The impact of elevated atmospheric CO2 on cadmium toxicity in Pyropia haitanensis (Rhodophyta)
Cadmium is one of the major heavy metal pollutions in coastal waters, and it is well known that cadmium at trace concentration is toxic to macroalgae. Change in marine carbonate system and ocean acidification caused by elevated atmospheric CO2 also alter physiological characteristics of macroalgae. However, less research is focused on the combined impacts of elevated CO2 and cadmium pollution on the growth and physiology in macroalgae. In this study, the maricultivated macroalga Pyropia haitanensis (Rhodophyta) was cultured at three levels of Cd2+ (control, 4 and 12 mg L−1) and two concentrations of CO2, the ambient CO2 (AC, 410 ppm) and elevated CO2 (HC, 1100 ppm). The results showed that 12 mg L−1 Cd2+ significantly suppressed the relative growth rate and superoxide dismutase activity in AC-grown P. haitanensis, while such inhibition extents by Cd2+ were alleviated in HC-grown algae. Cd2+ had no effects on efficiency of electron transport (α) and maximum electron transport rate (ETRmax), but α was increased by elevated CO2. Cd2+ dramatically suppressed the maximum net photosynthesis oxygen evolution rate (NPRm) and the minimum saturation irradiance (Ik) when the algal thalli were grown at AC, while such suppression of NPRm by Cd2+ was much decreased when the thalli were grown at HC. Collectively, our results suggested that elevated CO2 would alleviate Cd2+ toxicity on P. haitanensis.
KeywordsElevated CO2 Cadmium toxicity Growth Photosynthesis Antioxidant system Pyropia haitanensis Marine macroalgae
Pyropia haitanensis is one of the most important species for seaweeds aquaculture in China. This alga has been used as a traditional food material of southern China, being rich in amino acids and minerals such as calcium, magnesium, and zinc, while low in fat (Chen et al. 2011). In addition, P. haitanensis can extract porphyran, a sulfated polysaccharide which possesses excellent antioxidant activity (Zhang et al. 2010). Aquaculture area of P. haitanensis in Shantou City, China, was about 5.4 km2 with 32 million US dollars per year in value, and aquaculture area of Fujian province was ten times that of Shantou (Liu 2017). The macroalgal fields can not only provide various habitats for other plants and animals (Evans et al. 2014) but also prevent and remediate coastal eutrophication by taking up a large amount of N, P, and other nutrients (Chung et al. 2002; Yang et al. 2015). Meanwhile, macroalgae and other marine plants perform good effects on carbon sequestration, acting as an important carbon sink of atmosphere (Chung et al. 2011).
The urbanization and industrialization of coastal areas have brought severe heavy metal pollution to coastal waters, which is an increasingly serious threat to marine ecosystem (Ranjbar Jafarabadi et al. 2017; Qiao et al. 2013). For example, researches showed that Arabian Gulf and coastal areas of South China suffer severe metal contamination, especially cadmium pollution (Naser 2013; Wang et al. 2013). Li et al. (2015) reported that mercury, cadmium, and lead are the predominant heavy metal pollutants in Bohai Bay of northern China. And in southern China, marine cadmium pollution also poses a threat to aquatic organisms (Wang et al. 2013). Cadmium was proved to have toxic effects on marine plants. It was shown that, when treated with cadmium, DNA was damaged in phytoplankton (Babu et al. 2014).Two-hour 25 μM cadmium exposure could up-regulate the transcripts of both nuclear- and chloroplast-encoded genes (Rubinelli et al. 2002). Bouzon et al. (2012) reported that the growth rate and the contents of photosynthetic pigments were reduced in Hypnea musciformis when exposed to cadmium. Moreover, under Cd2+ treatment, Ulva linza showed reductions in Fv/Fm, relative growth rate, and actual photochemical efficiency of PSII (Jiang et al. 2013).
Frequent and intensive human activities have caused not only heavy metal pollution (Ranjbar Jafarabadi et al. 2017) but also CO2 elevation in the atmosphere (Bedir and Yilmaz 2016). Elevating atmospheric CO2 over the last century has changed the marine carbonate system and decreased pH of surface water, which further affected marine ecosystem (Fabry et al. 2008). Studies have shown that ocean acidification inhibited the growth of coralline algae (Ragazzola et al. 2012) but might be beneficial to other algae, thereby changing the benthic community structure in shallow-water ecosystem (Kuffner et al. 2008; Price et al. 2011). In addition, high concentration of CO2 might promote the photosynthesis process of marine macroalgae, but this promotion depends on the species (Zou and Gao 2002).
Cadmium pollution in coastal water and elevated CO2 in atmosphere may affect each other. Elevated CO2-induced ocean acidification released the cadmium and other heavy metals from sediment, elevating their concentrations in sea water (Millero et al. 2009). At the same time increasing cadmium induced the change the marine inorganic carbon system (Zheng et al. 2009). Researches also showed that cadmium may replace the role of zinc in carbonic anhydrase to convert HCO3− into CO2 (de Baar et al. 2017). How will marine macroalgae respond to the combined impacts of elevated atmospheric CO2 and cadmium pollution? Nevertheless, this interesting issue has mainly been remained as unexplored. As an economically important macroalgal species, P. haitanensis, the effects of CO2 elevation on growth and photosynthesis have been attracted much attention (Xu et al. 2017). However, none of the previous studies paid attention on the combined effects of cadmium pollution and elevated CO2 on macroalgae such as P. haitanensis. In this paper, we cultured P. haitanensis at two levels of CO2 and three cadmium concentrations, and the physiological indications such as the growth, photosynthesis, and antioxidant enzymes activity were measured, in an effort to shed light on the possible combined effects of atmospheric CO2 elevation and cadmium pollution on the growth and physiology of P. haitanensis.
Materials and methods
Algae collection and experimental design
Pyropia haitanensis was collected from Shen’ao Bay (117.1 E, 23.5 N), Shantou, China, and was transported to the laboratory in a clean sampling case under 4 °C within 6 h. The algal thalli were maintained in an intelligent artificial climate incubator (HP1000GS-D, Ruihua Instrument & Equipment, Wuhan, China) under constant temperature (18 °C) and irradiance (150 μmol photons m−2 s−1, cool white fluorescent tubes) of 12:12 light/dark cycle (8 a.m. to 8 p.m.), and were continuously aerated for 2 days before the experiment. Sterile seawater (salinity ~ 32‰, pH ~ 8.2) enriched with 200 μM NaNO3 and 50 μM Na2HPO4 was used as culture media as our previous study (Chen and Zou 2014).
Healthy thalli of P. haitanensis were quadrated by surgical scissors (about 35 mm × 35 mm). About 0.8 g (fresh weight, Fw) samples were randomly chose to culture in 2-L conical flask with 1.5-L culture media for 1 week, and media was renewed every 2 days. The pH of culture media was calibrated with 1 M HCl and 1 M NaOH solution, to maintain constant pH level of ~ 8.2, during each culture media renewal period. Two levels of CO2, 410 ppm (ambient CO2, AC) and 1100 ppm (high CO2, HC), were controlled by aerating ambient air and intelligent incubator, respectively. Three concentrations of Cd2+ (LCd, control; MCd, 4 mg L−1; HCd, 12 mg L−1) were prepared with CdCl2. Other conditions were retained as the conditions in preculture and three replicates were done for each treatment.
The growth of P. haitanensis was determined as relative growth rate (RGR): RGR = (InWt − InW0)/t × 100, where W0 was the initial Fw, while Wt was the Fw after t days (Hunt 1982). Before weighing, seawater was softly removed from the surface of P. haitanensis by blotting paper. The inhibition percentage of Cd2+ on RGR was calculated as follows: inhibition % = (RCRL − RCRt)/RCRL × 100, where RCRL is the RGR at AC-LCd, and RCRt is the RGR at MCd and HCd treatments (Gao et al. 2016).
Chlorophyll fluorescence measurement
The rapid light curves (RLCs) were generated by a pulse amplitude-modulated fluorometer (Junior PAM, Walz, Germany). RLCs were plots of electron transport rate (ETR) versus actinic irradiances (I: 0, 125, 190, 285, 420, 625, 820, 1150, and 1500 μmol photons m−2 s−1) applied for 10 s. ETR was calculated from chlorophyll fluorescence parameters automatically, ETR = Fv’/Fm’ × 0.5 × PFD, where Fv’/Fm’ is the effective PSII quantum yield and PFD is the photosynthetically active photon flux density. Efficiency of electron transport (α) and minimum saturating irradiance (Ik) were obtained from ETR = ETRmax × tanh(α × I/ETRmax) and Ik = ETRmax/α (Henley 1993).
Measurement of photosynthetic oxygen evolution
P. haitanensis was cut into 5~6-mm square and resumed in original culture media. Approximately 0.05 g (Fw) of algae (randomly chose) was placed in a transparent chamber filled with 15-mL original culture media. The chamber was sealed with a plug which a needle-type oxygen sensor (OXR50, PyroScience, Germany) was connected with, and a temperature controller (Cole-Parmer Polystat, USA) was used to ensure the whole process was proceeded at 18 °C. The net photosynthetic oxygen evolution rate (NPR) was determined under different irradiances (0, 30, 60, 100, 300, 500, and 700 μmol photons m−2 s−1, measured by a quantum sensor, SKP 200; ELE International, Leighton Buzzard, UK) supplied by a halogen lamp. The maximum photosynthetic oxygen evolution rate (NPRm) was obtained from NPR = NPRm × tanh(α × I/NPRm) + c (Henley 1993). The inhibition percentage of Cd2+ on NPRm was calculated as follows: inhibition % = (NPRL − NPRt)/NPRL × 100, where NPRL is the NPR at AC-LCd, and NPRt is the NPRm at MCd and HCd (Gao et al. 2016).
Measurement of SOD activity
For superoxide dismutase (SOD) activity, approximately 0.1 g (Fw) of P. haitanensis (randomly chose) was ground and incubated in 9 mL 0.1 M phosphate buffer (pH 7.0, containing 0.3% Triton X-100 and 4% PVPP) at 4 °C for 5 min (Neto et al. 2006). This extract was centrifuged (CT14RD, Techcomp, Shanghai, China) at 10,000 rpm, 4 °C for 10 min, and then, 30 μL of the supernatant was used to determine the SOD activity by SOD assay kit (A001-1-1, Nanjing Jiancheng Bioengineering Institute, China). The unit of SOD activity was expressed as U g−1 Fw.
Chlorophyll a and carotenoids
According to Wellburn (1994), approximately 0.05 g (Fw) of algae (randomly chose) was suspended by 8 mL 100% methanol at 4 °C for 24 h avoiding light. The extract was centrifuged at 10,000 rpm, 4 °C for 5 min. The absorbance values (A) of supernatant were determined by a UV spectrophotometer (UV-1800, Shimadzu, Japan) and the contents of chlorophyll a (Chl a) and carotenoid (Car) were calculated by
Chl a = (16.72A665.2 − 9.16A652.4) × 8/0.05 and Car = (4.53A470 − 16.26A652.4 − 7.38A665.2) × 8/0.05, mg g−1 Fw.
Phycoerythrin and phycocyanin
Approximately 0.08 g (Fw) of P. haitanensis was ground and incubated in 8 mL 0.1 M phosphate buffer (pH 6.8) at 4 °C, and then centrifuged at 10,000 rpm, 4 °C for 15 min. The content of phycoerythrin (PE) and phycocyanin (PC) were estimated according to Beer and Eshel (1985): PE = [(A564 − A592) − 0.20(A455 − A592)] × 0.12 × 8/0.08 and PC = [(A618 − A645) − 0.15(A592 − A645)] × 0.15 × 8/0.08, mg g−1 Fw.
Results were expressed as means of replicates ± standard deviation. One-way analysis of variance (ANOVA) was used to analyze the effects of Cd2+ and CO2 on RGR, SOD, Chl a, Car, PE, PC, ETRmax, α, Ik, and NPRm. Significant differences among the Cd2+ concentrations at the same CO2 levels were classified after post hoc comparison test (least significant difference test), while t tests were used to calculate the significant differences between the Cd2+ pollution alone or combined with elevated CO2 and the control. A P value of 0.05 was considered statistically significant. Statistical analyses were done using SPSS v.25 (IBM, USA).
The maximum electron transport rate (ETRmax), saturation irradiance (Ik), and electron transport efficiency (α) of Pyropia haitanensis cultured at different Cd2+ and CO2 conditions
129.63 ± 25.70a
125.39 ± 56.13a
116.89 ± 29.15a
116.18 ± 13.50a
112.48 ± 19.86a
109.48 ± 20.02a
0.1096 ± 0.0032b
0.1141 ± 0.0055a
0.1177 ± 0.0089a
0.1354 ± 0.0090a
0.1105 ± 0.0088a*
0.1274 ± 0.0163a
1145.70 ± 215.86a
893.97 ± 159.35a*
798.28 ± 25.88a*
947.08 ± 161.65a
1055.02 ± 66.98a
790.03 ± 193.78a
Net photosynthetic oxygen evolution
In AC treatment, no significant differences of the contents of Chl a (F2,6 = 0.010, P > 0.05) and Car (F2,6 = 1.068, P > 0.05) among the different Cd2+ treatments. Elevated CO2 significantly decreased the contents of Chl a at LCd (t = − 3.602, P < 0.05) and MCd (t = − 3.537, P < 0.05), but at HCd, it had no obvious effect on Chl a contents (HC-HCd vs. control: t = 1.519, P > 0.05). However, elevated CO2 concentration did not show effect on Car content of algae at LCd (t = − 2.539, P > 0.064). Car contents of HC-HCd were increased significantly compared to the pigment’s concentration of control (t = 3.657, P < 0.05).
The effect of cadmium on P. haitanensis
Coastal waters are mostly impacted by anthropogenic pollutants such as heavy metals. Being major primary producers and playing an important role in food chains in coastal waters, marine macroalgae are now being paid greater attention to the toxic effects of heavy metals such as cadmium. In the present study, 12 mg L−1 Cd2+ exerted a significant inhibition on the growth of P. haitanensis over our experiment period. Such significant suppression of growth by Cd2+ was also reported in the same algal species, P. haitanensis and Hizikia fusiformis previously (Zhu et al. 2011, 2017). A possible physiological reason for Cd2+ stress on growth might be the oxidative stress induced by cadmium exposure, as suggested by Kumar et al. (2010). It has been proved that Cd2+ induces oxidative stress like lipid peroxidation and H2O2 production in marine alga Nannochloropsis oculata (Mi and Shin 2003). SOD is an important antioxidant enzyme that can reduce oxidative stress; thereby, it plays a critical role in resisting cadmium stress (Fabrizio et al. 2003). In this study, 4 mg L−1 Cd2+ slightly increased SOD activity of P. haitanensis, while 12 mg L−1 Cd2+could dramatically reduce it. It means that the antioxidant ability was suppressed by high concentration of Cd2+. Similar results were also found in Chlorella vulgaris (a freshwater alga), in which SOD activity was increased by lower concentration of 0.5 mg L−1 Cd2+, but was dramatically decreased with higher concentration of 5 mg L−1 Cd2+ (Cheng et al. 2016).
No obvious changes were observed on contents of Chl a and Car in this experiment, even though previous studies exhibited that 10 mg L−1 Cd2+ could significantly inhibit Chl a and 8 mg L−1 Cd2+ could dramatically decrease Car of P. haitanensis (Zhu et al. 2017). The content of PE was significantly decreased by the highest concentration of Cd2+. These results displayed that the sensitivity of PE to Cd2+ were much higher than that of Chl a and Car, as the previous report (Xia et al. 2004). PE and PC are important accessory pigments, and PE is the highest content of pigment in red alga (Baghel et al. 2014), so that Cd2+ suppressed light absorption of P. haitanensis, although there was no corresponding reduction in PC. Our results showed that the maximum net photosynthetic rate NPRm and minimum saturating irradiance (Ik) of P. haitanensis were inhibited upon exposure to high level of Cd2+, even though ETRmax and α did not show significant changes that resulted from Cd2+. It has been suggested that Cd2+ may substitute Mg2+ as the central atom of chlorophyll, which thereby prevents photosynthetic light-harvesting and finally results in suppression of photosynthesis (Küpper et al. 2002). It might be the physiological reason why the content of Chl a did not decrease, but photosynthesis was negatively affected in P. haitanensis. Collectively, the present results showed that Cd2+ in high concentration (12 mg L−1) caused strong disturbance in the photosynthesis of P. haitanensis and induced oxidative stress. As a result, the thalli of P. haitanensis spent more energy on synthesizing metal-chelating proteins and peptides which reduce the bioavailability of Cd2+ to maintain cell viability (Moenne et al. 2016), making less energy available for growth.
Elevated CO2 alleviated the Cd2+ stress on P. haitanensis
Although the growth of P. haitanensis was not significantly promoted by HC (1100 ppm) at all Cd2+ levels, the inhibition of Cd2+ on growth was much less pronounced by HC (1100 ppm) in this study. Previous research showed that Cd2+ might replace the role of zinc in carbonic anhydrase to convert HCO3− into CO2 (de Baar et al. 2017), and Cd2+ in low concentration could enhance the utilization of HCO3− and CO2 in Ulca pertusa, making decrease of HCO3− and CO2 in seawater (Zheng et al. 2009). In addition, the decrease in pH induced by elevated CO2 would result in a raise in the concentrations of both HCO3− and CO2 in seawater (Feely et al. 2009). Cd2+ in low concentration and increased CO2 might have a synergistic effect on promoting growth of P. haitanensis. At the same time, the SOD activities of HC-grown algae (1100 ppm) were markedly higher than that of AC-grown algae (410 ppm) at all Cd2+ concentrations, and the inhibitory effect of Cd2+ on SOD activity was decreased in HC-grown algae, which means that 1100 ppm CO2 dramatically enhanced SOD activity of P. haitanensis. Moreover, SOD activity of algae grown in 1100 ppm was not inhibited at 12 mg L−1 Cd2+ compared to other Cd2+ levels, indicating that increased CO2 enhanced the antioxidant ability of P. haitanensis. Similar results were reported in Ginkgo biloba (Ruan et al. 2007) and Cocosnucifera L. (Sunoj et al. 2014), of which the SOD activities were dramatically increased by short-term CO2 elevation.
In present study, the contents of Chl a and Car in HC-grown algae (1100 ppm) decreased significantly at control and 4 mg L−1 Cd2+ compared to that in AC-grown algae (410 ppm). The content of Chl a in Porphyra leucosticte (Rhodophyta) was also reduced by elevated CO2 (Mercado et al. 1999). Such inhibition was likely due to the accumulation of excess polysaccharide which affected the normal structure and function of chloroplasts (Cave et al. 1981). However, the algae grown in HC-HCd (1100 ppm, 12 mg L−1 Cd2+) had similar contents of Chl a and Car with the one grown in AC-LCd (410 ppm, control). The increase of Chl a might be attributed to the synthesis of chlorophyll-Cd2+ (Küpper et al. 2002), and promotion of Car might result from the stress response to Cd2+ which enhanced the antioxidant ability of P. haitanensis (Strzałka et al. 2003).
Ik and ETRmax had no obvious changes at all Cd2+ levels in high CO2 concentration, but the efficiency of electron transport, α increased significantly at LCd by elevated CO2. A possible explanation is that P. haitanensis may improve the utilization efficiency of solar energy to offset the low contents of photosynthesis pigments in high CO2 environment. The notable higher NPRm in HC-grown algae also confirms this conjecture. The elevated CO2 might inhibit the photorespiration process and reduce oxygen fixation (Peterhansel et al. 2010), increasing NPRm in HC-grown algae. This can be supported by the fact that NPRm of Porphyra leucosticte (Mercado et al. 1999) and U. Prolifera (Gao et al. 2016) were increased in high CO2 level.
The defense against Cd2+ is energy-intensive. When the excess Cd2+ break through the cell membrane, phytochelatin (PCs) are produced to combined with Cd2+, and then, these Cd2+ chelates are wrapped to many small vesicles (Andrade et al. 2010; Talarico 2002). In addition, the synthetics of SOD enzyme and chlorophyll-Cd2+ are the energy-costing processes. Therefore, more energy would be used to maintain essential metabolism and cellular integrity, resulting in less energy for growth. Excess polysaccharide synthesized in high CO2 concentration might be a sufficient energy source (Andrade et al. 2010), so that the inhibition of Cd2+ on growth and antioxidant ability of HC-grown P. haitanensis were decreased in this research. In addition, the inhibitory effect of Cd2+ on NPRm of algal thalli was decreased by elevated CO2.
Collectively, the present study showed that high CO2 concentration (1100 ppm) reduced the toxicity of Cd2+ on the alga. We suggested that the predicted high CO2 level in the future might be in favor of P. haitanensis aquaculture when facing with the aggravation of marine cadmium pollution. Further studies are needed to address the time course of physiological characteristics and the underlying molecular mechanisms with the combined effects of future anthropogenic CO2 and heavy metal pollution.
This study was supported by the National Natural Science Foundation of China (Grant Nos. 31741018 and 41706136) and Guangdong Province (Grant No. 2018B030311029).
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