辽河口沉积物反硝化相关功能基因丰度和多样性分析

发布时间：2018-04-22 11:18

本文选题：氮循环 + 反硝化　；参考：《大连海洋大学》2017年硕士论文

【摘要】：随着工业现代化程度的提高以及沿海地区人口的逐渐增多,大量的生活污水以及工业、农业废水被排入海洋,由此导致了陆源无机氮的过量输入,已成为近海富营养化的主要原因之一,由此引起的环境危害亦呈现出逐年增加的趋势。反硝化过程可以有效地去除河口中的硝酸盐与亚硝酸盐,对水体中多余氮素的去除有着十分积极的意义。为深入了解辽河口的沉积物中反硝化过程与环境因子之间的关系,本研究于2014年5月采集该地区沉积物样品,测定其理化性质(TN、TOC、Sal、NO2-、NO3-、NH4+、ORP和DO);以亚硝酸盐还原酶基因(nirS、nirK)以及一氧化二氮还原酶基因(nosZ)作为分子标记,采用实时定量PCR和illumina Miseq高通量测序技术测定了采集沉积物中亚硝酸盐还原酶基因和一氧化二氮还原酶基因的丰度和nirK、nirS和nosZ 3种基因型反硝化细菌菌群多样性;分别分析了这三种功能基因的丰度与TN、TOC、Sal、NO2-、NO3-、NH4+、ORP和DO的相关性,同时分析了3种基因型反硝化细菌与上述理化因子的相关性。结果表明:从岸向海方向,盐度以及溶解氧逐渐升高,无机氮营养盐(硝酸盐、亚硝酸盐和铵盐)的浓度以及总有机碳的含量沿河口向海方向整体呈现下降的趋势;辽河口沉积物中nirS型基因的丰度为5.61×105copies/g~3.67×107copies/g,nirK的基因丰度为1.47×105copies/g~5.61×106copies/g,nosZ的基因丰度为1.44×103 copies/g~1.37×106copies/g。不同功能基因丰度受到不同环境理化因子的影响,其中nirK基因丰度与盐度及其他环境因素无明显的相关性;nirS基因的丰度与盐度成显著负相关(P=0.008,r=0.924),与NO2-呈显著正相关(P=0.007,r=0.933),与TOC也呈显著正相关的关系(P=0.007,r=0.93),与其他理化因子无显著相关性。nosZ基因的丰度与盐度呈显著负相关(P=0.044,r=-0.823),而与NO2-呈显著正相关(p=0.013,r=0.905),与TOC也呈显著正相关关系(P=0.031,r=0.852)与其他理化因子无显著相关性;nirK基因型反硝化菌的属水平样品聚类树与柱状图组合分析得到其优势属为中华根瘤菌属(Sinorhizobium),所占比例达到了23.55%,与根瘤菌属含量以及NO3-浓度显著负相关(P=0.019,r=-0.885,P=0.09,r=-0.921),推测中华根瘤菌属多样性的影响因子可能为NO3-。对nirS基因型反硝化菌属水平样品聚类树与柱状图组合分析得到其优势属为假单胞菌属(Pseudomonas),所占比例达到了19.64%,其与慢生根瘤菌属呈现显著负相关性(P=0.000,r=-0.985)。nosZ基因反硝化菌属水平样品聚类树与柱状图组合分析图得到其优势属为固氮螺菌属(Azospirillum)所占比例达25.69%,为主要优势菌;nirK、nir S以及nosZ基因型反硝化细菌的主要环境因子分别为NH4+和NO3-、Sal和NH4+、Sal和NO2-。nirK的OUT数量同环境因子并无显著相关性,而nirS的OUT数量与ORP、DO和NH4+呈显著相关性(P0.05),说明nirS在环境梯度下的变化比nirK更明显。而nosZ的多样性则与pH、ORP以及DO呈显著相关性。结果表明:1.辽河口由陆向海方向,盐度、无机氮营养盐和有机碳的含量整体呈现下降趋势。2.不同功能基因丰度受到不同环境理化因子的影响。3.辽河口的反硝化细菌由陆向海方向,含有nirK、nir S以及nosZ基因的反硝化细菌的多样性逐渐降低。4.NH4+、NO3-、NO2-以及盐度与含有这三种功能基因的细菌的关系最为密切。
[Abstract]:With the improvement of industrial modernization and the increasing population of coastal areas, a large number of domestic sewage and industrial wastewater have been discharged into the ocean, resulting in excessive input of inorganic nitrogen from land source, which has become one of the main causes of eutrophication in the coastal areas, and the resulting environmental hazards are also increasing year by year. Nitrification process can effectively remove nitrate and nitrite in estuaries. It is of great significance to remove the excess nitrogen in the water body. In order to understand the relationship between the denitrification process and environmental factors in the sediments of the Liaohe estuary, this study collected the sediment samples in this area in May 2014 and measured its physical and chemical properties (TN, TOC,) Sal, NO2-, NO3-, NH4+, ORP and DO), using the nitrite reductase gene (nirS, nirK) and the two nitrogen reductase gene (nosZ) as molecular markers, the abundance and the abundance of the nitrite reductase gene and the two N reductase gene in the sediments were measured by real-time quantitative PCR and Illumina Miseq high throughput sequencing technology. The diversity of 3 genotypes of denitrifying bacteria in nosZ, and the correlation between the abundances of the three functional genes and the TN, TOC, Sal, NO2-, NO3-, NH4+, ORP and DO were analyzed respectively. The correlation between the 3 genotypic denitrifying bacteria and the above physicochemical factors was analyzed. The results showed that the salinity and the dissolved oxygen increased gradually from the shore to the sea, and the inorganic nitrogen was gradually increased. The concentration of nutrients (nitrate, nitrite and ammonium salt) and the total organic carbon content declined along the direction of the estuary to the sea. The abundance of nirS gene in the Liaohe deposit was 5.61 x 105copies/g~3.67 x 107copies/g, the gene abundance of nirK was 1.47 x 105copies/g~5.61 * 106copies/g, and the gene abundance of nosZ was 1.44 * 103. The abundance of different functional genes in copies/g~1.37 x 106copies/g. was affected by different environmental physical and chemical factors, and there was no significant correlation between the abundance of nirK gene and salinity and other environmental factors, and the abundance of nirS gene was negatively correlated with salinity (P=0.008, r=0.924), and was positively correlated with NO2- (P=0.007, r=0.933), and was also positively correlated with TOC. P=0.007 (r=0.93) and no significant correlation with other physical and chemical factors, the abundance of.NosZ genes was negatively correlated with salinity (P=0.044, r=-0.823), but positively correlated with NO2- (p=0.013, r=0.905), and there was a significant positive correlation with TOC (P=0.031, r=0.852) without significant correlation with other physical and chemical factors; the genera of the nirK genotypic denitrifying bacteria. The combination of the horizontal sample cluster tree and the histogram analysis obtained that the dominant genus was Sinorhizobium, the proportion of which was 23.55%, which was negatively correlated with the content of rhizobia and NO3- (P=0.019, r=-0.885, P=0.09, r=-0.921). The influence factor of the diversity of Rhizobium in China may be NO3-. against nirS genotypes. The dominant genus is Pseudomonas (Pseudomonas), which has a dominant genus of 19.64%. It has a significant negative correlation with the genus chronic rhizobia (P=0.000, r=-0.985).NosZ gene denitrifying bacteria in the level of the group tree and histogram analysis map to obtain the advantage of nitrogen fixation. The proportion of Azospirillum is 25.69%, which is the dominant dominant bacteria. The main environmental factors of nirK, NIR S and nosZ genotype denitrifying bacteria are NH4+ and NO3- respectively. Sal and NH4+, Sal and NO2-.nirK OUT quantities have no significant correlation with environmental factors. The variation of the environmental gradient is more obvious than that of nirK, while the diversity of nosZ has a significant correlation with pH, ORP and DO. The results show that 1. Liaohe estuary is from land to sea, salinity, inorganic nitrogen, and organic carbon content as a whole is decreasing,.2. of different functional genes abundances are affected by different environmental physical and chemical factors in the.3. Liaokou estuary. The diversity of denitrifying bacteria containing nirK, NIR S and nosZ genes gradually decreased.4.NH4+, NO3-, NO2-, and salinity were most closely related to the bacteria containing these three functional genes.

【学位授予单位】：大连海洋大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：X52;Q933

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