Visualising the Emerging Platform of Using Microalgae as a Sustainable Bio-Factory for Healthy Lipid Production through Biocompatible AIE Probes

Nowadays, a particular focus is using microalgae to get high-valued health beneficiary lipids. The precise localisation of the lipid droplets (LDs) and biochemical changes are crucial to portray the lipid production strategy in algae, but it requires an in vivo tool to rapidly visualise LD distribution. As a novel strategy, this study focuses on detecting lipid bioaccumulation in a green microalga, Chlamydomonas reinhardtii using the aggregation-induced emission (AIE) based probe, 2-DPAN (C24H18N2O). As the messenger molecule and stress biomarker, hydrogen peroxide (H2O2) activity was detected in lipid synthesis with the AIE probe, TPE-BO (C38H42B2O4). Distinctive LDs labelled with 2-DPAN have elucidated the lipid inducing conditions, where more health beneficiary α-linolenic acid has been produced. TPE-BO labelled H2O2 have clarified the involvement of H2O2 during lipid biogenesis. The co-staining procedure with traditional green BODIPY dye and red chlorophyll indicates that 2-DPAN is suitable for multicolour LD imaging. Compared with BODIPY, 2-DPAN was an efficient sample preparation technique without the washing procedure. Thus, 2-DPAN could improve traditional fluorescent probes currently used for lipid imaging. In addition, the rapid, wash-free, multicolour AIE-based in vivo probe in the study of LDs with 2-DPAN could advance the research of lipid production in microalgae.


Introduction
Lipid droplets (LDs) are well-connected subcellular organelles synthesised through complex pathways in a wide range of organisms. They are involved in vital biological functions, such as cell membrane protection, signalling molecules, cell division, insulation and energy storage. The majority of the lipid compounds have fatty acids (FA) structures, the source of which is either de novo synthesis or exogenous sources [1]. Deregulation in the lipid homeostasis can lead to different pathophysiological conditions in organisms, while specific FAs, such as polyunsaturated fatty acids (PUFAs), are health beneficiaries for higher vertebrates [2]. Therefore, expanding knowledge on lipidomics is a major target for revolution in medical research and system biology [3]. In humans, PUFAs are the precursors of eicosanoids that play pivotal roles in preventing chronic diseases, such as cancers [4] and diabetes [5], through modulating the pro-and anti-inflammatory activities [6]. Longchain, omega-3 fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been linked to the recovery of asthma, cognition, inflammatory bowel disease and brain development [7,8]. Due to the lack of desaturase enzymes, a human cannot In this study, as a model species of microalgae containing carbohydrate cell walls, we used Chlamydomonas reinhardtii to identify the lipid inducing conditions with lipid-specific AIEgen, 2-DPAN. We evaluated the effects of light, nutrient starvation, supplemented carbon source on lipid biosynthesis in this microalga. A new H 2 O 2 detection technique in the lipid-induced C. reinhardtii cells was also established with H 2 O 2 -specific AIEgen, TPE-BO. Furthermore, the effect of direct H 2 O 2 supplementation on lipid production was analysed. More importantly, we have developed a novel method to visualise LDs and H 2 O 2 in a complex carbohydrate cell wall containing microalga using the lipid and H 2 O 2 -specific AIEgen to understand the LD enriching mechanism in different environments.

Study of the Fluorescence Properties of Chlamydomonas reinhardtii and AIEgens
Different natural biomolecules in photosynthetic organisms (e.g., different chlorophyll and accessory pigments) exhibit autofluorescence properties that must be determined to avoid interference during fluorescent staining. The autofluorescence properties of C. reinhardtii were detected at different excitation-emission wavelengths using a fluorescence spectrophotometer (Cary Eclipse, MY17180002, Agilent Technologies, CA, USA) ( Figure S1). AIEgen, 2-DPAN was obtained from the South China University of Technology, China [32]. The absorption and photoluminescence (PL) spectra of 2-DPAN in DMSO/water mixtures were determined using the fluorescence spectrophotometer with quartz cuvettes of 1 cm path length. Fluorescence spectra of 10 µM 2-DPAN in DMSO (Sigma-Aldrich, St. Louis, MO, USA) and 50-90% water fractions (fw) were measured at room temperature.

Determination of Algal Growth in Different Concentrations of AIEgens and H 2 O 2
To determine the effects of AIEgens on C. reinhardtii growth, cells at the density of 1.0 ± 0.015 (×10 4 ) cells / mL were introduced to different concentrations (10, 50 and 100 µM) of 2-DPAN and TPE-BO ( Figures S2a and S5). The experiment was conducted in an MBL medium and cultured under the previously described condition. The growth rate was determined by counting the cells with a haemocytometer at regular intervals for up to 6 days.

Determination of Hydrogen Peroxide Content
For the colorimetric measurement of H 2 O 2 content in C. reinhardtii, cells cultured in different nutrient altered conditions were harvested by centrifugation for 10 min at 10,000× g. The weight of 0.5 g of fresh algae was then homogenised in 5 mL of 0.1% w/v trichloroacetic acid (TCA) solution. The supernatant was then collected by centrifugation at 15,880× g for 10 min. An aliquot of 0.5 mL of the supernatant was then mixed with 0.5 mL phosphate buffer (10 mM, pH 7.0) and 1 mL potassium iodide (1 M). The absorbance of the solution was measured at 390 nm. The H 2 O 2 concentration (µmol H 2 O 2 /g fresh weight (FW)) in the sample was then determined using a calibration curve prepared with the known concentrations of H 2 O 2 [36].

Study of Algal Growth during Hydrogen Peroxide Supplementation
Prior to studying the effects of H 2 O 2 supplementation on lipid biosynthesis in C. reinhardtii, the algal growth at different concentrations of H 2 O 2 was determined. 1.0 ± 0.015 (×10 4 ) cells/mL were introduced to different concentrations (0 mM, 0.4 mM, 0.6 mM, 0.8 mM and 1.0 mM) of H 2 O 2 and the experiment was conducted in an MBL medium and cultured under the previously described condition. To determine the H 2 O 2 utilisation by algal cells, 1.0 ± 0.05 (×10 5 ) cells/mL were cultured in 0.4 and 0.6 mM H 2 O 2 supplemented with the MBL medium up to 40 h. H 2 O 2 concentration in the culture medium was determined colorimetrically as previously described. A control group without algal cells was set against each H 2 O 2 concentration treatment and the reduction rate of H 2 O 2 content at each time point in the presence of algae was normalised against the values of the control groups.

Sample Preparation for Fluorescent Staining of Lipid and H 2 O 2
A stock solution of 1.0 mM BODIPY™ 505/515 in dimethylsulfoxide (DMSO) (Thermo Fisher Scientific Inc., Australia) was prepared and stored at -20 • C in the dark. The stock solution was diluted to 10 µM in DI water and the final concentration of DMSO was adjusted to 0.1% in each sample. Fluorometric quantification of the lipids was done according to the modified method of Cooper et al. [37]. Briefly, the cultured algae were adjusted to 10 6 cells/mL and the cells were collected by centrifugation at 2000× g for 60 sec. Subsequently, the cells were resuspended in 200 µL DI water, gently mixed with 200 µL of the prepared 10 µM BODIPY™ 505/515 solution and incubated in the dark for 5 min. The cells were then washed three times with DI, followed by centrifugation at 2000× g for 60 sec and resuspended in water. The stained cells were then protected from light for further analysis.
To determine the fluorescence intensities of different concentration of 2-DPAN (5 µM, 10 µM, 20 µM and 30 µM), C. reinhardtii cells (10 6 ) of Treatment 4 was used. A 1.0 mM stock solution of 2-DPAN was prepared and was stored at 4 • C in the dark. Fluorescence intensities were determined using the fluorescence spectrophotometer with quartz cuvettes of 1 cm path length. Fluorometric quantification of lipids in C. reinhardtii cells with confocal microscopy was done by adjusting the cells at 10 6 cells/mL. Subsequently, 20 µM 2-DPAN was added, the samples were vortexed at 100 rpm for 30 sec and then stored in the dark for 30 min. The final concentration of the DMSO was adjusted to 0.1% for all the studies. The wash free performance of BODIPY and 2-DPAN was determined by analysing the samples before and after the washing process (wash with DI three times).  [33]. To determine the H 2 O 2 activity in C. reinhardtii, 1 mM stock solution of probe TPE-BO was prepared in DMSO and was stored at 4 • C in the dark. Algal cells were consequently adjusted to 10 6 cells/mL and were incubated with 100 µM TPE-BO for 15 min. The final concentration of DMSO was adjusted to 0.1%.

Imaging of Chlamydomonas reinhardtii with Confocal Microscope
The C. reinhardtii cells were imaged under a Zeiss LSM 880 Airyscan confocal microscope using ZEN 2.6 software (Carl Zeiss, Australia). The excitation wavelength for BODIPY™ 505/515 and 2-DPAN was 488 nm and the emission wavelengths for BODIPY™ 505/515 and 2-DPAN were 490-517 nm and 526-570 nm, respectively. Excitation and emission wavelength for TPE-BO were set at 405 nm and 428-499 nm, respectively. Autofluorescence of the chlorophyll was detected with the excitation wavelength at 488 nm and emission wavelength of 685-758 nm.

Flow Cytometric Analysis of Lipid Content
Cytometric analysis was done using a flow cytometer (model CytoFLEX Flow Cytometer, Beckman Coulter, Inc. USA). Microalgae from different treatments were taken in three replicates and were stained with BODIPY™ 505/515 (5.0 µM) and 2-DPAN (20 µM) according to the previously described methods. FITC (488 nm laser) and KO525-A (405 nm laser) channels were used to detect the fluorescence from BODIPY™ 505/515 and 2-DPAN, respectively. The mean fluorescence intensity from a minimum of 10,000 cells per sample was acquired. Relative fluorescence was determined using the population cell percentile, with cells cultured in the MBL medium serving as the control.

Lipid Extraction and Analysis of Fatty Acids
The total lipid was extracted according to the adapted method of Bligh and Dyer [38]. Briefly, a sample volume was taken from each growing C. reinhardtii culture to provide approximately 500 mg of dry algal biomass. The supernatant was discarded after centrifugation at 10,000× g and 4 • C for 10 min. The collected pellet was then washed three times with an equal volume of potassium phosphate buffer (pH: 7.2). After that, the pellet was resuspended in DI water and subsequently transferred and weighed in a weighing dish. The samples were dried for 48 h at 60 • C and stored at −20 • C for further analysis. An approximate 50 mg of dried algal biomass was then taken into a pre-washed (with hexane) mortar. Using a Pasteur pipette, the weighing dish was entirely washed with 1 mL of hexane to transfer the samples completely into the mortar. The algal biomass was then grounded into a fine, smooth paste using a pestle. During the grinding process, hexane was added to replenish the evaporated hexane and the resulting slurry was mixed with the pestle until complete homogenisation. All the process was done under the fume hood. After that, the homogenised hexane-cell mass mixture was centrifuged at 10,000× g (4 • C, 20 min) and the supernatant was collected in a pre-weighed vial. The pellet was then resuspended with hexane (3 mL) by vigorous vortexing for 1 min and centrifuged at 10,000× g (4 • C, 30 min) to ensure the removal of all the cell debris. After complete evaporation of the hexane, the extracted oil mass was then determined gravimetrically. All the lipid extracts were stored at −20 • C for further analysis. Quantitative analysis of fatty acids was done with a Perkin Elmer GC-MS (Clarus 500 and 560S) using internal standards. Crude lipids were extracted from the sample using solvents. The lipids were transmethylated and the profiles of different fatty acids were obtained with gas chromatography. Results were expressed as % of fatty acid methyl esters [39].

Data Analysis
Data from the confocal microscopy were analysed with ImageJ 1.52a [40]. Briefly, the raw images were exported in "tiff" format in ImageJ. Using "Sliding paraboloid", the Biosensors 2022, 12, 208 6 of 24 background was subtracted from each image and the fluorescence channel was calculated. Subsequently, "Area", "Mean grey value", "Integrated density" and "Area fraction" were determined. Fluorescence intensity per cell was then calculated from the total cells by subtracting the background IntDen value from the IntDen value.
The flow cytometry data were analysed using CytExpert v2.4 software and presented as histogram overlays. To detect BODIPY™ 505/515 labelled cells, samples were gated on FITC-A vs. SSC-A. For 2-DPAN, samples were gated on KO525-A vs. SSC-A. Confocal microscopy and flow cytometry data were exported to Excel 2010 to prepare the graphs. The means and medians of different treatments were compared by the relative fluorescence intensity of the gated population, with setting cells from the MBL medium as the control group. SPSS statistics software (version 23) calculated the differences between control and other treatments at the p < 0.05 level through the paired sample t-test.

Effects of Nutrient and Light Manipulation on Algal Growth
Except the MCM medium in continuous light, a slower growth rate was found in all treatments ( Figure 1). After 8 days of continuous culture, maximum growth of 1.14 ± 0.38 (10 6 ) cells/mL was observed in Treatment 1. Nitrogen starvation decreased the algal growth by 16.4% in Treatment 2 than in Treatment 1. A maximum reduction of 57.1% in growth was observed in nitrogen and calcium starved, but sodium acetate was supplemented in the dark condition of Treatment 5. Exposure to light under similar nutrient manipulation increased the algal growth by 26.5% in Treatment 4, while the presence of calcium in nitrogen starved but sodium acetate supplemented culture medium showed the same growth trend in Treatment 3. Data from the confocal microscopy were analysed with ImageJ 1.52a [40]. Briefly, the raw images were exported in "tiff" format in ImageJ. Using "Sliding paraboloid", the background was subtracted from each image and the fluorescence channel was calculated. Subsequently, "Area", "Mean grey value", "Integrated density" and "Area fraction" were determined. Fluorescence intensity per cell was then calculated from the total cells by subtracting the background IntDen value from the IntDen value.
The flow cytometry data were analysed using CytExpert v2.4 software and presented as histogram overlays. To detect BODIPY™ 505/515 labelled cells, samples were gated on FITC-A vs. SSC-A. For 2-DPAN, samples were gated on KO525-A vs. SSC-A. Confocal microscopy and flow cytometry data were exported to Excel 2010 to prepare the graphs. The means and medians of different treatments were compared by the relative fluorescence intensity of the gated population, with setting cells from the MBL medium as the control group. SPSS statistics software (version 23) calculated the differences between control and other treatments at the P < 0.05 level through the paired sample t-test.

Effects of Nutrient and Light Manipulation on Algal Growth
Except the MCM medium in continuous light, a slower growth rate was found in all treatments ( Figure 1). After 8 days of continuous culture, maximum growth of 1.14 ± 0.38 (10 6 ) cells/mL was observed in Treatment 1. Nitrogen starvation decreased the algal growth by 16.4% in Treatment 2 than in Treatment 1. A maximum reduction of 57.1% in growth was observed in nitrogen and calcium starved, but sodium acetate was supplemented in the dark condition of Treatment 5. Exposure to light under similar nutrient manipulation increased the algal growth by 26.5% in Treatment 4, while the presence of calcium in nitrogen starved but sodium acetate supplemented culture medium showed the same growth trend in Treatment 3.

Fluorescent Properties of 2-DPAN
The photoluminescence (PL) spectra of 2-DPAN (10 µM) in DMSO was analysed under 405 nm excitation. In the DMSO solution, 2-DPAN showed weak emissions. An absorption peak was observed at 380 nm in a 90% water fraction (Figure 2a). Increasing the water percentage from 50 to 90% also increased the PL intensity of 2-DPAN, which signified the AIE attributes of 2-DPAN ( Figure 2b). 2-DPAN was biocompatible compared to the control group and no significant difference was found in the growth pattern of algae even at a very high concentration of 100 µM of 2-DPAN exposure ( Figure S2a). Following incubation at different concentrations of 2-DPAN (5 µM, 10 µM, 20 µM and 30 µM), an increase in the fluorescence intensities was observed when cells were treated with 20 µM probes compared to that of the 5 µM and 10 µM probes. The differences between the fluorescence intensity of 20 µM and 30 µM were very insignificant ( Figure S2b). Therefore 20 µM 2-DPAN was used to label the LDs in this experiment. The fluorescence intensity was found to be slightly lower in 10 min incubation than in 30 min incubation ( Figure S2c). As no significant difference was observed between the 30 min and 60 min incubation, it was assumed that 2-DPAN could completely label lipid drops in C. reinhardtii cells within 30 min of the incubation period.

Fluorescent Properties of 2-DPAN
The photoluminescence (PL) spectra of 2-DPAN (10 µM) in DMSO was analysed under 405 nm excitation. In the DMSO solution, 2-DPAN showed weak emissions. An absorption peak was observed at 380 nm in a 90% water fraction (Figure 2a). Increasing the water percentage from 50 to 90% also increased the PL intensity of 2-DPAN, which signified the AIE attributes of 2-DPAN ( Figure 2b). 2-DPAN was biocompatible compared to the control group and no significant difference was found in the growth pattern of algae even at a very high concentration of 100 µM of 2-DPAN exposure ( Figure S2a). Following incubation at different concentrations of 2-DPAN (5 µM, 10 µM, 20 µM and 30 µM), an increase in the fluorescence intensities was observed when cells were treated with 20 µM probes compared to that of the 5 µM and 10 µM probes. The differences between the fluorescence intensity of 20 µM and 30 µM were very insignificant ( Figure S2b). Therefore 20 µM 2-DPAN was used to label the LDs in this experiment. The fluorescence intensity was found to be slightly lower in 10 min incubation than in 30 min incubation ( Figure  S2c). As no significant difference was observed between the 30 min and 60 min incubation, it was assumed that 2-DPAN could completely label lipid drops in C. reinhardtii cells within 30 min of the incubation period.

Flow Cytometric Analysis of Lipid Content
The plots from the flow cytometry indicated the efficient labelling of lipid drops in C. reinhardtii with BODIPY™ 505/515 and 2-DPAN (Figure 4a (Figure 4d,j), which was followed byTreatment 3 (N2 deprived, but sodium acetate (2.0 g/L) supplemented light condition) (Figure 4c,i). Moreover, greater side scatters in the nutrient-starved population indicated the obese phenotypes [42,43] caused by the lipid production and accumulation of probes within the LDs (Figure 4c,d,i,j), which was consistent with the higher relative lipid fluorescence per cell ( Figure 4m). As it was evident that the nutrient-starved dark condition of Treatment 5 (Figure 4e,k,m) could not induce lipid production in C. reinhardtii cells, this treatment was deduced from the later confocal analysis.

Flow Cytometric Analysis of Lipid Content
The plots from the flow cytometry indicated the efficient labelling of lipid drops in  (Figure 4d,j), which was followed byTreatment 3 (N 2 deprived, but sodium acetate (2.0 g/L) supplemented light condition) (Figure 4c,i). Moreover, greater side scatters in the nutrient-starved population indicated the obese phenotypes [42,43] caused by the lipid production and accumulation of probes within the LDs (Figure 4c,d,i,j), which was consistent with the higher relative lipid fluorescence per cell ( Figure 4m). As it was evident that the nutrient-starved dark condition of Treatment 5 (Figure 4e,k,m) could not induce lipid production in C. reinhardtii cells, this treatment was deduced from the later confocal analysis.

Confocal Analysis of the Stress-Induced Lipid Droplets in Chlamydomonas reinhardtii
Altering the physical and chemical parameters are well-known strategies for lipid induction in microalgae [16,44]. This experiment studied the effects of nitrogen and calcium deprivation, sodium acetate supplementation and light on lipid biosynthesis in C. reinhardtii. As the rapid and efficient scanning approach for identifying lipid inducing conditions, the lipid-specific AIE probe, 2-DPAN, was directly introduced to the growing media of different treatment groups ( Figure S3). Distinguished LDs from different treatments were clearly visible in the 2-DPAN labelled confocal images ( Figure 5). It was evident that Treatment 4 (MBL, (−) N 2 , (−) Ca 2+ , (+) sodium acetate (2.0 g/L)) showed the highest lipid accumulation ( Figure 5D,E), followed by those in Treatment 3 (MBL, (−) N 2 , (+) sodium acetate (2.0 g/L), (Figure 5C,E) and Treatment 2 (MBL, (−) N 2 ) ( Figure 5B,E). The lowest amount of lipid was produced in the nutrient-enriched condition of Treatment 1 (MBL medium) ( Figure 5A). Autofluorescence from the chlorophyll was lowest in Treatment 4 ( Figure 5D,E), where maximum lipid was produced ( Figure 5A,E).
The present study results suggest that 2-DPAN had reasonable lipid specificity and higher sensitivity and penetration abilities to this complex carbohydrate cell wall containing microalgae. It was also evident that without washing, the fluorescence intensity of the BODIPY dye reached outside the meaningful range of the spectrometer ( Figure S4a), whereas almost no difference in the PL intensities was observed between the wash and wash-free samples with 2-DPAN ( Figure S4b). Furthermore, confocal images of the wash free BODIPY samples also showed photobleaching of the samples (Figure S4c-f). Therefore, the photostable properties of AIE molecules, wash free and easy sample preparation techniques ( Figure S3) could make 2-DPAN superior to traditional BODIPY™ 505/515 dye for rapid visualisation and quick screening of lipid inducing conditions in microalgae.

Hydrogen Peroxide Content in the Chlamydomonas reinhardtii Cells
ROS was associated with the increased metabolite production in microalgae and high levels of H 2 O 2 inside the microalgal cells were observed in a stress condition [45]. We determined the amount of H 2 O 2 in lipid-induced C. reinhardtii cells. It was apparent that a maximum amount of 38.57 µM H 2 O 2 was produced in 1 g of fresh microalgal cells of Treatment 4, where the maximum lipid was produced. Compared to the 6.38 µM H 2 O 2 /g of fresh microalgal cells of Treatment 1, about a 3.2-fold and 3.8-fold rise in H 2 O 2 production was found in the cells of Treatment 2 and Treatment 3, respectively ( Figure 6). whereas almost no difference in the PL intensities was observed between the wash and wash-free samples with 2-DPAN ( Figure S4b). Furthermore, confocal images of the wash free BODIPY samples also showed photobleaching of the samples (Figure S4c-f). Therefore, the photostable properties of AIE molecules, wash free and easy sample preparation techniques ( Figure S3) could make 2-DPAN superior to traditional BODIPY™ 505/515 dye for rapid visualisation and quick screening of lipid inducing conditions in microalgae.

Hydrogen Peroxide Content in the Chlamydomonas reinhardtii Cells
ROS was associated with the increased metabolite production in microalgae and high levels of H2O2 inside the microalgal cells were observed in a stress condition [45]. We determined the amount of H2O2 in lipid-induced C. reinhardtii cells. It was apparent that a maximum amount of 38.57 µM H2O2 was produced in 1 g of fresh microalgal cells of Treatment 4, where the maximum lipid was produced. Compared to the 6.38 µM H2O2/g of fresh microalgal cells of Treatment 1, about a 3.2-fold and 3.8-fold rise in H2O2 production was found in the cells of Treatment 2 and Treatment 3, respectively ( Figure 6).

Confocal Analysis of H2O2 Activity in Nutrient-Starved Chlamydomonas reinhardtii Cells
We used an H2O2-specific AIE probe, TPE-BO, to check H2O2 in C. reinhardtii with different cell membrane structures from an animal cell model [33]. Additionally, labelling LDs with AIE probe 2-DPAN under the same assay condition enabled us to visualise the H2O2 activities in the cells during lipid induction. From the confocal images, H2O2 activities were detected in all microalgae cells, whereas more activities were evident in the lipid accumulated (orange arrow) and autophagic cells (white arrow) (Figure 7). It was apparent that with the increase of lipid production in the cells, H2O2 activities were increased. In comparison to Treatment 1 (Figure 7d,u), a 2.5-and 3.5-fold rise in the H2O2 production was observed in Treatment 2 (Figure 7i,u) and Treatment 3 (Figure 7n,u), respectively.

Confocal Analysis of H 2 O 2 Activity in Nutrient-Starved Chlamydomonas reinhardtii Cells
We used an H 2 O 2 -specific AIE probe, TPE-BO, to check H 2 O 2 in C. reinhardtii with different cell membrane structures from an animal cell model [33]. Additionally, labelling LDs with AIE probe 2-DPAN under the same assay condition enabled us to visualise the H 2 O 2 activities in the cells during lipid induction. From the confocal images, H 2 O 2 activities were detected in all microalgae cells, whereas more activities were evident in the lipid accumulated (orange arrow) and autophagic cells (white arrow) (Figure 7). It was apparent that with the increase of lipid production in the cells, H 2 O 2 activities were increased. In comparison to Treatment 1 (Figure 7d,u), a 2.5-and 3.5-fold rise in the H 2 O 2 production was observed in Treatment 2 (Figure 7i,u) and Treatment 3 (Figure 7n,u), respectively. Maximum 3.8-fold higher H 2 O 2 was found in Treatment 4, where the maximum lipid was biosynthesised (Figure 7s,u).

Effects of H 2 O 2 Supplementation on Chlamydomonas reinhardtii
Our analysis showed a clear relation to lipid biosynthesis and intracellular H 2 O 2 accumulation under nutrient-depleted conditions. As H 2 O 2 can act as a messenger molecule [26], many complex biological systems have been linked to control the surge of H 2 O 2 [25,27]. In this study, we studied the effects of direct supplementation of H 2 O 2 on the growth and lipid accumulation in C. reinhardtii. It was apparent that the concentration of H 2 O 2 is very critical for cells as almost no change in algal growth was observed until 0.6 mM of H 2 O 2 exposure, while a slight increase in H 2 O 2 of 0.2 mM from 0.6 mM inhibited the cell growth by 60% ( Figure S6a). Cells did not survive above 1.0 mM H 2 O 2 (data are not presented). While monitoring the cells exposed to 0.4 mM ( Figure S6b) and 0.6 mM ( Figure  S6c) H 2 O 2 , it was obvious that the utilisation of H 2 O 2 also increased with cell growth, showing a declining trend during the exponential growth period.

Effects of H 2 O 2 Supplementation on Lipid Bioaccumulation
In comparison to the control (0.0 mM H 2 O 2 ) group ( Figure S7a (Figure 8m). Interestingly, despite a significantly higher amount of lipid accumulation (p < 0.01), there was not any noticeable change in the chlorophyll autofluorescence among the groups (Figure 8m).

Fatty Acid Analysis
Altering the cultural conditions significantly increased the total fatty acid (TFA) content in the C. reinhardtii cells. Among the nutrient altered conditions, the maximum amount of TFA (~11% of DW) was found in the cells of Treatment 3 (MBL, (−) N 2 , (−) Ca 2 + ) and Treatment 4 (MBL, (−) N 2 , (−) Ca 2+ , (+) sodium acetate (2.0 g/L)) that was followed by the 8.4% TFA content of Treatment 2 (MBL, (−) N 2 ). However, supplementation of H 2 O 2 in the MBL medium almost increased TFA content by twofold, reaching~13% in Treatment 1 (MBL medium) ( Figure S8). Almost the same trend in the rise of PUFAs was observed in the lipid-induced C. reinhardtii cells ( Table 1). The PUFAs content was found to be the most at 55.

Discussion
Although the current levels of lipid research are extremely promising, many research questions are still pending [46]. Many aspects of lipid biosynthesis in microalgae differ among the species due to the evolutionary diversity and the complex regulatory pathways of lipid metabolism [47]. Even significant differences in lipid accumulation have been observed in the same species under the same assay conditions [48]. Therefore, deliberate identification of the lipid-inducing conditions, screening and selecting suitable candidates are the keys to making progress in microalgal lipid research. In this study, using AIE-based techniques, we have identified the lipid inducing conditions in a carbohydrates-based cell wall containing microalgae, C. reinhardtii. With lipid-specific AIE probe, 2-DPAN, we determined the impact of different nutrient starvation, carbon source and light on lipid bioaccumulation in this photosynthetic organism. Following the detection of the H 2 O 2 activities with the H 2 O 2 -specific AIE probe, TPE-BO in the nutrient-starved cells, we further investigated the effects of direct supplementation of H 2 O 2 on growth, lipid accumulation and fatty acid composition in this microalga.
The results showed the importance of the light, nitrogen and supplemented carbon sources for the growth and lipid biosynthesis in C. reinhardtii. Regarding the nutrientenriched condition, lipid production in the nutrient-starved cells significantly increased, whereas cell growth decreased. The dark condition could not induce lipid synthesis in the nutrient-starved cells (Figure 4). Nitrogen starvation increased lipid production. However, supplementation of sodium acetate as the primary carbon source in nitrogen starved cells showed a marked increase in lipid biosynthesis (Figures 4 and 5). This possibly happened due to the redirection of the metabolic pathway of acetate towards fatty acid biosynthesis instead of acting as the cellular building block precursors through the glyoxylate cycle and gluconeogenesis [49]. All the nitrogen starved conditions showed decreased chlorophyll content (Figures 5 and 7). To adjust the altered cellular metabolism and changed energy content of the cells during nutrient limitation, downregulation of the protein synthesis and photosynthetic apparatus might occur [49,50]. Reduction in the chlorophyll content was, therefore, associated with their utilisation as the internal nitrogen reserve and decreased light uptake rate to minimise excess ROS accumulation [49,51].
While excessive ROS could be lethal for the cells, a certain level can be the secondary messengers in various signal transduction mechanisms, including lipid biosynthesis [52,53]. Among the ROS, H 2 O 2 can easily pass across the phospholipid bilayer via the aquaporins [24] and, as a key signalling molecule, interact directly with receptor proteins and redox-sensitive transcriptional factors [54,55]. Correlation among H 2 O 2 -induced lipid accumulation, enhanced calcium (Ca 2+ )-ATPase and glutathione activity has been evident in microalgae from previous studies [44,56,57]. Higher lipid production in C. reinhardtii cells during N 2 and Ca 2+ starvation in this study was possible due to the modulation of H 2 O 2 and Ca 2+ pathways, which was also evident from the increased H 2 O 2 levels in the lipid-induced cells (Figures 6 and 7). H 2 O 2 perhaps triggered Ca 2+ signals and transferred the extracellular stimuli to the algal cells to regulate the response mechanisms further. At the point of Ca 2+ deficiency in the culture media, intracellular Ca 2+ stores perhaps maintained the cytosolic Ca 2+ levels [58]. During nutrient starvation, while the synthesis of the membrane components was reduced, photosynthesis continued; hence biosynthesis of the fatty acids was carried on [59]. Generally, photosynthesis generates ATP and NADPH for cell growth and proliferation. While the C. reinhardtii cells were starved by the nutrients, growth decreased and NADPH was utilised for FA synthesis [60,61].
To further analyse the effects of H 2 O 2 on the lipid accumulation and growth of C. reinhardtii, we supplemented the nutrient-enriched culture medium with different concentrations of H 2 O 2 . Although exposure of the algal cells to a higher concentration of exogenous H 2 O 2 (>0.6 mM) significantly decreased the growth ( Figure S6a), interestingly, the supplementation of 0.4 and 0.6 mM H 2 O 2 maximised the lipid bioaccumulation without affecting the algal growth and chlorophyll content (Figure 8 and Figure S6a). This indicates that the H 2 O 2 concentration is critical. Therefore, prior to application, there is a need to study the results in different species. Perhaps a specific concentration of H 2 O 2 (0.4 and 0.6 mM) supplementation induced the cytosolic Ca 2+ level in C. reinhardtii in this study and consequently alleviated the oxidative stress by triggering glutathione (GSH) activity, modulated the calmodulin and MAPK expression levels and resulted in increased lipid production [52,56,62]. Simultaneously, improved cellular signalling promoted cytokinesis by persuading the signal transduction cascades without affecting the nucleic acids content, genes expression, cell size and photosynthetic activity that accelerated the growth [63].
We also have reported a novel, rapid and wash-free strategy in vivo to visualise LDs and H 2 O 2 in C. reinhardtii using AIE-based fluorophores. With the increase in water fraction from 50% to 90% in DMSO, the PL intensity of 2-DPAN significantly increased ( Figure 2) due to the AIE attributes and the formation of the insoluble nanoaggregates of 2-DPAN in the aqueous media [64]. The successful labelling of LDs with AIEgen, 2-DPAN in C. reinhardtii cells was visualised from the confocal images, whereas complete co-localisation with the traditional BODIPY dye was also observed in this study ( Figure 3). Therefore, it appeared that penetration of the AIE fluorophore, 2-DPAN, was not hindered by this algal species' complex carbohydrate cell wall structure. The structure of the lipidspecific AIE probe with negatively charged oxygen atoms attached to the electron acceptor carbonyl group perhaps makes lipid-specific AIE probe hydrophobic [65]. Additionally, the cheap raw material benzophenone to synthesise the keto-salicylaldehyde hydrazine (KSA) derivative, 2-DPAN, is also hydrophobic to increase the hydrophobicity of this AIE probe [32,66]. Being uniquely constituted hydrophobic structure among different organelles, LDs allowed this nano-fluorophore to target and accumulate inside the algal cells strongly. Upon aggregation, KSA formed six-membered ring structures to restrict the intramolecular motion and increase the fluorescence. Additionally, the presence of KSA retained some hydrophilic ability to enhance the probe efficiency in the cells and successfully label the target molecules [32,67,68]. Furthermore, confocal images of the C. reinhardtii cells in this study revealed the superior performance of 2-DPAN to the BODIPY (Figure 3), which is possibly due to the presence of the 2-naphthalene group in 2-DPAN to enhance the luminous efficiency of this AIE probe [32]. We also used the AIE probe, TPE-BO, to rapidly detect H 2 O 2 in C. reinhardtii during nutrient manipulation. The simple structure with phenylboronic ester moiety enabled this probe to transform into the phenol group in the presence of H 2 O 2 [33]. This restricted the intramolecular rotation and turned on the fluorescence, allowing TPE-BO to detect H 2 O 2 rapidly in this study (Figure 7). Results of this study are also supported by a former study, where TPE-BO detected H 2 O 2 within seconds in living mice macrophage (RAW264.7) cells and TPE-BO was established as a very low toxic, photostable, highly-effective and very first AIEgen for in vivo H 2 O 2 research [33].
This study also demonstrated a relatively easy and rapid application method of AIEgen compared to the traditional dye, BODIPY ( Figure S3). Using 2-DPAN required fewer steps than the BODIPY, whereas no significant differences in the PL intensities have been observed between the wash and wash-free samples with 2-DPAN ( Figure S4b). Conversely, BODIPY dye showed unacceptable results and photobleaching in the washfree samples ( Figure S4a). Therefore, the dye acquisition techniques with the traditional probes, regardless of BODIPY and Nile Red, require utmost care. Furthermore, using lower concentrations of these commercial probes can cause self-decomposition, while slightly higher concentration or lower-level competencies during washing steps might result in marked background signals. Having the AIE properties, 2-DPAN is free from these limitations. Additionally, 2-DPAN was found biocompatible at much higher concentrations since no negative impact on the growth of C. reinhardtii was observed even after exposure to 100 µM of 2-DPAN ( Figure S2). Moreover, a previous study suggested that BODIPY could lose 90% of the signals at 2% laser power energy under confocal microscopy within 5 min of irradiation [66]. BODIPY can also unselectively label the mitochondria and nuclear membranes [69].
Besides, the non-fluorogenic nature of BODIPY can produce more background signals, while limited photostability with a slight stokes shift can cause self-absorption of this traditional dye [29]. Another study demonstrated that under 99% laser density, Nile Red could lose more than 95% signal after 20 min and 60 scans without affecting the fluorescent intensity of 2-DPAN [32]. Therefore, these studies indicated excellent photostability of 2-DPAN than the traditional fluorophores. It also appeared that 2-DPAN and TPE-BO did not interfere with the red channel of chlorophyll autofluorescence ( Figure S1) or the green channel of BODIPY dye. In addition, a wash-free, rapid and easy experimental procedure allowed in vivo detection of LDs and H 2 O 2 under the same assay condition. Thus, this study signifies the effectiveness of these AIEgens as multicolour visualising tools for the target molecules in microalgae.
Moreover, FAs can affect human health and well-being through their direct and indirect association with different cellular functions. The effects of saturated fatty acids (SAFAs), monounsaturated fatty acids (MUFAs) and PUFAs are distinct. Thus, the FAs classes influence various diseases, including cardiovascular disease, metabolic disorder, type 2 diabetes, inflammatory diseases and cancer [70,71]. In this study, the FA profile of C. reinhardtii consisted of a range of SAFAs, MUFAs and PUFAs, whereas palmitic acid, linoleic acid and α-linolenic acids were predominant ( Table 1). As Chlamydomonas and higher plants retained the same ancestry [72], the FA composition of Chlamydomonas is almost similar to that of higher plants like Arabidopsis thaliana [73]. However, cultural conditions can significantly affect FAs content and composition [74], which is also evident in this experiment as lipid-induced conditions of Treatment 3 (MBL, (−) N 2 , (−) Ca 2+ ), Treatment 4 (MBL, (−) N 2 , (−) Ca 2+ , (+) sodium acetate (2.0 g/L)), 0.4 and 0.6 mM H 2 O 2 supplemented MBL medium produced more ALA than the control group (MBL medium).
As an essential fatty acid, the beneficiary effects of ALA have been suggested by former cohort studies since it could have anti-inflammatory [75,76], anti-arrhythmic [77], anti-thrombotic [77,78] and neuroprotective effects [79] in humans. Other prospective studies suggested improved effects of ALA intake on fatal coronary heart disease (CHD) and gene programming for preventing metabolic diseases [80][81][82]. ALA is easier to burn by the body than palmitic, stearic, oleic, or linoleic acid and, therefore, is expected to induce fat utilisation as energy and reduce obesity [83]. In a study on adult males and females, ingestion of 2.6 g/day ALA reduced systolic and diastolic blood pressure without any negative impact on oxidation or blood coagulation [84]. Moreover, ALA can also be converted into eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in humans. EPA can be utilised to improve conditions like asthma and atopic diseases. As the building block precursors of prostaglandins, leukotrienes, thromboxanes and lipoxins, EPA also plays important roles in different bio-physiological processes [85,86]. DHA can support cell membrane and signalling, specifically in the brain and retina, allowing improved visual activities, immunity and cognitive functions in humans [87,88]. However, the conversion occurs to a limited extent, lower for DHA than EPA and is higher in women than men [89,90].

Conclusions
This study identified N 2 and Ca 2+ starvation; N 2 and Ca 2+ starvation, but sodium acetate (2.0 g/L) supplementation; 0.4 and 0.6 mM H 2 O 2 supplementation in MBL medium under 24 h light as the health beneficiary lipid-inducing conditions in C. reinhardtii. We also detected the H 2 O 2 activities in the cells and determined that a certain amount of H 2 O 2 supplementation could effectively induce the production of bio-functional components in microalgae. We have introduced a rapid, wash-free, multicolour imaging technique to visualise LDs and H 2 O 2 in C. reinhardtii with AIE-based bioprobes. These AIE probes are cheap, biocompatible and convenient for high contrast visualisation of LDs and H 2 O 2 in in situ conditions. Due to the AIE properties, 2-DPAN surpassed the performances of the traditional fluorophore, BODIPY. The AIE probes were able to efficiently localise the LDs and H 2 O 2 by penetrating the complex carbohydrate-based cell wall of C. reinhardtii. As reliable and easy bioimaging tools, these probes are more auspicious to identify the optimum lipid production conditions at early cultural stages in microalgae, which would be more cost-effective, timesaving and labour-efficient. In this study, we have also reported cultural conditions that can produce more health beneficiary α-linolenic acid, which is important for preventing life-threatening diseases in humans. This new technique will significantly ease microalgal lipid research. However, more studies focusing on other cell types, such as diatoms with silica cell walls, are recommended for further assurance of the reliability of these probes for all kinds of microalgae. Thus, it will broaden the horizon of these probes for biological research.

Data Availability Statement:
The data supporting the findings of this study are available from the corresponding author upon request.