Abstract
Fermentation of Phaffia rhodozyma is a major method for producing astaxanthin, an important pigment with industrial and pharmaceutical application. To improve astaxanthin productivity, single factor and mixture design experiments were used to investigate the effects of nitrogen source on Phaffia rhodozyma cultivation and astaxanthin production. Results of single factor experiments showed nitrogen source could significantly affect P. rhodozyma cultivation with respect to carbon source utilization, yeast growth and astaxanthin accumulation. Further studies of mixture design experiments using (NH4)2SO4, KNO3 and beef extract as nitrogen sources indicated that the proportion of three nitrogen sources was very important to astaxanthin production. Validation experiments showed that the optimal nitrogen source was composed of 0.28 g/L (NH4)2SO4, 0.49 g/L KNO3 and 1.19 g/L beef extract. The kinetic characteristics of batch cultivation were investigated in a 5-L pH-stat fermentor. The maximum amount of biomass and highest astaxanthin yield in terms of volume and in terms of biomass were 7.71 mg/L and 1.00 mg/g, respectively.
Keywords: Phaffia rhodozyma, Astaxanthin, Nitrogen source, Optimization
INTRODUCTION
Astaxanthin (3,3-dihydroxy-β,β-carotene-4,4′-dione) is a novel carotenoid with high market value for the pharmaceutical and feed industries (Wang and Li, 1997; Guerin et al., 2003; Johnson, 1991). Astaxanthin is currently chemically synthesized and added into some animal feeds for pigmentation of animals, especially for marine fishes. However, synthetic astaxanthin is expensive. It was reported (Johnson, 1991) that synthetic astaxanthin accounts for approximately 10% of the total cost of fish feed. There is growing interest in the use of astaxanthin as a pigment for the aquaculture and poultry industries (Verdoes et al., 1999). In addition, other diverse biological functions of astaxanthin have attracted more and more interest because of its health benefits to human beings due to its roles in cancer prevention, enhancement of immune response, and as free radical quencher (Paul et al., 1997).
Studies showed that fermentation of Phaffia rhodozyma is one of the most promising methods for producing natural astaxanthin (Johnson, 1991; 2003). Therefore, the optimal fermenting conditions should be very important for the industrialization of astaxanthin production. Nitrogen source is one of the necessary components in the fermentation medium, and different components of the nitrogen source could greatly affect the yield of astaxanthin (Du et al., 2005). In this study, different nitrogen sources were used in the fermentation of P. rhodozyma and the optimal nitrogen source for high astaxanthin yield was obtained by regressive analysis.
MATERIALS AND METHODS
Microorganism and inoculums development
Phaffia rhodozyme 7B12 originated from P. rhodozyma Past-1 that was generously provided by Professor Ulf Stahl, Berlin Industrial College, Germany. The yeast was maintained and activated on yeast/malt (YM) agar slants containing 10.0 g/L glucose, 5.0 g/L bacto-peptone, 3.0 g/L malt extract, 3.0 g/L yeast extract and 20.0 g/L agar, and inoculated in 250 ml shake flasks containing 25 ml sterilized YM medium and cultured on a rotary shaker at 22 °C, 200 r/min for 72 h (Johnson, 1991).
Reagents and chemicals
Astaxanthin and β-carotene were purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA) and stored at −20 °C. Yeast extract, peptone and beef extract were purchased from Shanghai Biochemical Agent Co. (China). Methanol used for determination of astaxanthin was HPLC grade. Other chemicals used in the study were analytical grade.
Experimental design
Single factor design experiments were used to analyze the influence of several nitrogen sources on astaxanthin production by the yeast. Mixture design experiments (Montgomery, 1997) were adopted to optimize the complex nitrogen compositions for P. rhodozyma fermentation of astaxanthin production.
The basic medium for P. rhodozyma fermentation was composed of 20 g/L glucose, 1 g/L (NH4)2SO4, 1 g/L KH2PO4, 0.5 g/L MgSO4·7H2O, 0.1 g/L CaCl2·H2O, 0.5 mg/L MnSO4·H2O, 0.14 mg/L ZnCl2, 0.27 mg/L FeCl3·6H2O, 1.25 mg/L CuSO4·5H2O and pH was initially controlled at 6.0. To investigate effects of different nitrogen sources on astaxanthin synthesis, (NH4)2SO4 was replaced by various nitrogen sources. The optimal experiments were carried out in 250 ml flasks containing 25 ml medium, inoculated with 1% inoculum and then incubated on a rotary shaker at 22 °C, 200 r/min for 96 h (Zhu and Liang, 2005).
Kinetics experiments on P. rhodozyma cultivation were carried out in a 5 L pH-stat fermentor (Bio. Braun Biotech International Co., Germany) containing 3.5 L fermentation medium and inoculated with 1% (v/v) inoculum and cultured at 22 °C, 200 r/min, 1.2 VVM, pH 5.0 (Hu et al., 2005).
Analytical methods
Yeast cells were harvested by centrifugation and biomass was calculated by dry weight (g/L). Residual sugar was determined by the 3,5-dinitrosalicylic acid (DNS) method (Zhang et al., 1997) and carotenoid was extracted by dimethylsulfoxide method (Sedmak et al., 1990; Ni et al., 2004).
Waters HPLC system (Waters Corporation, Milford, MA, USA) including the 1 525 pump, Nova-Pak C18 reverse phase column (3.9 mm×150 mm, 4 μm) and 2 478 UV detector were used to analyze carotenoids. Carotenoids were isolated in a Nova-Pak C18 reverse phase column under the conditions listed in Table 1 with flow rate at 1.2 ml/min, column pressure at 0~3000 psi and column temperature at 40 °C. Astaxanthin concentration was detected at the wavelength of 474 nm.
Table 1.
HPLC condition for astaxanthin analysis
| Time (min) | Methanol (%) | Water (%) |
| 0.00 | 80.0 | 20.0 |
| 3.00 | 80.0 | 20.0 |
| 5.00 | 100.0 | 0.0 |
| 8.00 | 100.0 | 0.0 |
| 9.00 | 80.0 | 0.0 |
RESUTLS
Single nitrogen sources affected the astaxanthin yield
Different nitrogen sources listed in Table 2 were used to investigate their effects on P. rhodozyma growth and pigmentation. The total amount of nitrogen of these sources was equal to the amount of (NH4)2SO4 in the basic medium. Biomass and the yield of astaxanthin were measured and are summarized in Table 2. As shown in Table 2, using beef extract can result in the highest biomass yield, but the astaxanthin yield in terms of broth volume or biomass were quite lower compared to the yields when using KNO3 or (NH4)2SO4 as nitrogen sources. It is indicated that the yield of astaxanthin was not proportional to the biomass. Using (NH4)2SO4 as the nitrogen source produced the highest astaxanthin yield in terms of biomass compared to use of other nitrogen sources. And using KNO3 produced the highest astaxanthin yield in terms of broth volume. Thus, KNO3 and (NH4)2SO4 were selected to do the following mixture experiments. Beef extracts were also selected in the further study since the largest amount of biomass was obtained when beef extracts were used as the nitrogen source.
Table 2.
Nitrogen source significantly affects growth and pigmentation of Phaffia rhodozyma
| Nitrogen sources | Commercial values ($/kg) | Average biomass of three repeats (g/L) | Average astaxanthin yield of three repeats (mg/L) | Average astaxanthin yield in terms to biomass of three repeats (mg/g yeast) |
| Carbamide | 2.11 | 3.74c | 3.44d | 0.92b |
| Ammonium acetic | 2.83 | 2.88e | 1.93h | 0.67c |
| NH4Cl | 1.84 | 4.95b | 5.19b | 1.05ab |
| KNO3 | 2.47 | 3.06d | 3.75d | 1.23a |
| (NH4)2SO4 | 1.96 | 4.96b | 5.51a | 1.11a |
| Ammonium oxalic | 2.86 | 3.13d | 2.92e | 0.93b |
| Peptone | 10.06 | 4.00c | 4.16c | 1.04ab |
| Beef extract | 13.23 | 5.62a | 2.29g | 0.41d |
| Yeast extract | 8.21 | 3.14d | 2.62f | 0.83bc |
The same letters behind the values in the same column mean P>0.05, different letters mean P<0.05
Different proportions of three selected nitrogen sources affected the astaxanthin yield
In order to optimize nitrogen source for astaxanthin production by P. rhodozyma cultivation, mixture experiment design was carried out by using the three selected nitrogen sources (i.e. beef extract, potassium nitrate and ammonium sulfate) at different levels. The results of mixture experiments showed that the yield of astaxanthin and biomass changed with the proportion of three nitrogen sources (Table 3). Table 4 shows the ANOVA results for the mixture experiment design. It is indicated that the proportion of these three nitrogen sources could significantly affect the amounts of the residual sugar, biomass and astaxanthin. Pro>F were 0.06%, 4.31% and 0.09%, respectively. The astaxanthin yield in terms of volume was selected as response variable for regressive analysis of the influence of the three nitrogen sources. Results in Table 5 demonstrated that X 1 ((NH4)2SO4, P>T=0.29%), X 2 (KNO3, Pro>T=3.11%), X 3 (beef extract, P>T=0.43%) and X 2×X 3 (P>T=4.38%) could significantly affect the response variables, compared to X 1×X 2 (P>T=62.81%) and X 1×X 3 (P>T=74.77%). Therefore, the effects of X 1×X 2 (P>T=62.81%) and X 1×X 3 can be neglected. According to the parameters listed in Table 5, Eq.(1) in a second-order polynomial prediction model was established. This model can be used to predict the influence of various proportions of these three nitrogen sources on astaxanthin yield.
Table 3.
Results of mixture experiment by using compounding nitrogen sources
| Runs | X1 | X2 | X3 | Average residual sugar of three repeats (g/L) | Average biomass of three repeats (g/L) | Average astaxanthin yield of three repeats (mg/L) |
| 1 | 1 | 0 | 0 | 0.12 | 3.94 | 4.43 |
| 2 | 0 | 1 | 0 | 1.79 | 3.43 | 2.61 |
| 3 | 0 | 0 | 1 | 0.46 | 6.84 | 4.53 |
| 4 | 0.333 | 0.667 | 0 | 0.17 | 3.90 | 2.78 |
| 5 | 0.333 | 0 | 0.667 | 0.40 | 4.86 | 4.18 |
| 6 | 0.333 | 0.333 | 0.333 | 0.30 | 3.88 | 4.71 |
| 7 | 0.667 | 0.333 | 0 | 0.18 | 3.82 | 5.51 |
| 8 | 0.667 | 0 | 0.333 | 0.23 | 5.38 | 4.77 |
| 9 | 0 | 0.333 | 0.667 | 0.35 | 6.66 | 5.67 |
| 10 | 0 | 0.667 | 0.333 | 0.20 | 6.01 | 5.68 |
X 1, X 2, X 3 are the proportions of the nitrogen content from ammonium sulfate, potassium nitrate and beef extract respectively in all nitrogen sources experimentation
Table 4.
Proportion of nitrogen sources significantly affects the astaxanthin biosynthesis by ANOVA analysis
| Biomass | Residual sugar | Astaxanthin production velocity | |
| F-value | 67.22 | 6.73 | 54.34 |
| Pro>F (%) | 0.06 | 4.31 | 0.09 |
| C.V. | 16.14 | 91.12 | 17.86 |
| R2 | 0.990 | 0.910 | 0.988 |
Table 5.
Regressive analysis of the mixture experiments
| Estimate | T for H0 | Pro>T (%) | |
| X1 | 4.88 | 6.47 | 0.29 |
| X2 | 2.46 | 3.26 | 3.11 |
| X3 | 4.41 | 5.84 | 0.43 |
| X1×X2 | 1.75 | 0.52 | 62.81 |
| X1×X3 | −1.15 | −0.34 | 74.77 |
| X2×X3 | 9.70 | 2.91 | 4.38 |
| Y=4.88X1+2.46X2+5.48X3+0.52X1×X2−0.34X1×X3+2.91X2×X3 | (1) |
Validation experiments for high astaxanthin yield using compounding nitrogen source
Eight compounding nitrogen sources were predicted maximum response values by Eq.(1), so they were selected to confirm if the predicted results were biased to the practical values. The results of validation experiment are shown in Table 6. The response value (astaxanthin yield in terms of volume) reached its maximum value when X 1=0.284, X 2=0.323 and X 3=0.393, and the maximum response values was 6.40 mg/L. Thus, for high astaxanthin yield by P. rhodozyma cultivation, the optimal nitrogen source was composed of 13.11% (NH4)2SO4, 22.82% KNO3 and 64.07% beef extract (containing 6% nitrogen). To make same amount of nitrogen equal to 0.1% (NH4)2SO4, the optimal nitrogen sources were composed of 0.28 g/L (NH4)2SO4, 0.49 g/L KNO3 and 1.19 g/L beef extract. In addition, a quadratic model showed as Eq.(2) was proposed to predict the response values of the stationary area.
Table 6.
Validation experiments on composed nitrogen sources for P. rhodozyma 7B12 cultivation
| Runs | X1 | X2 | X3 | Astaxanthin yield (mg/L) |
|
| Predicted values | Average values of three repeats | ||||
| 1 | 0.333 | 0.333 | 0.333 | 5.06 | 5.79b |
| 2 | 0.284 | 0.323 | 0.393 | 5.17 | 6.40a |
| 3 | 0.236 | 0.326 | 0.440 | 5.29 | 5.48c |
| 4 | 0.183 | 0.334 | 0.482 | 5.41 | 6.11ab |
| 5 | 0.134 | 0.345 | 0.522 | 5.54 | 5.84 |
| 6 | 0.084 | 0.356 | 0.559 | 5.68 | 6.06ab |
| 7 | 0.035 | 0.368 | 0.596 | 5.83 | 5.90b |
| 8 | 0 | 0.333 | 0.667 | 5.12 | 5.54c |
| Y=−527.28+1313.16X1+4732.54X2−1167.49X3−1034.77X12−3363.25X1×X2−6545.85X22+1015.82X32 | (2) |
High astaxanthin yield was obtained using the optimized nitrogen source
The optimal compounding nitrogen source was used to culture P. rhodozyma as a batch in a 5 L pH-stat bioreactor. As shown in Table 7, the sugar consumption was synchronous with the growth of the yeast, but astaxanthin synthesis lagged sugar consumption and yeast growth. The exponential phase began at 12 h and ended at 42 h after inoculation. Sugar consumption and growth of yeast were so fast in the exponential phase that the biomass reached to the maximum point (8.36 g/L) at 42 h, and then no significant sugar consumption was detected. Astaxanthin was mainly synthesized during 18~54 h after inoculation. The astaxanthin yields in terms of broth volume and in terms of biomass were 7.71 g/L and 1 mg/g, respectively after 54 h cultivation.
Table 7.
High astaxanthin yield was obtained using optimized nitrogen source
| Time (h) | pH | Residual sugar (g/L) | Biomass (g/L) | Astaxanthin in terms of batch volume (mg/L) | Astaxanthin in terms of biomass (mg/g) |
| 6 | 6.02 | 19.06 | 2.20 | 0.11 | 0.05 |
| 12 | 5.06 | 18.97 | 2.36 | 0.16 | 0.07 |
| 18 | 3.13 | 11.87 | 5.26 | 0.36 | 0.07 |
| 24 | 2.50 | 6.55 | 6.87 | 2.73 | 0.40 |
| 30 | 2.42 | 4.74 | 7.08 | 5.28 | 0.75 |
| 36 | 2.41 | 3.05 | 7.58 | 6.61 | 0.87 |
| 42 | 2.42 | 1.04 | 8.36 | 6.91 | 0.83 |
| 48 | 2.47 | 0.48 | 8.06 | 7.45 | 0.92 |
| 54 | 2.48 | 0.44 | 7.71 | 7.71 | 1.00 |
| 60 | 2.51 | 0.43 | 7.64 | 7.42 | 0.97 |
| 66 | 2.53 | 0.44 | 7.59 | 7.29 | 0.96 |
| 72 | 2.54 | 0.38 | 7.21 | 6.78 | 0.94 |
| 78 | 2.56 | 0.53 | 7.62 | 7.33 | 0.96 |
| 84 | 2.57 | 0.50 | 7.66 | 7.49 | 0.98 |
| 90 | 2.58 | 0.56 | 7.51 | 7.40 | 0.99 |
| 96 | 2.58 | 0.55 | 7.48 | 7.31 | 0.98 |
DISCUSSION AND CONCLUSION
Some studies showed that Phaffia rhodozyma can utilize many kinds of nitrogen sources including ammonium, nitrate and organic nitrogen sources (Johnson, 1991; Du et al., 2005). In order to test the effects of nitrogen sources on the growth of P. rhodozyma and yield of astaxanthin, single factor experiments were carried out and three nitrogen sources—(NH4)2SO4, KNO3 and beef extract were selected for further studies of compounding nitrogen sources.
Mixture experiment design is the most important method for optimal medium composition (Zhang et al., 1997). Using this method, compounding nitrogen sources composed of three nitrogen source components [i.e. (NH4)2SO4, KNO3 and beef extract] were used for cultivation of P. rhodozyma. Results of mixture experiments in Tables 3 and 4 indicated that different proportions of three components could affect P. rhodozyma cultivation and astaxanthin production significantly. After regressive analysis and validation experiments, the most effective nitrogen source was confirmed to be composed of 0.28 g/L ammonium sulfate, 0.49 g/L potassium nitrate and 1.19 g/L beef extract.
P. rhodozyma was batch cultivated in a 5 L pH-stat fermentor to investigate the kinetic characteristics. The results shown in Table 7 showed that highest astaxanthin yield in terms of broth volume and biomass were 7.71 mg/L and 1.00 mg/g, respectively. The maximum amount of biomass was 7.71 g/L. Compared with other fermentation experiments using the same strain, although the amount of biomass was less than that of original medium, astaxanthin yields in terms of volume and biomass were much higher than those of original nitrogen source (Ni et al., 2005a; 2005b; 2005c). Additionally, the kinetic characteristics of growth, carbon source consumption and astaxanthin synthesis were consistent with other researches reported (Ni et al., 2005a; 2005b; 2005c; Kusdiyantini et al., 1998; Jesus et al., 2001).
Footnotes
Project supported by the National Natural Science Foundation of China (No. 30571450) and the Foundation for Young Professors of Jimei University of Xiamen, China
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