Abstract
Astaxanthin, 3, 3’-dihydroxy-b, b-carotene-4, 4’-dione is a red-orange carotenoid pigment which is widely-applied to aquaculture, poultry feeds, nutraceutical market and cosmetics market. Astaxanthin consists of conjugated double bonds to provide its red color and strong antioxidative ability by donating the electrons to scavenge free radicals. Recently, Haematococcus pluvialis, Phaffia yeast, and chemical synthesis are major sources of astaxanthin in commercial applications. However, natural astaxanthin from H. pluvialis only occupies a small part of total sources. The needs for natural feeds and high cost of synthetic astaxanthin have raised the demand of natural astaxanthin from microalgae. Also, Haematococcus pluvialis is a well-known microalga accumulating a richer amount of astaxanthin than the other microalgae. In commercial production of microalgae, how to reduce the cost of cultivation and product extraction are two well-concerned problems. This study was aimed at screening the most suitable strains applying to commercial production, such as high astaxanthin content, high astaxanthin productivity or weak cell wall of cysts which is easily extracted at harvest. In this study, we used ethyl methanesulfonate (EMS) to carry out mutagenesis. Twenty-one wild-type strains were collected from many collection centers located in many countries. After random mutation, we screened the potential mutants by using five selection compounds which are herbicides or inhibitors in astaxanthin biosynthesis pathway. These potential mutants were cultivated with Rudic’s medium under continuous blue LED light (55 μEm-2s-1) at 20~25°C. We monitored the microalgae growth by measuring absorption wavelength of 674 nm which is usually estimating the amounts of microalgae. After harvest, we extracted the pigments from H. pluvialis and analyzed astaxanthin content to screen high productivity mutants. Moreover, we used calcofluor white to stain the cell wall of H. pluvialis. We assumed that thinner cell wall would present low fluorescent intensity than thicker one after calcoflour white staining with the same cell density. After random mutation, we got 11 mutants to cultivate and compare with each other in liquid culture. According to statistics, the positive correlation between cell size and astaxanthin content/ concentration was presented. In comparison of mutants, the astaxanthin content of No.13 mutant#18 was 25% higher than wild-type after 14-days cultivation. Also, the astaxanthin contents of No.19 mutant#2 and mutant#3 were 2.98% and 2.53% respectively. They were higher than the astaxanthin content of No.19 wild-type (1.71%) after 28-days cultivation. Moreover, the astaxanthin contents of No.10 mutant#2 and mutant#3 were 4.05% and 3.81% respectively. They were higher than the astaxanthin content of No.10 wild-type (2.11%) after 35-days cultivation. However, No.18 strains got lower cell density and biomass compared with No.18 wild-type in first screening and resulted in lower astaxanthin content and concentration. In calcofluor white staining assay, No.19 mutant#2, mutant#3, No.10 mutant#2 and mutant#3 got lower fluorescent intensity on unit sphere surface area of cell wall. It indicated that the cell wall of these strains were weaker than others. But No.19 mutant#2 had high viscosity and easily attached to the surface of flask. It might cause some problems on harvest and extraction. We assume that the mutant strain No.10 mutant#2 was cultivated with the ideal culture systems and conditions published in review paper in 2011 by Jian Li. The astaxanthin content of No.10 mutant#2 increased as time went by while the strain published only got 2.5% astaxanthin content after 14-days cultivation. If we extend the cultivation duration, No.10 muntant#2 was a more suitable strain to apply to commercial production. It can reduce about 17% to 62% cost of production depending on different cultivation periods.