Microalgal-Based Carbon Sequestration by Converting LNG-Fired Waste CO 2 into Red Gold Astaxanthin: The Potential Applicability

: The combinatorial approach of anthropogenic activities and CO 2 sequestration is becoming a global research trend to alleviate the average global temperature. Although microalgae have been widely used to capture CO 2 from industrial flue gas, the application of bioproducts was limited to bioenergy due to the controversy over the quality and safety of the products in the food and feed industry. Herein, the waste CO 2 emitted from large point sources was directly captured using astaxanthin-hyperproducing microalgae Haematococcus pluvialis . Astaxanthin production was successfully carried out using the hypochlorous acid water-based axenic culture process under highly contamination-prone outdoor conditions. Consequently, after 36 days of autotrophic induction, the productivity of biomass and astaxanthin of H. pluvialis (the mutant) reached 0.127 g L − 1 day − 1 and 5.47 mg L − 1 day − 1 under high summer temperatures, respectively, which was 38% and 48% higher than that of wild type cell. After grinding the wet astaxanthin-enriched biomass, the extract was successfully approved by compliance validation testing from Korea Food and Drug Administration. The assorted feed improved an immune system of the poultry without causing any side effects. The flue gas-based bioproducts could certainly be used for health functional food for animals in the future.


Introduction
The energy generated by the combustion of traditional energy sources is the driving force of a nation's development, however the world is facing the urgent environmental issue of global warming due to the increased carbon dioxide (CO 2 ) emissions, which mostly result from burning excessive fossil fuel forms, like coal, oil and natural gas [1,2].Therefore, it would be an inevitable demand that power generation must be combined with CO 2 sequestration process.Although CO 2 capture and storage (CCS) technology can reduce a considerable amount of CO 2 at once, it is not a perfect technology yet because there are many demerits, such as difficulty of choosing a location for stable CO 2 storage, the risk of leakage, the high cost for installation of infrastructures including pipeline and facilities of injection, and large scale storage [3].On the contrary, CO 2 capture and utilization (CCU) technology would be an alternative solution for CO 2 sequestration, which brings the striking and instant utilization of stored CO 2 and effectively solves the challenges of leakage and space for long-term storage.
In recent years, microalgae cultivation system is considered as an eco-friendly CCU technology and one of potentially viable models for capturing waste CO 2 emitted from large point sources [4][5][6][7][8][9].Microalgae efficiently convert inorganic CO 2 into organic biomass via photosynthesis and the biomass contains various value-added biochemicals, such as high energy density biofuels and high value pharmaceutical ingredients [10][11][12][13], which could reduce the overall cost of CCU-hybrid system.Astaxanthin is a high value keto-carotenoid pigment ($7000 kg −1 USD) [14] and a super antioxidant because its antioxidant activity surpasses that of other carotenoids and antioxidants in the market.Astaxanthin has a 550-times more powerful activity than vitamin E [15].Among the various microalgal astaxanthin producers, Haematococcus pluvialis is the richest source of natural astaxanthin (3-5%, w/w) [16].Above all, the H. pluvialis extracts predominantly consist of 3S, 3'S stereoisomer which is the most effective isomer for human and animal application as compared to the other optical isomer forms (i.e., 3R, 3'R and 3R, 3'S) [17].
During Haematococcus cultivation, separation of the first stage ("green" stage) for cellular multiplication (vegetative growth) and the second stage ("red" stage) for encystment (inductive growth) and carotenogenesis (astaxanthin accumulation) are necessary steps to achieve the high level of biomass production and astaxanthin yield [14,18], particularly for an industrial scale production [14, [18][19][20].In the green stage, the number of green motile flagellates increases under low-stress conditions, whereas in the red stage, cell proliferation is stopped due to exposure to high stress, but the single cell begins to increase in mass and color via morphological transformation from immature green cells to mature red cysts (aplanospore) to accumulate astaxanthin.
In the present study, H. pluvialis was cultured via employing a two-stage culture strategy under autotrophic conditions using natural sunlight and waste CO 2 (3-5%, v/v) resulting from the combustion of liquefied natural gas (LNG) in combined heat and power plants.Consequently, 3S, 3'S-astaxanthin was produced from H. pluvialis under tough or harsh outdoor conditions, lipid (bio-oil), which can be used for renewable energy generation, was also co-produced.Moreover, a remarkable increase in CO 2 removal rate, as well as biomass and astaxanthin productivity, were confirmed in outdoors conditions using previously developed highly photosensitive and photoinducible Haematococcus mutant (PP-PS 160), by adopting red cyst inoculation and iron (II) supplementation methods.
In the microalgae industry, the constant and stable supply of the waste CO 2 emitted from large point sources is regarded as a critical issue for the economical production of microalgae biomass [18,21].Although the waste CO 2 from flue gas has been broadly used for microalgae research, the produced biomass was mainly applied in the field of biofuel production, such as bio-diesel or solid fuels [4][5][6][22][23][24][25][26][27][28], not used in human food or animal feed, due to safety concerns over the waste CO 2 .The main focus of this study is to identify the potential application of the biomass derived from industrial flue gas in the animal feed industry.The H. pluvialis biomass containing valuable astaxanthin was successfully produced in an outdoor facility using natural sunlight and LNG-fired flue gas as gaseous carbon feedstock, which is relatively clean compared to other coal combustion flue gases.The astaxanthin extract was validated and approved by Korea Food and Drug Administration (KFDA), to meet KFDA-recognized consensus standards for application to the animal feed industry.After that, physiological studies were carried out to monitor the health benefits of the astaxanthin extract powder on the poultry feeding.The assorted feed improved the immune system of the poultry without unintended side effects.

Establishment of Outdoor Microalgal Culture System Using LNG-Fired Flue Gas
For effective conversion of waste CO 2 from industrial flue gas into high value products, such as bio-oil (omega-3 fatty acids) or astaxanthin, microalgal culture process was installed near the LNG combustion stack of the Korea District Heating Corporation (KDHC) located at Baekhyeon-dong, Bundang-gu, Seongnam-si, Gyeonggi-do, in South Korea [7][8][9].In the system, H. pluvialis cells were cultured autotrophically by using a transparent polymeric thin-film (polyethylene-nylon hybrid) 100 L-PBR [9] with the technical specifications of 120 cm diameter (D) and 180 cm height (H), which is consisted of serially connected six bubble columns (D of 15 cm, H/D ratio of 9:1), for outdoor mass production of the biomass packed with astaxanthin.In the PBR, biomass production was carried out using solar energy (heat and light) and LNG-fired flue gas at a flow rate of 0.1 vvm (vessel volumes per min).Composition of flue gas was 3.5 ± 0.5% (v/v) CO 2 , 3 ± 1.15 ppm CO, 10.15 ± 0.71% O 2 , and 21.57± 3.48 ppm NOx [7][8][9].

Pure Culture System of H. pluvialis Without Microbial Contamination
The slightly acidic hypochlorous acid (HClO) water-based axenic culture method was developed to cultivate contamination-free H. pluvialis under highly contamination-prone outdoor conditions, as Haematococcus cells are highly sensitive to the biotic stress, thus likely to contaminate easily (Figure 1).In this study, the pH of the culture medium was maintained within a constant range via a dissolved inorganic carbon (DIC) buffer system using 10 mM KOH and waste CO 2 from the LNG-fired flue gas during long periods of outdoor autotrophic cultivation.In addition, to minimize the biotic contamination during medium transfer, the nutrient medium (NIES-C or NIES-N) was directly injected in the PBR in the form of a concentrated stock solution (500×).
The entire microalgae culture process for contamination control can be described as follows: (1) Manufacturing polymeric thin-film PBR, (2) washing and filling the PBR using HClO water, (3) injecting flue gas in the PBR to degas Cl 2 for 2-3 days (HClO water → pure water), (4) injecting waste CO 2 containing flue gas in the PBR filled with 10 mM KOH to facilitate improved DIC level as well as buffered the solution (bicarbonate system), ( 5) supply of appropriate concentration of nutrients NIES-C medium stock solution (500×) in the PBR and inoculation of microalgal seed for biomass cultivation.
Energies 2018, 11, x FOR PEER REVIEW 3 of 18 L-PBR [9] with the technical specifications of 120 cm diameter (D) and 180 cm height (H), which is consisted of serially connected six bubble columns (D of 15 cm, H/D ratio of 9:1), for outdoor mass production of the biomass packed with astaxanthin.In the PBR, biomass production was carried out using solar energy (heat and light) and LNG-fired flue gas at a flow rate of 0.1 vvm (vessel volumes per min).Composition of flue gas was 3.5 ± 0.5% (v/v) CO2, 3 ± 1.15 ppm CO, 10.15 ± 0.71% O2, and 21.57± 3.48 ppm NOx [7][8][9].

Pure Culture System of H. pluvialis Without Microbial Contamination
The slightly acidic hypochlorous acid (HClO) water-based axenic culture method was developed to cultivate contamination-free H. pluvialis under highly contamination-prone outdoor conditions, as Haematococcus cells are highly sensitive to the biotic stress, thus likely to contaminate easily (Figure 1).In this study, the pH of the culture medium was maintained within a constant range via a dissolved inorganic carbon (DIC) buffer system using 10 mM KOH and waste CO2 from the LNG-fired flue gas during long periods of outdoor autotrophic cultivation.In addition, to minimize the biotic contamination during medium transfer, the nutrient medium (NIES-C or NIES-N) was directly injected in the PBR in the form of a concentrated stock solution (500×).
The entire microalgae culture process for contamination control can be described as follows: (1) Manufacturing polymeric thin-film PBR, (2) washing and filling the PBR using HClO water, (3) injecting flue gas in the PBR to degas Cl2 for 2-3 days (HClO water → pure water), (4) injecting waste CO2 containing flue gas in the PBR filled with 10 mM KOH to facilitate improved DIC level as well as buffered the solution (bicarbonate system), ( 5) supply of appropriate concentration of nutrients NIES-C medium stock solution (500×) in the PBR and inoculation of microalgal seed for biomass cultivation.

Algal Strains and Culture Conditions
The H. pluvialis NIES-144 (wild type) was purchased from the National Institute for Environmental Studies (NIES) in Tsukuba, Japan.The partially PSII-deficient and photosensitive (PP-PS) Haematococcus mutant PP-PS 160, which was derived from H. pluvialis NIES-144, was obtained from the Nanobiotechnology Laboratory at Korea University in Seoul, South Korea.The two H. pluvialis strains were cultured in two media of NIES-C (nitrogen-replete) and NIES-N (nitrogendeplete) medium for cell proliferation and astaxanthin induction, respectively [29].

Algal Strains and Culture Conditions
The H. pluvialis NIES-144 (wild type) was purchased from the National Institute for Environmental Studies (NIES) in Tsukuba, Japan.The partially PSII-deficient and photosensitive (PP-PS) Haematococcus mutant PP-PS 160, which was derived from H. pluvialis NIES-144, was obtained from the Nanobiotechnology Laboratory at Korea University in Seoul, South Korea.The two H. pluvialis strains were cultured in two media of NIES-C (nitrogen-replete) and NIES-N (nitrogen-deplete) medium for cell proliferation and astaxanthin induction, respectively [29].
NIES-C medium is comprised of 0.15 g L −1 Ca(NO 3 ) 2 , 0.10 g L −1 KNO 3 , 0.05 g L −1 β-glycerophosphoric acid disodium salt pentahydrate, 0.  A two-stage strategy was introduced to improve photoautotrophic astaxanthin production from H. pluvialis using waste CO 2 under outdoor spring and summer conditions (Figure 2).In the green stage, the two strains were firstly grown for 15-20 days in nitrogen-replete NIES-C medium at 25-45 µmol photons m −2 s −1 of limiting light conditions.The initial cell density of all cultures was approximately 0.12-0.15g L −1 .
Energies 2018, 11, x FOR PEER REVIEW 4 of 17 A two-stage strategy was introduced to improve photoautotrophic astaxanthin production from H. pluvialis using waste CO2 under outdoor spring and summer conditions (Figure 2).In the green stage, the two strains were firstly grown for 15-20 days in nitrogen-replete NIES-C medium at 25-45 µmol photons m −2 s −1 of limiting light conditions.The initial cell density of all cultures was approximately 0.12-0.15g L −1 .In particular, during summer experiments, the two methods of cyst inoculation (germination) and Iron(II) (Fe 2+ ) supplementation were consecutively employed for vegetative growth in the green stage and astaxanthin production in the red stage, respectively, to cope with harsh high-temperature environments [7,30].Nitrogen deplete NIES-N medium stock solution (500×) and 50 µM Fe 2+ were additionally injected into the photobioreactor (PBR) at the end of green stage (initial red stage), to accelerate nitrogen starvation and heat stress-driven Haber-Weiss reaction, respectively, thereby enhancing astaxanthin production under high temperature conditions [7,30].
Cells were cultured at high solar irradiance of 305-360 µmol photons m −2 s −1 and 315-380 µmol photons m −2 s −1 in spring and summer, respectively.During the outdoor spring and summer seasons, all cultivations were conducted in almost clear sunny days, thereby ensuring the intensity of solar light radiation.Nonetheless, natural light does not meet the requirements of constant and continuous strength of light intensity during daytime.Accordingly, irradiance was roughly controlled by using synthetic shading sheet, but not for the culture medium temperatures.

Analysis of Cell Growth
The cell density was determined by measuring DCW using pre-weighed filter paper via filtering aliquots of samples.Then, the cell suspensions (10 mL) followed by DI water (as flow through) were filtered using GF/F glass microfiber filters (Whatman, Cambridge, UK), and dried at 105 °C overnight.Then, the weight of dried cells was determined by the difference between the mass of the biomasscontaining filter paper and that of pre-weighed filter paper [17].

Analysis of Astaxanthin
In order to assay intracellular astaxanthin, cell suspensions (5-10 mL) were collected by centrifuging the cell culture fluid at 5590 g, and 4 °C, for 5 min, decanting the supernatant, and then rinsing the cell pellet with pre-chilled TE buffer (pH 7.5).The pellet was dissolved in pre-chilled 100% methanol with glass beads to extract pigments and homogenized with Tissue Lyser II (Qiagen, Valencia, CA, USA) using pre-chilled Tissue Lyser adaptors.The extraction procedure was repeated until the cell debris was colorless [30].The homogenized lysate was centrifuged at 5590 g, at 4 °C, for 10 min to separate the supernatant from the cell debris, and the fresh extracts were collected and In particular, during summer experiments, the two methods of cyst inoculation (germination) and Iron(II) (Fe 2+ ) supplementation were consecutively employed for vegetative growth in the green stage and astaxanthin production in the red stage, respectively, to cope with harsh high-temperature environments [7,30].Nitrogen deplete NIES-N medium stock solution (500×) and 50 µM Fe 2+ were additionally injected into the photobioreactor (PBR) at the end of green stage (initial red stage), to accelerate nitrogen starvation and heat stress-driven Haber-Weiss reaction, respectively, thereby enhancing astaxanthin production under high temperature conditions [7,30].
Cells were cultured at high solar irradiance of 305-360 µmol photons m −2 s −1 and 315-380 µmol photons m −2 s −1 in spring and summer, respectively.During the outdoor spring and summer seasons, all cultivations were conducted in almost clear sunny days, thereby ensuring the intensity of solar light radiation.Nonetheless, natural light does not meet the requirements of constant and continuous strength of light intensity during daytime.Accordingly, irradiance was roughly controlled by using synthetic shading sheet, but not for the culture medium temperatures.

Analysis of Cell Growth
The cell density was determined by measuring DCW using pre-weighed filter paper via filtering aliquots of samples.Then, the cell suspensions (10 mL) followed by DI water (as flow through) were filtered using GF/F glass microfiber filters (Whatman, Cambridge, UK), and dried at 105 • C overnight.Then, the weight of dried cells was determined by the difference between the mass of the biomass-containing filter paper and that of pre-weighed filter paper [17].

Analysis of Astaxanthin
In order to assay intracellular astaxanthin, cell suspensions (5-10 mL) were collected by centrifuging the cell culture fluid at 5590 g, and 4 • C, for 5 min, decanting the supernatant, and then rinsing the cell pellet with pre-chilled TE buffer (pH 7.5).The pellet was dissolved in pre-chilled 100% methanol with glass beads to extract pigments and homogenized with Tissue Lyser II (Qiagen, Valencia, CA, USA) using pre-chilled Tissue Lyser adaptors.The extraction procedure was repeated until the cell debris was colorless [30].The homogenized lysate was centrifuged at 5590 g, at 4 • C, for 10 min to separate the supernatant from the cell debris, and the fresh extracts were collected and saponified using the alkali-method [30,31].For saponification of esterified astaxanthin from H. pluvialis, 0.01 M Energies 2019, 12, 1718 5 of 17 KOH was used for 12 h to convert the esterified forms (10-50 mg L −1 ) into the free form.Under the conditions, the selective separation of free astaxanthin by alkaline hydrolysis of astaxanthin-esters can be efficiently performed in methanol by adding hydroxyl groups (OH − ).

Analysis of Lipid
Total cell lipid was determined using the modified Bligh and Dyer method [32].The mixed solution of methanol and chloroform (2:1, v v −1 ) was directly used for cell lysis and extraction of lipid molecules from the wet cells without using the dried cells.After that, the extracted wet lipid molecules were dried and then the total lipid was examined by measuring the weight.

Analysis of Carbohydrate
Total cell carbohydrate was measured via a colorimetric assay with an anthrone reagent [33].To analyze the intracellular quantity of carbohydrate, cell suspensions (3-5 mL) were harvested by centrifuging the culture fluid at 5600 g for 4 min at 4 • C, discarding the supernatant, and rinsing the cell pellet with pre-chilled TE buffer (pH 7.5).The pellet was homogenized with Tissue Lyser II (Qiagen, Valencia, CA, USA) in 1 mL of pre-chilled 100% methanol with glass beads in the pre-chilled Tissue Lyser adaptors.After evaporation of methanol in drying oven, 1 mL of 72% perchloric acid was added to the samples, which were subsequently reacted for 6 h at 4 • C.After that, each sample was mixed with 2 mL of 67% sulfuric acid by vortexing for 30 min and subsequently reacted with anthrone reagent at 100 • C in the water bath for 5 min.The quantitative amount of carbohydrate of each sample was measured at 630 nm.

Analysis of Protein
Total cell protein was examined by the Bradford method [34].To assay intracellular protein quantity, cell suspensions (3-5 mL) were collected by centrifuging the culture fluid at 5600 g for 5 min at 4 • C, discarding the supernatant, and then rinsing the cell pellet with pre-chilled PBS buffer (pH 7.5).The pellet was homogenized with Tissue Lyser II (Qiagen, Valencia, CA, USA) in 2 mL of pre-chilled PBS buffer (pH 7.5) with glass beads in the pre-chilled Tissue Lyser adaptors, the supernatant was colored by mixing with Bradford reagent (Sigma Chemical Co., St. Louis, MO, USA), and The quantitative amount of protein of each sample was measured at 595 nm.
2.4.6.Ball Milling Method to Rupture H. pluvialis Red Cysts and KFDA Approval of the Extracts After centrifugation of the suspensions containing astaxanthin-enriched H. pluvialis biomass, the harvested red cell cake (150-200 g L −1 of red cell density) was subsequently grinded by ball mill device containing zirconium oxide milling ball (Zirconia, 10 mm, Tencan) for 24 h and the extracts were sent to KFDA for compliance testing.

Physiological Studies of H. pluvialis Biomass on Poultry Feeding
After drying the mixture of ruptured H. pluvialis biomass and astaxanthin extract, resulting in the powder, the biomass-astaxanthin extracts powder were directly used to feed poultry.A total of Energies 2019, 12, 1718 6 of 17 270 number of egg-laying hens (eight days old) were employed in this trial.Dietary treatments in this experiment consisted of feeding a corn-soybean meal control diet alone or supplemented with 0, 0.1, and 0.2% ruptured H. pluvialis powder for a period of seven weeks.Three replicate groups of 30 hens were randomly dosed to each of the three treatments.

Outdoor Biomass and Astaxanthin Production Using LNG-fired Flue Gas During Spring and Summer Seasons
Wild type and highly photosensitive Haematococcus mutant PP-PS 160 were compared for the production of biomass and astaxanthin under outdoor transient culture conditions of light intensity and temperature using the flue gas from the combustion of LNG during spring and summer seasons.Consequently, the CO 2 removal rate of the mutant was 36.8% and 37.3% in spring and summer, respectively, compared to that of wild type cell (spring: 0.125 g L −1 day −1 , summer: 0.171 g L −1 day −1 ).Output details are summarized in Table 1.
In spring season, as shown in Figure 3, the production of biomass and astaxanthin from wild type cell reached to 3.989 g L −1 and 134.04 mg L −1 after 72 days of autotrophic induction (two-stage) respectively.The productivity of biomass and astaxanthin was 0.055 g L −1 day −1 and 1.861 mg L −1 day −1 in the wild type cell, respectively.Remarkably, the highly photosensitive mutant showed enhanced biomass and astaxanthin production in spring compared to that of wild type cell.After 57 days of autotrophic induction (two-stage), the production yield of biomass and astaxanthin of the mutant reached up to 4.310 g L −1 and 158.52 mg L −1 , respectively.Overall, the productivity of biomass and astaxanthin by the mutant was 36% and 49% higher than that of wild type cell.
Energies 2018, 11, x FOR PEER REVIEW 6 of 18 experiment consisted of feeding a corn-soybean meal control diet alone or supplemented with 0, 0.1, and 0.2% ruptured H. pluvialis powder for a period of seven weeks.Three replicate groups of 30 hens were randomly dosed to each of the three treatments.

Outdoor Biomass and Astaxanthin Production Using LNG-fired Flue Gas During Spring and Summer Seasons
Wild type and highly photosensitive Haematococcus mutant PP-PS 160 were compared for the production of biomass and astaxanthin under outdoor transient culture conditions of light intensity and temperature using the flue gas from the combustion of LNG during spring and summer seasons.Consequently, the CO2 removal rate of the mutant was 36.8% and 37.3% in spring and summer, respectively, compared to that of wild type cell (spring: 0.125 g L -1 day -1 , summer: 0.171 g L -1 day -1 ).Output details are summarized in Table 1.
In spring season, as shown in Figure 3, the production of biomass and astaxanthin from wild type cell reached to 3.989 g L -1 and 134.04 mg L -1 after 72 days of autotrophic induction (two-stage) respectively.The productivity of biomass and astaxanthin was 0.055 g L -1 day -1 and 1.861 mg L -1 day - 1 in the wild type cell, respectively.Remarkably, the highly photosensitive mutant showed enhanced biomass and astaxanthin production in spring compared to that of wild type cell.After 57 days of autotrophic induction (two-stage), the production yield of biomass and astaxanthin of the mutant reached up to 4.310 g L -1 and 158.52 mg L -1 , respectively.Overall, the productivity of biomass and astaxanthin by the mutant was 36% and 49% higher than that of wild type cell.When we compared the yield of biomass and astaxanthin within the wild type strain, the summer season was more productive as shown in Figure 4, the yield of biomass and astaxanthin was higher than that of spring season, reaching to 4.161 g L −1 and 166.43 mg L −1 after 45 days of autotrophic induction (two-stage), respectively, the productivity was 0.092 g L −1 day −1 and 3.698 mg L −1 day −1 respectively.However, the highly photosensitive mutant exhibited improved performance on the biomass and astaxanthin production compared to that of wild type cell during both seasons, in particular summer was a better yielding season.After 36 days of autotrophic induction (two-stage), the production yield of biomass and astaxanthin of the mutant reached to 4.564 g L −1 and 196.88 mg L −1 , respectively.Consequently, the productivity of biomass and astaxanthin in the mutant was 38% and 48% higher than that of wild type cell.When we compared the yield of biomass and astaxanthin within the wild type strain, the summer season was more productive as shown in Figure 4, the yield of biomass and astaxanthin was higher than that of spring season, reaching to 4.161 g L -1 and 166.43 mg L -1 after 45 days of autotrophic induction (two-stage), respectively, the productivity was 0.092 g L -1 day -1 and 3.698 mg L -1 day -1 respectively.However, the highly photosensitive mutant exhibited improved performance on the biomass and astaxanthin production compared to that of wild type cell during both seasons, in particular summer was a better yielding season.After 36 days of autotrophic induction (two-stage), the production yield of biomass and astaxanthin of the mutant reached to 4.564 g L -1 and 196.88 mg L -1 , respectively.Consequently, the productivity of biomass and astaxanthin in the mutant was 38% and 48% higher than that of wild type cell.

Outdoor Lipid and Carbohydrate Production Using LNG-Fired Flue Gas During Spring and Summer Seasons
The intracellular contents of protein, carbohydrate, and lipid of the two H. pluvialis cells were also measured at the end of each stage during outdoor cultivation in spring and summer.As a result, under the moderate temperature conditions in spring, the protein content decreased by half, but carbohydrate and lipid contents were increased in wild type cell at the end of the induction stage (Figure 5).Although the change in patterns for the contents of the mutant was similar to the wild type cell, the increase in the lipid content of the mutant was much higher than that of wild type cell.The phenomenon of high lipid content at the end of the induction stage was more prominent under the high temperature conditions in summer.

Outdoor Lipid and Carbohydrate Production Using LNG-Fired Flue Gas During Spring and Summer Seasons
The intracellular contents of protein, carbohydrate, and lipid of the two H. pluvialis cells were also measured at the end of each stage during outdoor cultivation in spring and summer.As a result, under the moderate temperature conditions in spring, the protein content decreased by half, but carbohydrate and lipid contents were increased in wild type cell at the end of the induction stage (Figure 5).Although the change in patterns for the contents of the mutant was similar to the wild type cell, the increase in the lipid content of the mutant was much higher than that of wild type cell.The phenomenon of high lipid content at the end of the induction stage was more prominent under the high temperature conditions in summer.

KFDA Approval and Physiological Studies of H. pluvialis Extracts
The astaxanthin-enriched wild type cells were harvested by centrifugation and the red cyst biomass cake was collected and ground by ball mill with zirconia beads.As a result, the cell wall of wet H. pluvialis was easily ruptured by treatment of ball mill for one day (Figure 6), thereafter astaxanthin was extracted without the use of organic solvent.As shown in Table 2, the H. pluvialis biomass and astaxanthin-mixed extract derived from the LNG-fired flue gas was approved by KFDA (Appendix A), and thereafter used for successful physiological studies on poultry.The characteristic of the extract was a dark reddish brown viscous fluid without unpleasant flavor and odor.The astaxanthin content of the extract was closely meet the standards.As the microalgal cells were derived from flue gas, thus heavy metal contamination was expected in the final cell biomass.All the contents of the heavy metals such as Pb, Cd, Hg, and As were tested and determined as lower than the detection limits suggested by KFDA.Moreover, microbial contaminants, as well as organic solvents, were not detected as expected.
In order to confirm the toxicity and other side effects of the extracts, the mixed powder of homogenized and dried H. pluvialis biomass and astaxanthin extract was tested by feeding to poultry.As a result, the immune system of poultry was boosted by feeding the mixture powder of ruptured H. pluvialis biomass and astaxanthin extract without specific side effects (Table 3).Accordingly, the mortality rate of the egg-laying hens fed with 0.1% and 0.2% ruptured H. pluvialis powder was decreased by 17% (8.3%) and 15% (8.5%) compared to that of the egg-laying hens fed without the astaxanthin powder (10%), respectively.

KFDA Approval and Physiological Studies of H. pluvialis Extracts
The astaxanthin-enriched wild type cells were harvested by centrifugation and the red cyst biomass cake was collected and ground by ball mill with zirconia beads.As a result, the cell wall of wet H. pluvialis was easily ruptured by treatment of ball mill for one day (Figure 6), thereafter astaxanthin was extracted without the use of organic solvent.As shown in Table 2, the H. pluvialis biomass and astaxanthin-mixed extract derived from the LNG-fired flue gas was approved by KFDA (Supplementary Materials), and thereafter used for successful physiological studies on poultry.The characteristic of the extract was a dark reddish brown viscous fluid without unpleasant flavor and odor.The astaxanthin content of the extract was closely meet the standards.As the microalgal cells were derived from flue gas, thus heavy metal contamination was expected in the final cell biomass.All the contents of the heavy metals such as Pb, Cd, Hg, and As were tested and determined as lower than the detection limits suggested by KFDA.Moreover, microbial contaminants, as well as organic solvents, were not detected as expected.
In order to confirm the toxicity and other side effects of the extracts, the mixed powder of homogenized and dried H. pluvialis biomass and astaxanthin extract was tested by feeding to poultry.As a result, the immune system of poultry was boosted by feeding the mixture powder of ruptured H. pluvialis biomass and astaxanthin extract without specific side effects (Table 3).Accordingly, the mortality rate of the egg-laying hens fed with 0.1% and 0.2% ruptured H. pluvialis powder was decreased by 17% (8.3%) and 15% (8.5%) compared to that of the egg-laying hens fed without the astaxanthin powder (10%), respectively.

Discussion
In order to capture waste CO2 from large point sources in outdoor cultivation, the freshwater microalgae Chlorella sp., Scenedesmus acutus and Desmodesmus abundans, have been commonly used for flue gas mitigation because they often exhibit an outstanding tolerance to biotic and abiotic stresses [4−6] compared to the other microalgae species.Astaxanthin production can be the most attractive option from a corporate standpoint to maximize the viability of CO2-emiting companies, even situated in a space-limited urban area.
Although astaxanthin-producing microalgae Chlorella zofingiensis was commonly an interesting host in the microalgal industry, the intracellular content of astaxanthin is still low and the individual carotenoids pool includes high levels of canthaxanthin [35], which could be harmful to the human cells, compared to H. pluvialis.Undoubtedly, the green microalga H. pluvialis is regarded as the richest source of natural astaxanthin in nature and cultivated on an industrial scale [14, [18][19][20].
However, the production of H. pluvialis biomass enriched with astaxanthin often fails in outdoor cultivations due to the microbial contamination [36,37].Typically, four species of contaminants, such as the chytrid zoosporic, Paraphysoderma sedebokerensis (fungus), Poterioochromonas malhamensis (golden algae), Scenedesmus sp.(microalga), and the Bacillus sp.(bacteria), have primarily led Haematococcus culture failure in our process.The Bacillus sp.attack to eat the encysted H. pluvialis cells by persistent drilling of the rigid cell walls.The Poterioochromonas malhamensis produces dangerous toxins and covers the encysted H. pluvialis cells to be starved and died.The Paraphysoderma sedebokerensis is an extremely detrimental predator of H. pluvialis.Once the PBR is contaminated by the fungus, all the encysted H. pluvialis cells are unmercifully destroyed in one to three days resulting in a color change of the PBR from red to white or yellow-brown.Although the microalga Scenedesmus sp.does not eat or kill H. pluvialis, there is no way to separate the two microalgal cells produced, thereby losing the quality of products.Therefore, the HClO water-based pure culture system was designed for the stable production of astaxanthin from H. pluvialis in this study.The slightly acidic electrolyzed water was harmless for the human but swiftly fatal for the contaminants.The concentration of Cl2 in the HClO water was 30 ppm, which was an effective concentration to kill the contaminants.Intriguingly, the HClO water can be simply converted into pure water by bubbling to degas dissolved Cl2 as the column reactor was efficient for Cl2 degassing (data not shown).
The buffer system is necessary for sustaining the required pH, particularly at neutral or mildly alkaline pH (7.5-8.0), in the microalgal culture medium because high or low pH is detrimental for the vegetative growth as well as the production of value-added products by microalgae [38,39].In general, Tris and HEPES have been widely used as a buffer for microalgae cultivation [12,29], which are very costly.It was previously reported that DIC buffer system using sodium hydroxide (NaOH) and CO2 has remarkable buffer potential for microalgal cultivation [40,41].In addition, our previous study showed that the productivity of biomass and astaxanthin by H. pluvialis was slightly enhanced by DIC buffer system under the optimal conditions of 10 mM of KOH and LNG-fired flue gas containing 3-5% waste CO2 [8].It is noteworthy that an aqueous environment is normally a CO2limited condition [42], hence the improved yield from such environment can be partially attributed

Discussion
In order to capture waste CO 2 from large point sources in outdoor cultivation, the freshwater microalgae Chlorella sp., Scenedesmus acutus and Desmodesmus abundans, have been commonly used for flue gas mitigation because they often exhibit an outstanding tolerance to biotic and abiotic stresses [4][5][6] compared to the other microalgae species.Astaxanthin production can be the most attractive option from a corporate standpoint to maximize the viability of CO 2 -emiting companies, even situated in a space-limited urban area.
Although astaxanthin-producing microalgae Chlorella zofingiensis was commonly an interesting host in the microalgal industry, the intracellular content of astaxanthin is still low and the individual carotenoids pool includes high levels of canthaxanthin [35], which could be harmful to the human cells, compared to H. pluvialis.Undoubtedly, the green microalga H. pluvialis is regarded as the richest source of natural astaxanthin in nature and cultivated on an industrial scale [14, [18][19][20].
However, the production of H. pluvialis biomass enriched with astaxanthin often fails in outdoor cultivations due to the microbial contamination [36,37].Typically, four species of contaminants, such as the chytrid zoosporic, Paraphysoderma sedebokerensis (fungus), Poterioochromonas malhamensis (golden algae), Scenedesmus sp.(microalga), and the Bacillus sp.(bacteria), have primarily led Haematococcus culture failure in our process.The Bacillus sp.attack to eat the encysted H. pluvialis cells by persistent drilling of the rigid cell walls.The Poterioochromonas malhamensis produces dangerous toxins and covers the encysted H. pluvialis cells to be starved and died.The Paraphysoderma sedebokerensis is an extremely detrimental predator of H. pluvialis.Once the PBR is contaminated by the fungus, all the encysted H. pluvialis cells are unmercifully destroyed in one to three days resulting in a color change of the PBR from red to white or yellow-brown.Although the microalga Scenedesmus sp.does not eat or kill H. pluvialis, there is no way to separate the two microalgal cells produced, thereby losing the quality of products.Therefore, the HClO water-based pure culture system was designed for the stable production of astaxanthin from H. pluvialis in this study.The slightly acidic electrolyzed water was harmless for the human but swiftly fatal for the contaminants.The concentration of Cl 2 in the HClO water was 30 ppm, which was an effective concentration to kill the contaminants.Intriguingly, the HClO water can be simply converted into pure water by bubbling to degas dissolved Cl 2 as the column reactor was efficient for Cl 2 degassing (data not shown).
The buffer system is necessary for sustaining the required pH, particularly at neutral or mildly alkaline pH (7.5-8.0), in the microalgal culture medium because high or low pH is detrimental for the vegetative growth as well as the production of value-added products by microalgae [38,39].In general, Tris and HEPES have been widely used as a buffer for microalgae cultivation [12,29], which are very costly.It was previously reported that DIC buffer system using sodium hydroxide (NaOH) and CO 2 has remarkable buffer potential for microalgal cultivation [40,41].In addition, our previous study showed that the productivity of biomass and astaxanthin by H. pluvialis was slightly enhanced by DIC buffer system under the optimal conditions of 10 mM of KOH and LNG-fired flue gas containing 3-5% waste CO 2 [8].It is noteworthy that an aqueous environment is normally a CO 2 -limited condition [42], hence the improved yield from such environment can be partially attributed to the increased uptake of DIC (bicarbonate, HCO 3 − ), which is converted to CO 2 by intracellular carbonic anhydrase and then can be used for biomass and astaxanthin production [8].In this study, it was also revealed that under outdoor moderate (spring) or high (summer) temperature conditions the DIC buffer system based on NaOH and LNG-fired flue gas successfully improved biomass and astaxanthin production from both H. pluvialis cells of the highly photosensitive mutant PP-PS 160 as well as the wild type NIES-144.Consistent with many other studies [7,16,29,30], which were conducted under nutrient stress, H. pluvialis cells were exposed to starvation, which rapidly stopped cellular multiplication but exhibited a considerable increase in volumetric growth (mass per single cell) in this study.Therefore, N-deficient medium was additionally used for rapid consumption of intracellular nitrogen remnant, thereby enhancing photoautotrophic induction in H. pluvialis.Although it is worth measuring the minimum inhibitory concentration (MIC) of intracellular nitrogen source to affect astaxanthin production in H. pluvialis, the rapid switching effect of transcription pools for carotenoid biosynthesis by the addition of N-deplete medium might be more contributable to the astaxanthin induction than the amount of intracellular nitrogen source.In Chlorella pyrenoidosa, under N-and P-deplete conditions, the biomass increased by as much as 112% compared to an initial cell density [43].In Neochloris oleoabundans, under N-deplete conditions, the biomass increased by as much as 127% compared to an initial cell density [44].
In microalgae, carotenogenesis is mainly regulated by biotic or abiotic stressors, such as nutrient deprivation, high irradiance, salinity, drought and moderate temperature [45,46].Reactive oxygen species (ROS) are mediators between abiotic stress and astaxanthin synthesis, whenever there is excess reducing power for photosynthesis [30].Therefore, the second stage of a two-stage strategy is highly dependent on the light intensity in H. pluvialis, [7,16,29,30,45,46].The more biomass increases, the more intensive light is needed for rapid induction.Therefore, the selection of photosensitive mutant would be one of the most important issues for economic astaxanthin production [16,17].In our previous study, we found highly photo-inducible H. pluvilais mutants derived from increased photo-sensitivity and proposed the mutant library to improve the output of the total astaxanthin production under moderate light conditions [16,17], which is limiting for efficient astaxanthin accumulation in the wild type cell.In the mutant, partial photosystem II (PSII) deficiency primarily contributed to the extreme generation of ROS, thereby accelerating both encystment for inductive growth and carotenogenesis for astaxanthin accumulation.
Although it has been previously reported that the H. pluvialis mutant, which is less sensitive to ROS, also showed enhanced astaxanthin production under high-light condition (250 µmol photons m −2 s −1 ) [47], the main downside of the mutant might be the difficulty of the efficient induction under the conditions of high cell density cultivation.In this regard, the highly photosensitive astaxanthin producer could be more useful when performing high cell density culture for achieving high astaxanthin concentration because it showed outstanding astaxanthin-accumulating performance under moderate light condition.Above all, under outdoor thermal conditions in summer, in which variations of the diurnal temperature occur, the mutant PP-PS 160 showed incredible inducibility of high-titer astaxanthin production.It clearly supports that in the PP-PS mutant, the enhanced photo-inducibility, which was arisen by a partially defective PSII, was related with the high-light and high-temperature stresses, hence accelerating an intracellular ROS generation.
In our system, the temperature in the culture medium was not controlled during outdoor spring or summer cultivation because the temperature control process is usually costly and energy consuming in a microalgal industry.The cost might be reduced using heat-utilizing technology.It has been reported that excessive heat stress negatively affects the autotrophic astaxanthin production in H. pluvialis [7,30,48,49], particularly during outdoor cultivation in the summer [7,30].Under the summer conditions, the vegetative growth was severely interrupted in the green stage and the inductive growth and inducibility for the biosynthesis of astaxanthin were inhibited in the red stage.Our previous study solved this problem by inoculating the red cyst cells to maintain relatively moderate inner carotenoid levels during the green stage and supplementing 50 µM Fe 2+ to induce the heat stress-driven Haber-Weiss reaction by converting less reactive oxygen species (O 2 •− and H 2 O 2 ) into more reactive oxygen species ( 1 O 2 and OH•) via the iron (II)-catalyzed Haber-Weiss reaction during the red stage to promote lipid peroxidation, thereby enhancing astaxanthin production under photoautotrophic high-temperature conditions [7,30].Although many types of PBR, such as flat panel, tubular reactor, and bubble columns, have been extensively proposed as outdoor closed PBR system in the field of microalgal industry, only bubble columns and tubular PBRs have proven to be capable of scaling up to high volumes [50].In the present study, the plastic film bubble column PBR, which is made from cheap materials, is cost-effective, easy to manufacture, and could be recycled several times if it is not contaminated yet.However, the scalability of the thin-film PBR could not be higher than the other bubble column PBRs made from more robust plastic or glass materials because the height of the reactor was normally limited to a relatively low level due to the low durability.The leakage of culture medium often occurred when the height of the reactor was higher than 180 cm.The height limit of the PBR might lead to an increase in the land lease fee, thereby restricting scalability of the microalgal process.
Although optimal autotrophic conditions for microalgal cultivation using the simulated gas containing CO 2 have been thoroughly studied, articles about the actual application of industrial flue gas to microalgal cultivation are still scarce, paticularly about photobiroeactor for maximizing the mass transfer of CO 2 to cells in an aqueous environment, as shown in Table 4. Various types of photobireactors, such as airlift, column, and the thin-film column, have been proposed to mitigate CO 2 from flue gas [4][5][6]8,[22][23][24][25][26][27][28]51].The variation in the unit working volume of each PBR was 1.5 to 1000 L for airlift PBR, 0.18 to 50 L for column PBR, and 5 L to 100 L thin-film column PBR.As mentioned previously, the majority of targeted products based on the flue gas strategy was biofuels, such as biodiesel, because microalgae could easily absorb and adsorb toxic molecules, such as heavy metals derived from the flue gas.In this study, the LNG-fired flue gas was directly used without further purification in this study.Nonetheless, the critical amount of heavy metals detrimental to health was not detected from the Haematococcus extracts (Table 2).
Although various combined physical/chemical methods have been proposed to extract astaxanthin from H. pluvialis due to the unique structure of robust cell wall and rigid cell membrane in cyst cells [52,53], the cells were easily homogenized by ball mill treatment using zirconia beads without an organic solvent.Therefore, acetone could not be detected by compliance testing from KFDA.Above all, microbial contaminants also could not be detected.It is implied that the HClO water-based contamination control technology is highly efficient for quality control of the outdoor astaxanthin production process of H. pluvialis.
The mixture powder of ruptured H. pluvialis biomass and astaxanthin extract, which was derived from the LNG-fired flue gas, successfully improved the immune system of poultry without exhibiting any side effects in this study.It has been previously reported that when the egg-laying hens uptake the microalgal biomass, omega-3 fatty acids (ALA, EPA, and DHA) as well as astaxanthin, they are not easily digested, but accumulate in the yolk of an egg laid by the poultry [54][55][56][57][58].The recommended daily intake of astaxanthin is about 5 mg day −1 for human.The astaxanthin and omega-3 containing eggs could be a popular choice for everyone at dining tables, thereby increasing a probable profit of the related corporations with large industrial point sources emitting considerable waste CO 2 .

Figure 1 .
Figure 1.Overall process scheme for mass cultivation of microalgae without microbial contamination.

Figure 1 .
Figure 1.Overall process scheme for mass cultivation of microalgae without microbial contamination.

Figure 2 .
Figure 2. Schematic illustration and actual photographs of two-stage strategy.

Figure 2 .
Figure 2. Schematic illustration and actual photographs of two-stage strategy.

Figure 3 .
Figure 3.Comparison of (A) biomass production (diamond), (B) astaxanthin content (square), and (C) astaxanthin concentration (circle) from H. pluvialis cells of wild type (white) and the mutant PP-PS 160 (black) under moderate temperature conditions in spring.(D) Photosynthetic photon flux density (PPFD) (black triangle) and medium temperature (white triangle) during the daylight period in spring (March-May).Each value is mean ± standard deviation of three replicates.

Figure 3 .
Figure 3.Comparison of (A) biomass production (diamond), (B) astaxanthin content (square), and (C) astaxanthin concentration (circle) from H. pluvialis cells of wild type (white) and the mutant PP-PS 160 (black) under moderate temperature conditions in spring.(D) Photosynthetic photon flux density (PPFD) (black triangle) and medium temperature (white triangle) during the daylight period in spring (March-May).Each value is mean ± standard deviation of three replicates.

Figure 4 .
Figure 4. Comparison of (A) biomass production (diamond), (B) astaxanthin content (square), and (C) astaxanthin concentration (circle) from H. pluvialis cells of wild type (white) and the mutant PP-PS 160 (black) under high-temperature conditions in summer.(D) Photosynthetic photon flux density (PPFD) (black triangle) and medium temperature (white triangle) during the daylight period in spring (June-August).Each value is mean ± standard deviation of three replicates.

Figure 4 .
Figure 4. Comparison of (A) biomass production (diamond), (B) astaxanthin content (square), and (C) astaxanthin concentration (circle) from H. pluvialis cells of wild type (white) and the mutant PP-PS 160 (black) under high-temperature conditions in summer.(D) Photosynthetic photon flux density (PPFD) (black triangle) and medium temperature (white triangle) during the daylight period in spring (June-August).Each value is mean ± standard deviation of three replicates.

Figure 5 .
Figure 5.Comparison of the contents of protein, carbohydrate, and lipid from H. pluvialis cells of wild type and the mutant PP-PS 160 at the end of each stage of green and red during spring and summer conditions.Each value is mean ± standard deviation of three replicates.

Figure 5 .
Figure 5.Comparison of the contents of protein, carbohydrate, and lipid from H. pluvialis cells of wild type the mutant PP-PS 160 at the end of each stage of green and red during spring and summer conditions.Each value is mean ± standard deviation of three replicates.

Figure 6 .
Figure 6.Morphological change of H. pluvialis cysts after mechanical pretreatment of ball mill for 12 h.

Figure 6 .
Figure 6.Morphological change of H. pluvialis cysts after mechanical pretreatment of ball mill for 12 h.

Table 1 .
Comparison of biomass production, astaxanthin production, and CO2 removal rate between H. pluvialis cells of wild type and mutant under outdoor spring and summer conditions.

Table 2 .
Korea Food and Drug Administration (KFDA) approval and compliance testing for the H. pluvialis extracts derived from the LNG-fired flue gas.

Table 3 .
Effect of dried H. pluvialis extracts on the physiological activity and immune system of poultry.