【作者】 孙书勤；

【导师】 孙铭一； 王江涛；

【作者基本信息】 中国海洋大学 ， 应用化学， 2009， 硕士

【摘要】 本论文以室内培养的方式,研究指数生长前期与稳定期后期收获的两种海洋微藻中,中性脂与脂肪酸分子在天然海水中的降解行为与组成变化规律,以及稳定期后期收获的两种海洋微藻细胞膜与细胞内脂肪酸在有氧降解过程中组成与含量的变化。主要结果如下:1.不同生长期收获的两种海洋微藻脂类分子组成指数生长前期与稳定期后期收获的两种海洋微藻,在有氧微生物参与的条件下,中性脂部分都检出植醇（phytol）与4种脂肪醇（12-ROH、14-ROH、16-ROH、18-ROH）,但甾醇部分组成两种微藻稍有不同,亚心型扁藻检出11种甾醇,分别是27△⁵（27△⁵, Cholest-5-En-3β-ol）,27△⁰（27△⁰, 5α-cholest-22-en-3β-ol）,28△^5,22(28△^5,22, 24-methylcholest-5,22-Dien-3β-ol),28△²²(28△²², 24-methylcholest-22-en-3β-ol),28△⁵（28△⁵, 24-methylcholest-5-en-3β-ol）,28△⁰（28△⁰, 23, 24-dimethyl-5α-cholest-22-en-3β-ol）,29△^5,22(29△^5,22, 24-Ethylcholestt-5, 22-Dien-3β-ol),29△²²(29△²², 23, 24-dimethyl-5α-cholest-22-en-3β-ol),29△⁵（29△⁵, 24-Ethylcholestt-5-en-3β-ol）,29△⁰（29△⁰, 23, 24-dimethyl-5α-cholest-22-en-3β-ol）,30△²²（4α,23,24-trimethyl-5α-cholest-22-en-3β-ol）,而等鞭金藻3011中没有检出27△⁰,其余组分与亚心型扁藻检出的相同。但是脂肪酸组成因收获时期而异。指数生长前期收获的两种海洋微藻都检出12种脂肪酸,包括7种饱和脂肪酸12:0、14:0、15:0、16:0、17:0、18:0、20:0,3种单不饱和脂肪酸18:1（n-9）、18:1（n-7）、20:1,2种多不饱和脂肪酸18:2与20:5。而稳定期后期收获的两种微藻,除与指数生长前期相同的脂肪酸分子外,亚心型扁藻还检出了16:1（n-9）与22:6,而等鞭金藻3011包含16:1（n-9）、16:1（n-7）与22:6。2.指数生长前期收获的两种海洋微藻细胞中脂类物质在有氧条件下的变化规律在有氧微生物条件下,亚心型扁藻总脂肪酸前一个月内浓度变化不大,一个月后浓度明显升高。等鞭金藻总脂肪酸在前一周内发生明显降解,此后浓度略有升高。相对于脂肪酸,指数生长前期收获的两种海洋微藻中性脂含量较少,浓度变化趋势大体一致:在培养的前一个月中,发生明显降解,等鞭金藻3011的降解速率比亚心型扁藻的降解速率快;而一个月后中性脂的总量略有升高。两种微藻中,主要饱和脂肪酸组分都包含16:0（一般占总脂肪酸的50%）与18:0（一般占总脂肪酸的20%）,但变化情况随藻种而异。亚心型扁藻中,16:0在前一个月内发生降解浓度降低,一个月后浓度升高;18:0在整个培养期浓度持续升高,后半个月的增加速率较快。而等鞭金藻中,16:0与18:0都在培养的前半个月内发生降解,16:0降解速率比18:0略快,半个月后两种脂肪酸浓度都呈增加趋势。在亚心型扁藻体内主要不饱和脂肪酸组分为18:1（n-9）,随着时间的变化,浓度明显降低,培养结束时几乎完全降解。而等鞭金藻3011中18:1（n-9）与18:2浓度变化情况相对复杂。中性脂分子包括脂肪醇、植醇、甾醇,其中主要组分为植醇与甾醇。两种微藻的植醇降解模式一致,在培养期内均发生显著降解,培养结束时几乎完全降解。各微藻所含甾醇种类较多,变化情况复杂。3.稳定期后期收获的两种海洋微藻细胞中脂类物质在有氧条件下的变化规律与指数生长前期收获的微藻相比,稳定期后期收获的微藻总脂肪酸的初始浓度较高,且降解模式也不同。亚心型扁藻在前14内总脂肪酸浓度略有升高,但14天到21天之间浓度迅速下降,随后浓度稍有升高。等鞭金藻3011与亚心型扁藻的变化情况大不相同,在培养过程中,等鞭金藻3011的总脂肪酸发生明显降解,但在培养的最后1周内浓度略有升高。两种微藻的总中性脂含量都少于各自的总脂肪酸含量。亚心型扁藻总中性脂浓度在前3天内迅速下降,此后浓度变化不大。等鞭金藻3011总中性脂在培养的前一个月发生降解,一个月后浓度几乎不变。亚心型扁藻中,饱和脂肪酸16:0与18:0含量较大。在培养的前两周内16:0浓度变化不大而18:0浓度却迅速升高,半个月后二者都发生明显降解。等鞭金藻3011中,饱和脂肪酸以14:0,16:0,18:0为主,三种组分降解情况各异。14:0在培养的前一个月内迅速降解,一个月后浓度基本不变。16:0是在培养的前35天内发生降解,35天以后浓度略有波动。18:0变化较为复杂。亚心型扁藻中,18:1（n-9）为含量较高的不饱和脂肪酸,在培养的前三周内发生降解,21天后浓度迅速升高。等鞭金藻3011中,18:1（n-9）在培养的第一周内迅速降解,此后浓度变化不大。与指数生长前期收获的微藻相比,稳定期后期收获的两种微藻细胞中植醇初始浓度较高,且在培养期内均发生显著降解。亚心型扁藻中主要甾醇组分28△⁵在培养前2周内迅速降解,浓度几乎降到0,14天后浓度发生波动上升。等鞭金藻3011中29△^5,22、29△⁵、30△²²含量较多。29△^5,22与29△⁵在培养的前2周内浓度变化不大,2周后发生降解,但29△⁵降解程度较大。30△²²在前7天内浓度稍有增加,此后发生缓慢降解。4.稳定期后期收获的两种海洋微藻中细胞内与细胞膜脂肪酸组成与含量变化稳定期后期收获的亚心型扁藻,在有氧降解的不同时期细胞内和细胞膜脂肪酸含量及分子组成明显不同。在第0天,亚心型扁藻细胞膜与细胞内脂肪酸含量分别为14.03%,与85.97%,细胞内组分主要是16:0（39.08%）、18:1（n-9）（18.41%）、18:0（10.87%）。而在培养的第7天,组成发生一定变化,细胞膜脂肪酸含量极少（3.7%）,而细胞内16:0（49.46%）与18:0（28.32%）含量接近90%。稳定期后期收获的等鞭金藻3011,在第0天,细胞膜与细胞内脂肪酸含量分别为20.70%与79.30%,细胞膜中主要组分为18:1（n-7）（5.97%）、18:0（5.73%）,细胞内主要包括16:0（26.19%）、14:0（13.04%）、16:1（n-9）（11.09%）。而在培养的第42天,脂肪酸组成与第0天恰好相反,细胞膜脂肪酸含量多（75.61%）,而细胞内脂肪酸较少（24.39%）。细胞膜中以16:0（36.03%）、18:0（30.22%）为主,细胞内主要是16:0（8.77%）、14:0（4.16%）。更多还原

【Abstract】 Two marine algae were cultured and harvested at early exponential growth phase and late stationary phase. The compositions and concentrations of algal lipids（neutral lipids and fatty acids） during decomposition in naturl oxic seawater were examined. Also via this study, the distributions and contents of membrane and intracellular fatty acids of algae from late stationary stage were investigated. The main findings were as follows:1. The lipid compositions of microalgae at different growth stage during decomposition in naturl oxic seawaterNeutral lipids were composed of phytol, four kinds of alcohols （12-ROH , 14-ROH , 16-ROH, 18-ROH） and sterols of microalgae at early exponential growth phase and late stationary phase. But the sterols were different between two marine algae. The sterols of Platymonas subcordiforus contained 27△⁵ （27△⁵,Cholest-5-En-3β-ol）,27△⁰ （27△⁰,5α-cholest-22-en-3β-ol）,28△^5,22 (28△^5,22, 24-methylcholest-5, 22- Dien-3β-ol), 28△²² (28△²², 24-methylcholest-22- en-3β-ol), 28△⁵ （28△⁵, 24-methylcholest-5- en-3β-ol）, 28△⁰ （28△⁰, 23, 24-dimethyl-5α-cholest-22-en-3β-ol）, 29△^5,22 (29△^5,22, 24-Ethylcholestt-5, 22-Dien-3β-ol),29△²² (29△²², 23, 24-dimethyl-5α-cholest-22-en-3β-ol),29△⁵ （29△⁵, 24-Ethylcholestt-5-en-3β-ol）, 29△⁰ （29△⁰, 23, 24-dimethyl-5α-cholest-22-en-3β-ol）, 30△²² （4α, 23, 24-trimethyl-5α-cholest-22-en-3β-ol）. Isochrysis galbana included ten kinds of sterols except 27△⁰ . However, fatty acid composition varied due to harvest time. The algae at exponential stage included seven saturated （12:0, 14:0, 15:0, 16:0, 17:0, 18:0, 20:0）, three monounsaturated （18:1（n-9）, 18:1（n-7）, 20:1）, and two polyunsaturated （18:2, 20:5） fatty acids. In addition to the same fatty acids as before, 16:1（n-9） and 22:6 were included in Platymonas subcordiforus harvested at late stationary stage, and Isochrysis galbana 3011 also contained 16:1（n-9）, 16:1（n-7） and 22:6. 2. The lipid compositions and concentrations of two marine algae harvested at early exponential growth phase during decomposition in naturl oxic seawaterTotal fatty acids concentration of Platymonas subcordiforus changed little during aerobic incubation in the first month , one month after the concentration increased apparently. After significant decrease in the first week, total fatty acids concentration of Isochrysis galbana 3011 enhanced. Compwered with the fatty acids, the contents of algal neutral lipids were less. Total concentration of algal neutral lipids reduced obviously in the first month during the incubation, and one month after the concentration increased slightly.16:0 （ 50% of total fatty acids） and 18:0 （²0% of total fatty acids） were the dominating saturated fatty acids of two microalgae, but the components varied with algal species. For Platymonas subcordiforus, the concentration of the 16:0 fatty acid increased rapidly from the thirtieth day although the concentration decresed slightly in the first month. The 18:0 fatty acid have exhibited an increasing trend during the incubation with a faster rate in the second half month. The concentration of 16:0 and 18:0 fatty acids reduced in the first half month for Isochrysis galbana 3011, and 16:0 decreased more rapidly. After decreasing the concentrations of two compounds showed a accumulation. The major unsaturated fatty acid 18:1（n-9） in Platymonas subcordiforus degraded with time ,and the concentration dropped to zero at the end of the incubation. But 18:1（n-9） and 18:2 in Isochrysis galbana 3011 changed with complicated patterns.Neutral lipids included alcohols, phytol and sterols. Among them phytol and sterols were major components. Phytol of two microalgae showed a dramatic decomposition degradation and were completely degraded after fifty days. Several sterols were found in two microalgae and they changed with diversities of patterns.3. The lipid compositions and concentrations of two marine algae harvested at late stationary phase during decomposition in naturl oxic seawaterThe initial concentrations of total fatty acids in microalgae harvested at late stationary phase were higher than that of early exponential growth phase and the degradation patterns were different. The concentration of total fatty acids of Platymonas subcordiforus increased slightly within the former 14 days, but during the 14th day to 21th day the concentration decreased rapidly, then followed by a slight increase in concentration. Apparently distinct with Platymonas subcordiforus, total fatty acids of Isochrysis galbana 3011 degraded remarkably but in the last week of incubation the concentration raised gently. The total neutral lipids contents of two algae were less than their total fatty acids contents of each. For Platymonas subcordiforus, total neutral lipids concentration decreased rapidly within three days, after which little change in concentration. The total neutral lipids of Isochrysis galbana 3011 degraded in the first month, one month after the concentration was almost unchanged.The concentrations of 16:0 and 18:0 were higher than the other fatty acids in Platymonas subcordiforus. In the first two weeks of incubation 16:0 changed little but 18:0 increased rapidly in concentration, two weeks after both of them degraded clearly. 14:0, 16:0 and 18:0 were the dominating saturated fatty acids of Isochrysis galbana 3011, but they showed different degradation patterns. In the first month of incubation the concentration of 14:0 fatty acid reduced rapidly, after one month the concentration remained constant. 16:0 fatty acid degraded witnin 35 days followed by a little fluctuation in concentration. 18:0 fatty acid has exhibited a complex mode. 18:1（n-9） is the most abundant unsaturated fatty acid of Platymonas subcordiforus and it degraded in the first three weeks then followed by a significant enhancement in concentration. For Isochrysis galbana 3011, 18:1（n-9） fatty acid showed no obvious change after the first week although it degraded rapidly at the beginning of the incubation.The initial concentrations of phytol in microalgae harvested at late stationary phase were higher than that of early exponential growth phase, and they degraded significantly with time, respectively. 28△⁵, the major sterol of Platymonas subcordiforus presented rapid degradation and its concentration almost dropped to zero until the 14th day. But after the first two weeks 28△⁵ exhibited an obvious increasing. Isochrysis galbana 3011 was abuntant in 29△^5,22、29△⁵、30△²². The concentrations of 29△^5,22 and 29△⁵ remained almost constant in the first two weeks, while both compounds degraded after two weeks with 29△⁵ decreasing faster. 30△²² showed an increasing in concentration in the first week , then followed by slow degradation.4. The distributions and contents of membrane and intracellular fatty acids of two algae harvested at late stationary stageFor Platymonas subcordiforus, the components and contents of membrane and intracellular fatty acids were apparently distinct in the aerobic degradation of the different periods of incubation. At the beginning, the contents of membrane and intracellular fatty acids were 14.03% and 85.97%, respectively. The main intracellular fatty acids were 16:0 （39.08%）, 18:1（n-9） （18.41%） and 18:0 （10.87%）. In the 7th day, fatty acids in membrane and intracellular components showed different distributions. The membrane fatty acid content is extremely low （3.7%）, while the contents of intracellular 16:0 （49.46%） and 18:0 （28.32%） were close to 90%.In the first, the contents of membrane and intracellular fatty acids in Isochrysis galbana 3011 were 79.30% and 20.70% respectively. The major fatty acids in membrane were 18:1 （n-7） （5.97%） and 18 : 0 （5.73%）, while 16:0 （26.19%）, 14:0 （13.04%） and 16:1（n-9） （11.09%） were the dominating intracellular components. The fatty acid composition was contrast to initial distribution in the 42th day with more membrane content （75.61%） and less intracellular fatty acids （24.39%）. The main membrane fatty acids were 16:0 （36.03%） and 18:0 （30.22%）, but 16:0 （8.77%） and 14:0 （4.16%） were the major intracellular components.更多还原