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The short-term effects of elevated CO2 and ammonium concentrations on physiological responses in Gracilariopsis lemaneiformis (Rhodophyta)

The short-term effects of elevated CO2 and ammonium concentrations on physiological responses in... Ocean acidification (OA) and coastal eutrophication affect coastal marine organisms. We studied the physiological responses of Gracilariopsis lemaneiformis (Gracilariales, Rhodophyta) to increased concentrations of CO and NH . 2 4 Incubation treatments were applied at two different pH units (low, 7.5; high (control), 7.9) and three different NH concentrations (low, 10; medium, 50; high, 100 μM). Growth, rates of photosynthetic oxygen evolution, and NH uptake rates were affected by both elevated CO and NH conditions. The changes in the pH of culture media 2 4 were influenced by elevated CO or NH treatments. However, chlorophyll fluorescence was affected only by the 2 4 level of NH . These results indicate that the physiological responses of G. lemaneiformis might be enhanced when the concentrations of CO and NH rise. Therefore, cultures of this alga could provide a good mitigation solution 2 4 against ongoing problems with OA and coastal eutrophication. Keywords: CO , Eutrophication, Gracilariopsis lemaneiformis,NH , Ocean acidification (OA) 2 4 Background seaweeds (Hinga 2002; Gao and Zheng 2010; Cornwall In coastal regions, marine organisms face serious environ- et al. 2012; Kram et al. 2016). Although OA can have a mental problems caused by anthropogenic eutrophication negative impact on the metabolism of calcifying organisms and, more recently, ocean acidification, or OA (Fei 2004; (Hofmann and Bischof 2014; Kram et al. 2016), many Doney et al. 2009; Boyd 2011). The combinations of ocean macroalgae respond positively under acidified condi- acidification and changing states of eutrophication affect tions (Zou 2005; Sarker et al. 2013; Kram et al. 2016). the physiology of marine species (Reymond et al. 2013; For example, many macroalgal species utilize carbon- Chen et al. 2016). A report by the Intergovernmental concentrating mechanisms (CCMs) that enable them Panel on Climate Change (IPCC 2014) revealed that, since to acquire CO from HCO . When concentrations of 2 3 the Industrial Revolution, atmospheric CO concentra- dissolved inorganic carbon (DIC) are elevated, macro- tions have increased from 280 to 400 ppm while pH values algaetakeupthisDIC andconserveenergybydown- have decreased 0.1 units. Orr et al. (2005) have predicted regulating their CCMs (Beardall et al. 1998; Sarker that pH values will decline by 0.3 to 0.4 units if atmos- et al. 2013). In this process, the growth of seaweeds pheric CO concentrations reach above 800 ppm. This might be enhanced under higher CO concentrations 2 2 OA phenomenon influences the physiology of marine (Beardall et al. 1998; Sarker et al. 2013). biota such as calcifying species, phytoplankton, and Coastal eutrophication is a severe challenge that has resulted from human activities such as aquaculture, industrialization, and urbanization. Higher concentra- * Correspondence: ikchung@pusan.ac.kr tions of nutrients, e.g., nitrogen (N) and phosphorus (P), Division of Earth Environmental System Oceanography major, Pusan can improve photosynthetic rates in primary producers, National University, Busan 46241, Republic of Korea Department of Oceanography, Pusan National University, Busan 46241, including seaweeds (Chen et al. 2016; Xu et al. 2017b). Republic of Korea However, excessive inputs of those nutrients can change Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Kang et al. Fisheries and Aquatic Sciences (2017) 20:18 Page 2 of 8 the function and structure of a marine ecosystem 116° 55′ E), in March of 2016. Samples were trans- (Valiela et al. 1997; Wu et al. 2015). In addition, ported to the field station near the farm and washed eutrophication could also cause ocean acidification in several times to remove any epiphytes. They were ac- the coastal zones because of the active respiration of climated for 24 h before the experiments under 20 ° −2 −1 bacteria (Cai et al. 2011). C, 100 μmol photons m s (12:12 light/dark; a LI- Gracilariopsis lemaneiformis (Bory de Saint-Vincent) 250 light meter, LI-COR, USA). For each treatment, E.Y.Dawson, Acleto & Foldvik (Gracilariales, Rhodo- fresh samples (2 g) were placed in culture vessels phyta, formerly Gracilaria lemaneiformis) is distributed containing 250 mL of filtered seawater. All experi- in tropical and warm-temperate regions, and is culti- ments utilized a factorial design that included two pH vated worldwide, especially in Chile, China, and Taiwan conditions (low 7.5; high 7.9, control) and three am- (Armisen 1995; Tseng 2001; Yang et al. 2015). In China, monium concentrations (low 10; medium 50; high this alga is cultivated on a large scale because it is an 100 μM). The low NH level was based on the am- economically important source of agar (Fei 2004; Wang bient NH (8.84 ± 1.42 μM) concentration at the et al. 2010). Furthermore, this species has been studied sampling site. The medium and high NH concentra- as a valuable component of Integrated Multi-Trophic tions were assumed severe eutrophication conditions and Aquaculture and as a bioremediation agent (Yang et al. were made by adding to ambient NH level, respectively. 2006, 2015; Wu et al. 2015). The low-pH condition indicated elevated CO concentra- The physiological responses of G. lemaneiformis have tion in the future condition and high-pH condition has previously been monitored in experiments that combined shown the ambient CO level. The six culture treat- various CO levels with a range of nutrient concentrations ments (four replicates each) were identified as follows: or light intensities (Zou and Gao 2009; Xu et al. 2010; Xu L L ,low pH +low NH ;H L , high pH + low pH A 4 pH A + + and Gao 2012; Chen et al. 2017). The results from those NH ;L M , low pH + medium NH ;H M , 4 pH A 4 pH A studies indicated that plant growth, photosynthesis, rate of high pH + medium NH ;L H , low pH + high 4 pH A + + nutrient uptake, and levels of biochemical components and NH ;and H H ,highpH+high NH .The low- 4 pH A 4 amino acids are increased under elevated CO conditions, pH medium was prepared by mixing filtered seawater light intensities, or nutrient concentrations (Zou and Gao with pure CO -enriched seawater from a CO tank. 2 2 2009; Xu et al. 2010; Xu and Gao 2012; Chen et al. 2017). Gattuso et al. (2010) indicated that mixing of high However, that earlier research did not focus on the physio- CO seawater is the reasonable method as similar as logical reactions of G. lemaneiformis underhigherCO and CO bubbling. The medium and high NH concen- 2 2 4 ammonium (NH ) concentrations. Therefore, we looked trations were obtained by adding 50 or 100 μM at whether this species has the capacity to mitigate the ef- NH Cl, respectively, to the medium. All media were fects of OA and coastal eutrophication by measuring changed every day. Total alkalinity (TA) was mea- growth rates, pH changes in the culture medium, oxygen sured by the electro-titration method (Gran 1952; −1 evolution by photosynthesis, the rate of ammonium uptake precision ±4 μmol kg ). The amounts of DIC and (an indicator of plant capacity for nutrient removal), and the pCO values were calculated by the CO2SYS pro- chlorophyll fluorescence (for assessing photosynthetic effi- gram (Lewis and Wallace 1998) (Table 1). The dis- ciency). We then analyzed the interactions among different sociation constant and KSO values were defined by combinations of CO and NH levels. Millero et al. (2006) and Dickson (1990). 2 4 The growth of G. lemaneiformis was quantified by Methods measuring the change in fresh weight (FW) after 3 days −1 We collected Gracilariopsis lemaneiformis at culture of incubation. The relative growth rate (RGR; % day ) farm near Nanao Island, Shantou, China (23° 20′ N, was calculated as follows: Table 1 Parameters of seawater carbonate system under tested pH conditions 2− − Culture conditions TA pCO CO CO HCO DIC 2 2 3 3 L L 2012.75 ± 1.77 1420.09 ± 1.27 46.11 ± 0.04 52.60 ± 0.05 1878.61 ± 1.67 1977.32 ± 1.76 pH A L M 2017.33 ± 1.87 1423.37 ± 1.34 46.22 ± 0.04 52.72 ± 0.05 1882.94 ± 1.77 1981.88 ± 1.87 pH A L H 2015.42 ± 6.29 1405.93 ± 3.86 45.60 ± 0.13 53.76 ± 0.13 1879.16 ± 5.03 1978.52 ± 5.29 pH A H L 2022.16 ± 2.60 394.69 ± 0.53 12.82 ± 0.02 146.20 ± 0.20 1651.11 ± 2.21 1810.13 ± 2.42 pH A H M 2021.86 ± 2.40 394.63 ± 0.49 12.81 ± 0.02 146.18 ± 0.18 1650.86 ± 2.04 1809.85 ± 2.23 pH A H H 2019.51 ± 5.00 389.79 ± 1.35 12.69 ± 0.04 148.36 ± 0.28 1645.46 ± 4.43 1806.51 ± 4.76 pH A −1 −1 2− −1 − Values are means ± SD. Both pH and TA (μmol kg ) were measured directly for each scenario, whereas pCO (μatm), CO (μmol kg ), CO (μmol kg ), HCO 2 2 3 3 −1 −1 (μmol kg ), and DIC (μmol kg ) were calculated according to the CO2SYS program of Lewis and Wallace (1998) Kang et al. Fisheries and Aquatic Sciences (2017) 20:18 Page 3 of 8 –1 Table 2 Results from two-way ANOVA of physiological activities RGR ¼ðÞ lnW – lnW 100  T 2 1 −1 (relative growth rate (% day ), extent of variation in pH, −1 −1 where W is the initial fresh weight, W is the final fresh 1 2 photosynthetic oxygen evolution rate (μmol O g FW h ), + −1 −1 weight, and T is the incubation period (3 days). ammonium uptake rate (μmol NH g FW h ), and photosynthetic Over a 12-h period, we monitored changes in pH in efficiency (F /F )) by samples of Gracilariopsis lemaneiformis v m the culture media, the rate of oxygen evolution, and Parameter/source of variation df MS F value p value NH uptake. An Orion-250A meter (Thermoscientific, Relative growth rate USA) was used to measure pH at 0, 2, 4, 6, and 12 h. pH level 1 0.69 21.50 <0.01 We also assessed the changes in pH in vessels contain- NH concentration 2 4.26 132.54 <0.01 ing media but no specimens (blanks), under low- and pH level × NH concentration 2 0.12 3.66 0.04 high-pH conditions. Extent of variation in pH Rates of photosynthetic oxygen evolution (μmol O g −1 −1 FW h ) were recorded with a Clark-type microelec- pH level 1 1.09 166.48 <0.01 trode oxygen sensor (Unisense, Denmark), which was NH concentration 2 0.38 57.63 <0.01 calibrated with a mixture of C H NaO (sodium ascor- 6 7 6 + pH × NH 2 0.01 0.10 0.90 bate) of 0.4 g and NaOH (sodium hydroxide) of 2 g in Photosynthetic oxygen evolution rate the 100-mL dilution water. + + −1 −1 pH level 1 198.72 68.85 <0.01 The rate of NH uptake (μmol NH g FW h ) 4 4 was calculated as the amount lost from each culture NH concentration 2 3678.60 1274.53 <0.01 medium over 12 h. The measurement method of NH 4 pH × NH 2 22.50 7.80 <0.01 uptake rates were described by Parsons et al. (1984). The NH uptake rate following equation was used in the calculation: pH level 1 0.17 24.72 <0.01 –1 –1 NH concentration 2 85.78 12,108.59 <0.01 V ¼ðÞ S –S  vol  W  T 4 i f pH × NH 2 0.13 18.95 <0.01 + 4 where S is the initial concentration of NH , S is the i 4 f Photosynthetic efficiency final concentration after T hours of incubation, vol is the pH level 1 <0.01 0.58 0.46 volume of the medium, and W is the fresh weight of each algal specimen. NH concentration 2 0.13 51.60 <0.01 Chlorophyll fluorescence was determined after 3 days pH × NH 2 <0.01 0.92 0.42 with a Plant Efficiency Analyzer (PEA, Hansatech, UK). −1 The maximum quantum yield (F /F ) of Photosystem II v m maximum RGR was 2.95 ± 0.20% day under L H con- pH A −1 was calculated as follows: ditions while the minimum RGR, 1.07 ± 0.21% day ,was achieved under H L conditions (Fig. 1). At the high NH pH A 4 F =F ¼ðÞ F −F =F v m m o m levels, RGR was greater at pH 7.5 than at pH 7.9 (H : where F is maximum fluorescence after dark adapta- F =6.04, p = 0.04). tion and F is minimum fluorescence after dark adapta- tion. Algal samples were placed in leaf-clip holders and F /F was measured by applying a saturating pulse after v m the samples were dark-adapted for 15 min. One- and two-way ANOVA were conducted with all experimental data. Normality and homogeneity were investigated before the statistical analysis began. After that, Tukey’s tests were used to compare among treatments, and the threshold for statistically significant differences was set at p < 0.05. All ana- lyses were performed with the SPSS software pro- gram (version 23.0). Results Results of physiological responses of Gracilariopsis lemanei- + −1 formis under elevated CO and NH treatments were sum- Fig. 1 Relative growth rates (% day )of Gracilariopsis lemaneiformis 2 4 at different pH and NH treatments. Bars not labeled with same letter marized in Table 2. During the incubation period, the indicate significant differences among culture conditions at p <0.05. relative growth rate of Gracilariopsis lemaneiformis in- Values are means ± SD (n =4) creased under elevated CO and NH treatments. The 2 4 Kang et al. Fisheries and Aquatic Sciences (2017) 20:18 Page 4 of 8 Alterations in pH in the culture media containing algal low than at high pH but there was no significant differ- specimens were significantly affected by initial pH or ence at the low NH level (L : F= 0.26, p =0.63; M : 4 A A NH levels. The extent of variation in pH ranged from F= 7.94, p =0.03; H : F= 7.67, p =0.03). 4 A 0.44 ± 0.02 to 1.26 ± 0.18 units, and the maximum and The rate of NH uptake was significantly affected by minimum changes in pH were associated with L H elevated concentrations of CO and NH , with rates pH A 2 4 and H L , respectively (Fig. 2; Table 3). At each pH ranging from 0.84 ± 0.01 to 7.43 ± 0.03 μmol NH g pH A 4 + −1 −1 level, the medium and high NH concentrations were FW h (Fig. 4). Uptake was more rapid under L H 4 pH A significantly different from the low NH condition conditions and slowest under H L treatment. When 4 pH A (L : F= 17.08, p = 0.01; H : F= 78.98, p < 0.01). At those rates were compared at the same pH, values in- pH pH + + individual NH levels, the change in pH was signifi- creased under higher NH concentrations (L : 4 4 pH cantly greater in the low-pH treatment than in the high- F= 3230.83, p < 0.01; H : F= 25,898.16, p < 0.01). Fur- pH pH treatment (L : F= 6.65, p = 0.04; M : F= 6.94, thermore, at the same level of NH , uptake was more A A 4 p = 0.04; H : F= 138.86, p < 0.01). The pH values in rapid under L H than under H H treatment (H : A pH A pH A A blank culture media were remain constant over 12 h that F= 6.50, p = 0.04). pH values were affected only photosynthesis oxygen evo- When values of the chlorophyll fluorescence were used lution of G. lemaneiformis. as a proxy to represent photosynthetic efficiency, they Photosynthetic oxygen evolution was affected by ele- ranged from 0.55 ± 0.22 (H L ) to 0.64 ± 0.02 (L H ) pH A pH A vated levels of CO and NH . Rates were (Fig. 5). Although values of F /F were significantly af- 2 4 v m −1 −1 + 62.28 ± 1.71 μmol O g FW h under H L and fected under high NH levels and under both pH con- 2 pH A 4 −1 −1 111.48 ± 0.95 μmol O g FW h under L H (Fig. 3). ditions (L : F= 44.64, p < 0.01; H : F= 15.91, 2 pH A pH pH When compared at the same initial pH, those rates in- p < 0.01), they were not significantly influenced by NH + + creased significantly as the NH level rose (L : conditions (L : F= 0.60, p = 0.47; M : F= 1.23, 4 pH A A F= 479.22, p <0.01; H : F= 854.92, p <0.01).Under p = 0.31; H : F= 0.92, p = 0.37). pH A medium and high NH conditions, the rate was greater at Discussion Growth of Gracilariopsis lemaneiformis was affected by elevated CO and NH treatments and there were in- 2 4 teractions of both factors. Previous studies have revealed similar results with G. lemaneiformis, Hypnea spinella, Chondrus crispus, Pyropia haitanensis, and Ulva pertusa (Yu and Yang 2008; Suárez-Álvarez et al. 2012; Sarker et al. 2013; Chen et al. 2016; Kang and Chung 2017). When DIC concentrations rise in the ocean, many macroalgal species conserve energy by regulating their CCMs, thereby improving their growth performance (Sarker et al. 2013). For example, red algae within the Gracilaria genus used external carbonic anhydrase to in- crease their capacity for HCO utilization, which boosts their growth under elevated CO conditions (Israel and Beer 1992; García-Sánchez et al. 1994; Zou et al. 2004). Greater availability of nutrients can also enhance the growth of macroalgae (Yu and Yang 2008). Although greater accumulations of nitrogen can stimulate growth, Xu et al. (2010) reported that G. lemaneiformis develop- ment was influenced by either CO or the level of phos- phorus, but those two factors did not show synergistic effects. Our results indicated that RGR were lower than those of other experiments. We speculated that short- term incubation period and high stocking density could be the reason of lower RGR. Kim et al. (2013) indicated that high stocking density might cause lower growth rates than those of light shading of the culture vessels. Fig. 2 Extent of variation in pH over time in response to different We assumed the high stocking density conditions of culture treatments. Data are means ± SD (n =4) aquaculture farm to find realistic growth of G. Kang et al. Fisheries and Aquatic Sciences (2017) 20:18 Page 5 of 8 Table 3 Maximum pH values and extent of variation in response over 12-h period to different treatment combinations Maximum pH values Extent of variation L (low pH) H (high pH) L (low pH) H (high pH) pH pH pH pH + a a a b L (low NH ) 8.36 ± 0.03 8.34 ± 0.02 0.86 ± 0.03 0.44 ± 0.02 A 4 + b b c a M (medium NH ) 8.66 ± 0.01 8.61 ± 0.04 1.16 ± 0.01 0.71 ± 0.04 A 4 + b b c a H (high NH ) 8.78 ± 0.18 8.77 ± 0.07 1.28 ± 0.18 0.87 ± 0.07 A 4 Data are means ± SD (n = 4). Within a column, values not followed by the same letter are significantly different at p < 0.05 lemaneiformis. In addition, Xu et al. (2017a) found that Photosynthetic oxygen evolution in our Gracilariopsis the RGRs of short-time cultivation period were higher lemaneiformis samples was also increased by elevated than those of long-time cultivation time. They explained CO and NH levels. The same has been reported for 2 4 that decreased of RGRs resulted from the acclimation to Hizikia fusiforme (currently, Sargassum fusiforme), culture conditions. Hypnea spinella,and Pyropia haitanensis (Zou 2005; The pH values in the culture medium could be used Suárez-Álvarez et al. 2012; Chen et al. 2016). Zou et al. as an indicator of physiological characteristics of the (2004) showed that the photosynthesis of G. lemaneifor- macroalgae (Maberly 1990; Murru and Sandgren 2004). mis is already saturated under normal DIC concentra- The pH of our culture medium was altered in response tions found in natural seawater. However, we noted that to increases in CO or NH concentrations. The this species was also influenced by the synergism be- 2 4 addition of CO gas caused pH values to decline while tween CO and NH . Similarly, Xu et al. (2010) has 2 2 4 the concentration of DIC, such as CO (aq), and HCO shown that the maximum net photosynthetic rate for G. 2 3 increased. This change in pH was more dramatic in the lemaneiformis is influenced by increases in both CO low-pH medium than under high-pH conditions. The and phosphorus concentrations. DIC concentrations were elevated in the culture The uptake of NH was influenced by treatment with medium, which lead to increases in pH levels by photo- exogenous CO and NH . Research with Gracilaria sp., 2 4 synthesis (Zhang et al. 2012). In addition, we did not G. chilensis, Hizikia fusiforme, Gracilariopsis lemaneifor- find any inhibition of photosynthesis. When pH values mis, Pyropia yezoensis, P. haitanensis, and U. pertusa are >9.0, photosynthesis of some species is more likely has demonstrated that all respond to elevated levels of to be inhibited (Maberly 1990; Björk et al. 2004). Zou CO or NH (Gao et al. 1993; Zou 2005; Xu et al. 2010; 2 4 et al. (2004) indicated that inorganic carbon (Ci) affinity Kang et al. 2014; Chen et al. 2016; Kang and Chung and photosynthetic rates at pH 9.0 were decreased in G. 2017). Increasing the concentration of CO can enhance lemaneiformis. In case of pH values over 9.0, this alga nitrogen uptake and the activity of nitrate reductase does not accumulate CO because of poor capacity of (Gordillo et al. 2001; Xu et al. 2010; Hofmann et al. HCO utilization (Zou et al. 2004). 2013; Liu and Zou 2015). In various seaweeds, the up- + + take of NH is greater when the initial NH 4 4 + −1 −1 Fig. 4 Ammonium uptake rates (μmol NH g FW h )of −1 + Fig. 3 Rates of photosynthetic oxygen evolution (μmol O g FW h Gracilariopsis lemaneiformis under different pH levels and NH 2 4 −1 + )of Gracilariopsis lemaneiformis at different pH and NH treatments. treatments. Bars not labeled with same letter indicate significant Bars not labeled with same letter indicate significant differences differences among culture conditions at p < 0.05. Values are means among culture conditions at p < 0.05. Values are means ± SD (n =4) ±SD (n =4) Kang et al. Fisheries and Aquatic Sciences (2017) 20:18 Page 6 of 8 2015). Yang et al. (2015) have suggested that large-scale cultivation of Gracilaria can improve water quality by uptake of excessive nutrients and potential carbon sink absorber along the coast of China. Therefore, this alga could provide a good solution for mitigation against the problems associated with such marine challenges. If we are to achieve practical results, future investigations will require more large-scale, long-term experiments. Those projects must also focus on the synergistic effects of sev- eral environmental factors, e.g., temperature, light inten- sity, level of salinity, and nutrient concentrations. Conclusions The combined treatment of elevated CO and NH 2 4 heightened the physiological reactions of G. lemaneifor- Fig. 5 Photosynthetic efficiency (F /F )of Gracilariopsis v m mis, as demonstrated by changes in relative growth lemaneiformis at different pH levels and NH treatments. Bars not labeled with same letter indicate significant differences among rates, rates of photosynthetic oxygen evolution, and the culture conditions at p < 0.05. Values are means ± SD (n =4) uptake of ammonium. The pH values were affected each elevated CO or NH treatments. In contrast, we noted 2 4 that chlorophyll fluorescence was affected only by alter- concentration is high (Dy and Yap 2001). In addition, ing the concentration of NH . when more nutrients are available, they are more easily Abbreviations absorbed (Runcie et al. 2003; Pérez-Mayorga et al. 2011). OA: Ocean acidification; RGR: Relative growth rates Therefore, because NH uptake by our G. lemaneifor- mis samples was more rapid under the elevated CO and Acknowledgements NH treatment, we speculate that this was due to the as- This work was supported by a grant from the National Research Foundation of Korea (NRF), funded by the Korean Government (MSIP) (NRF-2016R1A2B10 sociated rise in photosynthesis. 13637), to IKC. Chlorophyll fluorescence, which can reflect photosyn- thetic efficiency, is enhanced by increased N concentra- Funding This work was supported by a grant from the National Research Foundation tions (Dawes and Koch 1990). However, the role of CO of Korea (NRF), funded by the Korean Government (MSIP) (NRF-2016R1A2B10 is debatable with some studies showing that F /F is not v m 13637), to IKC. affected by elevated CO levels (Hofmann et al. 2012; Olischläger et al. 2013; Kram et al. 2016) and others in- Availability of data and materials All datasets in this study are available from the corresponding author on dicating that chlorophyll fluorescence increases under reasonable request. high CO concentrations (Chen et al. 2015). We found here that the photosynthetic efficiency of G. lemaneifor- Authors’ contributions mis was more strongly affected by greater NH concen- All authors conducted the field survey in the sampling stations. JW, Cicilia, and IK conducted experiments and wrote manuscript. All authors read and trations than by higher CO levels. Because of these approved the final manuscript. contrasting reports, direct measurement of oxygen evo- lution is now considered a more appropriate method Ethics approval and consent to participate than chlorophyll fluorescence for monitoring changes in Not applicable. efficiency (Kram et al. 2016). Consent for publication Our data provided evidence that the physiological ac- Not applicable. tivities of G. lemaneiformis are enhanced under elevated CO and NH treatments. We predict that mass pro- Competing interests 2 4 The authors declare that they have no competing interests. duction of this species in China will increase in response to OA and eutrophication. Kim and Yarish (2014) indi- cated that productivity of Gracilaria sp. was enhanced Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in under elevated CO levels with high stocking density published maps and institutional affiliations. and irradiance. 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The short-term effects of elevated CO2 and ammonium concentrations on physiological responses in Gracilariopsis lemaneiformis (Rhodophyta)

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Copyright © 2017 by The Author(s)
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Life Sciences; Fish & Wildlife Biology & Management; Marine & Freshwater Sciences; Zoology; Animal Ecology
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2234-1757
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10.1186/s41240-017-0063-y
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Abstract

Ocean acidification (OA) and coastal eutrophication affect coastal marine organisms. We studied the physiological responses of Gracilariopsis lemaneiformis (Gracilariales, Rhodophyta) to increased concentrations of CO and NH . 2 4 Incubation treatments were applied at two different pH units (low, 7.5; high (control), 7.9) and three different NH concentrations (low, 10; medium, 50; high, 100 μM). Growth, rates of photosynthetic oxygen evolution, and NH uptake rates were affected by both elevated CO and NH conditions. The changes in the pH of culture media 2 4 were influenced by elevated CO or NH treatments. However, chlorophyll fluorescence was affected only by the 2 4 level of NH . These results indicate that the physiological responses of G. lemaneiformis might be enhanced when the concentrations of CO and NH rise. Therefore, cultures of this alga could provide a good mitigation solution 2 4 against ongoing problems with OA and coastal eutrophication. Keywords: CO , Eutrophication, Gracilariopsis lemaneiformis,NH , Ocean acidification (OA) 2 4 Background seaweeds (Hinga 2002; Gao and Zheng 2010; Cornwall In coastal regions, marine organisms face serious environ- et al. 2012; Kram et al. 2016). Although OA can have a mental problems caused by anthropogenic eutrophication negative impact on the metabolism of calcifying organisms and, more recently, ocean acidification, or OA (Fei 2004; (Hofmann and Bischof 2014; Kram et al. 2016), many Doney et al. 2009; Boyd 2011). The combinations of ocean macroalgae respond positively under acidified condi- acidification and changing states of eutrophication affect tions (Zou 2005; Sarker et al. 2013; Kram et al. 2016). the physiology of marine species (Reymond et al. 2013; For example, many macroalgal species utilize carbon- Chen et al. 2016). A report by the Intergovernmental concentrating mechanisms (CCMs) that enable them Panel on Climate Change (IPCC 2014) revealed that, since to acquire CO from HCO . When concentrations of 2 3 the Industrial Revolution, atmospheric CO concentra- dissolved inorganic carbon (DIC) are elevated, macro- tions have increased from 280 to 400 ppm while pH values algaetakeupthisDIC andconserveenergybydown- have decreased 0.1 units. Orr et al. (2005) have predicted regulating their CCMs (Beardall et al. 1998; Sarker that pH values will decline by 0.3 to 0.4 units if atmos- et al. 2013). In this process, the growth of seaweeds pheric CO concentrations reach above 800 ppm. This might be enhanced under higher CO concentrations 2 2 OA phenomenon influences the physiology of marine (Beardall et al. 1998; Sarker et al. 2013). biota such as calcifying species, phytoplankton, and Coastal eutrophication is a severe challenge that has resulted from human activities such as aquaculture, industrialization, and urbanization. Higher concentra- * Correspondence: ikchung@pusan.ac.kr tions of nutrients, e.g., nitrogen (N) and phosphorus (P), Division of Earth Environmental System Oceanography major, Pusan can improve photosynthetic rates in primary producers, National University, Busan 46241, Republic of Korea Department of Oceanography, Pusan National University, Busan 46241, including seaweeds (Chen et al. 2016; Xu et al. 2017b). Republic of Korea However, excessive inputs of those nutrients can change Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Kang et al. Fisheries and Aquatic Sciences (2017) 20:18 Page 2 of 8 the function and structure of a marine ecosystem 116° 55′ E), in March of 2016. Samples were trans- (Valiela et al. 1997; Wu et al. 2015). In addition, ported to the field station near the farm and washed eutrophication could also cause ocean acidification in several times to remove any epiphytes. They were ac- the coastal zones because of the active respiration of climated for 24 h before the experiments under 20 ° −2 −1 bacteria (Cai et al. 2011). C, 100 μmol photons m s (12:12 light/dark; a LI- Gracilariopsis lemaneiformis (Bory de Saint-Vincent) 250 light meter, LI-COR, USA). For each treatment, E.Y.Dawson, Acleto & Foldvik (Gracilariales, Rhodo- fresh samples (2 g) were placed in culture vessels phyta, formerly Gracilaria lemaneiformis) is distributed containing 250 mL of filtered seawater. All experi- in tropical and warm-temperate regions, and is culti- ments utilized a factorial design that included two pH vated worldwide, especially in Chile, China, and Taiwan conditions (low 7.5; high 7.9, control) and three am- (Armisen 1995; Tseng 2001; Yang et al. 2015). In China, monium concentrations (low 10; medium 50; high this alga is cultivated on a large scale because it is an 100 μM). The low NH level was based on the am- economically important source of agar (Fei 2004; Wang bient NH (8.84 ± 1.42 μM) concentration at the et al. 2010). Furthermore, this species has been studied sampling site. The medium and high NH concentra- as a valuable component of Integrated Multi-Trophic tions were assumed severe eutrophication conditions and Aquaculture and as a bioremediation agent (Yang et al. were made by adding to ambient NH level, respectively. 2006, 2015; Wu et al. 2015). The low-pH condition indicated elevated CO concentra- The physiological responses of G. lemaneiformis have tion in the future condition and high-pH condition has previously been monitored in experiments that combined shown the ambient CO level. The six culture treat- various CO levels with a range of nutrient concentrations ments (four replicates each) were identified as follows: or light intensities (Zou and Gao 2009; Xu et al. 2010; Xu L L ,low pH +low NH ;H L , high pH + low pH A 4 pH A + + and Gao 2012; Chen et al. 2017). The results from those NH ;L M , low pH + medium NH ;H M , 4 pH A 4 pH A studies indicated that plant growth, photosynthesis, rate of high pH + medium NH ;L H , low pH + high 4 pH A + + nutrient uptake, and levels of biochemical components and NH ;and H H ,highpH+high NH .The low- 4 pH A 4 amino acids are increased under elevated CO conditions, pH medium was prepared by mixing filtered seawater light intensities, or nutrient concentrations (Zou and Gao with pure CO -enriched seawater from a CO tank. 2 2 2009; Xu et al. 2010; Xu and Gao 2012; Chen et al. 2017). Gattuso et al. (2010) indicated that mixing of high However, that earlier research did not focus on the physio- CO seawater is the reasonable method as similar as logical reactions of G. lemaneiformis underhigherCO and CO bubbling. The medium and high NH concen- 2 2 4 ammonium (NH ) concentrations. Therefore, we looked trations were obtained by adding 50 or 100 μM at whether this species has the capacity to mitigate the ef- NH Cl, respectively, to the medium. All media were fects of OA and coastal eutrophication by measuring changed every day. Total alkalinity (TA) was mea- growth rates, pH changes in the culture medium, oxygen sured by the electro-titration method (Gran 1952; −1 evolution by photosynthesis, the rate of ammonium uptake precision ±4 μmol kg ). The amounts of DIC and (an indicator of plant capacity for nutrient removal), and the pCO values were calculated by the CO2SYS pro- chlorophyll fluorescence (for assessing photosynthetic effi- gram (Lewis and Wallace 1998) (Table 1). The dis- ciency). We then analyzed the interactions among different sociation constant and KSO values were defined by combinations of CO and NH levels. Millero et al. (2006) and Dickson (1990). 2 4 The growth of G. lemaneiformis was quantified by Methods measuring the change in fresh weight (FW) after 3 days −1 We collected Gracilariopsis lemaneiformis at culture of incubation. The relative growth rate (RGR; % day ) farm near Nanao Island, Shantou, China (23° 20′ N, was calculated as follows: Table 1 Parameters of seawater carbonate system under tested pH conditions 2− − Culture conditions TA pCO CO CO HCO DIC 2 2 3 3 L L 2012.75 ± 1.77 1420.09 ± 1.27 46.11 ± 0.04 52.60 ± 0.05 1878.61 ± 1.67 1977.32 ± 1.76 pH A L M 2017.33 ± 1.87 1423.37 ± 1.34 46.22 ± 0.04 52.72 ± 0.05 1882.94 ± 1.77 1981.88 ± 1.87 pH A L H 2015.42 ± 6.29 1405.93 ± 3.86 45.60 ± 0.13 53.76 ± 0.13 1879.16 ± 5.03 1978.52 ± 5.29 pH A H L 2022.16 ± 2.60 394.69 ± 0.53 12.82 ± 0.02 146.20 ± 0.20 1651.11 ± 2.21 1810.13 ± 2.42 pH A H M 2021.86 ± 2.40 394.63 ± 0.49 12.81 ± 0.02 146.18 ± 0.18 1650.86 ± 2.04 1809.85 ± 2.23 pH A H H 2019.51 ± 5.00 389.79 ± 1.35 12.69 ± 0.04 148.36 ± 0.28 1645.46 ± 4.43 1806.51 ± 4.76 pH A −1 −1 2− −1 − Values are means ± SD. Both pH and TA (μmol kg ) were measured directly for each scenario, whereas pCO (μatm), CO (μmol kg ), CO (μmol kg ), HCO 2 2 3 3 −1 −1 (μmol kg ), and DIC (μmol kg ) were calculated according to the CO2SYS program of Lewis and Wallace (1998) Kang et al. Fisheries and Aquatic Sciences (2017) 20:18 Page 3 of 8 –1 Table 2 Results from two-way ANOVA of physiological activities RGR ¼ðÞ lnW – lnW 100  T 2 1 −1 (relative growth rate (% day ), extent of variation in pH, −1 −1 where W is the initial fresh weight, W is the final fresh 1 2 photosynthetic oxygen evolution rate (μmol O g FW h ), + −1 −1 weight, and T is the incubation period (3 days). ammonium uptake rate (μmol NH g FW h ), and photosynthetic Over a 12-h period, we monitored changes in pH in efficiency (F /F )) by samples of Gracilariopsis lemaneiformis v m the culture media, the rate of oxygen evolution, and Parameter/source of variation df MS F value p value NH uptake. An Orion-250A meter (Thermoscientific, Relative growth rate USA) was used to measure pH at 0, 2, 4, 6, and 12 h. pH level 1 0.69 21.50 <0.01 We also assessed the changes in pH in vessels contain- NH concentration 2 4.26 132.54 <0.01 ing media but no specimens (blanks), under low- and pH level × NH concentration 2 0.12 3.66 0.04 high-pH conditions. Extent of variation in pH Rates of photosynthetic oxygen evolution (μmol O g −1 −1 FW h ) were recorded with a Clark-type microelec- pH level 1 1.09 166.48 <0.01 trode oxygen sensor (Unisense, Denmark), which was NH concentration 2 0.38 57.63 <0.01 calibrated with a mixture of C H NaO (sodium ascor- 6 7 6 + pH × NH 2 0.01 0.10 0.90 bate) of 0.4 g and NaOH (sodium hydroxide) of 2 g in Photosynthetic oxygen evolution rate the 100-mL dilution water. + + −1 −1 pH level 1 198.72 68.85 <0.01 The rate of NH uptake (μmol NH g FW h ) 4 4 was calculated as the amount lost from each culture NH concentration 2 3678.60 1274.53 <0.01 medium over 12 h. The measurement method of NH 4 pH × NH 2 22.50 7.80 <0.01 uptake rates were described by Parsons et al. (1984). The NH uptake rate following equation was used in the calculation: pH level 1 0.17 24.72 <0.01 –1 –1 NH concentration 2 85.78 12,108.59 <0.01 V ¼ðÞ S –S  vol  W  T 4 i f pH × NH 2 0.13 18.95 <0.01 + 4 where S is the initial concentration of NH , S is the i 4 f Photosynthetic efficiency final concentration after T hours of incubation, vol is the pH level 1 <0.01 0.58 0.46 volume of the medium, and W is the fresh weight of each algal specimen. NH concentration 2 0.13 51.60 <0.01 Chlorophyll fluorescence was determined after 3 days pH × NH 2 <0.01 0.92 0.42 with a Plant Efficiency Analyzer (PEA, Hansatech, UK). −1 The maximum quantum yield (F /F ) of Photosystem II v m maximum RGR was 2.95 ± 0.20% day under L H con- pH A −1 was calculated as follows: ditions while the minimum RGR, 1.07 ± 0.21% day ,was achieved under H L conditions (Fig. 1). At the high NH pH A 4 F =F ¼ðÞ F −F =F v m m o m levels, RGR was greater at pH 7.5 than at pH 7.9 (H : where F is maximum fluorescence after dark adapta- F =6.04, p = 0.04). tion and F is minimum fluorescence after dark adapta- tion. Algal samples were placed in leaf-clip holders and F /F was measured by applying a saturating pulse after v m the samples were dark-adapted for 15 min. One- and two-way ANOVA were conducted with all experimental data. Normality and homogeneity were investigated before the statistical analysis began. After that, Tukey’s tests were used to compare among treatments, and the threshold for statistically significant differences was set at p < 0.05. All ana- lyses were performed with the SPSS software pro- gram (version 23.0). Results Results of physiological responses of Gracilariopsis lemanei- + −1 formis under elevated CO and NH treatments were sum- Fig. 1 Relative growth rates (% day )of Gracilariopsis lemaneiformis 2 4 at different pH and NH treatments. Bars not labeled with same letter marized in Table 2. During the incubation period, the indicate significant differences among culture conditions at p <0.05. relative growth rate of Gracilariopsis lemaneiformis in- Values are means ± SD (n =4) creased under elevated CO and NH treatments. The 2 4 Kang et al. Fisheries and Aquatic Sciences (2017) 20:18 Page 4 of 8 Alterations in pH in the culture media containing algal low than at high pH but there was no significant differ- specimens were significantly affected by initial pH or ence at the low NH level (L : F= 0.26, p =0.63; M : 4 A A NH levels. The extent of variation in pH ranged from F= 7.94, p =0.03; H : F= 7.67, p =0.03). 4 A 0.44 ± 0.02 to 1.26 ± 0.18 units, and the maximum and The rate of NH uptake was significantly affected by minimum changes in pH were associated with L H elevated concentrations of CO and NH , with rates pH A 2 4 and H L , respectively (Fig. 2; Table 3). At each pH ranging from 0.84 ± 0.01 to 7.43 ± 0.03 μmol NH g pH A 4 + −1 −1 level, the medium and high NH concentrations were FW h (Fig. 4). Uptake was more rapid under L H 4 pH A significantly different from the low NH condition conditions and slowest under H L treatment. When 4 pH A (L : F= 17.08, p = 0.01; H : F= 78.98, p < 0.01). At those rates were compared at the same pH, values in- pH pH + + individual NH levels, the change in pH was signifi- creased under higher NH concentrations (L : 4 4 pH cantly greater in the low-pH treatment than in the high- F= 3230.83, p < 0.01; H : F= 25,898.16, p < 0.01). Fur- pH pH treatment (L : F= 6.65, p = 0.04; M : F= 6.94, thermore, at the same level of NH , uptake was more A A 4 p = 0.04; H : F= 138.86, p < 0.01). The pH values in rapid under L H than under H H treatment (H : A pH A pH A A blank culture media were remain constant over 12 h that F= 6.50, p = 0.04). pH values were affected only photosynthesis oxygen evo- When values of the chlorophyll fluorescence were used lution of G. lemaneiformis. as a proxy to represent photosynthetic efficiency, they Photosynthetic oxygen evolution was affected by ele- ranged from 0.55 ± 0.22 (H L ) to 0.64 ± 0.02 (L H ) pH A pH A vated levels of CO and NH . Rates were (Fig. 5). Although values of F /F were significantly af- 2 4 v m −1 −1 + 62.28 ± 1.71 μmol O g FW h under H L and fected under high NH levels and under both pH con- 2 pH A 4 −1 −1 111.48 ± 0.95 μmol O g FW h under L H (Fig. 3). ditions (L : F= 44.64, p < 0.01; H : F= 15.91, 2 pH A pH pH When compared at the same initial pH, those rates in- p < 0.01), they were not significantly influenced by NH + + creased significantly as the NH level rose (L : conditions (L : F= 0.60, p = 0.47; M : F= 1.23, 4 pH A A F= 479.22, p <0.01; H : F= 854.92, p <0.01).Under p = 0.31; H : F= 0.92, p = 0.37). pH A medium and high NH conditions, the rate was greater at Discussion Growth of Gracilariopsis lemaneiformis was affected by elevated CO and NH treatments and there were in- 2 4 teractions of both factors. Previous studies have revealed similar results with G. lemaneiformis, Hypnea spinella, Chondrus crispus, Pyropia haitanensis, and Ulva pertusa (Yu and Yang 2008; Suárez-Álvarez et al. 2012; Sarker et al. 2013; Chen et al. 2016; Kang and Chung 2017). When DIC concentrations rise in the ocean, many macroalgal species conserve energy by regulating their CCMs, thereby improving their growth performance (Sarker et al. 2013). For example, red algae within the Gracilaria genus used external carbonic anhydrase to in- crease their capacity for HCO utilization, which boosts their growth under elevated CO conditions (Israel and Beer 1992; García-Sánchez et al. 1994; Zou et al. 2004). Greater availability of nutrients can also enhance the growth of macroalgae (Yu and Yang 2008). Although greater accumulations of nitrogen can stimulate growth, Xu et al. (2010) reported that G. lemaneiformis develop- ment was influenced by either CO or the level of phos- phorus, but those two factors did not show synergistic effects. Our results indicated that RGR were lower than those of other experiments. We speculated that short- term incubation period and high stocking density could be the reason of lower RGR. Kim et al. (2013) indicated that high stocking density might cause lower growth rates than those of light shading of the culture vessels. Fig. 2 Extent of variation in pH over time in response to different We assumed the high stocking density conditions of culture treatments. Data are means ± SD (n =4) aquaculture farm to find realistic growth of G. Kang et al. Fisheries and Aquatic Sciences (2017) 20:18 Page 5 of 8 Table 3 Maximum pH values and extent of variation in response over 12-h period to different treatment combinations Maximum pH values Extent of variation L (low pH) H (high pH) L (low pH) H (high pH) pH pH pH pH + a a a b L (low NH ) 8.36 ± 0.03 8.34 ± 0.02 0.86 ± 0.03 0.44 ± 0.02 A 4 + b b c a M (medium NH ) 8.66 ± 0.01 8.61 ± 0.04 1.16 ± 0.01 0.71 ± 0.04 A 4 + b b c a H (high NH ) 8.78 ± 0.18 8.77 ± 0.07 1.28 ± 0.18 0.87 ± 0.07 A 4 Data are means ± SD (n = 4). Within a column, values not followed by the same letter are significantly different at p < 0.05 lemaneiformis. In addition, Xu et al. (2017a) found that Photosynthetic oxygen evolution in our Gracilariopsis the RGRs of short-time cultivation period were higher lemaneiformis samples was also increased by elevated than those of long-time cultivation time. They explained CO and NH levels. The same has been reported for 2 4 that decreased of RGRs resulted from the acclimation to Hizikia fusiforme (currently, Sargassum fusiforme), culture conditions. Hypnea spinella,and Pyropia haitanensis (Zou 2005; The pH values in the culture medium could be used Suárez-Álvarez et al. 2012; Chen et al. 2016). Zou et al. as an indicator of physiological characteristics of the (2004) showed that the photosynthesis of G. lemaneifor- macroalgae (Maberly 1990; Murru and Sandgren 2004). mis is already saturated under normal DIC concentra- The pH of our culture medium was altered in response tions found in natural seawater. However, we noted that to increases in CO or NH concentrations. The this species was also influenced by the synergism be- 2 4 addition of CO gas caused pH values to decline while tween CO and NH . Similarly, Xu et al. (2010) has 2 2 4 the concentration of DIC, such as CO (aq), and HCO shown that the maximum net photosynthetic rate for G. 2 3 increased. This change in pH was more dramatic in the lemaneiformis is influenced by increases in both CO low-pH medium than under high-pH conditions. The and phosphorus concentrations. DIC concentrations were elevated in the culture The uptake of NH was influenced by treatment with medium, which lead to increases in pH levels by photo- exogenous CO and NH . Research with Gracilaria sp., 2 4 synthesis (Zhang et al. 2012). In addition, we did not G. chilensis, Hizikia fusiforme, Gracilariopsis lemaneifor- find any inhibition of photosynthesis. When pH values mis, Pyropia yezoensis, P. haitanensis, and U. pertusa are >9.0, photosynthesis of some species is more likely has demonstrated that all respond to elevated levels of to be inhibited (Maberly 1990; Björk et al. 2004). Zou CO or NH (Gao et al. 1993; Zou 2005; Xu et al. 2010; 2 4 et al. (2004) indicated that inorganic carbon (Ci) affinity Kang et al. 2014; Chen et al. 2016; Kang and Chung and photosynthetic rates at pH 9.0 were decreased in G. 2017). Increasing the concentration of CO can enhance lemaneiformis. In case of pH values over 9.0, this alga nitrogen uptake and the activity of nitrate reductase does not accumulate CO because of poor capacity of (Gordillo et al. 2001; Xu et al. 2010; Hofmann et al. HCO utilization (Zou et al. 2004). 2013; Liu and Zou 2015). In various seaweeds, the up- + + take of NH is greater when the initial NH 4 4 + −1 −1 Fig. 4 Ammonium uptake rates (μmol NH g FW h )of −1 + Fig. 3 Rates of photosynthetic oxygen evolution (μmol O g FW h Gracilariopsis lemaneiformis under different pH levels and NH 2 4 −1 + )of Gracilariopsis lemaneiformis at different pH and NH treatments. treatments. Bars not labeled with same letter indicate significant Bars not labeled with same letter indicate significant differences differences among culture conditions at p < 0.05. Values are means among culture conditions at p < 0.05. Values are means ± SD (n =4) ±SD (n =4) Kang et al. Fisheries and Aquatic Sciences (2017) 20:18 Page 6 of 8 2015). Yang et al. (2015) have suggested that large-scale cultivation of Gracilaria can improve water quality by uptake of excessive nutrients and potential carbon sink absorber along the coast of China. Therefore, this alga could provide a good solution for mitigation against the problems associated with such marine challenges. If we are to achieve practical results, future investigations will require more large-scale, long-term experiments. Those projects must also focus on the synergistic effects of sev- eral environmental factors, e.g., temperature, light inten- sity, level of salinity, and nutrient concentrations. Conclusions The combined treatment of elevated CO and NH 2 4 heightened the physiological reactions of G. lemaneifor- Fig. 5 Photosynthetic efficiency (F /F )of Gracilariopsis v m mis, as demonstrated by changes in relative growth lemaneiformis at different pH levels and NH treatments. Bars not labeled with same letter indicate significant differences among rates, rates of photosynthetic oxygen evolution, and the culture conditions at p < 0.05. Values are means ± SD (n =4) uptake of ammonium. The pH values were affected each elevated CO or NH treatments. In contrast, we noted 2 4 that chlorophyll fluorescence was affected only by alter- concentration is high (Dy and Yap 2001). In addition, ing the concentration of NH . when more nutrients are available, they are more easily Abbreviations absorbed (Runcie et al. 2003; Pérez-Mayorga et al. 2011). OA: Ocean acidification; RGR: Relative growth rates Therefore, because NH uptake by our G. lemaneifor- mis samples was more rapid under the elevated CO and Acknowledgements NH treatment, we speculate that this was due to the as- This work was supported by a grant from the National Research Foundation of Korea (NRF), funded by the Korean Government (MSIP) (NRF-2016R1A2B10 sociated rise in photosynthesis. 13637), to IKC. Chlorophyll fluorescence, which can reflect photosyn- thetic efficiency, is enhanced by increased N concentra- Funding This work was supported by a grant from the National Research Foundation tions (Dawes and Koch 1990). However, the role of CO of Korea (NRF), funded by the Korean Government (MSIP) (NRF-2016R1A2B10 is debatable with some studies showing that F /F is not v m 13637), to IKC. affected by elevated CO levels (Hofmann et al. 2012; Olischläger et al. 2013; Kram et al. 2016) and others in- Availability of data and materials All datasets in this study are available from the corresponding author on dicating that chlorophyll fluorescence increases under reasonable request. high CO concentrations (Chen et al. 2015). We found here that the photosynthetic efficiency of G. lemaneifor- Authors’ contributions mis was more strongly affected by greater NH concen- All authors conducted the field survey in the sampling stations. JW, Cicilia, and IK conducted experiments and wrote manuscript. All authors read and trations than by higher CO levels. Because of these approved the final manuscript. contrasting reports, direct measurement of oxygen evo- lution is now considered a more appropriate method Ethics approval and consent to participate than chlorophyll fluorescence for monitoring changes in Not applicable. efficiency (Kram et al. 2016). Consent for publication Our data provided evidence that the physiological ac- Not applicable. tivities of G. lemaneiformis are enhanced under elevated CO and NH treatments. We predict that mass pro- Competing interests 2 4 The authors declare that they have no competing interests. duction of this species in China will increase in response to OA and eutrophication. Kim and Yarish (2014) indi- cated that productivity of Gracilaria sp. was enhanced Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in under elevated CO levels with high stocking density published maps and institutional affiliations. and irradiance. 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Fisheries and Aquatic SciencesSpringer Journals

Published: Aug 16, 2017

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