一株耐低温异养硝化-好氧反硝化菌的分离鉴定及其脱氮特性

doi:10.13560/j.cnki.biotech.bull.1985.2023-0452

摘要/Abstract

摘要：

为了提高低温废水的生物脱氮效率，从寒冷地区冬季土壤和底泥中分离筛选耐低温异养硝化-好氧反硝化细菌，研究其脱氮特性及途径。通过菌落和细胞形态特征观察、16S rRNA基因序列分析鉴定菌种。分别以NH₄⁺-N、NO₃^--N、NO₂^--N为唯一氮源，以NH₄⁺-N和NO₃^--N为混合氮源，考察菌株在低温条件（10℃）的硝化、反硝化以及同步硝化反硝化性能。采用聚合酶链式反应（polymerase chain reaction, PCR）对菌株的脱氮功能酶基因扩增，推测低温脱氮途径。结果表明，从河水底泥中筛选出一株异养硝化-好氧反硝化菌，经鉴定为Pseudomonas veronii，命名为P. veronii DH-3。该菌分别以相同初始含氮量（105 mg/L）的NH₄⁺-N、NO₃^--N和NO₂^--N为唯一氮源，在10℃好氧培养48 h时，氮的去除率分别为99.07%、96.89%和90.29%，且在脱氮过程中几乎无亚硝酸盐的累积。以NH₄⁺-N和NO₃^--N为混合氮源时，NH₄⁺-N在48 h内被完全去除，NO₃^--N的去除率为87.09%；氮平衡分析结果表明，以NO₃^--N和NO₂^--N为唯一氮源时含氮气体和细胞内生物氮的转化率均低于NH₄⁺-N，表明该菌株的异养硝化能力强于好氧反硝化能力。脱氮功能基因hao、napA、nirS、nirK、cnorB和nosZ的成功表达，进一步证实该菌株具有硝化反硝化能力。根据上述研究结果，推测该菌株低温脱氮的主要途径为异养硝化-好氧反硝化作用和同化作用。菌株P. veronii DH-3具有良好的异养硝化-好氧反硝化性能，为低温含氮废水的生物净化提供了理论支持。

关键词: 耐低温菌, 生物脱氮, 异养硝化, 好氧反硝化, 分离鉴定, 脱氮功能基因, 假单胞菌

Abstract:

In order to improve the biological nitrogen removal efficiency of low-temperature wastewater, a psychrotolerant heterotrophic nitrification and aerobic denitrification bacterium was isolated from soil and river sediment in cold regions, and its nitrogen removal characteristics and pathway were investigated. The isolated bacterium was identified based on its colony and cell morphological observation as well as 16S rRNA gene sequence analysis. The nitrification, denitrification, and simultaneous nitrification and denitrification performance of the strain were investigated with NH₄⁺-N, NO₃^--N, and NO₂^--N as the sole nitrogen source and NH₄⁺-N and NO₃^--N as mixed nitrogen sources. The nitrogen-removing functional enzyme genes of the strain were amplified by polymerase chain reaction（PCR）to speculate the nitrogen-removing pathway at low-temperature. A heterotrophic nitrification and aerobic denitrification bacterium was screened from the river sediment and identified as Pseudomonas veronii, named P. veronii DH-3. When strain P. veronii DH-3 was cultured aerobically for 48 h at 10℃ with NH₄⁺-N, NO₃^--N, and NO₂^--N as the sole nitrogen sources（N 105 mg/L）, the corresponding nitrogen removal efficiencies were 99.07%, 96.89%, and 90.29%, respectively, without accumulation of nitrite. When NH₄⁺-N and NO₃^--N were used as mixed nitrogen sources, NH₄⁺-N was completely removed in 48 h and the NO₃^--N removal rate was 87.09%, indicating that strain P. veronii DH-3 had excellent simultaneous nitrification and denitrification performance at 10℃. The nitrogen balance analysis results showed that when NO₃^--N and NO₂^--N were the sole nitrogen sources respectively, the conversion rates of nitrogenous gas and intracellular nitrogen were lower than those of NH₄⁺-N, indicating that the heterotrophic nitrification ability of the strain DH-3 was stronger than that of aerobic denitrification. Meanwhile, the successful expressions of denitrification functional genes hao, napA, nirS, nirK, cnorB, and nosZ further confirmed that the heterotrophic nitrification and aerobic denitrification ability of strain DH-3. Based on the above results, it was speculated that the main nitrogen-removing pathway of the strain was heterotrophic nitrification, aerobic denitrification, and assimilation. These findings indicate that the psychrotolerant P. veronii DH-3 possesses excellent heterotrophic nitrification and aerobic denitrification capability, which provides the theoretical support for biological purification of low-temperature nitrogen wastewater.

Key words: psychrotolerant bacterium, biological nitrogen removal, heterotrophic nitrification, aerobic denitrification, isolation and identification, nitrogen removal functional genes, Pseudomonas

董怡华, 王凌潇, 任涵雪, 陈峰. 一株耐低温异养硝化-好氧反硝化菌的分离鉴定及其脱氮特性[J]. 生物技术通报, 2023, 39(12): 237-249.

DONG Yi-hua, WANG Ling-xiao, REN Han-xue, CHEN Feng. Isolation and Identification of a Psychrotolerant Heterotrophic Nitrification-aerobic Denitrification Bacterium and Its Nitrogen-removing Characteristics[J]. Biotechnology Bulletin, 2023, 39(12): 237-249.

图/表 11

表1 PCR引物序列表

Table 1 PCR primer sequences list

引物名称 Primer name	引物序列 Primer sequence（5'-3'）	用途 Usage	参考文献 Reference
27F 1492R	AGTTTGATCMTGGCTCAG GGTTACCTTGTTACGACTT	16S rRNA基因序列扩增	[21]
hao-F hao-R	GTTTCGGCATGGATACGCT CCAGGCATCAAAGAATACCC	hao基因序列扩增	[22]
napA-F napA-R	CATTGCCGACTATGAAGCTCAGG AAAATATAGCGACTATTGATCTCTTGCAG	napA基因序列扩增	[23]
nirS-cd3aF nirS-R3cd	GTSAACGTSAAGGARACSGG GASTTCGGRTGSGTCTTGA	nirS基因序列扩增	[24]
nirK-F nirK-R	CGAACAAGGCAAGGATTTG ACTGTCAACACACCGCTCA	nirK基因序列扩增	[16]
cnorB-F cnorB-R	CGNGARTTYCTSGARCARCC CRTADGCVCCRWAGAAVGC	cnorB基因序列扩增	[25]
nosZ-F nosZ-R	CCCGCTGCACACC（A/G）CCTTCGA CGTCGCC（C/G）GAGATCTCGATCA	nosZ基因序列扩增	[26]

表2 水质指标及分析方法

Table 2 Analysis methods for water quality indicators

水质指标 Water quality indicators	分析方法Analytical methods
NH₄⁺-N	纳氏试剂分光光度法
NO₃^--N	紫外分光光度法
NO₂^--N	N-（1-萘基）-乙二胺分光光度法
TN	过硫酸钾氧化-紫外分光光度法

图1 菌株DH-3的菌落和细胞形态学特征 A：固体培养基的菌落形态；B：革兰氏染色结果；C：细胞形态SEM照片

Fig. 1 Colony and cell morphological characteristics of strain DH-3 A: Colony morphology of solid medium. B: Gram staining results. C: SEM cell morphology

图2 基于16S rRNA基因序列同源性构建的菌株DH-3系统发育树

Fig. 2 Phylogenetic tree of strain DH-3 based on 16S rRNA homologous sequences

图3 以NH4+-N为唯一氮源时菌株DH-3的生长及异养硝化特性

Fig. 3 Growth and heterotrophic nitrification performance of strain DH-3 with NH4+-N as the sole nitrogen source

表3 脱氮过程中的氮平衡分析

Table 3 Nitrogen balance analysis during nitrogen removal

氮源 Nitrogen resource	培养时间 Culture time/h	氮质量浓度Nitrogen mass concentration/（mg·L^-1）
氮源 Nitrogen resource	培养时间 Culture time/h	NH₄⁺-N	NO₃^--N	NO₂^--N	细胞内生物氮Intracellular-N	含氮气体Gas-N
NH₄⁺-N	0	105.49 ± 2.27	-	-	-	-
NH₄⁺-N	48	0.98 ± 0.17	0.37 ± 0.04	-	47.24 ± 1.32	56.83 ± 1.24
NO₃^--N	0	-	105.43 ± 2.22	-	-	-
NO₃^--N	48	2.47 ± 0.18	3.28 ± 1.04	-	42.76 ± 2.07	55.46 ± 1.57
NO₂^--N	0	-	-	105.53 ± 2.28	-	-
NO₂^--N	48	2.48 ± 0.23	0.25 ± 0.01	10.24 ± 1.06	38.58 ± 2.31	53.21 ± 1.88
NH₄⁺-N+NO₃^--N	0	52.58 ± 1.48	52.61 ± 2.03	-	-	-
NH₄⁺-N+NO₃^--N	48	-	6.79±0.93	-	44.59 ± 2.03	53.82 ± 1.46

图4 NO3--N为唯一氮源时菌株DH-3的生长及好氧反硝化特性

Fig. 4 Growth and aerobic denitrification performance of strain DH-3 with NO3--N as the sole nitrogen source

图5 NO2--N为唯一氮源时菌株DH-3的生长及好氧反硝化特性

Fig. 5 Growth and aerobic denitrification performance of strain DH-3 with NO2--N as sole nitrogen source

图6 NH4+-N和NO3--N为混合氮源时菌株DH-3的生长及脱氮特性

Fig. 6 Growth and SND performance of strain NR-5 with NH4+-N and NO3--N as mixed nitrogen source

图7 菌株DH-3功能基因的扩增

Fig. 7 PCR amplification results of functional genes in strain DH-3

图8 菌株DH-3的氮代谢途径

Fig. 8 Proposed nitrogen metabolism pathway of strain DH-3

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