Theories and Applications of Chem. Eng., 2002, Vol. 8, No. 1

Applications of Chem. Eng., 2002, Vol. 8, No. 1

Theories and Applications of Chem. Eng., 2002, Vol. 8, No. 1

Theories and Applications of Chem. Eng., 2002, Vol. 8, No. 1

Theories and Applications of Chem. Eng., 2002, Vol. 8, No. 1

RSM을 통한 효모의 카로티노이드생산의 최적화

Theories and Applications of Chem. Eng., 2002, Vol. 8, No. 1

박 평규, 김 의용

서울시립대학교 화학공학과

Optimization of Carotenoid Production by Yeast through RSM

Applicatins of Chem. Eng., 2002, Vol. 8, No. 1

Applicatins of Chem. Eng., 2002, Vol. 8, No. 1

Applicatins of Chem. Eng., 2002, Vol. 8, No. 1

Applicatins of Chem. Eng., 2002, Vol. 8, No. 1

Applicatins of Chem. Eng., 2002, Vol. 8, No. 1

Applicatins of Chem. Eng., 2002, Vol. 8, No. 1

Theories and Applications of Chem. Eng., 2002, Vol. 8, No. 1

화학공학의 이론과 응용 제8권 제1호 2002년

Pyoung Kyu Park, Eui Yong Kim

Dept. of Chemical Engineering, Seoul City University

Theories and Applications of Chem. Eng., 2002, Vol. 8, No. 1

INTRODUCTION

In nature, carotenoids were mainly composed of colours, red, yellow and orange. In foods, in addition to their function as the natural pigments and provitamin A procursor role of carotenoid. These carotenoids used as food additives for colouring. Futhermore, diets rich in fruits and vegetables containing carotenoids have been of interest because of their potential health benefit against chronic diseases. There has been growing interest in the use of astaxanthin as a pigment for a aquaculture and the poultry industry(1-2). In green plant tissues, the colour of carotenoids is masked by the more dominant pigments, chlorophyll. Also, In microorganism, carotenoids acts to important properties (anti-oxidant, free radical scanvengers etc). Algae such as Dunaliella and Haematococcus, yeasts of the genra Phaffia, Rhodotorula and Sporobomyces, fungi like Blakeslea trispora and bacteria such as Flavobacterium and Micrococcus are reported to produce carotenoids. Yeasts are more convenient than algae or fungi for large-scale production in fermentors, due to thier unicellular nature and high growth rate. P. rhodozyma is known to produce astaxanthin normally.

Theories and Applications of Chem. Eng., 2002, Vol. 8, No. 1

Culture conditions are traditionally optimized by the one-at-time strategy, i.e., varying one factor while keeping all others constant. Although the strategy is simple and easy, without the need of statistical analysis, it involves a relatively large number of experiments and the interaction among factors is often ignored. In contrast, statistically based experimental designs are more efficient approaches that can deal with a large number of variables simultaneously. Morever, the interaction among different variables can be estimated. To date, this technique has been used by some researchers to optimized medium components and environmental factors. Literature reports little about the use of experimental design in optimizing the culture conditions of carotenoid production microorganisms(3-4).

In this study, to maximize carotenoid production by R. glutinis, determining the optimal culture conditions(including medium components and environmental factors) is needed. The first design (factorial design) analyzes the main factors which can be influencing volumetric carotenoid production. The second (central composite design) determines the optimum conditon for the carotenoid production.

MATERIAL AND METHODS

Strains and cultivation

Rhodotorula glutinis was cultivated in YM medium(glucose; 1%, peptone; 0.5%, yeast extract; 0.3%, malt extract; 0.3%). this was incubated on a rotary shaker(125rpm) for 48hr at 22℃. The cell density was determined by measuring the culture optical density at 660nm with a HP 8452(USA, Co) spectrophotometer. Culture samples were diluted with an appropriate amount of 0.9% sodium chloride solution to obtain an OD between 0.2 and 0.7. A standard curve was then used to convert the OD to dry cell weight. For the analysis of yeast carotenoids, 0.1g of freezed cell and dissolved in dimethy sulfoxide. 10 ml each of acetone, petroleum ether and 20%(w/v) NaCl solution was serially added with vortexing. The absorbance of the petroleum ether layer was read at 490nm with a Spectrophotometer.

Experimental Design

Our objective was to study the influence of several culture condition factors. We used a screening design(factorial design) as the first step in the opimization process. This design assesses the influence of the main factors, as well as their interaction. The nine factors studied and their levels were: 1 and 2% maltose; 0.02 and 0.25%(w/v) valine; 0.1 and 0.6% KH2PO4; 0.01 and 0.1% MgSO4; 0.01 and 0.06% CaCl2; 0.01 and 0.1% NaCl; 4 and 6 pH; 20 and 30℃ temperature; 100 and 200ml working volume (Table 1).

The next stage in medium optimization was to determine the optimum level of each of key independent variable as identified by the factorial design. An experimental design such as the response surface methodology, which is a fraction of the full factorial was used. This design evaluated the quadratic effects and two-way interactions among the variables and thus determined the nonlinear nature of the response, if any. For statistical calculations, the variables [eqn: X_i] were coded as [eqn: x_i] according to the following equation.

[eqn: x_i ~=~(X_i - X_o )/□X]

where, [eqn: x_i] = the dimensionless coded value of the variable [eqn: X_i]; [eqn: X_o]= the value of the [eqn: X_i]  at the centre point; □X= step change. The behaviour of the carotenoid production was explained by the following quadratic equation:

[eqn: Y=-□_o + SUM □_i x_i +SUM□_ii { x}`_{i } ^{2 }+ SUM□_ij x_i x_j]

where, [eqn: Y]=predicted response; [eqn: □_o]= offset term; [eqn: □_i]=linear effect; [eqn: □_ii]=square effect, and [eqn: □_ij]=interaction effect.

The design for the three independent variables and each row represents an experimental trial. The elements, (+), (0) and (-) represent the high middle and low levels respectively of each variable. The coefficients of the polynomial model were calculated using standard regression techniques.

RESULT AND DISCUSSIONS

In general, The carotenoid production was varied according to the culture condition. so, We considered important culture factors; maltose, valine, KH2PO4, MgSO4, CaCl2, NaCl, pH, temperature and working volume. The results from the first experiment on cell mass and volumetric carotenoid production are shown in past paper(1). Significant cell mass and carotenoid production was observed with maxima 10.06gl-1 and 9.94mgl-1. With this first design, the most important factors were determined (maltose, valine, temperature and pH) that were used in the second experimental strategy; the response surface method was applied in order to rapidly approach the optimum(Table 1). It was observed that in the basal medium, C/N ratios between 4 and 50. A low C/N ratios resulted in a high volumetric carotenoid production of carotenoid, due to high cell mass yield(Run.5, 9, 13.)  Higher C/N ratio there will be production of lipid in preference to carotenoid. Kim etc(5) reported that higher of the C/N ration could be effectively used for obtaining higher yields of fat synthesis. Also, Temperature affected carotenoid synthesis. In this study, When the temperature of incubation decreased from 35℃ to 15℃. Higher carotenoid production was observed at the 25℃. It was shown that R. glutinis increased cell mass at low temperature, compared to the cell mass at high temperature. Low temperature is ensured by enhanced biosyntheis of carotenoids that increase membrane fluidity. Higher carotenoid production was observed at the temperature of 25℃ empolyed. The volumetric carotenoid formation is known to be increased by low C/N ratio and by 1.41 times as Temperature decreased.

REFERENCE

1. Andrew Yong and George Britton, Carotenoids in Phtosynthesis, Chapman & Hall, (1993)

2. Helga Gerster, Internat. J. Vit. Nutr. Res., 63, 93-121, (1993)

3. Box, G. E., Hunter, W. G., Hunter, J. S., Statistics for Experimenters, Wiley, New York, NY. (1978)

4. Montagomery, D. C., Design and Aanlysis of Experiments. Wiley, New York.

5. Kim E. Y and Park. P. K and Chae Hee Jeong, Optimization of Culture Conditions for Extracellular Lipid Production from Rhodotorula glutinis K-501, Korean. J. Biotechnol. Bioeng., 13(1), 58-64, (1998)

Theories and Applications of Chem. Eng., 2002, Vol. 8, No. 1

Table 1 Response surface method matrix and experimental results of carotenoid production.

Run No.	C/N	pH	Temp	Cell mass (g/l)	Volumetric carotenoid (mg/L)
1	50	4.0	35	0.17	0.05
2	27	5.5	25	5.26	2.00
3	27	5.5	25	5.39	2.58
4	50	7.0	15	3.29	1.56
5	4	7.0	15	7.98	3.14
6	27	5.5	25	5.70	2.91
7	4	4.0	35	0.22	0.09
8	4	7.0	35	0.36	0.11
9	4	4.0	15	8.31	4.32
10	50	4.0	15	2.98	1.05
11	50	7.0	35	0.36	0.11
12	27	5.5	35	0.23	0.07
13	4	5.5	25	11.64	8.24
14	27	5.5	25	5.60	2.17
15	27	4.0	25	5.39	3.23
16	50	5.5	25	3.91	1.50
17	27	5.5	25	5.52	2.34
18	27	5.5	25	5.63	2.59
19	27	5.5	15	1.97	0.68
20	27	7.0	25	5.47	2.22

Fig. 1. Three-dimensional surface plots of       volumetric carotenoid as the function of pH,     Temperature according to the C/N ratio.