酸性旱地红壤无机氮同化菌株筛选及其全基因组分析

Isolation and whole genome analysis of bacterial strains assimilating inorganic nitrogen in acidic dryland red soils

摘要:

微生物执行的无机氮同化作用可固定施入土壤后未被作物直接吸收的化学氮肥，有效减少化学氮肥损失、降低环境氮素污染风险。土壤无机氮同化作用不是由大量冗余微生物共同执行的，而是由一小部分功能微生物优先执行。【目的】对酸性旱地红壤中的优势无机氮同化细菌进行富集、菌株分离鉴定及全基因组测序，并明确菌株在土壤中的氮同化能力，为酸性土壤化学氮肥应用及其转化过程研究提供菌株资源和理论依据。【方法】在酸性旱地红壤中添加KNO₃或(NH₄)₂SO₄作为无机氮源，以葡萄糖作为碳源，在好氧条件下进行富集预培养，采用稀释分离法筛选出优势无机氮同化细菌菌株；将菌株回接至土壤中从而验证其无机氮同化能力，并通过全基因组测序分析菌株的氮素代谢途径及相关功能基因。【结果】酸性旱地红壤经富集预培养一周后，优势无机氮同化微生物的16S rRNA基因相对丰度从0.20%-0.94%增长至20.2%-30.2%；分离筛选后得到的3株优势无机氮同化细菌菌株，鉴定为伯克霍尔德氏菌(Burkholderia sp.) M6-3、索状芽孢杆菌(Bacillus funiculus) M2-4和节杆菌(Arthrobacter sp.) M7-15。灭菌土壤中菌株M6-3、M2-4和M7-15的无机氮同化速率分别为(1.28±0.61)、(0.17±0.07)和(0.16±0.02) mg/(kg·d)。氮素代谢通路分析显示，菌株M6-3具备更高效的氮同化代谢通路，其氮同化相关功能基因数量显著高于其他2株细菌。氮素代谢通路与功能活性结果均显示菌株Burkholderia sp. M6-3在酸性旱地红壤无机氮同化过程中占据优势。【结论】本研究证实在酸性旱地红壤无机氮同化过程中低丰度微生物类群发挥重要功能，并从菌株基因组水平上揭示了其无机氮同化代谢过程。上述结果可为酸性旱地红壤农业利用过程中化学氮肥应用及其转化过程研究提供菌株资源和理论依据。

Abstract:

Inorganic nitrogen assimilation performed by microorganisms can immobilize chemical fertilizer nitrogen that is not directly absorbed by crops after application to the soil, which can reduce the losses of chemical nitrogen fertilizer and the risk of environmental nitrogen pollution. Soil inorganic nitrogen assimilation is performed by functional microbial populations rather than a large number of redundant microorganisms. [Objective] The enrichment, isolation, identification, and whole genome sequencing of dominant inorganic nitrogen-assimilating bacteria in acidic dryland red soil and clarification of the nitrogen assimilation capacity of the strains in soil can provide strain resources and a theoretical basis for the application of chemical nitrogen fertilizer in acidic soil and the research on the nitrogen transformation process. [Methods] We added KNO₃ or (NH₄)₂SO₄ as the inorganic nitrogen source and glucose as the carbon source into the acidic dryland red soil. Then, we performed strain enrichment under aerobic conditions and screened the dominant bacterial strains assimilating inorganic nitrogen by the gradient dilution isolation method. We verified the inorganic nitrogen assimilation ability of the strains by soil recolonization experiments, and employed whole genome sequencing to analyze the nitrogen metabolic pathways of different strains. [Results] The relative abundance of 16S rRNA genes of dominant inorganic nitrogen-assimilating microorganisms in acidic dryland red soils increased from 0.20%-0.94% to 20.2%-30.2% after one week of enrichment. We isolated three dominant inorganic nitrogen-assimilating strains, which were identified as Burkholderia sp. M6-3, Bacillus funiculus M2-4, and Arthrobacter sp. M7-15. The inorganic nitrogen assimilation rates of strains M6-3, M2-4, and M7-15 in sterilized soil were (1.28±0.61), (0.17±0.07), and (0.16±0.02) mg/(kg·d), respectively. M6-3 possessed a more complete metabolic pathway and more functional genes related to nitrogen assimilation than the other two strains. In terms of nitrogen metabolic pathways and functional activity, Burkholderia sp. M6-3 was dominant in the assimilation of inorganic nitrogen in acidic dryland red soils. [Conclusion] This study confirmed that low-abundance microbial taxa play a dominant role in the inorganic nitrogen assimilation of acidic dryland red soil, and revealed the metabolic process of inorganic nitrogen assimilation at the genomic level of the strains. The above results provide strain resources and a theoretical basis for the study of chemical nitrogen fertilizer application and transformation process in the acidic dryland red soil.