固氮红细菌对模拟水体中无机三态氮去除特性的研究

发布时间：2018-07-25 18:50

【摘要】：目前,海水养殖水体污染问题日益严重,其中氮污染是影响海水养殖业发展的首要问题,因此消除养殖水体氮污染、恢复微生态平衡,是解决养殖水体污染问题的关键。不产氧光合细菌(Anoxygenic phototrophic bacteria,APB)已在去除水体中的无机三态氮,恢复水体微生态平衡及提高养殖动物免疫力等方面显示出重要作用。然而,APB处理的实际水体环境复杂,探求复杂水体环境中APB的除氮效果,具有重要的实际应用价值。目前,关于APB去除亚硝氮的能力、以及对养殖水体中三氮的利用和转化研究报道较少。APB的实际应用中会受到水体中多种小分子有机氮源、碳源及盐度等的影响。针对以上问题,本文以固氮红细菌(Rhodobacter azotoformans)R7为材料,首先通过条件优化提高生物量,为其应用奠定基础;在此基础上,进一步探究了该菌株对三种无机态氮的去除特性、对无机三态氮的利用与转化能力以及无机三态氮对菌株生长和光合色素合成的影响;为了提高菌株的实际应用效果,又探究了复杂的水体环境因素,包括无机和有机氮化合物、有机碳化合物和盐度对菌株生长以及除氮效果的影响。主要结果如下:通过单因子轮换试验法(One-factor-at-a-time,OFAT)优化获得提高菌体生物量及Car产量的最适条件为:装样量为80%、3 g/L酵母粉、5 g/L蛋白胨、2.5 g/L葡萄糖。在最适条件下,菌体生物量由3.34 g/L提高到17.59 g/L,提高了4.27倍;菌体类胡萝卜素含量由优化前2.43 mg/L提高到30.31 mg/L,提高了11.47倍,主要积累球形烯、羟基球形烯及球形烯酮3种类胡萝卜素。Car光稳定性研究表明,含酮基的球形烯酮稳定性较高,在0 lux、500 lux及3000 lux光照条件下其半衰期依次为:568.53 h、405.56 h及112.64 h。R7菌株对无机三态氮的去除与转化结果显示,R7菌株能利用氨氮、硝氮及亚硝态氮良好的生长,对硝氮和亚硝氮具有较强的耐受和去除能力。培养8天内,R7菌株对氨氮、硝氮和亚硝氮最大去除量分别为:6.10 mmol/L、21.36mmol/L和16.86 mmol/L,最大耐受浓度分别为43.47 mmol/L、52.17 mmol/L和34.78 mmol/L;在4.54 mmol/L氨氮、34.35 mmol/L硝氮和34.64 mmol/L亚硝氮共存的体系中,该菌株对氨氮、硝氮及亚硝氮的去除率分别为54.38%、31.48%和99.95%;对无机三态氮的利用和转化的结果分析显示,分别以氨氮、硝氮和亚硝氮为唯一氮源培养的过程中,均出现了另外两种无机氮的暂时性积累,随着培养时间的延长,逐渐被去除。上述结果表明,R7菌株存在多种氮代谢作用,具有反硝化作用、厌氧硝化作用和亚硝酸盐厌氧氧化作用。氨氮、硝氮及亚硝氮对菌株生长和菌体光合色素合成的影响,结果表明:水体中氨氮(≤43.48 mmol/L)或硝氮(≤52.17 mmol/L)对菌体生物量及Car含量影响不明显,低浓度氨氮(≤8.70 mmol/L)或硝氮(≤8.66 mmol/L)对菌株生长具有促进作用;上述结果表明,水体中氨氮和硝氮对菌株色素含量的影响不明显,水体中亚硝氮浓度越高,对R7菌体色素的合成抑制越明显。环境中可能存在的小分子碳(糖类、有机酸或醇)、有机氮(酵母浸出粉、蛋白胨或尿素)和盐度影响菌株对无机三态氮的去除,结果表明:环境中的不同碳源对R7去除硝氮和氨氮的影响较大;丙酸钠能够提高菌株去除无机三态氮的能力,去除率均在87%以上;含丙酸钠或乙酸钠的环境中,利于菌株对氨氮和亚硝氮的去除,去除率分别在85%及98%以上;含蔗糖、丙酸钠或甘露醇的环境中,利于菌株对硝氮的去除,去除率均在80%以上;酵母粉(≤1.43mmol/L)的环境中,菌株对氨氮、硝氮和亚硝氮的最大去除率分别为97.61%、99.50%及99.08%;蛋白胨(≤3.21 mmol/L)的环境中,菌株对氨氮、硝氮和亚硝氮的最大去除率分别为90.57%、94.36%及99.54%;尿素(≤0.71 mmol/L)环境中,菌株对氨氮、硝氮和亚硝氮的最大去除率分别为98.26%、50.68%及99.69%;不同盐浓度(≤2 g/L)环境中,氨氮、硝氮及亚硝氮的最大除率分别为55.03%、96.62%及83.53%以上。R7对实际养殖水体中三态氮的去除效果结果表明,微好氧条件下该菌株能够有效地去除复杂的自然养殖水体中的三态氮。综上所述,经过条件优化,R7菌体生物量明显增加,且积累抗氧化性强的含酮基的类胡萝卜素,为该菌株在养殖水体中的实际应用奠定了基础。R7菌株对氨氮、硝氮及亚硝氮均具有较强的去除能力,对高浓度无机三态氮共存水体中的三态氮也具有较高去除及耐受能力。在含有小分子有机糖类、有机酸或醇、有机氮和不同盐度等复杂水体环境中,该菌株适应性强,能够去除水体中的无机三态氮。R7菌株的营养功能及去除无机三态氮的高效性具有潜在应用价值,为进一步开发环境适应能力强、针对性强的新型高效APB微生物生态制剂提供了理论参考。
[Abstract]:At present, the pollution problem of aquaculture water body is becoming more and more serious, and nitrogen pollution is the primary problem affecting the development of marine aquaculture. Therefore, eliminating nitrogen pollution in aquaculture water and restoring microecological balance are the key to solve the problem of aquaculture water pollution. Anoxygenic phototrophic bacteria (APB) has been removed from the water of water. Three state nitrogen, restoring the microecological balance of water body and improving the immunity of cultured animals, showed an important role. However, the actual water environment of the APB treatment was complex. It was of great practical value to explore the nitrogen removal effect of APB in the complex water environment. At present, the ability of APB to remove nitrite and the benefit of three nitrogen in the aquaculture water. The application and transformation of the research reports that less.APB will be affected by a variety of organic nitrogen sources, carbon sources and salinity in the water body. Aiming at the above problems, this paper uses the nitrogen fixing red bacteria (Rhodobacter azotoformans) R7 as the material to optimize the biomass by optimizing the conditions and lay the foundation for its application. The effects of the strain on the removal of three inorganic nitrogen, the utilization and conversion of inorganic three state nitrogen and the effect of inorganic three nitrogen on the growth and synthesis of photosynthetic pigments were investigated. In order to improve the practical application effect of the strain, the complex water environmental factors were also explored, including inorganic and organic nitrogen compounds, organic carbon compounds and the organic nitrogen compounds. The main results were as follows: the optimum conditions for improving the biomass and Car production by single factor rotation test (One-factor-at-a-time, OFAT) were 80%, 3 g/L yeast, 5 g/L peptone, 2.5 g/L glucose. Under the optimum conditions, the biomass of the bacteria was 3.34 g/L The increase to 17.59 g/L, increased by 4.27 times, the content of carotenoids increased from 2.43 mg/L to 30.31 mg/L, increased 11.47 times, and the main accumulation of globose, hydroxyl and spherical carotene.Car photostability showed that the stability of ketone based spherical ketones was higher, 0 lux, 500 lux and 3000 lux illumination strips. The removal and transformation of 568.53 h, 405.56 h and 112.64 h.R7 strains to inorganic three nitrogen show that R7 strain can grow well with ammonia nitrogen, nitrate nitrogen and nitrite nitrogen, and have strong tolerance and removal ability to nitrate nitrogen and nitrite nitrogen. The maximum removal of ammonia nitrogen, nitrate nitrogen and nitrite nitrogen by R7 strain in 8 days The maximum tolerable concentrations of 6.10 mmol/L, 21.36mmol/L and 16.86 mmol/L were 43.47 mmol/L, 52.17 mmol/L and 34.78 mmol/L, respectively. In the system of 4.54 mmol/L ammonia nitrogen, 34.35 mmol/L nitrate nitrogen and 34.64 mmol/L nitrite nitrogen, the removal rate of ammonia nitrogen, nitrate nitrogen and nitrite nitrogen were 54.38%, 31.48% and 99.95%, respectively, and inorganic three nitrogen nitrogen, respectively. The results of utilization and transformation showed that in the process of cultivation of ammonia nitrogen, nitrate nitrogen and nitrite as the sole nitrogen source, two other kinds of inorganic nitrogen were accumulated temporarily and gradually removed with the prolongation of the incubation time. The results showed that the R7 strain had a variety of nitrogen metabolism, denitrification and anaerobic nitrification. The effect of ammonia nitrogen, nitrite and nitrite on the growth and photosynthetic pigment synthesis of strains showed that ammonia nitrogen (less than 43.48 mmol/L) or nitrate nitrogen (less than 52.17 mmol/L) had no obvious effect on the biomass and Car content in the water, and the low concentration of ammonia nitrogen (less than 8.70 mmol/L) or nitrate nitrogen (less than 8.66 mmol/L) grew to the strain growth. The results showed that the effect of ammonia nitrogen and nitrate nitrogen on the pigment content of the strain was not obvious. The higher the concentration of nitrite nitrogen in the water body, the more obvious inhibition of the synthesis of R7 bacterial pigment. The possible small molecular carbon (saccharide, organic acid or alcohol), organic nitrogen (yeast extract, peptone or urea) and salinity affected bacteria in the environment The removal of inorganic three state nitrogen shows that the different carbon sources in the environment have great influence on the removal of nitrogen and ammonia nitrogen by R7. Sodium propionate can improve the ability of removing three nitrogen of inorganic nitrogen by the strain, and the removal rate is above 87%. In the environment containing sodium propionate or sodium acetate, the removal rate of ammonia and nitrite is 85% and 98, respectively. In the environment containing sucrose, sodium propionate or mannitol, the removal rate of nitrite was more than 80%, and the maximum removal rate of ammonia nitrogen, nitrite and nitrite was 97.61%, 99.50% and 99.08% in the yeast (less than 1.43mmol/L) environment, and the strain to ammonia nitrogen, nitrite and nitrite in the environment of egg white peptone (less than 3.21 mmol/L) The maximum removal rates of nitrogen were 90.57%, 94.36% and 99.54%, respectively, and the maximum removal rates of ammonia nitrogen, nitrite and nitrite were 98.26%, 50.68% and 99.69% in urea (less than 0.71 mmol/L) environment, and the maximum removal rate of ammonia nitrogen, nitrite and nitrite in different salt concentrations (less than 2 g/L) was 55.03%, 96.62% and 83.53%.R7 were used for actual cultivation. The results of the removal of three state nitrogen in water show that the strain can effectively remove three states of nitrogen in the complex natural aquaculture water under the condition of microaerobic. In summary, the biomass of R7 bacteria increases obviously and accumulates the ketone like Hu Luo, which has strong antioxidant activity, and is the practical application of the strain in the aquaculture water. The base.R7 strain has strong removal ability to ammonia nitrogen, nitrate nitrogen and nitrite nitrogen. It also has high removal and tolerance to three state nitrogen in high concentration inorganic three state nitrogen. It has strong adaptability in the complex water environment containing small molecular organic sugar, organic acid or alcohol, organic nitrogen and different salinity. It is of potential application value to remove the nutritional function of the inorganic three state nitrogen.R7 strain in the water and the efficiency of removing the inorganic three nitrogen nitrogen. It provides a theoretical reference for the further development of the environmental adaptation ability of the new high efficiency APB microorganism ecologic agent.
【学位授予单位】：华侨大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：X52;X714

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