Mixed culture of oleaginous yeast Rhodotorula glutinis and microalga Chlorella vulgaris for lipid production from industrial wastes and its use as biodiesel feedstock

A mixed culture of oleaginous yeast Rhodotorula glutinis and microalga Chlorella vulgaris was performed to enhance lipid production from industrial wastes. These included effluent from seafood processing plant and molasses from sugar cane plant. In the mixed culture, the yeast grew faster and the lipid production was higher than that in the pure cultures. This could be because microalga acted as an oxygen generator for yeast, while yeast provided CO(2) to microalga and both carried out the production of lipids. The optimal conditions for lipid production by the mixed culture were as follows: ratio of yeast to microalga at 1:1; initial pH at 5.0; molasses concentration at 1%; shaking speed at 200 rpm; and light intensity at 5.0 klux under 16:8 hours light and dark cycles. Under these conditions, the highest biomass of 4.63±0.15 g/L and lipid production of 2.88±0.16 g/L were obtained after five days of cultivation. In addition, the plant oil-like fatty acid composition of yeast and microalgal lipids suggested their high potential for use as biodiesel feedstock.     

Effects of nitrogen concentration and media replacement on cell growth and lipid production of oleaginous marine microalga Nannochloropsis oceanica DUT01

Cell growth and lipid production of a marine microalga Nannochloropsis oceanica DUT01 were investigated, and fresh medium replacement with different ratios to promote long term cell growth and lipid accumulation was also tested. The highest lipid content reached 64% in nitrogen deplete f/2 medium containing 37.5 mg/L NaNO₃ combined with 1/5 fresh medium replacement, however, the highest lipid titer (0.6 g/L) and lipid productivity (31 mg/L/d) were achieved using BG11 medium containing 1.5 g/L NaNO₃, taking advantage of 1/5 fresh medium replacement as well, which corresponded to the maximum biomass production of 1.4 g/L, highlighting the importance of high biomass accumulation for efficient lipid production. When biomass compositions were monitored throughout the culture, decreased protein content was found to be coupled with increased lipid production, whereas relatively stable carbohydrate content was observed. The fatty acids in the lipid of N. oceanica DUT01 comprise over 65% saturated fatty acids and monounsaturated acids (i.e. palmitic acid (C16:0) and oleic acid (C18:1)), suggesting that N. oceanica DUT01 is a promising candidate for biodiesel production. Interestingly, very high content of hexadecadienoic acid (C16:2, about 26–33%) was produced by DUT01, which distinguished this microalga with other microalgae strains reported so far.     

Carbon concentration and oxygen availability affect lipid and carotenoid production by carob pulp syrup‐grown Rhodosporidium toruloides NCYC 921

The simultaneous effect of oxygen availability and carbon source concentration on yeast lipid and carotenoid production has never been studied before. In this work, a Doehlert distribution design was used to study the simultaneous effect of carbon concentration and oxygen availability on Rhodosporidium toruloides NCYC 921 carotenoid and lipid production. A cheap industrial byproduct was used as carbon source (carob pulp syrup). A total sugar concentration of 106.3 g/L and a medium volume of 0.120 L induced the highest total carotenoid and total fatty acid productivities (4.60 μg/Lh and 0.029 g/Lh, respectively). Flow cytometry was used to assess yeast stress response under different cultivation conditions. The highest proportion of cells with permeabilised membrane (>20%) was induced when the cultivations were carried out at the highest sugar concentration studied (130.0 g/L) or when the culture reached the minimum final medium pH (4.60). The results showed that the total sugar concentration had a positive influence on the yeast biomass and carotenoid content, while the oxygen availability had little influence on the biomass concentration, but had a slight positive influence on the carotenoid content. Regarding the fatty acids, the two factors had a negative impact on the synthesis of these compounds.     

The effect of light, salinity, and nitrogen availability on lipid production by Nannochloropsis sp

We examined responses of batch cultures of the marine microalga Nannochloropsis sp. to combined alterations in salinity (13, 27, and 40 g/l NaCl) and light intensity (170 and 700 μmol photons/m²·s). Major growth parameters and lipid productivity (based on total fatty acid determination) were determined in nitrogen-replete and nitrogen-depleted cultures of an initial biomass of 0.8 and 1.4 g/l, respectively. On the nitrogen-replete medium, increases in light intensity and salinity increased the cellular content of dry weight and lipids due to enhanced formation of triacylglycerols (TAG). Maximum average productivity of ca. 410 mg TFA/l/d were obtained at 700 μmol photons/m²·s and 40 g/l NaCl within 7 days. Under stressful conditions, content of the major LC-PUFA, eicosapentaenoic acid (EPA), was significantly reduced while TAG reached 25% of biomass. In contrast, lower salinity tended to improve major growth parameters, consistent with less variation in EPA contents. Combined higher salinity and light intensity was detrimental to lipid productivity under nitrogen starvation; biomass TFA content, and lipid productivity amounted for only 33% of DW and ca. 200 mg TFA/l/day, respectively. The highest biomass TFA content (ca. 47% DW) and average lipid productivity of ca. 360 mg TFA/l/day were achieved at 13 g/l NaCl and 700 μmol photons/m²·s. Our data further support selecting Nannochloropsis as promising microalgae for biodiesel production. Moreover, appropriate cultivation regimes may render Nannochloropsis microalgae to produce simultaneously major valuable components, EPA, and TAG, while sustaining relatively high biomass growth rates.     

Factors affecting lipid accumulation by <Emphasis Type="Italic">Nannochloropsis oculata</Emphasis> in a two-stage cultivation process

Reserve lipids of microalgae are promising for biodiesel production. However, optimization of cultivation conditions for both biomass yield and lipid production of microalgae is a contradictory problem because required conditions for both targets are different. In this study, a two-stage cultivation strategy is proposed to enhance lipid production of the microalga Nannochloropsis oculata. Biomass growth and lipid production were carried out in two separate and non-interacting stages. In first-stage cultivation, microalgae were cultivated in optimal conditions for cell growth. Then, microalgae were harvested and transferred into a growth-limited environment, thus enhancing lipid production of microalgae. Here, optimization of the lipid production stage (second stage) with respect to different levels of inoculum concentration, salinity of culture broth, and intensity of irradiance was performed. The results show that irradiance exhibits a significant influence on lipid production. The highest lipid productivity of 0.324 g L⁻¹ day⁻¹ was obtained with an inoculum concentration of 2.3 g L⁻¹, a salinity of 35 g L⁻¹, and an irradiance of 500 μmol photons m⁻² s⁻¹. The final yield of lipid obtained from the two-stage process was 2.82-times higher than that from traditional single-stage batch cultivation systems.     

Effect of light supply and carbon source on cell growth and cellular composition of a newly isolated microalga Chlorella vulgaris ESP‐31

Light supply is one of the most important factors affecting autotrophic growth of microalgae. This study investigated the effect of the type and light intensity of artificial light sources on the cell growth of an indigenous microalga Chlorella vulgaris ESP‐31 obtained from southern Taiwan. In addition, a dissolved inorganic carbon source (i.e. sodium bicarbonate) was used to improve the biomass production of strain ESP‐31. The results show that a new fluorescent light source (TL5) was effective in indoor cultivation of microalgae. Better overall productivity of 0.029 g dry cell weight/L‐d was obtained when using TL5 lamps as the light source with a light intensity of 9 W/m². A carbon source (sodium bicarbonate) concentration of 1000 mg/L was found to be optimal for the growth of strain ESP‐31 in terms of both biomass production and carbon source utilization. Under the optimal growth conditions, the resulting microalgal biomass consisted of 25–30% protein, 6–10% carbohydrate, and 30–40% lipid.     

Outdoor microalgae cultivation in airlift photobioreactor at high irradiance and temperature conditions: effect of batch and fed-batch strategies,photoinhibition, and temperature stress

The microalgae Scenedesmus abundans cultivated in five identical airlift photobioreactors (PBRs) in batch and fed-batch modes at the outdoor tropical condition. The microalgae strain S. abundans was found to tolerate high temperature (35–45 °C) and high light intensity (770–1690 µmol m^− 2 s^− 1). The highest biomass productivities were 152.5–162.5 mg L^− 1 day^− 1 for fed-batch strategy. The biomass productivity was drastically reduced due to photoinhibition effect at a culture temperature of > 45 °C. The lipid compositions showed fatty acids mainly in the form of saturated and monounsaturated fatty acids (> 80%) in all PBRs with Cetane number more than 51. The fed-batch strategies efficiently produced higher biomass and lipid productivities at harsh outdoor conditions. Furthermore, the microalgae also accumulated omega-3 fatty acid (C18:3) up to 14% (w/w) of total fatty acid at given outdoor condition.

     

Effects of cassava starch hydrolysate on cell growth and lipid accumulation of the heterotrophic microalgae <Emphasis Type="Italic">Chlorella protothecoides</Emphasis>

Heterotrophic fermentation of microalgae has been shown to accumulate high amounts of microalgal lipids, which are regarded as one of the most promising feedstocks for sustainable biodiesel production. To increase the biomass and reduce the cost of microalgal culture, the purpose of this study was to evaluate the possibility of using cassava starch hydrolysate (CSH) instead of glucose as carbon source for heterotrophic culture of Chlorella protothecoides in flasks. First, the two-step enzymatic process of hydrolysis of cassava starch by α-amylase and glucoamylase was optimized; the conversion efficiency for cassava starch was up to 97.7%, and over 80% of CSH was glucose. Subsequently, we compared heterotrophic cultures of C. protothecoiedes using glucose or CSH as carbon source. The results demonstrated that when using CSH as the organic carbon source, the highest biomass and the maximum total lipid yield obtained were 15.8 and 4.19 g/L, representing increases of 42.3 and 27.7%, respectively, compared to using glucose as the organic carbon source. This suggests that CSH is a better carbon source than glucose for heterotrophic Chlorella protothecoides.     

High‐yield lipid production from lignocellulosic biomass using engineered xylose‐utilizing Yarrowia lipolytica

Lignocellulosic biomass shows high potential as a renewable feedstock for use in biodiesel production via microbial fermentation. Yarrowia lipolytica, an emerging oleaginous yeast, has been engineered to efficiently convert xylose, the second most abundant sugar in lignocellulosic biomass, into lipids for lignocellulosic biodiesel production. Yet, the lipid yield from xylose or lignocellulosic biomass remains far lower than that from glucose. Here we developed an efficient xylose‐utilizing Y. lipolytica strain, expressing an isomerase‐based pathway, to achieve high‐yield lipid production from lignocellulosic biomass. The newly developed xylose‐utilizing Y. lipolytica, YSXID, produced 12.01 g/L lipids with a maximum yield of 0.16 g/g, the highest ever reported, from lignocellulosic hydrolysates. Consequently, this study shows the potential of isomerase‐based xylose‐utilizing Y. lipolytica for economical and sustainable production of biodiesel and oleochemicals from lignocellulosic biomass.     

Pressurized extraction of unsaturated fatty acids and carotenoids from wet Chlorella vulgaris and Phaeodactylum tricornutum biomass using subcritical liquids

The objective of this study was to investigate the extraction of lipids, for example, mono‐ and polyunsaturated fatty acids (PUFA) as well as carotenoids, from wet microalgae biomass using pressurized subcritical extraction solvents, which meet the requirements of food and feed applications. To demonstrate the effect of the solvent and temperature on the lipid yield, we chose two microalgae species, viz. Chlorella vulgaris and Phaeodactylum tricornutum, differing in their biochemical composition fundamentally. In case of P. tricornutum, ethanol showed the highest fatty acid yield of 85.9% w/w. In addition to eicosapentaenoic acid (EPA), the ethanolic extracts contained exceptional amounts of fucoxanthin (up to 26.1 mg/g d. w.), which can be beneficial to protect unsaturated fatty acids from oxidation processes and in terms of human nutrition. For C. vulgaris, a fatty acid yield of 76.5% w/w was achieved from wet biomass using ethyl acetate at 150°C. In general, an increase in the extraction temperature up to 150°C was found to be important in terms of fatty acid yield when extracting wet microalgae biomass. The results suggest that it is possible to efficiently extract both fatty acids and carotenoids from wet microalgae by selecting suitable solvents and thus circumvent energy‐intensive drying of the biomass.     

Productivity correlated to photobiochemical performance of <Emphasis Type="Italic">Chlorella</Emphasis> mass cultures grown outdoors in thin-layer cascades

This work aims to: (1) correlate photochemical activity and productivity, (2) characterize the flow pattern of culture layers and (3) determine a range of biomass densities for high productivity of the freshwater microalga Chlorella spp., grown outdoors in thin-layer cascade units. Biomass density, irradiance inside culture, pigment content and productivity were measured in the microalgae cultures. Chlorophyll-fluorescence quenching was monitored in situ (using saturation-pulse method) to estimate photochemical activities. Photobiochemical activities and growth parameters were studied in cultures of biomass density between 1 and 47 g L⁻¹. Fluorescence measurements showed that diluted cultures (1–2 g DW L⁻¹) experienced significant photostress due to inhibition of electron transport in the PSII complex. The highest photochemical activities were achieved in cultures of 6.5–12.5 g DW L⁻¹, which gave a maximum daylight productivity of up to 55 g dry biomass m⁻² day⁻¹. A midday depression of maximum PSII photochemical yield (F _v/F _m) of 20–30% compared with morning values in these cultures proved to be compatible with well-performing cultures. Lower or higher depression of F _v/F _m indicated low-light acclimated or photoinhibited cultures, respectively. A hydrodynamic model of the culture demonstrated highly turbulent flow allowing rapid light/dark cycles (with frequency of 0.5 s⁻¹) which possibly match the turnover of the photosynthetic apparatus. These results are important from a biotechnological point of view for optimisation of growth of outdoor microalgae mass cultures under various climatic conditions.     

The potential of extracellular biopolymer production by Mesorhizobium sp. from monosaccharide constituents of lignocellulosic biomass

Objective

The effects of monosaccharide constituents of lignocellulosic materials on exopolysaccharide (EPS) production by Mesorhizobium sp. Semia 816 were studied.

Results

According to the results, by using sugars commonly found in lignocellulosic biomass as carbon sources (glucose, arabinose and xylose), no significant differences were observed in the production of EPS, reaching 3.39 g/L, 3.33 g/L and 3.27 g/L, respectively. Differences were observed in monosaccharide composition, mainly in relation to rhamnose and glucuronic acid contents (1.8 times higher when arabinose was compared with xylose). However, the biopolymers showed no differences in relation to rheological properties, with EPS aqueous-based suspensions (1.0% w/v) presenting pseudoplastic behavior, and a slight difference in degradation temperatures. Using soybean hulls hydrolysate as carbon source, slightly higher values were obtained (3.93 g/L).

Conclusion

The results indicate the potential of the use of lignocellulosic hydrolysates containing these sugars as a source of carbon in the cultivation of Mesorhizobium sp. Semia 816 for the production of EPS with potential industrial applications.

     

产油微藻自养培养条件调控

作为新兴生物燃料的生物柴油近年来发展迅速,以微藻为代表的第二代生物能源是解决能源危机的长远之计,但如何提高其产量仍是研究的热点问题。以提高产油自养微藻生物量和油脂含量为目的,在气升式光反应器中运用均匀设计实验方法进行了条件优化试验。分别得出了氮原子浓度、通气速率、二氧化碳体积浓度和光照强度4个因素对小球藻C2生物量积累和油脂含量影响的显著回归方程和反应器优化培养条件。以生物量为指标的优化培养条件是:氮原子浓度0.178 g/L,通气速率5 L/min,二氧化碳体积浓度3%(V/V),光照强度6000 lx。该优化条件下,生物量为2.11 g/L,即生产速率为0.352 g/(L.d),比测试实验中检测到的最高生物量[1.88 g/L,即生产速率为0.313 g/(L.d)]提高了12.2%;以油脂含量为指标的优化培养条件是:进气速率0.400 L/min,二氧化碳体积浓度1.94%(V/V),得到油脂含量为22.4%,比测试实验中检测到的最高油脂量(20.7%)提高7.7%。     

Effects of yeast extract on the production of phenylpropanoid metabolites in callus culture of purple basil (Ocimum Basilicum L. var purpurascens) and their in-vitro evaluation for antioxidant potential

Ocimum basilicum L. var. purpurascens is an enriched reservoir of pharmaceutically important compounds with plenty of health and therapeutic attributes such as phenolic acids and anthocyanins. However, the inefficient production of aforementioned metabolites in wild has restricted its commercial utilization. Herein, commercially viable phytochemicals have been enhanced through elicitation of in-vitro cultures of O. basilicum using yeast extract.The impact of various concentrations (YE 1 mg/L,YE 10 mg/L, YE 25 mg/L, YE 50 mg/L, YE 100 mg/L, YE 200 mg/L and YE 400 mg/L) of yeast extract on biomass accumulation, phytochemical production, and antioxidant activities were assessed in callus cultures. Moderate concentration of yeast extract (100 mg/L) enhanced biomass accumulation i.e. fresh weight (FW 216.28 g/L) and dry weight (DW 15.49 g/L) up to 1.5 folds as compared to control (FW 167.14 g/L and DW 10.25 g/L). Similarly, yeast extract (100 mg/L) increased total phenolic and flavonoid contents as well as enhanced antioxidant activities such as ABTS (2,2 azinobis 3-ethylbenzthiazoline-6-sulphonic acid), FRAP (ferric reducing antioxidant power) and DPPH (2,2-diphenyl-1-picryhydrazyl). High performance liquid chromatography (HPLC) analysis was elucidated for further phytochemical investigation. HPLC analysis showed an increase of almost 1.9 folds as compared to control in rosmarinic acid (15.19 mg/g DW), chicoric acid (2.13 mg/g DW), peonidin (2.70 mg/g DW) and cyanidin (1.57 mg/g DW). Likewise, 1.8 fold and 2.4 folds increase was observed in eugenol essential oils (0.25 mg/g DW) and chavicol (0.037 mg/g DW), respectively. For cellular antioxidant activity, reactive oxygen specie or reactive nitogen specie (ROS/RNS) was induced in yeast cells and the effect of O. basilicum callus culture was further investigated in stressed yeast cells. A positive correlation exists between the antioxidant activities, TPC and TFC analysis. In short, these results showed that yeast extract could act as an efficient elicitor to enhance pharmacologically important metabolites in callus cultures of Ocimum basilicum.

Graphical abstract

     

Distribution of Marine Red Yeasts in Shrimps and the Environments of Shrimp Culture

Populations of marine red yeast from shrimps and the environments of shrimp culture were investigated from various areas at Zhanjiang in China. All strains were studied for the production of biomass and carotenoids. We isolated 88 marine red yeast strains and the average populations of marine red yeast in seawater and the water from shrimp culture ponds were 70.0 and 172.4 CFU per 100 ml water, respectively. For shrimp samples, average populations of marine red yeast from gills, intestines, and stomachs were 178.0, 15.0, and 8.0 CFU per shrimp, respectively. The isolates were grouped into nine species belonging to three genera as follows: Rhodosporidium, Rhodotorula, and Sporidiobolus. R. sphaerocarpum had the highest average biomass yield (10.3 ± 0.88 g/l), followed by S. ruineniae (10.1 g/l) and Rh. mucilaginosa (9.9 ± 1.75 g/l). R. paludigenum had the highest average carotenoid yield (2.83 ± 0.589 mg/l), followed by S. pararoseus (2.72 mg/l) and R. sphaerocarpum (2.59 ± 0.454 mg/l). The results showed that marine red yeasts were normal microbial components in the environments of shrimp culture and shrimps, and carotenoids are abundant in these marine red yeast.     

Productivity and selective accumulation of carotenoids of the novel extremophile microalga <Emphasis Type="Italic">Chlamydomonas acidophila</Emphasis> grown with different carbon sources in batch systems

Cultivation of extremophile microorganisms has attracted interest due to their ability to accumulate high-value compounds. Chlamydomonas acidophila is an acidophile green microalga isolated by our group from Tinto River, an acidic river that flows down from the mining area in Huelva, Spain. This microalga accumulates high concentrations of lutein, a very well-known natural antioxidant. The aim of this study is to assess use of different carbon sources (CO₂, glucose, glycerol, starch, urea, and glycine) for efficient growth of and carotenoid production by C. acidophila. Our results reveal that growth of the microalga on different carbon sources resulted in different algal biomass productivities, urea being as efficient as CO₂ when used as sole carbon source (~20 g dry biomass m^–2 day^–1). Mixotrophic growth on glucose was also efficient in terms of biomass production (~14 g dry biomass m^–2 day^–1). In terms of carotenoid accumulation, mixotrophic growth on urea resulted in even higher productivity of carotenoids (mainly lutein, probably via α-carotene) than obtained with photoautotrophic cultures (70% versus 65% relative abundance of lutein, respectively). The accumulated lutein concentrations of C. acidophila reported in this work (about 10 g/kg dry weight, produced in batch systems) are among the highest reported for a microalga. Glycerol and glycine seem to enhance β-carotene biosynthesis, and when glycine is used as carbon source, zeaxanthin becomes the most accumulated carotenoid in the microalga. Strategies for production of lutein and zeaxanthin are suggested based on the obtained results.     

利用乙醇发酵副产CO2培养富含淀粉亚心型四爿藻作为其补充原料

建立了乙醇发酵耦联微藻培养系统,研究了利用酿酒酵母Saccharomyces cerevisiae乙醇发酵副产CO2为碳源,培养富含淀粉的亚心形四爿藻Tetraselmis subcordiformis,作为乙醇发酵补充原料的可行性。在连续光照培养条件下,间歇式培养7 d,反应器中藻细胞密度达到2.0 g/L左右,胞内淀粉含量约45％。微藻细胞收集后,经超声处理和酶法水解,葡萄糖释放量为胞内淀粉总量的71.1％。S. cerevisiae发酵微藻生物质水解液生产乙醇,其得率达到理论值的87.6％。表明乙醇发酵耦联微藻培养可行,既减少了CO2向环境的排放,又收获了富含淀粉的微藻生物质作为乙醇发酵的补充原料,节省粮食类淀粉质原料的消耗。     

Production of <Emphasis Type="Italic">Chlorella</Emphasis> biomass enriched by selenium and its use in animal nutrition: a review

Feedstuffs are routinely supplemented with various selenium sources, where organic forms of Se are more bio-available and less toxic than the inorganic forms (selenites, selenates). When the algae are exposed to environmental Se in the form of selenite, they are able as other microorganisms to incorporate the element to different levels, depending on the algae species. Technology of heterotrophic fed-batch cultivation of the microalga Chlorella enriched by organically bound Se was developed, where the cultivation proceeds in fermentors on aerated and mixed nutrient solution with urea as a nitrogen and glucose as a carbon and energy source. High volumetric productivity and high cell concentrations (about 70–100 g Chlorella dry mass l⁻¹) can be attained if nutrients and oxygen are adequately supplied. Addition of a small quantity of a new selenoprotein source-spray-dried Se-Chlorella biomass to the diet of farm animals had better effects on specific physiological and physical parameters of animals than selenite salt and was comparable with Se yeast added to the diet. This review introduces the importance of selenium for humans and animals, methods of Se determination, heterotrophic production of selenium-enriched Chlorella biomass in a fed-batch culture regime on organic carbon, and use of the biomass in animal nutrition.     

Monitoring microalgal growth of Chlorella minutissima with a new all solid-state contact nitrate selective sensor

As third generation feedstock, microalgae are microorganisms that can grow only in the optimum conditions. There are parameters including the concentration of macro and microelements in nutrient solution, pH, temperature and light intensity that have significant impact on microalgal growth. In recent years, various sensing devices have been developed for sensitive measurement of these parameters during microalgal growth. In this study, a new potentiometric nitrate selective sensor was developed to indicate the nitrate uptake of microalgae and the effect of nitrate nutrient on microalgal growth, specifically, and this sensor was successfully applied to determine nitrate concentration in medium during microalgal growth. Moreover, the effects of nitrate, carbonate and phosphate concentration in the growth medium on biomass production of Chlorella minutissima were investigated by using Box–Behnken design method, and optimum conditions were determined for the highest biomass production of microalgae. As a result of the experiments, it was seen that the highest C. minutissima production was achieved using the medium consist of 2.63 g/L NaNO₃, 0.35 g/L Na₂CO₃ and 0.4 g/L KH₂PO_4. Statistically, it was observed that there was a proportional relationship between the microalgae production and investigated parameters such as carbon, nitrogen and phosphate amounts of culture mediums. The electrode showed a wide linear range between 1.0 × 10⁻¹ and 5.0 × 10⁻⁵ M with a detection limit of the 5 × 10⁻⁶ M and the response time was found as 10 s. The results showed that developed nitrate selective sensor could be successfully applied for continuous measurement of nitrate in microalgal productions at reduced cost.     

Enhanced astaxanthin production from microalga,Haematococcus pluvialis by two-stage perfusion culture with stepwise light irradiation

For efficient astaxanthin production from the culture of green microalga, Haematococcus pluvialis, a two-stage mixotrophic culture system was established with stepwise increased light irradiance. By perfusion process, high density biomass (2.47 g/L) was achieved during the vegetative stage due to no detrimental effect of inhibitory metabolites, which was 3.09 and 1.67 times higher than batch and fed-batch processes, respectively. During the induction stage, biomass and astaxanthin were subsequently produced to the very high level 12.3 g/L and 602 mg/L, under stepwise increased light irradiance (150–450 μE/m²/s), respectively. These results indicate that the combinatorial approach of perfusion culture during the vegetative stage and stepwise light irradiation during the induction stage is a promising strategy for the simultaneous production of high concentration of biomass and astaxanthin in microalgae including H. pluvialis.