油脂微生物突變篩選及以本土Staphylococcus sp. TSP01生產生質柴油料源之研究

Study of lipid production from selected oleaginous microorganism applied as biodiesel feedstock

王懷俐(Hwai-Li Wang)

指導教授 ： 藍祺偉

元智大學/工程學院/化學工程與材料科學學系/碩士(2009年)

https://doi.org/10.6838/YZU.2009.00323

摘要

本研究目的在找出可以利用甘油以產生油脂（neutral lipids）之菌株。首先利用尼羅紅（Nile red）螢光染色法快速篩選出油脂微生物Rhodococcus opacus 16327之突變菌株Rhodococcus opacus M15009，以及辨識本土菌株Staphylococcus saprophyticu TSP01油脂累積之潛力。而後限制氮源，採用兩階段方式培養菌株以提升其油脂累積含量。在不同碳源為基質之測試下，Rhodococcus opacus M15009以葡萄糖酸鈉培養後，其脂肪酸產量可達61.62%(w/w)；而以甘油培養，其脂肪酸含量僅3.74%。另外，同以甘油為碳源培養Staphylococcus saprophyticu TSP01，經尼羅紅（Nile red）染劑辨識後有顯著之螢光反應，因此進行兩階段之最適化策略探討，首先以高細胞密度培養為目的，改變葡萄糖濃度及添加甘油以討論基質濃度對細胞生長之影響，環境條件上分別探討溫度、pH值、溶氧量，找出高細胞密度培養之最適化條件：T=30C、pH=8.3、stirring speed=200 rpm且TSB培養液中不添加額外碳源，其細胞乾重達3.43 g L-1。第二階段以第一階段找出之最佳化條件為前培養，限制氮源以利油脂累積，以MSM為培養液，相繼測試不同基質、基質濃度、溶氧量、pH及溫度。最終以甘油為碳源，在pH5操作條件之下，Staphylococcus sp. TSP01之油脂累積含量可達48.3%。

關鍵字

高細胞密度 ； 油脂微生物 ； 尼羅紅 ； 腐生葡萄球菌 ； 混濁紅球菌 ； 脂肪酸甲酯

並列摘要

The Nile-red staining method was applied to screen potentially strains of high lipid content. A mutant Rhodococcus opacus M15009 derived from random mutagenesis of wild-type strain was found to achieve 62% w/w of lipid accumulation through two-stage cultivation. However, this mutant was indicated to accumulate low concentration of lipid with glycerol supplement. A local oleaginous microorganism was discovered capably to yield high lipid content. It was identified using 16s rRNA and 97% alliance to Staphylococcus sp. The optimization study of high cell density and lipid accumulation of Staphylococcus sp. TSP01 was carried out in respect of temperature, pH, carbon source and dissolved oxygen. A 3.43 g L-1 of biomass can be reached in the first-stage of cultivation conditions (i.e. temperature at 30℃, pH value of 8.3 and 200 rpm of stirring speed). Using glycerol as carbon source was found to yield 48.3% w/w of lipid in Staphylococcus sp. TSP01 at pH value of 5. Both of Rhodococcus opacus M15009 and Staphylococcus sp. TSP01 are concluded in characteristics as high lipid accumulation bacteria and could be applied for production of lipid feedstock in biofuel industry.

並列關鍵字

High cell density ； oleaginous Rhodococcus ； Staphylococcus sp. ； lipid production ； fatty acid methyl esters ； Nile red

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