Phycobiliproteins Production Enhancement and Lipidomic Alteration

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Bioactive Constituents, Metabolites, and Functions

Phycobiliproteins production enhancement and lipidomic alteration by titanium dioxide nanoparticles in Synechocystis sp. PCC 6803 culture Zahra Zahra, Seok-Young Kim, Hye-Youn Kim, Hwanhui Lee, Heayyean Lee, JunYeong Jeon, Dong-Min Kim, Dong-Myung Kim, Seong-Joo Hong, Byung-Kwan Cho, Hookeun Lee, Choul-Gyun Lee, Muhammad Arshad, and Hyung-Kyoon Choi J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01522 • Publication Date (Web): 17 Jul 2018 Downloaded from http://pubs.acs.org on July 23, 2018

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Journal of Agricultural and Food Chemistry

[Research Article] Phycobiliproteins production enhancement and lipidomic alteration by titanium dioxide nanoparticles in Synechocystis sp. PCC 6803 culture

Zahra Zahraa,b,∥, Seok-Young Kima,∥, Hye-Youn Kima, Hwanhui Leea, Heayyean Leea, Jun-Yeong Jeona, Dong-Min Kima, Dong-Myung Kimc, Seong-Joo Hongd, Byung-Kwan Choe, Hookeun Leef, Choul-Gyun Leed, Muhammad Arshadb, Hyung-Kyoon Choia,*

a

College of Pharmacy, Chung-Ang University, Seoul 06974, Republic of Korea.

b

Institute of Environmental Sciences and Engineering, School of Civil and Environmental

Engineering, National University of Sciences and Technology, Sector H-12, Islamabad 44000, Pakistan c

Department of Chemical Engineering and Applied Chemistry, Chungnam National

University, Daejeon 34134, Republic of Korea. d

Institute of Industrial Biotechnology, Department of Biological Engineering, Inha

University, Incheon 22212, Republic of Korea. e

Department of Biological Sciences, KAIST, Daejeon 34141, Republic of Korea.

f

College of Pharmacy, Gachon University, Incheon 13120, Republic of Korea.

∥

These authors equally contributed to this work.

*Corresponding author Tel: +82-2-8205605; Fax: 82-2-8123921; E-mail: [email protected], College of Pharmacy, Chung-Ang University, Seoul 06974, Republic of Korea

1 ACS Paragon Plus Environment


1

ABSTRACT

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This study aimed to improve the production of phycobiliproteins using TiO2

3

nanoparticles (NPs) in Synechocystis sp. PCC 6803. The growth characteristics of

4

Synechocystis cells were not affected by TiO2 NPs treatment, but this treatment increased

5

the chlorophyll content significantly by 62.2% (14.6 mg/L) compared to that of control

6

(9.0 mg/L) on day 16. Phycocyanin production was increased by 33.8% (29.3 g/L)

7

compared to that of control (21.9 g/L) on day 8. Allophycocyanin production was

8

increased by 55.0% (6.2 g/L) compared to that of control (4.0 g/L) on day 8, and by 22.4%

9

(16.4 g/L) compared to that of control (13.4 g/L) on day 16. DI-MS revealed that TiO2

10

NPs treatment significantly increased the levels of major thylakoid membranes of

11

monogalactosyldiacylglycerols (18:2/18:3, 18:2/18:2, 18:1/18:2), phosphatidylglycerol

12

(16:0/16:1), and sulfoquinovosyldiacylglycerols (16:0/16:1, 16:0:18:4) on day 8. These

13

findings indicate that TiO2 NPs have potential for commercial applications in

14

Synechocystis species or other microalgal strains.

15

KEYWORDS: phycobiliproteins; TiO2 nanoparticles; Synechocystis sp. PCC 6803; DI-

16

MS


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INTRODUCTION

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Microalgae are considered suitable organisms for producing commercially valuable

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compounds. They have received considerable attraction recently due to their potential to

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grow autotrophically and provide atmospheric oxygen. An important aspect of using

21

microalgae is their enhanced photosynthesis efficiency compared to major crops.1 This

22

inherent characteristic of microalgae increases the productivity of various metabolites

23

within a relatively short time frame.2 The culture conditions that microalgae require for

24

their growth, such as light, carbon dioxide (CO2), and other inorganic nutrients, are

25

simpler than those required by animal and higher plant cells. Microalgae contain various

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pigments such as carotenoids, carotenoproteins, tetrapyrroles, quinones, azulenes, and

27

indigoids.3 Chlorophyll is a tetrapyrrole that plays a major role in photosynthesis, while

28

phycobiliproteins are tetrapyrroles that assist chlorophyll by broadening the absorption

29

wavelength of the photosynthesis antenna pigments.4 Phycobiliproteins may constitute

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50% of the soluble proteins in cyanobacteria, and representative members are

31

phycocyanin (PC; blue), allophycocyanin (APC; bluish green), and phycoerythrin (PE;

32

red).5 Synechocystis sp. PCC 6803 (Synechocystis 6803 hereafter) is the unicellular

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facultative heterotrophic cyanobacterium, and its entire genome sequence was first

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reported in cyanobacteria.6-8 It has been reported that Synechocystis 6803 is able to

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synthesize high-value biomaterials such as bioethanol,9 isoprene,10 ethylene,11 and

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phycobiliproteins.12

37

Phycobiliproteins have several pharmacological properties such as antioxidant,

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antiinflammatory, anticancer and hepatoprotective.13 They have been widely used in

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analytical reagents, immunodiagnostics,2 and played an important role as fluorescence

40

probes in fluorescence-based detection systems, especially in flow cytometry.13,14 They



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can also be utilized as biomarkers for electrophoretic methods because of their low

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molecular weight and visibility in all steps of the purification.15 Phycobiliproteins can be

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also employed as colorants for foods and cosmetics due to their non-toxic coloring

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property.13

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However, using phycobiliproteins is expensive because of certain limitations such as

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their low yield, long processing time, and difficulty in implementing large-scale

47

production. A breakthrough approach to overcome such limitations is to increase the

48

content of valuable products in cells. The importance of nanomaterials has increased

49

enormously in recent decades, and titanium dioxide (TiO2) nanoparticles (NPs) are now

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among the most widely used nanomaterials. Song et al. found that TiO2 NPs at low

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concentrations stimulated the growth of duckweed (Lemna minor L.) while inhibiting

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their growth at high concentrations. However, the chlorophyll content was increased in

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response to increased concentration of TiO2 NPs in duckweed cells.16 TiO2 NPs also

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increased the chlorophyll content in spinach leaves by 58% compared to the control.17

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The wide range of applications of phycobiliproteins have made them highly desirable to

56

explore the ways to increase their production. However, there have been no reports on the

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application of TiO2 NPs to enhance the production of phycobiliproteins using cultivation

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of Synechocystis 6803 cells. The present study hypothesized that the production of high-

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value biomaterials including phycobiliproteins in Synechocystis 6803 could be increased

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by treatment with a very small amount of TiO2 NPs. Comprehensive metabolic and

61

lipidomic profiling using gas chromatography mass spectrometry (GC-MS) and direct

62

infusion mass spectrometry (DI-MS) were also performed to investigate the mechanism

63

underlying how TiO2 NPs could modulate phycobiliproteins production. In addition,

64

inductively coupled plasma mass spectrometry (ICP-MS) was employed to investigate


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translocation of TiO2 NPs into Synechocystis 6803 cells.

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MATERIALS AND METHODS

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Culture conditions and TiO2 NPs treatment

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Synechocystis sp. PCC 6803 was obtained from the Pasteur Culture Collection, Pasteur

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Institute (Paris, France). Shaking incubator (NEX220SRL, Nexus Technologies, Seoul,

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Republic of Korea) was used to incubate the cells at 30 ± 1 °C and 120 rpm. During

71

incubation, fluorescent lamps at 40 μmol photons m−2 s−1 were employed for a 16:8-h

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day/night cycle. Cells were cultured in Blue-Green medium (BG11, Sigma-Aldrich, St.

73

Louis, MO, USA) supplemented with 1 mL/L of trace metal mix A5 with cobalt (Sigma-

74

Aldrich), 6 mg/L ammonium iron citrate, 20 mg/L Na2CO3 and 30.5 mg/L K2HPO4, and

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were routinely subcultured into fresh medium every week such that 500 µL stock culture

76

was inoculated to 40 mL medium in a 200 mL Erlenmeyer flask. For the TiO2 NPs

77

treatment experiment, 500 µL stock culture was inoculated to 100 mL medium (including

78

2.5 mL TiO2 NPs stock solutions and equal amount of distilled water for control) in a 250

79

mL Erlenmeyer flask.

80

TiO2 NPs used in the present study were obtained from Sigma-Aldrich (CAS No. 1317-

81

70-0, particle size: < 25 nm, anatase). TiO2 NPs stock solution (1 g/L) prepared in distilled

82

water was sonicated for 1 h using ultrasonicator (JAC-2010, Kodo, Hwaseong, Korea) to

83

make NPs dispersion. On day 0, NPs dispersion (2.5 mL) was added to TiO2 NPs treated

84

group (to make TiO2 NPs concentration of 25 mg/L), whereas an equal amount of distilled

85

water was added to the untreated control group. The experiment was performed in

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biological triplicate for each treatment group. After TiO2 NPs treatment, Synechocystis

87

6803 cells were harvested on day 0 (control), day 8 and day 16. The harvested samples



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were dried using a freeze-dryer (FDU-1200, EYELA, Miyagi, Japan) and stored at −80°C

89

for further analysis.

90

Measurements of cells growth

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To investigate the effects of TiO2 NPs (0 and 25 mg/L) on Synechocystis 6803 cells

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growth, 200 µL of the cell cultures were transferred into 96 well plate (Corning Inc.,

93

Corning, NY, USA) and their optical densities (OD) were measured at 730 nm using a

94

microplate spectrophotometer (xMarkTM, BIO-RAD, California, USA). OD was

95

measured on a daily basis and calculated based on an average of biological and analytical

96

replicates in triplicate.

97

Determination of chlorophyll content

98

The chlorophyll content in Synechocystis 6803 culture were measured according to the

99

method described in previous report.18 Briefly, the cell suspension of 1 mL from each

100

sample was centrifuged at 10,000 × g for 10 min at 4°C and the supernatant was removed.

101

To the obtained pellet, 0.9 mL of methanol was added followed by refrigeration for 1 h at

102

4°C, and then centrifuged at 10,000 × g for 10 min at 4°C. Chlorophyll content was

103

evaluated from the absorbance of methanolic extract measured at 665 nm using the

104

following equation (1).

105 106

Chlorophyll (mg/L) = OD665nm ⅹ13.9

(1)

107 108 109

Determination of phycobiliproteins content The phycobiliproteins content in Synechocystis 6803 culture (using 1 mL cell suspension)

110

were measured according to the method described in previous report.19 The contents of

111

PC, APC, and PE were calculated using the following equations (2), (3) and (4).


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112 OD620nm −0.7×OD650nm

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Phycocyanin, PC (g/L) =

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Allophycocyanin, APC (g/L) =

115

Phycoerythrin, PE (g/L) =

7.38 OD650nm −0.19×OD620nm 5.65

OD565nm −2.8[PC]−1.34[APC] 12.7

(2) (3) (4)

116 117

Comprehensive metabolite analysis by GC-MS

118

Samples from freeze-dried cultures of Synechocystis 6803 were weighed, and 10 mg

119

of each were used for comprehensive metabolite analysis by GC-MS according to the

120

method in previous report.20

121

Intact lipid species analysis by DI-MS

122

Samples from freeze-dried cultures of Synechocystis 6803 were weighed, and 5 mg of

123

each were used for intact lipid species analysis by DI-MS using the previously described

124

method.21

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Trace elements analysis by ICP-MS

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The concentrations of Fe, Mg, Ti, Mn and Zn in Synechocystis 6803 cells were measured

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by an inductively coupled plasma mass spectrometry (ICP-MS) using NexION 350D

128

ICP-mass spectrometer (PerkinElmer, Inc., MA, USA), equipped with an argon plasma

129

operating at 6,000 K. For each analysis, 10 mg of freeze-dried Synechocystis 6803 cells

130

were digested by adding 8 mL of nitric acid for 30 min and final weight of sample

131

pretreatment was adjusted to 15 g. Finally, all samples were diluted 100-fold for Fe and

132

Mg, and 10-fold for Ti, Mn and Zn prior to the analysis by ICP-MS in order to minimize

133

matrix effects and avoid salt formation. Each sample was measured in duplicate and the



134

data were presented as mean ± standard deviation (SD).

135

Data processing and statistical analysis

136

IBM SPSS Statistics 23 software (IBM, NY, USA) was used to analyze the results from

137

all the experiments. Statistically significant differences were assessed by Mann-Whitney

138

test or Student’s t-test at the level of P < 0.05.

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RESULTS AND DISCUSSION

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Effects of TiO2 NPs treatment on growth and chlorophyll content of Synechocystis 6803

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The effects of TiO2 NPs treatment on the growth of Synechocystis 6803 was investigated

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every alternate day over a 16-day period (Figure 1). The growth characteristics of TiO2

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NPs treated cells were indistinguishable from those of untreated control cells, which

144

suggests that exposure to TiO2 NPs had no adverse effects on Synechocystis 6803 growth.

145

This was consistent with a study on the responses of 10 species of algae in North America

146

to TiO2 NPs exposure finding no effect on algal growth.22

147

The chlorophyll content of Synechocystis 6803 did not change significantly during the

148

first 8 days after TiO2 NPs treatment (Table 1). However, there was a 62.2% increase in

149

the chlorophyll content after 16 days of treatment. Chlorophyll in plants converts CO2

150

and water into oxygen and glucose in the presence of sunlight. Hong et al. reported a

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practical approach for using TiO2 NPs in the agricultural sector based on studying their

152

effects on the photochemical reaction of chloroplasts in Spinacia oleracea. They reported

153

that treatment with TiO2 NPs increased chloroplast activity and photosynthesis.23

154

Microalgal cells could be entrapped by the large aggregates of TiO2 NPs, limiting the

155

light availability to them and eventually inhibiting algal cells growth.24 Treatment with

156

TiO2 NPs in the culture of Synechocystis 6803 in our study might play a major role in


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improving the function of chloroplast activity, which result in increasing the chlorophyll

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content of Synechocystis 6803.

159

Effects of TiO2 NPs on the phycobiliproteins content of Synechocystis 6803

160

The phycobiliproteins production (PC, APC and PE) in Synechocystis 6803 culture by

161

TiO2 NPs treatment on day 8 and 16 are listed in Table 1. In this study, production of

162

phycobiliproteins was enhanced when cultured in TiO2 NPs treatment. Among

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phycobiliproteins, the content of PC and APC increased significantly (by 33.8% and

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55.0%, respectively) after 8 days of TiO2 NPs treatment, whereas only the APC content

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increased significantly (by 22.4%) after 16 days (Table 1). However, no significant

166

increase was observed in the PE content after 8 and 16 days of TiO2 NPs treatment. PC

167

and PE, located in the phycobilisomes rods, produce photons and transfer the energy into

168

the APC. APC is the major core component of phycobilisomes having alpha and beta

169

subunits, where collected energy is transferred to chlorophyll in thylakoid membrane.25

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It seems that TiO2 NPs treatment mainly affected the energy transfer process to

171

chlorophyll by increasing the APC content in our study. PE has been reported as the most

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flexible phycobiliprotein, which facilities adaptation to environmental variations.26

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However no significant change in PE content by TiO2 NPs treatment in our study was

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observed, which needs further elucidation in future studies.

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There are some possible explanations how TiO2 NPs could increase the

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phycobiliproteins content in microalgae cultivation. Firstly, the protective response

177

against oxidative stress by TiO2 NPs in Synechocystis 6803 might result in increased

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content of phycobiliproteins as antioxidants. Phycobiliproteins, the major photosynthetic

179

pigment-proteins, present in microalgae and cyanobacteria are known to function as an

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antioxidant.27 Since TiO2 NPs induce oxidative stress in microalgae cells by producing



181

reactive oxygen species, microalgae synthesize antioxidant compounds such as pigments,

182

glutathione, ascorbic acid and phenolic compounds to prevent cell damages.28-30

183

Secondly, the compensatory increase of phycobiliproteins content with respect to

184

photosynthesis inhibition might be caused by light-shading effect of the TiO2 NPs on

185

Synechocystis 6803 cells. It is supported by the reports that internalization or cell surface

186

binding of NPs could reduce light availability for the photosynthesis, while the content

187

of photosynthetic pigments responsible for light absorption increased to optimize the light

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availability.31,32

189

Modulatory action of TiO2 NPs on promoting protein utilization might also promote the

190

enhanced production of phycobiliproteins in Synechocystis 6803 cells. Yang et al.

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reported that 52% increase of protein content in spinach leaves after TiO2 nano-anatase

192

treatment for 20 days. They demonstrated that TiO2 NPs treatment could enhance the

193

promotion of nitrogen metabolism in spinach, which accelerate nitrate absorption and

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transformation of inorganic nitrogen to organic nitrogen forms, such as amino acid,

195

proteins, and chlorophyll with the activation of the enzymes in NH4+ assimilation.17 It is

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suggested that TiO2 NPs treatment might affect the nitrogen metabolism in Synechocystis

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6803, which enhance the production of phycobiliproteins via modulation of protein

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utilization.

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Some studies have attempted to identify the diverse factors regulating the structure,

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function, and content of phycobiliproteins and phycobilisomes in Synechocystis 6803

201

cells. Mao et al. reported that a high concentration of glycerol led to the disassembly of

202

phycobilisomes and the dissociation of PC, and that a high temperature also induced APC

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disassembly in a mutant Synechocystis 6803 strain lacking the chlL gene.33 Liu et al.

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reported that the cores of phycobilisomes (APC) rather than the rods of phycobilisomes


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(PC) acted as targets for oxidative stress or silver ion induced damage.34 In contrast with

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those results, TiO2 NPs treatment in our study might not affect the disassembly or

207

dissociation of phycobilisomes in Synechocystis 6803, but rather increasing the overall

208

productivity. Schubert et al. reported that the content of chlorophyll and PC were higher

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for NaCl at 342 mM than for NaCl at 1,026 mM in Synechocystis 6803 cells.35 These

210

previous studies have revealed that applying several types of treatment to Synechocystis

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6803 cells can regulate the function and content of APC and PC in phycobilisomes.

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However, there were no previous reports regarding increase of phycobiliproteins content

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in microalgae by TiO2 NPs. Future studies could be performed to optimize those factors

214

combined with TiO2 NPs for enhanced production of each phytobiliprotein (such as PC

215

and APC).

216

Effect of TiO2 NPs on profiles of comprehensive metabolites and intact lipid species of

217

Synechocystis 6803

218

Table 2 demonstrates the comprehensive metabolic profiles of Synechocystis 6803 in

219

response to TiO2 NPs treatment. The metabolites of Synechocystis 6803 cells cultivated

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under control conditions and with the addition of TiO2 NPs were quantified

221

comprehensively using GC-MS. The 27 metabolites identified were classified into

222

different categories such as alcohols, amino acids, fatty acids, organic acids, and sugars.

223

Relative levels of the following eight metabolites differed significantly between the

224

control and TiO2 NPs treated samples: pyroglutamic acid, threonine, linoleic acid,

225

linolenic acid, pentadecanoic acid, lactic acid, succinic acid, and melibiose. In the results

226

obtained by GC-MS analysis, there were no consistent changes in each group of

227

metabolites by TiO2 NPs treatment in Synechocystis 6803 culture.

228

DI-MS was also employed to analyze the variations of intact lipid species in



229

Synechocystis 6803 in response to TiO2 NPs treatment. Total 34 lipid species were

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identified as like follows; 5 phosphatidylglycerols (PGs), 1 phosphatidylinositol (PI), 7

231

monogalactosyldiacylglycerols (MGDGs), 3 digalactosyldiacylglycerol (DGDGs), 16

232

sulfoquinovosyldiacylglycerol (SQDGs), and 2 phytyl derivatives. The relative levels of

233

most intact lipid species were higher in the control and TiO2 NPs treated groups on day 8

234

and 16 than that of day 0 (Table 3). After 8 days of TiO2 NPs treatment, the relative levels

235

of MGDG, PG and SQDG species increased. Especially, significant increase in relative

236

levels of MGDGs (18:2/18:3, 18:2/18:2, 18:1/18:2), PG (16:0/16:1), and SQDGs

237

(16:0/16:1, 16:0:18:4) were observed on day 8.

238

In cyanobacteria and higher plants, MGDG, DGDG, SQDG and PG were reported as

239

the main components of thylakoid membrane (main site of light-dependent

240

photosynthesis).36 It was also reported that MGDG has been responsible for construction

241

of photosynthetic apparatus in Arabidopsis thaliana.37 SQDG contributed to the activity

242

of photosystem II rather than photosystem I in green algae, Chlamydomonas

243

reinhardtii.38,39 SQDG was also reported as a player for replication of chromosomal DNA

244

in Synechocystis 6803.40 PG was known as an indispensable lipid species for the

245

Synechocystis to maintain photosynthetic activity, thus depletion of PG decreased

246

chlorophyll content and suppressed oxygen-evolving photosystem II in Synechococcus

247

sp. PCC 7942/ΔcdsA mutant.41,42 Similarly, the significant increase in chlorophyll

248

contents of Synechocystis 6803 after 16 days of TiO2 NPs treatment appears to be

249

influenced by an increase in PG of TiO2 NPs treated cells in the present study. The

250

increase in relative levels of MGDG, PG and SQDG at 8 days of cultivation were also

251

observed to be correlated with the increasing content of phycobiliproteins on day 8 in our

252

study. Photosynthesis was to attribute in the complicated interaction between


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phycobilisomes, acidic lipids existed in thylakoid membrane and sets of cofactors.43 TiO2

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NPs treatment could play an important role in increasing photosynthesis in Synechocystis

255

6803 culture by increasing phycobiliproteins (APC, PC) and acidic lipids (MGDG,

256

SQDG, and PG). This is the first report observing the changes in intact lipid species and

257

the relationship between these lipid species and phycobiliproteins affected by TiO2 NPs

258

treatment in Synechocystis 6803.

259

Translocation of Ti and variations in Fe, Mg, Mn, and Zn concentrations induced by

260

TiO2 NPs

261

The effects of TiO2 NPs treatment on the intracellular concentrations of Ti, Fe, Mg, Mn,

262

and Zn were determined on day 8 and 16 using ICP-MS (Table 4). The intracellular Ti

263

level increased significantly after 8 and 16 days of TiO2 NPs treatment in the culture

264

medium, which suggests that Ti was translocated to Synechocystis 6803 cells by TiO2

265

NPs treatment. In recent studies, TiO2 NPs aggregation and their translocation into cells

266

were also reported in microalgae.44 In our study, 8 days of TiO2 NPs treatment induced

267

significant decrease in the concentrations of Fe, Mg, Mn, and Zn, while those of Fe, Mn,

268

and Zn were decreased after 16 days.

269

Fe has been considered as an important element to keep the cell growth and

270

phycobiliproteins synthesis in microalgae.45 Kuchmina et al. reported ferrochelatase as an

271

essential enzyme in heme biosynthesis for phycobiliproteins.46 Mg was also regarded as

272

an important element in increasing fatty acids, and biosynthesis of chlorophyll and

273

MGDG in microalgae.47,48 It was also known that Mn and Zn play an important role in

274

photosystems of plant and microalgae.49,50 Based on those researches, TiO2 NPs treatment

275

could facilitate the utilization of Fe, Mg, Mn, and Zn in Synechocystis 6803 cells for

276

increasing chlorophyll, phycobiliproteins, intact lipid species (mainly MGDG) level, and



277

photosystem activity. The roles of Mg, Mn, and Zn in the biosynthesis of

278

phycobiliproteins in Synechocystis 6803 cells still needs to be investigated in future

279

studies.

280

In summary, this is the first report on enhanced phycobiliproteins production in

281

Synechocystis 6803 treated with TiO2 NPs. It was revealed that TiO2 NPs (25 mg/L)

282

treatment significantly increased the content of phycobiliproteins (PC and APC by 33.8%

283

and 55.0%, respectively) on day 8, and chlorophyll content (by 62.2%) on day 16

284

compared to the control in Synechocystis 6803 culture, while the growth was not

285

influenced by the TiO2 NPs. We demonstrated that increase in MGDG, SQDG, and PG

286

on day 8 induced by TiO2 NPs treatment was attributed to the increased need for thylakoid

287

membrane lipids in photosynthesis function. The lipidomic profiling could be a useful

288

tool for elucidating enhanced production of phycobiliproteins in Synechocystis 6803

289

cultivation. The TiO2 NPs treatment is relatively low cost and could be easily applicable

290

to large scale cultivation systems of various microalgae. These findings could be applied

291

to facilitate the large-scale production of phycobiliproteins in Synechocystis species and

292

other microalgal strains.


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ABBREVIATIONS USED

294

PC, phycocyanin; APC, allophycocyanin; PE, phycoerythrin; TiO2, titanium dioxide; NPs,

295

nanoparticles; GC-MS, gas chromatography mass spectrometry; DI-MS, direct infusion

296

mass spectrometry; ICP-MS, inductively coupled plasma mass spectrometry; MGDG,

297

monogalactosyldiacylglycerol;

298

sulfoquinovosyldiacylglycerol; DGDG, digalactosyldiacylglycerol

299

Funding

PG,

phosphatidylglycerol;

SQDG,

300

This work was supported by the Basic Core Technology Development Program for the

301

Oceans and the Polar Regions of the National Research Foundation (NRF) funded by the

302

Ministry of Science, ICT & Future Planning (No. NRF-2016M1A5A1027464), and by

303

the National Research Foundation of Korea (NRF) grant funded by the Korean

304

government (MSIP) (NRF-2015R1A5A1008958), and by the Chung-Ang University

305

Graduate Research Scholarship in 2017.

306

Author Contribution

307

Zahra Zahra contributed to analysis and interpretation of the data and writing the

308

manuscript. Seok-Young Kim contributed to performing experiments, interpretation of

309

the data and revising the manuscript. Hye-Youn Kim, Hwanhui Lee, Heayyean Lee and

310

Jun-Yeong Jeon contributed to performing experiments, analysis and interpretation of the

311

data. Dong-Min Kim contributed to performing experiments and writing the manuscript.

312

Seong-Joo Hong contributed to performing experiments. Dong-Myung Kim, Byung-

313

Kwan Cho, Hookeun Lee and Choul-Gyun Lee contributed to supervising the research.

314

Muhammad Arshad contributed to conception of the experiments. Hyung-Kyoon Choi



315

contributed to conception of the experiments, critically revising the manuscript and

316

supervising the research.

317

Conflict of Interest

318

The authors declare no competing financial interest.


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320 321


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537–544.



470

FIGURE CAPTIONS

471

Figure 1. Growth of Synechocystis 6803 cultivated under control and TiO2 treated

472

conditions. Data are shown as the mean values with bars representing SD from biological

473

triplication.


Page 24 of 34

Page 25 of 34


Table 1. Chlorophyll and phycobiliproteins content of Synechocystis 6803 cultivated under TiO2 treatment over three different time periods Day 0a

Day 8a

Day 16a

Treatment

Chlorophyll (mg/L) PC PhycoAPC biliprotein (g/L) PE a

Control

Control

TiO2 (25 mg/L)

% Change

Control

TiO2 (25 mg/L)

% Change

7.4±0.4

9.2±1.0

9.2±0.5

0.0

9.0±1.1

14.6±0.5#

62.2

8.4±0.8

21.9±0.4

29.3±3.7*

33.8

57.8±4.4

66.7±3.8

15.4

#

1.8±0.2

4.0±0.1

6.2±0.9*

55.0

13.4±1.0

16.4±1.3

22.4

1.0±0.1

1.5±0.1

1.7±0.2

13.3

2.6±0.3

2.9±0.3

11.5

Data of chlorophyll are listed as the mean ± SD values (biological triplication and

experimental triplication). Data of phycobiliproteins are listed as the mean ± SD values (biological triplication). The hash (#) in chlorophyll content represents significant differences (P < 0.05) between two groups (control, TiO2 25 mg/L) on day 16 by MannWhitney test. The asterisk (*) in PC and APC contents represents significant differences (P < 0.05) between two groups (control, TiO2 25 mg/L) on day 8 by Student’s t-test. The hash (#) in APC content represents significant differences (P < 0.05) between two groups (control, TiO2 25 mg/L) on day 16 by Student’s t-test. PC, phycocyanin; APC, allophycocyanin; PE, phycoerythrin.



Page 26 of 34

Table 2. Comprehensive metabolic profiles of Synechocystis 6803 cultivated under TiO2 treatment on the 16th day of growth Compound Alcohols Glycerol-3-phosphate Myo-inositol Myo-inositol-2phosphate Amino acids Alanine Asparagine Glutamic acid Glycine Pyroglutamic acid Serine Serine Threonine Threonine Tyrosine Fatty acids Glycerol monostearate Linoleic acid Linolenic acid 1-Monopalmitin Pentadecanoic acid Organic acids Citric acid Lactic acid Malic acid Succinic acid Sugars Glucose-6-phosphate Melibiose Sucrose Others Heptadecane

RT

Ion fragment (m/z)

24.90 31.25

Day 16a

TMS

Control

TiO2 (25 mg/L)

299, 315, 357, 445 191, 217, 305, 318

36.7±9.4 2.2±1.1

37.8±1.8 1.1±0.5

4 6

36.80

299, 315, 318, 387

1.6±1.0

1.3±0.4

7

8.61 22.88 21.79 13.94 19.38 12.69 15.42 13.66 16.05 28.47

59, 116, 190, 218 116, 132, 188, 231 128, 246, 348, 363 86, 174, 248, 276 84, 156, 230, 258 103, 116, 132, 234 100, 204, 218, 306 117, 130, 158, 219 101, 117, 218, 291 100, 179, 218, 280

23.7±1.6 4.2±3.9 210.4±9.8 3.3±0.6 4.6±0.3 5.0±0.4

20.0±9.3 2.3±1.3 179.8±98.4 3.4±1.1 7.1±2.3* 6.3±2.3

1.2±0.2

1.6±0.2*

2.3±1.2

7.3±4.8

2 3 3 3 2 2 3 2 3 3

42.50

129, 205, 399, 487

74.0±14.7

67.8±2.8

2

33.57 33.68 39.69 28.73

75, 95, 129, 337 75, 95, 129, 335 129, 239, 371, 459 117, 132, 145, 299

32.1±2.1 20.3±1.5 104.5±16.3 0.5±0.1

19.3±6.2* 10.1±5.0* 86.6±2.3 ND

1 1 2 1

26.09 7.52 18.67 14.25

273, 347, 363, 465 117, 133, 191, 219 133, 233, 245, 335 55,129, 172, 247

5.3±0.5 6.9±0.1 0.3±0.1 1.4±0.1

4.1±2.4 3.7±0.9* 0.2±0.0 0.7±0.5*

4 2 3 2

36.49 47.07 40.30

204, 299, 357, 387 0.5±0.2 129, 204, 217, 361 70.3±3.7 217, 271, 361, 437 372.0±238.3

0.7±0.2 43.9±18.4* 264.0±55.3

6 8 8

77.1±4.2

0

23.64

57, 71, 85, 240

73.2±7.0


Page 27 of 34


Neophytadiene Phosphoric acid a

27.40 13.07

68, 82, 95, 278 133, 211, 299, 314

7.7±1.3 56.2±11.4

7.9±0.4 117.3±62.1

0 3

Values in the table represent the relative intensities of each compound normalized by

dividing the peak intensity of internal standard (IS) and multiplying it by 100. Data are shown as the mean ± SD values (biological duplication and experimental duplication). The asterisk (*) represents significant differences (P < 0.05) between two groups (control, TiO2 25 mg/L) on day 16 by Mann-Whitney test. RT, retention time; TMS, trimethylsilylation



Page 28 of 34

Table 3. Relative levels of lipid compounds in Synechocystis 6803 cultivated under TiO2 treatment over three different time periods

Ion mode

Lipid molecular species

Day 8a Ion species

Positive ion mode Monogalactosyldiacylglycerol (MGDG) (+) MGDG 16:0/16:1 [M+Na]+ (+) MGDG 16:0/18:3 [M+Na]+ (+) MGDG 16:0/18:2 [M+Na]+ (+) MGDG 18:3/18:3 [M+Na]+ (+) MGDG 18:2/18:3 [M+Na]+ (+) MGDG 18:2/18:2 [M+Na]+ (+) MGDG 18:1/18:2 [M+Na]+ Phytyl Derivatives (+) Pheophytin a [M+H] + (+) Chlorophyll a [M+H] + Digalactosyldiacylglycerol (DGDG) (+) DGDG 16:0/18:3 [M+Na]+ (+) DGDG 16:0/18:2 [M+Na]+ (+) DGDG 18:2/18:3 [M+Na]+ Negative ion mode Phosphatidylglycerol (PG) (-) PG 16:0/16:1

[M-H]-

m/z

Day 0

Day 16a

Control

TiO2 (25 mg/L)

Control

TiO2 (25 mg/L)

751 775 777 797 799 801 803

1.4±0.2 21.8±1.9 8.1±0.7 1.0±0.3 1.4±0.2 24.3±4.2 27.9±5.3

0.8±0.2 20.8±3.3 6.4±1.7 1.0±0.4 0.9±0.3 16.0±1.1 20.4±1.2

0.9±0.2 21.8±3.5 7.7±1.8 1.2±0.6 2.0±0.3* 28.1±4.0* 30.3±4.9*

1.1±0.2 17.2±4.0 6.2±1.0 1.7±0.9 1.7±0.2 15.5±7.6 18.1±6.9

1.9±0.9 22.5±5.8 9.9±4.1 1.6±0. 5 1.2±0.7 11.5±7. 7 14.4±9.1

871 893

2.4±0.2 17.2±1.4

3.7±0.4 23.2±4.9

3.4±1.8 19.2±5.5

6.1±2.2 23.9±4.6

3.6±0.4# 24.3±2.2

937 939 961

5.4±0.6 2.7±0.3 1.3±0.4

5.3±1.2 3.6±0.9 2.1±0.8

6.0±1.7 3.2±0.9 1.6±0.6

4.3±0.8 3.1±1.0 2.9±1.3

5.7±2.1 3.6±1.4 2.2±0.7

719

12.5±1.2

11.7±1.7

14.6±2.2*

18.1±0.8

18.6±1.9


Page 29 of 34


(-) PG 16:0/16:0 [M-H](-) PG 16:0/18:3 [M-H](-) PG 16:0/18:2 [M-H](-) PG 16:0/18:1 [M-H]Sulfoquinovosyldiacylglycerol (SQDG) (-) SQDG 14:0/18:3 [M-H](-) SQDG 14:0/18:2 [M-H](-) SQDG 16:0/16:1 [M-H](-) SQDG 16:0/16:0 [M-H](-) SQDG 16:0/17:2 [M-H](-) SQDG 16:0/17:1 [M-H](-) SQDG 16:0/17:0 [M-H](-) SQDG 16:0/18:4 [M-H](-) SQDG 16:0/18:3 [M-H](-) SQDG 16:0/18:2 [M-H](-) SQDG 16:0/18:1 [M-H](-) SQDG 16:0/18:0 [M-H](-) SQDG 16:0/19:0 [M-H](-) SQDG 18:3/18:3 [M-H](-) SQDG 18:2/18:3 [M-H](-) SQDG 18:2/18:2 [M-H]Phosphatidylinositol (PI) (-) PI 16:0/18:2 [M-H]a

721 743 745 747

11.3±2.5 25.5±4.7 76.9±11.1 19.7±3.2

10.7±2.9 34.3±9.4 76.9±19.5 21.7±5.8

787 789 791 793 803 805 807 813 815 817 819 821 835 837 839 841

17.1±2.5 14.6±2.2 231.8±26.5 288.1±26.8 2.6±0.3 14.8±1.2 21.0±1.2 50.4±7.3 39.9±8.2 182.2±21.5 73.6±8.0 12.8±1.9 5.8±0.8 4.4±0.8 2.3±0.3 1.8±0.3

6.4±1.7 7.8±1.4 10.8±3.5 12.7±1.8 140.2±26.4 172.7±23.3* 229.6±53.4 286.5±54.6 3.5±1.3 3.6±0.9 12.4±2.8 13.7±2.3 18.7±6.6 16.3±1.8 28.9±5.3 37.6±6.9* 50.9±15.3 56.7±15.3 183.3±42.7 207.8±44.5 103.6±24.5 135.0±29.0 18.1±4.8 23.8±5.5 5.6±2.1 5.4±0.3 5.3±1.8 4.6±1.1 8.2±2.7 7.7±2.9 7.0±2.7 6.6±2.1

833

6.0±1.6

8.3±2.4

12.0±4.0 35.3±13.5 90.1±25.1 27.3±8.4

7.0±1.3

21.8±3.7 50.7±15.9 107.0±29.7 29.2±5.7

16.2±2.0# 32.2±5.7# 88.0±10.9 26.6±4.3

8.9±1. 5 14.7±2.7 209.3±16.2 304.0±30.9 6.0±1.1 14.3±1.5 22.3±7.0 24.6±1.0 92.0±16.5 223.2±35.6 90.3±9.0 14.1±1.5 8.8±3.1 8.1±3.0 13.2±4.2 11.5±3.8

13.4±1.4# 14.8±1.9 228.1±34.5 335.1±46.5 4.4±0.7# 15.3±1.5 22.3±2.2 41.0±5.2# 71.1±11.8# 203.1±22.8 103.0±15.7 17.3±3.0# 9.2±1.2 6.8±1.2 8.7±1.8# 7.6±1.6#

11.2±3.9

9.3±1.4

Values in the table represent the relative intensities of each compound normalized by dividing the peak intensity of IS. Data are shown as the

mean ± SD values (biological triplication and experimental duplication). The asterisk (*) represents significant differences (P < 0.05) between 29 ACS Paragon Plus Environment


two groups (control, TiO2 25 mg/L) on day 8 by Mann-Whitney test. The hash (#) represents significant differences (P < 0.05) between two groups (control, TiO2 25 mg/L) on day 16 by Mann-Whitney test.


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Table 4. Contents (mg/kg) of iron, magnesium, titanium, manganese and zinc of Synechocystis 6803 cultivated under TiO2 treatment over two different time periods analyzed by ICP-MS

Elements Fe Mg Ti Mn Zn a

Day 8a Control TiO2 (25 mg/L) 3471.5 ± 362.8 5451.1 ± 233.0 72.2 ± 3.6 137.2 ± 5.0 262.8 ± 95.4

1535.7 ± 24.4* 4257.7 ± 102.4* 267.3 ± 63.5* 57.5 ± 4.1* 103.8 ± 2.9*

Day 16a Control TiO2 (25 mg/L) 1387.9 ± 177.3 4679.0 ± 558.6 23.1 ± 3.7 68.7 ± 6.5 134.9 ± 61.5

906.0 ± 109.1# 4706.9 ± 96.6 273.2 ± 76.2# 48.3 ± 5.6# 30.0 ± 12.0#

Data are listed as the mean ± SD values (biological triplication and experimental duplication).

The asterisk (*) represents significant differences (P < 0.05) between two groups (control, TiO2 25 mg/L) on day 8 by Mann-Whitney test. The hash (#) represents significant differences (P < 0.05) between two groups (control, TiO2 25 mg/L) on day 16 by Mann-Whitney test.



Figure. 1.


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Graphic for Table of Contents



Enhancement of pigments and intact lipid species by TiO2 NPs treatment in Synechocystis sp. PCC 6803 culture. 82x39mm (300 x 300 DPI)

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Phycobiliproteins Production Enhancement and Lipidomic Alteration

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