Nitrogen Starvation Induced Oxidative Stress in an Oil-Producing Green Alga Chlorella sorokiniana C3

Microalgal lipid is one of the most promising feedstocks for biodiesel production. Chlorella appears to be a particularly good option, and nitrogen (N) starvation is an efficient environmental pressure used to increase lipid accumulation in Chlorella cells. The effects of N starvation of an oil-producing wild microalga, Chlorella sorokiniana C3, on lipid accumulation were investigated using thin layer chromatography (TLC), confocal laser scanning microscopy (CLSM) and flow cytometry (FCM). The results showed that N starvation resulted in lipid accumulation in C. sorokiniana C3 cells, oil droplet (OD) formation and significant lipid accumulation in cells were detected after 2 d and 8 d of N starvation, respectively. During OD formation, reduced photosynthetic rate, respiration rate and photochemistry efficiency accompanied by increased damage to PSII were observed, demonstrated by chlorophyll (Chl) fluorescence, 77K fluorescence and oxygen evolution tests. In the mean time the rate of cyclic electron transportation increased correspondingly to produce more ATP for triacylglycerols (TAGs) synthesis. And 0.5 d was found to be the turning point for the early stress response and acclimation of cells to N starvation. Increased level of membrane peroxidation was also observed during OD formation, and superoxide dismutase (SOD), peroxide dismutase (POD) and catalase (CAT) enzyme activity assays suggested impaired reactive oxygen species (ROS) scavenging ability. Significant neutral lipid accumulation was also observed by artificial oxidative stress induced by H2O2 treatment. These results suggested coupled neutral lipid accumulation and oxidative stress during N starvation in C. sorokiniana C3.


Introduction
Extensive utilization of fossil fuels has led to global climate change, environmental pollution, health problems and an energy crisis, associated with irreversible depletion of traditional sources of fossil fuels [1]. Many countries are thus turning their attention to the development of new, clean, and sustainable energy sources [2]. Biofuel is expected to play a crucial role in the global energy infrastructure in the future, which would bring several benefits such as foreign oil independence, carbon neutral processes, and profits to local farmers [3]. Biodiesel, one of the most commonly used biofuels, has attracted much attention in recent years and is recognized as an ideal renewable energy carrier, and thus also as a possible primary energy source [4]. A variety of biolipids can be used to produce biodiesel, and vegetable oils are a renewable and potentially inexhaustible source of energy with an energy content close to diesel fuel [5]. However, extensive use of vegetable oils may cause significant problems such as starvation in developing countries, and it is important that productive and cultivated land is used for food instead of fuel production. In recent years, renewed interest in producing biodiesel from microalgae has arisen, as microalgae can grow rapidly and convert solar energy into chemical energy via CO 2 fixation and are thus now considered as one of the most promising sources of oil for making biodiesel [6,7].
The green microalga Chlorella (Chlorophyta), which consists of about 10 species that can grow photoautotrophically, mixotrophically and heterotrophically with high biomass concentration, appears to be a particularly good option for biodiesel production [8]. The oil content in some species of Chlorella varies from about 14 to 63% of dry weight, and the fatty acid composition has been reported to range from C-14:0 to C-20:0 [9,10].
Lipid accumulation occurs within the microalgal cells, and varies with growth conditions. N limitation or N starvation is an efficient environmental pressure used to increase lipid accumulation [11]. The general principle is that when there is insufficient N for protein synthesis required for growth, excess carbon from photosynthesis is channeled into storage molecules such as triglyceride or starch [12]. It has been reported that the lipid content in Chlorella could be doubled or even tripled under N depletion conditions [13,14], and a linear relationship between the N source concentration and the lipid content was observed [15]. In addition to the increase in total lipid content in microalgal cells as a result of cultivation in N depleted media, it was found that changing from normal nutrient to N depleted media will gradually change the lipid composition from free fatty acid-rich lipid to mostly triglyceride-containing lipid [16]. Therefore, N limitation or N starvation could increase both lipid and triglyceride content in microalgal cells.
Defined as the third generation biofuel, algal feedstock has become one of the most promising resources for biodiesel production, due to the much higher photosynthetic efficiency to produce biomass, thus resulting in a much higher growth rate and productivity as compared to conventional crops [7,17]. However, the physiological changes, especially photosynthesis during lipid accumulation and OD formation in oil-producing microalgae under N starvation have not yet been fully elucidated. In this study, an oil-producing microalga isolated from the wild, Chlorella sorokiniana C3, was tested to investigate the effects of N starvation on lipid accumulation and photosynthetic parameters. The results showed that N starvation resulted in neutral lipid accumulation accompanied by increased damage to PSII, and an increased level of membrane peroxidation which occurred during OD formation.

Growth Conditions, N2 and Exogenous Oxidative Stress Treatment
The N-sufficient medium (N+) used was full-strength BG11 medium [18]. The N-deficient medium (N2) was BG11 without NaNO 3 . C. sorokiniana C3 in the exponential phase was inoculated into a 1 liter Erlenmeyer flask containing 500 ml BG11 medium at 20uC with continuous illumination of 70 mmol m 22 s 21 and continuously bubbled with filtered air, the initial OD 700 is 0.05. For N2 treatment, cells were harvested by centrifugation at 6,0006g for 3 min at 20uC when they reached the midlogarithmic growth phase (OD 700 approximately 0.8), and were then washed and resuspended in N2 medium to OD 700 0.3. For exogenous oxidative stress treatment, cells at midlogarithmic growth phase were harvested by centrifugation at 6,0006g for 3 min at 20uC and were then washed and resuspended to OD 700 0.3 with N+ medium including 1.5 mM H 2 O 2 as the oxidant.

TLC Analysis of Lipid
10 ml culture at OD 700 = 1 was harvested at 6,0006g for 3 min, and the cell pellet was washed with fresh medium and centrifuged again. The harvested cell pellet was resuspended in 400 ml of methanol:chloroform mixture (1:1, v/v). The mixture was shaken for 2 minutes followed by phase separation using 120 ml of 1 M potassium chloride in 0.2 M phosphoric acid. Then the mixture was centrifuged at 12, 0006g at room temperature for 5 min, and the chloroform phase was transferred to a glass tube and dried under nitrogen. The residue was resuspended in a volume of 20 ml chloroform to get the lipid extracts. TLC analysis of lipid extracts from whole cells was performed according to Reiser and Somerville [19] with some modifications. TAGs were separated by developing the plates in hexane-ethyl ether (7.5:2.5, v/v). Samples were visualized by exposure to iodine vapor for approximately 10 min. 3 ml of each samples extracted at different time points were used for TLC analysis. Glyceryl trioleate (3 ml, 10 mg ml 21 ) was used as a reference substance for TAGs, and the neutral lipid content of C. sorokiniana C3 was then determined accordingly by using ImageJ (ver1.41, NIH) [20] and calculated as a percentage of dry cell weight [21].

FCM Analysis
Samples stained with Bodipy 505/515 were analyzed on a board using a FACSAria flow cytometer (Becton Dickinson, San Jose, CA, USA) equipped with a laser emitting at 488 nm and an optical filter FL1 (530/30 nm). The collected data were analyzed using FlowJo software (Tree Star, San Carlos, CA, USA).

Pigments Quantification
100% methanol was used to extract the pigments, and the concentrations were determined spectrophotometrically and calculated using the formula developed by Lichtenthaler [

Photosynthetic Oxygen Evolution and Dark Respiration Rates
Rates of steady state photosynthetic oxygen evolution and respiration were measured using a Clark-type oxygen electrode (Oxylab 2, Hansatech, UK) at 20uC as described by Gao and Xu [24]. Cell suspensions (2 ml) were illuminated at a quantum flux density of 300 mmol m 22 s 21 .

Chl Fluorescence Analysis
Chl fluorescence was measured using the Dual-PAM-100 Chl fluorescence photosynthesis analyzer (Walz, Germany). Cells were fully dark-adapted for 15 min prior to initial (F 0 ) and maximum (F m ) fluorescence level measurements. The maximum and effective quantum yields of PSII electron transport were calculated as F v / F m = (F m 2F 0 )/F m and F v 9/F m 9 = (F m 92F 0 9)/F m 9, respectively, according to Genty et al. [25]; the proportion of closed PSII center, 12qL, a parameter estimating the fraction of closed PSII center or excitation pressure of PSII was based on a lake mode and was calculated as 12qL = 12[(F m 92F9)/(F m 92F 0 9)6(F 0 9/F9)] [26]; the non-regulated energy dissipation Y(NO) was calculated as Y(NO) = F/F m [26]. Transient increases in Chl fluorescence after turning off actinic light (AL) were monitored according to Shikanai et al. [27].

77K Fluorescence
Thylakoid membrane preparation and the 77K fluorescence emission spectra were performed as described previously [28] using a liquid nitrogen device attached to a PTI Fluorometer (QM-4CW, Photon Technology International Inc., South Brunswick, NJ, USA). Both the excitation and emission slit widths were 1 nm, and thylakoid membranes were adjusted to a chlorophyll concentration of 15 mg ml 21 . Excitation wavelengths of 435 nm and 480 nm were used to excite chlorophyll and carotenoids, respectively.

Lipid Peroxidation Assessment and ROS Scavenging Enzyme Activity Assays
Malondialdehyde (MDA) level, CAT, POD and SOD activities were measured according to Shi et al. [29]. MDA, CAT and SOD kits were purchased from the Beyotime Institute of Biotechnology, China. The POD kit was purchased from the Nanjing Bioengineering Institute, China.

Statistical Analyses
Each result shown is the mean of at least three biological replicates. Statistical analysis of the data was performed using the program SPSS-13 and significance was determined at 95% or 99% confidence limits.

N starvation Stimulates Lipid Accumulation in C. sorokiniana C3
To determine the regular patterns of lipid accumulation and OD formation in C. sorokiniana C3, the intracellular lipid levels at various times of N starvation were examined using TLC. During the first 2 days of treatment, cells resuspended in either N+ (

Cell Morphology was Affected during OD Formation
To visualize the four key stages of oil-droplet formation, C. sorokiniana C3 cells stained with Bodipy 505/515 were observed by CLSM. In accordance with the TLC results ( Fig. 1), no Bodipy 505/515 fluorescence (green) was detected at the Cs ( Fig. 2A, 0 d) and PDFs ( Fig. 2A, 0.5 d), a weak green fluorescence was first detected at the ODFs ( Fig. 2A, 2 d), the continuously enhanced green fluorescence signal was detected with time prolonging ( Fig. 2A, 3 d-8 d), and a strong green fluorescence signal was observed at the LDFs ( Fig. 2A, 8 d). However, only a weak green fluorescence was detected at 8 d in cells in N+ medium, which was much later (Fig. 2B, 8 d). In addition, Chl autofluorescence (red) intensities in cells in both N+ and N2 medium were almost identical, whereas Chl autofluorescence at the LDFs showed a significant heterogeneity (Fig. 2). Cell morphology observed by CLSM showed that cell size decreased during N starvation ( Fig. 2A). Cells at the Cs showed a variety of cell sizes, suggesting that cells were actively cycling. In contrast, cells under N starvation were relatively concentrated in size, suggested that N starvation inhibited the cell cycle, similar to prochlorophytes [30]. Although cell size was concentrated slightly after 8 days, cells in N+ medium have no significant variation in size (Fig. 2B).
To further characterize the lipid accumulation and cell morphology during N starvation, large numbers of C. sorokiniana C3 cells (.10000) at the four stages were analyzed using FCM. Fluorescence intensity of Bodipy 505/515 in cell populations at the PDFs, ODFs and LDFs increased 1.8-fold, 15.0-fold and 262.8fold, respectively, compared with the Cs, indicating a constant increase in cell neutral lipid content (Fig. 3F). In contrast, no significant difference of fluorescence intensity of Bodipy 505/515 could be observed in cells in N+ medium before 2 days followed a slight increase after 8 days (Fig. 3F9). In FCM, forward scatter (FSC) is normally assumed to be proportional to cell size or cell volume, because the signal intensity increases linearly with the square of the cell diameter to cross-sectional area [31]. At the Cs, FSC was distributed widely, whereas FSC distribution at other stages was more concentrated (Fig. 3A-E, G). The means of FSC at the Cs, PDFs, ODFs, and LDFs were 74.1 K, 72.9 K, 59.8 K, and 62.6 K respectively, which showed that cell volume tended to decrease after N starvation. In contrast, Latasa and Berdalet [32] reported that the cell size of Heterocapsa sp. increased under N starvation, whereas there was no clear differences in cell size in the marine diatom, Ditylum brightwellii, during N starvation [33]. The side scatter (SSC) of FCM has been shown to be affected by cell morphology, and especially by intracellular structures that are determined by chemical composition (e.g. starch content) [34]. A higher SSC intensity is usually obtained from cells with a higher level of cytoplasmic granularity [35]. An increase in SSC was detected, the means of which at the Cs, PDFs, ODFs, and LDFs were 50.7 K, 86.5 K, 99.9 K, and 122 K, respectively (Fig. 3A-E), indicating increased cell morphological complexity caused by damage due to N starvation stress. However, the changes of both FSC and SSC were minor in cells in N+ medium, and only small differences could be detected even after 8 days (Fig. 3A9-E9, G9). These results suggested that N starvation had an adverse effect on C. sorokiniana C3 cell survival and growth, and the alga accumulated and stored lipids in cells for recovery and re-growth under the appropriate conditions.

Photosynthetic Pigments Composition during OD Formation
Although lipid accumulation can be accelerated by N starvation, as a stressor, N starvation can also lead to depression of photosynthesis. To evaluate the variation in C. sorokiniana C3 photosynthesis during OD formation, pigment contents were determined spectrophotometrically. As shown in Table 1, Chl a, Chl b, Chl a+b and Car contents in cells in N+ medium all increased with time, and the variation in Chl a/Chl b and Car/ Chl a+b were minor. In contrast, Table 1 indicates that there were significant changes in pigment composition in cells in N2 medium during OD formation. Chl a, Chl a+b and Car contents in cells appeared to be similar, and all had a transient increase at the PDFs and then decreased, whereas Chl b content was inversely correlated to N starvation time. Chl a/Chl b increased up to the ODFs, which indicated reduced chloroplast photosynthetic phosphorylation activity [36] and acclimation of photosynthesis when first subjected to the stress condition.

Steady State Photosynthetic Rate and Dark Respiration Rate during OD Formation
To further investigate the variation in photosynthesis of C sorokiniana C3 during OD formation, the steady state photosynthetic rate and dark respiration rate of cells were examined. Compared with cells cultured in N+ medium, both steady state photosynthetic rate and dark respiration rate of cells under N starvation were reduced significantly (Fig. 4). As shown in Figure 4, photosynthetic oxygen evolution rate dropped sharply to 50.7% at the PDFs, and remained at the same level until the ODFs, then decreased to 23.6% at the LDFs, indicating that permanent damage to photosynthetic apparatus had occurred. In addition, an approximately linear decrease in dark respiration rate during OD formation was observed, and statistical analysis showed that the dark respiration rate had a significant inversely correlation with OD formation (correlation test, r = 20.978,0, P,0.05).

Chl Fluorescence Change Under N Starvation
To gain more insight into the photosynthetic activities of C sorokiniana C3 during OD formation, various parameters of PSII activity were determined using variable fluorescence measurements. In microalgae, nutrient stress is generally detected by a decrease in F v /F m [37], and the F v /F m value in cultures without stress has been found to be relatively constant, but decreases in cultures under nutrient stress. Figure 5A shows that the maximum photochemical efficiency of PSII (F v /F m ) declined linearly during OD formation (negative correlation by correlation test, r = 20.992,0, P,0.01). Although the effective quantum yields of PSII (F v 9/F m 9) declined with stagnation at the PDFs and ODFs (Fig. 5B), statistical analysis also showed a significant negative correlation between F v 9/F m 9 and OD formation (correlation test, r = 20.986,0, P,0.05). A decrease in both F v /F m and F v 9/F m 9 indicated increasing damage to PSII. 1-qL, a parameter which estimates the fraction of closed PSII center or excitation pressure of PSII [26], increased 20.76% at the PDFs, but decreased by 26.84% at the PDFs and subsequently at the ODFs, and then increased again at the LDFs (Fig. 5C), suggesting an immediate response, acclimation, and permanent damage to PSII at the PDFs, ODFs and LDFs, respectively, which corresponded with the pigments analysis and oxygen evolution test results, but appeared to be more sensitive. In contrast, an increase in damaging nonphotochemical quenching Y(NO), an indicator of the extent of damage to PSII [26], was observed, which was positively correlated to OD formation (correlation test, r = 0.992.0, P,0.01 ) (Fig. 5D). Different from the cells cultured in N2 medium, F v /F m , F v 9/F m 9, 1-qL and Y(NO) in cells cultured in N+ medium were relatively stable among all stages (Fig. 5).
The post-illumination transient increase in Chl fluorescence represents the NAD(P)H dehydrogenase (NDH)-dependent reduction in the plastoquinone pool in darkness and was considered to be a measurement of the cyclic electron flow [27]. A typical induction curve of Chl fluorescence in C. sorokiniana C3 is shown in Figure 6A, and the representative induction curves at different stages are shown in Figure 6B-E. As shown in the inserts of Figure 6A, the transient post-illumination increase in Chl fluorescence was undetectable at the Cs and PDFs, while a slight and a remarkable increase were detected at the ODFs and LDFs, respectively. All these results indicated increasing cyclic electron flow around PSI with prolonged N starvation, which supplies ATP to TAG synthesis when the rate of both photophosphorylation and respiratory oxidative phosphorylation dropped (Fig. 4).

77K Fluorescence Spectroscopy Under N Starvation
The impact of N starvation on excitation energy distribution and the stoichiometry of photosystems were also diagnostically investigated by 77K fluorescence spectroscopy. Thylakoid membranes were isolated from cells cultured in N2 medium at the four stages, and 77K fluorescence emission spectra were recorded in liquid nitrogen with excitation wavelengths at 435 nm (Fig. 7A) and 480 nm (Fig. 7B), to excite Chl and Car, respectively, and both showed very similar patterns.
The typical emission spectra of the Cs thylakoids showed a major peak at 723 nm (F723), which corresponded to PSI, and two smaller peaks at 687 nm (F687) and 697 nm (F697), which originated mainly from PSII. When normalized at 687 nm, both the amplitude and bandwidth of the PSI fluorescence (F723) increased simultaneously with treatment time (Fig. 7A and 7B), and a 3 nm blue shift (from 723 nm to 720 nm) was observed. No peak shifts were detected in PSII bands (F687 and F697), except for the PDFs (F687 blue shifted to 685 nm, F697 red shifted to 702 nm), furthermore, there was a dramatic increase in the amplitude of F697 at the PDFs. These exceptions at the PDFs corresponded with the pigments (Table 1) and Chl fluorescence (Fig. 5) results, suggesting that the PDFs could be the turning point for the early stress response and acclimation to N starvation. The FPSI/FPSII ratio generally correlates well with the relative content of PSI and PSII [38], interestingly, it was found that when excited at either 435 nm or 480 nm, the FPSI/FPSII ratios were significantly positively correlated to OD formation (correlation test, 435 nm, r = 0.971.0, P,0.05; 480 nm, r = 0.985.0, P,0.05, respectively). The increased FPSI during N starvation correspondence with the increased cyclic electron flow around PSI measured by chlorophyll fluorescence transient (Fig. 6).

Lipid Peroxidation and Activities of the ROS Scavenging Enzymes
In oxygenic photosynthetic organisms, most stress conditions could ultimately be described as oxidative stress [39]. Lipid peroxidation, the most commonly accepted indicator of oxidative stress, was estimated by measuring the contents of MDA in cells during OD formation. As shown in Figure 8A, although stagnation between 0.5 d and 2 d was detected, N starvation induced a significant increase in the MDA level in cells, indicating serious membrane system damage during OD formation caused by N starvation-induced oxidative stress.
During oxidative stress, ROS, including N O 2 2 , 1 O 2 and H 2 O 2 , with corresponding levels will be generated [40], and the scavenging enzymes including SOD, CAT, and POD will be induced or activated in cells [41]. Figure 8 shows that the relative activities of SOD increased during OD formation and tripled at the LDFs, i.e. 8 days after N starvation. The relative activities of CAT increased to 312.94% at the PDFs and dropped slightly to  241.16% at the LDFs. In contrast, the relative activities of POD declined first to 14.91% and 15.02% at the PDFs and ODFs, respectively, and then dramatically increased to 217.44% at the LDFs. Therefore, SOD, CAT and POD played important roles in removing ROS in C. sorokiniana C3 under N starvation at different stages.

Exogenous Oxidative Stress Improves Lipid Accumulation in C. sorokiniana C3
To further confirmed the possible connection between oxidative stress and neutral lipid accumulation, H 2 O 2 was used as the exogenous oxidant and neutral lipid accumulation in cells was detected by using CLSM with Bodipy 505/515. As shown in Figure 9, after 1 d treatment, neutral lipid was undetectable in cells under both N+ and N2 conditions, while a significant accumulation was observed under exogenous oxidative stress induced by H 2 O 2 , indicated that exogenous oxidative stress could be a more effective factor for neutral lipid induction in C. sorokiniana C3.

Discussion
Whether conditions are favorable or adverse, the intricate metabolic pathways of a cell are heavily influenced by its environment. In the case of microalgae, specialized cultivation can stimulate changes in metabolism [42]. It has been reported that N starvation enhances TAGs accumulation in many microalgal strains such as Neochloris oleoabundans, Chlorella vulgaris and Chlorella protothecoides [43][44][45]. The TLC profiles showed that the green alga C. sorokiniana C3 accumulated significant amounts of TAGs after N starvation (Fig. 1). In particular, it was found that in our algal cells, ODs were first detected after 2 days of N starvation (Fig. 1A, lane 7), and significant accumulation was observed after 8 days of treatment (Fig. 1A, Lane 8), which corresponded with our further tests by CLSM (Fig. 2) and FCM (Fig. 3). Thus, 0 d, 0-0.5 d, 0.5-2 d and 2-8 d after N starvation could be defined as the Cs, PDFs, ODFs, and LDFs, respectively, accordingly to the four key stages of lipid accumulation in C. sorokiniana C3.
In C. sorokiniana strains, various attempts to improve lipid production have been reported during the past few years. By using UV mutagenesis, four C. sorokiniana mutants contained elevated total lipid and TAGs content were selected [46]. In particular, it was found that culture condition was important for lipid accumulation and the studied on maximize lipid production by changing culture condition got more attention. For example, a study found that C/N ratios had a significant effect on cell lipid content in heterotrophic C. sorokiniana, and low C/N ratios favored high cell lipid content [21]. Wan et al. [47] revealed that a C. sorokiniana strain under heterotrophic condition could accumulate 56% (w/w) dry weight lipid content and much higher than photoautotrophic culture. Ngangkham et al. [48] also revealed that C. sorokiniana under mixotrophic conditions recorded more lipid productivity. In the present study, 18.7% neutral lipid content was detected in cells under N limitation, which was much lower than heterotrophic or mixotrophic culture cells mentioned above. However, as more cost in microalgae culture under heterotrophic or mixotrophic conditions, photoautotrophic culture for microalgae grow and lipid accumulation is economically more feasible. Thus, the study for improving neutral lipid accumulation in microalgae cells under photoautotrophic condition is critical. In addition, in our previous study the cell viability of C. sorokiniana C3 has not reduce significantly under N starvation as compared to N+ culture (data not published), so it was no significant influence on the overall lipid yield as well, which gives this strain an obvious applicable value as a test organism in studying neutral lipid accumulation mechanism under N starvation for improving biofuel production.
Higher ratios of Chl a/Chl b have been considered to decrease light collection in relation to the rate of PSII photochemistry [36], and Car could have an antioxidative role in protecting unsaturated lipids in oil bodies from peroxidation, and could participate in the screening and trapping of excessive light otherwise absorbed by the chloroplast [49]. In the present study, increased Chl a/Chl b and Car/Chl a+b ratios were found in C. sorokiniana C3 during N starvation, indicating that the light-harvesting complexes in C. sorokiniana C3 cells decreased and led to a decrease in the PSII photochemistry rate, which was further indicated by Chl fluorescence (Fig. 5) and 77K fluorescence (Fig. 7).
During OD formation under N starvation, the efficiency of PSII in C. sorokiniana C3 decreased initially, as the consequences of the reduced photosynthetic pigments, the increased proportion of closed PSII center, the reduced light collection capability and the intensified photo damage, all of which induced serious PSII damage, decreased the rate of photosynthesis leading to inhibition of oxygen evolution and respiration rate, and resulted in less ATP synthesis. In the mean time the accumulation of TAGs requires more ATP, thus the rate of cyclic electron flow around PSI increased significantly to compensate (Fig. 6). The up-regulation of PSI fluorescence peak resulted in a higher F 723 /F 687 ratio (Fig. 7), further proved that N starvation affected the stoichiometry of the photosystems, and thus the excited energy distribution between the two photosystems. The damage of N starvation to photosynthesis  in C. sorokiniana has also been reported in a study by Vona et al. [50], in which a marked loss of photosynthetic activity and the lower value of respiratory activity in cells after a 24 h of N starvation were exhibited.
Interestingly, the significant changes at the PDFs could be detected in many photosynthetic parameters. For example, Chl a, Chl a+b and Car contents all showed a transient increase at the PDFs and then decreased (Table 1), and 1-qL increased at the PDFs, but subsequently decreased at the ODFs (Fig. 5C), all suggested that the PDFs could be the turning point for the early stress response and acclimation to N starvation. At the initial stage of N starvation, C. sorokiniana C3 tried to acclimate to the stress by regulating photosynthesis after the immediate response, but failed with aggravated stress when the N starvation extended, and thus permanent damage occurred at the later stages. Therefore, the PDFs, ODFs and LDFs during OD formation corresponded to the immediate response, acclimation of C. sorokiniana C3 to N starvation stress, and permanent damage to cells, respectively.
The lipid peroxidation, a commonly accepted indicator of oxidative stress, was assayed by measuring the level of MDA. In the present study, it was found that MDA increased significantly in C. sorokiniana C3 cells with N starvation during OD formation, which indicated that oxidative stress was generated due to N starvation (Fig. 8). To mitigate and repair the damage caused by ROS, antioxidants including free radical scavenging nonenzymes of low molecular mass (e.g., b-carotene) and enzymes such as SOD, POD, and CAT usually accumulate in cells [51]. In the present study, SOD converted N O 2 2 into H 2 O 2 and O 2 at all four stages, and CAT scavenged H 2 O 2 effectively and kept the oxidation damage at a relatively lower level (Fig. 8A) at earlier stages (PDFs and ODFs), but failed at LDFs indicated by a 3 fold increase of the MDA level (Fig. 8A), although the activity of POD increased significantly to compromise. According to the changes of neutral lipid (Fig. 1, Fig. 2A and Fig. 3F) and MDA level (Fig. 8A) during N starvation, a linear relationship could be found between neutral lipid accumulation and the MDA level. These results suggested a possible connection between the N starvation induced oxidative stress and neutral lipid accumulation, which was further confirmed by the result that artificial oxidative stress (H 2 O 2 ) could significantly induce neutral lipid accumulation (Fig. 9). Moreover, the H 2 O 2 induced exogenous oxidative stress seemed like a more effective factor for neutral lipid induction in C. sorokiniana C3, 1 d induction by H 2 O 2 ( Fig. 9) resulted even more neutral lipid accumulated in cells than 5 to 6 d under N starvation ( Fig. 2A). Considering the time and oil content, exogenous oxidative stress supposed to be a more noteworthy method in the attempts to improve neutral lipid accumulation in microalgae.
In summary, N starvation resulted in neutral lipid accumulation in C. sorokiniana C3 cells, and OD formation and significant neutral lipid accumulation in cells occurred on 2 d and 8 d after N starvation, respectively. During OD formation, reduced photosynthetic rate, oxygen evolution, respiration rate and photochemistry efficiency accompanied by increased damage to PSII were observed. In the mean time the cyclic electronic flow increased to produce more ATP for TAGs synthesis, with increased proportion and energy distribution to PSI. The PDFs was considered to be the turning point for the early stress response and acclimation to N starvation, and the PDFs, ODFs and LDFs corresponded to the immediate response, acclimation of cells to stress and the permanent damage to cells during OD formation, respectively. Aroused oxidative stress indicated by increased level of membrane peroxidation was also observed during OD formation, together with the results that exogenous oxidant stress with H 2 O 2 induced significantly of the neutral lipid accumulation, which suggested a coupling between neutral lipid accumulation and oxidative stress during N starvation. As compared with N starvation, the more effective of neutral lipid induction in cells by exogenous oxidative stress may provide new insights into cost effectively microalgae lipid production.