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Apr 3, 2017 - Dunaliella tertiolecta, a halotolerant alga, can accumulate large amounts of neutral lipid, which makes it a potential biodiesel feedsto...
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Effects of salt concentrations, nitrogen and phosphorus starvations on neutral lipid contents in the green microalga Dunaliella tertiolecta Ming-Hua Liang, Xiao-Ying Qv, Hui Chen, Qiang Wang, and Jian-Guo Jiang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b00552 • Publication Date (Web): 03 Apr 2017 Downloaded from http://pubs.acs.org on April 5, 2017

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Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Journal of Agricultural and Food Chemistry

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Effects of salt concentrations, nitrogen and phosphorus starvations on

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neutral lipid contents in the green microalga Dunaliella tertiolecta

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Ming-Hua Liang 1 #, Xiao-Ying Qv 1 #, Hui Chen 2, Qiang Wang 2, Jian-Guo Jiang 1*

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1 College of Food Science and Engineering, South China University of Technology, Guangzhou, 510640,

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China

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2 Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, 430072, China

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These authors contributed to the work equally and should be regarded as co-first authors.

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*Author (Jian-Guo Jiang) for correspondence (e-mail: [email protected]; phone +86-20-87113849; fax:

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+86-20-87113843)

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Abstract

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Dunaliella tertiolecta, a halotolerant alga, can accumulate large amounts of neutral lipid, which makes it a

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potential biodiesel feedstock. In this study, neutral lipids of D. tertiolecta induced by different salinities, N

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or P starvation were analyzed by TLC, FCM and CLSM. High salinities, N or P starvation resulted in a

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decrease in cell growth and chlorophyll contents of D. tertiolecta. Neutral lipid contents increased

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markedly after 3~7 days of N starvation or at low NaCl concentrations (0.5~2.0 M). N starvation had a

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more dramatic effect on the neutral lipid contents of D. tertiolecta than P starvation. Four putative ME

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isozymes in different conditions can be detected by using isozyme electrophoresis. Two alternative

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acetyl-CoA producers, ACL and ACS genes were up-regulated under low salinities and N starvation. It was

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suggested that low salinities and N starvation are considered as the efficient ways to stimulate lipid

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accumulation in D. tertiolecta.

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Keywords: Dunaliella tertiolecta, nitrogen starvation, phosphorus starvation, neutral lipid, flow cytometry.

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Journal of Agricultural and Food Chemistry

Introduction

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Oleaginous microalgae have been considered as a kind of promising alternative sources for next

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generation of renewable fuels. Microalgae are abundant in triacylglycerol (TAG, neutral lipid), which can

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be used for biodiesel production, under stress conditions, especially during nitrogen (N) starvation. The

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advantages of exploiting microalgae as biofuel feedstock mainly due to their high lipid contents, high

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photosynthetic efficiency, short life cycle, less affection by venue, less labor required, and easier to scale up.

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1, 2

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per dry cell weight), which made it a potential biodiesel feedstock. 3 Numerous reports have indicated that

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N deprivation improves lipids accumulation in many microalgae, for example Chlamydomonas reinhardtii,

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Chlorella vulgaris, Chlorella zofingiensis, Micractinium pusillum and D. tertiolecta. 4-7 It was reported that

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lipid content increased under nitrogen starvation condition with time, and phosphate deprivation had little

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effect on lipid accumulation in D. tertiolecta. 7 However, little is revealed about the molecular mechanisms

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of lipid production under N or P starvation and other cultivation conditions in D. tertiolecta.

Dunaliella tertiolecta, a halotolerant alga, was reported to contain large amount of lipids (up to 71.0%

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In microalgae, lipids biosynthetic pathway includes fatty acid synthesis and TAG synthesis. To

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accumulate fatty acids, it is important to have a sufficient supply of NADPH in the cytosol and a

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continuous provision of acetyl-CoA as an essential precursor for fatty acid synthetase.

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(ME) plays an important role in regulating lipid accumulation process in many organisms. ME catalyzes

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the synthesis of pyruvate from the decarboxylation of malate with the formation of NADPH, which is

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necessary for the synthesis of fatty acids.

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Malic enzyme

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In plants and algae, as for the precursor (i.e. acetyl-CoA) for fatty acids synthesis, it was reported that

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there are three pathways involved in the formation of acetyl-CoA: acetyl-CoA synthetase (ACS),

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ATP-citrate lyase (ACL), and pyruvate dehydrogenase (PDH)

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accumulation of high TAG mainly relies on the enhancement of acetyl-CoA production. 11 Algal ACLs are

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composed of two different subunits, ACLA and ACLB. 9 It was reported that two alternative acetyl-CoA

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producers, ACS and ACL were up-regulated before TAG accumulation in the oleaginous species Chlorella

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desiccate under nitrogen deprivation. 10

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It was recommended that the

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The traditional gravimetric method has been used for detection of lipid content in microalgae, but it is

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time-consuming and needs relatively large algal sample size. 12 In contrast, the fluorescent staining method 3

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provides an inexpensive and efficient way to detect neutral lipid content, and only requires small sample

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volume. However, the Nile red staining method would be greatly influenced by many factors including

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algal species and different measurement conditions. 13 The emitting light of the Nile red is at 590~640 nm,

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which may interfere with the detection of chlorophyll fluorescence (detected at 650~700 nm). Compared

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with Nile red, a lipophilic bright green fluorescent dye BODIPY 505/515, has a high oil/water partition

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coefficient and sharp emission bands.

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detected at 505~515 nm, which is spectrally different from algal chloroplasts.

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BODIPY 505/515 is stimulated by a blue 488 nm laser and

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In this study, neutral lipids induced by different NaCl concentrations and N or P starvation were

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analyzed by thin-layer chromatography (TLC), flow cytometry (FCM), and confocal laser scanning

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microscopy (CLSM). The effects of different conditions on algal biomass and photosynthetic pigments

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synthesis were also discussed. The isozyme activity of ME in different conditions was investigated by using

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isozyme electrophoresis. The transcript levels of ACL and ACS genes from D. tertiolecta (DtACLA,

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DtACLB and DtACS) in response to N or P deficiency and different salinities were analyzed by qRT-PCR.

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Materials and methods

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Microalgal strain and culture conditions

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Dunaliella tertiolecta FACHB-821 was obtained from the Institute of Hydrobiology, Chinese Academy of

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Sciences. D. tertiolecta cells were grown in a Dunaliella medium

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condition) at 26 °C and 8000 lx provided by cool-white fluorescent lamps under a 16/8 h dark/light cycle

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for 14~16 days (d). The optical density (OD) of the algal samples was detected at 630 nm (OD630) by a

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spectrophotometer (Agilent, USA). After 7 d of normal growth (2.0 M NaCl) in N-sufficient medium (N+)

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(OD630=07~0.8), the algal cells were harvested by centrifugation at 2,000 g for 5 min and resuspended

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twice with a fresh N-deficient medium (N-) without NaNO3 for a second period of N starvation cultivation

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for 7 d. For phosphorus (P) starvation treatment, algal cells in P-sufficient medium (2.0 M NaCl) (P+) were

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harvested by centrifugation at 2,000 g for 5 min and resuspended twice with a fresh P-deficient medium (P-)

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without NaH2PO4·2H2O for P starvation cultivation for 7 d. In order to study lipid accumulation of D.

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tertiolecta in different salt concentrations, algal cells were grown in Dunaliella media with 0.5 M, 1.0 M,

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1.5 M, 2.0 M, 2.5 M, 3.0 M, 3.5 M and 4.0 M NaCl for 14~16 d, respectively.

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Algal Biomass Analysis

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containing 2.0 M NaCl (as normal

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D. tertiolecta cells were grown in a defined medium containing 2.0 M NaCl for 14 d. The OD630 of the

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algal samples (using blank medium to dilute into different proportions, 1:1, 1:2, 1:5, 1:10, and 1:15) were

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detected at 630 nm by a spectrophotometer (Agilent, USA), and the blank medium without algal cells was

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used as the control sample. For the measurement of algal dry cell weight (DCW), culture samples (100 mL)

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were harvested by centrifugation at 8000 r/min for 5 min, washed twice with distilled water, and dried at

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80 °C to a constant weight for 24 h. Then the DCW was obtained by determining OD630, y= 993.21x –

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16.359, R² = 0.9988, where y= DCW (mg/L) and x=OD630 value (0.05