胁迫因子对杜氏藻（Dunaliella）生长及其β-胡萝卜素积累的影响研究

【作者】 王培磊；

【导师】 张学成；

【作者基本信息】 中国海洋大学 ， 海洋生物学， 2005， 博士

【摘要】 盐生杜氏藻（Dunaliella salina）在人工控制和胁迫条件下,能大量积累β-胡萝卜素（占干重的10～14%）,被公认为是生产β-胡萝卜素的理想天然资源。早在1966年,Massyuk就提出杜氏藻可作为产品化的β-胡萝卜素的来源。杜氏藻还含有约40%的蛋白质,其氨基酸组成与植物蛋白相似,可用作动物饲料蛋白源。杜氏藻在适宜的条件下具有很高的生物量产量,最高可达15-20g/（m²·d）,大规模生产的平均水平亦可达到10-15g/（m²·d）,高于大多数其它藻类的产量,因此,杜氏藻是一种很好的单细胞蛋白源。甘油是重要的有机化工原料,目前主要从石油中提取,其售价在3～5美元/Kg,但随石油资源的耗尽,其价格不断上涨,利用杜氏藻生产甘油具有潜在的发展前景。大量积累甘油的杜氏藻种类,在适当的条件下,其甘油含量可达干重的50%。综合提取胡萝卜素和甘油,可使甘油生产成本降至0.752美元/Kg,低于石油生产甘油的成本（0.88美元/Kg,1980年价格）。另外,杜氏藻有很宽的盐度适应范围,D.salina可在1%～35%的盐度范围内生长,是自然界最耐盐的真核生物之一,它是生物学工作者研究生物机体对高渗环境适应机制的模式生物。有鉴于此,美国、澳大利亚、以色列、日本、西班牙、加拿大等国较早就开展了杜氏藻的形态、生理、生态和医学研究并投入生产和工业化养殖。我国具有漫长的海岸线（18000Km）和星罗棋布的内陆盐湖（仅1Km²以上的就有500多个）,具有培养杜氏藻生产β-胡萝卜素得天独厚的自然条件。 C、N、P、Fe源及pH值是影响杜氏藻生长的重要环境因子之一,明确杜氏藻对不同营养盐的吸收规律和杜氏藻生长的二阶段对营养盐种类和浓度的不同要求对优化杜氏藻培养基,提高生长速度,促进杜氏藻和β-胡萝卜素产业的发展有重要意义。本文研究了乙酸、C、N、P比例、氮盐、铁盐、磷盐、盐度、碳盐对杜氏藻（Dunaliella）生长、色素积累和生化组成的影响,建立了杜氏藻对主要营养盐吸收利用的动力学方程,并研究了杜氏藻对氨（（NH₄）₂CO₃）毒性的耐受性。 主要结果是: ●对D.salina,乙酸调节pH值可明显促进生长。细胞密度最大值120×10⁴cell/mL（pH≤8.5）,β-胡萝卜素最大值102mg/g（pH≤8.0）,叶绿素a含量达到104mg/g（pH≤8.5）,三者与对照组相比均有显著差异。乙酸调节pH值还可以提高单不饱和脂肪酸18:1和多不饱和脂肪酸18:2ω6的含量。蛋白含量随pH升高而提高;对D.parva,乙酸调节pH值对生长无明显促进作用,也不能提高β-胡萝卜素含量,但明显提高叶绿素a含量,最大值达144mg/g（pH≤9.0）,还可提高蛋白含量,达到33.5%（pH≤9.0）。 ●D.salina OUN04最大细胞密度114.2×10⁴cell/mL、最大β-胡萝卜素含量100.4mg/g、最大叶绿素a含量144.6mg/g分别出现在C、N、P比例为12/1/0.05、12/2/0.1、12/1/0.05的处理组中,且均与对照组差异显著。不同C、N、P比例对D.salina OUN04主要脂肪酸（16:0、18:1、18:2ω6）组成有显著影响;对D.salina OUN04蛋白含量也有显著影响,最大蛋白含量32.44%出现在6/1/0.05的处理组中,但与对照组（31.46%）差异不显著;D.salina OUN05最高细胞密度101.2×10¹ cell/mL、最高β-胡萝卜素含量更多还原

【Abstract】 The marine microalga Dunaliella salina is under commercial exploitation because of its high content of nature β -carotene (up to 10-14% of dry weight ). At 1966, Massyuk put forword the idea that D.salina should be cultured in order to obtain 3 -carotene. D.salina also can be used as food of animals because of its high content of protein (up to 40%). D.salina had higher yield than that of other microalgae, reaching 10-15 even 15-20 g/m~2·d. It is also a potential source of glycerol, although the cost of producing β -carotene by D.salina is higher than that from soil. Since it had the highest ability of anti-salinity in all life, D.salina is also used as model life by biologist. So, America, Australia, Israel, Japan, Spain and Canada had already studied D.salina and massive cultured it. Owing long coastline and many inland salinity lakes, our country had excellent conditions to culture D.salina.C, N, P, Fe, pH, acetic acid and salinity are important factors influencing the growth of D. salina and making clear the effects of these factors on the growth and 3 -carotene of D.salina is helpful to accelerate the growth rate of D.salina and optimize the culture media prescriptions. In this paper, The effects of C, N, P, Fe, pH, acetic acid and salinity of media on growth, β -carotene accumulation and cell biochemical composition of cells have been investigated and the results indicated that acetic acid can significantly enhance the growth rate of D. salina. The maximum cell density of 120 × 10~4cell/mL, β -carotene content of 102 mg/g and Chl a content of 104 mg/g were observed in treated groups of pH≤8.5, pH≤8.0 and pH≤8.5, respectively. Three of them significantly differed from those of the control group. Acetic acid also enhanced both the contents of monounsaturated fatty acid of 18:1 and the content of polyunsaturated fatty acid of 18:2ω6. The higher the pH values, the higher the contents of protein. To D. parva, acetic acid was found neither to enhance the growth rates, nor to enhance the contents of 3 -carotene of the culture media, though it was found to enhance both the content of Chl a, reaching 144 mg/g ( pH≤9.0), and the content of protein, reaching 335 mg/g ( pH≤9.0).The right ratio among C, N and P can promote the growth of D. salina OUN04, accumulation of β -carotene and biosynthesis of chl a. To D. salina OUN04, the maximum cell density of 114.2 × 10~4 cell/mL, the maximum β -carotene content of 100.4 mg/g and the maximum Chl a content of 144.6 mg/g were detected in the treated group of 12/1/0.05, 12/2/0.1 and 12/1/0.05, respectively, and all of them were significantly different from those of the control group. Right ratios among C, N and P can promote the contents of total fatty acids. The maximum content of total fatty acids was recorded to be 86.20% and it was significantly different from that of the control group of 81.02%. Ratios among C, N and P had significantly effect on the contents of protein in D. salina OUN04. The maximum protein content of 32.44% was detected in 6/1/0.05 group, but it was insignificantly different from that of the control group. To D. salina OUN05, the maximum cell density of 101.2×10~4 cell/mL, the maximum 3-carotene content of 109.2 mg/g and the maximum Chi a content of 108.7 mg/g were detected in treated groups of 6/2/0.05, 6/0.5/0.1 and 12/2/0.1, respectively, and all of them were significantly different from those of the control group. The maximum protein content of 34.41% was observed in the group of 6/1/0.05, but it was insignificantly different from that of the control group (33.17%).0.75mmol/L urea was the optimum nitrogen source to growth of D.salina OUN07 and the maximum cell density was 105 X 104cell/mL, whereas the control group only had the cell density of 59 X 104cell/mL. The maximum P-carotene content of 110.6 mg/g in D.salinaOUN07 was detected in the medium with 0.125/0.125mmo] NH4NO3/ urea as nitrogen source, but it was undifferentiated compared with that of the control group and the low nitrogen was found to be benefit to .8 -carotene accumulation. The maximum Chi a of 48.5 mg/g was found in the medium with 1.0 mmol/L urea, but it was undifferentiated compared with that of the control group and high nitrogen was found to be benefit to biosynthesis of Chi a. The main fatty acids of D.salina OUN07 were composed of 16^ 18:1 and 18:2"6 and the highest fatty acid content was detected in the medium with urea of 0.75mmol/L as nitrogen source. The marked effect of different nitrogen sources on the protein contents was also detected and the highest protein content of 33.61% was recorded in the medium with NUjNO.Vurea as nitrogen sources.For D.salina OUN04, the maximum cell density of 111.5 X 104 cell/mL was detected in 0.05mmol/L iron group, followed by 0.01 mmol/L iron group. The control group had cell density of 82.9 X 104 cell/mL. The 0.25mmol/L iron group had the minimum cell density of 70 X 104 cell/mL in all test group, suggesting that the growth of D.salina was inhibited by high iron content. The highest P -carotene content of 83.2 mg/g was observed in 0.25mmoi/L iron group, followed by 75.8 mg/L of 0.05mmol/L iron group and the control group had the minimum of 63.4 mg/L. In the first 1-3 days, the absorption rate of iron was slow, fast in 4-7 days, then declined in the last 3-4 days. For D.salina OUN09, the maximum cell density of 131X10 cell/mL was observed in 0.05mmol/L iron group. The control group had the cell density of 118.5 X 104 cell/mL and the 0.25 mmol/L iron group had the minimum cell density of 102.3 X 104 cell/mL. The maximum 3 -carotene content of 130.2 mg/g was found in 0.05mmol/L iron group and that of 70.4 mg/g in control group. The maximum Chi a content of 64.2 mg/g was observed in 0.05 mmol/L iron group and that of 37.4 mg/g in control group. The variety pattern of pH and the absorbted pattern of iron were similar to that of D.salina OUN04, respectively.The maximum cell density of 118 X 104 cell/mL was found in the medium with 0.1 mmol/L KH2PO4 and the minimum in the medium with no KH2PO4; The maximum B -carotene was detected in the medium with 0.1 mmol/L KH2PO4, followed by 0.05 mmol/L; The KH2PO4 of 0.1 mmol/L was assimilated completely within 7 days and the higher the KH2PO4 levels, the longer the lasting time of KH2PO4; Too high KH2PO4 was found to inhibit the growth of D.salina and the biosynthesis of P -carotene in D.salina and the function between the assimilated amount of KH7PO4 by D.salina and the culture time was set up. In the scope of test, low salinity was found to be benefit either to the cell growth, P -carotene accumulation and chl a biosynthesisor to raising contents of protein in D.saiina, and there were significant differences between the treated groups and the control group.In the scope of test, low salinity was found to be benefit either to the cell growth, P -carotene accumulation and chl a biosynthesis or to raising contents of protein in D.saiina, and there were significant differences between the treated groups and the control group. During the culture time, it was detected that the pH values rose up in the first 6 days, then declined in the followed several days. The maximum pH value of the media was found to be 9.31 and the minimum to be 8.23.NaHCOaof 12 mmol/L was optimum to growth of D.saiina and the maximum cell density was recorded to be 84.6 X 104cell/ml, whereas the control group only had the cell density of 36.7 X 104cell/ml. In the test scope, the higher the NaHCO3 levels, the higher the P -carotene contents and the maximum (3 -carotene contents of 104.6 mg/g was detected in medium with 15 mmol/L, whereas the control group only had 3 -carotene of 60.8 mg/g. The maximum chl a of 135 mg/g was also detected in the medium with 15mmol/L NaHC03. NaHCC^was consumed rapidly in the first 5 days after inoculation, but slowly in the followed 5-10 days and the assimilated model of NaHCC>3 by D.saiina was set up.The durable ability of D. salina to (NT-L^CC^, a kind of chemical which was harmful to D.saiina, was investigated. The results suggested it that in the test scope, the higher the dozen of (NH^CC^ and the longer the treated time, the lower the survival rates of D.saiina cells. The half survival rate in 12 hs was recorded to be 0.6g/L of (NH^CC^. The pH values rose up immediately when (NH^CChwas put in the culture media. The highest pH value reached 9.38, then gradually declined in 12 hs, and continued to decline in 12-48 hs, but the rate was slower. The dead, deformed and rotten cells were observed after (NFL^CC^ being put in, and the higher the dozen of (NH^CC^ and the longer the treating time, the more this kind deformed cells. The test showed it that in the massive culture of D.saiina, treating the culture media with 0.6 g/L (NH^CCh in 4 hs or 0.3 g/L in 6-8 hs was right to oppressing protozoa which polluted the culture media.更多还原