In situ carbon dioxide fixation in the process of natural astaxanthin production by a mixed culture of Haematococcus pluvialis and Phaffia rhodozyma
Introduction
Carbon dioxide is considered as a major greenhouse gas causing the global warming problem. Mankind has now recognized both the necessity and increasing urgency to reduce the CO2 content in the atmosphere. To find a feasible solution, many attempts have been made through out the world, among which, biological CO2 fixation (BCF) is an environment friendly way to remove CO2 using micro-algal photosynthesis.
Micro-algae are unique and valuable microscopic algae containing chlorophyll and other photosynthesis-related antenna pigments such as carotenoids, which enable them to absorb and utilize CO2 as the principal carbon source in the growth process. These microscopic algae usually are unicellular and are found growing nearly every biotope due to their ecological diversity and physiological adaptability, especially in water system. The major reason for the utilization of micro-algae in CO2 fixation is that they can tolerate up to 12% CO2 at a temperature of 35 °C, while most plants can only live with up to 0.1% CO2. Unlike higher plants, micro-algae cannot capture CO2 directly from the air. Therefore conventional technique for micro-algae cultivation needs to transfer CO2 and/or air into the culture broth, resulting in lower CO2 conversion rate [1], but higher concentration of O2, which inhibits the growth of algal cells [2].
Up to now, micro-algae biotechnologies have been successfully practiced to convert CO2 emitted from power plant [3] and lime production plant [4] into algal biomass. In the yeast fermentation, nearly 50% of the input carbon substrate, typically glucose is used for cell growth and maintenance, releasing high level of CO2 into the environment [5]. Nevertheless, no information is available regarding conversion of CO2 derived from microbial fermentation.
Furthermore, it is also economical to combine CO2 fixation with useful metabolite production. There are over ten thousands species of micro-algae that have been recognized, however, up to date, only a few are commercially cultivated: including Spirulina, a filamentous blue green alga, Chlorella, Dunaliella and Haematococcus, the last three are unicellular green algae. The first two species are used for food supplements, while the last two for their pigment content, especially beta-carotene and astaxanthin [6].
Astaxanthin is a high value carotenoid (US$ 2500 kg) and strong biological antioxidant, which has widespread applications in nutraceutical, cosmetic, food and feed industries. Haematococcus pluvialis and Phaffia rhodozyma are the two main microorganisms used in the natural astaxanthin production and have attracted attention through out the world. Of the two strains, H. pluvialis is a ubiquitous unicellular green alga that utilizes CO2 and produce O2 in photosynthesis, as the algal cells synthesize and accumulate astaxanthin in response to environmental stress such as high irradiance, high temperature [7], deficiency of nitrogen or phosphate [8], [9], [10]. While, P. rhodozyma is a sort of red yeast that can use a vast variety of organic materials as fermenting substrates, in particular carbohydrates, generating CO2 and organic acids [11], [12], which restrain both cell growth and astaxanthin formation of the red yeast. On the contrary, O2 is required both for the primary metabolic activity and astaxanthin synthesis in P. rhodozyma [13]. Thus, H. pluvialis and P. rhodozyma are good candidates for symbiosis. Nevertheless, at present, both of the two species are always cultivated purely in separated cultures in academic and commercial astaxanthin production [14], [15], [16], [17], and under the traditional separate culture strategy, astaxanthin production declines due to the changes of pH, CO2 and O2 concentrations in the broth throughout the culture process.
Mixed cultures of microorganisms are common in natural ecology system. In recent years, exploration of mixed cultures became critical to many key biochemical processes [18]. Mixed cultures have been used in various aspects, such as complementary biotransformations, multistep biotransformations, in situ enzyme regeneration, in situ oxygen generation, waste degradation and remediation. In the present study, in order to combine CO2 fixation with astaxanthin production, we have, for the first time, mix-cultivated two different astaxanthin-producing strains, H. pluvialis and P. rhodozyma, on a simple well-defined medium to make good use of the complementary effects between the two independent microbial metabolic activities.
Section snippets
Microorganisms
H. pluvialis and P. rhodozyma (AS2-1557) were obtained from Institute of Hydrobiology and Institute of Microbiology of Chinese Academy of Science respectively. H. pluvialis was maintained at 4 °C in a liquid BBM medium (Bold's Basal Medium) [19]; P. rhodozyma was maintained at 4 °C on a slant of yeast malt agar (YM agar, Difco) containing (per liter) 10 g of glucose, 5.0 g of Bacto peptone, 3.0 g of malt extract, 3.0 g of yeast extract, and 20 g of agar.
Culture media and conditions
The seed culture of H. pluvialis was prepared by
Results and discussion
In order to evaluate the effect of mixed culture on CO2 fixation and astaxanthin formation, mixed cultures were compared with pure cultures of H. pluvialis and P. rhodozyma on five aspects: biomass concentration, cell yield, glucose conversion rate, nitrogen conversion rate as well as astaxanthin production.
Conclusions
Mixed culture of H. pluvialis and P. rhodozyma presents a new strategy for combining CO2 fixation with high value co-product (astaxanthin) formation, promoting glucose and nitrogen conversion rate, keeping pH constant as well as increasing cell yield and astaxanthin production. This culture scheme lays a ground for developing a new technology using carbon dioxide fixation.
Acknowledgements
Supports from the National Natural Science Foundation of China (Grant No. 20036010) and National Project of Key Fundamental Research (Grant No. 2003cb716003) are very appreciated.
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