Denitrification Characteristics and Application of Psychrotolerant and Alkali-tolerant Denitrifying Bacterium Stutzerimonas stutzeri Strain 2B

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

Abstract

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

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.

Figures/Tables 8

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

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

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

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

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

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

Fig. 6 Nitrogen metabolism pathways of strain 2B

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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