复合湿地养殖系统中厌氧脱氮菌的菌群生态及其调控因子研究

Anoxic biological nitrogen removal (ABNR) is an important component in the aquatic nitrogen cycle. It is typically used for the removal of nitrogen from wastewater. As a modern freshwater aquaculture technology, the integrated wetland aquaculture system (IWAS) is a cost-effective natural alternative to the conventional fishpond. According to former researches, IWAS was proven to be a promising way to reduce nitrogen load and control nitrate concentration in water. However, there are still many unresolved issues in the mechanisms of the nitrogen cycle in this system, especially the ABNR processes. In this proposal, a suite of bacterial 16S rRNA sequencing, N15 stable isotope tracer method and isotope pairing technique will be combined to explore 3 bacterial clades directly involved in anoxic nitrogen removal in IWAS: the denitrifier, the anammox bacteria and the anaerobic methane oxidizers. The eco-distribution and species composition of these bacterial clades will be revealed. The environmental factors related with ABNR will be tested, such as nitrogen and carbon substrates, oxygen concentration, pH, temperature, hydraulic retention time and so on. Combining with one-factor laboratory experiments, the key factors controlling ABNR bacteria activity will be identified. Furthermore, the key environmental factors will be modified both in IWAS and in a laboratory mimic system. The change of the eco-distribution and species composition of the 3 bacterial clades will be tracked. The data will be analyzed and used to establish a predictive model of anaerobic nitrogen removal bacteria eco-distribution, function and relationship in IWAS. The project will provide a theoretical basis for practical measures to control anoxic nitrogen removal bacteria clades in IWAS and design self-sustainable ecological aquaculture systems.

厌氧脱氮作用是水生态系统氮循环的重要环节，也是水体含氮污染物的主要去除方式。复合湿地养殖系统作为新兴的高效淡水生态养殖模式，较好的解决了传统池塘养殖系统水体氮负荷高，硝酸盐不断累积的问题，但其生物脱氮机理尚不明确。本课题拟采用16S rRNA鉴定技术，分析主要厌氧脱氮菌类群（厌氧反硝化菌、厌氧氨氧化菌和反硝化型甲烷厌氧化菌）在复合湿地养殖系统中的空间分布和菌种构成，分析对应的环境理化条件；采用N15同位素示踪、气态产物测定及同位素配对等技术进行单因子实验，分析氮底物浓度、碳源构成、溶氧、酸碱度、水力停留时间等因子对3类厌氧脱氮菌群活性的影响，分析影响菌群生态的关键理化因子，并建立关键理化因子调控模型；在实际养殖系统和模拟系统中调节关键理化因子，追踪各菌群的变动情况，分析各菌群在系统氮循环中的作用和相互关系，探讨耦合三类厌氧脱氮菌的理化调控方法，为构建高效生态养殖系统提供理论依据。

为阐明复合湿地养殖系统中反硝化机制，采用16SrRNA 等技术，分析了厌氧反硝化菌、厌氧氨氧化菌和反硝化型甲烷厌氧化菌在复合湿地养殖系统中的空间分布和菌种构成，分析了碳源、温度度等因子对厌氧脱氮菌群的影响特征。结果表明，在复合湿地养殖系统中，反硝化细菌的丰度最高，其功能基因丰度范围在3.51×109 copies g-1~2.67×1010 copies g-1之间。不同季节复合湿地养殖系统中厌氧氨氧化菌（ANAMMOX）的丰度范围为8.0×104~9.4×106 copies·g-1，在潜流湿地中ANAMMOX的丰度存在明显的空间差异，但这种差异没有随时间变化的规律性。亚硝酸盐型甲烷厌氧氧化菌（DAMO）丰度较低，所属NC10门，占总细菌的比例为0.027%~0.050%之间。多样性分析表明，养殖池塘、生态塘和生态沟渠沉积物中的优势反硝化细菌属于未知菌类，潜流湿地中优势反硝化菌则为β-和γ-变形菌。ANAMMOX菌的优势种群为类brocadia属和类kuenenia属。在复合湿地系统中，与之密切关联的氨氧化微生物（AOA和AOB）的AOA amoA基因丰度比AOB高1个数量级，表明AOA是池塘沉积物中数量优势氨氧化微生物。但在0~10 cm深的表层沉积物中，AOB amoA mRNA表达水平要比AOA高2.5~39.9倍，表明表层沉积物中AOB是主要的氨氧化功能执行者。AOA和AOB多样性随沉积物加深而降低。AOB主要由亚硝化螺菌（Nitrosospira）和亚硝化单胞菌（Nitrosomonas）组成，后者在沉积物中占主导地位。0~2 cm深沉积物中AOA属于Nitrososphaera菌，而更深沉积物（10~15和20~25 cm）中AOA则属于Nitrosopumilus菌。.在底物影响方面，添加有机碳源能够显著促进反硝化细菌生长，提高沉积物中NO2--N、NO3--N的去除效率；但对于NH4+-N，则是当添加淀粉浓度超过50 mg·L-1，随着淀粉浓度的增加其去除效率降低，额外添加有机碳源可在一定程度上抑制ANAMMOX生长。温度对底泥反硝化菌群有一定的影响作用，当超过28 ℃时随着温度的升高其去除率程不断下降趋势，其去除效率受底物本底浓度值影响。本研究为池塘高效生态养殖水环境调控提供了理论依据。