【作者】 孙颖颖；

【导师】 王长海；

【作者基本信息】 大连理工大学 ， 生物化工， 2007， 博士

【摘要】 本文以球等鞭金藻为对象，运用一系列单因素实验和正交实验，分析了常量元素，维生素和微量元素等营养条件以及温度，光照，pH和通气速率等环境条件与球等鞭金藻生长的关系，确定了球等鞭金藻培养的优化营养条件和环境条件；利用柱状光生物反应器和平板式光生物反应器培养球等鞭金藻，研究了培养条件以及补加营养，流加培养和半连续培养等培养模式对球等鞭金藻生长的影响，确定了球等鞭金藻高密度培养的工艺条件；采用乙酸乙酯萃取，Sephadex G-15凝胶过滤，制备薄层层析和C₁₈反相高效液相色谱等分离纯化方法成功地分离出一种微藻生长抑制物，确定了其化学结构，并对其性质和抑藻活性进行了定性探讨；此外，还在抗氧化剂对生长抑制物抑藻效应的抵制以及环境因子对球等鞭金藻胞外碳酸酐酶活性的调节，质膜氧化还原活性与胞外碳酸酐酶活性的关系以及某些抑制剂对球等鞭金藻无机碳利用的抑制作用等方面进行了探索，为球等鞭金藻高密度大规模培养提供理论依据。主要研究结果如下：1、获得优化培养基配方和培养条件培养基配方：NaNO₃，45.5 mg；KH₂PO₄，2.195 mg；Na₂SiO₃，0.8714 mg；NaHCO₃，20 mg；微量元素溶液1 mL（MnCl₂·2H₂O，15.8336 mg；CuSO₄·5H₂O，14.9808 mg；ZnSO₄·7H₂O，0.2013 mg；Na₂MoO₄·2H₂O，0.7295 mg；CoCl₂·6H₂O，23.795 mg；FeCl₃·6H₂O，482.7 mg；Na₂EDTA，0.665 g；1 L蒸馏水配制），维生素溶液1 mL(维生素B₁，100 mg；维生素B₁₂，0.5 mg，1 L蒸馏水配制)；1 L海水配制。培养条件：温度25℃，pH 8.0，光强7500 lx，通气速率0.2 vvm。采用上述条件，在PhR-L20C光生物反应器中对球等鞭金藻进行了培养，其生物量产量达到1.3 g／L。2、确定了优化的培养工艺条件通过一系列的单因素实验和正交实验，在平板式光生物反应器中，光径，光强和通气速率分别为5 cm，7000 lx和0.375 vvm时是最佳培养条件。采用该优化条件对球等鞭金藻进行一次性培养，生物量产量达到2.2 g／L。同时还对流加培养，半连续培养和补加营养等培养工艺对球等鞭金藻生长的影响进行了研究，发现流加培养和半连续培养两种培养方式更适合该藻的培养。3、获得了一种生长抑制物并确定了其化学结构以球等鞭金藻高密度培养物的无细胞滤液对球等鞭金藻和其它微藻进行培养，发现了一种生长抑制物，研究表明该抑制物具有广泛的抗藻活性，不仅抑制自身藻细胞生长，而且抑制牟氏角毛藻等其它微藻的生长。随后对该生长抑制物的分离提取工艺进行了研究，其最适提取工艺为：pH2-3，提取时间3 h，提取料液比10：1，提取溶剂乙酸乙酯。在此基础上，运用乙酸乙酯萃取，Sephadex G-15凝胶柱层析过滤，硅胶G制备薄层层析和C₁₈反相高效液相色谱等分离纯化方法获得了该生长抑制物的纯品；采用紫外光谱，质谱，核磁共振碳谱和氢谱以及核磁共振二维谱等手段确定了其化学结构为1-羟基，丙二酸二乙酯-十二烯酸异丙酯(C₂₂H₃₈O₇)，分子量为414。深入研究发现，该抑制物能明显降低球等鞭金藻和牟氏角毛藻等微藻细胞内叶绿素含量，硝酸还原酶，过氧化物酶和超氧化物歧化酶的活性，提高细胞内丙二醛含量和胞外可溶性蛋白质与多糖含量的比值，从而通过影响细胞的光合作用，细胞内营养平衡，细胞内活性氧浓度以及细胞表面的疏水性，对微藻细胞的生长产生强烈的抑制效应。此外，发现抗氧化剂能够明显抵制该生长抑制物对球等鞭金藻的抑制效应，其主要机理可能是抗氧化剂能够有效地清除生长抑制物胁迫下藻细胞内积累的活性氧，减轻藻细胞的膜脂过氧化伤害。4、探索了细胞外碳酸酐酶活性与球等鞭金藻无机碳利用的关系碳酸酐酶（CA）是CO₂浓缩机制组成部分，在光合CO₂固定中起着较重要的作用。我们的研究结果表明，氮，磷，Zn²⁺，无机碳，光强，通气速率和pH等因素均能调节球等鞭金藻胞外碳酸酐酶活性。此外，我们注意到细胞质膜氧化还原活性，乙酰唑磺胺（AZ）和乙氧苯唑胺（EZ）等对球等鞭金藻无机碳的利用有直接的影响，同时还发现DIDS （4′4′-diisothiocyanatostilbene-2，2-disulfonic acid）和SITS（4-acetamido-4’-isothiocyano-2，2’-stibene-disulfonate）等对球等鞭金藻无机碳的利用也有一定的影响，进而我们获知球等鞭金藻具有依赖胞外CA，膜ATP酶，带Ⅲ蛋白以及Na⁺／HCO₃^-协同转运系统间接和直接利用HCO₃^-的能力。更多还原

【Abstract】 Isochrysis galbana, a species of marine microalgae, which is of substantial interest inaquaculture for its good nutritive characteristics, especially to mollusks larvae, fish andcrustaceans in the early stages of growth. Inorder to explore ways of high density culture, aseries of researches were carried out from the basic cultures in small bottles, high densitycultures in the photobioreactors, the isolation and identify of the growth-inhibitor to themechanism of dissolved inorganic carbon （DIC） utilization in Isochrysis galbana. The mainresults are summarized as following.1. The optimal medium and culture conditions of Isochrysis galbanaThe optimum medium: NaNO₃ 45.5 mg/L, KH₂PO₄ 2.195 mg/L, Na₂SiO₃ 0.8714 mg/L,NaHCO₃ 20 mg/L, MnCl₂·2H₂O 15.8336μg/L, CuSO₄·5H₂O 14.9808μg/L, ZnSO₄·7H₂O0.2013μg/L, Na₂MoO₄·2H₂O 0.7295μg/L, CoCl₂·6H₂O 23.795μg/L, FeCl₃·6H₂O 482.7μg/L, Na₂EDTA 0.665 mg/L, VB₁ 100μg/L and VB₁₂ 0.5μg/L were added to natural seawater.The optimum culture conditions: tempature 25℃, pH 8.0, light intensity 7500 1x andaeration rate 0.2 vvm. 1.3 g/L of dry cell biomass was obtained in eight days culture under theoptimal cultural conditions.2. The mode of high density culture in photobioreactorsThe optimal culture conditions and the fed-batch culture, semi-continuous culturesmode was established in the plate photobioreactors. Under the conditions of 5 cm light-path,7000 1x light intensity, 0.375 vvm aeration rate and semi-continuous culture mode, themaximal dry cell biomass of 2.2 g/L was determined in 8 days in plate photobioreactors. Theresults showed that the fed-batch culture, especially fed-batch culture with various feed rateand semi-continuous cultures, was more favorable for the growth of Isochrysis galbana.3. Isolation and identified a kind of microalgal growth-inhibitorA type of antialgal substanceswas found by the cultures of cell-free filtrates whichgotten from culture suspension at the three growth phases （exponential, stationary and deathphase） of Isochrysis galbana. Results showed that, as the age of Isochrysis galbana culturesadvances, the concentration of the antialgal substances in cultural liquid increases, and whichsuggested that Isochrysis galbana produced gradually antialgal substances from the exponential growth phase up to the death phase. The growth inhibitory rates of Isochrysisgalbana by cell-free filtrates at the three phases were 7.17%, 34.2% and 64.1% in day 10,respectively. The crude,antialgal substance （CEAE） was extracted out from the old culturalliquid by ethyl acetate, which also showed significant inhibitory effects on growth ofIsochrysis galbana. When CEAE concentrations was from 3.6 mg/L to 21.6 mg/L, the growthinhibitory rate of Isochrysis galbana by CEAE were 12.9%, 27.5%, 29.3%, 59.5% and 67.2%in day 6, respectively. The CEAE could significantly （P＜0.05） inhibit the cell growth of thesix species of marine feed microalgae too, at the concentration of 21.6 mg/L, the growthinhibitory rate of Chaetoceros muelleri, Chaetoceros gracilis, Phaeodactylum tricornutum,Nitzschia closterium, Platymonas elliptica, Dunaliella salina was 63.3%, 59.6%, 61.1%,66.2%, 68.3% and 48.5% in day 6, respectively. And the biochemical compositions in thecells of Isochrysis galbana and the six species of marine feed microalgae also could beaffected by CEAE.The growth-inhibitor was successful isolated and purified from CEAE. Molecularformula of the growth-inhibitor, C₂₂H₃₈O₇, was determined by FABMS, m/z 414 [M-H]-. InUV spectrum the maximum absorption was at 278 nm that suggested the presence ofO=C-C=C functionality. The ¹³C-NMR spectrum of the growth-inhibitor showed 22 carbonsignals, including three carbonyl carbon signals （6167.9, 167.9 and 167.89）, threeoxymethylene carbon signals （δ72.0, 65.7 and 65.7）, four methyl carbon signals （δ30.8, 30.8,19.8 and 19.8）, one ethylene carbon signal （δ132.5 and 129.1）, one quaternary carbon signal（δ77.4）, one methine carbon signal （δ27.9） and eight methylene carbon resonances. Fourmethyl proton signals （δ1.72, 1.72, 0.99 and 0.99） were observed in the ¹H-NMR spectrum.The ¹H-NMR spectrum of the growth-inhibitor exhibited two doublets for two couplingprotons atδ7.53 andδ7.12, a OH group atδ3.11, a isopropyl group atδ4.08 （1H） andδ0.99（6H）. According above information, the chemical structure of the growth-inhibitor wasidentified as 1-[hydroxyl-diethyl malonate]-isopropyl dodecenoic acid.The further investigation found that exogenously added GI significantly decreased thechlorophyll contents in Isochrysis galbana and the six species of microalgae （Chaetocerosmuelleri, Chaetoceros gracilis, Phaeodactylum tricornutum, Nitzschia closterium,Plytymonas elliptica and Dunaliella salina）. And it had a very significant effect on the rationof extracellular protein to polysaccharide and activities of NR （nitrate reductase）, SOD（superoxide dismutase） and POD （peroxidase） in the cells of all test microalgae. But it wasobvious to increase MDA （malonyldialdehyde） content in algal cells. The results gave hints toelucidate the species-specific antialgal mechanisms of GI. Inhibition mechanism of GI onmicroalgae is to decrease the chlorophyll contents, and decrease or increase activities of NR,SOD and POD of algal cells, and change the ratio of extracellular protein to polysaccharide, thus increase in ratio of extracellular protein to polysaccharide leads to the increase inhydrophobicity of algal cells, finally algal cells flocculate and subside. Affect uptake ofinorganic nitrogen by cells and photosynthesis might play a major in the inhibitorymechanism. And the excess active oxygens were produced under GI stress and active oxygenparticipated in the damage of GI to microalgae. It indicated that the physiological metabolismof microalgae was inhibited by GI.4. Relationship betweenthe the activity of carbonic anhydrase and utilization ofCO₂Carbonic anhydrase, a ubiquitous mental enzyme that can catalyze the transformationreaction between HCO₃^- and CO₂, is one of enzymes whose catalytic velocity is confirmed tobe the fastest. It has been taken as a component of CO₂ concentrating mechanism （CCM） andplays a role in photosynthetic CO₂ fixation. Extracellular carbonic anhydrase （CA） activity inIsochrysis galbana was influenced by several external factors. Our research demonstrated thatlight intensity, pH and ions such as NO₃^-, NH₂^-, NH₄⁺, H₂PO₄^- and HCO₃^- were importantexternal factors that could regulate the extracellular CA activity in the cells of Isochrysisgalbana. The activity of CA significantly depends on the light intensity, when the lightintensity increased from 0 to 8000 lx, the CA activity increased rapidly and arrived its highestpoint at 6000 lx. With the increasing of pH values, the CA activity significant increased. Atrange of NO₃^- concentration from 0.5 mmol/L to 4 mmol/L, the CA activity was higher, andarrived to the maximal value at the concentration of 2 mmol/L; the CA activity decrease whenNO₃^- concentration was above 4 mmol/L, but the CA activity still was higher than that of thecontrol. The CA activity was pronounced increased with the increasing of NH₂^-concentrations and arrived to the highest value at the concentration of 3.5 mmol/L, thenslowly decreased when NH₂^- concentration over 3.5 mmol/L. Low NH₄⁺（0.5-2 mmol/L）concentrations caused significant increase in the CA activity, but over 2 mmol/L, the CAactivity sharply decreased. The CA activity was lower under the conditions of phosphatestarvation, and a significant increase in the CA activity was observed with the increasing ofH₂PO₄^- concentration, and a maximum value was obtained at H₂PO₄^- of 0.05 mmol/L. But,against with the above factors, HCO₃^- inhibited the CA expression.According our data, the cells of Isochrysis galbana can also transport HCO₃^- directlydependent-ATPase as viewed by DIDS inhibition. Band three proteins and Na⁺/ HCO₃^-co-transport system have been found involving in the direct transportation since SITS hasaffection on carbon acquisition. Hence, external carbonic anhydrase dehydrating HCO₃^- toCO₂ is the main pathway of DIC utilization by Isochrysis galbana. In addition, dissolved inorganic carbon （DIC） utilization is significantly inhibited by Ez （ethoxyzolamide）,suggesting internal CA is essential to photosynthesis in Isochrysis galbana.更多还原