【作者】 崔小华；

【导师】 杨素萍；

【作者基本信息】 山西大学 ， 微生物学， 2013， 博士

【摘要】 紫色硫细菌是不产氧光合细菌中的一个生境独特的类群,大约出现在3.5Ga年前,是深入理解生命起源、自然界碳氮硫在物质循环中的作用、以及揭示不产氧光合作用机理的良好模型生物,在去除污染水体有机物、氮和磷以及有毒物质硫化物中也具有重要的应用价值。因该类群大多数种类生境特殊,较难获得纯培养物、菌种生长不良易于丢失,因此对其研究远不如紫色非硫细菌深入和广泛。奥氏酮(Okenone)类胡萝卜素是迄今发现的最古老的生物标志之一,已被用作研究地球进化和环境适应机制的一个有效工具,目前发现它仅出现在紫色硫细菌的15个种类中。本课题组从青岛东风盐场分离获得一株含有独特光合色素-奥氏酮的嗜盐紫色硫细菌Marichromatium sp.283-1。本文对该菌株的生物学特性、耐盐特性和耐盐机制、光合色素组份分析、环境因子对色素合成的影响、尤其是Okenone的特性和理化性质进行了系统研究,研究结果显示出Okenone类胡萝卜素性能稳定,耐热性、抗光性良好,高色价且色泽鲜艳,具有良好的特性,因此本文又进一步对Okenone的产量进行了优化。主要研究结果如下：1.生物学特性研究结果表明,紫色硫细菌Marichromatium sp.283-1对氧气敏感,最高能够耐受150g/L NaCl,1.8g/L Na2S.9H2O,7.7g/L NaNO2,能在pH6-9,温度15～45℃,光照强度100-8000Lux的范围内生长。不同的碳源、氮源、盐度、pH、光照、温度对菌体形态和胞内硫粒数量都有影响。高盐胁迫下细胞形态多样,体积增大,比表面积减小；在碱性和低温环境中细胞呈棒状而且体积较大,胞内积累较多的硫粒；高温下,细胞呈长杆状,体积变大,胞内积累较多硫粒。2. Marichromatiun sp.283-1是一株厌氧中度嗜盐紫色硫细菌,对其耐盐特性和耐盐机制进行了初步研究。结果表明随着盐度增加菌株283-1生长的延滞期延长,对Na+有专一依赖性,而对C1-依赖性较弱；它主要通过在胞内积累相容性溶质甜菜碱来对抗胞外渗透压力,胞内甜菜碱的含量随培养基NaCl浓度的增加而增加,2.0mol/L NaCl浓度时胞内甜菜碱含量可达到156.4mg/g细胞干重。外源添加甜菜碱可以明显提高菌株283-1耐盐生长的能力。因此,菌株283-1是通过胞内积累甜菜碱来调节细胞内外渗透压平衡,外源添加甜菜碱可以明显提高该菌株对盐的耐受能力。3.为了解决紫色硫细菌生物量普遍较低的问题,对其生长培养基进行了优化。采用单因子试验、Plackett-Burman设计、最陡爬坡试验及中心组合设计响应面分析,研究了乙酸钠、碳酸氢钠、氯化铵、硫化钠、硫酸镁和无机盐6个因子对菌株283-1生物量的影响。结果表明,乙酸钠和硫化钠是影响该菌株生物量的主要因素,并获得了最佳培养基配方。优化后菌株生长速率提高,延迟期缩短,生物量提高了6倍,并且结果与理论预测值相近。4.采用丙酮甲醇法提取菌体色素,通过UV-VIS、TLC、HPLC、MS和图像灰度分析等方法全面分析了该菌株的光合色素。结果表明：光照厌氧条件下,菌株283-1主要积累8种光合色素,包括BChl aGG、BChl aDHGG、BChl aTHGG和BChl ap4种细菌叶绿素,1种BPhe和OH-R.g-ketoⅠ、R.g-ketoⅢ和Okenone3种类胡萝卜素,其中细菌叶绿素BChl aP和类胡萝卜素Okenone含量最高,分别占总色素含量的39.6%和47.4%。同时建立了菌株283-1全色素的TLC和HPLC指纹图谱,这两种指纹图谱分析方法均能进行光合色素的快速分析,适合于紫色硫细菌Okenone系合成途径中和BChl a合成途径中主要积累色素组分和含量变化规律的研究。5.利用构建的指纹图谱研究了环境因子光照、温度和盐度对菌株色素合成的影响,结果表明：光照强度的变化主要影响类胡萝卜素和细菌叶绿素单位细胞的含量,对色素组份影响不明显；温度的对色素组份影响较大,低温(20℃)时,积累的类胡萝卜素主要是Okenone,TLC指纹图谱未检测到另外两种类胡萝卜素。在30℃,2000Lux时,细菌叶绿素和类胡萝卜素单位细胞含量最高；而在45℃,5000Lux时,细菌叶绿素和类胡萝卜素单位细胞含量最低。盐度变化不影响菌株的色素组份,但对各组分的相对含量影响较大。高盐(11%NaCl)下,BChl ap和BPhe的积累量明显增加,尤其是BChl ap的相对含量增加约70%；但是,OH-R.g-ketoI的积累量下调了40%。低盐(1%NaCl)下,BPhe和Okenone的相对含量明显上调。6. Okenone类胡萝卜素作为地球进化过程一种重要的生物标志物。菌株283-1胞内可积累大量的Okenone系类胡萝卜素。在紫色硫细菌Marichromatium sp.283-1光合色素组成分析的基础上,通过丙酮甲醇提取、皂化、TLC和HPLC分析、制备得到纯化的Okenone类胡萝卜素,并研究了光(包括紫外线)、温度、pH、金属离子、氧化剂、还原剂、自由基和食品添加剂等对Okenone类胡萝卜素稳定性的影响。结果表明,在暗处、1000Lux光照强度和4℃、30℃、50℃的条件下,Okenone具有很好的稳定性,有很高的耐光和耐热能力；强酸和强碱对Okenone破坏较大；对食品添加剂和多数金属离子稳定,但是Fe3+、Mg2+、Al3+和Cu2+的影响较大,尤其是Fe3+,因此,Okenone存放时应避免与这些物质接触。Okenone类胡萝卜素吸收光谱形状和精细结构受到有机溶剂极性的影响,当溶剂由非极性转变为极性时,其精细结构减弱或消失,吸收峰趋于平滑。Okenone在石油醚溶液中呈鲜艳的橙红色,色价为40000。Okenone对紫外辐射具有较强的吸收作用,因此它可通过牺牲自己起到防护作用。以[DPPH·]作为自由基检测试剂,测定了该类胡萝卜素清除[DPPH·]自由基能力,结果表明它具有抗氧化生物活性,EC50=0.022mg/mL。7.在优化了生物量的基础上,继续通过响应面分析法研究了培养基中5种无机盐和微量元素对菌株283-1Okenone类胡萝卜素积累的影响。结果表明：培养基中微量元素、碳酸氢钠和磷酸二氢钾显著影响Okenone类胡萝卜素在胞内的积累,通过Behnken Box试验设计,得到了最佳培养基配方。在优化培养基中,Okenone产量由原来的1.454mg/L提高到了2.926mg/L,提高了101.2%更多还原

【Abstract】 Purple sulfur bacterium was one group of anoxygen photosynthetic bacteria living in the unique habitats and emerged on earth about3.5Ga years ago. It was an ideal model organism to study the origin of life, the role of the carbon, nitrogen and sulfur in material recycling, as well as anoxygenic photosynthesis mechanism. It played an important role in the removal of organic matter, nitrogen and phosphorus and the toxic sulfide in sewage. Compared with purple non-sulfur bacteria, its research was far less depth and extensive because of its special habitats, difficulty to obtain pure cultures and its low biomass.Okenone carotenoid is one of the most ancient biomarkers discovered so far, and has been as an effective tool for the study of earth’s evolution and environmental adaptation mechanism. Now15kinds of purple sulfur bacteria were found that they could product Okenone carotenoid. A halophilic purple sulfur bacteria Marichromatium sp.283-1was obtained by our group which contained the unique photosynthetic pigment Okenone from Qingdao Dongfeng saltworks. In this research, the biological characteristics, osmotolerant mechanism, the photosynthetic pigment composition analysis, effect of environmental factors on the synthesis of photosynthetic pigments and the separation, identification, preparation and physical and chemical properties, production optimization of the major carotenoid Okenone from the halophilic purple sulfur bacteria Marichromatium sp.283-1were studied.The main results were as follows:1. Results of biological characteristics showed that the strain283-1was sensitive to oxygen, and could grow at these condition of the highest15%NaCl,1.8g/LNa2S.9H2O,7.7g/L NaNO2, pH6-9, temperature from15to45℃, light intensity within the range of100～8000Lux. Therefore, it was anaerobic, halophilic, with tolerance of a high concentrations of sulphur, salt, alkali, NaNO2and fixing carbon. Carbon, nitrogen and salinity, pH, light, temperature had an effect on its morphology. On high salt stress conditions cell morphological diversity, increase volume, specific surface area decreased; in the alkaline environment cells was stick and a bigger volume. Under low temperature stress, the cells are rod-shaped, bigger volume, more intracellular accumulation of sulfur particles, under high temperature stress, the cells are long rod, bigger volume, more intracellular sulfur particles.2.Marichromatiun sp.283-1was a moderate halophilic photosynthetic purple sulfur bacterium and its osmotolerant property and mechanism were studied. The results shown that strain283-1could maximally tolerate2.3mol/L NaCl in improved purple sulfur bacterium medium. The growth of283-1specifically depended on anion Na+and anion Cl-was more suitable provide intracellular osmotic pressure than anion Br-.283-1accumulated betaine to equilibrate extracellulary osmotic pressure, while betaine, which was most important compatible solutes, increased with increasing of the NaCl concentration, reaching156.4mg/gby dry weight under a2.0mol/L NaCl concentration. Exogenous betaine was sufficient to promote the growth of283-1under high salinities and enhance its salinity tolerance.3.1n order to improve the biomass, medium composition of the strain283-1was optimized. Effects of the six factors CH3COONa, NaHCO3, NH4C1, Na2S.9H2O, MgSO4and Inorganic salt on the biomass were evaluated by using single-factor test, Plackett-Burman design, the steepest ascent experiment and central composite design combined with response surface analysis in this work. Results showed that sodium acetate and sodium sulfide were most significant factors and the optimal media were obtained. With the optimized media, the biomass improved6times compared with the control and the value was in agreement with the predicted.4. Pigments were extracted from cell pellets by acetone and methanol and its comprehensive analysis was carried out using UV-VIS absorption spectrophotometry, thin layer chromatography (TLC), HPLC and mass spectrometry (MS). The results were shown that under light and anaerobic conditions, eight kinds of photosynthetic pigments were obtained including four bacterochlorophylls, BChl aGG, BChl aDHGG, BChl aTHGG BChl aP and BPhe, and three carotenoids OH-Rg-ketoI the Rg-ketoⅢ and Okenone. BChlap and okenone were main components and their contents were39.6%and47.4%, respectively. And we developed two types of photopigment fingerprintings by TLC and HPLC. The fingerprintings analyses are suitable for rapid determination of photopigments of purple sulfur bacteria and have important application in control of regulation mechanism for photopigment synthesis.5. Effects of the environmental factors of light, temperature and salinity on pigment synthesis were studied using developed photopigment fingerprintings of TLC and HPLC. The results showed that different light intensity mainly affected the content of carotenoids and bacteriochlorophyll, while temperature had great influence on the pigment components. At20℃, accumulation of carotenoids only was Okenone, and TLC fingerprinting was not detected the other two carotenoids. At30℃and2000Lux contents of bacterochlorophyll and carotenoids were the highest. At45℃and5000Lux, contents of bacteriochlorophyll and carotenoids is the lowest. The salinity had almost effect on the pigment components, but a greater impact on the relative content of each component. At11%NaCl, accumulation of BChl aP and BPhe increased significantly, and especially more than70%increase of BChl aP. However, the accumulation of OH-Rg-ketoI was down more than40%. At1%NaCl, the relative content BPhe and Okenone both increased significantly.6.On the basis of photosynthetic pigment composition analysis, we obtain the purified Okenone carotenoid by the acetone methanol extract, saponification, TLC and HPLC analysis. Effect of light (including ultraviolet), temperature, pH, metal ions, oxidizing agents, reducing agents, free radicals, and food additives and so on was researched on Okenone stability. The results showed that under the dark or1000Lux, and4℃,30℃and50℃conditions, Okenone had good stability, high lightfastness and heat capacity. Strong acids and bases destroyed Okenone largely. Food additives and metal ion effected little on Okenone stability, while Fe3+, Mg2+, Al3+and Cu2+, especially Fe3+effected the stability. Therefore Okenone should be stored to avoid of contacting with these substances. The shape and fine structure of Okenone carotenoid absorption spectral was influenced by the polarity of organic solvent, the fine structure weakened or disappeared and the absorption peaks smoothed when it was in the polarity solvent. Okenone solution had a strong absorption of ultraviolet radiation, so it could be by sacrificing itself to play a protective role.[DPPH·] as a radical detection reagent could be used as determination of ability of okenone clearing [DPPH·] radical scavenging, the results showed that Okenone had the anti-oxidation biological activity and EC50=0.022mg/mL.7. Based on the optimized biomass, effects of five kinds of inorganic salts and trace elements on Okenone production was researched by the plackett Burman design and the response surface analysis. The results showed that trace elements, sodium bicarbonate and potassium dihydrogen phosphate had the significant impact on Okenone production. The optimal medium was obtained by the Box-Benhnken design. In optimal medium, the Okenone production was from1.454mg/L to2.926mg/L, and an increase of101.2%.更多还原