硫化物抑制潮土反硝化过程中氧化亚氮还原的菌群机制

Microbial mechanism of sulfide inhibiting N2O reduction during denitrification in fluvo-aquic soil

摘要:

【背景】土壤中的反硝化作用形成气态产物N2O和N2，会导致氮素的气态损失，并造成温室效应。硫化物对土壤的N2O还原具有抑制作用，但其对菌群和功能基因的影响机制还不清楚。【目的】研究有无外加碳源情况下，硫化物对反硝化作用中间产物(NO、N2O)的积累、反硝化功能基因(narG、nirS、nirK和nosZ)表达量以及菌群结构的影响。【方法】分别设置不同量葡萄糖(0和1 000 mg-C/kg干重土壤)和硫化钠(0和150 mg-S/kg干重土壤)添加的交叉处理，进行室内微宇宙培养实验，利用自动化培养与实时气体检测系统检测培养过程中NO、N2O和N2的积累量，通过反转录定量PCR测定反硝化功能基因表达量，利用Miseq技术平台基于16S rRNA基因序列的高通量测序分析样品的菌群结构。【结果】硫化钠的添加显著抑制N2O还原，但是其对于N2O积累量没有显著影响，却显著降低了NO的积累量。硫化钠的添加短时间内在转录水平上显著抑制N2O还原酶的活性，并且抑制固氮弧菌属(Azoarcus)、微枝形杆菌属(Microvirga)、剑菌属(Ensifer)、氮氢单胞菌属(Azohydromonas)、芽孢杆菌属(Bacillus)、斯科曼氏球菌属(Skermanella)、申氏杆菌属(Shinella)和西索恩氏菌属(Chthoniobacter)的基因转录，降低它们的转录本丰度，结合Kyoto Encyclopedia of Genes and Genomes (KEGG)数据库的查询结果，发现硫化钠的添加抑制了不产生N2O的N2O还原反硝化细菌的生长。【结论】堆肥或其他原因引起的土壤硫化物增加，导致反硝化过程N2O还原被抑制的原因是由于其对氧化亚氮基因转录的抑制和对不同反硝化菌的选择作用，研究结果有助于认识硫化物对氮代谢影响的微生物机制。

Abstract:

[Background] The products of denitrification are N2O and N2, thus it causes nitrogen loss and increases greenhouse effect. Sulfide has a significant inhibitory effect on N2O reduction, however, the effect of sulfide on denitrifying bacteria and functional genes of denitrification are unclear. [Objective] To study the effect of sulfide on the accumulation of intermediate products of denitrification (NO and N2O), transcription of functional gene of denitrification (narG, nirS, nirK and nosZ) as well as the structure of the microbial community structure in soil amended with or without carbon source. [Methods] Different levels of sodium sulfide (0 and 150 mg-S/kg-soil) combined different levels of glucose (0 and 1 000 mg-C/kg-soil) were set up in soil microcosms experiment. Robotized incubation system was used to monitor the amount of NO, N2O and N2 accumulated during the incubation, and quantitative reverse transcription PCR (RT-qPCR) was used to quantify the transcriptions of functional gene of denitrification (narG, nirS, nirK and nosZ) as well as the MiSeq technology platform based on 16S rRNA gene high-throughput sequencing was used to analyze microbial community structure. [Results] The addition of sodium sulfide significant inhibited the reduction of N2O, but it had no significant effect on the accumulated N2O. And the addition of sodium sulfide significantly reduced the accumulated NO. Sodium sulfide addition significantly inhibited the activity of N2O reductase at a transcription level in a short time. Sodium sulfide addition inhibited the transcription of Azoarcus, Microvirga, Ensifer, Azohydromonas, Bacillus, Skermanella, Shinella, and Chthoniobacte. According to the query results of the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, the addition of sodium sulfide inhibited the growth of N2O reducing denitrifying bacteria that cannot produce N2O. [Conclusion] The increase of soil sulfide caused by compost or other reasons leads to the inhibition of N2O reduction in denitrification process, which is due to the inhibition of nitrous oxide gene transcription and the selection impact of denitrifying bacteria. This study would be helpful to understand the microbial mechanism of the impact of sulfide on nitrogen metabolism.