A highly efficient two-stage cultivation strategy for lutein production using heterotrophic culture of Chlorella sorokiniana MB-1-M12
Introduction
Lutein is a primary xanthophyll carotenoid, serving as light-harvesting antenna pigment and antioxidant in microalgae, plants and other photosynthetic organisms (Pascal et al., 2005). Other than light harvesting, the major function of lutein is to protect the photosystems from oxidative damage at high light intensities, especially blue light (Roberts et al., 2009). The structure of lutein is composed of a long conjugated double bond backbone chain with aromatic rings at either end. Lutein is lipo-soluble and is capable of scavenging free radicals and singlet oxygen, acting as a natural antioxidant (Britton, 1995). In humans, lutein along with zeaxanthin, accumulates in the macula of the eye, as the macular pigment and protects the retina from blue light and aids in improving visual acuity (Krinsky et al., 2003). Lutein has been implicated in human health mainly because of its ocular-protective activity and antioxidant property against neurodegenerative diseases, cardio-vascular diseases, diabetic retinopathy (Zhang et al., 2014) and respiratory health (Melo van Lent et al., 2016). However, humans are incapable of de novo synthesis of lutein and are strictly dependent on dietary intake to fulfil their lutein requirement. A daily dose of 5 mg is recommended for patients with Age related Macular Degeneration (AMD), but the status of lutein as an essential nutrient has not been approved yet by pharmaceutical authorities (Fernandez-Sevilla et al., 2010). Other than this, lutein has also been used as colorant and food additive for human consumption (known as colorant E161b in the European Union) and also as a feed additive to deepen the yellow color of egg yolks (Lin et al., 2015). With all these applications, market demand for lutein is increasing and in 2015 the global lutein market was estimated at US$ 135 million and will continue to rise (Global Market Insights Lutein Market Report, 2016).
Currently, the market demand for lutein is met by the extraction of lutein from the bright yellow petals of the marigold flowers of the genus Tagetes (Fernandez-Sevilla et al., 2010). However, lutein extraction from Tagetes petals are challenged by the seasonal availability of the flowers, large requirement of land for the cultivation of the plants, requirement of skilled labor and the very low lutein content present in the flower petals (Lin et al., 2015). Lutein primarily exists in mono- and di-esterified form in marigold flowers (Del Campo et al., 2007), and chemical saponification is required to extract lutein from them. Microalgae are a potential source of lutein and the advantages are: i) high cellular lutein content at 0.5–1.2% by weight, ii) unaffected by seasonal variation as in the case of Tagetus, iii) very low land and water requirements, and iv) the possibility to generate other high value products from the microalgal biomass (Lin et al., 2015). However, the major bottleneck in the mass production of microalgae is the supply of optimal light intensity, which is the prime criteria to stimulate pigment production in photoautotrophic cultures. As a result, effective new photobioreactor designs are being implemented to cope with this issue; however, this usually causes a significant increase in the product costs. Therefore, in contrast to phototrophic culture systems, heterotrophic cultivation of microalgae for the mass production of high-value products has been considered an economically feasible and commercially favorable option since the heterotrophic growth could attain much higher microalgal biomass productivity without the need of light supply and CO2 aeration (Hu et al., 2017). It is easier to control the product quality in a heterotrophic culture due to being operated in a well-control closed system. Also, since the growth and metabolism of microalgae occurs in dark conditions for heterotrophic cultures, conventional bacterial fermenters can be used for microalgal cultures without much modifications (Bumbak et al., 2011).
Although microalgae are genetically capable of utilizing organic carbon sources and are endowed with a central carbon catabolic pathway, the predisposition for heterotrophy is mainly strain dependent (Perez-Garcia et al., 2011). The other potential problem associated with producing pigments (e.g., lutein) under heterotrophic conditions would be the absence of light, as light illumination is usually required for promoting the production of pigments (Hu et al., 2017). Therefore, selecting a suitable heterotrophic strain for efficient lutein production under optimal operating conditions would be a pivotal step to realize the idea of producing microalgal lutein heterotrophically. In this study, a heterotrophic Chlorella sorokiniana MB-1-M12, was examined for its effectiveness in lutein production and the culture conditions were optimized to achieve the maximal lutein producing performance. In particular, a novel two-stage cultivation strategy was devised to further improve the lutein productivities to make it suitable for industrial scale applications.
Section snippets
Microalga used and its culture conditions
Chlorella sorokiniana MB-1-M12, is a glucose tolerant mutant of C. sorokiniana MB-1, which is originally a photoautotrophic lutein-rich strain isolated in southern Taiwan. C. sorokiniana MB-1-M12 possesses a high lutein content and productivity under heterotrophic conditions, making it a potential candidate for mass production of lutein. The MB-1-M12 strain was cultivated on BG-11 medium consisting of (mg/L): NaNO3, 1.0; Na2CO3, 20; CaCl2·2H2O, 36; Citric acid, 6.0; MgSO4·7H2O, 75; K2HPO4, 40;
Results and discussion
Chlorella sorokiniana MB-1-M12 is capable of heterotrophic growth under dark conditions using glucose as the carbon source. Preliminary experiments showed that optimal growth of the MB-1-M12 strain was obtained with BG-11 medium, while other frequently used media (such as Basal medium and Bold’s basal medium) did not provide optimal growth (data not shown). Therefore, BG-11 medium was used for the experiments investigating the optimal carbon source, nitrogen source, pH and the C/N ratio as well
Conclusions
Chlorella sorokiniana MB-1-M12, a heterotrophic high lutein accumulating microalga, could grow well and accumulate lutein using glucose and urea as the carbon and nitrogen sources with maximal lutein content obtained at pH 7.5 and C/N ratio of 10. The lutein content and productivity were much improved when the fermentation mode was changed from batch mode to a novel two-stage process integrating fed-batch and semi-batch modes. High lutein content (5.88 mg/g biomass) and lutein productivity
Acknowledgements
This work was supported by Taiwan’s Ministry of Science and Technology (MOST) under grant numbers of MOST 106-3113-E-006-011, 106-3113-E-006-004-CC2, 106-2621-M-006-007, 106-3114-E-006-008, 104-2221-E-006-227-MY3, and 103-2221-E-006-190-MY3.
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