Carotenoid production by phaffia rhodozyma with industrial waste as substrate

Abstract

Carotenoids are yellow, orange and red pigments that are widely distributed in nature. Astaxanthin is an important carotenoid, which is a red pigment of great commercial value. It is the principal carotenoid pigment responsible for the characteristic pink to red colour of flesh of many marine animals such as salmon, shrimps and trout. In nature, aquatic animals obtain their astaxanthin via their diets by feeding on the marine bacteria and microalgae that synthesize astaxanthin. Nowadays, since many of these aquatic animals are cultured by man and fed on commercial feed rather than natural diet in the sea, these animals cannot obtain enough astaxanthin for their pigmentation. However, the pigmentation of their flesh is the major factor that determining their prices. As a result, aquaculture started to add astaxanthin into the diet of these cultured aquatic animals in order to yield animals with pigmentation that is the same as the natural ones. Besides usage in aquaculture, astaxanthin is also useful to act as a health supplement for humans. Astaxanthin has shown to be a potent lipid-soluble antioxidant in vitro that may delay aging and degenerative diseases in humans and animals. It has been proposed to prevent some cancers and to stimulate the immune system. It was shown to be the most effective in stimulating immune defences when different carotenoids were compared. The yeast Phaffia rhodozyma is a possible candidate for commercial production because of its high astaxanthin content. The objectives of this research project are first to develop processes in which Phaffia rhodozyma can produce large amounts of astaxanthin with industrial wastes, like malt waste, containing cellulose, starch or lactose as carbon source, which is an attractive and feasible approach to meet the market needs of astaxanthin. Since shake flask and fermenter cultures of Phaffia rhodozyma will be studied, astaxanthin is extracted from samples of those cultures. In addition, since astaxanthin may exert anti-tumor activity through the enhancement of immune response, the second objective consists of determining the effects of dietary astaxanthin on tumor growth and tumor immunity against tumor cells. In order to fulfil the objectives, the hydrolysis conditions for industrial waste (malt waste) were optimised first, then the fermentation conditions for the growth of Phaffia rhodozyma were optimised. Initially, shake flask cultures were studied. Results showed that 40%-70% hydrolysates, especially 50%, seem to be more supportive for both biomass and carotenoid production than other concentrations. Thus, they were considered to be the optimum concentration range of hydrolysates for growth and carotenoid production of Phaffia rhodozyma and were used in fermenter for scale up in the later experiments. Based on the results from shake flask experiments, growth of Phaffia rhodozyma in a fermenter was studied. Both batch and fed-batch fermentations with either glucose or malt waste as carbon source were carried out in a Bioengineering (3.7L) fermenter to characterize biomass yield and carotenoid yield of Phaffia rhodozyma under different modes of fermentation. Batch fermentation in the fermenter was first studied. Then, fed-batch fermentation, such as exponential and continuous feeding that use pre-determined feeding profiles and DO-stat fermentation that is controlled by an online feedback loop, were also studied. Fed-batch fermentation is normally used instead of batch fermentation because Phaffia rhodozyma is a Crabtree positive yeast, which means their growth is affected by sugar concentration. If the sugar content in the medium is too high, they will change their growth from aerobic to anaerobic. Therefore, fed-batch fermentation was used because it can fulfil an important criterion to maintain the limiting substrate at a very low concentration such that repressive effect of high substrate concentration can be avoided. With glucose as the carbon source, results show that DO-stat cultures attained the highest biomass and carotenoids yield among the different modes of fermentations studied, the best run attained 23.2 g/L biomass yield and 4202 ug/L carotenoid yield. This is because DO-stat feeds glucose to the medium only when cells have consumed all the glucose that was added previously. Thus, glucose accumulation in the medium is avoided and glucose can be used more efficiently to produce biomass and carotenoid. DO-stat cultures of Phaffia rhodozyma using malt waste as carbon source produced 16.2 g/L biomass and 3589 ug/L carotenoids, this was lower than that attained in DO-stat cultures using glucose as carbon source. The second part of my research is to determine the anti-tumor activities of astaxanthin. Rat-models have shown that astaxanthin has suppressive effects on several types of cancer models. In this study the effect of dietary astaxanthin on tumor growth and tumor immunity was determined on 8-10 weeks old C57 BL/6 male mice using B16/neu syngeniec mouse melanoma cells. These cells were transfected with the human HER-2 gene expressing the p185 human breast cancer antigen. The tumor cells were injected subcutaneously into the left footpad of all mice. Astaxanthin extracts were administered daily as long as the treatment lasted. All animals were monitored for survival as well as tumor growth. Tumor size was expressed in terms of swelling in the footpads. Two kinds of treatments were studied. In the first kind of treatment, tumor cells were injected at day 0. The treatment started at day 0 and lasted till the end of the experiment. In the second kind of treatment, the tumor cells were injected on day 22 after 3 weeks pre-treatment. Once tumor cells were inoculated, treatments were stopped. The first type of treatment showed that astaxanthin was effective in delaying tumor growth compared to an untreated group. The effectiveness of astaxanthin in the second type of treatment was less then in the first type, the tumor size was bigger. In both types of treatment, astaxanthin showed a significant effect in lowering tumor size and weight than the untreated group. Thus, tumor-therapeutic properties here looked promising. So it can be concluded that astaxanthin has a potential to be employed as a therapeutic supplement for the treatment of melanomas and breast cancer.