农村型黑臭水体形成的硫循环关键功能微生物及其与碳、氮、铁循环功能微生物间的互作关系研究

Black and odorous water (BOW) has a severe impact on daily life, especially in developing countries like China. BOW in the regions polluted by human urban activities (RU) is characterized with high levels of COD and ammonia, while BOW in the regions polluted by human agricultural activities (RA) is characterized with high levels of sulfate introduced from chemical fertilizer. Previous study showed that the abundance of sulfur cycling genes (dsrB and soxB) were significantly elevated in RU, and the ratio of abundance of dsrB to soxB was significantly elevated in RA compared with those in the protected regions (RP). These results indicated that the formation mechanisms of BOW differed between RU and RA. Meanwhile, the interactions between sulfur cycling microorganisms and the functional microorganisms involved in carbon and metal cycling was more complex in RA compared with that in RP. However, how the sulfur cycling microorganisms are influence by high levels of sulfate? Who are the key sulfur cycling microorganisms for the formation of BOW in RA? How the sulfur cycling microorganisms interacted with the functional microorganisms involved in carbon nitrogen and iron cycling and the coupling mechanisms of community and function? Based on these scientific issues, this program intended to screen the key sulfur cycling microorganisms and classify the interactions between sulfur cycling microorganisms and the functional microorganisms involved in carbon, nitrogen and iron cycling in BOW in RA by using molecular biology methods, such as DNA/RNA-SIP, metagenome sequencing and qPCR. This program will help clarify the mechanisms of the formation of hydrosphere microbial community and the interactions with environmental factors.

有别于城市黑臭水体的高COD、氨氮等特点，农村黑臭水体增加了由化肥施用引起的高硫酸盐显著特征。前期研究表明，城市型黑臭水体形成是以硫循环功能菌群丰度的增加为核心；而农村型黑臭水体形成是以硫循环功能菌群相对丰度的改变为核心，且农村型黑臭水体中微生物间的互作关系更为复杂。硫酸根离子对农村型黑臭水体中硫循环微生物产生怎样的影响？在农村黑臭水体中发挥作用的硫循环功能微生物关键物种是谁？硫循环与C、N、Fe循环功能微生物间的群落-功能耦合及互作关系如何？基于以上科学问题，本研究将以海河流域典型农村河流系统为研究目标，基于DNA/RNA-SIP、高通量测序、qPCR等分子生物学技术手段，靶定硫循环关键功能微生物种群，探明高浓度硫酸根离子介导的硫循环功能微生物菌群变化及其与C、N、Fe等元素循环功能微生物间的互作关系。本项目的科学意义是为水圈微生物群落形成及其与环境互作的机理提供一定的理论基础。

近年来，因大量有机物、氮、硫、磷、铁等的积累而形成的黑臭水体，成为我国现阶段最重要的环境治理攻坚目标之一。到目前为止，城市黑臭水已初步得到控制，但农村地区的水污染问题依然突出。为此，国家出台了推进污染控制主战场空间格局转移的政策，旨在解决农业污染问题，实施农村农业污染控制。.农村型黑臭水体的一个显著特征是硫酸盐浓度高，通过对两条典型农村型黑臭水体底泥的微宇宙培养实验及稳定同位素探针（DNA-Stable isotope probe，DNA-SIP）研究，我们初步揭示了黑臭水体沉积物中AOM的驱动机制及功能微生物类群。结果表明，沉积物中存在多种电子受体偶联的厌氧甲烷氧化（anaerobic oxidation of methane，AOM）过程，尤其是很少在淡水水体中发现的硫酸盐还原偶联的AOM过程，并发现了进化上有别于海洋沉积物中的ANME-3的淡水硫酸盐还原型ANME-3（OTU-368）。通过600天5代的富集培养，我们利用宏基因组测序技术获得了高效拼接的MAGs 28个，其中ANME-3共8个，将进一步解析淡水ANME与海洋种属的基因组和代谢差异。.农村黑臭水体的第二个特征是氨氮和化学需氧量（COD）浓度高，我们开发了基于游离亚硝酸盐(Free nitrous acid，FNA)强化的狐尾藻人工湿地技术，通过对硝化和反硝化过程的调控，显著提高了养猪废水中COD、铵态氮和硫化物的总去除效率，分别为81.2%、72.8%和92.8%。我们进一步对该技术的微生物调控过程进行了解析，结果表明该技术增强了碳氮硫循环功能微生物之间偶联关系，富集了反硝化基因（nirK、nosZ）、硫氧化基因（soxB、fccAB）和相应的微生物类群（Bacillus, Conexibacter和Clostridium sensu stricto）。.本项目的科学意义是为理解水圈微生物群落形成及其与环境互作的机理提供一定的理论基础，为农村型黑臭水体中碳硫偶联过程及其生物阻控技术的发展提供理论依据。

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