Irradiance Regulation of Photosynthesis and Respiration in Modern

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Tropical Lagoon (New Caledonia) .... Southwest lagoon of New Caledonia, especially on and in ..... intensities, and metabolic activities were estimated from.
Irradiance Regulation of Photosynthesis and Respiration in Modern Marine Microbialites Built by Benthic Cyanobacteria in a Tropical Lagoon (New Caledonia) Olivier Pringault1, Rutger de Wit2 and Gilbert Camoin3 (1) Unite´ de Recherche Came´lia, Centre IRD de Noume´a, BP A5, 98848 Noume´a, Nouvelle Cale´donie (2) CNRS-Universite´ Montpellier II, UMR 5119 ‘‘Ecosyste`mes lagunaires’’, Universite´ Montpellier II, Case 093, 34095 Montpellier Cedex 05, France (3) Cerege, UMR 6635 CNRS-Universite´ d’Aix-Marseille III, Europole Me´diterrane´en de l’Arbois, BP 80, 13545 Aix en Provence cedex 04, France Received: 5 May 2004 / Accepted: 27 July 2004 / Online publication: 28 July 2005

anobacterial stromatolites observed in other shallow environments. However, the microbialites studied here were characterized by a lower respiration / production ratio which indicates a higher growth efficiency.

Abstract

Microbialites are organosedimentary deposits that have built up as a result of the growth and binding of detrital sediment by a benthic microbial community. This study focuses on microbialites built by monospecific populations of cyanobacteria in the south-west lagoon of New Caledonia, where they have been observed down to 20–25 m depth. The aim was to study their photosynthetic and respiratory responses to various light intensities. The Phormidium sp. TK1 microbialite was collected at 19 m depth and the P. crosbyanum (Tilden) microbialite was collected at 0.5 and 13 m depth. Phormidium sp. TK1 showed all the characteristic features of a low-light adapted species. The initial slope of the Photosynthesis versus Irradiance curve for this microbialite was close to the maximum quantum yield indicating an efficient light absorption and utilization at low light. The photosynthesis maximum was located 0.2–0.4 mm below the surface and did not shift with changing light intensity. Respiration rates were low and not enhanced by light; photoinhibition was observed at higher light intensities. In Phormidium crosbyanum (Tilden) microbialites, the photosynthesis maximum shifted downward to lower depths with increasing light, probably as a result of phototactic migration of cyanobacterial filaments, and light-enhanced respiration was observed at light intensities above light saturation. The photosynthetic parameters measured in P. crosbyanum indicate that P. crosbyanum is capable of photo-acclimation at high light intensities. The gross productivity of the different microbialites was comparable to values measured in cy-

Introduction

Microbialites are organosedimentary deposits that have built up as a result of benthic microbial community growth in concert with trapping and binding of detrital sediment and/or forming the locus of mineral precipitation [12]. Microbialites can be separated according their internal structure, into stromatolites (laminated structures), thrombolites (clotted structures), cryptic microbialites (patchy texture), oncolites (concentric laminations), and spherulitic microbialites (spherular aggregates) [30, 45]. Microbialites are characterized by a close interaction between the building microorganisms, the colonized surface, and the surrounding environment. However, the nature and extent of the processes involved in the formation of microbialites have rarely been identified and even more rarely quantified [9, 21, 45]. Marine microbialites have been abundantly reported in the fossil record: in the Precambrian, which bears the earliest microfossils at the Earth’s surface [43], and throughout the Phanerozoic until Quaternary times [13, 14]. Based on the similarities between the microfossils included in Precambrian stromatolites and cyanobacteria, it has been suggested that oxygenic photosynthesis was carried out by cyanobacteria 3.5 billion years ago [24]. Modern microbialites, including stromatolites, which that are usually called ‘‘microbial mats,’’ have been reported in very diverse environments, from freshwater [35] to hypersaline systems [23], and can thrive under a wide range of different temperatures, from polar regions

Correspondence to: Olivier Pringault; E-mail: olivier.pringault@ noumea.ird.nc

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DOI: 10.1007/s00248-004-0102-y

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to very hot deserts. While in temperate climates, large living microbial mats and microbialites are mostly restricted to so-called extreme environments where grazing pressure is reduced [27], in brackish and marine tropical environments these microbial structures are probably more widespread. Indeed, microbial mats cover large surface areas in Polynesian brackish ponds [37], and microbialites built by cyanobacteria are common in tropical lagoons such as those described for French Polynesia [45] and in New Caledonia (Camoin and Golubic, unpublished results). The taxonomy of microbialite-forming cyanobacteria in French Polynesia has been studied using a polyphasic approach [1, 2]. As typical for benthic organisms, microbialite-forming cyanobacteria require a substratum to adhere to. In tropical environments, the development of microbialites has been observed on various substrates including sand, phanerogams, thalli of green algae (Halimeda), and dead or living corals [45]. In coral reef systems, microbialites may compete with corals, which have similar light requirements and a phototrophic growth mode. As a consequence, the development of microbialites may severely affect reef-building organisms (Camoin et al., unpublished results). Light is, of course, a major concern in studies devoted to natural photosynthetic assemblages. Many studies have been carried out in order to understand and analyze the light adaptations of the phototrophic stromatolite-building microbial communities: their responses to changes in light intensity [18, 51] and to variations in light quality, with particular emphasis on the effects of UV exposure [6, 20, 32]. However, little is known about the light adaptations of microbialiteforming communities in tropical lagoons [45]. These ecosystems are characterized by clear waters with a very low suspended particle load [31]. As a consequence, the photosynthetically active radiation (PAR: 400–700 nm) can penetrate very deep into the water column, thus allowing the growth of phototrophic organisms from the surface down to several meters depth. In very clear waters, corals can usually grow down to a depth of 50 m [48, 49], and microbialites have frequently been observed at depths of 20–25 m on the flanks of pinnacles and islets in the lagoon of Tikehau, French Polynesia [45]. The aim of this work was to study the responses of microbialites to various light conditions, with special attention to the processes affecting the oxygen dynamics, i,e., photosynthesis and respiration. For that purpose, oxygen dynamics were measured with oxygen microsensors under different light conditions for microbialites that were collected in various environmental conditions characterized by distinctive light intensities. Two microbialite-building cyanobacterial species were compared. Furthermore, microbialites built by Phormidium crosbyanum Tilden were observed both in shallow envi-

ronments (0.5 m depth) and at depths below 10 m, which allowed us to study the in situ light acclimatization of this species.

Materials and Methods Sampling Site. A variety of microbialites, including domes, shapeless gelatinous masses, and horizontally spreading mats of various shapes and sizes, occur in the Southwest lagoon of New Caledonia, especially on and in the vicinity of the flanks and the slopes of pinnacles and islets at depths ranging from 0 to 25 m (see Fig. 1). These structures differ in appearance, species composition, and mode of growth, as well as in their relationship to the substrate they grow on. Microbialtes were sampled in the field following macroscopic identification of the dome-shaped pigmented colonies (referred to as ‘‘thalli’’ in the phycological literature) based on descriptions by S. Golubic (Camoin and Golubic unpublished results). The microbialites studied were produced by monospecific populations of filamentous cyanobacteria belonging to the Phormidium genus, ie., Phormidium crosbyanum Tilden [22] and Phormidium sp.TK1 [1, 2]. Microbialite domes of Phormidium sp.TK1 and P. crosbyanum were collected in the southwest lagoon of New Caledonia, at depths of 13 m (Tabu Reef) and 19 m (M’Bo islet), respectively (Fig. 1). Another P. crosbyanum isolate was collected in a very shallow back-reef setting near Bourail (Poe beach), about 200 km north of Noume´ a. In these lagoonal and back-reef environments, microbial domes develop on sandy sediments and are associated with green algae (Halimeda), flocculose cyanobacterial mats, and sea grass (Halophilda) and, less commonly, on coral fragments and live massive coral colonies; they usually coat fronds of Halimeda opuntia. In the south-west lagoon of New Caledonia, the average scalar irradiance intensity is around 60 mol photons m)2 d)1 (J-P. Torre´ ton pers. comm.). This gives an average value of 1388 lmol photons m)2 s)1 considering 12 h of sun. The attenuation coefficient of the PAR at the sampling sites ranges from 0.07 to 0.1 m)1. Therefore, average scalar irradiance intensity at the depths where microbialites were sampled ranges from 1250 to 1300 lmol photons m)2 s)1 at 1 m depth (P. crosbyanum / Poe), from 378 to 560 lmol photons m)2 s)1 at 13 m depth (P. crosbyanum / M’Bo), and from 207 to 367 lmol photons m)2 s)1at 19 m depth (Phormidium sp. TK1 / Tabu). Microbialite samples were carefully collected by SCUBA-diving and then stored in seawater collected at the same place and depth. After sampling, microbialites were rapidly transferred to the laboratory for the microsensor measurements.

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Figure 1. Sampling map of the study site

Microelectrode Measurements of Oxygen and of Microbialites were posiOxygenic Photosynthesis.

tioned in a flow cell [36] and seawater was circulated through the chamber to create a smooth flow over the microbialite. Seawater was maintained at 26 ± 1C, which corresponded to the in situ temperature. The surface of the microbialite was illuminated by a collimated light beam using a 150 W fiberoptic tungsten halogen lamp. Various light intensities were obtained with an internal neutral density filter. Light intensities at the microbialite surface were determined with a photon irradiance meter (Licor LI 92). Oxygen concentration was measured using a Clark type oxygen microelectrode [40] manufactured by Unisense (Denmark). The electrode had a 90% response time < 1 s and a stirring sensitivity of 1%. The electrode was mounted on a motorized micromanipulator and oxygen vertical profiles were performed with a vertical resolution of 100 lm. The oxygen microprobe was manually positioned at the microbialite

surface; profiling and data acquisition were then controlled by computer. Linear calibration of the microprobe was determined from the electrode readings in air-saturated water above the microbialite and in the anoxic part of the microbialite. Oxygen concentration in air-saturated water was calculated from the solubility equation according to Garcia and Gordon [19]. Gross Photosynthesis (GP) was determined using the light-dark shift technique as described by Revsbech and Jørgensen [41]. The gross rate of photosynthesis was estimated as the initial decrease of oxygen at a specific depth during the first 4 s after dark shift. Depth profiles of photosynthesis were measured with a vertical resolution of 200 lm. Calculations of Oxygen Fluxes, Respiration, and Oxygen profiles and Parameters of Photosynthesis.

gross photosynthesis measurements were performed under different light intensities. From the steady-state

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profiles of oxygen, fluxes and respiration rates were determined according to the procedure described by Ku¨ hl et al. [33], with oxygen production rates and respiration rates expressed as areal rates. Areal rates of gross photosynthesis (AGP) were calculated by integrating over depth the vertical gross photosynthesis profile and then corrected with the porosity (/) of the microbialite that was assumed to be equal to 0.90. This value is typically observed in the top most millimeters of cyanobacterial biofilms [18, 51]. The Net Productivity of the biofilm (NP), which is equivalent to the flux of oxygen across the microbialite surface–water interface into the water column, was calculated from the oxygen gradient in the diffusion boundary layer at this interface: dC ; ð1Þ dz where dC/dz represents the oxygen concentration gradient in the diffusion boundary layer above the microbialite surface and D0 the diffusion coefficient of oxygen in water. D0 was calculated from Brocket and Peng [8] and was equal to 2.42 · 10)5 cm)2 s)1. The downward flux of oxygen from the photic zone toward the aphotic interior of the microbialite (Japhot) was calculated according to Fick’s first diffusion law: NP ¼ D0

dC ð2Þ dz where dC/dz represents the oxygen gradient directly below the photic zone and Ds represents the diffusion coefficient of oxygen in the microbialite. Because precise determination of Ds remains delicate, we have used the Ulmman and Aller [46] equation (Ds = /2 · D0). This gives a Ds value of 1.96 · 10)5 cm)2 s)1, which is comparable to other Ds coefficients determined for cyanobacterial biofilms [50]. The photic zone was determined from the depth gross photosynthesis profiles. The net productivity of the photic zone (Pn): ð3Þ Pn ¼ jNPj þ Japhot Japhot ¼ /Ds

the respiration of the photic zone (Rphot): Rphot ¼ AGP  Pn

ð4Þ

and the respiration of the microbialite in the (Rdark): dC ð5Þ dz where dC/dz represents the steady-state oxygen concentration gradient in the diffusion boundary layer above the microbialite after prolonged dark exposure. Rdark ¼ D0

The photosynthesis versus irradiance curves (P vs I curves) were fitted to the data according the Jasby and Platt model [25]. When photoinhibition occurred, the model of Platt et al. [38] was used. Parameters of photosynthesis following the nomenclature and definitions of Sakshaug et al. [42] were obtained from the fitted P vs I curves as follows: Pmax is the maximum areal rate of photosynthesis (lmol O2 m)2 s)1); dividing this value by the areal chlorophyll a content of the 4-mm top layer of the microbialite we obtained the maximum photosynthesis rate [42], P*m [mol O2(mg Chla))1 s)1]. Because both the areal rate of photosynthesis and the irradiance have the product of surface and time (m2Æs)1) in their denominators, the initial slope a of these P versus E curves is directly expressed in units mol O2 (mol photons))1. This a parameter is comparable to the maximum quantum yield /m, although it is important to realize that a is defined in terms of ambient light (irradiance), while /m is expressed in terms of photons absorbed by photosynthetic microorganisms. The maximum light utilization coefficient [42], a* was obtained by dividing the value of a by the areal chlorophyll a content of the 4-mm top layer of the microbialite and expressed in units mol O2 m)2 (mg Chla))1 (mol photons))1. The light saturation parameter Ek [42] was calculated as follows: Ek ¼ Pmax =a

ð6Þ

where Pmax, is the maximum areal rate of photosynthesis, and a is the initial slope of the P versus E curve. The compensation point irradiance, Ec, corresponds to the irradiance where NP = 0, and was calculated as follows: Ec ¼ Ek tanh1  ðR=Pmax Þ

ð7Þ

At irradiance values below Ec the microbialite acts as an oxygen sink, and above Ec the microbialite acts as an oxygen source. HPLC Analyses of Pigments. Immediately after microsensor measurements, microbialites were stored in the dark at )80C. The frozen microbialites were cored manually using 8-mm i.d. copper cores with cutting edges, and the 4-mm top layer slice comprising the photosynthetically active zone was sampled for pigment analyses by cutting with a razor blade. Three cores were processed for each microbialite sample. Pigments were extracted, after freeze-drying, by both grinding and sonicating each sample for 30 s in 10 ml of 100% methanol (high performance liquid chromatography [HPLC] quality, PROLABO, France) [10]. Two replicate extractions were pooled and the combined extract was filtered through polytetrafluoroethylene (PTFE) filters (0.2 lm). Pigments were separated by reverse-phase HPLC using

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Figure 2. Photographs of microbialites built by cyanobacteria. (A) A microbialite dome structure built by Phormidiwn crosbyanum Tilden developed around the stem of the green alga Halimeda opuntia. (B) Close-up of a purple-reddish microbialite dome built by Phormidiun crosbyanum Tilden. (C) Cross-section of a microbialite dome built by Phormidium crosbyanum Tilden. (D) A microbialite dome structure built by Phormidium sp. TK1. (1) Microbialite dome built by Phormidium crosbyanum Tilden;(2) green alga Halimedea; (3) superficial photosynthetically active layer; (4) elastic organic matter mainly comprising empty cyanobacterial sheaths; (5) blackish area indicating bacterial degradation of organic matter in the dome; (6) hollow open area in the center of the dome.

Thermo-Finnigan equipment (Thermo-Finnigan, France) on a Lichosphere RP8 (5 lm, 25 cm, 4-mm i.d) column (Cluzeau, Saint-Foy, France) following the method of Barlow et al. [4], using a binary gradient from A (70% methanol/ 30% 1 M aqueous ammonium acetate buffer, pH = 7.2) to B (methanol). The methanol extracts were diluted with aqueous ammonium acetate (final content 30%) four min prior to injection by a Thermo-Finnigan (TSP AS3000) sample preparation and automatic injector. A Thermo-Finnigan UV6000 diode-array spectrophotometer was programmed to obtain the on-line absorption spectra from 320 to 800 nm. Pigments were identified by comparing their retention time and absorption spectra with those of authentic standards (International Agency for 14C Determination) and quantified using the areas and response factors at 440 nm. Results

The microbialite domes used for microsensor studies were in a healthy state and showed only very localized areas of advanced degradation (Fig. 2) and little evidence of internal calcification. The studied microbialites correspond to domes of up to 10 cm in diameter and 8 cm in height. Figure 2 shows photographs of different microbialite domes in their natural setting and a cross-section of Phormidium crosbyanum / M’Bo. These domes comprise hemispherical

Microbialite Morphology.

and spherical monospecific cyanobacterial colonies, which usually start growing from a point source, from which they expand upward and outward in all directions. In the process, the active cyanobacterial population grows upward from the substratum into the water column. As in horizontally spreading microbial mats, the live trichomes migrate and concentrate along the surface of the structure. The interior of the structure (see Fig. 2C) is comprised mostly of empty cyanobacterial polysaccharide sheaths, heterotrophic bacteria, and entrapped and precipitated carbonate grains (Camoin et al., unpublished results). Phormidium crosbyanum colonies are externally soft; they are either rose-red or bluish-violet in color because of their phycoerythrin content (see Fig. 2). P. crosbyanum colonies exhibit fine, darkly colored vertical ridges terminating in numerous 1–3-mm vertically oriented tips. Older colonies are increasingly calcified, with central depressions and are still expanding outwardly. Live filaments are restricted to a 1–2-mm-thick surface layer. They are fairly rigid, oriented perpendicular to the surface of the structure, are about 1 lm wide, and contain single trichomes within thin, but well-defined sheaths, similar to those described for this species sampled from Tikehau lagoon [2]. According to the morphological classification system proposed by Anagnostidis and Koma´ rek. [3], this species belongs to the genus Leptolyngbya.

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Figure 3. Oxygen profiles in three microbialites under saturating light (open squares) and dark (solid squares) conditions. The light intensity indicated in each panel represents the saturating downwelling irradiance for the different microbialites. The bars represent the corresponding gross photosynthesis profile, average + standard deviation (n = 4).

The microbialite sampled at 19-m depth at Tabu showed a striking resemblance, both macroscopically and microscopically, to a Phormidium sp. studied in Tikehau Lagoon, which has provisionally been described as Phormidium sp. TK1 [1, 2]. This species forms reddishbrown colonies with a smooth velvety surface, similar in size (up to 15 cm in diameter) and consistency to those of P. crosbyanum. It differs from the latter in color and filament size and has a lesser degree of calcification. As in other cyanobacteria with globular colony formation, live filaments are 4–5 lm wide and are restricted to the colony surface. They are sheathed with single trichomes which are curved and intertwined [2]. Oxygen Profiles and Gross Photosynthesis under Oxygen was consumed by the three Saturation Light.

microbialites in the dark. Prolonged incubation in the dark resulted in a steady-state oxygen profile characterized by an anoxic interior and a diffusive penetration of oxygen from the water column into the dome to a depth of less than 1 mm (Fig. 3). Exposure to saturating light conditions led to a strong accumulation of oxygen below the microbialite surface, with concentrations of 1100 to 1350 lM, equivalent to 5.5 to 6.75 times air saturation, respectively. Below 3-mm depth, oxygen concentrations continued to rise with time, which indicated that in the interior of the dome, respiration did not compensate for diffusive oxygen delivery. Thus, during the light period, the interior of the dome builtup a reservoir of oxygen, which then functioned as the source of oxygen during the first hours of the dark period. As a result, the oxygen profiles in the light did not achieve full steady-state conditions, at least not for the lower part of the micro-

bialite, below the photic zone. Nevertheless, the profiles depicted in Figure 3 correspond to pseudo-steady-state conditions, particularly in the oxygen peak, and the variations of oxygen concentration (dc/dt) were negligible, which allowed us to apply the light–dark shift technique. The light indicated in each panel of Fig. 3 corresponds to a saturation light intensity for each microbialite. The corresponding gross photosynthesis profile is also shown. The highest oxygen values were observed for P. crosbyanum / Poe. Oxygen maxima were observed at around 0.5 mm depth for Phormidium sp. TK1 / Tabu and P. crosbyanum / M’Bo, whereas in P. crosbyanum / Poe oxygen peaked at 1.25 mm depth. For the three microbialites, below the oxygen maximum, oxygen decreased very slowly, indicating that respiration rates were low within the aphotic zone. At 3 mm depth oxygen concentration was still very high, circa 400 lM for Phormidium sp. TK1 / Tabu and P. crosbyanum / M’Bo and more than 1000 lM for P. crosbyanum / Poe. The shape of the oxygen profiles suggested that diffusion was the major process affecting oxygen distribution below 2.5 mm; this was subsequently confirmed by modeling analysis using the Berg model [7] (data not shown). Under saturating light conditions (Fig. 3), the gross photosynthesis maximum was at the same depth horizon as the maximum oxygen concentration. The photic zone was 1 mm and 1.4 mm for Phormidium sp. TK1 / Tabu and P. crosbyanum / M’Bo, respectively. In P. crosbyanum / Poe, gross photosynthesis was measurable down to 2.2 mm depth. The highest maximum gross photosynthesis rate was observed for Phormidium. sp. TK1 / Tabu, with a value of 5.5 nmol O2 cm)3 s)1 at 0.4 mm below the surface. The lowest maximum rate was observed in

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Figure 4. Gross photosynthesis profiles under different light intensities in three microbialites. Numbers on graphs represent the downwelling irradiance measured at the microbialite surface, in lmol photons m)2 s)1.

P. crosbyanum / Poe—i.e., 3.01 nmol O2 cm)3 s)1 at 1.2 mm below the microbialite surface. Oxygen Processes under Various Light Intensi-

Microbialites were exposed to various light intensities, and metabolic activities were estimated from oxygen steady-state profiles and gross photosynthesis depth profiles. Results of Pgross depth profiles are depicted in Figure 4 for the different light intensities. For Phormidium sp.TK1 / Tabu, the maximum of Pgross was found at 0.2–0.4 mm, depth and it varied very little among the light intensities used. In contrast, for both P. crosbyanum microbialites, the maximum of Pgross shifted downward with increasing light intensities—i.e., from 0.4 mm to 0.8 mm depth for P. crosbyanum / M’Bo and from 0.4 mm to 1.4 mm depth for P. crosbyanum / Poe. This was probably due to phototactic migration of the cyanobacterial filaments achieved to position themselves at the depth horizon where optimal light intensities occur. Areal rates of oxygen fluxes versus light intensities are presented in Figures 5, 6 and 7 for Phormidium sp.TK1 / Tabu, P. crosbyanum / M’Bo, and P. crosbyanum / Poe, respectively. In Phormidium sp. TK1 / Tabu, gross oxygen production was measurable at very low light intensity (i.e., 3 lmol photons m)2 s)1) within the upper surface. Increasing the light intensity to 100 lmol photons m)2 s)1 led to a regular increase of the volumetric rates of photosynthesis (Fig. 4). As a consequence, the net productivity (NP) and the productivity of the photic zone (Pn) also, increased to reach a plateau at 52 lmol photons m)2 s)1 with corresponding values of 17 and 20 nmol O2 cm)2 min)1, respectively. The compensation point irradiance (Ec), which represents the light intensity where consumption equals production (i.e., NP = 0), was approxties.

imately 6 lmol photons m)2 s)1. The difference between gross and net productivity (AGP-NP) did not vary significantly with light intensity, and the respiration within the photic zone remained low. Exposure at 170 lmol photons m)2 s)1 led to a significant decrease of gross photosynthesis production (Fig. 4). As a consequence, NP and Pn also decreased at this light intensity (Fig. 5). This result clearly indicates that photoinhibition occurred. For P. crosbyanum, response to light variations differed as a function of the sampling location. For the ‘‘deeper lagoonal’’ P. crosbyanum (M’Bo site), the compensation point irradiance Ec was approximately 14 lmol photons m)2 s)1, which was slightly higher than observed for Phormidium sp. TK1/Tabu. Below 100 lmol photons m)2 s)1, NP, Pn, and AGP rates were remarkably similar, indicating that respiration in the photic zone was virtually zero. Ek was 73 lmol photons m)2 s)1; nevertheless, while gross photosynthesis production was still stimulated at irradiance values above 100 lmol, photons m)2 s)1, net productivity decreased. This was due to the onset of respiration within the photic zone at light irradiances above 100 lmol photons m)2 s)1, after which Rphot increased with increasing light intensity. For the P. crosbyanum collected in the back reef setting ‘‘surface’’ type (Poe site), below 200 lmol photons m)2 s)1 gross photosynthesis and net production concomitantly increased with light. As also observed for the deep type. NP, Pn, and AGP rates were quite similar, therefore no significant respiration was measurable in the photic zone (Fig. 7). The compensation point irradiance Ec was much higher than the one measured for the lagoon specimen; 102 lmol photons m)2 s)1 versus 14 lmol photons m)2 s)1. Exposure to light above 200 lmol photons m)2 s)1 led to a strong stimulation of both AGP and Rphot. No photoinhibition was observed for gross

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Figure 5. Areal rates of oxygen production and consumption in

the Phormidium sp. TK1 sampled in Tabu reef. AGP: areal goss photosynthesis. Pn: net productivity of the photic zone. NP: net productivity of the whole biofilm. Rphot: respiration in the photic zone. Japhot: flux of oxygen from the photic to the aphotic zone of the microbialite. See Methods for details of the calculations. Vertical bars represent the standard deviation (n = 3).

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Figure 6. Areal rates of oxygen production and consumption in the Phormidium crosbyanum Tilden sampled in / M’Bo islet. See Fig. 5 for description of the parameters. Vertical bars represent the standard deviation (n = 3).

production and net productivity. However, we were limited by our artificial light supply and therefore we could not expose the microbialite to a light intensity greater than 1400 lmol photons m)2 s)1. As a consequence we cannot exclude that photoinhibition may occur above this light intensity. Pigments and Photosynthetic Parameters. The pigment composition and the photosynthetic parameters of the different microbialite samples are given in Table 1. Pmax, Ek, and a have been estimated from the fitting of AGP versus light intensity with the model of Jasby and Platt [25] (see Methods). The highest a (0.106 mol O2 mol photons)1) was observed for the rnicrobialite sampled at 19 m depth (Phormidium sp. TK1/Tabu), whereas the lowest value (0.009 mol O2 mol photons)1) was observed for the ‘‘shallow’’ P. crosbyanum microbialite sampled in the back reef setting (Poe site). The lowest value for the light saturation parameter (Ek) was observed for Phormidium sp. TK1 Tabu (34 lmol photons m)2 s)1) whereas the highest value was calculated for P. crosbyanum / Poe, 632 lmol photons m)2 s)1. P. crosbyanum / M’Bo was intermediate between Phormidium sp. TK1 / Tabu and P. crosbyanum / Poe for Ek a, and Ec parameter

Figure 7. Areal rates of oxygen production and consumption in the Phormidium crosbyanum Tilden sampled in / Poe beach. See Fig. 5 for description of the parameters. Vertical bars represent the standard deviation (n = 3).

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Table 1. Photosynthetic parameters and pigment composition of the different microbialites

Ek (lmol photons m)2 s)1), Light saturation parameter Ec (lmol photons m)2 s)1), Compensation poin irradiance a (mol O2 mol photons)1), Initial slope of AGP versus E curve a*[mol O2 m2 (mg Chla))1 mol photons)1] Maximum light utilization coefficient Pmax (lmol O2 m)2 s)1), Maximum value of AGP P*m [mol O2 (mg Chla))1 s)1) Maximum photosynthetie rate Chlorophyll a (mg m)2) Myxoxanthophyll (mg)2) Zeaxanthin (mg m)2) b-Carotene (mg m)2) Estimated in situ light intensity (lmol photons m)2 s)1)

Phormidium sp. TK1 / Tabu

Phormidium crosbyanum / M’Bo

Phormidium crosbyanum / Poe

34 (±0.90)

73 (±1.50)

632 (±33)

6 (±0.5)

14 (±0.9)

102 (±0.9)

0.106 (±0.002)

0.042 (±0.002)

0.009 (±0.001)

1.33 · 10)3

0.28 · 10)3

0.089 · 10)3

3.57 (±0.042)

3.10 (±0.092)

5.62 (±0.27)

44.6 · 10)9

20.5 · 10)9

55.6 · 10)9

80 (±1) — 2.9 (±0.3) 4.1 (±0.6) 207–367

151 (±9) 1.3 (±0.2) 4.5 (±0.3) 9.4 (±0.2) 4378–560

101 (±22) 5.7 (±1.1) 10.6 (±1.4) 7.7 (±2.4) 1250–1300

Values in parentheses represent the standard error (n = 3). Estimated ranges of light intensities during sunny days are included based on surface irradiance measures and water column attenuation coefficients (see Methods).

values, but, in contrast, P. crosbyanum / M’Bo showed the lowest Pmax value of all three samples. P. crosbyanum / M’Bo had the highest chlorophyll content (151 mg m)2 ) of all three samples. Phormidium sp. TK1 / Tabu had the lowest contents of chlorophyll (80 mg m)2) and of zeaxanthin and b-carotene, and it lacked myxoxanthophyll. Both P. crosbyanum samples contained that latter pigment, although in different forms. P. crosbyanum / M’Bo contained a more hydrophobia form of myxoxanthophyll than P. crosbyanum / Poe, as shown by their HPLC retention times of 25 min and 21.7 min for the samples from M’Bo and Poe, respectively. P. crosbyanum / Poe also had more than three times as much myxoxanthophyll and more than twice as much zeaxanthin, although P. crosbyanum / M’Bo, had a slightly higher b-carotene content (22%). The a/Chla ratio or a*, the maximum light utilization coefficient, was particularly high for Phormidium sp. TK1 / Tabu (i.e., 1.33 · 10)3 mol O2 m)2 (mg Chla))1) and 5 to 15 times lower in P. crosbyanum for the ‘‘deeper’’ M’Bo and ‘‘shallow’’ Poe samples, respectively. The maximum photosynthetic rate scaled to chlorophyll a content, Pm*, showed much smaller variations (2.5-fold among samples). It was lowest for P. crosbyanum / M’Bo (20.5 nmol O2 (mg Chla))1 s)1), intermediate for Phormidium sp. TK1 / Tabu, and highest for P. crosbyanum / Poe. Discussion Irradiance Regulation of Photosynthesis and RespiraCyanobacterial microbialites in tropical oligotion.

trophic marine environments, particularly those occurring in shallow environments, are exposed to high

light intensities (see Table 1). It is surprising that for the three microbialites studied, the estimated in situ light intensities were much higher than their light saturation parameter Ek (cf. Table 1). This indicates that under in situ conditions, microbialites would exhibit either their maximal productivity or a lower activity due to high light stress. Short-term incubations of Phormidium sp. TK1 at light intensities above 100 lmol photons m)2 s)1 showed a clear inhibition of gross productivity (AGP) and a parallel decrease of net productivity (Fig. 5), indicating that Phormidium sp. TK1 is susceptible to photoinhibition. In contrast, there was no decrease of AGP at the studied high light intensities for the two P. crosbyanum microbialites. Hence, the P versus E curve of Phormidium sp. TK1 / Tabu suggests that under average in situ conditions (i e., 207–307 lmol photons m)2 s)1), this microbialite would be exposed to photo-inhibiting light conditions. However, in our study, irradiance was supplied by a focused collimated light beam, which gives a strongly anisotropic light field, which can give rise to a 100% increase of scalar irradiance in the surface layers of a highly refractive medium such as cyanobacterial biofilms [34]. The in situ light conditions at 19 m are characterized by an isotropic light field of different spectral composition [20] that is probably less stressful for the photosynthetic apparatus. As a consequence, photoinhibition for Phormidium sp. TK1 / Tabu under in situ conditions may not occur. The two P. crosbyanum microbialites adopted similar short-term strategies when exposed to light intensities exceeding their light saturation parameter Ek; these strategies were not observed in Phormidium sp. TK1. First at light levels above Ek, AGP remained almost constant or

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continued to increase slightly (Figs. 6 and 7), the gross photosynthesis depth distribution (Pgross, see Fig. 4) indicated downward migration of cyanobacterial filaments at increasing light intensities. This migration is a way to position their photosynthetic apparatus in the light gradient at scalar irradiance values that are more optimal for photosynthesis. Migration of the filaments along the light gradient is facilitated by their perpendicular orientation with respect to the surface in this species. Similar migratory behavior has been observed in cyanobacterial mats exposed to UV-rich radiation [6]. Secondly, respiration in the photic zone (Rphot), which was extremely low at irradiance values below Ek, appeared at higher irradiance and continued to increase with increasing light intensities. Thus, P. crosbyanum shows light-enhanced respiration at high light intensities. Light-enhanced respiration may include photorespiration, a process that is stimulated at a high O2/CO2 ratio [39, 51, 52]. Photorespiration is catalyzed by RuBisCo and is not, despite its name, directly dependent on light. Thus, we expect that photorespiration continues during the first four seconds after the light to dark shift as used during the Pgross measurements, and that according to Eq. (4), Rphot thus includes photorespiration if it occurs (see Methods). Actually, under high light conditions above Ek, the oxygen concentrations were high in both microbialites (i.e., more than 4 times air saturation; see Fig. 3) and pH was near 9 at the horizon of the oxygen peak (data not shown). High pH leads to a low concentration of CO2. However, some observations indicate that photorespiration is not important in some cyanobacterial species that possess an inorganic carbon concentrating mechanism [16]. Light-enhanced respiration can be considered a protective mechanism at high light intensities because it counterbalances the photosynthetic accumulation of nuisance oxygen and the depletion of CO2. Lightenhanced respiration can also be a consequence of downward migration of cyanobacterial filaments in microbialites, because it increases the diffusion distance of oxygen to the water column. As a result, the residence time of oxygen in the photic zone is increased, which stimulates Rphot and thus reduces NP [17]. While downward migration appears as a very good strategy to evade the potentially inhibiting surface light intensities, the trade-off is that cells become increasingly separated from the water column, which acts as a sink for oxygen. Light-enhanced respiration in the ‘‘surface’’ type P. crosbyanum / Poe resulted in stabilization of Pn and NP between 700 and 1400 lmol photons m)2 s)1 (see Fig. 7), whereas for the ‘‘deeper’’ P. crosbyanum / M’Bo the same phenomenon resulted in a decrease of Pn and NP with increasing light intensities above 200 lmol photons m)2 s)1 (see Fig. 6). This shows that P. crosbyanum / Poe was acclimated to the high light intensities to which it was

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exposed in the field (cf. Table 1). Compared to P. crosbyanum / M’Bo, the maximum light utilization coefficient a* of P. crosbyanum / Poe was reduced threefold, while it showed a 2.5-fold higher Pm* than the former (see Table 1). This indicates a strong reduction of the Photosystem II (PS II) optical cross section rPSII for P. crosbyanum / Poe, which could be due to photochemical quenching and heat dissipation by the accessory pigments myxoxanthophyll and zeaxanthin and by increased packaging of chlorophylls [42]. In contrast, the ‘‘deeper’’ P. crosbyanum / M’Bo showed some typical features of low light acclimation, although it was less well adapted to low light than Phormidium sp. TK1 / Tabu. Phormidium sp. TK1 / Tabu showed a 5 to 15 times higher maximum light utilization coefficient than P. crosbyanum. The initial slope of the AGP versus E curve (a) was 0.106 mol O2 (mol photons))1, which is very close to the theoretical maximum quantum yield of 0.125 mol O2 (mol photons))1. This indicates that Phormidium sp. TK1 / Tabu very efficiently absorbed and photosynthetically processed most of the photons available at low light intensities. In addition, respiration rates were low in this microbialite, which adds further advantage at low light intensities. In conclusion, P. crosbyanum is able to cope with a large range of light intensities; i.e., it acclimates very well to high light at the surface (0–1 m depth), and it is also able to acclimate to lower light intensities at depths below 10 m. In contrast to P. crosbyanum, Phormidium sp. TK1 appears to be a typically low-light–adapted species that most likely is incapable of acclimation to the very high light intensities close to the surface. These ecophysiological data nicely explain the depth distributions of both species in the lagoon. Hence, P. crosbyanum has been found between 0 and 18m deep, but it is rare below 13 m water depth. Phormidium sp. TK1 was found between 13 and 20 m deep (maximum abundance between 13 and 15 m deep), with an occasional observation at 8 m in a more turbid area of the lagoon (Camoin et al., unpublished results). Microbialite Productivity. Microbialites also include so-called stromatolites (see the original definition by Kalkowsky [29]), which form planar laminated structures [23]. Stromatolites or microbial mats are known to be among the most productive aquatic systems [15]. The productivity of the three microbialite domes has been compared to cyanobacterial stromatolites from different habitats (Table 2). The microbialite P. crosbyanum sampled in the shallow back reef zone of Poe exhibits similar productivity rates (gross and net) and respiration rates to hypersaline cyanobacterial mats sampled in Guerrero Negro. Solar Lake, and Chiprana Lake (Table 2). These microbial mats are formed in very shallow environments, showing light conditions that are similar to those observed for the formation of the mi-

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Table 2. Gross production (AGP), net production (NP), and respiration (AGP-NP) of different cyanobacterial biofilms

Cyanobacterial biofilms Cyanobacterial mat of Ebro Delta (Spain)b Cyanobacterial mat of Solar lake (Egypt) Cyanobacterial mat of Chiprana lake (Spain) Cyanobacterial mat of Guerrero Negro (Mexico) Epilithic Cyanobacteria biofilm (Denmark) Microbialite Phormidium sp. TK1/Tabu Microbialite P. crosbyanum/M’Bo Microbialite P. crosbyanum/Poe

Gross productivity nmol O2 cm)2 min)1 36 24 26 30 19 19.7 14.1 30

Net productiona nmol O2 cm)2 min)1 28 6 8 16 10.5 17 10.5 13

(77%) (25%) (31%) (53%) (55%) (86%) (75%) (44%)

Respirationa nmol O2 cm)2 min)1

Reference

22 (61%) 18 (75%) 18 (69%) 14 (47%) 8.5 (45%) 2.7 (14%) 3.6 (25%) 17 (56%)

[18] [51] [27] [17] [33] This study This study This study

a

Numbers in parentheses represent the fraction of gross productivity (in %). Data from this study were obtained from Fig. 3. For the mat of the Ebro Delta, the gross productivity was underestimated [18].

b

crobialite in the Poe area. The ratio respiration / gross production can be used as an indicator of the internal carbon cycling. The cyanobacterial mats and microbialites formed in very shallow environments (i.e., a few meters depth) are characterized by a high respiration / gross production ratio, indicating high carbon recycling within the microbial structure. This high carbon recycling has an effect on the accretion rate. In fact, despite their high gross productivity, microbial mats are characterized by very low accretion rates, ranging from only 1 mm to 5 mm per year [5, 47]. Precise in situ accretion rate calculations remain, however, difficult, but the respiration / production ratio can be used as a proxy for the growth efficiency, which allows determination of the accretion rate. In contrast, microbialites sampled in deeper lagoonal environments (13 and 19 m depth) are characterized by lower respiration / gross production ratios, the lowest ratio (14%) being observed for the microbialite collected at 19 m deep (Phormidium sp. TK1 / Tabu). This suggests that these structures are characterized by high net growth accretion rates, which is consistent with field observations carried out in these environments. Regular monitoring carried out at 20 m depth in the south-west lagoon of New Caledonia has demonstrated that microbialites such as Phormidium sp. TK1 and P. crosbyanum can colonize solid substrates very rapidly, thus forming structures several centimeters in diameter within a few weeks or months (Camoin et al., unpublished results). The low respiration observed in the microbialite built by Phormidium sp. TK1 merits some attention because this microbialite exhibits high oxygen production rates that are of the same order of magnitude as those observed in laminated microbial mats (Table 2). In contrast to the microbialite Phormidium sp TK1, these microbial mats are characterized by high respiration rates (Table 2). In these highly productive systems, heterotrophic bacteria are stimulated by the excretion of low molecular weight compounds, such as glycolate, lactate, or propionate, excreted by the photosynthetic microorganisms [26, 27]. This tight coupling between heterotrophic respiration and oxygenic photosynthesis is not observed in the

Phormidium sp TK1 microbialite, a finding that could be explained by different hypotheses. First, cyanobacteria of the genus Phormidium are known to produce toxin [11, 28], which could have negative effects on heterotrophic bacteria [11]. Sellner suggests that the production of toxin by bloom-forming cyanobacteria may explain the inhibition of the bacterial activity observed during the bloom [44]. Second, this absence of stimulation of respiration in the Phormidium sp TK1 microbialite might be explained by a low production of dissolved organic matter by the cyanobacteria. Indeed, a recent study has shown that during blooms of the cyanobacterium Trichodesmium, (1) the production of dissolved organic matter is extremely low (around 1% of the total primary production) and (2) bacterial production (estimated with 3 H-thymidine incorporation) is not stimulated by the photosynthetic activity of Trichodesmium (Renaud et al, unpublished results). To conclude, the low respiration observed in the Phormidium sp. TK1 microbialite calls for further investigations to provide a better understanding of the interactions between the cyanobacterium and the associated heterotrophic community.

Acknowledgments

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