Two-stage Cultivation Strategy Research Articles

Selenite as a Lipid Inductor in Marine Microalga Dunaliella tertiolecta: Comparison of One-Stage and Two-Stage Cultivation Strategies.

Microalgae have emerged as one of the most promising alternative sources of biofuels due to their high lipid accumulation ability. High lipid content is of pivotal importance for biodiesel production. In order to obtain high lipid content, modifications of culture conditions and development of an efficient lipid induction method are called for. In the present study, the possibility of using selenium in a form of sodium selenite as a lipid inductor in marine microalga Dunaliella tertiolecta was investigated during one- and two-stage cultivation modes. The effects of selenite on algal growth, pigment content, oxidative stress, and neutral lipid content were determined during both cultivation modes. The results revealed that the two-stage cultivation on 10.00-40.00mg L-1 of selenite resulted in up to twofold higher algal cell density compared to the one-stage cultivation. Selenite concentrations from 2.50 to 20.00mg L-1 increased lipid peroxidation during both cultivation modes, emphasizing the selenite-induced oxidative stress accompanied by the increased lipid accumulation in microalgae cells. During one- and two-stage cultivation on 20.00mg L-1 of selenite, lipid content increased 2.39- and 5.73-fold at days 9 and 14 of cultivation, respectively. Moreover, the highest obtained neutral lipid content during the two-stage cultivation was 5.40-fold higher than lipid content obtained during the one-stage cultivation. Collectively, these results suggest that the two-stage cultivation strategy, initiated with optimal culture conditions for biomass production and followed by the addition of selenite as a stress inductor, can be successfully deployed to enhance the lipid content in D. tertiolecta.

Enhancement of lipid productivity in Chlorella pyrenoidosa by collecting cells at the maximum cell number in a two-stage culture strategy

Two-stage culture strategies have been widely applied to improve biomass growth and lipid accumulation in microalgae. Most studies on two-stage culture strategies collect microalgal biomass at the maximum biomass concentration in the first stage and then transfer this biomass into the second stage for subsequent culture. However, there is no scientific conclusion regarding the appropriate time for microalgal biomass collection in the first stage. In this study, the biomass of Chlorella pyrenoidosa was collected at the maximum cell number and maximum biomass concentration in the first stage, and then transferred to the second stage for lipid accumulation. The results show that superior lipid productivity was achieved when using algal cells collected at the maximum cell number than those collected at the maximum biomass concentration. This was due to the strong cell proliferation ability of C. pyrenoidosa at the maximum cell number in the first stage. Moreover, the culture time of the first stage was shortened from 96 h to 48 h for autotrophic culture and 96 h to 60 h for heterotrophic culture. The results presented herein are of great significance for optimizing the current two-stage culture strategies.

Low Crystallinity of Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) Bioproduction by Hot Spring Cyanobacterium Cyanosarcina sp. AARL T020.

The poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) derived from cyanobacteria is an environmentally friendly biodegradable polymer. The low yield of PHBV’s production is the main hindrance to its sustainable production, and the manipulation of PHBV production processes could potentially overcome this obstacle. The present research investigated evolutionarily divergent cyanobacteria obtained from local environments of Thailand. Among the strains tested, Cyanosarcina sp. AARL T020, a hot spring cyanobacterium, showed a high rate of PHBV accumulation with a fascinating 3-hydroxyvalerate mole fraction. A two-stage cultivation strategy with sole organic carbon supplementation was successful in maximizing cyanobacterial PHBV production. The use of an optimized medium in the first stage of cultivation provided a 4.9-fold increase in biomass production. Subsequently, the addition of levulinic acid in the second stage of cultivation can induce significant biomass and PHBV production. With this strategy, the final biomass production and PHBV productivity were increased by 6.5 and 73.2 fold, respectively. The GC-MS, FTIR, and NMR analyses confirmed that the obtained PHBV consisted of two subunits of 3-hydroxyvaryrate and 3-hydroxybutyrate. Interestingly, the cyanobacterial PHBV contained a very high 3-hydroxyvalerate mole fraction (94%) exhibiting a low degree of crystallinity and expanding in processability window, which could be applied to polymers for desirable advanced applications.

Synergistic bioconversion of lipids and carotenoids from food waste by Dunaliella salina with fulvic acid via a two-stage cultivation strategy

Dunaliella salina is a promising microbial host for the sustainable production of bioenergy and valuable compounds. D. salina can be mixotrophically cultivated using a pure organic carbon source. However, pure organic carbon sources remarkably enhance the cost of cultivation and constrain the development of upscale microalgal cultivation. This study examined the feasibility of using low-value food waste supplemented with fulvic acid in D. salina mixotrophic cultivation for lipid and carotenoid accumulation. The glucose-rich hydrolysate was first obtained via enzymatic hydrolysis from selected food waste. This hydrolysate was an appropriate source of organic carbon for microalgal mixotrophy based on a two-stage cultivation strategy for investigating the biomass and accumulation of valuable products. In addition, fulvic acid, which contains oxygen-rich and carbon-poor functional groups, was used in the second stage to maximise the production of biomass (3.1 ± 0.2 g/L), lipids (0.75 ± 0.03 g/L) and carotenoids (0.52 ± 0.03 g/L). The biodiesel quality of the lipid derived from D. salina biomass based on food waste hydrolysate met the previously approved standards. These data illustrate a sustainable and efficient algal-based food waste biorefinery strategy for the co-production of bioenergy and bioactive compounds, and offer key insights to address the issues of global food waste and algal-based resource utilisation.

Triton X-100 improves co-production of β-1,3-D-glucan and pullulan by Aureobasidium pullulans.

The effects of several surfactants on the biosynthesis of β-1,3-D-glucan (β-glucan) and pullulan by Aureobasidium pullulans CCTCC M 2012259 were investigated, and Triton X-100 was found to decrease biomass formation but increase β-glucan and pullulan production. The addition of 5g/L Triton X-100 to the fermentation medium and bioconversion broth significantly increased β-glucan production by 76.6% and 69.9%, respectively, when compared to the control without surfactant addition. To reveal the physiological mechanism underlying the effect of Triton X-100 on polysaccharides production, the cell morphology and viability, membrane permeability, key enzyme activities, and intracellular levels of UDPG, NADH, and ATP were determined. The results indicated that Triton X-100 increased the activities of key enzymes involved in β-glucan and pullulan biosynthesis, improved intracellular UDPG and energy supply, and accelerated the transportation rate of precursors across the cell membrane, all of which contributed to the enhanced production of β-glucan and pullulan. Moreover, a two-stage culture strategy with combined processes of batch fermentation and bioconversion was applied, and co-production of β-glucan and pullulan in the presence of 5g/L Triton X-100 additions was further improved. The present study not only provides insights into the effect of surfactant on β-glucan and pullulan production but also presents a feasible approach for efficient production of analogue exopolysaccharides. KEY POINTS: • Triton X-100 increased β-glucan and pullulan production under either batch fermentation or bioconversion. • Triton X-100 increased the permeability of cell membrane and accelerated the transportation rate of precursors across cell membrane. • Activities of key enzymes involved in β-glucan and pullulan biosynthesis were increased in the presence of Triton X-100. • Intracellular UDPG levels and energy supply were improved by Triton X-100 addition.

Strategic implementation of phosphorus repletion strategy in continuous two-stage cultivation of Chlorella sp. HS2: Evaluation for biofuel applications

Lipid production in microalgae under nitrogen (N) starved condition can be enhanced by excess phosphorus (P) supply in the second stage of two-stage cultivation strategy. However, implementing two-stage cultivation is difficult in large-scale cultivation system as it requires high energy of transferring large algal biomass from first stage to second stage. To address this problem, we have optimized a continuous two-stage (CTS) cultivation strategy using Chlorella sp. HS2, where nitrogen in the growth environment is depleted naturally via consumption. To enhance both biomass and lipid productivity this strategy explored supplementation of additional P from 50% to 2500% of the initial concentration at the start of N-limited second stage of growth. The results of the optimization study in photobioreactor (PBR) showed that supplementing 500% of initial P and 100% of initial other nutrients (O) (N0–P500–O100) on 5th day showed the maximum biomass productivity of 774.4 mg L−1 d−1. It was observed that Chlorella sp. HS2 grown in PBR yielded higher biomass (3.8 times), lipid (6.1 times) and carbohydrate (5.5 times) productivity in comparison to the open raceway ponds (ORP) study, under optimum nutrient and carbon supply condition. The maximum lipid (289.6 mg L−1 d−1) and carbohydrate (219.2 mg L−1 d−1) productivities were obtained in TPBR-3, which were 1.9 and 1.3 times higher than that of TPBR-2 (+ve control) and 9.6 and 3.7 times higher than that of TPBR-1 (-ve control), respectively. Fatty acid mainly composed of C16/C18 (84.5%–85.7%), which makes the microalgal oil suitable for biofuel production. This study concluded that feeding excess amount of P is an effective and scalable strategy to improve the biomass and lipid productivity of CTS cultivation.

An unconventional two-stage cultivation strategy to increase the lipid content and enhance the fatty acid profile on Chlorella minutissima biomass cultivated in a novel internal light integrated photobioreactor aiming at biodiesel production

Biodiesel quality is dependent upon the fatty acids distribution of the raw material used in the trans/esterification reactions. While microalgae cells are regarded as potential oil suppliers for the biodiesel production chain, a common criticism is related to the low lipid productivities and often to a poor fatty acid distribution. In the present work we propose to submit microalgal cultivation to a second stage with nutritional stress by a simple addition of a 70% nitrate source depleted medium at the beginning of stationary phase, without harvesting cells, saving time and water. Chlorella minutissima was cultivated in a novel unconventional photobioreactor, named Internally Illuminated Integrated Photobioreactor (ILI-PBR), to verify the efficiency of the proposed strategy. Biomass concentration, lipid content and volumetric lipids productivity increased 24.9%, 120.7% and 55.6%, respectively, from Stage 1 to Stage 2, when ILI-PBR was illuminated by constant blue LED and 0.5 vvm of aeration. The fatty acid profiles, evaluated in terms of LED illumination (white, red and blue), showed a predominance of palmitic acid in all cases (59.49%, 57.28% and 46.51%, respectively), which allowed to enhance some biodiesel properties such as cetane number, cold filter plugging point, density and kinematic viscosity, according to international specifications.

Two-stage cultivation strategy for the improvement of pigment productivity from high-density heterotrophic algal cultures

Herein, a two-stage cultivation process was devised to overcome low pigment content of algal biomass grown in heterotrophy. Post-treatment conditions (i.e., light intensity, temperature, pH and salinity) were initially tested for dense heterotrophically-grown Chlorella sp. HS2 cultures in a multi-channel photobioreactor (mcPBR), and the results clearly indicated the influence of each abiotic factor on algal pigment production. Subsequently, the optimal post-treatment conditions (i.e., 455 μmol m−2 s−1, 34.8℃, pH 8.23 and 0.7% (w/v) salinity), in which highest accumulation of algal pigments is expected, were identified using Response Surface Methodology (RSM). Compared to the control cultures grown in mixotrophy for the same duration of entire two-stage process, the results indicated a significantly higher pigment productivity (i.e., 167.5 mg L–1 day−1) in a 5-L fermenter following the post-treatment at optimal conditions. Collectively, these results suggest that the post-treatment of heterotrophic cultures can be successfully deployed to harness the nascent algae-based bioeconomy.

Synergistic Effect of Nutrient and Salt Stress on Lipid Productivity of Chlorella vulgaris Through Two-Stage Cultivation

Lipid productivity of microalgae depends considerably on the method of its cultivation. In the present study, a two-stage culture strategy was conducted to cultivate Chlorella vulgaris to enhance lipid productivity. In the first stage, C. vulgaris was grown in the synthetic medium to obtain maximum biomass concentration. Wastewater is used as a medium for growing microalgae to have commercial and environmental aptitudes for biomass production. The nutrient removal efficiencies were 80.5% (COD), 70% (TN), and 78% (TP). In the second stage, the synergistic effects of nitrogen and NaCl concentration on the lipid productivity was investigated by response surface methodology (RSM) for 2 days. The optimum conditions for relatively high lipid productivity (up to 80 mg L−1 day−1) were 2.6 mg L−1 of nitrogen, 6.3 g L−1 of NaCl, and 4.9 g L−1 of biomass concentration. The value which was predicted by model was in good agreement with the experimental value, as it was determined by the validation experiments. Saturated fatty acid composition was increased by 27.3% under optimized medium compared with synthetic medium, which is very suitable for biodiesel production. According to the above results, it is concluded that the combination of multiple stress conditions can lead to a cost-effective technology of microalgae lipid production.

Shake flask methodology for assessing the influence of the maximum oxygen transfer capacity on 2,3-butanediol production

BackgroundProduction of 2,3-butanediol from renewable resources is a promising measure to decrease the consumption of fossil resources in the chemical industry. One of the most influential parameters on biotechnological 2,3-butanediol production is the oxygen availability during the cultivation. As 2,3-butanediol is produced under microaerobic process conditions, a well-controlled oxygen supply is the key parameter to control biomass formation and 2,3-butanediol production. As biomass is on the one hand not the final product, but on the other hand the essential biocatalyst, the optimal compromise between biomass formation and 2,3-butanediol production has to be defined.ResultsA shake flask methodology is presented to evaluate the effects of oxygen availability on 2,3-butanediol production with Bacillus licheniformis DSM 8785 by variation of the filling volume. A defined two-stage cultivation strategy was developed to investigate the metabolic response to different defined maximum oxygen transfer capacities at equal initial growth conditions. The respiratory quotient was measured online to determine the point of glucose depletion, as 2,3-butanediol is consumed afterwards. Based on this strategy, comparable results to stirred tank reactors were achieved. The highest space–time yield (1.3 g/L/h) and a 2,3-butanediol concentration of 68 g/L combined with low acetoin concentrations and avoided glycerol formation were achieved at a maximum oxygen transfer capacity of 13 mmol/L/h. The highest overall 2,3-butanediol concentration of 78 g/L was observed at a maximum oxygen transfer capacity of 4 mmol/L/h.ConclusionsThe presented shake flask approach reduces the experimental effort and costs providing a fast and reliable methodology to investigate the effects of oxygen availability. This can be applied especially on product and by-product formation under microaerobic conditions. Utilization of the maximum oxygen transfer capacity as measure for the oxygen availability allows for an easy adaption to other bioreactor setups and scales.

A novel two-stage culture strategy used to cultivate Chlorella vulgaris for increasing the lipid productivity

A novel two-stage culture strategy was used to cultivate Chlorella vulgaris (ESP-6) for increasing the lipid productivity. In the first stage, ESP-6 was grown in the cathode chamber of Air-lift-type microbial carbon capture cell (ALMCC) to obtain maximum biomass productivity (nutrient-sufficient condition). In the second stage, the culture was transferred into the air-lift-type photobioreactor (ALP) to cultivate continuously for increasing the lipid content of ESP-6 (nutrient-starvation condition). The maximum CO2 fixation rate (887.49 mg L−1 d−1) was achieved on day 7, and the maximum lipid content (54.23%) was achieved on day 12, while the maximum lipid productivity was 190.45 mg L−1 d−1 on day 11, which was 1.7 folds than that cultivated in nutrient-sufficient condition. In this study, the lipid productivity was improved by the two-stage culture strategy. Moreover, the microalgae suspension could be directly transferred to the second stage without the traditional pre-centrifugation treatment, which could thereby reduce the energy consumption and greenhouse gas emissions.

Fabrication and in vitro evaluation of a packed-bed bioreactor based on an optimum two-stage culture strategy

A packed-bed (PB) bioreactor for bioartificial liver (BAL) was fabricated based on an optimum two-stage culture strategy and evaluated invitro in this research. Human induced hepatocytes (hiHeps) were first expanded using Cytodex 3 microcarriers and the choice of microcarrier concentration and fetal bovine serum (FBS) content was optimized. Then, the cells expanded under the optimum expansion condition were perfused into a perfusion system containing Fibra-Cel (FC) disks to fabricate a PB bioreactor. Operating parameters including flow rate and seeding density for perfusion culture were optimized, respectively. Results indicated that during suspension culture, rapid cell proliferation and favorable amino acid metabolism were achieved at 3mg/mL microcarriers combined with 1% FBS. While for the perfusion culture, the most effective flow rate and seeding density were 2mL/min and 1×106cells/mL, respectively. Under this optimum perfusion condition, hiHeps showed good proliferation ability, high viability, homogeneous distribution, high metabolism activities and efficient albumin secretion as well as high liver-specific genes expression. Therefore, the two-stage culture strategy based on operating parameters optimization provides a new method for the development of PB bioreactors.

Development of a strategy for the production of docosahexaenoic acid by Schizochytrium sp. from cane molasses and algae-residue

The aim of this work was to reduce the algae-residue emission and make use of cane molasses as fermentation materials for docosahexaenoic acid (DHA) fermentaion by Schizochytrium sp., which further could cut the cost of DHA production. Algae-residue and cane molasses were respectively used as nitrogen and carbon sources to replace yeast extract and glucose. A significant DHA yield of 18.58 g/L was obtained using algae-residue, while cane molasses could not be used well as sole carbon source due to the presence of undesirable substance. A two-stage culture strategy with glucose followed by pretreated cane molasses as carbon source was developed, resulting in a final DHA yield of 15.22 g/L. This study therefore offers an economical and green strategy for DHA production by Schizochytrium sp.

Pilot-scale production of poly-β-hydroxybutyrate with the cyanobacterium Synechocytis sp. CCALA192 in a non-sterile tubular photobioreactor

The biopolymer poly-β-hydroxybutyrate (PHB) can be used as a promising bioplastic. It has a broad range of applications and is degraded relatively rapidly by soil organisms. Like many prokaryotes, the cyanobacterium Synechocystis sp. CCALA192 produces this biopolymer as a storage compound, especially under nutrient limitation.In a 200-L tubular photobioreactor, we cultivated Synechocystis sp. CCALA192 semi-continuously over a period of 75 days with CO2 as sole carbon source. A two-stage cultivation strategy was performed, where after 5–7 days nitrogen was depleted and the culture started to produce PHB and gradually turned from blue-green to yellow. After 16–20 days, 90% of the culture were harvested and the residual 10% were used as inoculum for the following cycle. The harvested culture had an average biomass concentration of 1.0 g/L with an average PHB content of 12.5% of cell dry weight. After restarting with fresh nutrients, the yellow culture turned blue-green again and degraded the PHB within 24–48 h. When nitrogen of the medium was consumed, PHB was produced again and the cycle continued. In the late stage of each production cycle, a ripening process was observed, where no CO2 was consumed but the PHB concentration was still rising at the expense of the existing glycogen rich biomass.Establishing a stable Synechocystis sp. CCALA192 culture under non-sterile conditions turned out to be difficult, as this small unicellular organism is very sensitive and easily grazed by protozoa. Therefore, a special cultivation strategy with partially anoxic conditions was necessary.

A Hetero-Photoautotrophic Two-Stage Cultivation Process for Production of Fucoxanthin by the Marine Diatom Nitzschia laevis.

There is currently much interest in fucoxanthin due to its broad beneficial health effects. The major commercial source of fucoxanthin is marine seaweed, which has many shortcomings, and has thus restricted its large-scale production and more diversified applications. In this study, growth characteristics and fucoxanthin accumulation were evaluated to explore the potential of the marine diatom Nitzschia laevis in fucoxanthin production. The results suggested that heterotrophic culture was more effective for cell growth, while the mixotrophic culture was favorable for fucoxanthin accumulation. A two-stage culture strategy was consequently established. A model of exponential fed-batch culture led to a biomass concentration of 17.25 g/L. A mix of white and blue light significantly increased fucoxanthin content. These outcomes were translated into a superior fucoxanthin productivity of 16.5 mg/(L·d), which was more than 2-fold of the best value reported thus far. The culture method established herein therefore represents a promising strategy to boost fucoxanthin production in N. laevis, which might prove to be a valuable natural source of commercial fucoxanthin.

A highly efficient two-stage cultivation strategy for lutein production using heterotrophic culture of Chlorella sorokiniana MB-1-M12

A heterotrophic mutant of Chlorella sorokiniana MB-1-M12 was evaluated for its ability to produce lutein using organic carbon and nitrogen sources and without light irradiation. In batch fermentation, the maximal lutein content (3.67 mg lutein/g biomass) and productivity (2.84 mg/L/d) could be obtained when cultivated in BG-11 medium with 7.5 g/L glucose, 0.75 g/L urea, pH 7.5 and a C/N ratio of 10. A novel two-stage cultivation strategy that integrates fed-batch and semi-batch operations was applied to enhance the lutein production performance. When growing MB-1-M12 strain in a 5L fermenter using the optimal operation strategies, the maximum biomass concentration, biomass productivity, lutein content and lutein productivity could reach 25 g/L, 4.88 mg/L/d, 5.88 mg/g and 16.2 mg/L/d, respectively. This high lutein productivity could significantly reduce the cultivation time and the associated costs, indicating the potential of using MB-1-M12 strain for heterotrophic lutein production in commercial scale.

Two-stage mixotrophic cultivation for enhancing the biomass and lipid productivity of Chlorella vulgaris

This study proposes a two-stage mixotrophic process for cultivating Chlorella vulgaris. Heterotrophic growth is the dominant step in Phase I (to increase microalgal biomass) and photoautotrophic growth occurs in Phase II (to improve biomass concentration and lipid production). The results show that the addition of the low-cost antioxidant sodium erythorbate (8 g L−1) significantly accelerates the growth of microalgae in the first stage with air aeration. Furthermore, a higher CO2 fixation rate was obtained in the second stage (at least 344.32 mg CO2 L−1 day−1) with 10% CO2 aeration. This approximately corresponds to an increase of 177% over simple photoautotrophic cultivation with 10% CO2 aeration during the whole period. The two-stage cultivation strategy achieved a maximum C. vulgaris biomass concentration of 3.45 g L−1 and lipid productivity of 43.70 mg L−1 day−1, which are 1.85 and 1.64 times those arising due to simple photoautotrophy, respectively. Moreover, an analysis of the product’s fatty acid profile indicates that C. vulgaris might be an ideal candidate for two-stage mixotrophic cultivation of a renewable biomass for use in biodiesel applications. Another interesting point to note from the study is that it is an insufficiency of N and CO2 that probably limits the further growth of C. vulgaris.

Impact of nutrient starvation on intracellular biochemicals and calorific value of mixed microalgae

A two-stage cultivation strategy was applied to mixed microalgae, which were first cultured in complete nutrient medium then switched to different nutrient-free mediums in order to assess the impact of nutrient starvation on intracellular biochemical components of mixed microalgae. The effects of nitrogen, sulfur and phosphorus starvation on cell counts, chlorophyll, carotenoid, protein, starch and lipid content of the mixed microalgae are compared in this study. The obtained results revealed that starch, as a dominant storage compound, was the highest in the nitrogen-free medium up to 49% of dry weight (DW). Protein and chlorophyll content declined slightly from 512 to 472.96mgL−1 and 29.43 to 26.58mg.L−1 only in the case of nitrogen starvation. Nitrogen starvation showed the best result as it ceased cell division immediately whereby in the case of sulfur and phosphorus starvation, cell division was not interrupted as microalgae are a pool of phosphorus and store sulfur. Calculation of starch and lipid energy content on the basis of electron equivalent and J/mg dry weight showed the higher energy content of lipid in compare with that of starch for all nutrients starvation. Nitrogen deprivation condition represents the superlative energy value of storage compounds content with the value of 17.61J/mg dry weight. The finding proves the potential of attractive and economically feasible mixed microalgae cultivation for high percentages of storage compounds production under nitrogen starvation

Two-stage (photoautotrophy and heterotrophy) cultivation enables efficient production of bioplastic poly-3-hydroxybutyrate in auto-sedimenting cyanobacterium

Sustainable production of bioplastics by heterotrophic microbes has been restricted by the limited resources of organic substrates and the energy required for biomass harvest. Here, the easy-to-harvest cyanobacterium (Chlorogloea fritschii TISTR 8527), from which the biomass instantaneously settled to the bottom of liquid culture, was utilized to produce poly-3-hydroxybutyrate (PHB) using a two-stage cultivation strategy. The cells were first pre-grown under normal photoautotrophy to increase their biomass and then recultivated under a heterotrophic condition with a single organic substrate to produce the product. Through optimization of this two-stage cultivation, the mass conversion efficiency of acetate substrate to PHB was obtained at 51 ± 7% (w/w), the comparable level to the theoretical biochemical conversion efficiency of acetate to PHB. This two-stage cultivation that efficiently converted the substrate to the product, concurrent with a reduced culture biomass, may be applicable for the production of other biopolymers by cyanobacteria.

Cultivation of four microalgae for biomass and oil production using a two-stage culture strategy with salt stress

A two-stage culture strategy was used for maximum biomass production under nutrient-sufficient conditions, followed by cultivation under low-salt stress, to cause the accumulation of oil in the biomass. Controlled conditions of nitrate, salt concentration, and time to exposure to stress were optimized for oil production with four species of microalgae, Isochrysis galbana, Nannochloropsis oculata, Dunaliella salina, and Dunaliella tertiolecta. Using conditions with addition of nitrate to 24.0 mg/L, I. galbana and N. oculata showed higher biomass productions than D. salina and D. tertiolecta. The oil contents of the microalgae increased from 24.0% to 47.0% in I. galbana with 10 psu for 2 days, from 17.0% to 29.0% in N. oculata with 0 psu for 3 days, from 22.0% to 43.0% of D. salina with 10 psu for 1 day, and from 23.0% to 40.0% (w/w) in D. tertiolecta with 0 psu for 2 days as the second stage culture with low-salt stress. Thus, I. galbana could be a suitable candidate microalga for oil production.