耐冷耐碱反硝化细菌Stutzerimonas stutzeri 2B的脱氮特性及应用

doi:10.13560/j.cnki.biotech.bull.1985.2026-0059

摘要/Abstract

摘要：

目的针对北方冬季海水水产养殖中NO₃^--N去除效率低的问题，筛选高效耐冷耐碱反硝化菌株，为养殖尾水处理提供菌种资源。方法从渤海海域水产养殖尾水中筛选到菌株2B。通过形态学、生理生化特征及16S rRNA基因序列分析，鉴定菌株为施氏假单胞菌Stutzerimonas stutzeri。通过单因素试验考察菌株的反硝化性能，以NH₄⁺-N、NO₃^--N和NO₂^--N为单一或混合氮源，研究其脱氮性能和氮代谢途径。结果菌株以淀粉为最优碳源，在C/N=5、盐度3%、pH 8的最佳条件下，低温10 ℃培养96 h，NO₃^--N去除率达99.80%；菌株能耐受pH 11的强碱环境，96 h内NO₃^--N去除率达87.72%，属于中度嗜碱菌。此外，菌株也能以NH₄⁺-N或NO₂^--N为单一氮源生长，在混合氮源中进行异养硝化或好氧反硝化。全基因组分析表明，菌株具有完整的反硝化途径：NO₃^--N→NO₂^--N→NO→N₂O→N₂，同时具有耐冷基因katG和合成海藻糖、谷氨酸等小分子物质的耐盐碱相关基因。在大菱鲆海水养殖尾水中加入菌株2B，NO₃^--N被完全降解且无其他含氮化合物产生。结论菌株2B在低温、低C/N、中高盐度和碱性条件下展现了优异的反硝化性能，为北方冬季海水养殖尾水的生物脱氮处理提供潜在的菌种资源与技术依据。

关键词: 耐冷耐碱菌, 低C/N, 好氧反硝化, 脱氮途径, 海水养殖尾水

Abstract:

Objective To overcome the low NO₃^--N removal efficiency in northern seawater aquaculture during winter, psychrotolerant and alkali-tolerant denitrifying strains were screened for aquaculture wastewater treatment applications. Method Strain 2B was isolated from aquaculture wastewater in the Bohai Sea. Based on morphological, physiological, biochemical characteristics, and 16S rRNA gene sequence analysis, the strain was identified as Stutzerimonas stutzeri. Single-factor experiments were conducted to evaluate the denitrification performance of strain 2B, and its nitrogen removal capabilities and metabolism pathways were investigated with NH₄⁺-N, NO₃^--N, and NO₂^--N as single or mixed nitrogen sources. Result Starch was the optimal carbon source for the strain. Under optimal conditions of C/N=5, salinity 3%, and pH 8, a 96-hour culture achieved a NO₃^--N removal rate of 99.80% at a low temperature of 10 ℃. Notably, the strain achieved 87.72% NO₃^--N removal in 96 h at pH 11, indicating that it is a moderately alkaliphilic bacterium. Moreover, the strain could grow with NH₄⁺-N or NO₂^--N as the sole nitrogen source and perform heterotrophic nitrification and aerobic denitrification in mixed nitrogen sources. Whole-genome analysis demonstrated that the strain possessed a complete denitrification pathway: NO₃^--N → NO₂^--N → NO → N₂O → N₂, and also harbored the cold-tolerance gene katG, along with genes related to salinity-alkalinity tolerance involved in the synthesis of small molecules such as trehalose and glutamic acid. When applied to the seawater aquaculture wastewater of Larimichthys crocea, NO₃^--N was completely degraded without the accumulation of other nitrogenous compounds. Conclusion Strain 2B exhibits excellent nitrogen removal performance under low temperature, low C/N ratio, moderate salinity, and alkaline conditions, providing a potential microbial resource and technical foundation for the biological nitrogen removal treatment of mariculture wastewater in northern regions during winter.

Key words: psychrotolerant and alkali-resistant bacterium, low C/N ratio, aerobic denitrification, nitrogen metabolism pathway, seawater aquaculture wastewater

刘晋雅, 张莹, 张媛, 车佳轩, 郭艾英, 秦保平, 蔡爱军, 郑会会. 耐冷耐碱反硝化细菌Stutzerimonas stutzeri 2B的脱氮特性及应用[J]. 生物技术通报, 2026, 42(11): 340-350.

LIU Jin-ya, ZHANG Ying, ZHANG Yuan, CHE Jia-xuan, GUO Ai-ying, QIN Bao-ping, CAI Ai-jun, ZHENG Hui-hui. Denitrification Characteristics and Application of Psychrotolerant and Alkali-tolerant Denitrifying Bacterium Stutzerimonas stutzeri Strain 2B[J]. Biotechnology Bulletin, 2026, 42(11): 340-350.

图/表 8

图1 菌株2B形态观察与鉴定A：菌落形态；B：扫描电镜图片；C：基于16S rRNA基因序列构建菌株2B的系统发育树

Fig. 1 Morphological observation and identification of strain 2BA: Colony morphology; B: Scanning electron microscope images; C: Phylogenetic tree of strain 2B based on 16S rRNA gene sequences

图2 不同条件对菌株2B的反硝化特性影响A：碳源；B：温度；C：转速；D：接种量

Fig. 2 Denitrification characteristics of the strain under different conditionsA: Carbon sources; B: Temperatures; C: Rotation speeds; D: Inoculum size

表1 菌株的氮平衡分析

Table 1 Nitrogen balance of the strain

氮源

Nitrogen resource

初始总氮浓度TN_i（mg/L）

培养液总氮浓度TN_t（mg/L）

上清液总氮浓度

TN_s（mg/L）

胞内氮转化率

Re_in（%）

气态氮转化率

Re_g（%）

NO₃^--N

NO₂^--N

NH₄⁺-N

图3 三因素交互作用对NO3--N去除率影响的响应面分析A：C/N和pH；B：C/N和盐度；C：pH和盐度

Fig. 3 Response surface analysis of the effects of three-factor interactions on NO3--N removal efficiencyA: C/N and pH; B: C/N and salinity; C: pH and salinity

图4 菌株2B在不同培养基上的生长及脱氮特性A： NM； B： DM-1； C： DM-2； D： DM-3

Fig. 4 Growth and denitrification characteristics of strain 2B on different media

图5 菌株2B的生长及异养硝化好氧反硝化特性

Fig. 5 Growth and simultaneous nitrification and denitrification characteristics of strain 2B

图6 菌株2B氮代谢途径

Fig. 6 Nitrogen metabolism pathways of strain 2B

图7 菌株2B对不同大菱鲆尾水处理结果A：未额外添加氮源；B：30 mg/L NO3--N；C：50 mg/L NO3--N

Fig. 7 Nitrogen removal from different turbot effluents by strain 2BA: Without added nitrogen source; B: 30 mg/L NO3--N; C: 50 mg/L NO3--N

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