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Title: Investigation of major biochemical factors for improved carotenoid (astaxanthin) production by the yeast xanthophyllomyces dendrorhous
Authors: Liu, Yuanshuai
Degree: Ph.D.
Issue Date: 2007
Abstract: Astaxanthin, a carotenoid pigment, has been increasingly used as a feed in the aquaculture industry, and is also a potential functional food and pharmaceutical supplement because of its excellent antioxidant activity. Xanthophyllomyces dendrorhous is a high astaxanthin-producing yeast, though its commercial application is still hampered by the relatively low astaxanthin productivity compared with the chemical and other biological processes. In order to understand the physiology of the yeast and improve the astaxanthin production by X. dendrorhous yeast culture, we carried out this project to examine and optimize the major nutrients and cultural factors, use various stimulants to enhance astaxanthin biosynthesis, and develop more efficient culture processes. The X. dendrorhous yeast strain used was ENM 5, and its astaxanthin content accounts for more than 80% of the total carotenoid pigment. Most of the experiments were performed in small volume, with 25~50 mL liquid medium in each shake-flask. Oxygen supply was identified as an important factor on the carotenoid production by X. dendrorhous. Poor oxygen transfer in the shake-flasks resulted in constantly lower DO level than the critical DO during culture process. Based on linear regression of the experimental data, the oxygen transfer coefficient (KLa) was correlated to the shaker speed (N) and liquid volume (VL) as, KLa = 0.141 N0.88(VL/VO)-0.80 (VO=flask volume) The carotenoid production showed a strong linear correlation with the oxygen transfer rate. The addition of a suitable hydrocarbon liquid as an oxygen vector can relieve the oxygen insufficiency during X. dendrorhous shake-flask culture, thus to improve carotenoid production. In particular, n-hexadecane was proved the most beneficial oxygen vector, and the addition of 9% (v/v) n-hexadecane increased the volumetric carotenoid yield by 58%. The major nutrients and cultural conditions were evaluated and optimized for X. dendrorhous growth and carotenoid biosynthesis through statistical experiment design. From these culture factors, glucose, ammonium sulfate and pH were first screened out and identified as the significant factors using a Plackett-Burman design. Then these significant factors were optimized by response surface methodology, and the optimal levels for cell growth or carotenoid accumulation were determined separately. Carotenoid biosynthesis favors for high C/N ratio, low carbon and nitrogen concentrations and a slightly acidic condition. Based on the optimization study, a two-stage culture process was exercised with optimal conditions tailored for cell growth and carotenoid accumulation, achieving 95.8% higher volumetric carotenoid yield. Carotenoid biosynthesis in X. dendrorhous is partially growth-associated, which is mainly synthesized when the cell growth is suppressed or the cells are under stress (such as nutrient limitation). The occurrence of Crabtree effect in X. dendrorhous is a consequence of its limited respiratory capacity and overflow metabolism, but not glucose repression. The addition of TCA cycle intermediates only improved the carotenoid biosynthesis slightly as precursors. Various metal ions, osmotic stress and reactive oxygen species H2O2 were tested as stimulants of astaxanthin biosynthesis in X. dendrorhous. The metal ions and osmotic stress only showed slight effect, while H2O2 nearly doubled the astaxanthin content in the yeast cells. The results suggest that astaxanthin is a supplement of catalase deficiency in X. dendrorhous, and its biosynthesis can be greatly stimulated by H2O2 as an antioxidative response. A semicontinuous perfusion process was evaluated for astaxanthin production in shake-flask cultures, which gave rise to the maximum biomass and astaxanthin production due to the elimination of inhibitory metabolites during culture. Furthermore, the combined use of this process and 30 mM H2O2 stimulation achieved an even higher volumetric astaxanthin yield 62.4 mg/L and productivity 4.46 mg/L-d, which are 6.7-fold and 2.4-fold of those in the batch cultures, respectively. In conclusion, the evaluation and optimization of the major cultural factors, the exploration of yeast physiology and special stimulants, and the development of new culture processes are fruitful for improving astaxanthin production. These will make meaningful contributions to future research and commercial application of X. dendrorhous fermentation for astaxanthin production.
Subjects: Hong Kong Polytechnic University -- Dissertations.
Pages: xvii, 185 p. : ill. ; 31 cm.
Appears in Collections:Thesis

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