Green Microalga Chlorella Zofingiensis Research Articles

Differential responses of the green microalga Chlorella zofingiensis to the starvation of various nutrients for oil and astaxanthin production

Chlorella zofingiensis has been proposed as a potential producer of lipids and the high-value carotenoid astaxanthin. In this study, the responses of photoautotrophic C. zofingiensis with respect to growth, lipid profiles and astaxanthin accumulation were investigated upon the starvation of N (NS), P (PS) and S (SS). NS and SS stimulated triacylglycerol (TAG) accumulation, which reached 27% and 21% of dry weight (DW), respectively. Stresses also stimulated astaxanthin accumulation greatly, reaching 3.9 mg/g DW by NS. SS led to the highest TAG productivity (52.4 mg L−1 d−1) while NS gave rise to the highest astaxanthin productivity (0.624 mg L−1 d−1). In combination with transcriptional analysis, a working model for stress-associated TAG and astaxanthin biosynthesis was proposed. Taken together, these detailed data shed light on the elucidation of differential responses to nutrient stresses and may provide insights into future engineering of this promising alga for improving TAG and astaxanthin production.

Enhanced Accumulation of Carbohydrate and Starch in Chlorella zofingiensis Induced by Nitrogen Starvation

Though less attention has been paid to microalgae as a feedstock for bioethanol production, many microalgae seem to have this potential since they contain no lignin, minor hemicellulose, and abundant carbohydrate. The objective of this study was to investigate the effect of nitrogen starvation on carbohydrate and starch accumulation in green microalga Chlorella zofingiensis and assess the feasibility of using this microalga as a bioethanol feedstock. The results showed that the specific growth rate under nitrogen starvation (0.48 day(-1)) was much lower than that under nitrogen repletion (1.02 day(-1)). However, nitrogen starvation quickly induced the accumulation of carbohydrate, especially starch. After merely 1 day of nitrogen starvation, carbohydrate and starch increased 37 % and 4.7-fold, respectively. The highest carbohydrate content reached 66.9 % of dry weight (DW), and 66.7 % of this was starch. In order to obtain enough carbohydrate productivities for bioethanol production, two-stage cultivation strategy was implemented and found to be effective for enhancing biomass, carbohydrate, and starch simultaneously. The optimal biomass, carbohydrate, and starch productivities of C. zofingiensis were obtained after 5 days of cultivation, and their values were 699, 407, and 268 mg L(-1) day(-1), respectively.

Efficient Heterologous Transformation of Chlamydomonas reinhardtii npq2 Mutant with the Zeaxanthin Epoxidase Gene Isolated and Characterized from Chlorella zofingiensis

In the violaxanthin cycle, the violaxanthin de-epoxidase and zeaxanthin epoxidase catalyze the inter-conversion between violaxanthin and zeaxanthin in both plants and green algae. The zeaxanthin epoxidase gene from the green microalga Chlorella zofingiensis (Czzep) has been isolated. This gene encodes a polypeptide of 596 amino acids. A single copy of Czzep has been found in the C. zofingiensis genome by Southern blot analysis. qPCR analysis has shown that transcript levels of Czzep were increased after zeaxanthin formation under high light conditions. The functionality of Czzep gene by heterologous genetic complementation in the Chlamydomonas mutant npq2, which lacks zeaxanthin epoxidase (ZEP) activity and accumulates zeaxanthin in all conditions, was analyzed. The Czzep gene was adequately inserted in the pSI105 vector and expressed in npq2. The positive transformants were able to efficiently convert zeaxanthin into violaxanthin, as well as to restore their maximum quantum efficiency of the PSII (Fv/Fm). These results show that Chlamydomonas can be an efficient tool for heterologous expression and metabolic engineering for biotechnological applications.

Enhancement of carotenoids biosynthesis in Chlamydomonas reinhardtii by nuclear transformation using a phytoene synthase gene isolated from Chlorella zofingiensis.

The isolation and characterization of the phytoene synthase gene from the green microalga Chlorella zofingiensis (CzPSY), involved in the first step of the carotenoids biosynthetic pathway, have been performed. CzPSY gene encodes a polypeptide of 420 amino acids. A single copy of CzPSY has been found in C. zofingiensis by Southern blot analysis. Heterologous genetic complementation in Escherichia coli showed the ability of the predicted protein to catalyze the condensation of two molecules of geranylgeranyl pyrophosphate (GGPP) to form phytoene. Phylogenetic analysis has shown that the deduced protein forms a cluster with the rest of the phytoene synthases (PSY) of the chlorophycean microalgae studied, being very closely related to PSY of plants. This new isolated gene has been adequately inserted in a vector and expressed in Chlamydomonas reinhardtii. The overexpression of CzPSY in C. reinhardtii, by nuclear transformation, has led to an increase in the corresponding CzPSY transcript level as well as in the content of the carotenoids violaxanthin and lutein which were 2.0- and 2.2-fold higher than in untransformed cells. This is an example of manipulation of the carotenogenic pathway in eukaryotic microalgae, which can open up the possibility of enhancing the productivity of commercial carotenoids by molecular engineering.

Differential lipid and fatty acid profiles of photoautotrophic and heterotrophic Chlorella zofingiensis: Assessment of algal oils for biodiesel production

The objective of this study was to document and compare the lipid class and fatty acid composition of the green microalga Chlorella zofingiensis cultivated under photoautotrophic and heterotrophic conditions. Compared with photoautotrophic cells, a 900% increase in lipid yield was achieved in heterotrophic cells fed with 30 g L(-1) of glucose. Furthermore heterotrophic cells accumulated predominantly neutral lipids (NL) that accounted for 79.5% of total lipids with 88.7% being triacylglycerol (TAG); whereas photoautotrophic cells contained mainly the membrane lipids glycolipids (GL) and phospholipids (PL). Together with the much higher content of oleic acid (C18:1) (35.2% of total fatty acids), oils from heterotrophic C. zofingiensis appear to be more feasible for biodiesel production. Our study highlights the possibility of using heterotrophic algae for producing high quality biodiesel.

Sugar-based growth, astaxanthin accumulation and carotenogenic transcription of heterotrophic Chlorella zofingiensis (Chlorophyta)

The green microalga Chlorella zofingiensis can grow and produce the ketocarotenoid astaxanthin in the dark with glucose as sole carbon and energy source. In the present study, we reported that glucose, mannose, fructose, sucrose, galactose and lactose could differentially support the cell growth and astaxanthin biosynthesis. Of the sugars surveyed, glucose and mannose were the best carbon sources for the algal growth in the dark, as indicated by the relatively high specific growth rates (ca. 0.03 h −1) and high cell dry densities (ca. 10 g l −1). Furthermore, the algal cells cultured with glucose and mannose accumulated the highest amounts of astaxanthin (ca. 1 mg g −1), indicating a correlation between cell growth and astaxanthin formation. In addition, various sugars differentially regulated the transcription of three carotenogenic genes encoding phytoene desaturase (PDS), β-carotene ketolase (BKT), and β-carotene hydroxylase (CHYb) respectively. By using glucose fed-batch fermentation, high cell dry weight concentration (ca. 53 g l −1) and high astaxanthin production (ca. 32 mg l −1) were obtained, which are much higher than those ever reported in the alga. The present study suggests that C. zofingiensis is suitable for the production of natural astaxanthin on a massive scale.

Growth-associated biosynthesis of astaxanthin in heterotrophic Chlorella zofingiensis (Chlorophyta)

The green microalga Chlorella zofingiensis can produce the ketocarotenoid astaxanthin under heterotrophic culture conditions. Here we report the growth-associated biosynthesis of astaxanthin in this biotechnologically important alga. With glucose as sole carbon and energy source, C. zofinginesis grew fast in the dark with rapid exhaustion of nitrogen and carbon sources from media, leading to a high specific growth rate (0.034 h(-1)). Cultures started at a cell concentration of about 3.4 x 10(9) cells l(-1) reached, after 6 days, standing biomass values of 1.6 x 10(11) cells or 8.5 g dry weight l(-1). Surprisingly, the biosynthesis of astaxanthin was found to start at early exponential phase, independent of cessation of cell division. A general trend was observed that the culture conditions benefiting cell growth also benefited astaxanthin accumulation, indicating that astaxanthin was a growth-associated product in this alga. The maximum cell dry biomass and astaxanthin yield were 11.75 g l(-1) and 11.14 mg l(-1) (about 1 mg g(-1)), simultaneously obtained in the fed-batch culture with a combined glucose-nitrate mixture addition, which were the highest ever reported in dark-heterotrophic algal cultures. The possible reasons why dark-heterotrophic C. zofingiensis could produce astaxanthin during the course of cell growth were discussed.

Peroxynitrite and nitryl chloride enhance astaxanthin production by the green microalga Chlorella zofingiensis in heterotrophic culture

The fast growing unicellular green microalga Chlorella zofingiensis has attracted interest as a promising organism for commercial production of the high-value carotenoid astaxanthin [Del Campo JA, Rodriguez H, Moreno J, Vargas MA, Rivas J, Guerrero MG. Accumulation of astaxanthin and lutein in Chlorella zofingiensis. Appl Microbiol Biotechnol 2004;64:848–54; Ip PF, Wong KH, Chen F. Enhanced production of astaxanthin by the green microalga Chlorella zofingiensis in mixotrophic culture. Process Biochem 2004;39:1761–6; Ip PF, Chen, F. Production of astaxanthin by the green microalga Chlorella zofingiensis in the dark. Process Biochem 2005;40:733–8.]. In this study, the reactive nitrogen species (RNS) peroxynitrite and the reactive nitrogen intermediate (RNI) nitryl chloride were applied to induce carotenogenesis leading to enhanced astaxanthin formation in C. zofingiensis in dark heterotrophic culture. Both the RNS and RNI when applied at an appropriate concentration stimulated astaxanthin biosynthesis in C. zofingiensis. Cultures treated with 1 mM peroxynitrite led to an enhanced yield of astaxanthin from 9.9 to 11.78 mg l −1. Nitryl chloride generated from the reaction mixture of 0.1 mM sodium nitrite with 0.5 mM sodium hypochlorite also increased the astaxanthin yield to 10.99 mg l −1. The present study is the first report demonstrating the possibility of using RNS and RNI for the enhancement of astaxanthin production in heterotrophic microalgal culture.

Employment of reactive oxygen species to enhance astaxanthin formation in Chlorella zofingiensis in heterotrophic culture

The advantages of using heterotrophic cultivation for commercial production of the high-value ketocarotenoid astaxanthin by microalgae have been emphasized in recent years [Chen F. High cell density culture of microalgae in heterotrophic growth. Trends Biotechnol 1996;14:421–5; Chen F, Chen H, Gong X. Mixotrophic and heterotrophic growth of Haematococcus lacustris and rheological behaviour of the cell suspensions. Bioresour Technol 1997;62:19–24; Ip PF, Chen F. Production of astaxanthin by the green microalga Chlorella zofingiensis in the dark. Process Biochem 2005;40:733–8]. In a heterotrophic culture where light is not provided, the employment of oxidative stress is essential for promoting the formation of secondary carotenoids including astaxanthin. In the present study, oxidative treatment imposed by chemically generated reactive oxygen species (ROS) including the hydroxyl radicals ( OH) and singlet oxygen ( 1O 2) for inducing carotenogenesis and astaxanthin production by C. zofingiensis in heterotrophic culture was investigated. OH stimulated secondary carotenoids accumulation significantly, whereas 1O 2 did not. The addition of 0.1 mM hydrogen peroxide to the culture leading to the generation of OH enhanced the yield of astaxanthin from 9.9 mg l −1 to 12.58 mg l −1. These results indicate that the application of a suitable type and dosage of ROS is potentially useful for mass production of astaxanthin by C. zofingiensis in heterotrophic culture.

Production of astaxanthin by the green microalga Chlorella zofingiensis in the dark

Dark-heterotrophic growth of microalgae eliminates the requirement for light and therefore, facilitates large-scale cultivation of microalgae, especially in denser cultures [Trends Biotechnol. 14 (1996) 421]. In this study, the heterotrophic production potential of the secondary carotenoid astaxanthin by the green microalga Chlorella zofingiensis was investigated. The alga showed excellent growth on glucose-supplemented media in batch culture. The maximum specific growth rate and astaxanthin yield were 0.031 h −1 and 10.3 mg l −1, respectively obtained at glucose concentrations of 20 and 50 g l −1, which were the highest ever reported in heterotrophic algal cultures. In the absence of light, formation of secondary carotenoids was mostly dependent on the initial carbon and nitrogen balance in the medium; enhanced biosynthesis of secondary carotenoids including astaxanthin was found in the medium with a high C/N ratio (i.e. C/N ratio=180). The light-independent astaxanthin-producing ability of C. zofingiensis suggested that the alga might be potentially employed for commercial production of astaxanthin on a large-scale.

Enhanced production of astaxanthin by the green microalga Chlorella zofingiensis in mixotrophic culture

Mixotrophic culture may be used as an efficient means for enhanced production of light-induced pigments in microalgae. The growth and pigments formation of the green microalga Chlorella zofingiensis cultivated at various glucose and nitrate concentrations in mixotrophic culture were investigated. High glucose and low nitrate concentrations favoured the production of astaxanthin in the algal culture. The highest yield of astaxanthin was 12.5 mg l −1, which was obtained in the medium containing 30 g l −1 glucose and 0.55 g l −1 nitrate. The results indicated that supplementation of glucose and nitrate in the culture medium could be effectively manipulated to enhance astaxanthin production by C. zofingiensis in mixotrophic culture.