Effects of pH and nitrogen form on Nitzschia closterium growth by linking dynamic with enzyme activity

Journal Pre-proof Effects of pH and nitrogen form on Nitzschia closterium growth by linking dynamic with enzyme activity Keqiang Li, Min Li, Yunfeng He, Xingyan Gu, Kai Pang, Yunpeng Ma, Dongliang Lu PII:

S0045-6535(20)30347-7

DOI:

https://doi.org/10.1016/j.chemosphere.2020.126154

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CHEM 126154

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Received Date: 24 November 2019 Revised Date:

16 January 2020

Accepted Date: 7 February 2020

Please cite this article as: Li, K., Li, M., He, Y., Gu, X., Pang, K., Ma, Y., Lu, D., Effects of pH and nitrogen form on Nitzschia closterium growth by linking dynamic with enzyme activity, Chemosphere (2020), doi: https://doi.org/10.1016/j.chemosphere.2020.126154. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.

Credit Author Statement Keqiang Li: Writing draft; Writing-Reviewing and Editing; Software Min Li: Writing draft; Methodology; data analysis Yunfeng He: Software Xingyan Gu: Writing-Original draft preparation Kai Pang: data analysis Yunpeng Ma: data analysis Dongliang Lu: Methodology,

1

Effects of pH and nitrogen form on Nitzschia closterium growth

2

by linking dynamic with enzyme activity

3

Keqiang Lia,b*, Min Lia, Yunfeng Hea, Xingyan Gua, Kai Panga, Yunpeng Maa,c, Dongliang Lud

4

a

5

Chemistry and Chemical Engineering, Ocean University of China, Qing Dao 266100, China

6

b

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Marine Science and Technology, Qingdao 266071, China

8

c

9

d

Key Laboratory of Marine Chemistry Theory and Technology, Ministry of Education, College of

Laboratory for Marine Ecology and Environmental Science, Qingdao National Laboratory for

Qingdao National Laboratory for Marine Science and Technology, Qingdao 266071, China

Guangxi Key Laboratory of Marine Disaster in the Beibu Gulf, Beibu Gulf University. Qinzhou

10

535011, China

11

*

Corresponding authors. E-mail: [email protected] TEL: +86 532 66786355

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Abstract: In this study, Nitzschia closterium was incubated in seawater at different

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pH values (8.10, 7.71, and 7.45) and using different nitrogen forms (NO3-N and

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NH4-N) in the laboratory. The results showed that the growth of N. closterium was

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inhibited by ocean acidification, with individuals under lower pH levels showing

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lower growth rates and lower nitrogen uptake rates for both nitrogen forms. The

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Vmax/Ks ratio decreased with decreasing pH, indicating the inhibition of nitrogen

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uptake, whereas the ratios for NH4-N cultures were higher than those for NO3-N

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cultures, implying the highly competitive position of NH4-N. Acidification might

21

induce reactive oxygen species based on the result that the maximum enzyme 1

22

activities of SuperOxide Dismutase (SOD) and CATalase (CAT) increased under

23

lower pH levels. The SOD and CAT activities for the NO3-N cultures were higher

24

than those for NH4-N cultures at the low pH level, indicating that acidification might

25

cause more oxidative stress for NO3-N cultures than for NH4-N cultures. Thus, ocean

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acidification might have a more detrimental effect on the growth of N. closterium

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under NO3-N conditions than NH4-N conditions, with a lower ratio (γ) of the

28

maximum growth rate to the maximum nutrient uptake rate, and a drop in nitrate

29

reductase activity under lower pH levels.

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Key words: Ocean acidification; Nitzschia closterium; Nitrogen; Antioxidant Enzyme

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Activity; Nitrate Reductase

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1 Introduction

34

The ocean is a significant carbon sink for the earth, and it plays a crucial role in

35

the global carbon cycle (Zeebe et al., 2008). Approximately one-third of the

36

atmospheric CO2 released by anthropogenic activities (fossil fuel burning, cement

37

production, and others) has been absorbed by the ocean (Sabine et al., 2004), slowing

38

global warming. Anthropogenic CO2 entering the ocean is altering the seawater

39

chemistry and carbonate system and reducing the pH. The process is commonly

40

referred to as ocean acidification (OA). OA has the potential to detrimentally effect

41

organisms, species, and communities (Caldeira and Wickett, 2003; Kurihara and

42

Shirayama, 2004). Since the beginning of the industrial revolution, the pH of surface

43

seawaters has decreased by approximately 0.1 units (pH: 8.10). This is equivalent to 2

44

an approximately 30% increase in H+ concentrations, making the ocean more acidic

45

(Orr et al., 2005). At present, the rate of change is the fastest observed in the past 300

46

million years because humans are rapidly changing the atmospheric composition and

47

marine chemical properties (Hönisch et al., 2012). According to the Intergovernmental

48

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

49

atmosphere is predicted to further increase to 800-1000 ppm by the end of the 21st

50

century, and the pH of surface seawaters will further decrease by 0.3-0.4 units (pH:

51

7.80-7.70), equivalent to an approximately 150% increase in H+ concentrations

52

(Caldeira and Wickett 2005; Hönisch et al., 2012). Researchers (e.g., the World Ocean

53

Circulation Experiment [WOCE]) have detected the anthropogenic signal in the open

54

ocean, but similar research has not been conducted until recently in coastal oceans

55

(Andersson et al., 2015). Acidification driven by atmospheric CO2 in the open ocean

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may be minor compared to the internal processes in coastal ecosystems, particularly

57

within eutrophic regions (Wallace et al., 2014).

58

With the development of societies and economies, humans have discharged large

59

amounts of sewage, and industrial and agricultural wastewater into coastal waters,

60

resulting in increased nutrient concentrations and a change in the nutrient structure

61

(NH4-N, NO3-N). Excessive nutrient loading promotes algal productivity and triggers

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widespread hypoxia during the summer. There is a relationship between coastal

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hypoxic zones and the pCO2 in seawater, providing the foundation for studying

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acidification and eutrophication (Howarth et al., 2011). The pH will drop by 0.47 units

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(pH: 7.30-7.20) under the action of eutrophication by the end of the 21st century (Cai 3

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et al., 2011). Meanwhile, coastal waters undergo natural pH fluctuations on daily and

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seasonal scales, and coastal species undergo seasonal acidification arising from

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increased respiration due to higher amounts of organic matter produced by primary

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production related to eutrophication. The geological record of OA events during

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Earth’s history suggests that many marine organisms have been affected by

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environmental changes (Hönisch et al., 2012). Meanwhile, ocean acidification may

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develop to pose an unprecedented threat to marine life, and our understanding of the

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processes that underling its observed effects on ecosystems and biogeochemistry is

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still rudimentary (Riebesell, 2008). Essentially, algae, that play a basal role in the

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marine ecosystem food chain, may be unable to adapt to this change (Riebesell et al.,

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2013). Therefore, algae populations will be confronted with a great survival crisis.

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Thus, the transition from an experimental strategy that examines the effect of a single

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driver to multiple drivers has to deal with many challenges (Boyd et al., 2018).

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Marine phytoplankton are the primary producer affecting the ocean carbon cycle

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on a global scale, consuming anthropogenic CO2 dissolved in the ocean through

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photosynthesis and producing CO2 by respiration. As an important species of

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phytoplankton, diatoms contribute about 20% of the world's organic carbon through

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photosynthesis every year (Field et al., 1998). Increased atmospheric pCO2 due to

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anthropogenic activities could affect the growth of marine primary producers through

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photosynthesis (Riebesell and Tortell, 2011), and numerous studies have indicated the

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toxic effect of OA on marine organisms. For instance, some calcified ecosystems,

87

such as coral reefs, could be negatively affected due to the dissolution of CaCO3 4

88

caused by acidification (Orr et al., 2005; Hendriks et al., 2010). Also, the composition

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of diatoms living in different pCO2 has changed and copepods are biologically

90

influenced by eating the affected diatoms (Rossoll et al., 2012). According to recent

91

studies, OA promotes the growth of diatoms Phaeodactylum tricornutum, Attheya sp.

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and Pseudo-nitzschia multiseries (Wu et al., 2010; King et al., 2011; Sun et al., 2011),

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the growth of diatom Nitzschia spp. and Chaetoceros brevis are not affect by OA

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(Kim et al., 2006; Boelen et al., 2011), while the growth rate of the diatom

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Skeletonema costatum and Thalassiosira pseudonana show no increase after

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acclimation to OA (Chen & Gao, 2003; Crawfurd et al., 2011). OA would also have

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an effect on other algal growth factors. Ocean acidification treatments of lowered pH

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with elevated CO2 stimulate diatom growth under low to moderate levels of light, but

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lead to inhibition of diatom growth when combined with excess light (Gao and

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Campbell, 2014). Another study showed that OA decreased the iron uptake rate of

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diatoms (Shi et al., 2010). In addition, the C/N of Phaeodactylum tricornutum was in

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nitrogen limited and nitrogen replete conditions both increased under elevated pCO2

103

conditions (Li et al., 2012). These results indicate that the effects of OA are different

104

on algal growth, which raises the uncertainty of the effect of marine ecosystem safety

105

(Kroeker et al., 2013). According to one forecast, OA will mainly occur at depths of a

106

few hundred meters below the sea surface at the end of the 21st century (Caldeira and

107

Wickett, 2003), therefore, phytoplankton that live in the euphotic layer will be the first

108

affected by OA. At the same time, as the dominant species in marine ecosystems,

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diatoms are sensitive to pH changes and CO2 availability, which are the most 5

110

important factors affecting diatom survival, and can reflect living conditions in the

111

aquatic environment (Carpenter and Waite, 2000).

112

Over the last few decades, considerable research has shown that reactive oxygen

113

species (ROS) play a critical role in the pathophysiological pathways of algae (Zhang

114

et al., 2013). Under normal circumstances, there is a set of complete antioxidant

115

systems in the algal body, and the production and clearance of ROS are always in a

116

state of dynamic balance (Seel et al., 1992). When algae are exposed to acidified

117

environment, the acid-base imbalance can activate the antioxidant system to eliminate

118

the overproduction of oxygen free radicals and ROS accumulation in order to

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maintain normal algal physiological activities (Li et al., 2015). Superoxide dismutase

120

(SOD) and catalase (CAT) are the main antioxidant enzymes that play a key role in

121

alleviating the oxidative stress caused by acidification (Sies and Stahl, 1995). Jahnke

122

and White (2003) noted that SOD activity increased with increasing salinity when

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Dunaliella grew under salinity stress. The ROS level increased when Phaeodactylum

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tricornutum was exposed to ethyl 2-methyl acetoacetate, and the SOD and CAT

125

activities increased with the exposure concentration and decreased with prolonged

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exposure time (Yang et al., 2011). Generally, diatoms are selective about the type

127

nitrogen they use and prefer to be in water with rich ammonium-nitrogen (NH4-N)

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because this compound can directly synthesize amino acids by transamination under

129

the action of GS/GAGOT enzymes (Clayton and Ahmed, 1986). Other nutrients, such

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as NO3-N, should be reduced to NH4-N by nitrate reductase (NR) (Berges and

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Mulholland, 2008). NR can control the primary step of NO3-N assimilation, and 6

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indicates the nitrogen forms used by diatoms. This is a useful tool in that a positive

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result indicates the utilization of NO3-N (Eppley et al., 1970). Xia and Gao (2005)

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noted that the NR activities of green algae (Chlorella pyrenoidosa and

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Chlamydomonas reinhardtii) were significantly decreased with CO2 enrichment

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(3-186 µmol·L-1). At present, researchers are mainly focusing on the OA response of

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calcareous organisms, such as foraminifera, coccolithophores and coral reefs

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(Riebesell et al., 2000; Sciandra et al., 2003; Hoegh-Guldberg et al., 2007; Burns,

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2008). However, marine phytoplankton have not been given sufficient research

140

attention, especially related to different nitrogen form effects, although some

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investigators have ventured into these (Gu et al., 2017; Gazeau et al., 2017; Wang et

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al., 2017). Thus, understanding the OA response of marine phytoplankton, especially

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the dominant diatom species, to different nitrogen forms is essential.

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In this study, we selected the common coastal diatom N. closterium and

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subjected it to culture experiments in the laboratory. We aimed to determine the effect

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of pH and different nitrogen forms on phytoplankton growth, while the growth,

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nutrient-uptake kinetics, and enzymatic activity of N. closterium were assessed. This

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study might provide an experimental basis for further studies about the potential

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impacts of OA on phytoplankton and marine ecosystems.

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2 Materials and Methods

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2.1 Cultures

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The N. closterium culture was obtained from the algal species room at the Key

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Laboratory of Marine Chemistry Theory and Technology, Ministry of Education,

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Ocean University of China. For preparing the culture medium, seawater was collected

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from the Yellow Sea coast (36°5.707′ E, 120°15.676′ N) with a DOC concentration of

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3.94 mg· L-1, pH of 8.10, and a salinity of 27. The seawater was successively filtered

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through 2, 0.45, and 0.22 µm membrane filters to remove particulate matter and

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bacteria. Then, 5 L transparent glass vessels covered by qualitative filter paper (Φ11

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cm) were used for the experiment, and 70 mL of the N. closterium suspension was

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added to 3.5 L of sterile seawater where the carbonate chemistry equilibrium had been

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reached before the experiment began. The phytoplankton biomass chlorophyll a (Chl

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a) concentration was ~8.36 µg·L-1. Nutrients were added, including PO4-P (1.5 µM,

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KH2PO4; Sinopharm Chemical Reagent Co., Ltd. (SCRC)), SiO3-Si (30 µM,

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Na2SiO3·9H2O, SCRC), and two types of nitrogen forms (NO3-N (30 µM, NaNO3;

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SCRC) and NH4-N (30 µM, NH4Cl; SCRC). The cultures were maintained at room

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temperature (20±1 °C) and a simulative light environment using light-emitting diodes

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(LEDs) (~191 µmol·m-2·s-1 under 12 h:12 h of light: dark cycle), with double samples

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(Table 1). The experimental period was 5-10 d, with sampling frequencies of 1-2

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times/d.

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Samples for the analysis of dissolved inorganic nitrogen (NO3-N, NO2-N,

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NH4-N), dissolved organic nitrogen (DON), Chl a, particulate organic nitrogen (PON),

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and the antioxidant enzyme and NR activities were collected in four portions at all 8

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time points. The water samples were filtrated by a Whatman GF/F 0.70 µm glass fiber

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membrane (450 ℃, firing for 5 h), and collected in 50 ml acid washed (HCl)

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polyethylene bottles. The filtrate and filter were kept frozen (-20 °C) until

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measurement in the laboratory.

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2.2 pH setting and manipulation

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Three 8.10, 7.71, and 7.45 pH gradients were set that represent the present day

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seawater pCO2 conditions (pCO2: 400 ppm), and two future scenarios (pCO2: 800

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ppm and pCO2: 1500 ppm = medium and high treatments), respectively. The pCO2

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levels in the high treatments were greater than the end of the 21st century ‘rapid

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economic growth’ projections (IPCC ‘A1’ scenarios, IPCC 2007, 2014). However, the

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pH will drop under the action of eutrophication and undergo natural pH fluctuations

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on daily and seasonal scales in coastal waters (Cai et al., 2011). To manipulate

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targeted pH values in the culture medium, prepared air/CO2 gas was bubbled through

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samples, with the HEPES (2-[4-(2-Hydroxyethyl)-1-piperazinyl] ethanesulfonic acid,

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Sigma Ultra) buffer, which has no effect on biological cells (Charles, 1969; Gu et al.,

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2017), and the dissolved inorganic carbon (DIC) and total alkalinity (TA) of the

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seawater were measured (Table 2). Under pH conditions of 8.10, 7.71, and 7.45, the

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DIC concentrations were 1828, 1860, and 1904 µmol·L-1, respectively. Concurrently,

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the TA values were 2314, 2354, and 2366 µmol·L-1, respectively (Table 2). The initial

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NO3-N and NH4-N values in their cultures were 30.11, 30.44, and 33.00 µmol·L-1, and

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30.02, 31.16, and 29.83 µmol·L-1, respectively, under pH conditions of 8.10, 7.71, and

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7.45.

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2.3 Analytical methods

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The nitrate, nitrite, and ammonium concentrations were determined using an

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autonomous spectrophotometric nutrient analyzer (Bran-Lubbe AA℃

Germany)

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(Grasshoff et al., 2009). The Chl a concentration was measured using UV

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spectrophotometry (UV2550, Shimadzu) after extraction with 90% acetone (Jeffrey

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and Humphrey, 1975). The particulate organic nitrogen (PON) was determined by a

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nitration process using potassium persulfate containing boric acid (Grasshoff et al.,

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2009). The DIC was obtained by the coulometric method (Johansson et al., 1982) and

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the TA was determined by automated potentiometric titration (Anderson and

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Wedborg, 1985). Spectrophotometry with m-cresol purple as an indicator (Clayton

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and Byrne, 1993) was used to determine the seawater pH.

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The enzymatic activity samples were always immediately analyzed after

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collection. The SOD activity was determined using the nitro blue tetrazolium (NBT)

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method by measuring its ability to inhibit the NBT photochemical reduction and the

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change in absorbance was measured using UV spectrophotometry (UV2550,

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Shimadzu). At 560 nm, NBT was photoactivated by 50% as one enzyme activity unit

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(U·µg-1) (Beauchamp and Fridovich, 1971). The CAT activity was determined by a

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decline in absorbance at 240 nm from the H2O2 decomposition using UV

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spectrophotometry (UV2550, Shimadzu) that decreased 0.01 by D240nm per minute as

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one CAT enzyme activity unit (U·mg-1·min-1) (Jiang and Huang, 2001). The NR

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activity was measured by the absorbance at 540 nm using UV spectrophotometry

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(UV2550, Shimadzu) employing an improvement to the Radin assay (Radin, 1973).

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The NR activity was expressed as the total amount of NO2-N produced per gram of

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fresh algae cells per hour by catalytic reaction (µg·mg-1·h-1).

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2.4 Calculation methods

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The growth curve of N. closterium can be described by the Slogistic2 model with

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periods of exponential phase of growth and a stationary phase (Zhang et al., 2002).

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The final biomass (Bf) and the maximum growth rate (µmax) can be regressed

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according to the Slogistic2 model using the Marquardt–Levenberg algorithm

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implemented in the Origin 9.0 software (OriginLab Corporation) (Gu et al., 2017).

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Nitrogen uptake is often calculated using the Michaelis-Menten equation (Eppley

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and Thomas, 1969). However, to cover both the uptake and growth situations, the

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Monod equation will be used here (Healey, 1980). For more a more genuine

228

representation, we modified the equation as follows:

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dC V max ( C − C = dt C + Ks − C

min

)

min

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where dC dt is the nutrient uptake rate, Vmax is the maximum nutrient uptake rate, C is the

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substrate concentration, Cmin is the minimum concentration needed to support

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phytoplankton growth that also represents the final concentration during the death

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period, and Ks is the half-saturation value or the substrate concentration supporting

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half the maximum nutrient uptake rate. The nonlinear differential coefficient

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simulation method was used to fit the modified Monod equation with the least-squares

11

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method based on MATLAB (R2015b, Mathworks Inc.). The V max/ Ks ratio is the

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slope of the Monad equation at the lowest substrate concentrations that can be

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considered as competition drives the nutrient concentrations down. A higher ratio

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indicates a higher rate at the lowest nutrient concentrations (Healey, 1980).

240 241 242

The ratio (γ) was used to evaluate the growth and nitrogen uptake of N. closterium (Gu et al., 2017) as:

γ = β * µmax /V

max

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where β is the mean ratio of the PON per uniform unit to the chlorophyll mass (PON:

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Chl a ratio) of the exponential phase of algae growth that is a sensitive indicator of the

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algae physiological state in (Manny and Bruce, 1969; Geider, 1987). Based on the N.

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closterium culture results, the PON: Chl a ratios (β) were determined during the

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exponential phase of growth. The 3 PON: Chl a ratios in the 8.10, 7.71, and 7.45 pH

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treatments were 0.82 µmol·µg-1, 1.03 µmol·µg-1, and 1.07 µmol·µg-1 for NO3-N and

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1.13 µmol·µg-1, 1.16 µmol·µg-1, and 1.69 µmol·µg-1 for NH4-N, respectively. Mou et

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al., (2018) stated that the elevated pCO2 enhanced the PON: Chl a ratio during the

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exponential phase. Meanwhile, for the effect of elevated pCO2 and acidification, more

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algae might die at the lower pH that enhanced the PON: Chl a ratio. Thus, we used the

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NO3-N and NH4-N β values with no acidification effect treatment that were 0.82 and

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1.13 µmol·µg-1, respectively. Our data support previous studies (Jiao and Wang, 1994;

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Hessen et al., 2003; Frigstad et al., 2011) that were based on the PON to Chl a ratio in

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Jiaozhou Bay (~2.0 µmol·µg-1 for net plankton), Norwegian shelf in Skagerrak

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(fluctuating around 1.0 µmol·µg-1), and 109 temperate lakes (~0.81 µmol·µg-1 on 12

258

average).

259

Significance tests were carried out using the T Test, with the level of significance

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set as α = 0.05. The Pearson product-moment correlation (PPMC) was used to test the

261

correlation between the two factors. Statistical analyses were performed with SPSS

262

(IBM Corp. Version 19.0. Armonk, NY: IBM Corp., USA).

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3 Results

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3.1 Growth and nitrogen uptake conditions

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The concentrations of Chl a increased quickly during the first 2 days, then

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decreased after one day plateau. This indicated that there were three periods for N.

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closterium during all the pH treatment batches (Fig. 1A, B, C), namely, the

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exponential growth period, plateau period, and death period. Different nitrogen forms

269

(NO3-N and NH4-N) had apparent effects on the population growths between the three

270

pH treatments. The maximum growth rate and the final biomass of the NH4-N

271

conditions were higher than those of the NO3-N conditions that were significant (p <

272

0.05) for the former at pH 7.45 and for the latter at pH 8.10 and 7.71 (Fig. 2). With the

273

lower pH, the exponential growth period rate decreased and the maximum population

274

density decreased (Fig. 1). The Chl a and PON concentrations were higher at pH 8.10

275

than at the medium pH and high treatments during the plateau period, decreasing

276

~14―15% and ~19―38%, respectively. The final biomass and maximum growth

277

rates at pH 8.10 were significantly (p < 0.05) higher than those at the low pH

278

treatment, and there were no significant differences with the medium pH treatment

13

279

(Table 3).

280

The NO3-N and NH4-N concentrations decreased quickly at the first 2 days

281

(exponential growth period), then tended to be stable during the later experimental

282

phase (Fig. 3) that was associated with the growth of N. closterium. In addition, the

283

rates of nitrogen concentration decline in the NH4-N conditions were faster than those

284

in the NO3-N conditions, while the rates under normal conditions were also faster than

285

those under the acidified conditions. The Cmin of the nitrogen uptake for N. closterium

286

increased with lower pH. The Cmin of the NO3-N and NH4-N conditions at pH 8.10

287

had the lowest values (~3.65 and ~2.62 µmol·L-1, respectively), followed by those at

288

pH 7.71 (~3.72 and ~2.77 µmol·L-1, respectively), and the highest values were

289

detected at pH 7.45 (~4.22 and ~3.88 µmol·L-1, respectively) with no significance

290

(p > 0.05). The Cmin of the NO3-N conditions were significantly (p < 0.05) higher than

291

those of the NH4-N conditions at pH 8.10 and 7.71 (Fig. 4C).

292

3.2 Dynamics of growth and nitrogen uptake

293

The results showed that the maximum uptake rates of the NO3-N and NH4-N

294

conditions were obviously different under the different pH treatments. There were no

295

significant (p > 0.05) differences for the Ks values with the different nitrogen forms,

296

while the Ks values could change with an acclimation phase. The maximum uptake

297

rates for the NH4-N conditions were significantly (p < 0.05) higher than those of the

298

NO3-N conditions at pH 8.10 and 7.71, with no significant differences at pH 7.45 (Fig.

299

4A). With the lower pH, the maximum uptake rates decreased and the Ks increased,

14

300

indicating that N. closterium had a lower affinity for the environmental conditions

301

(Fig. 4A, B). As the pH decreased, the environment was not conducive to the growth

302

of N. closterium, with the maximum uptake rates for the NO3-N and NH4-N

303

conditions decreasing by ~13―25% and ~22―39%, respectively. The Ks values

304

increased by ~1.2―1.4 times and ~1.8―2.4 times, respectively. The maximum

305

uptake rates for the NH4-N conditions were significantly (p < 0.05) higher at pH 8.10

306

than those at pH 7.71 and pH 7.45, and there were no significant differences for the

307

NO3-N conditions.

308

The Vmax/Ks ratio, thought to represent the sum of the total affinity at all nutrient

309

uptake sites, involves a single algae and the comparison of rates of a particular

310

process under different conditions. A higher Vmax/Ks ratio indicates a condition that

311

would tend to improve the competitive position of the algae in the process being

312

considered (Healey, 1980; Smayda, 1997). The Vmax/Ks ratios for the NH4-N

313

conditions were higher than those of the NO3-N conditions, with a decreasing trend as

314

the pH decreased (Fig. 5B). More specifically, the Vmax/Ks ratio for the NO3-N and

315

NH4-N conditions were the highest at pH 8.10, followed by those at pH 7.71, and

316

lowest values were detected at pH 7.45 (Fig. 5B). The ratios decreased by ~47.8%

317

(pH 7.45) and ~29.0% (pH 7.71) for the NO3-N cultures, while they decrease by

318

~76.0% (pH 7.45) and ~58.4% (pH 7.71) for the NH4-N cultures. According to the

319

significance analysis, the Vmax/Ks ratio for the NH4-N conditions at pH 8.10 were

320

significantly (p < 0.01) higher than those at pH 7.71 and 7.45. The ratio of the

321

maximum growth rate to the maximum nitrogen uptake rate of N. closterium (γ) was 15

322

used to evaluate the effect of algal growth and nitrogen uptake under different pH

323

conditions and nitrogen forms. The γ values for the NO3-N conditions decreased as

324

the pH decreased, whereas the trend was first increased and then decreased for the γ

325

values under the NH4-N conditions (Fig. 5A). More specifically, the γ value for the

326

NO3-N conditions was highest at pH 8.10, less at pH 7.71, and lowest at pH 7.45,

327

while the γ value for the NH4-N conditions was highest at pH 7.71, followed by pH

328

7.45 and 8.10. According to the significance analysis, the γ values were not

329

significantly (p > 0.05) different under different pH conditions, except for NO3-N

330

conditions at pH 7.45.

331

3.3 Biological enzymes activities

332

The maximum enzymatic activities (SOD and CAT) were greatly different in all

333

of the cultures due to the effect of acidification, and the SOD and CAT activities

334

increased with decreasing pH under the NO3-N and NH4-N conditions. The SOD

335

maximum activities for the NO3-N and NH4-N cultures were ~2.36 and ~2.51 U·mg-1

336

at pH 8.10, respectively. They increased as the pH decreased, ~5.29 and ~4.90 U·mg-1

337

at pH 7.71, and ~7.56 and ~5.76 U·mg-1 at pH 7.45 for the NO3-N and NH4-N

338

cultures, respectively (Fig. 6A). Similarly, the SOD and CAT activities for the NO3-N

339

and NH4-N cultures were highest at pH 7.45, followed by those at pH 7.71 and pH

340

8.10 (Fig. 6B). The changes in the SOD and CAT enzyme activities indicate that the

341

acidified environment had a significant negative impact on the survival of N.

342

closterium at the 0.05 level (2-tailed) for most cases. The SOD and CAT activities for

16

343

the NO3-N cultures were higher than those for the NH4-N cultures at pH 7.71,

344

especially at pH 7.45 (p<0.05 for SOD activity), while they were reversed at pH 8.10

345

(Fig. 6), indicating that acidification might cause more oxidative stress in the NO3-N

346

cultures than in the NH4-N cultures.

347

The NR activities increased in the NO3-N cultures during the growth period, and

348

after the peaks were reached in 48 h they decreased (Fig. 7). The NR activity was the

349

highest, with a peak value of ~54.56 µg·mg-1·h-1 at pH 8.10, significantly (p < 0.05)

350

higher than those at pH 7.71 and 7.45 with peak values of ~32.40 and ~24.04

351

µg·mg-1·h-1, respectively.

352

4 Discussions

353

In this study, the growth of N. closterium was constrained by the OA, with final

354

biomass and maximum growth rates decreasing under lower pH levels (pH 7.71 and

355

7.45) associated with elevated pCO2. Consistent with our study, extensive research

356

indicated that the concentrations of Chl a fell by 5.6% with a decline in the pH

357

(pCO2:500 ppm) for N. closterium var. minutissima (Xia and Yu, 2009). The growth

358

rate of diatom is usually limited by pCO2 levels in seawater (Riebesell et al., 1993),

359

that could result in changes to the physiological regulation mechanism of algae (e.g.,

360

metabolism) and exert a negative influence (Riebesell et al., 2013; Sobrino et al.,

361

2014). Light and nutrients available are both limiting factors for algal growth. With

362

increasing light levels, the grown cells of diatom in the high pCO2 environment

363

increase faster than those in the ambient pCO2 (Gao et al. 2012b), and OA has a

364

positive effect on cell division in diatoms (Gervais and Riebesell, 2001). The 17

365

photosynthetic carbon fixation rate and the maximum carbon sequestration rate of the

366

diatom increased under acidified conditions (Wu et al., 2010; Sun et al., 2011; Yang

367

and Gao, 2012). The self-shading of chlorophyll at high cell densities might inhibit

368

photosynthesis of algae (Zhang and Liu, 2003). Other research suggests that the

369

growth rate of diatoms is closely related to the size of the cells and it decreases almost

370

linearly with the log of increasing cell size (Finkel et al., 2010). Algae would face an

371

acid-base imbalance living in an acidified environment, seriously affecting the growth

372

and metabolism of the algae (Hervé et al., 2012). The main effect driving algal

373

physiology is the downregulation of the photosynthetic apparatus by OA, and ROS is

374

released from the mitochondria, leading to cell structure damage (Li and Ji, 2015).

375

ROS production was significantly induced by acidification stress for organisms, e.g.

376

Mytilus edulis and Crassostrea gigas that had an inhibitory effect on certain

377

antioxidant enzyme activities and might damage physiological mechanisms (Wang et

378

al., 2016; Sun et al., 2017). OA would promote ROS in marine organisms and trigger

379

cellular antioxidant defense mechanisms, stimulating antioxidant reductase (e.g. SOD

380

and CAT) to maintain the intracellular balance and normal physiological function by

381

cleaning the oxygen free radicals (Sies and Stahl, 1995; Liu et al., 2016). Aksmann

382

and Tukaj (2004) noted that the SOD activity of Scenedesmus armatus increased with

383

increasing pCO2 that was cultured in an Anthropocene environment. The SOD and

384

CAT activities increased with decreasing pH in the N. closterium culture experiments,

385

revealing a strong defense capability for ROS in algal cells in order to cope with the

386

stress of an acidified environment (Liu et al., 2017). This can also be found in 18

387

Diopatra neapolitana (Polychaete, Onuphidae), which presented significantly higher

388

enzyme activity under lower pH levels (Freitas et al., 2016).

389

Linking the algal growth and nitrogen uptake of N. closterium under lower pH

390

levels, the limit of algal growth relates to the negative effects on the nitrogen uptake.

391

This shows a significant correlation at the 0.01 level (2-tailed). The uptake rates of the

392

NO3-N cultures were not obviously affected by OA, growth inhibition was slightly

393

higher, implying that more NO3-N might be used for alleviating the oxidative stress

394

caused by acidification. The maximum growth rate ratio to the maximum nutrient

395

uptake rate (γ) can be used to describe the phenomenon and indicate the assimilation

396

efficiency of nitrogen in the OA environment that decreased with a lower pH in the

397

NO3-N cultures. In terms of physiological activities, the NR activity of N. closterium

398

decreased with a lower pH in the NO3-N cultures, indicating that less NO3-N was used

399

for growth and the assimilation was inhibited by OA for N. closterium. When the pH

400

was more than a certain concentration, algal cells could induce the overload of

401

hydroxyl radicals, which then inhibited the activities of NR, glutamate-tRNA synthase,

402

and NADPH (i.e. the reduced species of nicotinamide adenine dinucleotide phosphate)

403

(Rouco et al., 2013; Liu et al., 2016). OA might destroy the acid-base balance of the

404

algae cells, even leading to metabolic disorders (Raven, 1991; Matsuda et al., 2001;

405

Doney et al., 2009; Hervé et al., 2012), thereby weakening their physiological

406

behavior or the ability to tolerate environmental changes (e.g. decrease in growth and

407

reproduction rate) (Gao, 2011). Therefore, algae need consume more energy to resist

408

acidified environmental stress to maintain normal physiological activity. 19

409

Nitrogen is a key nutrient for the growth of marine algae (Ryther and Dunstan

410

1971). Many scholars have shown that reduced nitrogen is the priority species used by

411

algae because the reduction of nitrogen can directly synthesize amino acids by

412

transamination under the action of GS/GAGOT enzymes (Clayton and Ahmed, 1986;

413

Berges and Mulholland, 2008). Thus, using one of these reduced nitrogen forms (e.g.

414

NH4-N) consumes less energy for absorption and utilization by algae, and less

415

nitrogen might be needed at lower growth rate associated with lower uptake rate of

416

NH4-N for N. closterium with the pH decreasing. While NO3-N and NO2-N would

417

consume more energy for NH4-N conversion through the corresponding nitrate and

418

nitrite reductases (Gardner et al., 2004, Paerl et al., 2011). Which might be the reason

419

that the γ was the highest at pH 7.71 under NH4-N conditions, and then at pH 7.45 and

420

8.10, with the maximum growth rate (µmax) and the maximum nutrient uptake rate

421

(Vmax) both decreasing with the pH decreasing. In this study, the results showed that

422

acidified seawater has a more detrimental effect on the growth of N. closterium under

423

the NO3-N conditions, with the drop in the ratio (γ) of the maximum growth rate to

424

the maximum nutrient uptake rate, as well as individuals under lower pH levels

425

showing lower growth indications (final biomass and maximum growth rate), lower

426

V

427

conditions.

max

/ Ks ratios, and higher enzyme activities (SOD and CAT) than NH4-N

428

As previously reported, algae would be significantly influenced by rapid

429

increases in nitrogen in coastal areas (Zou et al., 2011), while the changes in nitrogen

430

supply would also affect the algal response to increased atmospheric pCO2 (Andria et 20

431

al., 1999). Eutrophication is generally regarded as the main cause of coastal

432

environmental degradation and marine ecosystems in OA environments will be

433

affected by eutrophication changes (Breitburg et al., 2015) that draws extensive

434

attention from the public community. Combined with our results, both the growth and

435

uptake of N. closterium will be restrained by two nitrogen forms by the end of this

436

century. However, for different nitrogen forms, the NO3-N inhibition for N. closterium

437

was higher than that of NH4-N in acidified environment, and the inhibition at pH 7.45

438

will be more serious than at pH 7.71 in the coastal areas by the end of the 21st century.

439

5 Conclusions

440

Incubation experiments using three pH treatments showed that the growth of N.

441

closterium was inhibited by OA. The final biomass and maximum growth rate were

442

lower and the half-saturation constant was higher under lower pH levels for both

443

NO3-N and NH4-N conditions. There was also inhibition of nitrogen uptake, with

444

lower Vmax/Ks ratios under lower pH levels for both nitrogen conditions. The enzyme

445

activities of SOD and CAT increased under lower pH levels that were associated with

446

the growth inhibition of N. closterium induced by OA.

447

Ocean acidification had a more detrimental effect on the growth of N. closterium

448

under NO3-N conditions than the NH4-N conditions. The Vmax/Ks ratios for NH4-N

449

cultures were higher than those for NO3-N cultures, implying the highly competitive

450

position of NH4-N. The enzyme activities (SOD and CAT) were higher under the

451

NO3-N conditions than the NH4-N conditions, which were associated with the growth

452

and uptake differences of both nitrogen forms. This can also be inferred from the 21

453

results of the drop in nitrate reductase activity and the drop in the ratio (γ) of the

454

maximum growth rate to the maximum nutrient uptake rate induced by OA under the

455

NO3-N conditions.

456

Ocean acidification has become a new challenge for the global ocean ecosystem,

457

which can adversely affect N. closterium based on our incubation experiments. The

458

eutrophication caused by nitrate is increasing in coastal water, which can be stressed

459

by ocean acidification. The stability of the marine ecosystem could experience an

460

enormous impact by the end of the 21st century. Increasingly more scholars are

461

proving that OA may be an important cause of serious damage to the marine

462

environment and ecology, while the related research work is still extremely

463

inadequate and requires further work.

464 465

Acknowledgments

466

This study was financially supported by the National Key R&D Program of

467

China (No. 2017YFC1404300), the Natural Science Foundation of China (No.

468

41676062 and U1706215), the Fundamental Research Funds for the Central

469

Universities (No. 201962008), and the NSFC-Shandong Joint Fund for Marine

470

Ecology and Environmental Sciences (No. U1606404).

471 472

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37

Tables Table 1. Experimental conditions of Nitzschia closterium Table 2. Setting conditions for pH Table 3. Growth and nitrogen uptake kinetics parameters for Nitzschia closterium (± confidence limits).

1

Table 1. Experimental conditions of Nitzschia closterium pH

8.10

7.71

7.45

Chl a concentration (µg·L-1) (NO3-N)

7.64

8.65

9.76

8.58

8.45

9.59

Chl a concentration (µg·L-1) (NH4-N)

8.69

8.73

8.66

8.66

6.22

6.67

Nutrient concentration (µmol·L-1)

NH4Cl/ NaNO3

30

KH2PO4

1.5

Na2SiO3·9H2O

30

Photoperiod

12 h:12 h

Temperature (°C)

20±1

Light (µmol·m-2·s-1)

191

Table 2. Setting conditions for pH pH

8.10

7.71

7.45

CO2 flux (ml·min-1)

0

0.04

0.04

Bubbling time of CO2 (min·day-1)

0

2

4

Bubbling time of air (min·day-1)

4

4

4

HEPES (g·L-1)

0.01

0.04

0.05

DIC (µmol·L-1)

1828

1860

1904

TA (µmol·L-1)

2314

2354

2366

2

Table 3. Growth and nitrogen uptake kinetics parameters for Nitzschia closterium (± confidence limits). NO3-N

pH 8.10

pH 7.71

pH 7.45

Final biomass (unit:µg·L-1)

31.11±0.17

26.68±2.28

25.19±0.01

Maximum growth rate (unit:µg·L-1·h-1)

0.82±0.01

0.64±0.01

0.46±0.03

Maximum nitrogen uptake rate (unit: µmol·L-1·h-1)

0.71±0.01

0.63±0.01

0.53±0.07

Half-saturation constant (unit: µmol·L-1)

0.29±0.09

0.36±0.04

0.41±0.40

Min-concentration threshold (unit: µmol·L-1)

3.65±0.07

3.72±0.01

4.20±1.84

0.95±0.02

0.84±0.01

0.72±0.06

V max /K s (unit: h-1)

2.45±0.72

1.74±0.17

1.28±1.09

Final biomass (unit:µg·L-1)

48.99±3.18

41.50±0.83

30.21±0.66

Maximum growth rate (unit:µg·L-1·h-1)

0.95±0.08

0.86±0.06

0.52±0.01

Maximum nitrogen uptake rate (unit: µmol·L-1·h-1)

1.03±0.01

0.80±0.01

0.63±0.03

Half-saturation constant (unit: µmol·L-1)

0.11±0.01

0.20±0.03

0.27±0.02

Min-concentration threshold (unit: µmol·L-1)

2.62±0.01

2.77±0.06

3.88±2.25

Ratio of maximum growth rate and maximum nitrogen uptake rate

1.04±0.07

1.21±0.06

0.94±0.02

V max /K s (unit: h-1)

9.61±0.76

4.00±0.55

2.31±0.06

Ratio of maximum growth rate and maximum nitrogen uptake rate

NH4-N

3

Figures Fig. 1. Growth curve (chl a (up) (A, B, C) and PN (down) (D, E, F)) of Nitzschia closterium under different nitrogen forms and different pH conditions (pH 8.10 (A, D), pH 7.71 (B, E), pH 7.45 (C, F)). Fig. 2. Terminative biomass (A) and maximum growth rate (B) of Nitzschia closterium under different nitrogen forms and pH conditions. Fig. 3. Uptake curve of NO3-N (A , B, C) and NH4-N (D, E, F, cited from Gu et al., 2017) for Nitzschia closterium under pH 8.10 (A, D), pH 7.71 (B, E) and pH 7.45 (C, F). Fig. 4. Maximum uptake rate (A), half-saturation constant (B) and min-concentration threshold (C) of Nitzschia closterium under different pH conditions and nitrogen forms. Fig. 5. The ratio (γ) of the maximum growth rate to the maximum nutrient uptake rate (A) and the ratio V max /K s (B) for Nitzschia closterium under different pH conditions and nitrogen forms. Fig. 6. Activity of superoxide dismutase (SOD) (A) and Activity of catalase (CAT) (B) of Nitzschia closterium under different pH conditions and nitrogen forms in 24 h. Fig. 7. Activity of nitrate reductase (NR) (A) and NR maximum activity (B) of Nitzschia closterium under different pH conditions for NO3-N culture.

1

Fig. 1. Growth curve (chl a (up) (A, B, C) and PN (down) (D, E, F)) of Nitzschia closterium under different nitrogen forms and different pH conditions (pH 8.10 (A, D), pH 7.71 (B, E), pH 7.45 (C, F)).

Fig. 2. Final biomass (A) and maximum growth rate (B) of Nitzschia closterium under different nitrogen forms and pH conditions.

2

Fig. 3. The concentration of NO3-N (A , B, C) and NH4-N (D, E, F) varies with time under pH 8.10 (A, D), pH 7.71 (B, E) and pH 7.45 (C, F)

Fig. 4. Maximum uptake rate (A), half-saturation constant (B) and min-concentration threshold (C) of Nitzschia closterium under different pH conditions and nitrogen forms.

Fig. 5. The ratio (γ) of the maximum growth rate to the maximum nutrient uptake rate (A) and the ratio Vmax /Ks (B) 3

for Nitzschia closterium under different pH conditions and nitrogen forms.

Fig. 6. Activity of superoxide dismutase (SOD) (A) and Activity of catalase (CAT) (B) of Nitzschia closterium under different pH conditions and nitrogen forms in 24 h

Fig. 7. Activity of nitrate reductase (NR) (A) and NR maximum activity (B) of Nitzschia closterium under different pH conditions for NO3-N culture.

4

Highlights
The growth of Nitzschia closterium was inhibited by ocean acidification with low growth indication
Acidification might induce ROS with the enzyme activities (SOD, CAT) increase under lower pH levels
Acidification has a more detrimental effect on the growth of N. closterium under NO3-N than NH4-N

Declaration of interests √ The authors declare that they have no known competing financialinterestsor personal relationships

that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: