苏北海滨盐土对3种耐盐植物种植的响应研究及其微生物资源化利用探索
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摘要
人口和经济的快速增长,使资源短缺问题成为一个全球关注的热点,中国的发展正面临资源短缺的瓶颈。如何开发新资源,为中国快速增长的经济提供资源保障,是实现可持续性发展的关键。从极端环境中开发新土壤资源、种质资源和能源成为焦点研究领域,海滨滩涂不仅是一种重要的土地资源,还是珍贵的生物资源宝库,具有大量尚未被开发的潜在药物、食品和工农业产品。盐生生物由于其特殊的生长环境,以特定的生理代谢途径,产生具有特殊生理功能的次生代谢产物,如酶、色素、抗生素等。因此,在海滨盐土上种植耐盐植物,发展盐生农业,开发盐生生物资源,成为新资源开发的一个热点和亮点。本文通过研究种植耐盐植物前后,盐土养分、生物化学活性和微生物群落结构变化,找出不同耐盐种植过程中各元素的变化规律,为发展盐土农业,种植耐盐植物提供理论依据。本文还对从盐土中筛选获得的一株产蓝色素的耐盐微生物,从菌种鉴定、培养条件、色素组分、色素稳定性和安全性等方面进行研究,考查该菌种能否用于工业化微生物产天然蓝色素的生产。本文的主要研究内容和结果如下：
(1)蓖麻,海滨锦葵和野大豆种植后,盐土性质得到了很大的改善,土壤肥力增加,微生物数量增加,生化活性增强。耐盐植物种植后,土壤的pH有所增加,电导率都显著下降,土壤环境改良,盐胁迫降低,更适合微生物生长,土壤中各类微生物数量增加。比较3种耐盐植物种植后盐土的肥力,种植海滨锦葵土壤的改良效果最明显,其导电率最低,阳离子交换量最大,有机质、全氮、全磷含量最高,说明海滨锦葵是一种适合盐土生长的先锋耐盐植物。
(2)蓖麻种植可以改良土壤,使土壤的电导率显著下降,交换量增加,全氮含量升高。但随着种植时间的加长,受蓖麻根际所分泌的大量酚酸类化感物质的影响,土壤肥力下降,电导率上升,交换量、有机质、全氮、速效磷和速效钾等下降。蓖麻具有发达的根系,在种植中根际分泌的大量有机酸,能分泌土壤中的矿物质,提供微生物生长所需矿质元素,促进根际微生物数量的增加,使得土壤微生物的活性和多样性增加,各种细菌、放线菌和各功能微生物数量显著增加。
(3)从金海农场筛选到一株产蓝色素菌株NJYS-02,研究发现该菌株为革兰氏阴性菌,微好氧,短杆状,无鞭毛、芽孢和荚膜。在PDA固体培养基上,菌落形态为蓝色,圆形凸起,有光泽,不透明,直径1.2mm；在营养琼脂培养基、高氏培养基和孟加拉红培养基上不生长。菌株可在28℃下PDA培养基上生长并产生蓝色素。在8%以下盐度菌体生长,在4%以下盐度产生色素。通过16S rDNA克隆测序,得到了1430bp左右的碱基序列,Blast比对显示该菌在分类学上属于假单胞菌属(Pseudomonas sp.)。
(4)对NJYS-02菌株产蓝色素的最佳发酵条件研究表明,该菌株产蓝色素的最适发酵条件是在20%的马铃薯汁中添加2.5%的葡萄糖,pH6.0,28℃静置培养3d。发酵液离心后,用超声破碎法和中性盐沉淀法协调作用,在室温下,中性pH,以乙醇溶液抽提7h,色素的得率最高。用薄层层析、纸层析、柱层析、高效液相色谱法分离色素表明,NJYS-02所产蓝色素粗提取物含有两个组分,且含量差别较大,蓝色素Ⅰ约占83%,,蓝色素Ⅱ约占15%。
(5)对NJYS-02所产蓝色素的稳定性研究表明,该色素有较强的耐光、耐高温特性；还原剂、柠檬酸钠和维生素B1对其影响不大；在中低浓度的糖溶液中,该色素也有较好的稳定性；但是氧化剂和防腐剂对其破坏较大。但本文实验显示NJYS-02菌株产生的蓝色素无抗菌性。小鼠毒性试验也研究表明,NJYS-02发酵所产生的蓝色素,是一种可应用于食品、化妆品和饲料添加剂的一种有极高开发前景和应用前途的微生物天然蓝色素。
The rapid increase of population and economy is making the resources shortage a hot spot with global concern, and the development of China is faced with a hard time of resources shortage. So, it's the key to achieve the sustainable development that how to exploit the new resources to provide the resources guarantee for the rapid economic development in China. To develop new soil resources, germ plasm resources and energy resources from extreme environment is now becoming the focus in the research. The coastal beach is not only one kind of important soil resources, but also precious biological resources, and there are abundant potential drug food and industrial-agricultural products, which haven't been developed yet. Because of the special living conditions of the halobionts, they will, in specific physiological and metabolic ways, produce some secondary metabolites with special physiological functions, such as enzymes, pigments, and antibiotics, etc. So, planting salt tolerant plants in the seaside solonchak, developing the halobiotic agriculture and exploiting the halobionts resources become the hot and bright spot in the new resources development. By studying the change of the nutrient, biochemical activity, and the structure of microbial colonies in the saline soil before and after planting the halophytes, we find the change rule of every element in the planting process of different halophytes, which provides the theoretic basis for the development of the saliniferous agriculture and the planting of salt tolerant plants. Also, we conducted the research on a strain of cyanine-producing and salt tolerant microorganism, which was selected from the solonchak. We studied the identification of strain, the condition of cultivation, the components, stability, and safety of the pigment, and tested whether this strain was useful for the industrial production of microorganism producing natural cyanine. The main studying details and results are as follows:
(1) After planting the Ricinus communis L., Kosteletzkya virginica L. and Glycine soja, the physicochemical property, biochemical activity and microbial amount in the saline soil changed. After planting the halophytes, the pH range of the soil increased, the conductivity of a variety of planting soil decreased remarkably, which showed that the saline content decreased, the texture of the soil loosed, thus, the soil was suitable for the living of microbes, and the quantity of many microbes in the saline soil grew. After planting the Kosteletzkya virginica L., the exchange capacity in the soil was up to the highest, while the conductivity was down to the lowest, and the number of halophilic fungi was the largest, that means the Kosteletzkya virginica L. has strong ability to desalinate. The fungi proliferated a lot, such as AM, etc, the soil environment improved, making the exchange capacity in the soil increase and the sum of the microbes rise. The Glycine soja provides the nitrogen element for the plants by nitrogen fixation effect, so, if the intensity of nitrogen fixation of soil is maximized, and the amount of nitrogen-fixing bacteria is the most, the content of total nitrogen and hydrolysable nitrogen will be the highest. After planting the Ricinus communis L, the hydrolysable nitrogen in the soil descended, the ammoniation enhanced, and the number of the ammonifying bacteria was more than that in the soil where other salt tolerant plants grew.
(2) The saline soil is improved after planting the Ricinus communis L. It has a significantly decrease on soil conductivity, but the exchange capacity and total nitrogen content increased observably. But along with the sowing time is lengthened, the influence of a large number of phenolic allelochemicals which is secreted by the castor rhizosphere made the soil and fertilizer decrease, the conductivity increase, exchange capacity, organic matter, total nitrogen, available phosphorus and available potassium decrease. Castor has developed root system, planted in the rhizosphere secrete large quantities of organic acids, can secrete the minerals in the soil, microbial growth required to provide mineral elements, to promote the rhizosphere microbial quantity increase, so the soil microbial activity and diversity increased, a variety of bacteria, actinomycetes and the significant increase in the number of functional microorganism.
(3) NJYS-02, a cyanine-producing strain, was screened from Gimhae farm. The strain was discovered to be gram-negative, microaerobic, short rhabditiform, aflagellar, sporeless and decapsulated. The colonial morphology on PDA solid media was blue, rounded raised, lustrous and opaque, with a diameter of1-2mm. The bacteria couldn't grow on nutrient agar, Gao's medium and rose Bengal medium. It could grow and produce cyanine on PDA medium at28℃. The strain could grow at the salinity under8%and produce pigment under4%. By16s rDNA cloning and sequencing, a base sequence of about1430bp was obtained. Blast comparison showed that the strain should belong to pseudomonas sp. in taxonomy.
(4)The most suitable fermentation conditions of the strain NJYS-02are20%potato juice with2.5%glucose solution, pH6.0,28℃for3days. The best extracting method was to combine supersonicbroken method and neutral salt precipitation and use neutral pH ethanol solution to extract for7hours at the room temperature.In this way,we obtained the highest pigment yield. By separating the crude extraction, it is obvious that the cyanine pigment contains two components whose contents differ largely. Cyanine I accounts for about85%while cyanine II accounts for about15%.
(5)The research on the stability of cyanine produced by NJYS-02indicates that the pigment has a relatively strong light resistance and thermotolerance and is stable in sucrose solution of both medium and low concentration. However, reducing agents, sodium citrate and vitamin B1could not damage its structure, but oxidizing agents and preservatives may cause the damage even worse. The cyanine pigment shows no bacterial resistance. The mice study demonstrates that cyanine generated by NJYS-02, is a kind of natural microbial pigment applicable to food, cosmetics, and fodder additives, with a good development prospect and wide applications.
(1)蓖麻,海滨锦葵和野大豆种植后,盐土性质得到了很大的改善,土壤肥力增加,微生物数量增加,生化活性增强。耐盐植物种植后,土壤的pH有所增加,电导率都显著下降,土壤环境改良,盐胁迫降低,更适合微生物生长,土壤中各类微生物数量增加。比较3种耐盐植物种植后盐土的肥力,种植海滨锦葵土壤的改良效果最明显,其导电率最低,阳离子交换量最大,有机质、全氮、全磷含量最高,说明海滨锦葵是一种适合盐土生长的先锋耐盐植物。
(2)蓖麻种植可以改良土壤,使土壤的电导率显著下降,交换量增加,全氮含量升高。但随着种植时间的加长,受蓖麻根际所分泌的大量酚酸类化感物质的影响,土壤肥力下降,电导率上升,交换量、有机质、全氮、速效磷和速效钾等下降。蓖麻具有发达的根系,在种植中根际分泌的大量有机酸,能分泌土壤中的矿物质,提供微生物生长所需矿质元素,促进根际微生物数量的增加,使得土壤微生物的活性和多样性增加,各种细菌、放线菌和各功能微生物数量显著增加。
(3)从金海农场筛选到一株产蓝色素菌株NJYS-02,研究发现该菌株为革兰氏阴性菌,微好氧,短杆状,无鞭毛、芽孢和荚膜。在PDA固体培养基上,菌落形态为蓝色,圆形凸起,有光泽,不透明,直径1.2mm；在营养琼脂培养基、高氏培养基和孟加拉红培养基上不生长。菌株可在28℃下PDA培养基上生长并产生蓝色素。在8%以下盐度菌体生长,在4%以下盐度产生色素。通过16S rDNA克隆测序,得到了1430bp左右的碱基序列,Blast比对显示该菌在分类学上属于假单胞菌属(Pseudomonas sp.)。
(4)对NJYS-02菌株产蓝色素的最佳发酵条件研究表明,该菌株产蓝色素的最适发酵条件是在20%的马铃薯汁中添加2.5%的葡萄糖,pH6.0,28℃静置培养3d。发酵液离心后,用超声破碎法和中性盐沉淀法协调作用,在室温下,中性pH,以乙醇溶液抽提7h,色素的得率最高。用薄层层析、纸层析、柱层析、高效液相色谱法分离色素表明,NJYS-02所产蓝色素粗提取物含有两个组分,且含量差别较大,蓝色素Ⅰ约占83%,,蓝色素Ⅱ约占15%。
(5)对NJYS-02所产蓝色素的稳定性研究表明,该色素有较强的耐光、耐高温特性；还原剂、柠檬酸钠和维生素B1对其影响不大；在中低浓度的糖溶液中,该色素也有较好的稳定性；但是氧化剂和防腐剂对其破坏较大。但本文实验显示NJYS-02菌株产生的蓝色素无抗菌性。小鼠毒性试验也研究表明,NJYS-02发酵所产生的蓝色素,是一种可应用于食品、化妆品和饲料添加剂的一种有极高开发前景和应用前途的微生物天然蓝色素。
The rapid increase of population and economy is making the resources shortage a hot spot with global concern, and the development of China is faced with a hard time of resources shortage. So, it's the key to achieve the sustainable development that how to exploit the new resources to provide the resources guarantee for the rapid economic development in China. To develop new soil resources, germ plasm resources and energy resources from extreme environment is now becoming the focus in the research. The coastal beach is not only one kind of important soil resources, but also precious biological resources, and there are abundant potential drug food and industrial-agricultural products, which haven't been developed yet. Because of the special living conditions of the halobionts, they will, in specific physiological and metabolic ways, produce some secondary metabolites with special physiological functions, such as enzymes, pigments, and antibiotics, etc. So, planting salt tolerant plants in the seaside solonchak, developing the halobiotic agriculture and exploiting the halobionts resources become the hot and bright spot in the new resources development. By studying the change of the nutrient, biochemical activity, and the structure of microbial colonies in the saline soil before and after planting the halophytes, we find the change rule of every element in the planting process of different halophytes, which provides the theoretic basis for the development of the saliniferous agriculture and the planting of salt tolerant plants. Also, we conducted the research on a strain of cyanine-producing and salt tolerant microorganism, which was selected from the solonchak. We studied the identification of strain, the condition of cultivation, the components, stability, and safety of the pigment, and tested whether this strain was useful for the industrial production of microorganism producing natural cyanine. The main studying details and results are as follows:
(1) After planting the Ricinus communis L., Kosteletzkya virginica L. and Glycine soja, the physicochemical property, biochemical activity and microbial amount in the saline soil changed. After planting the halophytes, the pH range of the soil increased, the conductivity of a variety of planting soil decreased remarkably, which showed that the saline content decreased, the texture of the soil loosed, thus, the soil was suitable for the living of microbes, and the quantity of many microbes in the saline soil grew. After planting the Kosteletzkya virginica L., the exchange capacity in the soil was up to the highest, while the conductivity was down to the lowest, and the number of halophilic fungi was the largest, that means the Kosteletzkya virginica L. has strong ability to desalinate. The fungi proliferated a lot, such as AM, etc, the soil environment improved, making the exchange capacity in the soil increase and the sum of the microbes rise. The Glycine soja provides the nitrogen element for the plants by nitrogen fixation effect, so, if the intensity of nitrogen fixation of soil is maximized, and the amount of nitrogen-fixing bacteria is the most, the content of total nitrogen and hydrolysable nitrogen will be the highest. After planting the Ricinus communis L, the hydrolysable nitrogen in the soil descended, the ammoniation enhanced, and the number of the ammonifying bacteria was more than that in the soil where other salt tolerant plants grew.
(2) The saline soil is improved after planting the Ricinus communis L. It has a significantly decrease on soil conductivity, but the exchange capacity and total nitrogen content increased observably. But along with the sowing time is lengthened, the influence of a large number of phenolic allelochemicals which is secreted by the castor rhizosphere made the soil and fertilizer decrease, the conductivity increase, exchange capacity, organic matter, total nitrogen, available phosphorus and available potassium decrease. Castor has developed root system, planted in the rhizosphere secrete large quantities of organic acids, can secrete the minerals in the soil, microbial growth required to provide mineral elements, to promote the rhizosphere microbial quantity increase, so the soil microbial activity and diversity increased, a variety of bacteria, actinomycetes and the significant increase in the number of functional microorganism.
(3) NJYS-02, a cyanine-producing strain, was screened from Gimhae farm. The strain was discovered to be gram-negative, microaerobic, short rhabditiform, aflagellar, sporeless and decapsulated. The colonial morphology on PDA solid media was blue, rounded raised, lustrous and opaque, with a diameter of1-2mm. The bacteria couldn't grow on nutrient agar, Gao's medium and rose Bengal medium. It could grow and produce cyanine on PDA medium at28℃. The strain could grow at the salinity under8%and produce pigment under4%. By16s rDNA cloning and sequencing, a base sequence of about1430bp was obtained. Blast comparison showed that the strain should belong to pseudomonas sp. in taxonomy.
(4)The most suitable fermentation conditions of the strain NJYS-02are20%potato juice with2.5%glucose solution, pH6.0,28℃for3days. The best extracting method was to combine supersonicbroken method and neutral salt precipitation and use neutral pH ethanol solution to extract for7hours at the room temperature.In this way,we obtained the highest pigment yield. By separating the crude extraction, it is obvious that the cyanine pigment contains two components whose contents differ largely. Cyanine I accounts for about85%while cyanine II accounts for about15%.
(5)The research on the stability of cyanine produced by NJYS-02indicates that the pigment has a relatively strong light resistance and thermotolerance and is stable in sucrose solution of both medium and low concentration. However, reducing agents, sodium citrate and vitamin B1could not damage its structure, but oxidizing agents and preservatives may cause the damage even worse. The cyanine pigment shows no bacterial resistance. The mice study demonstrates that cyanine generated by NJYS-02, is a kind of natural microbial pigment applicable to food, cosmetics, and fodder additives, with a good development prospect and wide applications.
引文
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