreased by 0.0116 pH units.h-1.cm-2. After calcification started ]]>
hr-1.cm-2) the pH remained nearly constant, decreasing at the much lower rate of 0.0011 pH units.hr-1.cm-2. In seawater with a mean pH of 8.173 when no calcification was taking place the mean pH change (n=10) was -0.013 pH units.hr-1.cm-2. After calcification started the mean calcification rate of the corals was 0.082mg.hr-1.cm-2 and the pH rate of change decreased to a mean of -0.0047 pH units.hr-1.cm-2.
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A piece of Agaricia placed in the balance chamber at 3pm and kept for 30 hours (Fig. 4B) showed calcification taking place (0.348mg.hr-1.cm-2 ) until 7 pm coupled with small changes in pH. Thereafter the coral lost weight (-0.94mg.hr-1.cm-2) and the pH decreased (-0.0019pH units.hr-1.cm-2) until about 3 am. Weight gain commenced again and continued until about 3pm, but this time the pH decreased at a higher rate. The weight after 24 hours did not reach its previous level. Similar results were obtained when the experiment was repeated.
Mean calcification ]]>
hr-1.cm-2 as compared to 0.098mg.hr-1.cm-2 under higher light (n=8, p=0.0027 ). At the same time the pH change under low light was -0.0076 pH units .hr-1 while under higher light was lower at -0.0030 pH units.hr-1 (n=8, p=0.0001).
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Dead coral: The relationship between calcification rate and pH was explored and the results presented in an earlier paper (Sandeman, 2012). When the experiments were repeated with pH monitoring the results obtained were exactly as expected from Zeebe and Wolf Gladrow (2000) and Fig. 1. When the rate of weight change was positive, i.e. when deposition of calcium carbonate was taking place, the concomitant pH change in the seawater ]]>
Fig. 1), in a negative direction and when dissolution of the coral skeleton took place the pH of the seawater medium always increased.
Discussion ]]>
Waterpiked coral: Freshly waterpiked coral provides a simpler system than live coral in that calcification takes place directly on the surface of the organic matrix/skeleton which contains the enzyme carbonic anhydrase and which is in direct contact with the sea water medium (Sandeman, 2012) and is not separated by layers of tissue. The very high initial rate of calcification (mean, 0.84mg.hr-1.cm-2) was always accompanied by relatively small changes in pH of the seawater medium (mean 0.0004 pH units.hr-1.cm-2). During the first 10 hours the calcification rate ]]>
CaCO3 was formed, the pH of the seawater would have decreased rather than remain constant as the CaCO3 arrow follows the reduction of DIC by one unit and TA by two units. Second, the rate of pH change would be proportional to the calcification, starting with a high rate and ]]>
CaCO3 reduces DIC by 1 mol and TA by 2mol independent of the carbon source (HCO3-, CO32-, or even CO2) used by the organisms for calcification”. A possible resolution to the problem is that the “CaCO3 formation” arrow (cf. Fig. 1) would follow the line for pH 8.2 if the DIC:TA ratio was 2:2. A possibility is that in the initial phase of ]]>
2CO2 + 2H2O + Ca2+ → Ca(HCO3)2 + 2H+ (Eq. 3)
The reaction of two moles of carbon with one mole of
Ca2+ would fulfill the requirement of a 2:2 ratio for DIC: TA to remain on the iso pH line (cf. Fig. 1).
After the initially very high rate of weight gain of the freshly waterpiked coral the rate decreases ]]>
hr-1.cm-2 and the mean rate of pH change was 0.003 pH units.hr-1.cm-2 which is an order of magnitude greater than the expected rate of pH change. However if, during this second phase, the calcium bicarbonate was slowly converting to calcium carbonate following the reaction:
Ca(HCO3)2 →CaCO3 + H2O + CO2 (Eq. 4)
Then the resulting release of
CO2 into the seawater would help to explain its high rate of pH decrease. The conversion of Ca(HCO3)2 to CaCO3 would mean a decrease in weight of the skeleton but this was not evident in the results and it seems ]]>
CaCO3 is taking place from seawater at the same time following Eq. 2. Calcium bicarbonate is only known in solution and has not previously been suggested as a precursor to carbonate formation in corals. It appears quite commonly in the geological literature as a precursor to calcium carbonate deposition in nature and its formation when carbonate solutions are bubbled with CO2 is widely used for the experimental deposition of calcium carbonate. For example it was used in crystallization studies (Reddy, Plummer & Busenberg, 1981) and in a study of the kinetics of aragonite formation (Romanek, Morse & Grossman, 2011). It has also used in investigations of the conditions under which vaterite, calcite or aragonite are deposited and the effect of metallic ions on deposition (Tokuyama, Kitana & Kanamori, 1973; Kitano, Tokuyama & Arakaki, 1979). Of some interest is their finding that the presence of Mg2+ ions inhibits calcite ]]>
The finding that ]]>
Agaricia. There is also a possibility that, if present, phosphate could have a role in calcification by enhancing the activity of carbonic anhydrase and amplifying the formation of skeleton where the quantities of carbonic anhydrase are highest. The results seen in
Fig. 4 also suggest the shape of the Michaelis-Menten curve reaching an asymptote may not be the result of a limitation set by the concentrations of the reactants.
Live coral. In early stages of experiments with living coral (Fig. 4A) the seawater pH decreased ]]>
hr-1.cm-2) while the coral was not calcifying. When calcification was active the mean pH rate of change decreased by an order of magnitude to 0.0011 pH units.hr-1.cm-2. If carbonate is the substrate for calcification and the ratio of DIC:TA 1: 2 then the pH of the seawater should decrease during calcification and not change when the coral is not calcifying as indicated by weight changes taking place.
While calcification is taking place the pH of the medium remains nearly constant. This would suggest that DIC is being taken up and the TA is changing with a ratio of 2:2 and ]]>
Fig. 1) the decrease in pH must be the result of release by the coral of CO2 into the seawater medium. Thus a possible explanation for the results obtained here is that calcium bicarbonate is deposited (Eq. 3) and that ]]>
CO2.
The results from experiments in which corals were kept in the balance chamber, without the water being changed for 30 hours showed weight gain in the daylight hours followed ]]>
Ca2+ in all corals exposed to dark. Tentori & Allemand (2006) reported decalcification and a daily cycle of sclerite formation in the soft coral Cladiella. While weight decrease took place (
Fig. 4B) the pH of the medium decreased indicating a transfer of CO2 from the coral into the seawater. This could be the CO2 produced as the result of conversion of Ca(HCO3)2 to CaCO3, in which case a loss of 38% in weight might be expected.
How the materials necessary for calcification to take place reach the ECF is suggested in Fig. 5. CO2 is uncharged and can diffuse through the plasma membranes of the coral tissues and be assisted in moving into the ECF by the CO2 gradient which results from the activity of the Ca2+ ATPase pump. Protons generated (Eq. 3) on the skeleton surface in the ECF (
Fig. 5) probably enter the calicoblastic layer via the Ca2+ ATPase pump which moves calcium out of the tissues while moving protons in the other direction. Cohen and McConnaughey (2003) suggested that the calcium pump is light sensitive and there is some experimental evidence to support this (Sandeman, 2008a). There are several possibilities for what happen to protons thereafter. Although charged and existing in the form of hydronium ions (H3O+) in solution, in their ]]>
P pistillata included a long poly-aspartate domaine with 36 aspartic residues. While Puverel et al. (2005) suggested a calcium binding function it is also possible that these amphoteric amino acids may have a role in the sequestration of protons. If protons are sequestered for any length of time TA may be affected. How sufficient calcium ions get into the calicoblastic layer is not clear, but toxins produced by zooxanthellae may open Ca2+ channels (McConnaughey, ]]>
The experiments with low and increased illumination indicated that mean calcification rate increased from 0.063mg.h-1.cm-2 to 0.098mg.hr-1.cm-2 with increased illumination which is as expected. The rate of change of pH in the seawater medium was from -0.0076 pH units.
hr-1.cm-2 to -0.0030 pH units.hr-1.cm-2 in increased illumination. A reduction of pH in the medium is the result of transfer of
CO2 into it, and it has to be from the coral. It is to be expected that if CO2 is being taken up by zooxanthellae and used for photosynthesis that the amount transferred would be less. A change of -0.0076 in pH of the seawater would result from an increase of 4.9μmol of CO2 and -0.0030 from an increase of 1.92μmol of CO2, so this would indicate an uptake of 3μmol of CO2 by the algae.
Calcium bicarbonate ]]>
Ca2+, 320-370 ppm) will precipitate CaCO3 while slowly releasing
CO2. If magnesium ions are present calcite formation is inhibited and aragonite precipitation favoured (Kitano et al., 1979). It seems possible therefore that if the right ions are present, a fairly concentrated solution of calcium bicarbonate could build up in the spaces of the organic matrix and slowly convert to a deposit of aragonite on the surface present. The unusual results obtained in this study are explainable by an initial formation of calcium bicarbonate and its slow conversion to carbonate. The temporal separation between ]]>
Ca2+ (change of TA) and movements in, then later out, of CO2 (changes in DIC), means that care should be applied in the application of the alkalinity anomaly technique for the measurement of calcification in corals.
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