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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
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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
