植物叶际好氧反硝化细菌的筛选及其脱氮性能研究

Aerobic denitrifying bacteria in the phyllosphere: screening and characterizing of nitrogen removal performance

摘要:

【目的】探索叶际微生物协同植物削减大气氮氧化物的机制，了解叶际可培养好氧反硝化细菌的存在及多样性，获得高效的叶际好氧反硝化细菌资源。【方法】采用富集培养结合格里斯试剂检测、溴百里酚蓝(bromothymol blue, BTB)培养基筛选的方法从景观植物叶际分离筛选好氧反硝化细菌，对好氧反硝化细菌的16S rRNA基因序列进行系统发育分析，并选取其中一株高效好氧反硝化细菌进行脱氮性能研究。【结果】从6种景观植物石楠、女贞、木樨、樟树、卫矛冬青、荷花玉兰的叶际中分离到好氧反硝化细菌13株，经16S rRNA基因序列分析发现，13株细菌分别属于4门7科7属，其中4株为肠杆菌属(Enterobacter)，3株为无色杆菌属(Achromobacter)，2株为假单胞菌属(Pseudomonas)，其余4株分别属于鞘氨醇杆菌属(Sphingobacterium)、不动杆菌属(Acinetobacter)、微杆菌属(Microbacterium)和假节杆菌属(Pseudarthrobacter)。定量分析发现菌株SF的反硝化效果较好。通过单因素试验和响应面设计试验，对菌株SF的脱氮性能进行了一系列研究，探究了碳源、温度、初始pH、碳氮比和转速等因素对菌株SF脱氮效果的影响。结果表明，菌株SF的最佳脱氮条件：碳源为葡萄糖，初始pH值为7.5，碳氮比为9.7，转速180 r/min，温度为33.5 ℃。在此条件下，当初始硝酸盐浓度为361 mg/L时，72 h总氮去除率可达到93.3%。【结论】景观植物叶际中存在较多种类的可培养好氧反硝化细菌，丰富了叶际氮循环相关微生物的类型，为探索叶际微生物协同削减大气氮氧化物的机制奠定了基础。通过高效脱氮菌株的筛选，为进一步应用微生物协同植物削减空气氮氧化物污染提供了候选菌株。

Abstract:

[Objective] To identify efficient aerobic denitrifying bacteria in the phyllosphere and provide new ideas for the prevention and control of air pollution by phytoremediation. [Methods] We used the enrichment culture method combined with the Griess reagent and bromothymol blue to isolate aerobic denitrifying bacteria from the phyllosphere and analyzed the phylogenetic relationship of the isolates based on the 16S rRNA gene sequences. Efficient aerobic denitrifying bacteria were screened out and their denitrification conditions were optimized. [Results] Thirteen strains were isolated from the phyllosphere of six landscape plant species: Photinia serratifolia, Ligustrum lucidum, Osmanthus fragrans, Camphora officinarum, Euonymus japonicus, and Magnolia grandiflora. According to the 16S rRNA gene sequences, the 13 strains were classified into 7 genera, 7 families of 4 phyla. Specifically, 4, 3, and 2 strains belonged to Enterobacter, Achromobacter, and Pseudomonas, respectively, and the remaining 4 strains belonged to Acinetobacter, Sphingobacterium, Microbacterium, and Pseudarthrobacter. The comparative analysis revealed that strain SF outperformed the other strains in denitrification. The factors (carbon source, temperature, initial pH, C/N ratio, and rotational speed) influencing the denitrification performance of SF were optimized by single factor tests and response surface methodology. The denitrification conditions of SF were optimized as follows: glucose as the carbon source, initial pH 7.5. C/N ratio of 9.7, rotational at 180 r/min, and temperature of 33.5 ℃. Under the optimized conditions, the total nitrogen removal rate reached 93.3% in 72 h when the initial nitrate concentration was 361 mg/L. [Conclusion] The rich culturable aerobic denitrifying bacteria in the phyllosphere of landscape plants laid a foundation for phytoremediation. It is feasible to synergistically reduce nitrogen oxide pollution in the air by screening efficient denitrifying strains and improve the denitrifying performance by optimizing the culture conditions.