Article International Journal of Modern Biology and Medicine, 2017, 8(1): 1-13 International Journal of Modern Biology and Medicine Journal homepage: www.modernscientificpress.com/journals/ijbiomed.aspx ISSN: 2165-0136 Florida, USA Investigation on the Production of Carotenoid from Molasses by Phaffia rhodozyma Fang Liu, Ming-Jun Zhu * School of Bioscience and Bioengineering, South China University of Technology, Guangzhou Higher Education Mega Center, Panyu, Guangzhou 510006, People s Republic of China * Author to whom correspondence should be addressed; E-Mail: mjzhu@scut.edu.cn; Tel.: +86-20- 39380623; Fax: +86-20-39380601. Article history: Received 3 February 2017, Received in revised form 15 March 2017, Accepted 21 March 2017, Published 28 March 2017. Abstract: This study investigated the sugarcane molasses as a carbon source and other important conditions for cell growth and carotenoid production of yeast Phaffia rhodozyma. Different molasses concentrations had a significant effect on the growth of Phaffia rhodozyma, and the dry cell weight reached 12.35 g/l, with the initial sugar concentration of 50 g/l. The higher initial sugar concentration was not beneficial to carotenoid accumulation, and 30 g/l of initial sugar concentration was determined as optimum initial sugar concentration for carotenoid accumulation with 10.71 g/l biomass. The effects of nitrogen source, C/N ratios and initial ph on the cell growth and carotenoid accumulation were also investigated, and yeast extract as nitrogen source, carbon to nitrogen ratio of 8, and initial ph of 5 were determined. Furthermore, the volume of medium had a significant effect on cell growth and carotenoid production and the medium volume of 25 ml in Erlenmeyer flasks (250 ml) was optimal with 11.81 g/l dry cell weight, and the production of carotenoid was about 32-fold that of the minimum one. The present study suggests that sugarcane molasses is a suitable carbon source for natural carotenoid production by Phaffia rhodozyma. Keywords: Phaffia rhodozyma; carotenoid; sugarcane molasses; medium volume; C/N ratio.
2 1. Introduction Carotenoid, a class of pigments widely distributed in nature, gives red, yellow and orange colors, and has been found in bacteria, fungi, and plants (Mezzomo and Ferreira, 2016). Astaxanthin (3, 3 - dihydroxy-β, β -carotene-4, 4 -dione), a kind of red lipid-soluble pigment, belongs to the family of the carotenoid, and is the oxygenated derivative of carotenoid (Higuera-Ciapara et al., 2006; Yuan et al., 2011). In fact, astaxanthin has been produced industrially on a large scale in aquaculture industry (Kim and Lee, 2005; Shan, 2012). Astaxanthin contains conjugated double bonds, hydroxyl and keto moieties on each ionone ring (Kishimoto et al., 2010, Ambati et al., 2014), and this molecular structure makes it an excellent antioxidant (Liu and Osawa, 2007; Dose et al., 2016), which is 10 times higher than that of other carotenoid such as zeaxanthin, lutein, canthaxanthin and β-carotene, and 100 times higher than that of α-tocopherol. The potential antioxidant power of astaxanthin has been reported to have beneficial effects on various diseases, such as cancer (Tanaka et al., 2012), cardiovascular diseases (Fassett and Coombes, 2011), diabetes (Otton et al., 2010), chronic inflammatory diseases (Arunkumar et al., 2012), liver diseases (Bhuvaneswari et al., 2010), and immune response (Park et al., 2011). With the growing public perception of the relationship between diet and human health, astaxanthin has become one of the important health-promoting ingredients in functional foods and beverages. As early as the 1960s, Phaffia rhodozyma was isolated from trees in the mountain areas of Japan and Alaska by Herman Phaff, during his pioneering studies of yeast ecology (Park et al., 2011). Since then, this yeast has attracted considerable biotechnological interest because of its ability to synthesize the economically important astaxanthin in 3R, 3 R-configuration. Compared with the green alga, Haematococcus pluvialis, another astaxanthin over-producing microorganism (Dong and Zhao, 2004), Phaffia rhodozyma has great industrial potential for natural astaxanthin production due to advantages of no illumination requirement, breeding rapidly, short growth cycle, a vast variety of organic materials as fermenting substrates and mature high-density fermentation process (Xiao et al., 2015). Cane molasses, a cheap by-product in sugar refinery, is widely used for the production of carotenoid by some microorganisms. It consists of sugars (mainly sucrose, glucose, and fructose), water, protein, fat, heavy metals, organic acids, vitamins and other substances, with a sugar content of about 30%-50% (w/w). In this study, molasses was used as a carbon source to produce carotenoid by yeast Phaffia rhodozyma. Through the investigation on the effects of different carbon source, nitrogen source and other conditions on the growth and yield of carotenoid of Phaffia rhodozyma, sugarcane molasses is a suitable carbon source for the growth of Phaffia rhodozyma and carotenoid production, and this might lay the foundation on the industrial production of carotenoid. 2. Materials and Methods
3 2.1. Pretreatment of Molasses Sugarcane molasses was a gift from Guangzhou Sugarcane Industry Research Institute (Guangdong Province, China) and it contained about 30.55% (w/w) sucrose, 14.78% (w/w) fructose, and 7.71% (w/w) glucose determined simultaneously by high performance liquid chromatography using maltose as internal standard with refractive index detector( Xu et al., 2014). The crude molasses was diluted with distilled water to four times, and then stirred thoroughly for 30 min after adjusted to ph 3.3~3.6 with concentrated sulfuric acid following overnight at 25. Next, after centrifugation at 12,000 rpm/min for 15 min, the supernatant was collected (Levi et al., 2004). 2.2. Microorganism, Medium and Inoculum Preparation Phaffia rhodozyma YM119 was stored in our laboratory and used as an inoculum. It was obtained by mutating and adapting Phaffia rhodozyma R29 by continuous transfer in Jerusalem artichoke extract medium consisting of (per liter) 20 g total sugar, 10 g peptone, and 5 g yeast extract. The culture refrigerated in -80 C was inoculated into 25 ml (YPD) media in a 250-mL Erlenmeyer flask. The flask was incubated at 22 C with 220 rpm/min orbital shaking for 36 h. Then 2.5 ml of this culture was inoculated to 250 ml flasks containing 22.5 ml of the same fresh medium for 12 h. After the 12 hours incubation, the cultures were used as seed culture. 2.3. Flask Fermentations In flask batch cultures, Erlenmeyer flasks (250 ml), each containing different media volumes supplemented with various sugar concentrations with sugarcane molasses and organic or inorganic nitrogen source and different initial ph (3.0 ~ 8.0) were inoculated with 10% (v/v) of exponentially growing inoculum and then incubated at 22 in an orbital shaker at 220 rpm/min. 2.4. Determination of Residual Sugar The anthrone-sulfuric acid colorimetric assay was used to determine the content of initial and residual reducing sugars in the broth (Lin and Chen, 2007; Song et al., 2009). Anthrone was dissolved in 80 % sulfuric acid to prepare anthrone-sulfuric acid reagent just before use. The calibration curve was constructed based on the various glucose concentrations and their corresponding absorbance determined by spectrophotometer at 620 nm. 2.5. Determination of Dry Cell Weight (DCW)
4 The biomass of Phaffia rhodozyma was calculated by dry cell weight (DCW). DCW was determined by centrifuging a 5-mL sample of the culture at 5,000 rpm/min for 10 min, washing three times with distilled water to remove residual media, and drying the washed cells at 105 C until constant weight and subsequently cooling down to room temperature in a desiccator before weighting (Hu et al., 2012; Bie and Zhu, 2015). 2.6. Extraction and Determination of Pigment The modified hot acid method was used to analyze carotenoid production (Hasegawa et al., 1990). Briefly, 5 ml of the cell suspension was centrifuged at 5,000 rpm for 10 min. The obtained cell pellets were washed three times with distilled water until the supernatant became colorless. Then 3 ml of 3M HCl solution was added into the sample. After 30 min at 25, the suspension was heated until boiling for 6 min and then, placed rapidly in an ice-bath. The samples were washed and centrifuged at 12,000 rpm for 10 min, and the supernatant was removed. The cell pellet was washed three times, and then 5 ml acetone was added and vortexed for 60 s. The upper layer containing carotenoid was separated by centrifugation for 10 min. The absorbance of the final sample solutions was measured at 474 nm using a UV/visible spectrophotometer (UNIC, 2802s UV/VIS, US). The concentration of carotenoid extracted by acetone was calculated by the following equations: OD474 D V1 Production of carotenoid (mg/l) = (1) 0.16 V2 OD474 D V1 Content of carotenoid (mg/g) = (2) 0.16 W Where OD474 is the absorbance at 474 nm; D is dilution ratio; V1 and V2 are the volumes of culture sample and acetone used for extraction, respectively; W is the cell weight for the volume of V1, and 0.16 is the extinction coefficient of organic solvent (Ni et al., 2004). 2.7. Specific Growth Rate Specific growth rate (μ, h -1 ) was calculated during the logarithmic phase of growth, using the following equation: lnx2 lnx1 Specific growth rate (h -1 ) = (3) t2 - t1 Where X2 and X1 represent DCW values at times t2 and t1, respectively.
5 2.8. Statistical Analysis The data were analyzed statistically by IBM SPSS software for Student s t test. The software SPSS 21.0 (SPSS Inc. Chicago) was used for all statistical analysis. Different small letters on the columns of figures indicated a statistically significant difference at P < 0.05 when compared with each other. 3. Results and Discussion 3.1. Effect of Different Molasses Concentration on Cell Growth and Carotenoid Accumulation Sugarcane molasses as a by-product of the sugar industry contains a certain amount of fermentable sugar. Six different gradient sugar concentrations were designed from 10 g/l to 60 g/l, coupled with ammonium sulfate as a nitrogen source and C/N ratio of 4. Time courses of dry cell weight, production of carotenoid, residual sugar and content of carotenoid in the culture process are shown in Fig. 1. Figure 1. Effect of different molasses concentrations on cell growth and carotenoid accumulation: (a) Dry cell weight, (b) Production of carotenoids, (c) Residual sugar, and (d) Content of carotenoids at different molasses concentration. Data are mean values ± S.D. of three independent measurements.
6 It was in the lag phases in the initial 12 h from Fig.1a. After that thalli grew rapidly and began to enter the logarithmic growth period. At 96 h, the cultures, the maximum value of dry cell weight of 12.35 ± 0.52 (96 h) was achieved at 50 g/l. The sugar concentration of molasses had a significant effect on the cell growth (P < 0.05) in spite of no significant difference at 40, 50 and 60 g/l at 96 h. The DCW increased with increasing molasses sugar concentration within a certain range, but the excess one inhibited the cell growth. As shown in Fig. 1 b, the carotenoid production initially gradually increased to reach a peak at 30 g/l and then reduced. The maximum value of 51.70 mg/l at 30 g/l (96 h) was achieved. When the sugar concentration was 40, 50 and 60 g/l, the production of carotenoid was particularly low and decreased to 6.66 mg/l at 60 g/l. High sugar concentration might affect the growth by the Crabtree effect even though the oxygen supply was sufficient, and the cell growth and the synthesis of astaxanthin would still be inhibited (Moriel et al., 2005). In Fig. 1 d, the content of carotenoid at 60 g/l was only 0.56 mg/g, which was 11.64% of the maximum value. The optimum initial sugar concentration was selected to be 30 g/l. 3.2. Effect of Different Nitrogen Sources with Molasses as Carbon source on Cell Growth and Carotenoid Accumulation The initial sugar concentration of molasses was selected as 30 g/l, and the five organic or inorganic nitrogen sources were investigated, including yeast extract, peptone, urea, KNO3 and (NH4)2SO4. A significant increase in DCW was observed only in the yeast extract group, while other experiment groups did not show obvious difference (Fig. 2 a & c). The highest dry cell weight was achieved for yeast extract followed by the groups of peptone, urea and the control. The minimum DCW was obtained for inorganic nitrogen source. The results showed that the organic nitrogen source is more favorable than the inorganic ones and the sugarcane molasses treated by cold-acid method could be used directly for the growth of yeast Phaffia rhodozyma without addition of other nitrogen source, inorganic salts and vitamins. The maximum value of the production of carotenoid was obtained for yeast extract (Fig. 2 b). The production of carotenoid was very low in the initial 12 h, and then began to accumulate quickly. At 96 h, production of carotenoid reached stable or began to decline except for yeast extract and urea groups. It could be seen from the Fig. 2 b & d that the yeast extract group might continue to accumulate carotenoid. The statistical analysis showed that there were no significant differences in carotenoid contents among the groups of peptone, urea, KNO3, (NH4)2SO4 and the control at 96 h. Thus, the yeast extract was selected as the optimum nitrogen source.
7 Figure 2. Effect of nitrogen sources on the cell growth and accumulation: (a) Dry cell weight, (b) Production of carotenoids, (c) Residual sugar, and (d) Content of carotenoids at different nitrogen sources with molasses as carbon source. Data are mean values ± S.D. of three independent measurements. 3.3. Effect of Different C/N Ratios on Cell Growth and Carotenoid Accumulation The effect of C/N ratios on cell growth and carotenoid accumulation were studied, and the results are shown in Fig. 3. A short lag phase at the initial 12 h was observed as shown in Fig. 3 a, and C/N ratios showed a significant effect (P < 0.05) on cell growth. When the ratio of C/N was 1, the highest value of DCW of 10.94 g/l (96 h) was achieved. C/N ratio had also a very significant effect on carotenoid synthesis as shown in Fig. 3 b. When the C/N ratio was 8, the production of carotenoid gained the highest value of 58.35 mg/l at 96 h, while the ratio of C/N had a very significant impact on the content of carotenoids. It was also at the ratio of 8 that the content of carotenoids reached the highest value of 5.88 mg/g as shown in Fig. 3 d. The ratio of 1 obtained the lowest value of 3.62 mg/g. So the optimal C/N ratio of 8 was determined. From the above results, it could be seen that the low C/N ratio was beneficial to the growth of Phaffia rhodozyma; while the high one was good for the accumulation of carotenoids. It is possible to
8 obtain a large number of cells by maintaining a low C/N ratio, and then increase the ratio to promote the accumulation of carotenoids. Yamane et al. (1997) reported that it was by a two-step method that a higher production of astaxanthin was obtained. Teresa et al. found that the cultivation of Rhodotorula glutinis was more favorable for the accumulation of lipids and carotenoids at higher C/N ratio (Braunwald et al., 2013). Figure 3. Effect of C/N ratios on cell growth and carotenoid accumulation: (a) Dry cell weight, (b) Production of carotenoids, (c) Residual sugar, and (d) Content of carotenoids at different C/N ratios. Data are mean values ± S.D. of three independent measurements. 3.4. Effect of Initial ph on Cell Growth and Carotenoid Accumulation Different microorganisms have their own optimum and tolerable ph range, and the optimum ph for yeast growth is from 3 to 6. The optimum ph for the microbial growth phase and the metabolite synthesis phase is often different. To study the effect of initial ph on the growth and carotenoids accumulation, the initial ph was adjusted from 3 to 8, and the results are shown in Fig. 4.
9 Figure 4. Effect of initial ph on cell growth and carotenoid accumulation: (a) Dry cell weight, (b) Production of carotenoids, (c) Residual sugar, and (d) Content of carotenoids at different initial ph. Data are mean values ± S.D. of three independent measurements. From Fig. 4 a, the initial ph significantly affected the growth of Phaffia rhodozyma. When the initial ph was 5, the highest value of DCW of 12.80 g/l was obtained (96 h), while the DCW of the other ph groups were much lower than this maximum one. For example, the DCW of 8.30 g/l at the initial ph of 8 was obtained, only 64.80% of the maximum one. The initial ph of 5 was optimal for cell growth. As shown in Fig. 4 b & c, the initial ph also significantly affected the consumption of sugar and synthesis of carotenoid. At 96 h, the production of carotenoid reached the highest value of 60.75 mg/l at ph 5 followed by 57.20 mg/l at ph 6. As shown in Fig. 4 d, the ph ranges from the initial 3.00 ~ 8.00 to 3.53 ~ 4.84 at 24 h. The decrease of ph was mainly resulted from the production of organic acids and the increase of ph from 3.00 to 3.3 was probably due to the corresponding adaptation to the harsh environment. Thus the optimal ph of 5 was determined.
10 3.5. Effect of Medium Volume on Cell Growth and Carotenoid Accumulation The influence of dissolved oxygen on cell growth and carotenoid accumulation of Phaffia rhodozyma was studied by varying the medium volume in the flask. The volume was adjusted over a range of 15-95 ml as shown in Fig. 5. The control group was that the seed liquid was inoculated to the anaerobic bottle of 100 ml containing the medium of 50 ml. As shown in Fig. 5 a & c, Phaffia rhodozyma couldn t grow without the oxygen. It could also be seen that cells were in the lag phase in the initial 24 h. After that, the strain entered the logarithmic growth phase, with a logarithmically increase of DCW, and then reached the stable phase after about 48 h. Overall, the DCW increased with the decrease of medium volume, probably because more dissolved oxygen could be supplied with a lower medium volume. The maximum value of DCW reached 11.81 g/l with a volume of 25 ml. However, the medium of 15 ml was different, and its DCW was not so high as expected, probably because a large sheer stress was adverse for cell growth. The rate of sugar consumption was the highest in the initial 48 h, especially during the period of 24 ~ 48 h with the fastest cell growth. Figure 5. The influence of medium volume on cell growth and carotenoid accumulation of Phaffia rhodozyma: (a) Dry cell weight, (b) Production of carotenoids, (c) Residual sugar, and (d) Content of carotenoids at different medium volume. Data are mean values ± S.D. of three independent
11 measurements. Carotenoid accumulation was greatly influenced by dissolved oxygen (Fig. 5 b). When the volume of medium was above 35 ml, the production of carotenoid was only 1.42-2.41 mg/l and the content of carotenoid was only 0.26-0.31 mg/g. Meanwhile the production of carotenoid increased rapidly when the liquid volume reduced to 25 ml, and reached the maximum value of 45.55 mg/l, which was a 31.08-fold increase over that of the 95 ml volume. The results indicated that the dissolved oxygen has a significant effect on cell growth and carotenoid accumulation and a certain volume of medium appeared to facilitate cell growth and carotenoid accumulation of Phaffia rhodozyma. Adequate dissolved oxygen made the accumulation of pigment in advance. For instance, the culture in 15 ml system began to accumulate carotenoid since the 24 h, while those in 35 ml did not quickly accumulate carotenoid until 72 h. However, the content of carotenoids remained unchanged in the other three experimental groups (55 ml, 75 ml and 95 ml), indicating that cells could not synthesize a large amount of carotenoids when the volume of medium was above 35 ml in the Erlenmeyer flask of 250 ml. Considering the cell growth and the accumulation of carotenoids, 25 ml of medium in the 250 ml flask was selected as the best volume. 4. Conclusions Sugarcane molasses is a suitable carbon source for cell growth and accumulation of carotenoids by P. rhodozyma. The key medium components were optimized, and the best medium (per liter) consists of 30 g sugar concentration of sugarcane molasses and yeast extract 3.75 g with initial ph 5.0. The medium volume had significant effect on cell growth and the synthesis of carotenoids. Acknowledgments The authors gratefully acknowledge the financial support of the Guangzhou Science and Technology Program [grant no. 2014Y2-00515]. References Arunkumar, E., Bhuvaneswari, S., and Anuradha, C. V. (2012). An intervention study in obese mice with astaxanthin, a marine carotenoid--effects on insulin signaling and pro-inflammatory cytokines. Food Funct., 3: 120-126. Bhuvaneswari, S., Arunkumar, E., Viswanathan, P., and Anuradha, C. V. (2010). Astaxanthin restricts weight gain, promotes insulin sensitivity and curtails fatty liver disease in mice fed a obesitypromoting diet. Process Biochem., 45: 1406-1414.
12 Bie, X. and Zhu, M. (2015). Effects of culture conditions on the growth and β-fructofuranosidase activity of Phaffia rhodozyma. Int. J. Modern Biol. Med., 6: 38-47. Braunwald, T., Schwemmlein, L., Graeff-Hoenninger, S., French, W. T., Hernandez, R., Holmes, W. E., and Claupein, W. (2013). Effect of different C/N ratios on carotenoid and lipid production by Rhodotorula glutinis. Appl. Microbiol. Biotechnol., 97: 6581-6588. Dong, Q. L. and Zhao, X. M. (2004). In situ carbon dioxide fixation in the process of natural astaxanthin production by a mixed culture of Haematococcus pluvialis and Phaffia rhodozyma. Catalysis Today, 98: 537-544. Fassett, R. G. and Coombes, J. S. (2011). Astaxanthin: a potential therapeutic agent in cardiovascular disease. Mar Drugs, 9: 447-465. Hasegawa, J., Ogura, M., Tsuda, S., Maemoto, S. I., Kutsuki, H., and Ohashi, T. (1990). High-yield production of optically active 1,2-diols from the corresponding racemates by microbial stereoinversion. Agric. Biol. Chem., 54: 1819-1827. Higuera-Ciapara, I., Felix-Valenzuela, L., and Goycoolea, F. M. (2006). Astaxanthin: A review of its chemistry and applications. Crit. Rev. Food Sci. Nutr., 46: 185-196. Hu, Z. C., Jiang, G. F., and Zheng, Y. G. (2012). A kinetic model for astaxanthin fermentation by Xanthophyllomyces dendrorhous Zjut46. Mater. Environm. Protec. Ener. Appl. Parts 1 and 2, 343-344: 397-402. Kim, J. D. and Lee, C. G. (2005). Systemic optimization of microalgae for bioactive compound production. Biotechnol. Bioproc. Engin., 10: 418-424. Levi, L., Gyura, J., Djuri, M., and Kuljanin, T. (2004). Optimization of ph value and aluminium sulphate quantity in the chemical treatment of molasses. Europ. Food Res. Technol., 220: 70-73. Lin, W.J. and Chen, M. H. (2007). Synthesis of multifunctional chitosan with galactose as a targeting ligand for glycoprotein receptor. Carbohydrate Polymers, 67: 474-480. Mezzomo, N. and Ferreira, S. R. S. (2016). Carotenoids functionality, sources, and processing by supercritical technology: A review. J. Chem., 2016: 1-16. Moriel, D. G., Chociai, M. B., Machado, I. M. P., Fontana, J. D., and Bonfim, T. M. B. (2005). Effect of feeding methods on the astaxanthin production by Phaffia rhodozyma in fed-batch process. Brazilian Archiv. Biol. Technol., 48: 397-401. Ni, H., He, G., Yang, Y., Wu, G., Chen, S., and Chai, L. (2004). Optimization of condition for extracting astaxanthin from phaffia rhodozyma. Transac. Chin. Soc. Agric. Engin., 20: 204-208. Otton, R., Marin, D. P., Bolin, A. P., Santos Rde, C., Polotow, T. G., Sampaio, S. C., and de Barros, M. P. (2010). Astaxanthin ameliorates the redox imbalance in lymphocytes of experimental diabetic rats. Chem. Biol. Interact., 186: 306-315. Park, J. S., Mathison, B. D., Hayek, M. G., Massimino, S., Reinhart, G. A., and Chew, B. P. (2011).
13 Astaxanthin stimulates cell-mediated and humoral immune responses in cats. Vet. Immunol. Immunopathol., 144: 455-461. Song, B., Zhang, W., Peng, R., Huang, J., Nie, T., Li, Y., Jiang, Q., and Gao, R. (2009). Synthesis and cell activity of novel galactosylated chitosan as a gene carrier. Colloids Surf. B Biointerfaces, 70: 181-186. Wu, S., Li, S., Xu, X. R., Deng, G. F., Li, F., Zhou, J., and Li, H. B. (2012). Sources and bioactivities of astaxanthin. Int. J. Modern Biol. Med., 1: 96-107. Xiao, A., Jiang, X., Ni, H., Yang, Q., and Cai, H. (2015). Study on the relationship between intracellular metabolites and astaxanthin accumulation during Phaffia rhodozyma fermentation. Electronic J. Biotechnol., 18: 148-153. Xu, W., Liang, L., and Zhu, M. (2014). Determination of sugars in molasses by HPLC following solidphase extraction. Int. J. Food Proper., 18: 547-557. Yamane, Y., Higashida, K., Nakashimada, Y., Kakizono, T., and Nishio, N. (1997). Astaxanthin production by Phaffia rhodozyma enhanced in fed-batch culture with glucose and ethanol feeding. Biotechnol. Lett., 19: 1109-1111. Yuan, J. P., Peng, J., Yin, K., and Wang, J. H. (2011). Potential health-promoting effects of astaxanthin: A high-value carotenoid mostly from microalgae. Mol. Nutr. Food Res., 55: 150-165.