Isolation and Identification of a Psychrotolerant Heterotrophic Nitrification-aerobic Denitrification Bacterium and Its Nitrogen-removing Characteristics

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

Abstract

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

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.

Figures/Tables 11

References 51

[1]	卢瑞朋, 徐文江, 李安峰, 等. 强化反硝化除磷的新型多级缺氧-好氧工艺[J]. 中国环境科学, 2022, 42(4): 1706-1713.
	Lu RP, Xu WJ, Li AF, et al. Enhanced denitrifying phosphorus removal in novel multistage anoxic-oxic process[J]. China Environ Sci, 2022, 42(4): 1706-1713.
[2]	郭超, 尹辉, 范奎, 等. 一株好氧反硝化菌的分离鉴定及脱氮特性研究[J]. 安全与环境工程, 2020, 27(4): 41-47.
	Guo C, Yin H, Fan K, et al. Isolation, identification and nitrogen removal characteristics of aerobic denitrifying bacteria[J]. Saf Environ Eng, 2020, 27(4): 41-47.
[3]	Ren JL, Cheng XW, Ma HJ, et al. Characteristics of a novel heterotrophic nitrification and aerobic denitrification bacterium and its bioaugmentation performance in a membrane bioreactor[J]. Bioresour Technol, 2021, 342: 125908. doi: 10.1016/j.biortech.2021.125908 URL
[4]	Angar Y, Kebbouche-Gana S, Djelali NE, et al. Novel approach for the ammonium removal by simultaneous heterotrophic nitrification and denitrification using a novel bacterial species co-culture[J]. World J Microbiol Biotechnol, 2016, 32(3): 36. doi: 10.1007/s11274-015-2007-y URL
[5]	何腾霞, 倪九派, 李振轮, 等. 1株Arthrobacter arilaitensis菌的耐冷异养硝化和好氧反硝化作用[J]. 环境科学, 2016, 37(3): 1082-1088.
	He TX, Ni JP, Li ZL, et al. Heterotrophic nitrification and aerobic denitrification of the hypothermia aerobic denitrification bacterium: Arthrobacter arilaitensis[J]. Environ Sci, 2016, 37(3): 1082-1088.
[6]	Xu Y, He TX, Li ZL, et al. Nitrogen removal characteristics of Pseudomonas putida Y-9 capable of heterotrophic nitrification and aerobic denitrification at low temperature[J]. Biomed Res Int, 2017, 2017: 1429018.
[7]	Robertson LA, Kuenen JG. Aerobic denitrification: a controversy revived[J]. Arch Microbiol, 1984, 139(4): 351-354. doi: 10.1007/BF00408378 URL
[8]	Zhang DY, Li WG, Huang XF, et al. Removal of ammonium in surface water at low temperature by a newly isolated Microbacterium sp. strain SFA13[J]. Bioresour Technol, 2013, 137: 147-152. doi: 10.1016/j.biortech.2013.03.094 URL
[9]	Liu YX, Wang Y, Li Y, et al. Nitrogen removal characteristics of heterotrophic nitrification-aerobic denitrification by Alcaligenes faecalis C16[J]. Chin J Chem Eng, 2015, 23(5): 827-834. doi: 10.1016/j.cjche.2014.04.005 URL
[10]	Wei R, Hui C, Zhang YP, et al. Nitrogen removal characteristics and predicted conversion pathways of a heterotrophic nitrification-aerobic denitrification bacterium, Pseudomonas aeruginosa P-1[J]. Environ Sci Pollut Res Int, 2021, 28(6): 7503-7514. doi: 10.1007/s11356-020-11066-7
[11]	Yang L, Ren YX, Liang X, et al. Nitrogen removal characteristics of a heterotrophic nitrifier Acinetobacter junii YB and its potential application for the treatment of high-strength nitrogenous wastewater[J]. Bioresour Technol, 2015, 193: 227-233. doi: 10.1016/j.biortech.2015.05.075 URL
[12]	高宇轩, 靳静晨, 徐利杉, 等. 耐盐异养硝化-好氧反硝化菌Bacillus megatherium N07的分离及脱氮特性[J]. 生物技术通报, 2022, 38(7): 247-257. doi: 10.13560/j.cnki.biotech.bull.1985.2021-1111
	Gao YX, Jin JC, Xu LS, et al. Isolation of halophilic heterotrophic nitrification-aerobic denitrification bacterium Bacillus megatherium N07 and its denitrification characteristics[J]. Biotechnol Bull, 2022, 38(7): 247-257.
[13]	Ge QL, Yue XP, Wang GY. Simultaneous heterotrophic nitrification and aerobic denitrification at high initial phenol concentration by isolated bacterium Diaphorobacter sp. PD-7[J]. Chin J Chem Eng, 2015, 23(5): 835-841. doi: 10.1016/j.cjche.2015.02.001 URL
[14]	杨静丹, 祝铭韩, 刘琳, 等. 异养硝化-好氧反硝化菌HY3-2的分离及脱氮特性[J]. 中国环境科学, 2020, 40(1): 294-304.
	Yang JD, Zhu MH, Liu L, et al. Isolation and denitrification characteristics of heterotrophic nitrification-aerobic denitrification bacterium HY3-2[J]. China Environ Sci, 2020, 40(1): 294-304.
[15]	Yang M, Lu DW, Qin BD, et al. Highly efficient nitrogen removal of a coldness-resistant and low nutrient needed bacterium, Janthinobacterium sp. M-11[J]. Bioresour Technol, 2018, 256: 366-373. doi: 10.1016/j.biortech.2018.02.049 URL
[16]	Xi HP, Zhou XT, Arslan M, et al. Heterotrophic nitrification and aerobic denitrification process: promising but a long way to go in the wastewater treatment[J]. Sci Total Environ, 2022, 805: 150212. doi: 10.1016/j.scitotenv.2021.150212 URL
[17]	He TX, Xie DT, Li ZL, et al. Ammonium stimulates nitrate reduction during simultaneous nitrification and denitrification process by Arthrobacter arilaitensis Y-10[J]. Bioresour Technol, 2017, 239: 66-73. doi: 10.1016/j.biortech.2017.04.125 URL
[18]	丁晓宇, 卢兴顺, 吕航, 等. 耐低温异养硝化-好氧反硝化菌的分离鉴定及脱氮特性[J]. 水处理技术, 2022, 48(12): 65-70, 76.
	Ding XY, Lu XS, Lv H, et al. Isolation, identification and nitrogen removal characteristics of heterotrophic nitrification-aerobic denitrification bacteria under low temperature[J]. Technol Water Treat, 2022, 48(12): 65-70, 76.
[19]	周英萍, 唐美珍, 曹彦雪, 等. 一株耐冷异养硝化菌的分离鉴定及脱氮特性[J]. 湖泊科学, 2019, 31(3): 746-754.
	Zhou YP, Tang MZ, Cao YX, et al. Isolation and denitrification characteristics of a psychrotrophic and heterotrophic nitrification bacterium[J]. J Lake Sci, 2019, 31(3): 746-754.
[20]	汤默然, 李茹莹. 异养硝化-好氧反硝化菌株的分离筛选及复配菌剂对河水的净化效果[J]. 环境科学学报, 2021, 41(7): 2657-2663.
	Tang MR, Li RY. Isolation of heterotrophic nitrification-aerobic denitrification bacteria strains and purification of river water by mixed cultures[J]. Acta Sci Circumstantiae, 2021, 41(7): 2657-2663.
[21]	Lane D J, Stackebrandt E, Goodfellow M. 16S/23S rRNA Sequencing. Nucleic Acid Techniques in Bacterial Systematics[M]. New York: Wiley, 1991: 115-175.
[22]	Xing CY, Fan YC, Chen X, et al. A self-assembled nanocompartment in anammox bacteria for resisting intracelluar hydroxylamine stress[J]. Sci Total Environ, 2020, 717: 137030. doi: 10.1016/j.scitotenv.2020.137030 URL
[23]	Ke X, Liu C, Tang SQ, et al. Characterization of Acinetobacter indicus ZJB20129 for heterotrophic nitrification and aerobic denitrification isolated from an urban sewage treatment plant[J]. Bioresour Technol, 2022, 347: 126423. doi: 10.1016/j.biortech.2021.126423 URL
[24]	Zhao TT, Chen PP, Zhang LJ, et al. Heterotrophic nitrification and aerobic denitrification by a novel Acinetobacter sp. TAC-1 at low temperature and high ammonia nitrogen[J]. Bioresour Technol, 2021, 339: 125620. doi: 10.1016/j.biortech.2021.125620 URL
[25]	Lei X, Jia YT, Chen YC, et al. Simultaneous nitrification and denitrification without nitrite accumulation by a novel isolated Ochrobactrum anthropic LJ81[J]. Bioresour Technol, 2019, 272: 442-450. doi: 10.1016/j.biortech.2018.10.060 URL
[26]	Xia L, Li XM, Fan WH, et al. Heterotrophic nitrification and aerobic denitrification by a novel Acinetobacter sp. ND7 isolated from municipal activated sludge[J]. Bioresour Technol, 2020, 301: 122749. doi: 10.1016/j.biortech.2020.122749 URL
[27]	国家环境保护总局. 水和废水监测分析方法[M]. 4版. 北京: 中国环境科学出版社, 2002.
	State Environmental Protection Administration. Water and Wastewater Monitoring Analysis Method[M]. 4th ed. Beijing: China Environmental Science Press, 2002.
[28]	Arpigny JL, Feller G, Gerday C. Cloning, sequence and structural features of a lipase from the Antarctic facultative psychrophile Psychrobacter immobilis B10[J]. Biochim Biophys Acta, 1993, 1171(3): 331-333. pmid: 7916627
[29]	Qin W, Li WG, Huang XF, et al. A proteomic analysis of heterotrophic nitrifying bacterium Acinetobacter sp. HITLi 7^T adaptation to low temperature using two-dimensional difference gel electrophoresis approach[J]. Int Biodeterior Biodegrad, 2016, 113: 113-119. doi: 10.1016/j.ibiod.2016.03.009 URL
[30]	He TX, Li ZL, Sun Q, et al. Heterotrophic nitrification and aerobic denitrification by Pseudomonas tolaasii Y-11 without nitrite accumulation during nitrogen conversion[J]. Bioresour Technol, 2016, 200: 493-499. doi: 10.1016/j.biortech.2015.10.064 URL
[31]	Cui Y, Cui YW, Huang JL. A novel halophilic Exiguobacterium mexicanum strain removes nitrogen from saline wastewater via heterotrophic nitrification and aerobic denitrification[J]. Bioresour Technol, 2021, 333: 125189. doi: 10.1016/j.biortech.2021.125189 URL
[32]	Yang Q, Yang T, Shi Y, et al. The nitrogen removal characterization of a cold-adapted bacterium: Bacillus simplex H-B[J]. Bioresour Technol, 2021, 323: 124554. doi: 10.1016/j.biortech.2020.124554 URL
[33]	Lang XD, Li QW, Ji MM, et al. Isolation and niche characteristics in simultaneous nitrification and denitrification application of an aerobic denitrifier, Acinetobacter sp. YS2[J]. Bioresour Technol, 2020, 302: 122799. doi: 10.1016/j.biortech.2020.122799 URL
[34]	Qu J, Zhao RJ, Chen YY, et al. Enhanced nitrogen removal from low-temperature wastewater by an iterative screening of cold-tolerant denitrifying bacteria[J]. Bioprocess Biosyst Eng, 2022, 45(2): 381-390. doi: 10.1007/s00449-021-02668-7
[35]	Jin RF, Liu TQ, Liu GF, et al. Simultaneous heterotrophic nitrification and aerobic denitrification by the marine origin bacterium Pseudomonas sp. ADN-42[J]. Appl Biochem Biotechnol, 2015, 175(4): 2000-2011. doi: 10.1007/s12010-014-1406-0 URL
[36]	Guo Y, Wang YY, Zhang ZJ, et al. Physiological and transcriptomic insights into the cold adaptation mechanism of a novel heterotrophic nitrifying and aerobic denitrifying-like bacterium Pseudomonas indoloxydans YY-1[J]. Int Biodeterior Biodegrad, 2018, 134: 16-24. doi: 10.1016/j.ibiod.2018.08.001 URL
[37]	Ye Q, Li KL, Li ZL, et al. Heterotrophic nitrification-aerobic denitrification performance of strain Y-12 under low temperature and high concentration of inorganic nitrogen conditions[J]. Water, 2017, 9(11): 835. doi: 10.3390/w9110835 URL
[38]	Chen PZ, Li J, Li QX, et al. Simultaneous heterotrophic nitrification and aerobic denitrification by bacterium Rhodococcus sp. CPZ24[J]. Bioresour Technol, 2012, 116: 266-270. doi: 10.1016/j.biortech.2012.02.050 URL
[39]	张宇红, 董先博, 刘香宇, 等. 新型异养硝化-好氧反硝化菌Paracoccus sp. QD-19的分离及脱氮特性研究[J]. 生物技术通报, 2023, 39(3): 301-310. doi: 10.13560/j.cnki.biotech.bull.1985.2022-0834
	Zhang YH, Dong XB, Liu XY, et al. Isolation of a novel heterotrophic nitrification-aerobic denitrification bacterium Paracoccus sp. QD-19 and its characterization of removing nitrogen[J]. Biotechnol Bull, 2023, 39(3): 301-310.
[40]	Chen SH, He SY, Wu CJ, et al. Characteristics of heterotrophic nitrification and aerobic denitrification bacterium Acinetobacter sp. T1 and its application for pig farm wastewater treatment[J]. J Biosci Bioeng, 2019, 127(2): 201-205. doi: 10.1016/j.jbiosc.2018.07.025 URL
[41]	Chen HJ, Zhou WZ, Zhu SN, et al. Biological nitrogen and phosphorus removal by a phosphorus-accumulating bacteria Acinetobacter sp. strain C-13 with the ability of heterotrophic nitrification-aerobic denitrification[J]. Bioresour Technol, 2021, 322: 124507. doi: 10.1016/j.biortech.2020.124507 URL
[42]	Huang F, Pan LQ, Lv N, et al. Characterization of novel Bacillus strain N31 from mariculture water capable of halophilic heterotrophic nitrification-aerobic denitrification[J]. J Biosci Bioeng, 2017, 124(5): 564-571. doi: S1389-1723(17)30267-0 pmid: 28716629
[43]	Rout PR, Bhunia P, Dash RR. Simultaneous removal of nitrogen and phosphorous from domestic wastewater using Bacillus cereus GS-5 strain exhibiting heterotrophic nitrification, aerobic denitrification and denitrifying phosphorous removal[J]. Bioresour Technol, 2017, 244(Pt 1): 484-495. doi: 10.1016/j.biortech.2017.07.186 URL
[44]	Yang JX, Feng L, Pi SS, et al. A critical review of aerobic denitrification: insights into the intracellular electron transfer[J]. Sci Total Environ, 2020, 731: 139080. doi: 10.1016/j.scitotenv.2020.139080 URL
[45]	Padhi SK, Tripathy S, Sen R, et al. Characterisation of heterotrophic nitrifying and aerobic denitrifying Klebsiella pneumoniae CF-S9 strain for bioremediation of wastewater[J]. Int Biodeterior Biodegrad, 2013, 78: 67-73. doi: 10.1016/j.ibiod.2013.01.001 URL
[46]	Throbäck IN, Enwall K, Jarvis A, et al. Reassessing PCR primers targeting nirS, nirK and nosZ genes for community surveys of denitrifying bacteria with DGGE[J]. FEMS Microbiol Ecol, 2004, 49(3): 401-417. doi: 10.1016/j.femsec.2004.04.011 pmid: 19712290
[47]	Carneiro Fidélis Silva L, Santiago Lima H, Antônio de Oliveira Mendes T, et al. Heterotrophic nitrifying/aerobic denitrifying bacteria: ammonium removal under different physical-chemical conditions and molecular characterization[J]. J Environ Manage, 2019, 248: 109294. doi: 10.1016/j.jenvman.2019.109294 URL
[48]	Yao S, Ni JR, Ma T, et al. Heterotrophic nitrification and aerobic denitrification at low temperature by a newly isolated bacterium, Acinetobacter sp. HA2[J]. Bioresour Technol, 2013, 139: 80-86. doi: 10.1016/j.biortech.2013.03.189 URL
[49]	Li YJ, Zhang LH, Liu WF, et al. Simultaneous removal of urea nitrogen and inorganic nitrogen from high-salinity wastewater by Halomonas sp. H36[J]. Environ Sci Pollut Res Int, 2023, 30(2): 2544-2554. doi: 10.1007/s11356-022-22018-8
[50]	Man QL, Zhang PL, Huang WQ, et al. A heterotrophic nitrification-aerobic denitrification bacterium Halomonas venusta TJPU05 suitable for nitrogen removal from high-salinity wastewater[J]. Front Environ Sci Eng, 2022, 16(6): 69. doi: 10.1007/s11783-021-1503-6
[51]	Qiao ZX, Wu YG, Qian J, et al. A lab-scale study on heterotrophic nitrification-aerobic denitrification for nitrogen control in aquatic ecosystem[J]. Environ Sci Pollut Res Int, 2020, 27(9): 9307-9317. doi: 10.1007/s11356-019-07551-3

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]

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]

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

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

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