从中药药渣堆肥中分离鉴定的一株异养硝化菌及其硝化反硝化特性研究

doi:10.13560/j.cnki.biotech.bull.1985.2024-1049

摘要/Abstract

摘要：

目的为实现中药药渣好氧堆肥中氮素的高效保留，旨在寻找具有高效硝化能力的微生物并揭示其硝化反硝化特性，以期为开发保氮微生物菌剂提供菌种资源与理论依据。方法采用富集培养法、涂布平板法和划线分离法从中药药渣堆肥中获得纯化菌株。通过异养硝化能力测定，筛选出1株能够高效转化铵态氮的硝化菌株。结合形态学特征及16S rDNA基因测序对所得菌株进行鉴定。通过调整碳源、碳氮比（C/N）、初始铵态氮浓度、pH、温度、转速6种培养条件，探究环境因素对菌株生长及硝化能力的影响。在以亚硝态氮或硝态氮为唯一氮源的培养基中培养该菌株，检测其反硝化能力。结果从堆肥中分离得到1株高效硝化菌，命名为YF-5，形态学观察及分子生物学鉴定为水生产碱杆菌（Alcaligenes aquatilis）。优化实验确定该菌株最佳硝化条件为：琥珀酸钠作碳源、C/N 20、初始铵态氮浓度100 mg/L、pH 7.0、温度35 ℃、转速160 r/min。硝化产物分析显示，菌株YF-5进行异养硝化作用，其过程中伴有亚硝态氮的积累。反硝化实验证实，菌株YF-5无法利用亚硝态氮或硝态氮进行反硝化。结论菌株YF-5在优化条件下能将铵态氮转化为稳定的亚硝态氮，且不具备反硝化能力。这一特性使其在中药药渣堆肥应用中能够通过定向转化氮素，同步减少氨挥发与反硝化作用，从而降低堆肥过程中的氮素损失，展现出良好的应用潜力。

关键词: 产碱杆菌属, 硝化细菌, 异养硝化, 中药药渣, 好氧堆肥, 反硝化, 氮素保留

Abstract:

Objective To achieve efficient nitrogen retention during the aerobic composting of Chinese medicinal herbal residues (CMHRs), this study is aimed to isolate and identify microorganisms with high nitrification capacity and to elucidate their nitrification-denitrification characteristics, thereby providing potential microbial resources and a theoretical basis for developing nitrogen-conserving microbial agents. Method Pure strains were obtained from CMHRs compost using the enrichment culture, spread plate, and streak plate methods. Screening based on heterotrophic nitrification capability led to the selection of the high-efficiency strain. Identification of the strain was performed based on morphological characteristics and 16S rDNA gene sequencing. The effects of environmental factors on strain growth and nitrification capacity were investigated by adjusting six cultivation conditions: carbon source, carbon to nitrogen ratio (C/N), initial ammonium nitrogen concentration, pH, temperature, and rotational speed. The denitrification capability of strain YF-5 was examined by culturing it in media with nitrite nitrogen or nitrate nitrogen as the sole nitrogen source. Result A highly efficient nitrifying bacterium was isolated from the compost and named YF-5, which was identified as Alcaligenes aquatilis by morphological observation and molecular biology. Optimization experiments determined that the optimal nitrification conditions for this strain included a carbon source of sodium succinate, a C/N of 20, an initial ammonium nitrogen concentration of 100 mg/L, a pH of 7.0, a temperature of 35 ℃, and a rotation speed of 160 r/min. Analysis of nitrified products indicated that strain YF-5 performed heterotrophic nitrification with the accumulation of nitrite nitrogen. Denitrification experiments confirmed that strain YF-5 was incapable of employing nitrite or nitrate nitrogen as substrates for denitrification. Conclusion Strain YF-5 converts ammonium nitrogen into stable nitrite nitrogen under optimized conditions and lacks denitrification capability. This characteristic enables it to reduce both ammonia volatilization and denitrification through targeted nitrogen conversion during CMHRs composting, thereby minimizing nitrogen loss and demonstrating promising application potential.

Key words: Alcaligenes, nitrifying bacteria, heterotrophic nitrification, Chinese medicinal herbal residues, aerobic composting, denitrification, nitrogen conservation

郑婉婷, 曾千诺, 傅嘉懿, 饶敏欣, 何国振, 张英. 从中药药渣堆肥中分离鉴定的一株异养硝化菌及其硝化反硝化特性研究[J]. 生物技术通报, 2026, 42(1): 305-314.

ZHENG Wan-ting, ZENG Qian-nuo, FU Jia-yi, RAO Min-xin, HE Guo-zhen, ZHANG Ying. Isolation and Identification of a Heterotrophic Nitrifying Bacterium from the Compost of Chinese Medicinal Herbal Residues and Study of Its Nitrification and Denitrification Capabilities[J]. Biotechnology Bulletin, 2026, 42(1): 305-314.

图/表 9

图1 菌株YF-5的形态学特征A：平板菌落形态；B：革兰氏染色结果；C：扫描电镜图

Fig. 1 Morphological characteristics of strain YF-5A: Plate colony morphology. B: Gram staining results. C: Scanning electron microscope image

图2 菌株YF-5基于16S rDNA基因序列构建的系统发育树

Fig. 2 Phylogenetic tree of strain YF-5 based on 16S rDNA gene sequences

图3 不同碳源下菌株YF-5 的生长状况以及硝化特性

Fig. 3 Growth status and nitrification characteristic of strain YF-5 under different carbon sources

图4 不同C/N下菌株YF-5的生长状况及其硝化特性

Fig. 4 Growth status and nitrification characteristic of strain YF-5 under different C/N

图5 不同初始铵态氮浓度下菌株YF-5的生长状况及其硝化特性

Fig. 5 Growth status and nitrification characteristic of strain YF-5 under different initial ammonium nitrogen concentration

图6 不同pH下菌株YF-5的生长状况及其硝化特性

Fig. 6 Growth status and nitrification characteristic of strain YF-5 under different pH

图7 不同温度下菌株YF-5的生长状况及其硝化特性

Fig. 7 Growth status and nitrification characteristic of strain YF-5 under different temperature

图8 不同转速下菌株YF-5的生长状况及其硝化特性

Fig. 8 Growth status and nitrification characteristic of strain YF-5 under different rotational speed

图9 菌株YF-5的反硝化特性A、B：亚硝态氮为氮源的菌体细胞密度（A）和质量浓度及降解率（B）；C、D：硝态氮为氮源的菌体细胞密度（C）和质量浓度及降解率（D）

Fig. 9 Denitrification characteristic of the strain YF-5A, B: Cell density (A) and mass concentration and degradation rate of NO2‒-N (B) while nitrite as nitrogen source. C, D: Cell density (C) and mass concentration and degradation rate of NO3‒-N (D) while nitrate as nitrogen source

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