Single-Cell Mass Spectrometry Analysis of Metabolites Facilitated by Cell Electro-Migration and Electroporation.

Single-cell metabolite analysis plays an important role in biological study. While mass spectrometry is a powerful tool for identification and quantitation of metabolites, the low absolute analyte amounts in single cell and difficulty in sampling represent significant challenges in single cell analysis. In this study, we developed an effective method with a simple sampling procedure for analyzing single cells. A single cell was driven to a capillary tip through electro-migration, followed by releasing the cell contents through electroporation, into a sealed small volume (∼1.5 pL) to prevent dilution. Subsequent mass spectrometry analysis was performed directly with nanoelectrospray ionization. This method was applied for analyzing a variety of cells and monitoring the metabolic changes in response to perturbed cell culturing conditions. This method opens a new avenue for easy and rapid analysis of single cells with high sensitivity.

A study into the species sensitivity of green algae towards imidazolium-based ionic liquids using flow cytometry.

The sensitivity of individual organisms towards toxic agents is an important indicator of environmental pollution. However, organism-specific quantification of sensitivity towards pollutants remains a challenge. In this study, we determined the sensitivity of Chlorella vulgaris (C. vulgaris) and Scenedesmus quadricauda (S. quadricauda) towards three ionic liquids (ILs), 1-alkyl-3-methyl-imidazolium chlorides [Cnmim][Cl] (n = 4,6,8). We kept all external parameters constant to identify the biotic parameters responsible for discrepancies in species sensitivity, and used flow cytometry to determine four conventional endpoints to characterise cell viability and cell vitality. Our results demonstrate that after exposure to the ILs, cell proliferation was inhibited in both species. At the same time, the cell size, complexity and membrane permeability of both algae also increased. However, while Chl a synthesis by S. quadricauda was inhibited, that of C. vulgaris was enhanced. S. quadricauda has evolved a metabolic defense that can counteract the decreased esterase activity that has been shown to occur in the presence of ILs. While it is likely that S. quadricauda was less sensitive than C. vulgaris to the ILs because of this metabolic defense, this alga may also exhibit better membrane resistance towards ILs.

Effect of algal surface area and species interactions in toxicity testing bioassays.

Single and multispecies algal bioassays were assessed using copper toxicity with three green algae (Scenedesmus subspicatus, Scenedesmus quadricauda and Ankistrodesmus angustus) and one blue-green algae species (Oscillatoria prolifera). Single and multispecies toxicity tests were conducted based on cell density as per standard toxicity testing, and on equivalent surface area. A higher copper sulfate toxicity was registered for O. prolifera, followed by S. subspicatus, S. quadricauda, and A. angustus in single-species toxicity tests based on cell density. Single species toxicity tests based on surface area showed increased copper toxicity with increasing algal surface area except for A. angustus. In multispecies control bioassays, the growth of A. angustus was inhibited in the presence of other species in surface area-based tests. As compared to single species bioassays, O. prolifera, and S. quadricauda showed a decreased sensitivity to copper sulfate in both cell density and surface area based multispecies tests. However, for the algae species with the smallest surface area, S. subspicatus, 96h-EC50 value decreased in multispecies bioassays based on surface area as compared to the single species test, while it increased in multispecies bioassays based on cell density. The difference in S. subspicatus sensitivity to copper between tests based on cell density and surface area supports the need to adopt multispecies toxicity testing based on surface area to avoid the confounding effect on copper toxicity of increased biomass for metal binding. 96h-EC50 values for all species combined in the multispecies test based on cell density and on surface area were significantly different from 96h-EC50 values obtained in single species bioassays. These results demonstrate that single-species bioassays may over- or underestimate metal toxicity in natural waters.

Combined Confocal Laser Scanning Microscopy Techniques for A Rapid Assessment of the Effect and Cell Viability of Scenedesmus sp. DE2009 Under Metal Stress.

Phototrophic microorganisms are the dominant populations in microbial mats, which play an important role in stabilizing sediments, such as happens in the Ebro Delta. These microorganisms are exposed to low metal concentrations over a long period of time. Distinct methods have been used to evaluate their toxic effect on the preservation of these ecosystems. Nevertheless, most of these techniques are difficult to apply in isolated phototrophs because (i) they usually form consortia with heterotrophic bacteria, (ii) are difficult to obtain in axenic cultures, and (iii) do not grow on solid media.In this study, and for the first time, a combination of fast, non-invasive, and in vivo Confocal Laser Scanning Microscopy (CLSM) techniques were applied in a consortium of Scenedesmus sp. DE2009 to analyze its physiological state and viability under metal stress conditions. Microalga was more resistant to Pb followed by Cr and Cu. However, in multimetal combinations, the presence of Cu negatively affected microalga growth. Additionally, the inhibitory concentration (IC) values were also calculated by CLSM pigment analysis. The result determines a higher degree of toxicity for Cu and Cr in comparison to Pb. The high sensitivity of these CLSM-methods to detect low concentrations allows consideration of Scenedesmus sp. DE2009 as a good bioindicator of metal pollution in natural environments.

A novel method to analyse in vivo the physiological state and cell viability of phototrophic microorganisms by confocal laser scanning microscopy using a dual laser.

Phototrophic microorganisms are very abundant in extreme environments, where are subjected to frequent and strong changes in environmental parameters. Nevertheless, little is known about the physiological effects of these changing environmental conditions on viability of these microorganisms, which are difficult to grow in solid media and have the tendency to form aggregates. For that reason, it is essential to develop methodologies that provide data in short time consuming, in vivo and with minimal manipulating the samples, in response to distinct stress conditions. In this paper, we present a novel method using Confocal Laser Scanning Microscopy and a Dual Laser (CLSM-DL) for determining the cell viability of phototrophic microorganisms without the need of either staining or additional use of image treating software. In order to differentiate viable and nonviable Scenedesmus sp. DE2009 cells, a sequential scan in two different channels was carried out from each same xyz optical section. On the one hand, photosynthetic pigments fluorescence signal (living cells) was recorded at the red channel (625- to 785-nm fluorescence emission) exciting the samples with a 561-nm laser diode, and an acousto-optic tunable filter (AOTF) of 20%. On the other hand, nonphotosynthetic autofluorescence signal (dead cells) was recorded at the green channel (500- to 585-nm fluorescence emission) using a 405-nm UV laser, an AOTF of 15%. Both types of fluorescence signatures were captured with a hybrid detector. The validation of the CLSM-DL method was performed with SYTOX green fluorochrome and electron microscopic techniques, and it was also applied for studying the response of distinct light intensities, salinity doses and exposure times on a consortium of Scenedesmus sp. DE2009.

Effect of subcellular distribution on nC60 uptake and transfer efficiency from Scenedesmus obliquus to Daphnia magna.

The potential uptake and trophic transfer ability of nanoparticles (NPs) in aquatic organisms have not been well understood yet. There has been an increasing awareness of the subcellular fate of NPs in organisms, but how the subcellular distribution of NPs subsequently affects the trophic transfer to predator remains to be answered. In the present study, the food chain from Scenedesmus obliquus to Daphnia magna was established to simulate the trophic transfer of fullerene aqueous suspension (nC60). The nC60 contaminated algae were separated into three fractions: cell wall (CW), cell organelle (CO), and cell membrane (CM) fractions, and we investigated the nC60 uptake amounts and trophic transfer efficiency to the predator through dietary exposure to algae or algal subcellular fractions. The nC60 distribution in CW fraction of S. obliquus was the highest, following by CO and CM fractions. nC60 uptake amounts in D. magna were found to be mainly relative to the NPs' distribution in CW fraction and daphnia uptake ability from CW fraction, whereas the nC60 trophic transfer efficiency (TE) were mainly in accordance with the transfer ability of NPs from the CO fraction. CW fed group possessed the highest uptake amount, followed by CO and CM fed groups, but the presence of humic acid (HA) significantly decreased the nC60 uptake from CW fed group. The CO fed groups acquired high TE values for nC60, while CM fed groups had low TE values. Moreover, even though CW fed group had a high TE value; it decreased significantly with the presence of HA. This study contributes to the understanding of fullerene NPs' dietary exposure to aquatic organisms, suggesting that NPs in different food forms are not necessarily equally trophically available to the predator.

The influence of ultrasound frequency and power, on the algal species Microcystis aeruginosa, Aphanizomenon flos-aquae, Scenedesmus subspicatus and Melosira sp.

We report on the effectiveness of sonication on controlling the growth of four problematic algal species which are morphologically different and from three algal divisions. Two cyanobacterial species Microcystis aeruginosa (unicellular) and Aphanizomenon flos-aquae (filamentous), one green alga Scenedesmus subspicatus (colonial) and lastly a diatom species Melosira sp. (filamentous) were subjected to ultrasound of selected low to high frequencies ranging from 20 to 1144 kHz. Microcystis aeruginosa and Scenedesmus subspicatus highest cell removal rates were 16 +/- 2% and 20 +/- 3% when treated with the same ultrasound frequency of 862 kHz but differing energy levels of 133 and 67 kWh m(-3), respectively. Aphanizomenon flos-aquae best removal rate was 99 +/- 1% after 862 kHz and 133 kWh m(-3) of energy, with Melosira sp. achieving its highest cell removal at 83% subsequent to ultrasound of 20 kHz and 19 kWh m(-3). Microcystis aeruginosa and Scenedesmus subspicatus are considered non-susceptible species to ultrasound treatment from a water treatment perspective due to their low cell removal rates; however, photosynthetic activity reduction of 65% for Microcystis aeruginosa does indicate the possible utilization of ultrasound to control bloom growth, rather than bloom elimination. Conversely, Aphanizomenon flos-aquae and Melosira sp. are deemed species highly susceptible to ultrasound. Morphological differences in shape (filamentous/non-filamentous) and cell wall structure (silica/peptidoglycan), and presence of gas vacuoles are probable reasons for these differing levels of susceptibility to ultrasound.

Development of a helical coagulation reactor for harvesting microalgae.

In this study, an innovative helical coagulation reactor (HCR) was developed for harvesting microalgae by sedimentation with polyaluminium chloride (PAC). The effects of construction and hydrodynamic characteristics on harvesting performance were investigated. Results showed that a higher harvesting efficiency, 96.37%, was achieved for the large and compact flocs generated by the HCR, and the settling rate of flocs was substantially influenced by the velocity gradient (G) and the Reynolds number (Re). When the Reynolds number closed to the transition between laminar and turbulent flow (4000), the flocs settled faster (20.51 m h-1), although settling slowed as the Reynolds number increased further because of ruptured flocs. The settling rate of flocs could be further improved to 23.27 m h-1 by a pulse flow field, mainly due to larger and more compact flocs forming in the plug pipe flow. Furthermore, a comparative investigation of a mechanically agitated vessel and the HCR with the same Camp number (Gt) showed that the HCR achieved higher settling rates and a shorter residence time than those with a mechanical agitator. The HCR provided a uniform dissipation of energy and high velocity gradient while avoiding electrical and mechanical energy consumption, suggesting this reactor is an efficient and economic option for microalgae harvesting.

Inductive and resonance effects of substituents adjust the microalgal biodegradation of toxical phenolic compounds.

The type and the position of the substituent in the phenolic ring, the bond dissociation energy and the exogenously supplied carbon source as well as the inductive and resonance effect phenomena of the substituents adjust the biodegradability of the phenolic compounds. The comparative biodegradation study of mono-nitrophenols (electron acceptors) and mono-methylphenols (electron donors) revealed that it is a completely photoregulated process. The closer the donor group (OH(-)) of the phenolic ring is to the acceptor group (NO(2)(-)), the higher the biodegradation values are (2-nitrophenol>3-nitrophenol>4-nitrophenol); the further the donor group (OH(-)) of the phenolic compound is from the second donor group (CH(3)(+)), the higher the biodegradation values are (2-methylphenol<3-methylphenol<4-methylphenol). However, there are compounds without a specific role of acceptor or donor such as mono-iodophenols, where a type of counteraction between the inductive and resonance effect determines the behavior of the substituent. This fact combined with the presence of the hydroxyl group in the phenolic ring gave the observed stabilization in the biodegradation results of mono-iodophenols (2-iodophenol approximately 3-iodophenol approximately 4-iodophenol).

Ammonia-nitrogen and orthophosphate removal by immobilized Scenedesmus sp. isolated from municipal wastewater for potential use in tertiary treatment.

Scendesmus sp. isolated from municipal wastewater, entrapped in calcium alginate as algal sheets was employed to remove inorganic nutrients (N and P) from artificial and real domestic secondary effluents in parallel-plate bioreactor after starvation. The key factors affecting the removal efficiency (NH4+-N and PO4(3-)-P), system stability and reuse efficiency of screens were studied and discussed. It has been shown that cell density in the mixture of algal gel was the key factor compared with the thickness of the gel and the cell density of the reactor. A complete removal of NH4+-N and PO4(3-)-P was achieved within 4h of treatment in parallel bioreactors with the optimal cell density in the mixture of algal (2 x 10(8) algae mL(-1)) and 3mm gel sheets after second cycle. Nine cycles of wastewater treatment in 21 days were accomplished, holding higher removal efficiency. NH(4)(+)-N removal efficiency was 99.1% after 105 min, 100% after 135 min, PO4(3-)-P removal efficiency was 100% after 15 min in domestic secondary effluents. Immobilized Scendesmus sp. is shown to have great potentialities for removal of inorganic nitrogen and phosphorus from treated effluents.

Toxic effect of serial perfluorosulfonic and perfluorocarboxylic acids on the membrane system of a freshwater alga measured by flow cytometry.

Flow cytometric measurements were used to investigate the toxic effect of perfluorobutanoic sulfonate (PFBS), perfluorooctane sulfonate (PFOS), perfluorohexanoic acid (PFHxA), perfluorooctanoic acid (PFOA), perfluorododecanoic acid (PFDoA), and perfluorotetradecanoic acid (PFTeA) on some membrane systems of the freshwater alga species Scenedesmus obliquus. Among the test compounds, PFOS, PFDoA, and PFTeA inhibited algal growth rate in a concentration-dependent manner while PFBS, PFHxA, and PFOA did not inhibit algal growth within the test concentration ranges. An enhancement of the mitochondrial membrane potential (MMP) and cell membrane permeability in S. obliquus was observed caused by exposure to PFOS, PFOA, PFDoA, and PFTeA. Both carbon chain length and acid group influenced the toxicity of PFAAs, where the toxicity increased with increasing carbon chain length for the compounds belonging to the same class. The observed effective concentrations lie in the micromole range and the test compounds disrupted membrane properties at concentrations below those associated with algal growth inhibition. Flow cytometry is proved to be a useful technique for toxicity testing with microalgae and provide additional information regarding the mode of action of PFAAs to algal species.

Effect of ionic liquids with different cations and anions on photosystem and cell structure of Scenedesmus obliquus.

The rapid increase in the production and practical application of ionic liquids (ILs) could pose potential threats to aquatic systems. In this study, we investigated the effects of four ILs with different cations and anions, including 1-hexyl-3-methylimidazolium nitrate ([HMIM]NO3), 1-hexyl-3-methylimidazolium chloride ([HMIM]Cl), N-hexyl-3-metylpyridinium chloride ([HMPy]Cl), and N-hexyl-3-metylpyridinium bromide ([HMPy]Br), on photosystem and cellular structure of Scenedesmus obliquus. The results indicated that ILs are phytotoxic to S. obliquus. The contents of chlorophyll a, chlorophyll b and total chlorophyll decreased with increasing ILs concentrations. The chlorophyll fluorescence parameters of photosynthetic system II (PSII), including minimal fluorescence yield (F0), potential efficiency of PSII (Fv/Fo), maximum quantum efficiency of PSII photochemistry (Fv/Fm), yield of photochemical quantum [Y(II)], and non-photochemical quenching coefficient without measuring F0' (NPQ), were all affected. This indicates that ILs could damage PSII, inhibit the primary reaction of photosynthesis, interdict the process of electron-transfer and lead to loss of heat-dissipating ability. ILs also increased cell membrane permeability of S. obliquus, influenced the cellular ultrastructure, changed the morphology of algae cells and destroyed the cell wall, cell membrane and organelles. The results indicated that imidazolium ILs had greater effect than pyridinium ILs, NO3--IL and Br--IL had greater effect than Cl--IL. To minimize threats to the environment, the structure of ILs should be taken into consideration.

Isolation and algae-lysing characteristics of the algicidal bacterium B5.

Water blooms have become a worldwide environmental problem. Recently, algicidal bacteria have attracted wide attention as possible agents for inhibiting algal water blooms. In this study, one strain of algicidal bacterium B5 was isolated from activated sludge. On the basis of analysis of its physiological characteristics and 16S rDNA gene sequence, it was identified as Bacillus fusiformis. Its algaelysing characteristics on Microcystis aeruginosa, Chlorella and Scenedesmus were tested. The results showed that: (1) the algicidal bacterium B5 is a Gram-negative bacterium. The 16S rDNA nucleotide sequence homology of strain B5 with 2 strains of B. fusiformis reached 99.86%, so B5 was identified as B. fusiformis; (2) the algal-lysing effects of the algicidal bacterium B5 on M. aeruginosa, Chlorella and Scenedesmus were pronounced. The initial bacterial and algal cell densities strongly influence the removal rates of chlorophyll-a. The greater the initial bacterial cell density, the faster the degradation of chlorophyll-a. The greater the initial algal cell density, the slower the degradation of chlorophyll-a. When the bacterial cell density was 3.6 x 10(7) cells/ml, nearly 90% of chlorophyll-a was removed. When the chlorophyll-a concentration was less than 550 microg/L, about 70% was removed; (3) the strain B5 lysed algae by secreting metabolites and these metabolites could bear heat treatment.

Long-term toxicity of ZnO nanoparticles to Scenedesmus rubescens cultivated in different media.

The aim of this work was to investigate the long-term toxic effect of zinc oxide (ZnO) nanoparticles (NPs) on freshwater microalgae, combined with the nutrient consumption in the culture. For this purpose, two common microalgae media (Blue-Green 11, BG-11, and Bold's Basal Medium, BBM) were used. Scenedesmus rubescens was used as freshwater microalgae model species and was exposed to ZnO NPs at different concentrations (0.081 to 810 mg/L) for a period up to 28 d. The experimental results revealed that microalgae growth was affected by the time of exposure and the NPs concentrations, but mainly the culture medium used. Differences in microalgae growth rates were observed and attributed to the selected culture medium. The toxic effect of ZnO NPs was higher on microalgae cultured in modified BG-11 compared to BBM, despite the fact that S. rubescens exhibited higher growth rate in modified BG-11 without the exposure of ZnO NPs.

The complete chloroplast genome sequence of the chlorophycean green alga Scenedesmus obliquus reveals a compact gene organization and a biased distribution of genes on the two DNA strands.

BACKGROUND: The phylum Chlorophyta contains the majority of the green algae and is divided into four classes. While the basal position of the Prasinophyceae is well established, the divergence order of the Ulvophyceae, Trebouxiophyceae and Chlorophyceae (UTC) remains uncertain. The five complete chloroplast DNA (cpDNA) sequences currently available for representatives of these classes display considerable variability in overall structure, gene content, gene density, intron content and gene order. Among these genomes, that of the chlorophycean green alga Chlamydomonas reinhardtii has retained the least ancestral features. The two single-copy regions, which are separated from one another by the large inverted repeat (IR), have similar sizes, rather than unequal sizes, and differ radically in both gene contents and gene organizations relative to the single-copy regions of prasinophyte and ulvophyte cpDNAs. To gain insights into the various changes that underwent the chloroplast genome during the evolution of chlorophycean green algae, we have sequenced the cpDNA of Scenedesmus obliquus, a member of a distinct chlorophycean lineage. RESULTS: The 161,452 bp IR-containing genome of Scenedesmus features single-copy regions of similar sizes, encodes 96 genes, i.e. only two additional genes (infA and rpl12) relative to its Chlamydomonas homologue and contains seven group I and two group II introns. It is clearly more compact than the four UTC algal cpDNAs that have been examined so far, displays the lowest proportion of short repeats among these algae and shows a stronger bias in clustering of genes on the same DNA strand compared to Chlamydomonas cpDNA. Like the latter genome, Scenedesmus cpDNA displays only a few ancestral gene clusters. The two chlorophycean genomes share 11 gene clusters that are not found in previously sequenced trebouxiophyte and ulvophyte cpDNAs as well as a few genes that have an unusual structure; however, their single-copy regions differ considerably in gene content. CONCLUSION: Our results underscore the remarkable plasticity of the chlorophycean chloroplast genome. Owing to this plasticity, only a sketchy portrait could be drawn for the chloroplast genome of the last common ancestor of Scenedesmus and Chlamydomonas.

Comparative study of zooid and non-zooid forming strains of Scenedesmus obliquus. Physiology and cytomorphology.

Two zooid forming strains and four non-zooid strains of the green chlorococcal alga Scenedesmus obliquus were compared in terms of growth, morphological and physiological characteristics. Large differences were observed among the strains grown under various growth conditions (light and temperature). The assumption that the zooid forming strains may be similar was not confirmed. Since they considerably differed in daughter cells morphology, photosynthesis, growth rate in batch culture or commitment to cellular division. Molecular-genetic comparison of 18S RNA/DNA might distinguish zooid forming strains from non-zooid ones.

Boosting TAG Accumulation with Improved Biodiesel Production from Novel Oleaginous Microalgae Scenedesmus sp. IITRIND2 Utilizing Waste Sugarcane Bagasse Aqueous Extract (SBAE).

This investigation utilized sugarcane bagasse aqueous extract (SBAE), a nontoxic, cost-effective medium to boost triacylglycerol (TAG) accumulation in novel fresh water microalgal isolate Scenedesmus sp. IITRIND2. Maximum lipid productivity of 112 ± 5.2 mg/L/day was recorded in microalgae grown in SBAE compared to modified BBM (26 ± 3 %). Carotenoid to chlorophyll ratio was 12.5 ± 2 % higher than in photoautotrophic control, indicating an increase in photosystem II activity, thereby increasing growth rate. Fatty acid methyl ester (FAME) profile revealed presence of C14:0 (2.29 %), C16:0 (15.99 %), C16:2 (4.05 %), C18:0 (3.41 %), C18:1 (41.55 %), C18:2 (12.41), and C20:0 (1.21 %) as the major fatty acids. Cetane number (64.03), cold filter plugging property (-1.05 °C), and oxidative stability (12.03 h) indicated quality biodiesel abiding by ASTM D6751 and EN 14214 fuel standards. Results consolidate the candidature of novel freshwater microalgal isolate Scenedesmus sp. IITRIND2 cultivated in SBAE, aqueous extract made from copious, agricultural waste sugarcane bagasse to increase the lipid productivity, and could further be utilized for cost-effective biodiesel production.

Points of commitment to reproductive events as a tool for analysis of the cell cycle in synchronous cultures of algae.

Methods for determining the points of commitment for cell division are described for species of green algae dividing by multiple fission, both forming coenobia (Scenedesmus quadricauda) and releasing single daughter cells (Chlamydomonas eugametos, Scenedesmus armatus). The timing of commitment points was followed in detail in synchronous cultures of S. quadricauda grown under various light intensities, illumination regimes, and temperatures. The pre-commitment periods were rate limiting, while the post-commitment periods remained more or less constant under various light intensity. Temperature, on the other hand, affected both periods in a similar manner and they were prolonged with decreasing temperature.

Microalgal cell disruption via ultrasonic nozzle spraying.

The objective of this study was to understand the effect of operating parameters, including ultrasound amplitude, spraying pressure, nozzle orifice diameter, and initial cell concentration on microalgal cell disruption and lipid extraction in an ultrasonic nozzle spraying system (UNSS). Two algal species including Scenedesmus dimorphus and Nannochloropsis oculata were evaluated. Experimental results demonstrated that the UNSS was effective in the disruption of microalgal cells indicated by significant changes in cell concentration and Nile red-stained lipid fluorescence density between all treatments and the control. It was found that increasing ultrasound amplitude generally enhanced cell disruption and lipid recovery although excessive input energy was not necessary for best results. The effect of spraying pressure and nozzle orifice diameter on cell disruption and lipid recovery was believed to be dependent on the competition between ultrasound-induced cavitation and spraying-generated shear forces. Optimal cell disruption was not always achieved at the highest spraying pressure or biggest nozzle orifice diameter; instead, they appeared at moderate levels depending on the algal strain and specific settings. Increasing initial algal cell concentration significantly reduced cell disruption efficiency. In all UNSS treatments, the effectiveness of cell disruption and lipid recovery was found to be dependent on the algal species treated.

Effects of nitrogen source availability and bioreactor operating strategies on lutein production with Scenedesmus obliquus FSP-3.

In this study, the effects of the type and concentration of nitrogen sources on the cell growth and lutein content of an isolated microalga Scenedesmus obliquus FSP-3 were investigated. With batch culture, the highest lutein content (4.61 mg/g) and lutein productivity (4.35 mg/L/day) were obtained when using 8.0 mM calcium nitrate as the nitrogen source. With this best nitrogen source condition, the microalgae cultivation was performed using two bioreactor strategies (namely, semi-continuous and two-stage operations) to further enhance the lutein content and productivity. Using semi-continuous operation with a 10% medium replacement ratio could obtain the highest biomass productivity (1304.8 mg/L/day) and lutein productivity (6.01 mg/L/day). This performance is better than most related studies.