【作者】 刘晓娟；

【导师】 段舜山； 李爱芬；

【作者基本信息】 暨南大学 ， 水生生物学， 2008， 博士

【摘要】 微藻的营养方式具有多样性,兼养和异养研究成为近年来国内外藻类生物学领域关注的热点问题。兼养和异养有利于提高微藻生物量和胞内活性物质产量,同时不同营养方式之间转化的叶绿体特征有助于光合作用机理的研究。但迄今为止,能够进行兼养和异养生长的微藻物种有限,并且制约微藻兼养和异养生长的机理尚不清楚。本文以硅藻门的典型藻三角褐指藻（Phaeodactylumtricornutum）为研究对象,从细胞生长、营养吸收、胞内生化组分、光合能力和细胞结构等方面,研究了三角褐指藻的自养、兼养和异养特性,同时探讨了异养生长的光需求机制。1.研究了有机碳种类、浓度和光照强度对三角褐指藻生长的影响。结果表明:三角褐指藻具有兼养和光异养生长的能力,不具备化能异养和光激活异养生长的能力,并且对有机碳的利用具有选择性;三角褐指藻在兼养下的生物量显著提高,50 mM甘油兼养下的最大生物量可达自养的1.46倍,光异养条件下的生物量低于自养;有机碳浓度对其生长的影响呈现正态分布特征;兼养生长不受高光强的限制,兼养和光合自养的饱和光强均为50μmol·m^-2·s^-1。2.通过对藻液中有机碳、无机氮和磷浓度的测定,研究了三角褐指藻在兼养和光异养下的营养吸收。结果表明:三角褐指藻对有机营养和无机营养的吸收与生长状态相对应,藻细胞快速繁殖的指数生长期,有机碳浓度直线下降;兼养较自养消耗更多的氮和磷来满足生长的需要,光异养消耗的氮和磷浓度低于自养。3.三角褐指藻在不同营养方式下的胞内生化组分存在显著差异,在兼养和光异养条件下,蛋白质含量明显下降,碳水化合物和总脂含量上升,饱和脂肪酸和单不饱和脂肪酸比例提高,多不饱和脂肪酸和EPA比例降低,乙酸钠兼养下的胞内EPA含量和产量分别是自养的1.10倍和1.40倍,甘油和葡萄糖兼养及光异养下的EPA含量和产量均低于自养。4.通过对光合色素含量和组成、常温吸收光谱、低温荧光光谱、光合放氧速率、呼吸速率、PSⅡ最大光能转化效率（Fv/Fm）和电子传递速率（ETR）等指标的测定,研究了三角褐指藻在不同营养方式下的光合能力特征。结果表明:三角褐指藻在兼养和光异养条件下,叶绿素a、叶绿素c和类胡萝卜素含量以及Chl a/Chl c均下降;相对荧光强度和PSⅠ/PSⅡ激发能比例均降低,光合放氧速率、Fv/Fm和ETR均下降,从而表明有机碳降低了三角褐指藻的光合能力,但是有机碳显著提高了三角褐指藻的呼吸速率。5.利用显微镜和透射电镜拍照,观察了三角褐指藻在不同营养方式下的细胞体积和细胞结构的变化。结果表明:在兼养和光异养条件下,三角褐指藻的细胞体积增大,细胞形态由自养条件下纤细的三角放射型转变为中心膨胀的三角短粗型;自养下的类囊体膜垛叠整齐有序,兼养下类囊体膜垛叠程度降低,光异养下类囊体膜呈现非垛叠现象。6.采用不同抑制剂对三角褐指藻生长的影响,以及气相-质谱联用法（GC-MS）测定有机酸,揭示了黑暗制约该藻生长的原因。结果表明:有机碳主要作为三角褐指藻兼养和光异养生长的碳源,光是能量的主要来源;藻细胞中存在琥珀酸脱氢酶和细胞色素氧化酶系统;在完全黑暗条件下,添加葡萄糖的藻液pH显著下降,GC-MS检测到了甲酸和乙酸,推测三角褐指藻利用葡萄糖进行异养生长的光需求机制在于,黑暗条件下末端氧化系统不起作用,从而使藻细胞利用葡萄糖转入了厌氧代谢途径。更多还原

【Abstract】 Trophic modes of microalgae are diversified.Over the last decades,significant attention has been drawed to mixotrophy and heterotrophy.Culturing microalgae in mixotrophic or heterotrophic conditions could potentially yield higher biomass concentration and algal products of high value.Meanwhile,many studies on photosynthetic mechanisms have been carried out with mixotrophic and heterotrophic strains.Up to now,few microalgae species have been found to possess the ability to grow mixotrophically and heterotrophically,and the reasons for the restrictions are still unknown.In this paper,a typical diatom ofPhaeodactylum tricornuturn was used as the material,characteristics of photoautotrophy,mixotrophy and heterotrophy were studied,parameters of cell growth,nutrient absorption,biochemical components, photosynthetic activity,and cell structure were examined,and mechanism of the light requirement for heterotrophic growth was also investigated.The main results are listed as follows:1.Effects of sources and concentration of organic carbon sources and light intensity on the growth of P.tricornutum were investigated.The results showed that P. tricornutum could grow mixotrophically and photoheterotrophically,and the utilization of organic carbon sources was selective.Under mixotrophic condition,the biomass of P.tricornutum was greatly enhanced.But under photoheterotrophic condition,the biomass maintained in the lower concentration level.Effects of organic carbon concentrations on the growth of P.tricornutum were in normal distribution. The mixotrophic growth was not inhibited by the high light.The saturation irradiance of mixotrophic and photoautotrophic growth was almost at the same level,which was 50μmol·m^-2·s^-12.To study the nutrient absorption of P.tricornutum under different trophic modes,concentrations of organic nutrient and inorganic nutrient in the culture were examined.The results indicated that the absorption of organic nutrient and inorganic nutrient were corresponding to the growth state.During the exponential growth phase, the growth rate was faster,and the concentration of organic carbon decreased dramatically.Under mixotrophic conditions,the absorption of nitrogen and phosphorus were more than those under photoautotrophic condition.However,the nitrogen and phosphorus absorption were lower under photoheterotrophic condition.3.The biochemical components of P.tricornutum under different trophic modes were significantly different.Compared with photoautotrophic condition,under mixotrophic and photoheterotrophic conditions,the content of soluble protein was obviously decreased,whereas the content of soluble carbohydrate and total lipid were increased.The proportions of saturated fatty acids and mono-saturated fatty acids were enhanced,however the proportion of polyunsaturated fatty acids and EPA （Eicosapentaenoic acid） were lowered.The EPA content and yield were 6.23%and 36.59 mg·L^-1 during mixotrophic culture with 100 mM acetate,which were 1.10-fold and 1.40-fold of those obtained under photoautotrophic condition,respectively. During mixotrophic culture with glycerol or glucose,and photoheterotrophic culture with glycerol,acetate,or glucose,the EPA content and yield were lower than those obtained under photoautotrophic condition.4.The experiment was conducted to determine the photosynthetic characteristics of P.tricornutum under different trophic modes.Parameters of photosynthetic pigment content and composition,absorption spectra,77 K fluorescence spectra, photosynthetic O₂ evolution rate,respiration rate,maximal photochemical efficiency of PSⅡ（Fv/Fm）,and electron transport rate（ETR） were examined.The results showed that under mixotrophic and photoheterotrophic conditions,the cell photosynthetic pigment content,values of Chl a/Ch1 c were reduced.The relative fluorescence intensity of PSⅠ,PSⅡand the values of F685/FT10 and F685/F738 were decreased.The net maximum photosynthetic O₂ evolution rate was also depressed. These were in consistent with Fv/Fm and ETR.Therefore,organic carbon sources reduced the photosynthesis efficiency of P.tricornuturn.However,organic carbon sources enhanced the respiration rate dramatically.5.Microscopy and transmission electron microscopy were conducted to investigate the cell volume and structure of P.tricornutum under different trophic modes.Under mixotrophic and photoheterotrophic condition,the cell volume was increased.The cell structure was finely triangular under photoautotrophic condition, but the cell center became bulgy,and the radiation was dumpy.Under photoautotrophic condition,the thylakoids were predominantly arranged in pairs. Chloroplast thylakoid pairs were less packed under mixotrophic and photoheterotrophic conditions.6.Organic carbon sources were mostly used as carbon sources ofP.tricornutum, and the main source of energy came from light.Effects of sodium malonate and sodium azide on the growth of P.tricornutum confirmed that there were succinate dehydrogenase and cytochrome oxidase system in the cell.Under chemoheterotrophic growth with glucose,the value of pH dramatically decreased,and there were formic and acetic acids in the culture.It can be assumed that an inoperative terminal oxidase system would adjust metabolism toward anaerobic metabolism pathway.更多还原