【作者】 纪丽丽；

【导师】 柳建设；

【作者基本信息】 东华大学 ， 环境科学与工程， 2014， 博士

【摘要】 马氏珠母贝珍珠指我国南海及其周边海域所产的海水珍珠,主要分布在广东、广西、海南三省。马氏珠母贝培育的珍珠多以白色、淡黄色为主,天然彩色珍珠极为稀少,且价格不菲。颜色是衡量珍珠价值的重要指标之一,因此,关于珍珠致色机理的研究一直是国内外研究热点。本论文基于构建的室内封闭育珠体系,以诱导合成的2-乙基-4,5-咪唑二甲酸金属配合物为传递物质,经食源藻传递,探讨其对马氏珠母贝珍珠层颜色的影响。首先,分析了室内封闭育珠体系中放养方式、养殖密度和循环水质指标,确定了该体系的最佳育珠条件。其次,采用水热法诱导合成了三种咪唑羧酸金属配合物,分别为锌配合物、铜配合物和镍配合物,并对其结构进行了表征。通过三种配合物对扁藻的急毒试验,确定最佳传递配合物；并对育珠贝进行急毒试验,确定传递配合物的最佳投喂浓度范围。然后,采用稳定氮同位素示踪标记结合元素分析方法,探讨镍配合物在育珠贝体内的传递过程及生化指标变化,分析镍配合物对育珠贝生长的影响。最后,采用CIE标准色度系统对传递后珍珠层的颜色进行表征,分析了镍配合物对珍珠层微结构的影响,确定了金属配合物对珍珠层的致色方式。采用Translight软件模拟珍珠层光子晶体结构,根据其反射率的变化,分析金属配合物对珍珠层的致色机理。本文对揭示马氏珠母贝珍珠层致色机理提供理论基础,为人工培育高值化彩色珍珠提供理论依据。本文的研究工作主要包括以下几个方面：(1)构建了马氏珠母贝室内封闭育珠体系(IRAS),分析了IRAS中育珠贝的放养方式、养殖密度对其生长状况和死亡率的影响,并分析了IRAS水体中亚硝氮、氨氮、BOD、COD、pH、Ca2+、盐度、悬浮物质8项指标的变化情况。结果发现,IRAS中底养和吊养两种放养方式对育珠贝生长状况的影响不显著,但底养的死亡率明显小于吊养,因此,确定了IRAS最佳的放养方式为底养。IRAS每只小缸中养殖密度较低(20、25、30只／缸)时,育珠贝的生长速率较高,且死亡率较低,综合考虑,选取IRAS每个小缸的养殖密度为30只。IRAS水体中盐度、pH、Ca2+的变化不明显；BOD、COD、亚硝氮的有小幅度变化；氨氮与悬浮物的波动比较显著。在90d的实验周期内,8项指标的含量变化,均符合养殖海水水质标准要求(GB3097-1997)。因此,IRAS中每三个月更换一次循环水即可。(2)采用水热合成法,以2-乙基-4,5-咪唑二甲酸为配体,首次诱导合成了锌、铜、镍三种配合物,并采用单晶衍射仪和元素分析仪对其结构进行了表征。锌配合物的化学结构式为[Zn(C7H7N2O4)2(H2O)2]·3H2O,属于三斜晶系,空间群为PT。其不对称单元由一个配体分子,一个中心金属Zn(Ⅱ)离子,一个结合水和一个半游离的水分子组成：铜配合物的化学结构式为[Cu(C7H7N2O4)2]n,属于单斜晶系,空间群为P21/n。其不对称单元由一个Cu(Ⅱ)离子和一个配体分子组成；镍配合物的化学结构式为[Ni(C7H7N2O4)2(H2O)2]·3H2O,属于三斜品系,空问群为P1,与锌配合物是同构化合物。其不对称单元由一个配体分子,一个中心金属Ni(Ⅱ)离子,一个结合水和一个半游离的水分子组成。红外分析发现锌配合物和镍配合物存在O-H、C-H、C-N键的振动吸收峰、金属氮键的吸收峰及羧基的不对称v as(COO-)和对称·v s(COO-)伸缩振动；热重分析发现两个配合物在700℃降解的最终产物为Zn0和NiO。铜配合物红外光谱中存在-NH的振动吸收峰、羧酸基团的特征吸收峰、羧基的不对称振动v as(COO-)和对称振动v s(COO-)吸收峰及金属氮键吸收峰；铜配合物的热重分析发现在700℃,降解的最终产物为Cu0。(3)三种配合物对扁藻生长的毒性效应分析发现锌配合物和铜配合物对扁藻生长的毒性效应曲线基本相似,呈现出先促进后抑制的趋势,而镍配合物对扁藻的毒性效应曲线,呈现出先促进后逐渐平稳的趋势,可知镍配合物的毒性相对较小。锌、铜、镍配合物对扁藻生长的96h-ECso分别是24.266、19.187、105.682mg/L,且镍配合物和锌配合物的剂量反应方程符合χ2检验精度要求。通过三种配合物对扁藻的96h-EC50,判断出三种配合物的毒性大小顺序是：铜配合物>锌配合物>镍配合物,因此,选择镍配合物作为食源性传递配合物。镍配合物对马氏珠母贝的急毒试验中,发现随试验时间的延长,镍配合物对育珠贝的毒性效应明显增强,且配合物的浓度越高,死亡率越高。镍配合物对马氏珠母贝48h、72h、96h的ECso分别是78.520mg/L、18.320mg/L和9.441mg/L,结合χ2检验,得出72h和96h急毒试验的剂量反应方程符合精度要求,对应的安全质量浓度分别为0.916mg/L和0.471mg/L。因此,镍配合物的投喂浓度需低于0.471mg/L。(4)采用稳定同位素标记示踪结合电感耦合等离子体原子发射光谱分析,发现15N和Ni元素在马氏珠母贝体内的传递顺序：外套膜—珍珠囊—珍珠层,镍配合物以部分或全部组分代谢到珍珠层,其中含氮基团和镍离子在育珠贝体内同步传递,传递后镍配合物的15N和Ni均参与了珍珠层有机质形成。镍配合物传递后,马氏珠母贝体内的碱性磷酸酶活性显著提高,而且与免疫相关的生化指标球蛋白、尿素氮均朝向有利于提升免疫力的方向变化,说明经食源性传递的镍配合物可促进珍珠质的分泌,并能提高马氏珠母贝的免疫力。(5)采用CIE标准色度系统计算出镍配合物传递组和对照组珍珠层的色度坐标分别为(0.4661,0.5272)、(0.4245,0.5323),主波长分别为577nm、560nm,对应的主色调分别为黄绿色和黄色,与肉眼观察到的主色调相一致。采用扫描电镜和原子力显微镜对珍珠层的微结构进行了表征,发现镍配合物经食源性传递后,对珍珠层的微结构具有修饰和优化作用,其珍珠层表面结构呈现出层状结构,晶体排列和形貌比较清楚,文石大小均匀、紧密,呈现出规则的六变形。珍珠层傅里叶红外光谱分析表明镍配合物传递后对珍珠层的组成影响不明显,其吸收峰的种类和吸收强度与对照组基本一致,只有一处有机质吸收峰发生小幅度红移。(6)珍珠层纵断面经扫描电镜分析,发现其呈现出典型的层状结构,由文石片和有机质层周期性交替排列组成,文石片生长区厚度约为300±35nm,有机质层厚度约为30±5nm,生长区的层数约为20层,符合典型的一维光子晶体结构。采用Translight软件计算珍珠层一维光子晶体结构,设计了三种不同膜层厚度一维结构a、b、c,经软件计算,在Vrml.wrl程序中输出了一维光子晶体的结构模型。该模型的建立,表明Translight软件参数设置和理论计算的正确性。珍珠层一维光子晶体TE端的五条反射率曲线表明,a、b、c三种光子晶体结构在可见光存在一个光子禁带,且发现随着珍珠层层数的增加,一维光子晶体的禁带特征逐渐增强,带隙边缘变陡,带隙特征更加明显,带隙中心发生蓝移。随着层数的增多,珍珠层光子禁带中心发生蓝移现象,珍珠层的颜色会相应的朝向短波颜色变化。本文发现镍配合物传递组珍珠层的反射率峰也发生显著蓝移,而且发现试验组珍珠层纵断面珍珠层层数显著增多,珍珠层的颜色由黄色变为黄绿色,符合上述推测的致色机理。因此,推测食源性传递镍配合物对珍珠层致色机理：经食源性传递的镍配合物提升了育珠贝ALP活性,而ALP活性与珍珠质分泌速度成正比例关系,即镍配合物促进了珍珠质的分泌,导致珍珠层层数增多,使珍珠层光子禁带中心蓝移到黄绿色波段。更多还原

【Abstract】 The pearls of Pinctada martensii are seawater pearls, mainly produced in the South China Sea and its surrounding waters, which are mainly distributed in Guangdong, Guangxi and Hainan Province. Their colors are mainly white and faint yellow, and those with other natural colors are rare and expensive. Color is an important indicator of measuring pearl’s value, which has attracted much attention in studying the mechanism of pearl’s color.In this study, we explored the coloring mechanism of2-ethyl-4,5-imidazole dicarboxylic acid metal complex to nacre in Pinctada martensii, based on indoor recirculating aquaculture system through the transmission of dietary algae. Firstly, we analyzed culture way, culture density and water quality in indoor recirculating aquaculture system, and determined the optimum conditions for culturing pearl. Secondly, three imidazole carboxylic acid metal complexes were synthetized by the hydrothermal method, namely zinc complex, copper complex and nickel complex, and their structures were characterized respectively. Then we analyzed acute toxicity tests of three complexes to Platymonas subcordiformis, which determined the optimum transmission complex is nickel complex. Acute toxicity tests of nickel complex to pearl oyster determined the feeding concentration range of nickel complex. Thirdly, the transmission process of nickel complex in pearl oyster was studied with nitrogen stable isotope tracer and element analysis. And biochemical indicators of pearl oyster were detected in the transmission process, which is to analyze influence on nickel complex to the growth of pearl oyster. Finally, the color of nacre after nickel-complex transmission was characterized with a CIE standard colorimetric system, microstructure of nacre was observed with SEM and AFM, and the coloring way of metal complex to nacre was determined. We had simulated the photonic crystal structure of nacre with translight software. And according to reflection change of nacre’s strucuture, the coloring mechanism of metal complex to nacre was further analyzed. The study will provide a theoretical basis for revealing the coloring mechanism of nacre in Pinctada marlensii and artificial cultivation of high-value color pearl. The primary subjects of which include six parts as follows:(1)Based on indoor recirculating aquaculture system (IRAS), the influences of culture way and culture density to the growth and mortality of pearl oyster were studied, and nitrite, ammonia, BOD, COD, pH, Ca2+, salinity and suspended matters, eight indicators of water were analyzed. The results show that the growth rate of pearl oyster is not significant in two kinds of culture way, bottom and hang, but mortality rate is lower in bottom culture, so we choose bottom culture as the optimum culture way. Under low culture density (20,25and30/vat) in IRAS, the growth rate is higher and mortality is lower, so we select30/vat pearl oysters in IRAS. We find salinity, pH and Ca2+in IRAS did not change significantly; BOD, COD and nitrite are small variations; but ammonia and suspended matters may change significantly. In the90d experimental period, the changes of eight indicators are in line with aquaculture water quality standards (GB3097-1997). Therefore, IRAS can replace ciculating water every three months.(2)We synthesized three2-ethyl-4,5-imidazole dicarboxylic acid metal complexes with a hydrothermal method, namely zinc, copper and nickel complex, whose structures were characterized with single-crystal diffractometer and elementary analyzer. Zinc complex’s chemical structural formula is [Zn (C7H7N2O4)2(H2O)2]·3H2O, belonging to triclinic system and Pi space group. The asymmetric unit consists of a ligand molecule, a central metal Zn(II) ion, a combination of water and a semi-free water molecule. Copper complex’s chemical structural formula is [Cu(C7H7N2O4)2]n, belonging to monoclinic system and P21/n space group. Its asymmetric unit consists of a Cu(II) ion and a ligand molecule. Nickel complex’s chemical structural formula is [Ni(C7H7N2O4)2(H2O)2]·3H2O, belonging to triclinic system and P1space group. Nickel complex is isomorphic compounds with zinc complex. The asymmetric unit consists of a ligand molecule, a central metal Ni(Ⅱ) ion, a combination of water and a semi-free water molecule. It can be seen through infrared analysis that zinc complex and nickel complex exist four main absorption peaks followed as, O-H, C-H and C-N bond vibration absorption peak, metal-nitrogen bond absorption peak, and asymmetric v as (COO-) and symmetric v s (COO-) stretching vibration of carboxyl group. TGA analysis shows that degradation final products of znic complex and nickel complex at700℃are respectively ZnO and NiO. Copper complex presents N-H bond absorption peak, carboxylic acid group characteristic absorption peak and asymmetric vibration vas (COO-) and symmetric vibrations vs (COO-) peaks of carboxyl group in the infrared spectrum. TGA of copper complex shows the degradation final product is CuO at700℃.(3)Acute toxicity effect of algae show that zinc complex and copper complex have a similar curve about growth of algae, presenting the trend of suppressing growing after promoting, but nickel complex’s curve showing the trend of gradual stability after promotion, which can be seen nickel complex has a lower toxicity to algae.96h-EC50of zinc, copper and nickel complex to algae are respectively24.266,19.187and105.682mg/L, nickel complex and zinc complex dose-response equation meet χ2test requirements, determining three complexes’ toxicity order is copper complex> zinc complex> nickel complex, and choosing nickel complex as transmission matter. Through the acute toxicity test of nickel complex to pearl oyster, it is found that with the increase of nickel complex’s concentration and test time, the mortality rate of pearl oyster is significantly increased.48h-EC50,72h-EC50and96h-ECso of nickel complex to pearl oyster are78.520mg/L,18.320mg/L and9.441mg/L, dose-response equation of72h and96h acute toxicity meet χ2test requirements, and corresponding safety concentrations are respectively0.916mg/L and0.471mg/L. Therefore, the feeding concentration of nickel complex must be less than0.471mg/L.(4) It is found15N and Ni elements can transmit into the vivo of pearl oyster with stable isotope labeled tracer and inductively coupled plasma atomic emission spectrometry, and metabolic pathway is mantle-pearl sac-nacre, indicating nickel complex transmists into nacre with some or all of the components, nitrogen-containing groups and nickel ions could synchronously transmit in vivo of pearl oyster, and15N and Ni involve the organic-matter formation of nacre. After transmission nickel complex could significantly increase alkaline phosphatase activity, and boost the immune system, indicating nickel complex can promote the secretion of nacre, and improve the immunity of pearl oyster.(5)It is calculated the nacre’s chromaticity coordinate in nickel complex transmission group and control group are respectively (0.4661,0.5272) and (0.4245,0.5323) with CIE standard colorimetric system, the main wavelengths are577nm,560nm, and the corresponding primary color are yellow-green and yellow, which is consistent with mian color of naked eye observing. Nacre microstructure is characterized with scanning electron microscopy and atomic force microscopy, founding nickel complex could modify and optimize nacre structure, which exhibiting a layered structure, arrangement crystal, more clearly morphology, aragonite in every layer uniform size, tightly and regular six deformation. It is found nacre’s composition have no obvious change after transmission with fourier transform infrared spectroscopy, and type and intensity of absorption peaks are almost same with control group, of which only an organic matter absorption peak have a small range red-shift. (6)Cross-section of nacre shows a typical layer structure through scanning electron microscopy, consisting of aragonite and organic matter periodically alternately arranged. The thickness of aragonite layer is about300±35nm, the one of organic matter layer is about30±5nm, and layer number is about20, which is in line with a typical one-dimensional photonic crystal structure. One-dimensional photonic crystal structure of nacre is calculated by translight software. We design three one dimensional structures with different film thickness, respectively structure a, b and c, and one-dimensional photonic crystal structure model is output in Vrml.wrl. The model indicates that parameter settings and theoretical calculations of translight software are correct. TE side’s five reflectance curves in one-dimensional photonic crystal indicate a, b, and c three photonic crystal structures in the visible presentof a photonic band gap. As the number of layers increase, the intensity of photonic crystal band gap gradually increase, the edge of band gap becomes steeper, the band gap is more obvious, and the center of band gap has a blue-shift. With the number of nacre’s layers increasing, its photonic band gap presents blue-shift, and the color of nacre changes toward short-wave color. In the article, we found reflection peak of nacre has a significant blue shift after nickel-complex transmission, the number of nacre’s layers significantly increase in SEM image, and nacre color changes from yellow to yellow-green. These results suggest that the mechanism of nacre coloring through nickel-complex dietary transmission:nickel complex can significantly enhance ALP activity in pearl oyster, which has a positive relation with secretion speed of nacre, namely nickel complex can promote the secretion of nacre, resulting in layer number of nacre increasing, and make photonic band gap of nacre shift to yellow-green wave band.更多还原