Simultaneous accumulation of neutral lipids and biomass in Nannochloropsis oceanica IMET1 under high light intensity and nitrogen replete conditions

Algal Research 11 (2015) 55–62

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Simultaneous accumulation of neutral lipids and biomass in Nannochloropsis oceanica IMET1 under high light intensity and nitrogen replete conditions Yan Xiao a,b, Jingtao Zhang a,b, Jiatao Cui a,b, Xingzhe Yao a,b,d, Zhijie Sun a,b, Yingang Feng a,b,c,⁎, Qiu Cui a,b,c,⁎ a

Qingdao Engineering Laboratory of Single Cell Oil, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao 266101, PR China Shandong Provincial Key Laboratory of Energy Genetics, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao 266101, PR China c Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao 266101, PR China d University of Chinese Academy of Sciences, Chinese Academy of Sciences, Beijing 100049, PR China b

a r t i c l e

i n f o

Article history: Received 19 November 2014 Received in revised form 22 May 2015 Accepted 27 May 2015 Available online xxxx Keywords: Nannochloropsis Neutral lipid Biodiesel High light intensity Nitrogen repletion

a b s t r a c t The difficulty in simultaneously obtaining a high production of neutral lipids and biomass in the oleaginous microalga Nannochloropsis is an attractive challenge that needs to be addressed. To overcome this challenge and unveil the underlying metabolic characteristics, the model oleaginous microalga Nannochloropsis oceanica IMET1, under high light intensity and nitrogen-replete conditions, was prepared via steady-state continuous culture in the present study. The simultaneous accumulation of lipids, particularly neutral lipids, and biomass in N. oceanica IMET1 was triggered at a high light intensity (331.2 μmol photons m−2 s−1) in the light saturation region accompanied by a sufficient nitrogen supply (2000 μmol L−1). Moreover, high amounts of valuable fatty acids (arachidonic acid and eicosapentaenoic acid) were also produced. The distribution of fatty acids and intracellular soluble metabolites, the membrane lipid composition and the photosynthetic pathway were associated with the concurrent accumulation of biomass and TAGs in N. oceanica IMET1. The common and different metabolic responses between high-light and nitrogen-depletion strategies for the accumulation of neutral lipids were also discussed. These results provide detailed metabolic clues for the genetic and metabolic engineering of Nannochloropsis for biofuel production. © 2015 Elsevier B.V. All rights reserved.

1. Introduction As one of the most promising feedstocks for biofuel production, microalgal triacylglycerols (TAGs) have been studied worldwide for decades. However, many technological bottlenecks (such as the maximization of lipids, cell harvesting, dewatering, lipid extraction and processing) have hindered microalgal biofuel production due to the high cost and energy consumption [12,18,34,41]. Microalgal TAG accumulation is commonly triggered in unfavorable environments or stressful conditions, resulting in low biomass production and leading to low final TAG yields [12,22,45]. Genetic engineering methods for stimulating the accumulation of TAGs while maintaining adequate microalgae growth have been less successful, primarily reflecting the cellular regulation mechanism of TAG accumulation, which depends on the microalgae species and is poorly understood [20]. Therefore, the selection of reference microalgae, the optimization of growth to achieve synchronous rapid growth and TAG accumulation and the characterization of the underlying ⁎ Corresponding authors at: Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, No. 189 Songling Road, Laoshan District, Qingdao 266101, PR China. E-mail addresses: [email protected] (Y. Feng), [email protected] (Q. Cui).

http://dx.doi.org/10.1016/j.algal.2015.05.019 2211-9264/© 2015 Elsevier B.V. All rights reserved.

cellular mechanisms are crucially important to ensure maximum sustainable lipid production and will guide the rational metabolic and genetic manipulation of microalgae to increase TAG production [34]. Nannochloropsis has been widely studied due to its high level of TAGs in nature and has now become one of the most promising reference microalgae, which has been successfully grown indoors and outdoors for large-scale biodiesel production [23,32,46]. Furthermore, genome sequences, nuclear transformation and high-efficiency homologous recombination transformation methods for multiple species of Nannochloropsis have been recently reported [14,16,27,29,39,40], and these methods provide important tools for improving the accumulation of TAGs via genetic and metabolic engineering in Nannochloropsis. In addition, the proteomic, metabolomic, transcriptomic and lipidomic dynamics in Nannochloropsis oceanica IMET1 under nitrogen (N) depletion conditions were also recently studied [5,19,40,43]. These studies provided additional clues concerning the mechanisms that regulate TAG accumulation, with new insights at the molecular level. Previous comprehensive Omics studies have indicated that N. oceanica is a good model for rational genetic and metabolic engineering to overproduce TAGs for biodiesel production. Recently, various biochemical and physical stresses alone or combined were utilized to maximize the production of TAGs from Nannochloropsis; however, none of these studies could simultaneously

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increase the TAGs and valuable components (e.g., EPA) [25,37,38,43]. N repletion triggered the remarkable production of biomass and EPA, with a sharp decrease in TAGs in Nannochloropsis [43], while high light intensity resulted in TAG accumulation [25,36]. Thus, conditions involving high light intensity with N-replete culture condition might synchronously increase the production of TAGs, biomass and EPA. To confirm this hypothesis, different light intensities with constant sufficient N supply at steady-state continuous culture were used in the present study. In continuous culture, fresh medium is continually added as the culture medium is withdrawn, facilitating the maintenance of a steadystate cell concentration [42]. Ho et al. reported that the higher productivity of the biomass of a photosynthetic microalgae Scenedesmus obliquus CNW-N was achieved when the light intensity was in the light saturation region (180–540 μmol photons m− 2 s−1) compared with the light limited (b180 μmol photons m−2 s−1) and light inhibition regions (N540 μmol photons m−2 s−1) [11]. Based on these findings, three light intensities were used in the present study, covering light saturation and limited regions: 331.2 μmol photons m−2 s−1 (designated as high light), 157.7 μmol photons m−2 s−1 (designated as medium light) and 71.5 μmol photons m−2 s−1 (designated as low light). Recent advances in microalgae showed that the TAG metabolism significantly differs between algae species and plants; thus, the study of TAG metabolism in N. oceanica IMET1 might provide new insights into genetic and metabolic engineering for improving biofuel production. Moreover, these insights might fill existing gaps in the understanding of lipid metabolism in plants [20]. In the present work, the effect of light intensity under N replete culture conditions on the simultaneous high production of biomass, TAGs and C20:5 in N. oceanica IMET1 via continuous culture and the corresponding metabolic response of N. oceanica IMET1 were investigated. Furthermore, the metabolic characteristics of TAG biosynthesis in N. oceanica IMET1 were identified to provide detailed clues for the genetic and metabolic engineering of Nannochloropsis for biofuel production. 2. Materials and methods 2.1. Microalgae and growth conditions N. oceanica IMET1 was maintained in annular glass columns, containing 700 mL of autoclaved sea water and modified F/2 medium, with the following composition (per liter): 400 mg KNO3, 50 mg NaH2PO4·2H2O, 10 mg FeCl3·6H2O, 4.5 g EDTA and 1 mL of trace elemental solution. The trace elemental solution has been previously described [43]. The pH was adjusted to 7.6. The cells were incubated at 20 ± 2 °C and illuminated continuously on one side using cool white fluorescent lamps. Pure CO2, from cylinders, was supplied continuously to the air stream (1.5% to 2%, v/v) to provide a carbon source for the culture. The mixed stream was filtered through 0.22-μm filters and injected into the columns. The cell density (OD750) was determined in triplicate with an UV/Vis spectrophotometer (Beijing Purkinje General Instrument Co., Ltd., Beijing, China). The culture experiment was performed as two-step cultivation. First, the cells were cultured for 6 days to an OD750 of 5 in a batch culture. Next, the cells were centrifuged at 3000 ×g for 10 min, washed three times to remove the nutrients, and resuspended with a fresh modified F/2 medium for a continuous culture. In the continuous culture, a stream of feeding medium was pumped from a storage tank at a certain rate into the 700 mL culture tube, while the culture medium and cells were pumped out at the same rate to the collection flask. The components of the feeding medium were the same as those of the culture medium, except the nitrate concentration, which was high up to 8000 μmol L−1 (808.8 mg L− 1). During the culture, the samples were collected every 4 h for nitrate and cells density quantification, and the dilution rates were adjusted according to the nitrate concentration of the samples till the nitrate concentration was constant to

2000 μmol L−1, and then they were set at a fixed dilution rate (D) of 0.0125 h−1. The nitrate concentrations in the media were fluctuated in first days, and the steady-state continuous cultures were established when the nitrate concentrations became constant at 2000 μmol L−1 in 12 days and the following days. The nitrate concentrations were detected using the Collos method [4]. The nitrate concentrations are shown in Fig. S1A. The optical density at 750 nm (OD750) of the culture was used to measure the cell density (Fig. S1B). The schematic view of continuous culture is shown in Fig. S2A. The following light intensities were chosen: 71.5 μmol photons m− 2 s−1 (designated as low light, LL), 157.7 μmol photons m− 2 s− 1 (designated as medium light, ML) and 331.2 μmol photons m−2 s−1 (designated as high light, HL). The pictures of the cultivation are shown in Fig. S2B. 2.2. Cell harvest and dry biomass measurement The 700-mL samples were harvested after 20 days of cultivation. The wet biomass was centrifuged (21,700 ×g) at 4 °C for 10 min and then washed three times with deionized water to remove the salt. The cells were intact after the wash according to the microscopy observation. The cell pellets were lyophilized using vacuum freeze-drying equipment (ALPHA1-2LD, Martin Christ, Osterode am Harz, Germany) for 24 h. After drying, the cell pellets were weighed and stored at 4 °C. 2.3. Lipids extraction and fractionation The lipids extraction was performed using the Bligh and Dyer's method [2]. The crude lipids extraction was fractionated followed according to the method of Gamian et al. [9]. Briefly, lyophilized algal powder was added into 6 mL chloroform:methanol (2:1, v/v) solution and was shaken at 37 °C and 180 rpm for 2 h. The solution was centrifuged at 12,000 ×g for 10 min. The extracted supernatant was collected, and the pellet was further repeatedly extracted twice. Approximately 5 mL of water was added into the combined supernatants and the mixture was centrifuged at 12,000 ×g for 10 min. The chloroform layer was evaporated under the protection of nitrogen gas and then freeze-dried for 24 h, and the extracts were weighted as total lipid weight. The lipid fractions were separated after passing the extracted lipids through a home-made silica column using chloroform, then acetone and finally methanol as eluents. Neutral lipids and polar lipids were washed out by chloroform and methanol, respectively. This process is shown in Fig. S2C–E. 2.4. GC–MS analysis for fatty acid methyl ester analysis Approximately 10 mg of the total lipids, NLs and PLs were converted into methyl esters using 2.5 mL of 2% H2SO4–methanol (v/v) [44] and spiked with an internal standard (nonadecanoic acid, C19:0) in duplicate. The detailed fatty acid methyl esters (FAMEs) analysis was performed using the Agilent 7890-5975 GC–MS system (Agilent Technologies Inc., Santa Clara, CA, USA) previously described [43]. The image shows the assignment of FAMEs in Fig. S3. 2.5. Extraction of water-soluble cellular metabolites Approximately 25 mg of the freeze-dried powder was used to extract the water-soluble cellular metabolites and each sample was analyzed in triplicate. The details of this process have been described in a previous study [43]. 2.6. NMR measurements of the water-soluble cellular metabolites Approximately 2 mg of the freeze-dried powder were used, and each sample was analyzed in triplicate. The detailed sample preparation and data collection procedures via NMR were previously described in a previous study [43].

Y. Xiao et al. / Algal Research 11 (2015) 55–62

2.7. Data reduction and multivariate NMR data analysis The 1H NMR spectra were manually corrected for phase and baseline distortions using TOPSPIN (Bruker Biospin GmbH, Karlsruhe, Germany), and the spectral region of 0.5–10.0 ppm was uniformly integrated into 3166 buckets with a width of 0.003 ppm (1.8 Hz) using the AMIX package (Bruker Biospin GmbH, Karlsruhe, Germany). The region of 4.70– 5.81 ppm was discarded to eliminate the effect of imperfect water suppression. The detailed analysis has been described in a previous study [43]. The image shows the assignment of metabolism in Fig. S4.

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DCW and NLs was obtained at two culture conditions involving N repletion and N depletion, respectively [43]. In the present study, the yields of biomass, lipid and NLs were successfully maximized under the same culture condition (HL). Moreover, the production of DCW (3 folds), lipids (4.8 folds) and NLs (15.8 folds) was increased compared with those of our previous study [43]. These data also indicated that the accumulation of lipids under the HL condition primarily reflected the NLs accumulation, and the biomass accumulation resulted from lipids accumulation. 3.2. The fatty acid profile of lipids

2.8. Carbohydrates quantification by ion chromatography Approximately 50 mg of the freeze-dried algae samples were added to 1 mL of double distilled H2O and agitated in a 2 mL EP tube with a vortex at room temperature for 10 s, followed by 15 min of intermittent ultrasonication (2 s sonication and 1 s break) in an ice bath. After centrifugation (12,300 ×g) for 2 min at 4 °C, the supernatants from the extracts were filtered using a 0.22-μm filter and subsequently diluted 100 times. After the extraction of total water-soluble carbohydrates, the carbohydrates were quantified using an ICS-3000 system (Dionex Corporation, Sunnyvale, USA). Each sample was performed in triplicate. The CarboPac PA10 (4 × 250 mm, Dionex) column set, including the PA10 guard (4 × 250 mm, Dionex) was used. The standards of 8 carbohydrates were measured and used to quantify the corresponding intracellular metabolites of the algae samples. 2.9. Elemental analysis Approximately 2 mg of the freeze-dried algae samples were used to calculate the cellular contents of C, H, and N in the biomass, and each sample was performed in triplicate in a Vario EL III Element Analyzer (Elementar Analysen Systeme GmbH, Germany). 3. Results and discussion 3.1. Effect of different light intensities on TAGs and biomass production To illustrate the effect of the irradiation intensity on the TAGs and biomass yield in N. oceanica IMET1, the culture condition with a sufficient nitrate supply was selected. The light intensity effects on the production of biomass, lipids and TAGs are shown in Table 1. The steadystate continuous culture was established after 12 days of cultivation (Fig. S1) and the cells were harvested after 20 days of cultivation. Notably, HL simultaneously maximized the yield of lipids and dry cell weight (DCW) compared with ML and LL. This result differed from a previous report on Nannochloropsis gaditana, which showed that the light intensity did not directly influence lipids accumulation [35]. The different results might be primarily due to the different concentration of nitrogen (N repletion vs. N depletion) and culture method (continuous vs. batch culture). In this work, the production of neutral lipids (NLs, mainly TAGs according to TLC data shown in Fig. S5) was also maximized at 11.83 mg L−1 h−1 in the HL condition, which was approximately 4- and 18-fold higher than that in the ML and LL conditions, respectively. The levels of lipids (54.3% of DCW) and NLs (34.7% of DCW) were also highest for the HL condition compared with the other irradiation conditions. In a previous study, we reported that the maximum production of

The fatty acids (FAs) profile plays a key role in influencing the biodiesel properties [17]. Medium- and long-chain FAs (e.g., C14–C18) are common FAs for biodiesel production [17], and polyunsaturated FAs (e.g., C20:4 and C20:5) with health-beneficial properties are potential co-products of biodiesel production [15]. The FAs profile of the total lipids of N. oceanica IMET1 is shown in Table 2. Overall, the level of monounsaturated FAs (C16:1 and C18:1) increased (particularly C18:1), and this trend is similar to that of a previous report [32,43], while the polyunsaturated FAs (C18:2, C20:4 and C20:5) decreased, and the other saturated FAs irregularly fluctuated in response to increasing the light intensity from LL to HL. The yield of short chain FAs (C14–C18) was maximized at 12.38 mg L−1 h−1 in response to the HL condition compared with the ML (4.00 mg L−1 h−1) and LL (1.75 mg L−1 h−1) conditions. The yield of high value-added FAs (C20) was also maximized at 1.50 mg L−1 h−1 in response to the HL condition compared with the ML (1.00 mg L−1 h−1) and LL (0.75 mg L−1 h−1) conditions, and this value benefitted from the synchronous accumulation of total FAs (13.88 mg L−1 h−1) and biomass (34.14 mg L−1 h−1). The content of C20 (10.8% of total FAs) was the noticeably minimized under the HL condition, demonstrating the presence of physiological adaptations to the high light intensity condition by decreasing the membrane fluidity [21]. Although the level of FAs (22% of DCW) was similar for the ML and LL conditions, the content of C20 was lower under the ML condition compared with the LL condition, illustrating that a higher light intensity could trigger the accumulation of C14– C18 FAs, instead of C20 FAs (Table 2). This finding was consistent with the results of a previous report [12]. The FAs content of TAGs is critical for biodiesel production. To thoroughly investigate the FAs profile of TAGs, the total lipids were separated into NLs (mainly TAGs) and polar lipids (PLs). The FAs profile of NLs and PLs is shown in Table 3. A remarkable accumulation of C18:1 both in the NLs and PLs was observed as the light intensity increased. In addition, with the light intensity increase, the level of C20:4 was increased in the NLs and PLs, but the level of C20:5 was increased only in the NLs. Recently, it was observed that a small fraction of C20:5 from membrane glycerolipids was recycled for TAG biosynthesis in N. oceanica IMET1 via the phospholipid:diacylglycerol acyltransferase (PDAT)-mediated conversion in response to N repletion and N depletion [19]. In addition to PDAT, the lipase-mediated membrane degradation contributed to the channeling of FAs from membrane lipids to TAG [19]. These results provided additional evidence demonstrating that C20:5 can be transferred from PLs to NLs. Recently, Jia et al. reported that the accumulation of C20:5 in TAG was associated with the degradation of several C20:5-containing membrane glycerolipids (i.e., DGTS, PG, and MGDS), suggesting that C20:5 in TAG was recycled from membrane glycerolipids [13].

Table 1 Light intensity effects on the DCW, lipids, and NLs. Condition

Light density (μmol photons m−2 s−1)

DCW (mg L−1 h−1)

Lipids (mg L−1 h−1)

NLs (mg L−1 h−1)

Lipids/DCW (%)

NLs/DCW (%)

NLs/lipids (%)

HL ML LL

331.2 157.7 71.5

34.14 23.61 11.57

18.53 ± 0.00 7.41 ± 0.06 3.09 ± 0.01

11.83 ± 0.85 2.82 ± 0.16 0.65 ± 0.01

54.28 ± 0.00 31.40 ± 0.23 26.70 ± 0.05

34.65 ± 0.61 11.96 ± 0.66 5.61 ± 0.00

65.43 ± 1.04 37.89 ± 1.29 20.99 ± 2.35

HL, high light; ML, medium light; LL, low light; DCW, dry cell weight; NLs, neutral lipids.

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synchronously maximize the yield of biomass (34.14 mg L−1 h−1), neutral lipids (11.83 mg L−1 h−1, Table 1), the high value C20 (AA plus EPA, 1.50 mg L− 1 h− 1, Table 2) and C14–C18 FAs in the NLs (12.38 mg L−1 h−1, Table 3) obtained from N. oceanica IMET1. The results demonstrated that a high light intensity in the light saturation region with N-replete culture condition in the culture of N. oceanica IMET1 might be a promising strategy to synchronously obtain the high production of TAGs, biomass and EPA. This strategy might be not easily extrapolated for outdoor cultivation because outdoor results would be influenced through many factors, such as fluctuant light intensity and temperature. Although additional studies with specific techniques, such as reactor design, are needed to realize the strategy for outdoor cultivation, the results of the present study provide a potential direction for the concurrent accumulation of biomass and TAGs.

Table 2 Fatty acids' profile. FAME

C14:0 C15:0 C16:0 C16:1Δ9 C17:0 C18:0 C18:1Δ9 C18:2Δ9,12 C20:4Δ5,8,11,14 C20:5Δ5,8,11,14,17 Total C14–C18 Total C20 Total SFA Total USFA (C14–C18) (mg L−1 h−1) C20 (mg L−1 h−1) Total FA (mg L−1 h−1) FA/DCW (%)

FA/total FA% HL

ML

LL

4.57 ± 0.44 0.63 ± 0.09 33.08 ± 0.36 26.71 ± 0.97 0.62 ± 0.06 3.67 ± 0.16 18.16 ± 0.15 1.73 ± 0.13 5.03 ± 0.05 5.80 ± 0.24 89.17 ± 0.30 10.83 ± 0.30 42.57 ± 0.38 57.43 ± 0.38 12.38 ± 0.19 1.50 ± 0.01 13.88 ± 0.50 40.82 ± 1.47

5.33 ± 0.40 0.56 ± 0.05 33.74 ± 0.77 24.74 ± 0.49 0.96 ± 0.07 5.82 ± 0.04 6.36 ± 0.04 2.19 ± 0.09 7.91 ± 0.19 12.41 ± 0.37 79.68 ± 0.57 20.32 ± 0.57 46.41 ± 0.22 53.61 ± 0.22 4.00 ± 0.20 1.00 ± 0.00 5.13 ± 0.20 21.52 ± 0.83

4.76 ± 0.46 0.44 ± 0.05 28.56 ± 0.34 21.60 ± 0.29 0.70 ± 0.05 5.87 ± 0.12 2.69 ± 0.01 3.96 ± 0.36 13.14 ± 0.24 18.28 ± 0.45 67.58 ± 0.26 32.42 ± 0.75 40.33 ± 0.32 59.67 ± 0.27 1.75 ± 0.13 0.75 ± 0.00 2.63 ± 0.13 22.25 ± 0.97

3.3. Metabolites response In a previous study, we showed that oil accumulation accompanied a reduction in the concentration of osmolytes in N. oceanica IMET1 cultured under N-deficient conditions [43]. The concentration of metabolites might to respond to the high light intensity and N replete conditions. To thoroughly examine the oil accumulation metabolic mechanism and confirm this hypothesis, the effect of light intensity on the concentration of metabolites was investigated. The metabolites were identified via nuclear magnetic resonance (NMR) spectroscopy and ion chromatography and showed a wide distribution in many metabolic pathways (Fig. 1). The concentrations of a majority of these metabolites significantly varied between the three light intensities (Table 4). Remarkably, the majority of the detected metabolites were lowest under the HL condition compared with the ML or LL conditions (Fig. 1 and Table 4). To our knowledge, this report is the first to show that high light intensity decreases the contents of osmoprotectants (e.g., mannitol, proline and trehalose) accompanied by the accumulation of TAGs. The results of a previous study suggested that nitrogen deficiency might reduce the contents of the osmolytes, accompanied by lipid accumulation [43]. Based on the findings obtained in the present study, reducing the contents of some osmoprotectants (e.g., mannitol, proline and trehalose) might be a common response to HL or N-limited stress [43]. The salt concentrations (autoclaved sea water was used for cultures) of the continuous cultivation were identical; therefore, the change in the membrane permeability likely compensated for the reduction in the osmoprotectants in response to HL or low nitrogen. This possibility

FA, fatty acid; FAME, fatty acid methyl ester; HL, high light; ML, medium light; LL, low light; SFA, saturated fatty acid; USFA, unsaturated fatty acid; DCW, dry cell weight.

The C20:5 FA was primarily detected in the membrane lipids fraction, with only a trace present in the TAGs fraction [39]. These results illustrated that the EPA yield in the TAGs fraction was increased in the PLs fraction in response to the HL (0.41 mg L−1 h− 1 in NLs vs. 0.34 mg L−1 h−1 in PLs, Table 3), although the C20:5 level was low in the TAGs fraction (Table 3). The yield of FAs, which was remarkably light dependent, and higher in the NLs (27.31% of DCW) than in the PLs (4.35% of DCW) under the HL condition, but this trend was reversed under the ML and LL culture conditions (Table 3). This finding suggests that the high light intensity induces the de novo biosynthesis of total FAs (Table 2), and also triggers the distribution of the total FAs to NLs synthesis instead of PLs synthesis (Table 3). Regarding the downstream processes for biodiesel production, the C16:1 and C18:1 in the TAGs fraction have been suggested as suitable FAs for enrichment in the FAs profile to improve the low-temperature, ignition and combustion qualities of biodiesel, respectively [17]. The TAGs obtained under the HL condition contained the highest fraction of C18:1 (20% of FAs), which might benefit the biodiesel properties. To the best of our knowledge, the present study is the first to show that a high light intensity combined with N replete conditions could Table 3 Fatty acids' profile of NLs and PLs. FAME

C14:0 C15:0 C16:0 C16:1Δ9 C17:0 C18:0 C18:1Δ9 C18:2Δ9,12 C20:4Δ5,8,11,14 C20:5Δ5,8,11,14,17 C14–C18 C20 Total SFA Total USFA C14–C18 (mg L−1 h−1) C20 (mg L−1 h−1) Total FA (mg L−1 h−1) FA/DCW (%)

NLs (FA/total FA%)

PLs (FA/total FA%)

HL

ML

LL

HL

ML

LL

3.92 ± 0.36 0.60 ± 0.08 34.34 ± 0.26 28.08 ± 0.39 0.60 ± 0.08 2.38 ± 0.12 20.31 ± 0.03 1.79 ± 0.07 3.54 ± 0.13 4.44 ± 0.05 92.03 ± 0.06 7.97 ± 0.08 41.84 ± 0.36 58.16 ± 0.36 8.58 ± 0.13 0.74 ± 0.01 9.38 ± 0.14 27.31 ± 0.41

3.87 ± 0.02 0.70 ± 0.00 41.80 ± 0.02 30.89 ± 0.16 1.64 ± 0.01 5.68 ± 0.08 8.40 ± 0.16 1.86 ± 0.05 2.72 ± 0.23 2.44 ± 0.05 94.84 ± 0.17 5.16 ± 0.17 53.69 ± 0.13 46.31 ± 0.13 1.74 ± 0.00 0.09 ± 0.00 1.88 ± 0.00 7.77 ± 0.00

4.00 ± 0.15 0.79 ± 0.04 43.09 ± 0.29 35.14 ± 0.26 2.08 ± 0.04 5.87 ± 0.09 4.11 ± 0.06 2.29 ± 0.05 0.68 ± 0.02 1.95 ± 0.03 97.37 ± 0.12 2.63 ± 0.06 55.83 ± 0.26 44.17 ± 0.38 0.44 ± 0.00 0.01 ± 0.00 0.50 ± 0.00 3.93 ± 0.00

5.73 ± 0.28 0.51 ± 0.03 29.70 ± 0.36 16.72 ± 0.55 – 7.67 ± 0.04 3.90 ± 0.50 1.70 ± 0.02 11.09 ± 0.04 22.96 ± 0.06 65.95 ± 0.10 34.05 ± 0.10 43.62 ± 0.43 56.38 ± 0.43 0.98 ± 0.00 0.51 ± 0.00 1.50 ± 0.00 4.35 ± 0.01

5.30 ± 0.32 0.35 ± 0.04 25.83 ± 0.50 17.92 ± 0.56 – 4.76 ± 0.03 2.02 ± 0.04 2.25 ± 0.09 14.37 ± 0.09 27.20 ± 0.20 58.43 ± 0.25 41.57 ± 0.45 36.25 ± 0.07 63.75 ± 0.07 1.17 ± 0.02 0.83 ± 0.01 2.00 ± 0.01 8.47 ± 0.05

4.45 ± 0.27 0.31 ± 0.03 27.74 ± 0.56 17.24 ± 0.34 – 5.44 ± 0.03 0.88 ± 0.03 2.04 ± 0.06 10.08 ± 0.09 31.83 ± 0.13 58.09 ± 0.09 41.91 ± 0.37 37.93 ± 0.26 62.07 ± 0.26 0.52 ± 0.02 0.37 ± 0.00 0.88 ± 0.01 7.67 ± 0.03

“–”: not detected. FA, fatty acid; FAME, fatty acid methyl ester; HL, high light; ML, medium light; LL, low light; SFA, saturated fatty acid; USFA, unsaturated fatty acid; DCW, dry cell weight; NLs, neutral lipids; PLs, polar lipids.

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Fig. 1. Responsiveness changes in the metabolite levels of N. oceanica IMET1. The proposed metabolic pathways were based on the KEGG database (http://www.kegg.jp). The level of significance was set at p b 0.05. The metabolites indicated in blue were detected, while those in black were not detected. For trehalose, glucose, FAs and lipids, the level of significance was defined as above or below 30% of the value obtained under the medium light or low light condition. The “a” and “b” significant differences were derived from a one-way ANOVA (p b 0.05) between the high- vs. medium-light intensity data and the high- vs. low-light intensity data, respectively. Metabolites: 3PG, 3-phospho-D-glycerate; alpha-KG, alpha-ketoglutarate; F6P, fructose-6-phosphate; FUM, fumarate; G6P, glucose-6-phosphate; PYR, pyruvate; Cit, citrate; DMA, dimethylamine; Frc, fructose; GABA, gamma-amino-n-butyrate; GB, glycinebetaine; Galac, galactose; Glc, glucose; Gln, glutamine; Glu, glutamate; Gly, glycine; Ile, isoleucine; MA, malate; Phe, phenylalanine; Pro, proline; Shik, shikimate; Succ, succinate; TMA, trimethylamine; Tyr, tyrosine; Uri, uridine; Val, valine; UMP, uridine monophosphate; IMP, inosine monophosphate; Aden, adenosine; His, histidine; PRPP, phosphoribosyl pyrophosphate; OA, oxaloacetic acid; P5C, 1-pyrroline-5-carboxylic acid; Ser, serine; FAs, fatty acids; TAGs, triacylglycerols; Acetyl-CoA, acetyl-coenzyme A; TCA, tricarboxylic acid cycle; PPP, pentose phosphate pathway; PEP, phosphoenolpyruvate; MA, malate; CCM, CO2 concentrating mechanism; ATP, adenosine triphosphate; ADP, adenosine diphosphate; NADPH, nicotinamide adenine dinucleotide phosphate hydrogen; NADP+, nicotinamide adenine dinucleotide phosphate; H+, hydrogen; Pi, phosphorus; C, carbon; CA, carbonic anhydrase; PEPC, phosphoenolpyruvate carboxylase; MDH, malate dehydrogenase; ME, malic enzyme; PPDK, pyruvate phosphate dikinase; HL, high light; ML, medium light; LL, low light. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

was supported through the observed recycling of C20:5 from membrane glycerolipids for TAG biosynthesis in N. oceanica IMET1 in response to nitrogen deplete [13,19] or HL conditions (Table 3), and this recycling likely resulted in membrane composition and permeability changes. The change in the membrane lipid composition is a mechanism for adaption to light intensity, and a decrease in the very-long-chain polyunsaturated FA 20:5 is typically observed when light intensity increases [24]. Table 3 shows that the very-long-chain polyunsaturated FAs (C20:5) in the PLs decreased in response to increasing light intensity, which might decrease the membrane fluidity. Moreover, the levels of proline and trehalose, stabilized membranes through interactions with phospholipids [33], and significantly decreased under HL conditions compared with LL conditions (Table 4), indirectly suggesting that the

membrane permeability was influenced through high light intensity. The acyl group of structural lipids (mainly membrane lipids) could contribute to TAG accumulation during N starvation [10], and a PDATmediated pathway might be involved [7]. In N. oceanica IMET1, the FAs for TAGs synthesis were partially cleaved from membrane lipids in response to N replete or deplete conditions [19]. The recycling of FAs from membrane lipids for TAGs accumulation likely constituted another common response to HL or N-limited stress [43]. Remarkably, the yield of carbohydrates was extremely high, up to approximately 28% of DCW in response to LL and ML, and remained at 16% of DCW in response to the HL conditions (Table 4). The carbohydrates primarily comprise trehalose (approximately 39%–46% of the carbohydrates and 7%–11% of DCW), mannitol, glucose and trace amounts of cellobiose. The N. oceanica CCMP1779 alcohol-insoluble

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Table 4 Quantification of the soluble cellular metabolites. Metabolites

Metabolite quantity (mean ± SD, mg g−1 DCW) HL

Amino acids Ile Val Ala GABA Pro Gln Tyr His Phe Trp

ML

LL

Differences

0.14 ± 0.00 0.30 ± 0.02 2.84 ± 0.35 1.64 ± 0.14 4.88 ± 0.51 0.81 ± 0.05 0.56 ± 0.04 0.43 ± 0.02 0.11 ± 0.00 0.28 ± 0.01

0.21 ± 0.01 0.87 ± 0.03 7.55 ± 0.12 4.74 ± 0.23 24.80 ± 0.36 2.10 ± 0.01 0.20 ± 0.01 0.12 ± 0.02 0.13 ± 0.01 0.11 ± 0.01

0.26 ± 0.01 0.91 ± 0.02 6.49 ± 0.12 4.55 ± 0.42 20.64 ± 0.44 3.55 ± 0.22 0.21 ± 0.02 0.12 ± 0.03 0.14 ± 0.01 0.11 ± 0.01

a, b, c a, b a, b, c a, b a, b, c a, b, c a, b a, b a, b a, b

72.54 ± 2.77 55.69 ± 4.90 29.39 ± 0.77 0.95 ± 0.04

107.69 ± 2.28 98.42 ± 2.02 67.67 ± 0.00 1.30 ± 0.06

107.25 ± 2.57 92.16 ± 2.07 73.26 ± 0.20 3.16 ± 0.05

a, b a, b, c a, b, c a, b, c

Organic acids/amine Succinate 1.17 ± 0.19 Fumarate 0.01 ± 0.00 Citrate 8.57 ± 0.50 Pyruvate 0.29 ± 0.02 Formate 0.05 ± 0.01 Lactate 0.82 ± 0.02

4.59 ± 0.27 0.01 ± 0.00 5.53 ± 0.13 0.95 ± 0.02 0.08 ± 0.01 0.36 ± 0.03

4.18 ± 0.34 0.01 ± 0.01 9.05 ± 0.64 0.98 ± 0.03 0.07 ± 0.01 0.36 ± 0.05

a, c a, b a, b a, b

Nucleotide derivatives Adenosine 0.07 ± 0.03 ADP 0.05 ± 0.02 uridine 0.16 ± 0.02 NMND 0.04 ± 0.01 0.02 ± 0.00 NAD+

0.07 ± 0.01 0.06 ± 0.00 0.14 ± 0.00 0.03 ± 0.00 0.10 ± 0.01

0.05 ± 0.01 0.06 ± 0.01 0.21 ± 0.00 0.07 ± 0.00 0.15 ± 0.02

a, b, c b, c a, b, c

Others Choline GB TMA DMA

0.60 ± 0.01 6.97 ± 0.09 0.12 ± 0.01 0.17 ± 0.00

1.56 ± 0.03 1.38 ± 0.04 0.12 ± 0.01 0.20 ± 0.00

a, b, c a, b, c a, b a, b, c

Carbohydrates Trehalose Mannitol Glucose Cellobiose

0.82 ± 0.03 4.22 ± 0.34 0.06 ± 0.01 0.10 ± 0.01

a, b

The “a”, “b” and “c” significant differences were derived from a one-way ANOVA analysis (p b 0.05) between the high- vs. medium-light intensity data, high- vs. low-light intensity data and medium- vs. low-light intensity data, respectively. Metabolites: Ile, isoleucine; Val, valine; Ala, alanine; GABA, gamma-amino-n-butyrate; Pro, proline; Gln, glutamine; Tyr, tyrosine; His, histidine; Phe, phenylalanine; Trp, tryptophan; ADP, adenosine-5-diphosphate; NMND, N-methylnicotinamide; NAD+, nicotinamide adenine dinucleotide; GB, glycinebetaine; TMA, trimethylamine; DMA, di-methyl-amine; HL, high light; ML, medium light; LL, low light.

DCW) might act as a stress protectant, a source of energy, or a reserve compound for spore germination in bacteria and lower eukaryotes [1]. In N. oceanica IMET1, similar trehalose contents (7% to 11% of DCW) might have similar functions. Further studies of the function of trehalose at HL conditions are required to verify this hypothesis. 3.4. Photosynthetic pathway and acclimation The yields of biomass and TAGs in N. oceanica IMET1 were simultaneously maximized under the HL condition. Therefore, identifying how cells acclimate to the different light intensities is extremely important. The yield of biomass (Table 1) obtained under the HL condition was the highest compared with the other conditions, suggesting that CO2 fixation was enhanced under the HL condition. The higher CO2 fixation rate under the HL condition also resulted in a higher ratio of C/N (18.94, Table 5) detected through the elemental analysis of biomass. This result was consistent with a previous report showing that CO2 fixation via photosynthesis was positively correlated with the carbon content and negatively correlated with the nitrogen content [28]. The higher heating value (HHV, gross calorific value), i.e., enthalpy of the complete combustion of fuels, is used to evaluate the properties of fuels and can be calculated based on the elemental composition of the biomass [8]. According to the elemental analysis of N. oceanica IMET1 biomass, the highest HHV of biomass was obtained for the HL condition, demonstrating that the FAs composition of TAGs obtained under the HL condition was more suitable for biodiesel. Both C3 and C4-like CO2 concentrating mechanisms (CCMs) have previously been reported in diatoms and Nannochloropsis [29–31,39]. Recently, the CCMs of N. oceanica IMET1 were studied based on the whole genome, in which all of the C4-like pathway genes (e.g., phosphoenolpyruvate carboxylase, PEPC; pyruvate phosphate dikinase, PPDK; malic enzyme, ME; malate dehydrogenase, MDH), carbonic anhydrase (CA) and inorganic carbon transporters were identified. The findings implied that a C4-like pathway was likely [19]. Moreover, the C4-like pathway might likely be involved in replenishing the TCA cycle intermediates for lipid biosynthesis instead of carbon fixation in N. oceanica IMET1 [19]. Based on the above reports, the coexistence of C3 and C4 photosynthetic pathways in N. oceanica IMET1 is highly likely. Thus, the effective combination of the C3 and C4 photosynthetic pathways might be a key point to obtain a high production of biomass and lipids in N. oceanica IMET1. 4. Conclusion

residue mainly consists of glucose and other 6 neutral monosaccharides of mannose, followed by trace amounts of rhamnose, fucose, arabinose, xylose and galactose [39]. N. oceanica CCALA 804 reportedly contains mannitol, followed by glucose and low amounts of trehalose and other 8 monosaccharides according to a recent report [26]. The different composition and concentration of carbohydrates might reflect the speciesspecific properties of N. oceanica under different culture conditions. The different composition of carbohydrates constitutes the first special metabolic characteristic of N. oceanica IMET1 at HL and N-replete conditions. Many metabolites of algae, which primarily function as osmoprotectants for algae cells against extracellular stresses, are multifunctional and remain poorly understood. Trehalose, a multifunctional molecule composing two glucose residues, is much more than a simple storage compound [6] and is now considered as a potential feedstock for bioethanol production [3]. Trehalose has been identified in a wide number of organisms, and the concentration of this molecule was only considerably lower in algae [39]. Interestingly, the level of trehalose in N. oceanica IMET1 is high, between 7% and 11% of DCW (Table 4). To the best of our knowledge, this report is the first to show that Nannochloropsis accumulates a high concentration of trehalose up to 11% of DCW, another special metabolic characteristic. Trehalose (10% of

Biomass and TAG simultaneously accumulated in N. oceanica IMET1 in response to HL in the light saturation region and a sufficient N supply conditions. Moreover, the FAs of TAGs in response to the HL condition were more suitable for biodiesel properties. The distribution of fatty acids and intracellular soluble metabolism (particularly carbohydrates), the membrane lipid composition and the photosynthetic pathway were associated with the concurrent accumulation of biomass, TAGs and valuable FAs (AA and EPA) in N. oceanica IMET1. Authors' contributions YX, YF, and QC designed the study; YX, JZ, JC, XY, and ZS performed the experiments; YX, JZ and JC analyzed the data; and YX, YF, and QC drafted the manuscript. All authors read and approved the final manuscript. Acknowledgments The authors would like to thank Dr. Jian Xu and Jing Li for providing the microalgae Nannochloropsis oceanica IMET1. This work was financially supported through grants from the “135” Projects Fund of the CAS-QIBEBT Director Innovation Foundation, the National High-tech R

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Table 5 Element analysis and quantification of the pigments. Condition

C (% of DCW)

H (% of DCW)

N (% of DCW)

C/N

HHVa (MJ kg−1)

HL ML LL

60.43 ± 0.05 55.25 ± 0.01 53.51 ± 0.06

8.91 ± 0.16 7.95 ± 0.01 7.67 ± 0.01

3.19 ± 0.01 6.20 ± 0.05 7.57 ± 0.00

18.94 ± 0.08 8.94 ± 0.03 7.05 ± 0.00

27.66 ± 0.10 24.19 ± 0.01 23.25 ± 0.02

HL, high light; ML, medium light; LL, low light; DCW, dry cell weight; C, carbon; H, hydrogen; N, nitrogen; HHV, higher heating value. a Heating value was calculated using the following formula [8] as HHV (MJ kg−1) = (3.55 C2 − 232 C − 2230 H + 51.2 C × H + 131 N + 20,600) × 103.

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