Long-Term Experiment on Physiological Responses to Synergetic

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Long-term experiment on physiological responses to synergetic effects of ocean acidification and photoperiod in Antarctic sea ice algae Chlamydomonas sp. ICE-L Dong Xu, Yitao Wang, Xiao Fan, Dongsheng Wang, Naihao Ye, Xiaowen Zhang, Shanli Mou, Zheng Guan, and Zhimeng Zhuang Environ. Sci. Technol., Just Accepted Manuscript • Publication Date (Web): 12 Jun 2014 Downloaded from http://pubs.acs.org on June 19, 2014

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Environmental Science & Technology

Figure 1. Cell growth of Chlamydomonas sp. ICE-L in different photoperiods and pCO2 during incubation period of 28 days. (a) Variation of cell concentration during the period. (b) Growth rate of the first 10 days. Treatment of low pCO2 and regular light and dark cycle marked as LLD, low pCO2 and continuous light marked as LLL, low pCO2 and continuous darkness marked as LDD, high pCO2 and regular light and dark cycle marked as HLD, high pCO2 and continuous light marked as HLL, and high pCO2 and continuous darkness marked as HDD. Vertical lines represent standard deviations of triplicate incubations. 90x146mm (300 x 300 DPI)

ACS Paragon Plus Environment


Figure 2. PSII photo-physiological responses of Chlamydomonas sp. ICE-L to different photoperiods and pCO2. (a) maximum quantum yield (Fv/Fm). (b) relative electron transport rate (rETR). (c) light utilization efficiency (α). Treatment of low pCO2 and regular light and dark cycle marked as LLD, low pCO2 and continuous light marked as LLL, low pCO2 and continuous darkness marked as LDD, high pCO2 and regular light and dark cycle marked as HLD, high pCO2 and continuous light marked as HLL, and high pCO2 and continuous darkness marked as HDD. Vertical lines represent standard deviations of triplicate incubations. 90x195mm (300 x 300 DPI)


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Figure 3. Nutrient analysis during incubation of Chlamydomonas sp. ICE-L in cultures grown in different photoperiods and pCO2. (a) Variation of N concentration over the time. (b) The average N uptake rate during the first 5 days. (c) Variation of P concentration over the time. (d) The average P uptake rate during the first 3 days. Treatment of low pCO2 and regular light and dark cycle marked as LLD, low pCO2 and continuous light marked as LLL, low pCO2 and continuous darkness marked as LDD, high pCO2 and regular light and dark cycle marked as HLD, high pCO2 and continuous light marked as HLL, and high pCO2 and continuous darkness marked as HDD. Vertical lines represent standard deviations of triplicate incubations. 168x112mm (300 x 300 DPI)



Figure 4. Variation of lipid content (%) of Chlamydomonas sp. ICE-L grown in darkness under low and high pCO2. Treatment in low pCO2 marked as L, and Treatment in high pCO2 marked as H. Vertical lines represent standard deviations of triplicate incubations. 80x56mm (300 x 300 DPI)


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Figure 5. Growth (a) and photosynthetic responses (b) of Chlamydomonas sp. to high light stress of 400 µmol photons m−2 s−1. LDDL and LDDH represent cells treated in LDD and HDD in EI and recovered under low and high pCO2, respectively. HDDH represent cells treated in HDD in EI and recovered under high pCO2. Vertical lines represent standard deviations of triplicate incubations. 80x119mm (300 x 300 DPI)



74x47mm (300 x 300 DPI)


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1

Long-term experiment on physiological responses to synergetic

2

effects of ocean acidification and photoperiod in Antarctic sea ice

3

algae Chlamydomonas sp. ICE-L

4

Dong Xu,

5

Zhang, † Shanli Mou, † Zheng Guan, § and Zhimeng Zhuang, †

6

†

7

China

8

‡

9

§

10

*

11

†

Yitao Wang,

‡

Xiao Fan,

†

Dongsheng Wang, ‡ Naihao Ye,

*,†

Xiaowen

Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071,

College of Marine Science and Engineering, Qingdao Agricultural University, Qingdao 266109, China College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China Corresponding author. Tel.: +86 532 85830360 E-mail address: [email protected] (N. Ye)

12 13 14 15 16 17 18 19 20 21 22

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ABSTRACT: Studies on ocean acidification have mostly been based on short-term

24

experiments of low latitude with few investigating the long-term influence on sea-ice

25

communities. Here, the combined effects of ocean acidification and photoperiod on

26

the physiological response of an Antarctic sea ice microalgae Chlamydomonas sp.

27

ICE-L were examined. There was a general increase in growth, PSII photosynthetic

28

parameters, N and P uptake in continuous light, compared to those exposed to regular

29

dark and light cycle. Elevated pCO2 showed no consistent effect on growth rate

30

(p=0.8) and N uptake (p=0.38) during exponential phrase, depending on the

31

photoperiod. But it had a positive effect on PSII photosynthetic capacity and P uptake.

32

Continuous dark reduced growth, photosynthesis, and nutrient uptake. Moreover,

33

intracellular lipid, mainly in the form of PUFA, was consumed by 80% and 63% in

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low and high pCO2 in darkness. However, long-term culture under high pCO2 gave a

35

more significant inhibition of growth and Fv/Fm to high light stress. In summary,

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ocean acidification may have significant effects on Chlamydomonas sp. ICE-L

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survival in polar winter. The current study contributes to an understanding of how a

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sea ice algae-based community may response to global climate change at high

39

latitudes.

40 41 42 43 44

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INTRODUCTION

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Ocean acidification, derived from anthropogenic CO2 emissions, is a major

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environmental issue of the 21st century.1–5 According to the Intergovernmental Panel

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on Climate Change (IPCC), the atmospheric partial pressure of carbon dioxide (pCO2)

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has increased from 285 ppm in the pre-industrial age to the current level of 384 ppm

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and will approach ~1000 ppm by 2100.6 While an ocean uptake of ~25% of

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anthropogenic CO2 provides an invaluable service by mitigating CO2-related global

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warming, unabated CO2 emissions are likely to cause a 100–150% increase in the

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hydrogen ion concentration and a 0.3–0.4 drop in the pH of surface waters.7,8 Ocean

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acidification, accompanied by substantial warming, is projected to threaten the

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biodiversity and function of marine ecosystems, from the deep sea to coastal estuaries

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and from the tropical oceans to polar regions.9–13

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Recent laboratory and field studies have revealed that organisms with calcareous

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shells or exoskeletons may be unable to function as oceans acidify over the next 100

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years.14–17 However, some non-calcifying phytoplankton taxa, such as nitrogen-fixing

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cyanobacteria or diatoms, may benefit from the increasing CO2 levels in seawater.18–20

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Minor effects of ocean acidification on the growth of several macroalgae were also

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found.21 Despite an increasing interest in the understanding of the impacts of elevated

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CO2 concentrations on a wide range of marine organisms, research has primarily

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focused on low latitude regions and on short time scale studies.22 It is still not possible

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to make meaningful projections of the impacts on high latitude marine environments,

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which are particularly vulnerable to ocean acidification due to the high solubility of

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CO2 in cold waters.22,23 Moreover, most results are based on single stressor studies

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and a comprehensive understanding of the synergistic effects of the main climate

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variables on ocean productivity during longer time scales is lacking.24–26

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Phototrophic organisms in Antarctic oceans usually experience strong seasonal light

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variation, which is characterized by long periods of continuous light during polar

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summer or total darkness during the polar winter.27 The steep variation in incident

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solar radiation requires considerable physiological adaptations for polar organisms to

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survive. 28,29 These organisms rely on a variety of species-specific strategies to survive

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the long periods of darkness. The production of cysts or resting cells, adjusting

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metabolic rates, or increasing metabolic diversity have all been recognized as

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important

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organisms.30,31 Although such acclimation mechanisms have been adapted by polar

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organisms to survive longtime darkness in winter, additional global climate change

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co-stressors may challenge them in the future.

overwintering

strategies for

both

heterotrophic

and

autotrophic

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McMinn and Martin (2013)32 reported that although Arctic and Antarctic

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phytoplankton are able to survive temperature increases of up to 6 °C in the dark,

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elevated temperatures in winter reduces the viability of some polar microalgae.32 Here

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we examine whether they can also survive in an increasingly acidified ocean.

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Shadwick et al. (2013) compared the complete annual cycles of the CO2 system

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between Arctic and Antarctic seasonal cycles and demonstrated that the Arctic system

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is more vulnerable to anthropogenic change due to lower alkalinity, enhanced

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warming, and nutrient limitation.33 Torstensson et al. (2013) also showed that polar

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oceans are particularly susceptible to ocean acidification and warming and that

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elevated pCO2 and temperature caused a synergistic effect on the growth and lipid

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profiling of the Antarctic sea ice diatom Nitzschia lecointei.23 Nevertheless, to our

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knowledge, the physiological response of polar organisms to survive winter darkness

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under ocean acidification is poorly understood.

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Chlamydomonas sp. ICE-L is one of the green microalgae which can thrive in

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extreme environments in Antarctic.34 Despite extensive research showed that it can

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undergo specific ecological and physiological adaptations to harsh habitats, including

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low temperature, intense solar irradiance, excessive UV radiation and nutrient

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depletion, its survival strategy to overwinter is mostly lacking.35,36 Even less

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investigation has been undertaken on the impacts of climate change on its adaptation

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mechanism during winter. Under this scenario, we aim to present a comprehensive

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overview of multi-aspect cellular events associated to prolonged dark treatment under

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current

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Chlamydomonas sp. ICE-L. Moreover, to test physiological differences of

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re-acclimated microalgae under light, we examined and compared photosynthetic and

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growth responses to stressor between samples treated in current and elevated CO2.

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EXPERIMENTAL DETAILS

and

ocean

acidification

conditions

in

the

Antarctic

microalgae

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Sampling and culture conditions. The Antarctic ice microalgae Chlamydomonas

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sp. ICE-L was isolated from pack ice near the Zhongshan Research Station of

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Antarctica (69.8°S, 77.8°E) and grown axenically in Provasoli seawater medium.37

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Cultures were incubated with aeration in a plant growth chamber (GXZ, Ruihua,

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Wuhan, China) at 5 ± 1°C with a 12 h: 12 h light: dark (L:D) photoperiod. During

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light periods, PAR which was provided by 17 W, cool-white lamp, averaged

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approximately 40 µmol photons m−2 s−1. When exponential growth phase was reached,

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the cultures were incubated in fresh culture medium which was bubbled with low

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(pCO2 = 390 µatm, current CO2 levels) or high CO2 (pCO2 = 1000 µatm, predicted

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CO2 levels in 2100). The corresponding pH of the culture medium was 7.68 and 8.02

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respectively, which was monitored with a pH meter (Orion ROSS, Thermo Electron

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Corp., Beverly, MA, USA). Parameters in carbonate system were calculated using the

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CO2SYS Package.38 based on the pH, temperature, salinity, and total alkalinity (TA).

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TA was measured using the 848 Titrino plus automatic titrator (Metrohm, Riverview,

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FL, USA) on 100 ml GF/F filtered samples.

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Experimental set-up. In the first experiment (Experiment I, EI), conducted to

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explore the combined effects of photoperiod and partial pressure of CO2 (pCO2) on

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the physiological responses of Chlamydomonas sp. ICE-L, six combination treatments

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were implemented: low pCO2 and regular light and dark cycle (LLD), low pCO2 and

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continuous light (LLL), low pCO2 and continuous darkness (LDD), high pCO2 and

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regular light and dark cycle (HLD), high pCO2 and continuous light (HLL), and high

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pCO2 and continuous darkness (HDD). The experimental treatments were set up with

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a flask and tubing system similar to Zou and Gao (2009).39 All treatments were

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administered in triplicate. Cultures in exponential growth phase were inoculated into

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5000 ml Erlenmeyer flasks containing 3000 ml of acidified Provasoli seawater

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medium. The initial cell concentration used for the experiment was 2.2 × 105 cells

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ml−1. The low and high pCO2 was manipulated in a CO2 chamber, which was

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programmed to supply 390 uatm and 1000 uatm CO2 by bubbling. The regular light

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and dark cycle (12h/12h) was switched at 9:00 am and 21:00 pm; the continuous light

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was set at 20 µmol photons m−2 s−1 to maintain the same irradiance as the ambient

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photoperiod. Flasks in continuous darkness were wrapped in aluminium foil to

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prevent light penetration. Six controls, prepared acidifyied Provasoli seawater

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medium but without algal cultures under each treatment combination, were used to

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monitor the stability of the pH in the culture systems. Experiment I ran for 28 days.

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During this period, physiological parameters including growth, PSII photosynthetic

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activity, and nutrient uptake rate, were determined by sampling at 0, 1, 3, 5, 7, 14, 21,

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and 28 days. In addition, a further 2 cultures (3 replicates) were incubated in darkness

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under low (LDD) or high pCO2 (HDD) to examine the lipid profile changes during the

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experiment. 400 ml cultures were sampled for lipid and fatty acid analysis at 0, 7, 14,

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and 28th day.

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A second experiment (Experiment II, EII) was set up to investigate the

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photosynthetic

responses of

Chlamydomonas

sp.

ICE-L to

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re-acclimation to light. HDD and LDD cultures in EI were supplemented with fresh

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culture medium and re-acclimated in regular light and dark cycle under low or high

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pCO2 conditions. Cells treated in LDD and re-acclimated in low pCO2 or high pCO2

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were marked as LDDL or LDDH, respectively. Cells treated in HDD were only

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re-acclimated in high pCO2 and marked as HDDH. All treatments were cultured

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semi-continuously for 28 days until they reached exponential phase at a cell

7


stressor after


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concentration of 8.6 × 105 cells ml−1, 8.9 × 105 cells ml−1, and 9.1 × 105 cells ml−1,

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respectively. 100 ml of each treatment were exposed to high light of 400 µmol

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photons m−2 s−1 for 12 hours. PSII photosynthetic performance and cell numbers were

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monitored during the treatments.

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Growth measurements. A 0.5 ml sample in EI was collected at each sampling

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point and preserved in Lugol’s solution to estimate microalgal growth by directly

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counting cells using a hemocytometer under an optical microscope (Nikon, Tokyo,

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Japan). In EII, cells were firstly dyed with trypan blue (Solarbio, Cat.No.C0040, Lot.

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No.20110505) and then counted microscopically using hemocytometer to check cell

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viability according to Gao et al. (2013)40.

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Chlorophyll

fluorescence

measurements.

Variable

in

vivo

Chlorophyll

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fluorescence was measured using Pulse Amplitude Modulated (PAM) technique and a

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Dual-PAM-100 (Walz, Effeltrich, Germany) instrument. The minimum (F0) and

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maximum fluorescence (Fm) was measured after a dark-acclimation period of 15 min.

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The Fm yield of illuminated sample was denoted as Fm’ and Ft was the real-time

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fluorescence yield. The PSII maximum quantum yield was calculated as Fv/Fm, where

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Fv is the variable fluorescence emission (Fm–F0).41 The relative electron transport rate

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(rETR) was calculated as: rETR = Y(II) × PAR × 0.5, where PAR is the

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photosynthetic actinic radiation and 0.5 assumes that half of the absorbed light is

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distributed to PSII.41 The rapid fluorescence light-response curve (LRC) was

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measured by exposing the samples to 10 step-wise increasing irradiance (8 up to 827

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µmol photons m−2 s−1) at intervals of 30 each seconds. From LRC, light utilization

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efficiency (α) was obtained.

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Inorganic nutrients. The concentration of dissolved inorganic nutrients, NO2−1,

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NO3−1, NH4+ and PO43− was analyzed photometrically using an AutoAnalyzer (BRAN

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and LUEBBE AA3, Germaby). Nutrient uptake rates were calculated as follows: NUR

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= (C0 – Ct)V/N/t, where NUR is the nutrient uptake rate (µmol cell−1 h−1); C0 and Ct

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are the nutrient concentrations (µmol L−1) at the beginning and the end of the

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experiment, respectively; V is the volume of water (L); N is the average cell numbers

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(cells), and t is the time interval (h). The nutrient removal efficiency (NRE, %) was

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calculated as follows: NRE = 100 – (100 ×Ct/C0), where C0 and Ct are the nutrient

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concentrations (µmol L−1) at the beginning and the end of the experiment,

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respectively.

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Total lipid and Fatty acid analysis. The total lipid contents of Chlamydomonas sp.

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ICE-L at sampling time under different pCO2 were analyzed by the gravimetric

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method42 and intracellular lipid droplets were strained and detected with lipophilic

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fluorescent

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(4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene; Invitrogen Molecular

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Probes, Carlsbad, CA) according to Xu et al. (2013).43 For the fatty acid analysis, it

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was performed as described by An et al. (2013).34

dye

BODIPY

505/515

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Statistical analysis. The significance of the variance between treatments was

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analyzed using two-way ANOVA or repeated measures ANOVA by the software SPSS

197

17.0 (SPSS, Chicago, IL, USA). Post hoc tests were examined using Tukey’s test for

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two-way ANOVA and Dunnett’s Test for multivariate ANOVA. The assumptions of

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homogeneity of variance and normality were assessed by scatter plots of residuals and

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normal curves of residuals, respectively. All tests were run using the software SPSS

201

17.0. The significance level was set at 0.05 for all tests unless otherwise stated.

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RESULTS

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Variation of carbonate system in Experiment I. The variation of parameters of

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seawater carbonate system including pH, DIC, HCO3-, CO32-, CO2, pCO2 under

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different treatments were monitored and presented in Fig. S1. The initial

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corresponding pH under pCO2 of 390 and 1000 ppm was 8.02 ± 0.08 and 7.68 ± 0.06.

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Due to the density of the cultures, the carbonate chemistry of culture was difficult to

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maintain. During the culture period, there was a variation of carbonate system within

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a certain degree. pH varied

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and 8.28 ± 0.01 under treatment of LLD, LLL, and LDD. Under high pCO2 treatment,

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it changed from

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treatment of HLD, HLL, and HDD. However, although these variations occurred, a

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clear difference of pH (df=18, F=35.370, p

Long-Term Experiment on Physiological Responses to Synergetic

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