Enhanced astaxanthin extraction efficiency from Haematococcus pluvialis via the cyst germination in outdoor culture systems

Accepted Manuscript Title: Enhanced astaxanthin extraction efficiency from Haematococcus pluvialis via the cyst germination in outdoor culture systems Author: Yoon Young Choi Min-Eui Hong Sang Jun Sim PII: DOI: Reference:

S1359-5113(15)30075-1 http://dx.doi.org/doi:10.1016/j.procbio.2015.09.008 PRBI 10511

To appear in:

Process Biochemistry

Received date: Revised date: Accepted date:

2-6-2015 4-9-2015 5-9-2015

Please cite this article as: Choi Yoon Young, Hong Min-Eui, Sim Sang Jun.Enhanced astaxanthin extraction efficiency from Haematococcus pluvialis via the cyst germination in outdoor culture systems.Process Biochemistry http://dx.doi.org/10.1016/j.procbio.2015.09.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Title: Enhanced astaxanthin extraction efficiency from Haematococcus pluvialis via

2

the cyst germination in outdoor culture systems

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Authors: Yoon Young Choia, 1, Min-Eui Hongb, 1, and Sang Jun Sima,c,*

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a

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South Korea

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b

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South Korea

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c

Department of Chemical and Biological Engineering, Korea University, Seoul 136-713,

Department of Chemical Engineering, Sungkyunkwan University, Suwon 440-746,

Green School, Korea University, Seoul 136-713, South Korea

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These authors contributed equally to this work

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*Corresponding author: Sang Jun Sim

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Tel.: +82 2 3290 4853; fax: +82 2 926 6102.

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E-mail: [email protected]

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Running title: Effect of germination on the astaxanthin extraction in H. pluvialis

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Graphical abstract

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2

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Highlights 

Cyst germination method was used for efficient astaxanthin extraction in outdoors.



Light was a limiting factor for thegermination rate under autotrophic conditions.



Astaxanthin concentration was highly stable during autotrophic germination.



Astaxanthin extraction efficiency was enhanced by dividing cysts.

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Abstract

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Haematococcus pluvialis is the richest source of natural astaxanthin (3S, 3′S), but the

5

rigid cell wall of mature red cyst (aplanospore) complicates the efficient extraction of

6

astaxanthin from the strain. Herein, the cyst germination method was developed by

7

using nitrate and light for practical application for an efficient astaxanthin extraction

8

from Haematococcus cells cultured in outdoor condition where flue gas, solar radiation

9

and photobioreactor were used. Notably, autotrophic germination rate was easily

10

regulated by light intensity. Under conditions of low germination rate, total astaxanthin

11

concentration was highly maintained and astaxanthin extraction efficiency was rapidly

12

enhanced during autotrophic germination. As a result, under homogenization (30 sec),

13

the extracted astaxanthin concentration in the cells treated with 1 mM KNO3–150

14

μE/m2/s was highly increased by 58% compared to the cells treated without germination.

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Our technical solution will definitely improve an astaxanthin extraction titer with the

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practical application in outdoor Haematococcus culture system.

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Keywords: Haematococcus pluvialis; Outdoor autotrophic culture; Astaxanthin; Cyst

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germination; nitrate and solar radiation

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1. Introduction

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Astaxanthin (3,3′-dihydroxy-β-carotene-4,4′-dione), which is one of the most

3

powerful antioxidants among carotenoids, is extensively used as a pigment source in

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aquaculture and also gained broad application in pharmaceutical and nutraceutical

5

industries due to the strong antioxidant property [1]; astaxanthin possesses 500-fold and

6

38-fold stronger free radical antioxidant activity of vitamin E and β-carotene,

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respectively [2–4]. Haematococcus pluvialis accumulates high levels of the red

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ketocarotenoid pigment, natural astaxanthin up to 4% of its dry mass [5]. The market

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price of natural astaxanthin from H. pluvialis is approximately $7000 per kg because it

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is preferred for human consumption [6].

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H. pluvialis has various phases in lifecycle of differentiating morphology [7]. Under

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low stress conditions, H. pluvialis exists as a flagellate (green motile cell) and a

13

palmelloid (green nonmotile cell) during vegetative growth. Under high stress

14

conditions such as high irradiance, nutrition deficiency, high salinity and drought,

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Haematococcus cells show morphological and physiological transformation [8–11] in

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which green flagellates are turned into red cysts (aplanospore) to accumulate

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astaxanthin. However, under favorable conditions, germination is occurred to form

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flagellated zoospores in order to start a new vegetative growth [7].

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Various methods of cell wall disruption such as bead-beating, sonication, microwaves,

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acid- and alkali-treatment, enzyme lysis and supercritical CO2 have been investigated

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for efficient astaxanthin extraction from astaxanthin-enriched aplanospore in H.

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pluvialis [12]. However, compared to a flagellate, an aplanospore has a rigid cell wall

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which is made up of sporopollenin-like material, algaenan, thereby hindering solvent

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extraction of astaxanthin [13–15]. To enhance the extraction efficiency of astaxanthin 5

1

from H. pluvialis, cell wall-deficient mutant was isolated by strain improvement of

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upstream process [16].

3

Herein, under outdoor autotrophic conditions in which flue gas and solar radiation

4

were used, astaxanthin extraction efficiency from H. pluvialis was successfully

5

improved via germination by controlling nitrogen supplement and light intensity.

6

Notably, in the conditions of high cell density of aplanospore, one of the most crucial

7

factors for an increase in germination rate was a light intensity. Consequently, under

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conditions of 1 mM KNO3 and 150 μE/m2/s solar irradiance, maximal astaxanthin

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extraction was obtained after 3 days of autotrophic germination. Above all, high amount

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of dividing cysts in the cells cultured with 1 mM KNO3 and 150 μE/m2/s solar

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irradiance were easily harvested without additional costs and energy prior to release of

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zoospores from mature red cyst (aplanospore). In large-scale outdoor Haematococcus

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culture system, the practical application of the germination strategy will surely reduce

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energy costs for astaxanthin extraction from H. pluvialis by efficiently weakening the

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rigid cell wall of aplanospore.

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2. Materials and Methods

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2.1. Algal strain and culture conditions

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H. pluvialis NIES-144 (wild-type) was purchased from the National Institute for

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Environmental Studies in Tsukuba, Japan. The strain was cultured photoautotrophically

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in NIES-C and NIES-N media during the green and red stages, respectively [17–18].

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NIES-C medium (pH 7.5) of green stage is comprised of 0.15 g/L Ca(NO3)2, 0.10 g/L 6

1

KNO3, 0.05 g/L β-glycerophosphoric acid disodium salt pentahydrate, 0.04 g/L

2

MgSO4∙7H2O, 0.50 g/L Tris-aminomethane, 0.01 mg/L thiamine, 0.10 μg/L biotin, 0.10

3

μg/L vitamin B12, and 3.00 mL/L PIV metal solution, which consisted of 1.0 g/L

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Na2EDTA, 0.196 g/L FeCl3∙6H2O and (in mg/L) 36.0 MnCl2∙4H2O, 22.0 ZnSO4∙7H2O,

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4.0 CoCl2∙6H2O, and 2.5 Na2MoO4∙2H2O. The NIES-N medium (pH 7.5) of red stage

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was prepared by excluding an N source from the NIES-C medium, by substituting (in

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gram per liter) CaCl2·2H2O 0.13 and KCl 0.07 for Ca(NO3)2 and KNO3, respectively,

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for supplement of calcium and potassium ions.

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In this study, a two-stage induction strategy was applied to improve astaxanthin

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production in H. pluvialis under outdoor photoautotrophic conditions. In the first

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(“green”) stage, the cultures were fully grown in the N-replete medium (NIES-C, pH

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7.5) under weak lights (~35 μE/m2/s) for 21 days (green stage). In the second (“red”)

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stage, the cells were transferred to N-deplete medium (NIES-N, pH 7.5) and cultured

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under strong lights (~300 μE/m2/s) over 42 days (red stage) for highly efficient

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induction by facilitation of rapid nitrogen starvation and photo-inducibility. After 42

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days of autotrophic induction, the concentration of biomass and astaxanthin was

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maximally reached to 3.82 g/L and 150.8 mg/L, respectively (Figure S1).

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After that, autotrophically induced red cyst cells (aplanospore) were germinated for

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efficient astaxanthin extraction from mature red cysts by weakening the rigid cell wall

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of aplanospore using nitrate and light. To demonstrate the effect of germination on the

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astaxanthin extraction efficiency, different levels of nitrate (KNO3) (1.0–2.0 mM) and light

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intensity (150–300 μE/m2/s) were used in outdoors for germinating the mature red cyst cell

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(aplanospore). Under the outdoor autotrophic conditions, Haematococcus cells experienced

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daily variations in the light intensity and temperature of solar radiation. Therefore, during 6 7

1

days of germination, cells were maximally exposed to light intensities of 150 μE/m2/s for

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moderate light conditions and 300 μE/m2/s for high light conditions (Figure S2).

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Cultures were also roughly maintained in outdoors between 17.5°C and 27.5°C without

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temperature control (Figure S2).

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The outdoor Haematococcus culture system was developed for natural astaxanthin

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production using flue gas and natural solar radiation. The outdoor Haematococcus

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culture system for natural astaxanthin production was installed near the thermal power

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plant, Korea District Heating Corporation located at Baekhyeon-dong, Bundang-gu,

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Seongnam-si, Gyeonggi-do, in the Republic of Korea (latitude: 37°00'00"North,

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longitude: 127°30'00"East) [19].

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In the system, our previously developed thin-film 25 L-photobioreactor (PBR)

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constructed from a polymer film (CPP; polypropylene-based cast polypropylene) was

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used for outdoor culture of H. pluvialis [20–21]. In the PBR, cells were cultured with

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flue gas, which was composed of 3.5 ± 0.5% (v/v) CO2, 1.36 ± 4.12 ppm CO, 10.17 ±

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0.73% O2, and 21.63 ± 3.54 ppm NOx, at a flow rate of 0.2 vessel volumes per min

16

(vvm). During the outdoor culture period, control of the photon flux density was applied

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by using shade.

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2.2. Analytical methods

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2.2.1. Measurement of dry cell weight and cell count

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Cell biomass was determined by filtering aliquots of samples using GF/F microfiber

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filter paper (Whatman, Cambridge, UK). 10 ml of cell suspensions were filtered with 8

1

pre-weighed filters and dried at 100°C dry oven overnight to determine the biomass.

2

Nitrates in the culture were analyzed by ion chromatography (DIONEX 500,

3

Chelmsford, MA, USA).

4

The cells in the germination stage were classified into 4 types, namely mature cyst,

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dividing cyst with cell divisions, dividing cyst with differentiated zooids, releasing cyst

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with differentiated zooids, and were counted at 1 day intervals using an improved

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Neubauer counting chamber (C-Chip, DHC-N01, iNCYTO, Korea) [22].

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2.2.2. Measurement for photosynthetic pigment (chlorophyll, carotenoid, and astaxanthin) analysis

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To assay intracellular pigments, cell suspensions (10 mL) were collected by

13

centrifuging the culture fluid at 5,000 rpm for 5 min at 4°C, discarding the supernatant,

14

and rinsing the cell pellet with pre-chilled TE buffer (pH 7.5). The pellet was

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homogenized with Tissue Lyser II (Qiagen, Valencia, CA, USA) using pre-chilled

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Tissue Lyser adaptors in pre-chilled 100% methanol to extract pigments. The extraction

17

procedure was repeated until the cell debris was colorless. The homogenized lysate was

18

centrifuged at 15,000 rpm for 10 min at 4°C to separate the supernatant and cell debris.

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The extracts (supernatants) were used to measure chlorophyll and astaxanthin. Total

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intracellular concentrations of chlorophyll were assayed using UV spectrophotometry

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[23].

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Astaxanthin concentrations were quantified by a Shimadzu high performance liquid

23

chromatography (HPLC) system equipped with two LC-10AD pumps and SPD-10A

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UV-Vis detector (Shimadzu, Japan) [17, 24–25]. The extracts were saponified with 0.01 9

1

M NaOH (in methanol) and separated using a 250x4.6 mm HS-303 hydrosphere C18

2

column (YMC, Japan). The mobile phase consisted of solvents A (dichloromethane :

3

methanol : acetonitrile : water, 5.0 : 85.0 : 5.5 : 4.5, v/v) and B (dichloromethane :

4

methanol : acetonitrile : water, 22.0 : 28.0 : 45.4 : 4.5, v/v). For the effective separation

5

of free astaxanthin, the following linear gradient from 0 to 100% B for 12 min, and

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100% B for 50 min. The flow rate was 1.0 ml/min and the peaks were measured at 480

7

nm [17, 25].

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2.2.3. Analysis of astaxanthin extraction efficiency

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In our study, 120 min of homogenization treatment was sufficient for completely

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breaking the cell wall of astaxanthin-enriched Haematococcus cells in 10 ml of the

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fermented sample containing about 40 mg of mature red cysts (Figure S3). Accordingly,

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we determined the astaxanthin concentration after 2 hour of homogenization treatment

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is a total astaxanthin (100%) of the cell. Subsequently, 30 min of homogenization was

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highly efficient for astaxanthin extraction from 10 ml of the fermented sample containing

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high amounts of the dividing cyst with differentiated zooids (Figure S3). Therefore,

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astaxanthin extraction efficiency was determined by comparing quantities of extracted

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astaxanthin after sufficient (120 min) and deficient (30 min) homogenization treatment;

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the percentage (%) of extraction efficiency was determined by the equation below.

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1 2

3. Results and discussion

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3.1. Effect of nitrate and light intensity on the cyst germination in H. pluvialis under

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outdoor autotrophic conditions

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After final stage of outdoor autotrophic induction, mature red cyst cells were exposed

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to strong light intensities (150–300 μE/m2/s) and nitrate-replete conditions with

9

supplementation of KNO3 (1.0–2.0 mM). Overall, after germination, biomass

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concentrations were increased, but astaxanthin contents were diminished (Figure 1).

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Accordingly, astaxanthin concentration was reduced gradually. As expected, the cells

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cultured with 2 mM nitrate showed a higher reduction rate of astaxanthin concentration than

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that of the cells cultured with 1 mM nitrate, but the reduction rate was also significantly

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affected by light intensity (Figure 1).

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It was previously demonstrated that light is essential for the life cycle of H. pluvialis,

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particularly for cell differentiation (encystment and germination) [7]. In our study, under

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initial conditions of high cell density of mature red cyst (aplanospore), nitrate consumption

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rate in the cells cultured at 300 μΜ/m2/s was much higher than in the cells cultured at 150

19

μΜ/m2/s (Figure 2). The results imply that light intensity was a limiting factor for efficient

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autotrophic germination than nitrate in H. pluvialis.

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Four days later, astaxanthin concentration was increased progressively in the cells

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cultured with 1.0 mM KNO3 and 300 μE/m2/s. As shown in Figure 2, 1 mM of nitrate

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was completely exhausted after 2 days of germination. Accordingly, it was estimated

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that the high amounts of germinated mature cyst cells (4.5–4.7 g/L) were easily exposed 11

1

to N-starvation after nitrate consumption, thereby augmenting astaxanthin accumulation

2

again.

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For germination to occur, both nitrogen source and light energy are essential. In the light-

4

exposed Haematococcus cells without nitrate supplementation, extraction efficiency and

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concentration of astaxanthin were nearly identical to that in the mature cysts without

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germination (data not shown). This was because completely dormant cysts did not go

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through an additional biosynthesis of astaxanthin even with the presence of light (150–300

8

μE/m2/s).

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3.2. Effect of the low germination rate on the astaxanthin extraction efficiency in H.

11

pluvialis under outdoor autotrophic conditions

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To evaluate astaxanthin extraction efficiency, the values of extracted astaxanthin

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concentration obtained by insufficient treatment of homogenization for 30 min were

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calculated. As shown in Figure 3a, astaxanthin extraction efficiency was highly light-

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dependent during autotrophic germination in H. pluvialis. After 2 days of germination,

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astaxanthin extraction efficiency in the cells exposed to 300 μE/m2/s was diminished

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gradually because released zoospores (flagellates) from mature cyst (aplanospores) are

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partially transformed into the immature or mature cysts (Palmelloids or aplanospores)

20

(data no shown).

21

The extracted astaxanthin concentration was maximally reached to 131.73 mg/L at 3

22

days of germination under conditions of 1 mM KNO3 and 150 μE/m2/s (Figure 3b);

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after that it was reduced progressively. As a result, the extracted astaxanthin

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concentration in the cells treated with 1 mM KNO3 and 300 μE/m2/s was highly 12

1

increased by 31% compared to that of the cells treated without germination (Figure 3b).

2

Moreover, the extracted astaxanthin concentration in the cells treated with 1 mM KNO3

3

and 150 μE/m2/s was remarkably increased by 58% compared to that of the cells treated

4

without germination (Figure 3b).

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3.3. Analysis of chlorophyll contents and relative distribution in three cell types (mature

7

cyst, dividing cyst and releasing cyst) during germination in H. pluvialis under outdoor

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autotrophic conditions

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Germination coincided with chlorophyll and protein syntheses and carotenoid

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degradation in H. pluvialis [7]. Overall, during autotrophic germination, chlorophyll

12

contents were increased, but an increase in chlorophyll contents was more rapid in the

13

cells exposed to 300 μE/m2/s compared to that of the cells exposed to 150 μE/m2/s

14

(Figure 4). These results imply that astaxanthin contents in the cells cultured under a

15

light intensity of 150 μE/m2/s were highly maintained during early stages of

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germination.

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As morphological changes occur during germination, cells were observed by light

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microscopy and classified into the following four groups: mature cyst, dividing cyst

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with cell divisions, dividing cyst with differentiated zooids, releasing cyst with

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differentiated zooids (Figure 5). During 3 days of autotrophic germination, the astaxanthin

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concentration in the cells exposed to 150 μE/m2/s was highly maintained, but, after that, the

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reduction rate in astaxanthin concentration was increased (Figure 1c). It was because that

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rapid generation of releasing cysts (zoospores or flagellates) from dividing cysts facilitated

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to degrade astaxanthin, but to synthesize chlorophyll (Figure 4 and 6). 13

1

The cyst wall of H. pluvialis is composed of three layers; primary wall is the most

2

external layer called as trilaminar sheath, second wall is the middle layer and tertiary wall is

3

the most internal layer of the cell wall. Notably, during the germination of aplanospore,

4

both trilaminar sheath and secondary wall normally break down in the dividing cysts [15,

5

22]. Therefore, under exposure to 150 μE/m2/s, astaxanthin extraction efficiency could be

6

successfully enhanced by a weakened cell wall of dividing cysts in comparison with that of

7

mature cysts.

8

In our study, during autotrophic germination of mature red cyst (aplanospore), an

9

intracellular content of astaxanthin was primarily sensitive to light intensity. Although the

10

cells cultured under exposure to 300 μE/m2/s exhibited a high reduction rate in

11

astaxanthin content, it was highly maintained under conditions of 150 μE/m2/s during the

12

early stages of germination. It was recently reported that the dividing cysts and zooid

13

cells could still retain their astaxanthin content for a short period before becoming

14

completely acclimatized to the favorable environment [22]. Therefore, when the cyst

15

germination method was used for efficient astaxanthin extraction, particularly in outdoors in

16

which solar irradiance is used, the light intensity should be controlled carefully.

17

Under conditions of 300 μE/m2/s solar irradiance, an increase in chlorophyll contents

18

was related to the rapid differentiation rate via the transformation of mature cysts to

19

dividing and releasing cysts (Figure 4 and 6). However, an increase of releasing cysts

20

(zoospores or flagellates) complicates cell harvest. Remarkably, when the germination

21

progressed slowly under exposure to 150 μE/m2/s, dividing cyst was much formed

22

instead of releasing cyst due to light limitation (Figure 6). This result clearly indicates

23

that majority of cells can be easily harvested by gravity without additional cost and

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energy. 14

1

Until now, due to its high efficiency, ‘supercritical carbon dioxide (SC-CO2)’ was

2

widely used in the industry for extraction of natural astaxanthin from H. pluvialis,

3

which has a tough cell wall. However, although SC-CO2 is a highly efficient strategy

4

for extracting astaxanthin from the mature red cysts (51– 97%), most studies of SC-CO2

5

experimented with disrupted cyst cells [26]. Generally, the pretreatment for breaking

6

rigid cell walled Haematococcus cysts is both energy-intensive and time-consuming,

7

whereas the cost-effective astaxanthin production will be achieved by using the

8

germination method with remarkable extraction efficiency under the conditions of

9

minimizing the loss in astaxanthin recovery yield.

10

In summary, cyst germination method was developed by using nitrate and solar

11

radiation for practical application in outdoor Haematococcus culture, particularly in a

12

large-scale closed photobioreactor system. Compared to the mature red cyst cells

13

without germination treatment, the total astaxanthin concentration was reduced by 4%

14

under the conditions of germination with 1 mM KNO3 and 150 μE/m2/s, but astaxanthin

15

extraction efficiency was enhanced by 56.8% under the conditions of homogenization

16

for 30 sec, thereby maximally achieving 87.3% and 78.6% (released zooids excluded)

17

of total astaxanthin recovery yield using the cyst germination method. Unfortunately,

18

although an economic viability of this strategy was not demonstrated in this study, we

19

can estimate that our germination methods will definitely help to reduce energy

20

consumption for pretreatment in disrupting enormous amounts of Haematococcus

21

biomass, thereby obtaining highly astaxanthin-enriched concentrate and making the

22

whole process economically feasible, in an industrial scale.

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Acknowledgements 15

1

This work was supported by the “Energy Efficiency & Resources Technology R&D”

2

project (grant No. 20152010201900) of the Korea Institute of Energy Technology

3

Evaluation and Planning (KETEP) grant funded by the Korea government Ministry of

4

Trade, Industry & Energy (MOTIE) and supported by the National Research

5

Foundation (NRF) grants (grant No. NRF-2013R1A2A1A01015644/2010-0027955),

6

University-Institute Cooperation Program (2013) and also supported by the Korea CCS

7

R&D Center (KCRC) grant (grant No. 2014M1A8A1049278), funded by the Korean

8

Government Ministry of Science, ICT & Future Planning (MSIP).

16

1

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Appl Microbiol Biotechnol 2005;68:237–241.

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[18] Kang CD, Lee JS, Park TH, Sim SJ. Complementary limiting factors of

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astaxanthin synthesis during photoautotrophic induction of Haematococcus

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pluvialis: C/N ratio and light intensity. Appl Microbiol Biotechnol 2007;74:987–

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994.

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[19] Yoon SY, Hong ME, Chang WS, Sim SJ. Enhanced biodiesel production in

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photobioreactors. Bioprocess Biosyst Eng 2015; DOI 10.1007/s00449-015-1383-x.

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[20] Yoo JJ, Choi SP, Kim BW, Sim SJ. Optimal design of scalable photo-bioreactor

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for phototropic culturing of Haematococcus pluvialis. Bioprocess Biosyst Eng

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2012;35:309–315.

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[21] Yoo JJ, Choi SP, Kim JYH, Chang WS, Sim SJ. Development of thin-film photo-

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bioreactor and its application to outdoor culture of microalgae. Bioprocess Biosyst

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Eng 2013;36:729–736.

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[22] Praveenkumar R, Lee K, Lee J, Oh Y-K. Breaking dormancy: an energy-efficient

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and Characterization by UV-VIS Spectroscopy. Curr Protoc in Food Analyt Chem

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[24] Gilmore AM, Yamamoto HY. Zeaxanthin formation and energy dependent 19

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cyclic electron transport. Plant Physiol 1991; 96: 635–643.

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[25] Han D, Wang J, Sommerfeld M, Hu Q. Susceptibility and protective mechanisms

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9

20

1 2

List of figures

3 4

Fig. 1. Comparison of (a) the cell density from dry weight, b) the astaxanthin content

5

and (c) the total astaxanthin concentration (sufficient extraction: 120 min) among the

6

four Haematococcus cells cultured under conditions of nitrate (KNO3) (1.0–2.0 mM) and

7

light intensities (150–300 μE/m2/s) during autotrophic germination for 6 days: 1 mM

8

KNO3–150 μE/m2/s (▲), 2 mM KNO3–150 μE/m2 /s (△), 1 mM KNO3–300 μE/m2/s (●),

9

2 mM KNO3–300 μE/m2/s (○). Each value represents the mean ± standard deviation of

10

three replicates.

11 12

Fig. 2. Comparison of residual nitrate concentration in the among the four

13

Haematococcus cells cultured under conditions of nitrate (KNO3) (1.0–2.0 mM) and light

14

intensities (150–300 μE/m2/s) during autotrophic germination for 6 days: 1 mM KNO3–

15

150 μE/m2/s (▲), 2 mM KNO3–150 μE/m2/s (△), 1 mM KNO3–300 μE/m2/s (●), 2 mM

16

KNO3–300 μE/m2/s (○). Each value represents the mean ± standard deviation of three

17

replicates.

18 19

Fig. 3. Comparison of (a) the astaxanthin extraction efficiency (insufficient extraction:

20

30 min), (b) the astaxanthin concentration (insufficient extraction: 30 min) among the

21

four Haematococcus cells cultured under conditions of nitrate (KNO3) (1.0–2.0 mM) and

22

light intensities (150–300 μE/m2/s) during autotrophic germination for 6 days: 1 mM

23

KNO3–150 μE/m2/s (▲), 2 mM KNO3–150 μE/m2 /s (△), 1 mM KNO3–300 μE/m2/s (●),

21

1

2 mM KNO3–300 μE/m2/s (○). Each value represents the mean ± standard deviation of

2

three replicate.

3 4

Fig. 4. Comparison of the chlorophyll content among the four Haematococcus cells

5

cultured under conditions of nitrate (KNO3) (1.0–2.0 mM) and light intensities (150–300

6

μE/m2/s) during autotrophic germination for 4 days: 1 mM KNO3–150 μE/m2/s (black

7

bar), 2 mM KNO3–150 μE/m2/s (white bar), 1 mM KNO3–300 μE/m2/s (Grey bar), 2

8

mM KNO3–300 μE/m2/s (dashed bar). Each value represents the mean ± standard

9

deviation of three replicate.

10 11

Fig. 5. Time-course differentiation of mature red cyst of H. pluvialis during germination

12

process. (a) Mature cyst. (b) Dividing cyst with cell divisions. (c) Dividing cyst with

13

differentiated zooids. (d) Releasing cyst with differentiated zooids. Scale bars = 10 μm.

14 15

Fig. 6. Comparison of relative distribution (%) in three cell types (mature cyst, dividing

16

cyst and releasing cyst) among the four Haematococcus cells cultured under conditions

17

of nitrate (KNO3) (1.0–2.0 mM) and light intensities (150–300 μE/m2/s) during

18

autotrophic germination for 4 days: mature cyst (black bar), dividing cyst (dashed bar),

19

releasing cyst (white bar). Data shown represent mean values obtained from three

20

independent experiments.

21 22 23 24 22

1

Fig. 1

6.0

Astaxanthin concentration (mg/L)

Astaxanthin content (mg/g biomass)

Biomass concentration (g/L)

(a) 5.5 5.0 4.5 4.0

(b) 40 35 30 25 20

(c) 150 140 130 120 110 100 0

2

1

2

3

4

Time after germination (day) 23

5

6

Residual nitrate concentration (mM)

Fig. 2

2.0

1.5

1.0

0.5

0.0 0

1

2

3

4

Time after germination (day)

24

5

6

Astaxanthin concentration (mg/L)

Astaxanthin extraction efficiency (%)

Fig. 3

(a)

90

80

70

60

140

(b)

120

100

80

0

1

2

3

25

4

Time after germination (day)

5

6

Fig. 4

Chlorophyll content (mg/g biomass)

25 20 15 10 5 0 0

1

2

3

4

5

Time after germination (day)

26

6

Fig. 5

27

0

300 μE/m /s 2

2 mM KNO3 1 mM KNO3

300 μE/m /s 2

28 150 μE/m /s 2

2 mM KNO3

150 μE/m /s

2

1 mM KNO3

6 day

5 day

4 day

3 day

2 day

1 day

0 day

6 day

5 day

4 day

3 day

2 day

1 day

0 day

6 day

5 day

4 day

3 day

2 day

1 day

0 day

6 day

5 day

4 day

3 day

2 day

1 day

0 day

Relative distribution (%)

Fig. 6

100

90

80

70

60

50

40

30

20

10