Astaxanthin is a carotenoid pigment with strong antioxidant activity and some essential biological functions. Among astaxanthin producers the interest of the scientific and business community has focused for years on yeast Xanthophyllomyces dendrorhous. Objective of the study is to find the optimal culture conditions for the yeast to obtain highest yield of astaxanthin. The study evaluated the effect of media with various composition and soybean oil as co-substrates. Moreover, fed-batch fermentation bioprocesses of astaxanthin have been investigated. pH control was employed in fed-batch fermentation. The obtained results showed that the medium containing diluted pineapple juice was found to be the optimal medium with best astaxanthin production. Soybean oil of 2% (v/v) supplemented in culture media resulted in even higher astaxanthin producing. In addition, the fed-batch fermentation with glucose enhanced astaxanthin and biomass yield significantly. pH shift strategy gave the maximum astaxanthin yield of 32.65 mg/l at 132 h.   

Astaxanthin (3,3’-dihydroxy-β,β’-carotene-4,4’-dione) is a carotenoid widely distributed in nature, found as the main pigment in some crustaceans (shrimp and lobster), fish (trout and salmon), birds (flamingo and scarlet ibis) and microorganisms (the yeast Xanthophyllomyces dendrorhous and the algae Haematococcus pluvialis).   Astaxanthin is proven that possesses a very high antioxidant capacity, over 550 times than that of vitamin E and 6000 times than that of vitamin C (Naguib, 2000). 

H. pluvialis has high concentrations of astaxanthin, but industrial application is limited by lengthily autotrophic cultivation in open freshwater ponds and necessity of disrupting the cell wall to liberate the carotenoid. Phaffia yeast Xanthophyllomyces dendrorhous has desirable properties and potential commercial value as a dietary source of natural astaxanthin, including rapid heterotropic metabolism and production of high cell densities in bioreactors. X. dendrorhous exhibits 100% free, non-esterified astaxanthin that consist virtually all in 3R, 3R’ form, an important astaxanthin source in nature having great bio-availability. 

The astaxanthin synthesis by X. dendrorhous was verified on media enriched with natural origin substances. Natural carbon sources, such as grape juice, pineapple juice and plant extracts such as carrot, Perilla frutescens leaf promote the synthesis of astaxanthin in X. dendrorhous (Stachowiak, 2012; Kim et al., 2007). A X. dendrorhous mutant enhanced production by 66% when pineapple juice was used as a medium at 10% dilution in replacement of YM medium (Jarasripongpun et al., 2008). Kim et al. 2007 showed that an addition of plant extract from P. frutescens significantly improved the efficiency of carotenoids in cultures of mutant X. dendrorhous G276 and reduced the cultivation time by 2 days. In cultures on the carrot medium the highest cellular and volumetric concentrations of astaxanthin were recorded for four out of five tested strains (Stachowiak, 2012).

Previous studies have shown that X. dendrorhous can grow on a variety of carbon sources. Among them, glucose was the most efficient carbon source for cell growth and astaxanthin production. However, rapid catabolism of this sugars could cause a decrease in the biosynthesis of many bioactive metabolites (Rokem et al., 2007). Nowadays slowly metabolized carbon, such as vegetable seed oils, is used as a part of the carbon source, which can reduce the glucose suppressive effect. 

X. dendrorhous showed the Crabtree effect during its fermentation process: high glucose concentration caused ethanol production rather than biomass during the exponential growth phase, as a result of overflow metabolism or fermentative catabolism of glucose, leading to significant reductions in biomass and astaxanthin production. The strategy for solving this problem is the fed-batch culture (Reynders et al., 1997). Trend in research is to develop the new culture technologies such as fed-batch fermentation and pH control, facilitating an intensified commercial-scale production of pigment by this red yeast. A kinetic model was applied to fed-batch culture to predict the optimum feeding scheme for astaxanthin production, leading to a volumetric yield of 28.6 mg/l (Liu and Wu, 2008). In another study, a pH-shift strategy on astaxanthin production by X. dendrorhous in batch fermentation obtained the maximum astaxanthin concentration of 27.05 mg/l, increased by 24.1% as comparison with that of constant pH fermentation (Hu et al., 2006). 

In this study, the effect of plant origin culture media and supplemented soybean oil on the biosynthesis of astaxanthin was investigated. Moreover, fed-batch fermentation and pH control strategy experiments were also carried out in order to increase the final astaxanthin yield as well as to extend the astaxanthin production period.

The microorganism, X. dendrorhous T1, employed in this study, was maintained at 4°C on YM agar slants with composition of 1% glucose, 0.3% malt extract, 0.3% yeast extract, 0.5% peptone and 2% g agar, and transferred monthly. Inoculum medium was YM liquid medium with pH adjusted to 6.0. The soybean oil used in this study was purchased from Cai Lan oils & fats industries company (Vietnam) and other chemicals were from Chemical and scientific technological materials joint stock company. Astaxanthin standard was purchased from Sigma-Aldrich Pte Ltd., Singapore.

Strain was cultivated on the following test culture media: 1% Glucose, 10% carrot extract, 5% Perilla frutescens leaf extract and 10% pineapple juice (v/v). All media were supplemented with 1% glucose, 0.8% yeast extract and 0.2% peptone. All media were autoclaving for 20 min at 121°C. Erlenmeyer flask (500 ml) containing 250 ml of an appropriate medium was inoculated with 5% spore suspension of X. dendrorhous T1 and incubated at 22°C for 5 days, agitation speed of 200 rpm, illumination of 600 lx. The biomass and astaxanthin yields were determined at the end of fermentation period. All experiments were performed triplicate.

Soybean oil was added to the above selected culture medium at the concentration of 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0% (v/v). The fermentation procedure was carried out 22°C for 5 days, agitation speed of 200 rpm, illumination of 600 lx. The biomass and astaxanthin yields were determined at the end of fermentation period. All experiments were performed triplicate.

The experiments were performed in a 14 liter Bioreactor containing 10 l of selected medium. The operational conditions during 6 days were: inoculum rate of 5% (v/v); 22°C; aeration ratio of 1 v/v/min; agitation speed of 200 rpm; pH was maintained at 5.0 with 2.0 M NaOH and 2.0 M HCl. The course of fermentation was constantly monitored by off-line sampling at 12h intervals, each sample subjected to test for total carbohydrate content, biomass content and astaxanthin yield. Two batches were repeated for each experiment. 

In fed-batch experiments, fermentation was first carried out in the batch model until the total carbohydrate content had dropped to around 5 g/l, then glucose of 10 g/l was added at 72, 96 and 120 h of the fermentation time in order to reestablish the total carbohydrate concentration in the medium to above 10 g/l. The stock solution of glucose (50%) was used as the feeding substrate in the fed-batch process. The main operating conditions of fed-batch fermentation were the same as those of the batch fermentation procedure.

In this experiment, the conditions of temperature, dissolved oxygen and agitation speed were the same as those of fed-batch fermentation. However, the pH-shift control strategy was as follows: first, the culture pH was maintained at pH 6 around 80 h with 2.0 M NaOH and 2.0 M HCl, and then it was shifted slowly to pH 4 with 2.0 M HCl. 

Yeast biomass was separated from the liquid medium by centrifuging and rinsed twice with double distilled water and then dried at 60°C to constant weight, yielding the Dry Cell Weight (DCW). Astaxanthin content was determined by HPLC according to Lu et al. (2010). Briefly, the dried yeast was disrupted with Dimethyl Sulphoxide (DMSO) and then extracted with acetone to 4.0 ml and freezing was done at -20°C to precipitate the colorless lipids. The defatted crude extracts were filtered through a 0.45 µm syringe filter and 10 µl aliquots were subjected to reversed-phase HPLC analysis. All samples and assays were carried out in triplicate and the results were determined as the average mean values ± Standard deviation (SD).

Previous studies showed that X. dendrorhous can grow well and produce astaxanthin with high content on several plant extracts (Jarasripongpun et al., 2008; Stachowiak, 2012). In this study, we used several media enriched with natural abundant plant sources including pineapple juice, carrot extract, P. frutescens leaf extract for culturing the yeast in comparison to 1% glucose medium. The results showed that the highest growth yield of yeast strain was obtained in cultures on glucose medium, on which biomass yield was 15.18 g/l (table 1).

However, the astaxanthin yield on this medium was the lowest. Meanwhile, the medium supplemented by 10% pineapple juice proved to be the medium most effectively promoting carotenogenesis, which had the highest astaxanthin yield of 12.79 mg/l. The astaxanthin content in cell reached 0.84 mg/g dry cell. This result was higher than that recorded by Jirasripongpun et al. (2007) and Stachwiak (2012) with 0.56 mg/g and 0.46 mg/g, respectively. Lower result reached for the medium added carrot extract and P.frutescens leaf extract, which were 11.18 mg/l and 8.19 mg/l, respectively. The carrot and pineapple media may contain some precursors of carotenoid synthesis. It was reported that pineapple juice and carrot extract contain beta-carotene, which could be used as intermediates or precursors for astaxanthin production by yeast. The nutrient composition of pineapple juice and carrot extract may be the source of vitamins such as B1, B2, C and minerals (Ca, Na, K, Fe, Mg, P). Moreover, the acidic pH at 4.0 of pineapple juice was also suitable for growth and carotenoid formation without any pH treatment (Jarasripongpun et al., 2008). Therefore, pineapple juice is a cheap and satisfactory medium for carotenoid production by X. dendrorhous T1. This would make the use of pineapple juice a feasible and an economical culture medium for mass and astaxanthin production at the large scale. 

Soybean oil has been reported to be beneficial to the biomass and to increase the production of bioactive metabolites in several fungi (Yang and He, 2008). The supplement of soybean oil as co-substrates was very effective in enhancing astaxanthin accumulation by X. dendrorhous (Wu and Yu, 2013). In this research, the effect of soybean oil on astaxanthin production of X. dendrorhous T1 was studied, where oil was added at the concentration of 1.0, 1.5, 2.0, 2.5, 3.0% (v/v). The results showed that the growth of the yeast increased in correlation with increase of soybean oil concentration. The maximum biomass (17.4 g/l) was achieved in medium supplemented by 3% soybean oil, which was 1.21 fold higher than the control. The stimulation of biomass by soybean oil might be caused by partial incorporation of lipids in the cell membrane, thereby facilitating the uptake of nutrients from the medium (Yang et al., 2000). However, the soybean oil concentration of 2% resulted in the maximum astaxanthin yield (15.39 mg/l), which was 1.2 fold higher than the control. These results suggested that soybean oil may have a beneficial influence on astaxanthin production of X. dendrorhous, but redundant fatty acids were suitable for biomass but not for astaxanthin production.  

To establish the optical conditions for the production of astaxanthin, the batch and fed-batch fermentation experiments were carried out in 10-litre fermentor scale. The time-courses of the batch and fed-batch fermentation at 22ºC, pH=5.0 were shown in Fig.2. 

In the batch fermentation, with the consumption of sugar, the astaxanthin formation was started after the beginning of cell growth (Fig.2a). Total carbohydrate content reduced during 72 first hours of fermentation process from 30 g/l down to 5.18 g/l. With this decrease, the cell growth sharply increased and the biomass content reached the maximum of 16.33 g/l at 108 h. Meanwhile, the maximum astaxanthin yield achieved 26.62 mg/l at 108 h that was higher than that reported by Hu et al. (2005).  

Since astaxanthin is one of the intracellular components, two factors, the cell density and astaxanthin content in the cell, both affect astaxanthin production. It was found that astaxanthin formation was observed after the cessation of cell growth. To obtain high astaxanthin production, it is necessary to attain a certain cell mass, and extend the astaxanthin formation period after the cessation of cell growth. The fermentation period might be separated into two stages: the cell growth stage and the astaxanthin production stage. Fed-batch experiments were carried out to achieve high astaxanthin production and to extend astaxanthin formation.     

Based on the results of batch fermentation experiment, the residual carbohydrate concentration of 5 g/l was selected. When the residual content dropped to 5 g/l, the feeding glucose was added to the fermentor. In this fed-batch fermentation, the substrate was added to the fermentor at various times including 72, 96 and 120 h of fermentation process. The results indicated that the biomass content and the astaxanthin yield in the fed-batch fermentation were higher than those in the batch fermentation (Fig.2b). The maximum biomass of 19.36 g/l was achieved at 132 h, which was higher by 18.5% than that of batch fermentation. The astaxanthin concentration reached the maximum of 29.35 mg/l, which was higher by 10.3% than that of batch fermentation. Thus, the utilization of fed-batch fermentation showed to be the efficient method for astaxanthin production.

Some reports proved that the optimal pH for cell growth and for astaxanthin formation was around 6.0 and 4.0, respectively. And, the optimal pH-shift control strategy in batch fermentation process was developed as follows: in the first stage, the culture pH was maintained at pH=6.0 around 80 h for cell growth, it was then shifted to pH=4.0 for astaxanthin production in the secondary stage (Hu et al., 2006). In this work, the same pH-shift control strategy was carried out in fed-batch fermentation of X. dendrorhous, and the results of the experiment were shown in Fig.4.

The obtained study results showed that production of astaxanthin clearly increased during the fermentation. The maximum astaxanthin concentration at fed-batch fermentation with pH-shift control strategy reached 32.65 mg/l at 132 h, which was higher by 22.6% and 11.2% than that of the batch and fed-batch fermentation, respectively, with constant pH = 5.0. However, the maximal biomass content at fed-batch fermentation with pH-shift control was 18.98 g/l, which was lower by 2.0% than that of fed-batch fermentation with constant pH = 5.0. These results were much higher than those reported by Hu et al. (2006), in which maximum astaxanthin content gained 27.05 mg/l from fed-batch fermentation with pH-shift control. 

Comparing the various media enriched on plant resources in this research, we found that medium supplemented with diluted pineapple juice could be sufficiently used by X. dendrorhous, resulting in high biomass and astaxanthin production. The addition of soybean oil (2%, v/v) in the culture medium as co-substrates was very effective in enhancing astaxanthin accumulation and it may be an area for further increasing astaxanthin production by supplying effective ingredients isolated from soybean oil. 

The obtained results showed that fed-batch fermentation with pH-shift control strategy is the best among all the experiments in this work.  A significant increase (22.6%) in production of astaxanthin was achieved at 132 h in fed-batch fermentation with pH-shift control as compared to batch process. The utilization of fed-batch fermentation with pH-shift control strategy for production of astaxanthin by the yeast X. dendrorhous could be an important alternative to increase the astaxanthin productivity. By fine tuning of operation conditions, there may be a scope for further enhancement of the astaxanthin yield.

The authors are grateful to Vietnam Ministry of Industry and Trade for financial support of this research.

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               Bui Kim Thuy*, Vu Kim Thoa, La Manh Tuan and Nguyen Duy Lam
Vietnam Institute of Agricultural Engineering and Postharvest Technology, Hanoi, Vietnam
*Corresponding author e-mail addresses: [email protected]