短期培养下硝化抑制剂对黑土氨氧化微生物丰度和硝化作用的影响

Nitrification inhibitors affect the abundance of ammonia-oxidizing microorganisms and nitrification in black soil in a short-term microcosm

摘要:

【背景】东北黑土区是我国重要的粮食生产基地，然而由于化肥的过量使用，已造成土壤氮素的大量损失和环境污染问题。硝化作用是土壤氮循环的核心环节，主要由微生物驱动。【目的】利用硝化抑制剂3,4-二甲基吡唑磷酸盐(3,4-dimethylpyrazole phosphate, DMPP)和乙炔区分低氮和高氮黑土壤氨氧化细菌、氨氧化古菌和完全氨氧化菌对土壤硝化作用的相对贡献。【方法】基于黑土长期定位实验研究平台，以不施加氮肥(低氮土壤)和施加氮肥(高氮土壤)的黑土为研究对象，采用微宇宙培养实验和定量PCR技术，分析抑制剂添加对土壤有效态氮、氨氧化微生物丰度的影响及两者的关系。【结果】低氮黑土氨氧化微生物主要由氨氧化古菌和完全氨氧化细菌Clade B占主导，而高氮黑土主要由氨氧化细菌和完全氨氧化细菌Clade A占主导。硝化抑制剂DMPP和乙炔均显著抑制土壤氨的氧化，且在高氮土壤中更为明显，乙炔的抑制效果大于DMPP。另外，添加乙炔显著降低了低氮土壤氨氧化古菌、氨氧化细菌和完全氨氧化菌Clade B的丰度；而在高氮土壤中，硝化抑制剂DMPP则对土壤氨氧化细菌和完全氨氧化菌Clade A有显著抑制作用。相关分析表明，低氮土壤中氨氧化细菌和古菌amoA基因丰度与土壤NO₃⁻-N含量显著正相关和NH₄⁺-N含量显著负相关，而高氮土壤中，土壤氨氧化微生物amoA基因丰度与土壤NH₄⁺-N和NO₃⁻-N含量相关性均不显著。【结论】氮的长期输入显著改变了黑土氨氧化微生物对环境变化的响应。

Abstract:

[Background] The black soil area in northeast China is an important grain production base in China. However, the excessive use of chemical fertilizer has caused serious environmental issues, including nitrogen loss and environmental pollution. Nitrification, one of the key steps in nitrogen cycling, is mainly driven by microorganisms. [Objective] To distinguish the relative contributions of ammonia-oxidizing bacteria, ammonia-oxidizing archaea, and comammox to soil nitrification by using nitrification inhibitors 3,4-dimethylpyrazole phosphate (DMPP) and acetylene in the black soil fields with low and high nitrogen content. [Methods] The soil samples applied with no nitrogen fertilizer (low-nitrogen soil) and nitrogen fertilizer (high-nitrogen soil) were collected from the black soil area in the long-term fertilization experiment. The effects of nitrification inhibitors on soil available nitrogen content, the abundance of ammonia-oxidizing bacteria, and their relationship were investigated by quantitative PCR of soil microcosms. [Results] Ammonia-oxidizing archaea and comammox Clade B predominated in the low-nitrogen soil, while ammonia-oxidizing bacteria and comammox Clade A predominated in the high-nitrogen soil. Both nitrification inhibitors, DMPP and acetylene, significantly inhibited ammonia oxidation, with more pronounced effects in high-nitrogen soil. Acetylene had a greater inhibitory effect than DMPP on ammonia oxidation. In addition, acetylene significantly reduced the abundance of ammonia-oxidizing archaea, ammonia-oxidizing bacteria, and comammox Clade B in low-nitrogen soil, while DMPP significantly inhibited ammonia-oxidizing bacteria and comammox Clade A. The correlation analysis showed that the amoA abundance of ammonia-oxidizing bacteria and archaea was positively correlated with NO₃⁻-N content and negatively correlated with NH₄⁺-N content in low-nitrogen soil. However, the amoA abundance of ammonia-oxidizing microorganisms had no significant correlation with NH₄⁺-N or NO₃⁻-N content in high-nitrogen soil. [Conclusion] The long-term application of nitrogen altered the responses of ammonia-oxidizing microorganisms to environmental changes in black soil.