蝦紅素為一種橘紅色的類胡蘿蔔素，被廣泛應用在飼料添加、著色劑、保健食品、化妝品產業中。其結構能提供電子給自由基或是吸引未配對的電子來達到去除自由基的作用，進而減緩因自由基產生所引起的氧化反應或是各種相關疾病。
目前蝦紅素的主要來源為：化學合成、酵母菌（Phaffia rhodozyma)，以及雨生紅球藻，但相對前兩者，微藻所生產的天然蝦紅素只佔了一小部分。化學合成及酵母菌發酵的蝦紅素大部分用於飼料添加以及著色劑，而隨著攝取天然食品的需求，以及為避免化學合成類胡蘿蔔素的高成本，微藻所生產的天然蝦紅素的需求日益漸高，再加上，雨生紅球藻被視為目前能累積最多蝦紅素的微藻類，故其已廣泛被研究，以用來作為天然蝦紅素的生產來源。然而，有別於酵母菌發酵，微藻養殖與色素萃取耗費相對較高的成本。如何降低養殖成本以及萃取成本成為微藻產業的主要課題。本篇研究目的主要在於篩選適合應於工業化量產的藻種，高蝦紅素含量、薄細胞壁的藻種，皆為此篇研究所關注的目標。
此篇研究蒐集二十一株來自不同國家藻種中心的雨生紅球藻，進行第一次液體培養的篩選，再利用甲基磺酸乙酯作為致變劑，使篩選出的雨生紅球藻進行隨機突變，並利用除草劑或蝦紅素合成的抑制劑篩選出十五株突變株，進行第二次液體培養的篩選。另外，利用calcofluor white staining細胞壁染色的方式，間接判別細胞壁的厚度差異。以等量細胞進行染色之後利用Victor 2偵測螢光強度，再依細胞大小計算出細胞壁單位面積所發出的螢光強度，作為厚度的參考依據。
經第一階段篩選後，我們選擇4株野生株進行突變，並篩選出11個突變株進行培養。實驗結果發現，雨生紅球藻細胞大小與蝦紅素含量以及蝦紅素濃度呈正相關。經14天培養後，我們得到No.13 mutant#18的蝦紅素含量相較野生株成長了25%。經28天培養後，No.19 mutant#2和mutant#3的蝦紅素含量分別為2.98%和2.53%，約兩倍於No.19野生株 (1.71%)。另外，No.10 mutamt#2和mutant#3經35天培養後，蝦紅素含量分別為4.05%和3.81%，與其他突變珠與野生株相比，含有相對較高的含量。然而，相較於第一階段篩選，No.18進行第二階段篩選時，獲得較少細胞濃度以及生物量，造成其蝦紅素含量遠低於其他藻種。
在螢光染色方面，細胞大小與單位面積細胞壁所發出的螢光強度呈負相關。No.10 mutamt#2、mutant#3、No.19 mutant#2、mutant#3此四株突變株的細胞壁單位面積所含的螢光強度皆低於野生株，推測其細胞壁厚度較薄。利用顯微鏡觀察No.10 mutant#2與mutant#3時，也出現許多細胞破掉並釋出色素的現象。此四株突變株的蝦紅素累積含量或乾重比隨培養時間延長而增加，相對於野生株及已發表的藻種在第21天已趨於飽和。若以發表在文獻 (Jian Li, et al 2011) 相同的理想培養系統及條件，No.10 mutant#2之蝦紅素乾重比分別為2.93% (21天)、3.51% (28天)、4.05% (35天)，依不同長度時間的培養，其乾重能降低約17%~62%的生產成本。

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.

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