Optimization of Culture Media for Large-Scale Lutein Production by Heterotrophic Chlorella vulgaris Jin Young Jeon Dept. of Biomaterials Science and Engineering, Yonsei University, Seoul 120-749, Korea Dept. of Health Food Research & Development of Daesang Corp., Icheon 467-813, Korea

Ji-Sue Kwon, Soon Tae Kang, Bo-Ra Kim, Yuchul Jung, Jae Gap Han, and Joon Hyun Park Dept. of Health Food Research & Development of Daesang Corp., Icheon 467-813, Korea

Jae Kwan Hwang Dept. of Biotechnology & Dept. of Biomaterials Science and Engineering, Yonsei University, Seoul 120-749, Korea DOI 10.1002/btpr.1889 Published online in Wiley Online Library (wileyonlinelibrary.com)

Lutein is a carotenoid with a purported role in protecting eyes from oxidative stress, particularly the high-energy photons of blue light. Statistical optimization was performed to growth media that supports a higher production of lutein by heterotrophically cultivated Chlorella vulgaris. The effect of media composition of C. vulgaris on lutein was examined using fractional factorial design (FFD) and central composite design (CCD). The results indicated that the presence of magnesium sulfate, EDTA-2Na, and trace metal solution significantly affected lutein production. The optimum concentrations for lutein production were found to be 0.34 g/L, 0.06 g/L, and 0.4 mL/L for MgSO47H2O, EDTA-2Na, and trace metal solution, respectively. These values were validated using a 5-L jar fermenter. Lutein concentration was increased by almost 80% (139.64 6 12.88 mg/L to 252.75 6 12.92 mg/L) after 4 days. Moreover, the lutein concentration was not reduced as the cultivation was scaled up to 25,000 L (260.55 6 3.23 mg/L) and 240,000 L (263.13 6 2.72 mg/L). These observations C 2014 American Institute of Chemical suggest C. vulgaris as a potential lutein source. V Engineers Biotechnol. Prog., 000:000–000, 2014 Keywords: fractional factorial design, central composite design, lutein, heterotrophic, Chlorella vulgaris

Introduction Carotenoids are colorful, fat-soluble pigments synthesized by plants and microorganisms that serve a variety of roles in cellular biology, primarily photosynthetic in nature. Carotenoids are members of a class of tetraterpenoid organic pigments with a 40-carbon backbone and a large conjugated double-bond system.1 Carotenoids are an important group of compounds because of their critical roles in photosynthesis and photoprotection in plants and algae; absorption of light energy for use in photosynthesis and protection of chlorophyll from photodamage,2 with wide applicability as foodcoloring agents,3 in addition to their physiological roles as antioxidants and blue light filters in human tissues.4,5 Over 600 known, naturally occurring carotenoids exist, and these are divided into two classes: xanthophylls (which contain oxygen)—of which lutein is a member6—and carotenes (which are solely hydrocarbons and contain no oxygen). Lutein is an antiAdditional Supporting Information may be found in the online version of this article. Correspondence concerning this article should be addressed to J.K. Hwang at [email protected]. C 2014 American Institute of Chemical Engineers V

oxidant for blue light absorption and has garnered increasing attention recently due to its potential role in ameliorating agerelated macular degeneration. The total value of the eye care market in the United States has been estimated at $140 million with an annual growth rate of 5%. The global lutein market is expected to reach $125 million within the next 3 years.7 Unlike secondary carotenoids, which are influenced by environmental factors, including other carotenoids (e.g., astaxanthin, canthaxanthin, and echinenone), lutein is a primary carotenoid directly associated with photosynthesis.8 Currently, lutein is produced from marigold oleoresin; however, large-scale farming of marigolds is impractical due to the large acreage required, as well as both seasonal and climactic variances. Compared with higher plants, microalgae possess a distinct advantage: they can be cultivated in bioreactors, thereby resulting in a continuous and reliable source of the product.9,10 Microalgae can be a source of considerable quantities of lutein, depending on two primary variables: lutein content and biomass productivity. Additional variables, such as the presence of a cell wall or the percent content of other carotenoids, may also be a consideration.7 Chlorella vulgaris is a type of microalgae that is an edible and particularly prolific source of lutein.11 1

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The aim of this research was to determine the optimal media for the accumulation of lutein in C. vulgaris during commercial-scale cultivation, to facilitate the high-yield production of lutein in C. vulgaris under heterotrophic media.

Materials and Methods Microalgal strains and culture conditions C. vulgaris was isolated from water samples aseptically collected from several reservoirs around Seoul, Gyeongsang-Do, and Cheongchung-Do in South Korea. One milliliter of each of the test samples was transferred to 500-mL flasks containing a 50-mL volume of autoclaved proprietary media and incubated for approximately 7–10 days in a shaking incubator at 30 C and 120 rpm under dark conditions. Subcultures were prepared by adding the inoculum into serial dilutions and onto petri plates containing proprietary media solidified with 2% (w/v) bacteriological agar (BD Diagnostics, Sparks Glencoe, MD, USA). The plates were incubated under the same conditions at the flask for up to 7–10 days. After incubation, colonies from the petri plates with a healthy, dark green color were selected. These steps were repeated three times for each of the original flasks and selected colonies. An isolated microalgal strain was deposited as C. vulgaris DSV77 (KCTC 11383BP) in the Korea Research Institute of Bioscience and Biotechnology (KRIBB, Daejon, South Korea). Stock cultures of C. vulgaris were maintained both in agar slants and liquid cultures of DS (Daesang Corp., Seoul, South Korea) medium, consisting of 10 g/L of glucose, 0.3 g/L of K2HPO4, 0.7 g/L of KH2PO4, pH 6.3, 0.3 g/L of MgSO47H2O, 0.015 g/L of FeSO47H2O, 0.05 g/L of EDTA2Na, 0.025 g/L of urea, and 1 mL of trace metals solution. Trace metal solution consists of 0.0029 g/L of H3BO3, 0.002 g/L of MnCl24H2O, 0.0022 g/L of ZnSO47H2O, 0.0008 g/L of CuSO45H2O, and 0.0005 g/L of (NH4)6Mo7O244H2O. Experimental setup Optimization of C. vulgaris using statistical methods was performed in 500-mL flasks. The media and flasks were sterilized in an autoclave for 20 min at 121 C. Incubation of cultures was performed using a shaking incubator (Dong-A Scientific Co. Ltd., Seoul, South Korea) under darkness for their growth. The cultivations were maintained at 28 C, pH 7.2, and 150 rpm. Fermentation in 5-L jars The validation of central composite design (CCD) results was performed in 5-L jar fermenters made of Pyrex glass (650 mm height, 35 mm internal diameter). The cultivation conditions in the fermenters were automated as follows: pH, 7.1 6 0.1; temperature, 28 C with filtered sterile air. Filtration was performed using glass fiber aerated into the jar fermenter through an air sparger at the bottom of the fermenter at a 1.0 v/v flow rate. Scale-up of C. vulgaris cultivation Commercial pilot-scale cultivation was performed in a 25,000-L stirred-tank bioreactor containing 12,000 L of medium. The components of the medium were identical to those prepared during the 5-L jar-scale cultivation. The aeration rate and the agitation speed were initially set at 200 m3/

min and 110 rpm, respectively. The inoculation and growth were accomplished in three stages. First, the C. vulgaris cells were grown in 50 L of medium. Second, the cells were transferred for inoculation and grown in a bioreactor with 3,000 L medium each. Finally, the cells were transferred for inoculation and grown in 9,000 L of sterilized medium with a final volume of approximately 12,000 L. For the commercial 240,000-L scale cultivation, cultured C. vulgaris from 25,000L pilot scale was inoculated to 105,000 L of sterilized fresh media. The other cultivation conditions in the bioreactor were automatically controlled as follows: pH, 7.1 6 0.1, and temperature, 28 C. After 32 h of aseptic culturing, the cells were harvested by centrifugation at 4,500 rpm for 10 min and spraydried with a rotary atomizer-type spray dryer. Analytical methods Biomass Estimation. Biomass concentration was estimated by measurements of dry cell weight (DCW). The DCW of algal cells was measured by filtering an aliquot of the culture suspension through pre-weighed GF/C filters (Whatman, Kent, UK). The cultures were harvested by centrifugation at 3,000 rpm for 10 min, and the cell pellets were washed twice with distilled water. Subsequently, the pellet was dried at 105 C for 12 h and re-weighed. The dry weight of algal biomass was determined gravimetrically, and growth was expressed in terms of dry weight (g/L). Parameter Etraction (Lutein). Lutein extraction from C. vulgaris was determined according to the method described by Shi and Chen.12 Analysis of Lutein. An HPLC system (System Gold; Beckman Instruments, Inc., Pasadena, CA, USA) was used to separate, identify, and quantify pigments. This system was equipped with a 150 3 4.6-mm reversed-phase YMC-C30 (3 lm) column, a model 125 solvent module pump, and a model 166 UV– VIS detector. The mobile phase consisted of methanol:methyl tert-butyl ether (95:5, v/v). The flow rate was set at 1.0 mL/ min. The column was maintained at a constant temperature of 35 C. The samples were filtered through a 0.22-lm filter (Millipore Co., Billerica, MA, USA) before injection through a Rheodyne model 7725 valve with a 10-lL fixed loop. The wavelength for detection was set at 445 nm. Lutein (X6250, Sigma–Aldrich, St. Louis, MO, USA), used as a standard, was dissolved in the methanol:methyl tert-butyl ether (95:5, v/v) mixture, and diluted prior to HPLC analysis. All solvents were of HPLC grade (BDH Chemical Co., PA, USA). The pigments were identified by comparing retention times against known standards. A Beckman System Gold data system was used to process the chromatographic data in which the peak areas of the pigments were calculated. All chromatographic data displayed are the mean of three trials.13 Statistical analysis Statistical optimization for growth media was performed to maximize the concentration of lutein. To optimize a culture medium, the variables that affect lutein production had to be identified. Seven variables (glucose, phosphate sources [K2HPO4 and KH2PO4], MgSO47H2O, FeSO47H2O, EDTA-2Na, urea, and trace metal solution) were evaluated with 10 center points in 42 runs of a resolution III design. The orthogonal experimental design of coded variables for each run and actual values of variables for fractional factorial design are shown in Table 1.

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Table 1. Variables to be Screened in Fractional Factorial Design (A) The Experimental Design of Coded Variables of Each Run Coded Variables

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

Coded Variables

A

B

C

D

E

F

G

2 1 2 1 2 1 0 1 2 0 0 1 0 1 2 0 2 1 2 2 1

1 2 2 1 1 1 0 2 2 0 0 2 0 1 2 0 1 2 1 2 1

2 1 1 2 1 1 0 2 2 0 0 1 0 2 2 0 2 2 1 1 1

2 2 1 1 2 1 0 2 1 0 0 2 0 1 1 0 2 2 2 1 1

1 1 2 2 2 1 0 2 1 0 0 1 0 2 1 0 1 2 2 2 1

2 2 2 2 1 1 0 1 1 0 0 2 0 2 1 0 2 1 1 2 1

1 2 1 2 2 1 0 1 2 0 0 2 0 2 2 0 1 1 2 1 1

22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

A

B

C

D

E

F

G

2 1 0 2 1 2 0 1 1 0 2 1 2 2 2 0 0 1 1 1 2

1 2 0 1 1 2 0 2 2 0 1 2 1 2 2 0 0 1 1 1 2

1 2 0 1 2 1 0 2 1 0 2 1 2 1 2 0 0 2 1 1 2

2 2 0 2 1 1 0 2 2 0 2 2 2 1 1 0 0 1 1 1 1

2 2 0 2 2 2 0 2 1 0 1 1 1 2 1 0 0 2 1 1 1

1 1 0 1 2 2 0 1 2 0 2 2 2 2 1 0 0 2 1 1 1

2 1 0 2 2 1 0 1 2 0 1 2 1 1 2 0 0 2 1 1 2

(B) Actual Values of Variables (units; A–E: g/L, F and G: mL/L) A

1 0 2

B

C

D

E

F

G

Glucose

KH2PO4

K2HPO4

MgSO47H2O

EDTA-2Na

FeSO47H2O

Urea

Trace metal solution

15 10 5

10.5 7 3.5

0.45 0.3 0.15

0.45 0.3 0.15

0.075 0.05 0.025

0.0225 0.015 0.0075

1.5 1 0.5

1.5 1 0.5

For the growth medium, lutein concentration was selected as the response, which can be calculated using Eq. 1. k X XX y5b0 1 bi xi 1 bij xi xj 1e

(1)

i

Optimization of culture media for large-scale lutein production by heterotrophic Chlorella vulgaris.

Lutein is a carotenoid with a purported role in protecting eyes from oxidative stress, particularly the high-energy photons of blue light. Statistical...
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