The microbial mechanism of denitrification associated with nitrification in paddy soils

水稻是世界上重要的粮食作物之一，水稻土不仅在农业生产中具有重要地 位，其生态效应也越来越受到关注。随着粮食需求的不断增长，大量氮肥的投 入，不仅降低了稻田生态系统的生产力，同时由硝化和反硝化作用造成的氮肥 利用效率低、土壤硝态氮淋失和温室气体 N2O释放等环境问题。因此，本研究 以水稻土为研究对象，通过微宇宙模拟培养，结合15N同位素标记技术和硝化 抑制剂乙炔添加，以及功能基因转录表达分析，研究了不同干湿条件下土壤硝 化、反硝化作用及其功能微生物的变化特征，以揭示土壤硝化/反硝化过程相互 作用发生的环境条件及其中的微生物学机理，阐明二者相互作用对 N2O产生的 贡献，为土壤氮素管理和温室气体排放预测等提供参考。取得研究结果如下： （1）在不同水分条件下对滨海盐渍土（BH）、哈尔滨黑土（HEB）和桃源 红壤（TY）三种类型的水稻土培养结果显示 BH土壤和 HEB土壤具有较高的 硝化活性，但这两种土壤中 N2O产生速率均较低。而在 TY土壤中，硝化作用 在 60%WHC和 90%WHC条件下均明显发生，以 90%WHC处理下最强烈，同 时，在 90%WHC处理条件下，TY土壤 N2O释放，显著高于 60%WHC处理和 淹水处理，以及其他两种土壤，推测该水分条件可能导致了硝化/反硝化过程相 互作用的发生。 （2）基于以上结果，利用15N同位素标记技术对桃源红壤水稻土进行了深入研究，在 55%和 90%WHC两个水分条件下，利用未标记的和NH4NO3分别对土壤进行培养，并设置相应的加 0.2%C2H2抑制剂处理以抑制硝化作用，结果发现培养第 1天 90%WHC处理土壤 （90%-15 N）中 N2O释放量最高，显著高于同等含水量条件下加 C2H2抑制剂处理（90%-C2H2）和 55%WHC处理土壤（55%- 15N）和 7天后，表明反硝化作用是 N2O释放的主要来源；15N同位素计算结果由硝化作用驱动的反硝化作用占整个反硝化作用的贡献为86.0%，表明 90%WHC- 15NH4+处理土壤发生了硝化/反硝化过程相互作用，即该水分条件下硝化作用驱动反硝化作用，导致 N2O大量产生。 3）在 DNA水平上，氨氧化细菌（AOB）amoA基因在加 C2H2抑制剂的处理（90%-C2H2）中显著低于无抑制剂处理土壤，古菌（AOA）amoA基因和反硝化微生物 nirK和 nosZ基因在不同处理间无明显差异，但在 RNA水平上均表现出明显差异。AOA和 AOB的 amoA基因表达被迅速激活，且在 55%WHC处理中最高，随时间延长表达活性降低，乙炔添加显著抑制了 amoA基因的表达。在 90%WHC处理下，AOB amoA基因表达在第 1天显著高于第 7天，与观察到的 N2O释放一致，而 AOA amoA基因表达则相反。相似地，反硝化微 生物 nirK基因的转录活性也在第 1天被快速激活，且在不加 C2H2的处理中显著高于加 C2H2处理。此外，在加入底物后 1天，nosZ基因表达则在 90%WHC处理最高，第 7天后显著降低，表明反硝化过程被迅速启动，与 N2O产生的趋势一致。 （4）对 amoA, nirK和 nosZ基因的 cDNA克隆测序分析表明，90%WHC 和 55%WHC处理土壤中 AOA amoA基因以 Soil SedimentⅡ簇为主导，AOB amoA基因以 Nitrosospira C12簇为主导，且 Nitrosospira C12簇在 90%WHC处 理中占绝对优势，是该水分条件下硝化作用的主要驱动者；nirK基因的 cDNA 序列主要为 Bradyrhizobium，Mesorhizobium sp.和 Nitrosospira tenuis strain三个 类群所代表，且以 Nitrosospira tenuis strain占主导；nosZ基因的 cDNA序列主 要为 Bradyrhizobium，Shinella，Castellaniella，Uncultured bacterium和 Azoarcus 五个类群所代表，且以 Bradyrhizobium占主导。Rice is one of the most important crops in the world. Paddy soil not only plays an important role in agricultural production, but also gets more and more attention with the respect to its ecological effect. Increasing demands of crops and vast nitrogen fertilizer addition not only declined the productivity of paddy soil ecosystem, but also caused environmental issues like low use efficiency of fertilizer, soil nitrate leaching and N2O emission due to nitrification and denitrification occurred. Therefore, in this research Hunan Taoyuan red soil paddy soil was collected as our research object, through microcosm incubation, combined with 15N isotope labeling technique and nitrification inhibitor acetylene addition, and the transcription of functional gene transcription and expression analysis, variation characteristics of soil nitrification, denitrification and their relative functional microbial effects of different wet and dry conditions were studied, so that we could reveal the soil condition occurred nitrification coupling of denitrification and its microbial mechanism, explained the contribution of N2O emission and provide scientific reference for the management of soil nitrogen and greenhouse gas emission prediction. The results are as follows: 1. Results of incubation of Binhai (BH) coastal saline soil, Harbin (HEB) black soil and Taoyuan (TY) red soil showed that nitrification of BH and HEB soil were strong with low N2O emission rate, whereas in TY soil, nitrificatioin occurred significantly in 60%WHC and 90%WHC treatments, in particular, nitrification in 90%WHC was most intense. In addition, obvious N2O emission rate was found in 90%WHC treatment than that of 60%WHC treatment, indicating that denitrification associated with nitrification could happen in 90%WHC condition. 2. Based on results above, N isotope labeling technology was used to deeply study TY red soil in 55%WHC and 90%WHC condition combined with the addition of 0.2%C2H2. Results showed that N2O emission rate in 90%WHC was the highest and significantly higher than that of 90%-C2H2 and 55%-N treatments, suggested that denitrification was the main source of N2O emission; Besides, 15N-isotope calculation results indicated that denitrification driven by nitrification accounted for 86% in total denitrification, suggesting that nitrification was the main reason driving denitrification occurs in the soil. 3. DNA PCR results showed that the abundance of ammonia oxidizing bacteria (AOB) amoA gene in the treatment of 90%-C2H2 was significantly lower than that of treatment without inhibitor addition. There was significant difference between treatments with respenct to the abundance of ammonia oxidizing archaea (AOA) amoA gene and denitrifying bacteria nirK and nosZ gene in RNA PCR results rather than DNA PCR results. In particular, the amoA gene expression of AOA and AOB was highest in the 55%WHC treatment after 8 hours of substrate addition, and decreased significantly after seventh days incubation, but the abundance in its corresponding C2H2 treatment was hardly detected. In 90%WHC treatment high amoA gene expression was also detected, but the expression of AOB amoA gene was significantly decreased after day 7, while the expression of AOA amoA gene increased. All these results indicated that AOA and AOB amoA gene expression was started quickly after the addition of isotopic substrate, which was consistent with nitrification activity observed. Similarly, the transcriptional activity of denitrifiers’ nirK gene was also rapidly activated in day 1 and was significantly higher than that of in its corresponding C2H2 treatments. In addition, the nosZ gene expression was highest in the 90%WHC treatment and decreased significantly after day 7 after 8 hours of substrate addition, indicating that the denitrification process was rapidly initiated, which was consistent with the trend of N2O production. 4. Results of clone sequencing demonstrated that Soil Sediment II cluster was dominant in AOA community in both 55%WHC and 90%WHC treatments. In AOB community, Nitrosospira C12 cluster was dominant and the only group in 90%WHC, suggested that Nitrosospira C12 cluster drove nitrification in 90%WHC condition; nirK genes sequences were mainly distributed in clusters of Bradyrhizobium, Mesorhizobium sp. and Nitrosospira tenuis strain, in which Nitrosospira tenuis strain cluster was dominant in both 90%WHC and 55%WHC treatments. Sequences of nosZ gene were distributed in five clusters, including Bradyrhizobium, Shinella, Castellaniella, Uncultured bacterium and Azoarcus, in particular, Bradyrhizobium cluster predominated in 90%WHC treatment, 90%WHC-C2H2 and 55%WHC treatment