Aquaculture 299 (2010) 140–148
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Aquaculture j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / a q u a - o n l i n e
Light acclimation strategies of three commercially important red algal species Ronny Marquardt a,⁎, Hendrik Schubert a, Daniel A. Varela b, Pirjo Huovinen b, Luis Henríquez b, Alejandro H. Buschmann b a b
University of Rostock, Institute of Biosciences, D-18055 Rostock, Germany Centro i-mar, Universidad de Los Lagos, Camino Chinquihue km 6, Puerto Montt, Chile
a r t i c l e
i n f o
Article history: Received 10 February 2009 Received in revised form 6 November 2009 Accepted 11 November 2009 Keywords: Integrated aquaculture Photosynthesis Seaweeds Solar irradiance Macroalgae Macrophytes Production Fluorescence Non-photochemical quenching
a b s t r a c t In order to test their usability in vertical structured, integrated aquaculture approaches, the light acclimation characteristics of three commercially important red algal species of southern Chile were investigated. Light afﬁnity, maximum photosynthesis and respiration rate were measured. Analyses of the non-photochemical quenching and pigment contents were conducted. This work is focused on the differences between ﬁeldgrown algae exposed to the full range of irradiance variability in the ﬁeld caused by tidal changes, and algae grown in buoyant long-line aquacultures at different depths (1, 3, 5 and 7 m). The acclimation patterns of the respective species were clearly related to the irradiance regime of their natural habitat. However, all species were also able to acclimate their photosynthesis characteristics to conditions exceeding the irradiance ranges of their natural habitat. The results revealed that the three species followed different acclimation strategies: Gracilaria chilensis was almost completely lacking physiological acclimation. Sarcothalia crispata exhibited a combination of both morphological as well as physiological acclimation processes, while Mazzaella laminarioides exclusively used physiological acclimation mechanisms. Further differences were found with respect to their ability to survive periods of supersaturating irradiation. As expected, the subtidal S. crispata was unable to acclimate to large irradiance ﬂuctuations in shallow water, whereas the other two species adjusted their capability for dynamic photoinhibition to the respective irradiance climate. The implications of these acclimation patterns for aquaculture purposes are discussed and recommendations for appropriate positioning of these species in vertical structured aquaculture are given. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Seaweed landings are an important economic activity in Chile, producing up to 350 000 metric tons per year, involving a broad variety of Phaeophyta and Rhodophyta species. The industry is mainly based on agar and carrageenan production (Buschmann et al. 2008a). In spite of extensive seaweed production, commercial seaweed farming has existed only since the mid-eighties, and this activity is still restricted to the agarophytic red alga Gracilaria chilensis (Buschmann et al. 1995, 2001a). Several studies on other commercial red algae like Sarcothalia crispata, Mazzaella laminarioides, Gigartina skottsbergii and Chondracanthus chamissoi as well as brown algae such as Macrocystis pyrifera, M. integrifolia, Lessonia nigrescens, L. trabeculata and Durvillaea antarctica have been carried out. However, the low market value has hampered the advancement of seaweed aquaculture of these species during the last decade (see Buschmann et al. 2008a for review). On the other hand, the potential of nutrient
⁎ Corresponding author. Fax: +49 381 498 6072. E-mail address: [email protected]
(R. Marquardt). 0044-8486/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2009.11.004
ﬁxation by algae in integrated farming approaches (Troell et al. 1997; 1999; Ahn et al. 1998; Fei, 2004; Kraemer et al. 2004; Buschmann et al. 2008b) has generated an alternative economic driver as the environmental standards for top carnivore aquaculture is strongly increasing (Buschmann et al. 2006). The integration of autotrophic and heterotrophic components in aquaculture in order to mimic nutrient cycles is a complex approach. Several aspects still require optimisation, in particular the efﬁciency of nutrient removal should be improved (Buschmann et al. 2001b; Chopin et al. 2001). A mass balance model indicates that every ton of ﬁsh produced requires a nitrogen input of 91 kg. From this input only 34 kg of the nitrogen will be retained in the ﬁsh tissues and harvested. This means that 62.6% of the nitrogen input is released into the environment. According to the estimations of Mente et al. (2006) a farm with a production capacity of 1500 tonnes of salmon per year therefore releases more than 65 tons of nitrogen. To remove this nitrogen by means of a seaweed farm, the irradiance input of about 100 ha is needed (Abreu et al. 2009). It is obvious, that for such a large surface, demand optimisation of the nitrogen remediation is needed. Because the main problem in this regard is the light utilization efﬁciency of seaweed biomass formation, Buschmann et al. (2008b) proposed an approach entailing species-speciﬁc vertical extension to
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reduce farm size. In this approach the culturing of several seaweeds with different depth (irradiance) optima increases the biomass production per farming area unit (Buschmann et al. 2008b) as well as the nitrogen removal effectiveness by choosing different species with complementary nitrogen source uptake capacities (Bracken & Stachowicz, 2006). A further increase in the efﬁciency is obtained by the cultivation of the macrophytes in open suspended culture systems. These systems reduce the light variability by eliminating the inﬂuence of tidal variation. Benthic algae at tidal coasts are subjected to a highly variable underwater light climate, where attenuation dependent irradiance changes caused by tides can play a major role (Sagert, 1999; Schubert et al. 2001). Moreover, the impact of high frequency irradiance changes caused by wave focusing is depth-dependent. Wave focusing therefore differs between ﬁeld sites and buoyant cultivation systems, independent of tidal water level changes (Schubert et al. 2001). With respect to these differences between irradiance conditions at ﬁeld sites and the conditions in buoyant cultivation systems, theoretical considerations are of limited value for choosing the appropriate species. The algae at different culture depths have to acclimate to the respective light climate, which often differs largely from the light climate they are subjected to in nature. In this study we test the light acclimation strategies of three commercially important red algal species (G. chilensis, M. laminarioides and S. crispata). By comparing the light acclimation characteristics of ﬁeld-grown and cultivated specimens, the following questions were addressed: I) Does elimination of tidal variation in buoyant long-line cultures inﬂuence the light acclimation status? II) To what extent are these species able to acclimate to depths beyond their natural distribution ranges? The information obtained will be discussed with respect to the possibility of complementary depth cultivation and in order to develop a more efﬁcient seaweed bio-ﬁltering culture integrated in a salmon farm.
incubated for at least 14 days in September. After this period, 3–5 randomly-chosen individuals were taken to the laboratory for pigment analysis, determination of their photosynthesis and nonphotochemical quenching characteristics. Similar measurements were performed simultaneously with algae from natural ﬁeld sites nearby. The term “ﬁeld samples“ is used as follows for all parameters of algae taken from natural growing sites whereas the term “aquaculture samples” is used for parameters of algae acclimated for at least two weeks in long-line cultures. 2.2. Photosynthesis measurements Oxygen evolution and PSII ﬂuorescence were measured simultaneously with an Illuminova light dispenser system (‘light pipette’ MKII, Uppsala, Sweden, described by Wolfstein and Hartig, 1998) equipped with a Clarke-type oxygen electrode (Microelectrodes, Bedford, USA) and a Diving-PAM (Pulse Amplitude Modulated Chlorophyll Fluorometer, Walz, Germany, Schreiber, 1994). The two instruments were connected to the same 2.4-ml incubation cuvette ﬁlled with ﬁltered seawater and kept at constant 14 °C by a water jacket. Algal pieces were taken from the ﬁeld and kept in darkness for 1–2 h at 14 °C prior to each measurement. The samples were exposed to 0, 3, 8, 22, 45, 80, 195, 435, 840 and 2135 µmol photon m− 2 s− 1 for at least 4 min at each irradiance. 2.3. Pigmentation After the photosynthesis measurements, the pigmentation of the samples was analysed. Water was removed by means of a paper towel and the fresh weight was determined. Samples were then transferred into 2 ml N, N-Dimethylformamide (DMF) and extracted at 4 °C for at least 24 h. The absorption values of the extracts were measured and the chlorophyll content was calculated using the formula and extinction coefﬁcients of Porra et al. (1989):
2. Materials and methods
Chl a = ½ðE662 nm −E748 nm Þ × 12ðE645 nm −E748 nm Þ × 3:11:
2.1. Field site and algal material
For the pigmentation analysis as presented in Fig. 2, ﬁve samples from each group were transferred directly into DMF without being used for photosynthesis measurements beforehand. After its processing for chlorophyll determination as described above, carotenoid determination was done by means of the following equation:
The investigation site was in the Seno de Reloncaví, southern Chile (41 °S) at “CEACIMA”, the marine ﬁeld station of the Universidad de Los Lagos, which is located at the Bahia Metri (41° 36′ S, 72° 43′ W). The seaweeds used in this study were the carragenophytic algae M. laminarioides (Bory) Fredericq, S. crispata (Bory) Leister, and the agarophytic alga G. chilensis (Bird, McLachlan & Oliveira). M. laminarioides forms conspicuous and almost monospeciﬁc stands in mid-intertidal levels exposed to highly variable environmental conditions. These can affect its production patterns at local and latitudinal scales (Varela et al. 2006). S. crispata is a low intertidal to upper subtidal (down to 5–6 m) species growing in high abundances (biomasses over 500 g dry weight m− 2 during spring and summer seasons). It coexists with other red algae, e.g. Grateloupia doryphora, Trematocarpus dichotomus, and C. chamissoi (Otaíza et al. 2001) and is also present as an under storey species in kelp stands (Almanza, 2007). Therefore, this species is considered to prefer lower light intensity regimes than M. laminarioides. Finally, the agarophytic alga G. chilensis is known as a species of the upper to mid-intertidal region, which is growing and cultivated on sandy bottoms (Buschmann et al. 1995). This species, being highly tolerant to environmental stress, survives in highly variable temperature and salinity environments. However, exposure to high intensities of solar UV radiation can be detrimental during the spring–summer period (Gómez et al. 2005). For the experiments, algae of these three species were cultivated at four different depths (1, 3, 5, and 7 m) by attaching them to vertically buoyed lines. Twenty individuals of each species per depth were
Carconc ½μg ml
= ðð1000 × A470 nm Þ−3:27½Chl a−104½Chl b 229Þ
(Küster et al. 2004). 2.4. Irradiance measurements Scalar underwater irradiance was measured with a high-resolution spectroradiometer (SR-9910, Macam Photometrics Ltd. Livingston, Scotland) equipped with a 10 m light guide and a spherical light collector of 0.7 cm diameter. Successive underwater irradiance scans at depth intervals between 20 and 400 cm were used to calculate the diffuse attenuation coefﬁcient, Ko(λ) (Smith, 1968). Calibration of the absolute sensitivity and wavelength accuracy was conducted at regular intervals against voltage-stabilized deuterium and tungsten standard lamps (Macam SR-990) traceable to the National Physical Laboratory, London, UK. Continuous measurements of the surface irradiance were done by means of the above-mentioned instrument equipped with a cosine-corrected light collector, and by a cosinecorrected PAR-sensor (LiCor Inc., USA). Both units are installed close to the investigation site. “Average irradiance at depth” was calculated from these continuous measurements and PAR-attenuation, which is given by the mean irradiance value for the respective depth and daylight period.
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2.5. Data analysis Photosynthesis vs. irradiance curves (P–E curves) were established using the following equation: Pnet = ðPmax · tanhðα·Irradiance = Pmax ÞÞ + Rd (Jassby and Platt, 1976; Henley, 1993). whereby Pnet is the net O2 evolution rate at the respective irradiance, Pmax is the maximum O2 evolution rate at saturating irradiances, α is the slope of the photosynthesis vs. irradiance curve at limiting irradiances and Rd the dark respiration rate. The saturation irradiance (Ek) was calculated as Pmax /α. From the Diving-PAM obtained ﬂuorescence data (see above) were used to calculate, non-photochemical ﬂuorescence quenching, NPQ= (Fm − Fm′) / Fm′ (Krause and Weis, 1984, 1991). Because the ﬂuorescence nomenclature in this paper follows the recommendations given by Kromkamp and Forster (2003), where Fm (MT) is deﬁned as “maximum ﬂuorescence after dark acclimation then measurement during exposure to multiple turnover ﬂash” (c.f. Kromkamp and Forster, 2003), negative values of NPQ appear in cases where the organisms hold a fair amount of NPQ in darkness. This has been shown to be a common feature of phycobilisome-containing organisms, including red algae (Delphin et al. 1996, 1998; Campbell and Öquist, 1996; Campbell et al., 1998). The characteristics of the NPQ vs. irradiance relation were analysed using a bi-exponential model. This model is consisting of a component (negative exponential) responsible for the description of the state-transition dependent negative slope of the curve at low irradiances (see Campbell et al. 1998) and a second component, NPQ r, which describes the “regular” increase of NPQ due to photoprotective mechanisms (Fig. 1), i.e. the contribution of “regular” NPQ increase after saturation of state-transition component NPQ st (Campbell et al. 1998; Schubert et al. 2004). This NPQ r is fully equivalent to the NPQ components of green algae and higher plants and is also exponential, but also includes a constant “C”, indicating the irradiance at which NPQ st becomes saturated (Fig. 1). Both components are adaptations of the P/I-algorithm ﬁrst described by Webb et al. (1974). The two components are calculated separately as: ð−alphaST IStmax Þ
NPQ st = STmax ð1−e
ð−alphaNPQ ðI−CÞNPQ max Þ
NPQ r = NPQ max ð1−e
with NPQ st as a measure of the irradiance (I) dependent effect of state1 transition in the low light range sensu Campbell and Öquist (1996); characterised by the slope of the decrease of NPQ (alphaST) and the maximum value reached after completion of state 1 transition (STmax) and NPQ r, as a measure of the irradiance dependent downregulation
Fig. 1. Representative graph of NPQ versus irradiance data shows the parameters of the NPQ characteristics. These parameters are estimated from the ﬁtted curve (circles), which was derived from measured values (triangles). NPQ max correspond to the maximum NPQ value, indicated by the upper dashed line: STmax, maximum state transition, indicated by the lower dashed line; C, irradiance at which the “regular” NPQ onset to increase; Ek(NPQ), irradiance at which NPQ is saturated.
of PSII ﬂuorescence yield characterised by the slope of increase of NPQ (alphaNPQr) after a certain trigger irradiance (C) has been exceeded and the maximum value was reached (NPQ max). Consequently, EkNPQ is calculated by NPQmax / alphaNPQr. All parameters (NPQ max, alphaNPQr, alphaST, STmax, and C) were calculated by means of the “Solver” add-in of Excel, using the least square method as described by Schubert and Forster 1997. Fig. 1 shows an example of a ﬁtted equation with indications of the respective parameters achieved. Hierarchical Analysis of Variance (ANOVAs) was done to test for differences in photosynthesis parameters and pigmentation between the depth incubation groups as well as between ﬁeld samples and aquaculture samples of the respective species. For light saturation irradiance (Ek) and pigmentation data ANOVA was done for differences between the species at a given depth. Tukey-HSD tests were used to discriminate among different incubations after signiﬁcant F-tests (p b 0.05). Signiﬁcantly different groups are denoted by lower case letters in the ﬁgures. All tests were done with SPSS 15 for Windows.
3. Results 3.1. Pigment concentration and pigment ratio Fig. 2 summarizes the pigmentation data obtained from ﬁeld samples as well as from aquaculture samples. For all three species pigment content and Chl a/Car ratio of the ﬁeld samples matched with the pigmentation of an aquaculture sample from a depth equal to the ﬁeld sample's position in the tidal gradient, except for the Chl a/Car ratio of S. crispata, which was slightly higher than expected. For M. laminarioides (Fig. 2A), a decreasing tendency in carotenoid and chlorophyll a content from the intertidal (ﬁeld samples) to the greatest depth at 7 m was observed. Carotenoid content dropped from 0.35 to 0.2 µg/g FW and chlorophyll a content from 0.93 to 0.77 µg/g FW. However, as also for G. chilensis, chlorophyll a as well as carotenoid contents varied largely between the individuals, hence no signiﬁcant differences could be detected between the depth groups. The variability itself most likely originates from the parameter fresh weight. When pigment ratios are calculated from eliminating fresh weight as an inﬂuencing parameter, then clear and signiﬁcant differences between the experimental groups become obvious. The chlorophyll a/carotenoid ratio of M. laminarioides, increased signiﬁcantly from the intertidal (ﬁeld samples) to 3 m depth from 2.8 to 4.0. After this depth the value remained almost constant until 7 m. In G. chilensis (Fig. 2B) the chlorophyll a/carotenoid ratio increased signiﬁcantly from 5 to 6.5 between 3 m and 7 m depth. There were no signiﬁcant differences between ﬁeld samples and aquaculture samples from 1 m and 3 m depth. The ﬁeld samples of this species showed a slightly higher Chl a concentration than expected from the aquaculture samples' pattern. However, this difference was not signiﬁcant. Chl a/Car ratio of G. chilensis was similar to the values calculated for aquaculture samples at 1 m as well as 3 m depth. A similar observation was made for S. crispata (Fig. 2C). In comparison with the 5 m aquaculture sample, representing the relative depth of origin of the ﬁeld samples, the carotenoid content of ﬁeld samples was signiﬁcantly higher than that of aquaculture ones. However, because no differences were found in chlorophyll a concentration, the chlorophyll a/carotenoid ratio was signiﬁcantly lower in ﬁeld-grown samples than in the 5 m aquaculture samples. In general, with increasing depth a signiﬁcant increase in chlorophyll a concentration and chlorophyll a/carotenoid ratio was found in S. crispata. Its chlorophyll a concentration increased from 1.5 µg/g FW at 1 m to 3.2 µg/g FW at 7 m, and its chlorophyll a/carotenoid ratio from 4.2 at 1 m to 15.5 at 7 m.
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Fig. 2. Pigment concentration and pigment ratio of Mazzaella laminarioides (A), Gracilaria chilensis (B) and Sarcothalia crispata (C). Samples were taken directly from the ﬁeld or cultivated for two weeks in buoyant long-line cultures at different constant depths. Carotenoid concentration (Car), chlorophyll a concentration (Chl a) and chlorophyll a/carotenoid ratio (Chl a/Car) are shown (mean ± S.D., n = 5). Signiﬁcantly different groups are denoted by lower case letters. Field samples were taken from individuals collected in the intertidal for M. laminarioides, whereas G. chilensis and S. crispata were sampled at depths 2 m and 5 m below mean low water level, respectively. Please note the different Y-axis scales in panel C.
3.2. Photosynthetic parameters The chlorophyll-speciﬁc photosynthetic parameters (Pmax; alpha; R) of the three species tested are shown in Fig. 3. As for pigment content and ratio (see above), the photosynthesis parameters of ﬁeld-grown individuals matched with the respective values of aquaculture samples from comparable depths. In both aquaculture and ﬁeld samples, Pmax of M. laminarioides showed a signiﬁcant decreasing trend (R2 = 0.38, F = 16.39, df = 25) from the intertidal (ﬁeld samples) to 7 m depth with 56 to 30 mmol O2 h− 1 g Chl a− 1. On the other hand the respiration rate (R) as well as alpha did not show a signiﬁcant relation o the depth. The same held true for G. chilensis, where Pmax decreased signiﬁcant with increasing depth from 90 mmol O2 h− 1 g Chl a− 1 at 1 m depth to 70 mmol O2 h− 1 g Chl a− 1 at 5 m depth. The slight
Fig. 3. Chlorophyll a-based photosynthetic parameters of Mazzaella laminarioides (A), Gracilaria chilensis (B) and Sarcothalia crispata (C). Samples were taken directly from the ﬁeld (left columns) or cultivated for two weeks in buoyant long-line cultures at constant depths as indicated on the X-axis. Shown are: maximum photosynthesis rate (Pmax: black bars); respiration rate (R: grey bars); photosynthesis light afﬁnity (alpha: empty bars, secondary Y-axis), are shown as mean values (± S.D.) of three replicates. Signiﬁcantly different groups are denoted by lower case letters. Field samples (Field) were taken from individuals collected in the intertidal for M. laminarioides, whereas G. chilensis and S. crispata were sampled at 2 m and 5 m depth below mean low water level respectively. Please note the different Y-axis scales.
increase of Pmax at 7 m was insigniﬁcant. Respiration rate as well as alpha values did not indicate signiﬁcant depth dependency. Similar to pigment content and ratio, the photosynthetic parameters of the ﬁeld samples were equivalent to those of aquaculture samples from comparable depths for both M. laminarioides and G. chilensis. Pmax of S. crispata (Fig. 3C) decreased signiﬁcantly with cultivation depth from 32 mmol O2 h− 1 g Chl a− 1 at 1 m, to 20 mmol O2 h− 1 g Chl a− 1 at 7 m. But no signiﬁcant differences were detected for its respiration rates. Alpha increased signiﬁcantly from 0.3 to 0.35 mmol O2 h− 1 g Chl a− 1 per µmol photon m− 2 s− 1 between 1 and 3 m to 0.55 mmol O2 h− 1 g Chl a− 1 per µmol photon m− 2 s− 1 at 7 m depth. Pmax and R of ﬁeld samples did not differ signiﬁcantly from their equivalent aquaculture samples at 5 m depth. The difference between the alpha values of the ﬁeld samples and the aquaculture samples proved to be not signiﬁcant. When related to fresh weight instead of chlorophyll a, and therefore reﬂecting the morphological rather than the physiological
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acclimation, the pattern described above does not change much for M. laminarioides (Fig. 4). Fresh weight based Pmax still decreased progressively with increasing depth, whereas alpha tended to increase and respiration remained constant at about 1 mmol O2 h− 1 g FW− 1 down to 3 m (Fig. 4A). However, a signiﬁcant difference was presented between the low respiration rate of the deepest sample (7 m) and the higher respiration rates of shallow water samples. For G. chilensis, fresh weight-based Pmax did not change with depth (15–17 mmol O2 h− 1 g FW− 1), nor their respiration, which varied between 3 and 5 mmol O2 h− 1 g FW− 1 irrespective of the depth. Only alpha, raised from 0.24 to 0.34 mmol O2 h− 1 g FW− 1 per µmol photon m− 2 s− 1 between 1 and 7 m depth and showed a depth dependency. But there were still no signiﬁcant differences between the groups detectable. For S. crispata, the highest Pmax values were measured close to the surface with values of 5.0 to 5.5 mmol O2 h− 1 g FW− 1 at 1 m and 3 m respectively. At greater depths the Pmax decreased signiﬁcantly to a minimum of 2.9 mmol O2 h− 1 cm− 2 at 7 m. Respiration of S. crispata decreased insigniﬁcantly with increasing depth, whereas alpha increased signiﬁcantly between 1 and 5 m (0.05 mmol O2 h− 1 g FW− 1 at 1 m to 0.1 mmol O2 h− 1 g FW− 1 at 5 m). However, the alpha value at 7 m was not different from the 5 and 3 m values. Again, for all species tested the photosynthesis parameters of the ﬁeld samples matched the long-line sample values at the respective depths. Fig. 5 shows a plot of the Ek values versus the calculated average irradiance at incubation depth for all 3 species. Ek of
Fig. 4. Fresh weight-based photosynthetic parameters of Mazzaella laminarioides (A), Gracilaria chilensis (B) and Sarcothalia crispata (C). For symbols and explanations see legend of Fig. 3.
Fig. 5. Ek versus irradiance (PAR) plot for aquaculture-grown Mazzaella laminarioides (dotted line, ﬁlled dots), Gracilaria chilensis (broken line, ﬁlled triangles) and Sarcothalia crispata (solid line, empty dots). Algae were acclimated for two weeks in buoyant long-line cultures at 1, 3, 5 and 7 m depth, resulting in an average daily light dose (calculated from surface irradiance measurements and attenuation) of 9.6, 6.5, 4.4 and 3.0 mol photon m− 2 d− 1 respectively. For comparison with irradiance value, the daily light dose was converted to µmol photon m−2 s− 1. Shown are mean values and standard deviation of 3 independent samples each.
M. laminarioides was reduced signiﬁcantly (b0.05) from approx. 80 µmol photon m − 2 s − 1 at 1 m to 42 µmol photon m −2 s − 1 at 7 m depth. (Ek of ﬁeld samples, not shown, was about 100 µmol photon m−2 s− 1). A similar pattern was observed in S. crispata with the highest values (approx. 110 µmol photon m−2 s− 1) at 1 m and lowest (∼ 30 µmol photon m−2 s− 1) at 7 m depth. The decreasing trend of Ek observed for G. chilensis was much less pronounced and tested as insigniﬁcant. Fig. 5 also clearly reveals the differences in the acclimation strategy between the species. S. crispata, originating from the subtidal zone, reached the steepest irradiance vs. Ek relationship, whereas the Ek of the upper intertidal M. laminarioides showed only a slight increase with acclimation irradiance. Trend analyses give for the Ek vs. ambient irradiance relation by the sublittoral species S. crispata (Y = 0.42X − 0.66; R2 = 0.97), whereas the intertidal species exhibited far lower slopes (Fig. 5) (Y = 0.22X + 24.5 and Y = 0.09X + 47.5 for M. laminarioides and G. chilensis respectively with R2 = 0.99 for both linear trends). Fig. 6 shows the fresh weight per area values of the two foliaceous species M. laminarioides and S. crispata. For M. laminarioides, area-
Fig. 6. Ratio of fresh weight by area (FW/area) of Mazzaella laminarioides (empty bars) and Sarcothalia crispata (ﬁlled bars). Samples were taken directly from the ﬁeld (left columns) or cultivated for two weeks in buoyant long-line culture at constant depths as indicated on the X-axis. Shown are mean values with their standard deviation derived from 3 replicates. Signiﬁcantly different groups are denoted by lower case letters. Field samples (Field) were taken from individuals collected in the intertidal for M. laminarioides, whereas S. crispata was sampled 5 m depth below mean low water level.
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speciﬁc fresh weight remained nearly unchanged throughout the whole depth range at approximately 0.06 g cm− 2, whereas in S. crispata the mean fresh weight was observed almost the doubled amount from 0.07 g cm− 2 at 1 m to 0.124 g cm− 2 at 7 m. 3.3. Light protective capacity The non-photochemical quenching parameters revealed the differences in the light protection capabilities between the three species tested (Fig. 7). In aquaculture, all three species exhibited only a slightly, insigniﬁcant increase of the non-photochemical quenching capacity (NPQ max) with increasing depth. But S. crispata showed a marked and signiﬁcant lower NPQ max in ﬁeld samples compared with aquaculture samples (Fig. 7A). The irradiance at which the non-photochemical quenching is onset (C) exhibited both differences between species as well as between ﬁeld samples and aquaculture samples (Fig. 7B). In aquaculture, C values of S. crispata were by far the highest of all three species, continuously and signiﬁcantly decreasing with depth. The lowest C values of S. crispata were observed in the ﬁeld samples, which are still similar to the 7 m aquaculture samples. For both M. laminarioides and G. chilensis the highest C value was observed in the ﬁeld samples. In these samples the non-photochemical quenching started at 100 and 45 µmol photon m−2 s− 1 respectively.
In both species C values in aquaculture samples were relatively low (i.e. around 10 µmol photon m−2 s− 1) and did not change with increasing depth (Fig. 7B). Ek(NPQ), the irradiance, at which the non-photochemical quenching becomes saturated is presented in Fig. 7C and gives an overview of the three species and all experimental groups. By far the highest values were reached in G. chilensis, where E k(NPQ) increased from 450 µmol photon m −2 s − 1 at 1 m to 650 µmol photon m−2 s− 1 at 7 m depth. In this species, the ﬁeld samples displayed lower values (350 µmol photon m−2 s− 1) than aquaculture samples. For M. laminarioides Ek(NPQ), values of ﬁeld samples were signiﬁcantly higher (200 µmol photon m−2 s− 1) than the aquaculture samples. The latter was almost constant between 60 to 75 µmol photon m−2 s− 1 irrespective of depth. Ek(NPQ) of S. crispata decreased signiﬁcantly from 175 µmol photon m−2 s− 1 at 1 m to 80 µmol photon m−2 s − 1 at 7 m depth. In ﬁeld samples E k(NPQ) reached 120 µmol photon m−2 s− 1, which is equivalent to aquaculture samples at 5 m depth. 4. Discussion The goal of this study was to describe the irradiance acclimation characteristics of economically important red algae in order to deﬁne their proper cultivation depth range. The experiments were designed to compare the acclimation status of ﬁeld-grown individuals, taken at the depth of their maximum occurrence, with individuals growing at different depths in long-line aquaculture. In this experimental approach two different kinds of changes in irradiance climate must be taken into account: i) the reduction of photon ﬂux density and spectral bandwidth with increasing cultivation depth and; ii) the changes in variability of the light climate between ﬁeld-grown algae, subjected to tidal changes, and long-line cultivated algae, growing at constant depths. 4.1. Photosynthesis acclimation strategies
Fig. 7. NPQ parameters of Mazzaella laminarioides (grey bars), Gracilaria chilensis (empty bars) and Sarcothalia crispata (black bars). Samples were taken directly from the ﬁeld (left columns) or cultivated for two weeks in buoyant long-line culture at constant depths as indicated on the X-axis. Field samples (Field) were taken from individuals collected in the intertidal for M. laminarioides, whereas G. chilensis and S. crispata were sampled at 2 m and 5 m depth below mean low water level respectively. Shown are: maximum NPQ at supersaturating irradiance (NPQmax, upper panel); irradiance at which NPQ onset (C, middle) and irradiance at which NPQ becomes saturated (Ek(NPQ), lower panel) as mean values with standard deviations derived from 3 independent samples. Signiﬁcantly different groups are denoted by lower case letters.
Regarding the photosynthesis parameters, all algae tested in aquaculture acclimated to the irradiance at the respective cultivation depth. These results present a clear depth dependency of the light saturation point, Ek and appear to be a useful general indicator for the light acclimation status in several studies (e.g. Hammer et al. 2002). The Ek of all species tested in this study decreased with increasing depth and agreed well with the decreased light availability. Comparing Ek values with the average irradiance at the respective depths, all species showed a quasi-linear dependency, but with large differences between the species in the slope of these linear trends. The steepest increase of the Ek vs. ambient irradiance relation was discovered by the sublittoral species S. crispata, whereas the intertidal species exhibited far lower slopes (Fig. 5) Those differences can be explained by the mechanisms employed for irradiance acclimation by the individual species. There are two very distinct ways macroalgae can achieve irradiance acclimation. One way is the adjustment of the photosynthetic apparatus itself via, e.g., changes of the reaction center ratio (e.g. Sonoike et al. 2001), changes of the relative size of the light harvesting complex (e.g. Sagert et al. 1997) or changes in the relative content of light protective pigments. All of these mechanisms inﬂuence primarily pigment ratios of the organisms and lead to pronounced acclimation-dependent differences, if photosynthesis parameters are expressed on a Chl a base. Another possibility to achieve acclimation are changes in morphology, such changes in e.g. thickness, branching, length, density of “photosynthesis units” etc. (e.g. Küster et al. 2004). This becomes most visible, when photosynthesis data are expressed on a morphological base such as weight or
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surface area (Johansson & Snoeijs 2002). Morphological and physiological acclimation may both take place. M. laminarioides, a species growing in the high-mid eulittoral, reached acclimation mainly by adjusting its photosynthetic apparatus. A pronounced decrease of Pmax, accompanied by a parallel increase of alpha was observed along the depth gradient for both parameters expressed on chlorophyll a basis. Chlorophyll a content per fresh weight did not show signiﬁcant differences over the whole depth gradient. Thus, the changes in chlorophyll a/carotenoid ratio are mainly due to a decreased carotenoid content with increasing depth. The data received from ﬁeld samples of this species agree well with a growing in the mid eulittoral and being exposed to atmospheric irradiance conditions for long periods each day. Field samples had higher chlorophyll a-based Pmax and lower chlorophyll a-based alpha values than all cultivated individuals. Similar variation has also been reported among plants at different intertidal levels particularly in spring and summer, where upper shore plants show lower photosynthetic efﬁciency than lower shore ones (Varela et al. 2006). Additionally, individuals can vary from greenish–yellowish to darker brownish–reddish depending on their location along the tidal gradient (Buschmann & Pizarro, 1986; Santelices, 1989). Because this change in pigmentation also reﬂects a physiological acclimation, it can be concluded that morphological acclimation is of minor importance for this species. In contrast to this is G. chilensis, a species which grows deeper, but is still intertidal and showed a very restricted physiological acclimation capability. Because only a slight reduction of Pmax at depths of more than 5 m occurred, the light saturation point Ek remained almost unchanged irrespective of the depth, where the samples were taken from (Fig. 5). The Pmax values of ﬁeld-growing individuals of this species may be placed between the 1 m and 3 m long-line cultivated specimens, whereas no clear conclusion can be drawn with respect to alpha due to strong variations. On the other hand, pigment ratios evidenced that at least part of the acclimation is achieved via physiological changes as demonstrated by the chlorophyll a/carotenoid ratio, which increased signiﬁcantly with depth. Consequently, G. chilensis keeps its photosynthetic potential per fresh weight constant throughout the whole depth range tested, whereas Pmax per fresh weight declined in the other species. The sublittoral species S. crispata, which is most abundant at around 5 m depth, seems to combine both types of acclimation. This species exhibited a very pronounced morphological acclimation as well as an efﬁcient physiological acclimation of the photosynthetic apparatus. Chlorophyll a content increased signiﬁcantly and steadily with increasing depth clearly indicating morphological acclimation. The increasing fresh weight/area relation, an additional morphologyrelated parameter, supports this assumption. In addition to this, a physiological acclimation of the photosynthetic apparatus took place. Chlorophyll a-based Pmax and alpha showed the classical opposite pattern, the former decreased with depth whilst the latter increased. This result is a very pronounced depth dependency of the light saturation point, which almost matched the average irradiance at depth. Fresh weight-based Pmax and alpha, reﬂecting the interplay of both principal acclimation mechanisms, showed a non-linear pattern with maxima for alpha and Pmax at 3 and 5 m respectively. On the other hand, chlorophyll a-based photosynthetic parameters of ﬁeld-grown individuals are comparable to the parameters of those growing at a similar depth (5 m) in long-line aquaculture. Alpha values are slightly lower but this difference was not signiﬁcant, because of large standard deviation. Moreover, which can be seen from the Ek values, the acclimation status of the ﬁeld-growing samples correspond perfectly to those exhibited by the long-line individuals cultivated at 5 m depth. 4.2. Differences in the light protective mechanism The above description seems to indicate, that there are no major differences in the light acclimation status of ﬁeld-grown individuals in
comparison with algae grown at assimilable depth in long-line aquaculture. However, this does not hold true with respect to nonphotochemical quenching parameters, summarizing the light protective capacities of the algae at supersaturating intensities. Two of the three species did not exhibit large differences between ﬁeld and aquaculture samples with respect to maximum NPQ (NPQmax). Only ﬁeld-grown samples of S. crispata showed signiﬁcantly lower NPQmax than aquaculture-grown samples. Regarding to Ek(NPQ), the irradiance at which NPQ becomes saturated, a conspicuous difference between ﬁeld-grown and longline cultivated algae was exhibited by M. laminarioides. Ek(NPQ) was signiﬁcantly, highest for ﬁeld samples, indicating a larger tolerance to supersaturating intensities. In comparison Ek(NPQ) with Ek values, the difference between them is very narrow for aquaculture individuals, whereas the large difference between the Ek(NPQ) and Ek of ﬁeldgrown algae indicates a broad range of tolerance of supersaturating intensities. Therefore ﬁeld-grown M. laminarioides can be expected to be much more resistant to irradiance ﬂuctuations than those grown in aquaculture. In S. crispata, Ek(NPQ) follows almost perfectly the shape of the Ek vs. acclimation irradiance curve. In addition, the constant distance between C and Ek(NPQ) (approx. 80 µmol photon m−2 s− 1) indicates, that this species retains a constant range of dynamic photoinhibition. Because the light intensity difference between onset (C) and saturation of the processes diminishing excitation energy pressure on PSII (EkNPQ) is constant, irrespective of the average intensity experienced during acclimation. However, with this strategy chronic photoinhibition (e.g. Altamirano et al. 2004) will likely occur in shallow depths, because the absolute amplitude of irradiance variability is depth-dependent and highest close to the surface (Kirk 1984, Schubert et al. 1996, 2001). In contrast to S. crispata, G. chilensis shows no signiﬁcant trend with respect to Ek(NPQ). As already shown for Ek, the acclimation status of G. chilensis seems to be almost constant with respect to both parameters. G. chilensis shows a distinct and signiﬁcant difference between ﬁeld and long-line growing samples only with respect to C. Field samples can switch on their NPQ formation far later and much closer to Ek than long-line grown samples. Because NPQ formation reduces the efﬁciency of light utilization in photosynthesis, ﬁeld-grown G. chilensis seems to improve its efﬁciency in light use by delaying the onset of this protective mechanism. The same pattern is exhibited by M. laminarioides in a more pronounced way, whereas S. crispata adapt C closely to Ek as well as Ek(NPQ) without differences between ﬁeld and long-line samples. 4.3. Light acclimation and aquaculture implications The three species tested represent different acclimation strategies. M. laminarioides relied almost exclusively on a physiological acclimation of the photosynthesis apparatus. Pmax as well as alpha values was adjusted mainly to average irradiance availability. NPQ onset and NPQ saturation were dependent on irradiance variability. Both NPQ parameters reacted with delay under the highly variable ﬁeld conditions compared to aquaculture conditions. This kept the photosynthetic efﬁciency high under variable ﬁeld conditions, even under circumstances in which temporarily supersaturating irradiances might hit the algae. This strategy of adjustment of the photosynthetic apparatus will allow efﬁcient cultivation also at higher depths. However, since this alga does not acclimate morphologically, lower biomass growth rates must be taken into account. G. chilensis was lacking any sign of acclimation of the photosynthesis main parameters. Neither Ek nor alpha values changed as a result of decreased irradiance availability along depth. Even the pigment ratios remained almost constant for the whole depth range. The ﬁeld samples matched with values of aquaculture samples of the same respective depths. Acclimation was only reached by increasing chlorophyll a concentration with a greater depth, and counteracting the effects of lower light availability. Signiﬁcant differences between
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ﬁeld and aquaculture samples were found with respect to the onset of NPQ, which was heavily delayed under variable ﬁeld conditions. G. chilensis therefore seems to be the most robust algae of the three species tested. This species can be cultivated near the surface (Troell et al. 1997; Halling et al. 2005) and also on traditional bottom cultures in shallow subtidal estuaries and bays, as well as on intertidal mudﬂats (Buschmann et al. 1995). The increasing pigment content will balance lower irradiance availability with increasing depth. By increasing Ek(NPQ) with depth, efﬁciency of photosynthesis is welladjusted to the requirements at the respective depth and efﬁciency is kept high even under ﬂuctuating irradiance regimes. S. crispata employed both mechanisms, physiological adjustment of the photosynthetic apparatus as well as morphological acclimation. As a result, this species almost perfectly adjusted its optimum point of photosynthesis to the ambient irradiance conditions. Considering the fresh weight-based photosynthesis, a clear optimum range was found between 3 and 5 m. However, this species adjusted its NPQ characteristics to the average irradiance rather than variability of irradiance. NPQmax was unchanged along the culture depth and only slightly lower in ﬁeld-grown samples. Irradiance for onset and saturation of NPQ decreased with depth. This indicates, that S. crispata is unable to acclimate to the variability of irradiance. In clear-sky conditions in shallow water this species will be subject to photodamage. This explains also, why cultivation requires the maintenance of these algae in deeper water during summer (Romo et al. 2001). S. crispata, with its pronounced optimum depth, should not be cultivated close to the surface, where fresh weight productivity will decrease and the potential danger of photodamage increases. From the results obtained, the following conclusion with respect to aquaculture purposes can be drawn, which with regard to the limited duration of the experiments must be still tested for a whole growth period: The optimal depth range of S. crispata is between 3 and 5 m, where the combined morphological and physiological acclimation strategies still enable high growth rates, whereas the missing acclimation to light variability does not harm the algae. The reliance of M. laminarioides on full physiological acclimation with highly active protective mechanisms strongly suggests its cultivation at a shallow depth. However, early onset of NPQ combined with low Ek(NPQ) lowers efﬁciency of growth in the uppermost zones. The robustness of G. chilensis, achieved through its efﬁcient morphological acclimation, allows good primary productivity at every depth tested. However, its high Ek(NPQ) favours this species as an overstorey species in a multilevel culture. Acknowledgement This study was supported by a grant of the German Academic Exchange Program (CHL 06/014) and FONDECYT 1050550 (Chile), which is gratefully acknowledged. We recognize the ﬁeld help by Luis Henríquez and Adrián Villarroel. We also thank the three unknown reviewers. References Abreu, M.H., Varela, D.A., Henriquez, L., Villarroel, A., Yarish, C., Sousa-Pinto, I., Buschmann, A.H., 2009. Traditional vs. integrated multi-trophic aquaculture of Gracilaria chilensis Bird, C.J., Mc Lachlan, J., Oliveira E.C. productivity and physiological performance. Aquaculture 293, 211–220. Ahn, O., Petrell, R.J., Harrison, P.J., 1998. Ammonium and nitrate uptake by Laminaria saccharina and Nereocystis luetkeana originating from a salmon sea cage farm. J. Appl. Phycol. 10, 333–340. Almanza, V., 2007. Importancia de huirales de Macrocystis pyrifera (L.) C. Agardh (Laminariales, Phaeophyta) en la abundancia, diversidad y asentamiento de organismos bentónicos asociados. Tesis de Magíster, Universidad de Los Lagos Osorno Chile p. 150. Altamirano, M., Murakami, A., Kawai, H., 2004. High light stress in the kelp Ecklonia cava. Aquat. Bot. 79, 125–135. Bracken, M.E.S., Stachowicz, J.J., 2006. Seaweed diversity enhances nitrogen uptake via complementary use of nitrate and ammonium. Ecology 87, 2397–2403.
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