一株异养硝化-好氧反硝化细菌的筛选及氮转化特性研究

doi:10.13560/j.cnki.biotech.bull.1985.2023-0614

摘要/Abstract

摘要：

水产养殖过程中氮元素超标易造成水质恶化，威胁水产动物的生长繁殖，亟需安全、高效的脱氮方法。从福建某水产养殖池底的污泥中筛选出一株氨氮降解菌，菌种鉴定后进行适应性驯化，并研究不同条件对菌株生长和氨氮降解的影响，随后又以亚硝态氮为唯一氮源检测菌株的好氧反硝化特性。菌株经16S rRNA鉴定为阿氏芽孢杆菌Bacillus aryabhattai，命名为JN01；驯化后，菌株的生长、氨氮去除率均有不同程度提高，初始NH₄⁺-N浓度为200 mg/L的氮去除率最高达87.29%；影响菌株脱氮的单因素实验结果表明，当NH₄⁺-N浓度为200 mg/L、丁二酸钠为碳源、碳氮比为15∶1、pH 7.5、培养温度为30℃时，菌株的氨氮降解率可以达到92.78%。在亚硝态氮为唯一氮源的条件下，菌株JN01也能进行好氧反硝化转化，NO₂^--N降解率为82.30%。B. aryabhattai JN01具有良好的异养硝化和好氧反硝化特性，具备同时解决养殖废水中氨氮和亚硝酸盐超标的应用潜力。

关键词: 阿氏芽孢杆菌, 异养硝化, 好氧反硝化, 氨氮, 亚硝态氮

Abstract:

The excessive nitrogen in aquaculture easily causes the deterioration of water quality and threatens the growth and reproduction of aquatic animals, thus a safe and efficient nitrogen removal method is urgently needed. An ammonia-degrading bacterium was selected from the sludge at the bottom of an aquaculture pond in Fujian province. After the strain identification, the adaptive domestication was carried out. The effects of different conditions on the growth of the strain and its ammonia-nitrogen degradation were studied. Subsequently, nitrite was used as the only nitrogen source to detect the aerobic denitrification characteristics of the strains. The strain was identified as Bacillus aryabhattai by 16S rRNA and named as JN01. After acclimation, the growth of the strain and removal rate of ammonia nitrogen increased at varied degrees, and the removal rate of nitrogen was up to 87.29% with the initial NH₄⁺-N concentration was 200 mg/L. The results of single factor experiment showed that the degradation rate of ammonia nitrogen of the strain reached 92.78% when NH₄⁺-N concentration was 200 mg/L, sodium succinate as carbon source, carbon to nitrogen ratio was 15∶1, pH 7.5 and culture temperature was 30℃. Under the condition that nitrite nitrogen was the only nitrogen source, strain JN01 also performed aerobic denitrification, and the degradation rate of NO₂^--N was 82.30%. In conclusion, B. aryabhattai JN01 has promising heterotrophic nitrification and aerobic denitrification characteristics, and has the potential to concurrently solve the excessive ammonia nitrogen and nitrite in aquaculture wastewater.

Key words: Bacillus aryabhattai, heterotrophic nitrification, aerobic denitrification, ammonia nitrogen, nitrite nitrogen

蒋慧慧, 王强, 付维来, 饶志明, 张显. 一株异养硝化-好氧反硝化细菌的筛选及氮转化特性研究[J]. 生物技术通报, 2023, 39(12): 250-260.

JIANG Hui-hui, WANG Qiang, FU Wei-lai, RAO Zhi-ming, ZHANG Xian. Screening of a Heterotrophic Nitrifying-aerobic Denitrifying Bacterium and Its Nitrogen Transforming Characterization[J]. Biotechnology Bulletin, 2023, 39(12): 250-260.

图/表 11

图1 异养-好氧反硝化微生物氮代谢途径

Fig. 1 Nitrogen metabolism pathways of HNAD microorganisms

图2 不同菌株的生长曲线（A）和NH4+-N的去除效率（B）

Fig. 2 Growth curves（A）and NH4+-N removal efficiencies（B）of different strains

图3 菌株JN01的系统进化树

Fig. 3 Phylogenetic tree of the strain JN01

图4 菌株JN01驯化前后的 OD600 nm（A）和 NH4+-N去除效率（B）的变化

Fig. 4 Variation of OD600 nm（A）and NH4+-N removal efficiency（B）before and after the domestication of B. aryabhattai JN01

图5 不同氮源浓度对菌株JN01的OD600 nm（A）和NH4+-N去除率（B）的影响

Fig. 5 Effects of N concentration on the growth characteristics OD600 nm（A）and NH4+-N removal efficiencies（B）of B. aryabhattai JN01

图6 不同碳源对菌株JN01的OD600 nm（A）和NH4+-N去除率（B）的影响

Fig. 6 Effects of different carbon sources on the growth characteristics OD600 nm（A）and NH4+-N removal efficiencies（B）of B. aryabhattai JN01

图7 C/N对菌株JN01的OD600 nm（A）和NH4+-N去除率（B）的影响

Fig. 7 Effects of C/N on the growth performance OD600 nm（A）and NH4+-N removal efficiencies（B）of B. aryabhattai JN01

图8 初始pH对菌株JN01的OD600 nm（A）和NH4+-N去除率（B）的影响

Fig. 8 Effects of pH on the growth performance OD600 nm（A）and NH4+-N removal efficiencies（B）of B. aryabhattai JN01

图9 温度对菌株JN01的OD600 nm（A）和NH4+-N去除率（B）的影响

Fig. 9 Effects of temperature on the growth performance OD600 nm（A）and NH4+-N removal efficiencies（B）of B. aryabhattai JN01

图10 菌株JN01在铵态氮（A）和亚硝态氮（B）为唯一氮源培养时的生长曲线及NH4+-N、NO3--N、NO2--N和TN 变化趋势

Fig. 10 Growth characteristics of strain JN01 and trends of NH4+-N, NO3--N, NO2--N, TN with initial NH4+-N (A) or initial NO2--N (B) as sole nitrogen source

表1 HNAD菌对NH4+-N、NO3--N、NO2--N和TN的去除情况

Table 1 Removal efficiencies of NH4+-N, NO3--N, NO2--N and TN by HNAD microorganisms

菌株名Strain	来源Source	NH₄⁺-N去除率NH₄⁺-N removal efficiency/%	NO₃^--N去除率NO₃^--N removal efficiency/%	NO₂^--N去除率NO₂^--N removal efficiency/%	总氮去除率Total nitrogen removal effciency/%	参考文献Reference
芽孢杆菌属Bacillus sp.
B. aryabhattai JN01	水产养殖池底污泥	92.78	/	82.30	80.73	本文
B. Subtilis Ab03	鱼虾养殖池塘	91.67	/	/	89.01	[10]
B. thuringiensis WXN-23	猪场猪皮饲料滤液	86.74	90.74	100.00	82.12	[11]
B. cereus GS-5	生物膜	87.10-93.10	69.40-88.40	/	/	[17]
B. litoralis N31	养殖水体	86.30	89.40	89.30	/	[18]
B. simplex H-b	土壤	82.16	67.29	78.69	/	[19]
其他种属微生物 Other species of microorganisms
H.venusta TJPU05	运城盐湖	86.12	95.68	100.00	84.57	[6]
P. alcaliphila	冬季水浸稻田土壤	100.00	100.00	100.00	/	[7]
Enterococcus sp. XH1	黄河三角洲海水	78.90	68.20	/	78.80	[12]
Acinetobacter johnsonii sp. N26	羊粪堆肥	95.50	93.60	/	/	[15]
Pseudomonas sp.GZWN4	养殖水体	98.62	82.54	99.72	/	[20]
Barnettozyma californica K1	海洋表层沉积物	99.11	98.84	99.13	90.16	[21]
Glutamicibacter sp. WS1	活性污泥	100.00	98.10	99.87	/	[22]

参考文献 32

[1]	Chen MP, He TX, Wu QF, et al. Enhanced heterotrophic nitrification and aerobic denitrification performance of Glutamicibacter arilaitensis EM-H8 with different carbon sources[J]. Chemosphere, 2023, 323: 138266. doi: 10.1016/j.chemosphere.2023.138266 URL
[2]	Yang T, Xin Y, Zhang L, et al. Characterization on the aerobic denitrification process of Bacillus strains[J]. Biomass Bioenergy, 2020, 140: 105677. doi: 10.1016/j.biombioe.2020.105677 URL
[3]	Chen JL, Xu J, Zhang SN, et al. Nitrogen removal characteristics of a novel heterotrophic nitrification and aerobic denitrification bacteria, Alcaligenes faecalis strain WT14[J]. J Environ Manage, 2021, 282: 111961. doi: 10.1016/j.jenvman.2021.111961 URL
[4]	Jetten MS, Logemann S, Muyzer G, et al. Novel principles in the microbial conversion of nitrogen compounds[J]. Antonie Van Leeuwenhoek, 1997, 71(1-2): 75-93. doi: 10.1023/A:1000150219937 URL
[5]	Huang MQ, Cui YW, Huang JL, et al. A novel Pseudomonas aeruginosa strain performs simultaneous heterotrophic nitrification-aerobic denitrification and aerobic phosphate removal[J]. Water Res, 2022, 221: 118823. doi: 10.1016/j.watres.2022.118823 URL
[6]	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, 2021, 16(6): 69. doi: 10.1007/s11783-021-1503-6
[7]	陈思宇, 刘晶, 杨正, 等. 低温异养硝化-好氧反硝化菌的分离及其除氮特性[J]. 湖南农业大学学报: 自然科学版, 2022, 48(6): 712-717.
	Chen SY, Liu J, Yang Z, et al. Isolation and nitrogen removal characteristics of a low temperature heterotrophic nitrification-aerobic denitrification bacterium[J]. J Hunan Agric Univ Nat Sci, 2022, 48(6): 712-717.
[8]	杨墨. 耐冷菌Janthinobacterium sp. M-11的异养硝化好氧反硝化特性及耐冷机制研究[D]. 哈尔滨: 哈尔滨工业大学, 2019.
	Yang M. Research on heterotrophic nitrification-aerobic denitrification characterisation and psychrotrophic mechanism of psychrotrophic strain Janthinobacterium sp. M-11[D]. Harbin:Harbin Institute of Technology, 2019.
[9]	李誉琦, 马佩钰, 刘涵, 等. 一株耐高温亚硝酸盐型反硝化细菌的鉴定及脱氮特性[J]. 生物技术通报, 2019, 35(9): 194-201. doi: 10.13560/j.cnki.biotech.bull.1985.2019-0130
	Li YQ, Ma PY, Liu H, et al. Identification and denitrification characters of a high-temperature-resistant nitrite-denitrifying bacterium[J]. Biotechnol Bull, 2019, 35(9): 194-201.
[10]	Wang Q, Fu WL, Lu RQ, et al. Characterization of Bacillus subtilis Ab03 for efficient ammonia nitrogen removal[J]. Syst Microbiol Biomanuf, 2022, 2(3): 580-588. doi: 10.1007/s43393-022-00088-6
[11]	Xu N, Liao M, Liang YQ, et al. Biological nitrogen removal capability and pathways analysis of a novel low C/N ratio heterotrophic nitrifying and aerobic denitrifying bacterium(Bacillus thuringiensis strain WXN-23)[J]. Environ Res, 2021, 195: 110797. doi: 10.1016/j.envres.2021.110797 URL
[12]	殷璐璐, 吕剑, 王建华, 等. 一株蔗糖偏好型海洋异养硝化-好氧反硝化菌的分离鉴定及性能研究[J]. 海洋环境科学, 2023, 42(3): 425-431.
	Yin LL, Lyu J, Wang JH, et al. Isolation, identification and characteristics of a sucrose-preferred heterotrophic nitrification-aerobic denitrification bacterium strain from seawater[J]. Mar Environ Sci, 2023, 42(3): 425-431.
[13]	杨景瑞, 王莹, 陈虎, 等. 一株好氧反硝化菌的脱氮特性及其氮代谢机理研究[J]. 工业水处理, 2023, 43(10):135-141. doi: 10.19965/j.cnki.iwt.2023-0192
	Yang J, Wang Y, Chen H, et al. Nitrogen removal characteristics and metabolism mechanism of an aerobic denitrifying bacteria[J]. Industrial Water Treatment, 2023, 43(10):135-141. doi: 10.19965/j.cnki.iwt.2023-0192
[14]	Fu WL, Wang Q, Chen SH, et al. Isolation and identification of an efficient aerobic denitrifying Pseudomonas stutzeri strain and characterization of its nitrite degradation[J]. Catalysts, 2021, 11(10): 1214. doi: 10.3390/catal11101214 URL
[15]	夏远舰, 杨小丽, 李海华, 等. 异养硝化-好氧反硝化菌Acinetobacter johnsonii sp. N26的脱氮性能及代谢途径[J]. 微生物学通报, 2023, 50(4): 1374-1395.
	Xia YJ, Yang XL, Li HH, et al. Optimization of nitrogen removal performance and metabolic pathway of a heterotrophic nitrifying-aerobic denitrifying bacterial strain Acinetobacter johnsonii sp. N26[J]. Microbiol China, 2023, 50(4): 1374-1395.
[16]	张宇红, 刘香宇, 董先博, 等. 好氧反硝化细菌SY-D-22的分离、优化及脱氮机理[J]. 微生物学通报, 2023, 50(5): 1815-1825.
	Zhang YH, Liu XY, Dong XB, et al. Aerobic denitrifying bacterium SY-D-22: isolation, optimization of denitrification conditions, and characterization of denitrification mechanism[J]. Microbiol China, 2023, 50(5): 1815-1825.
[17]	Rout PR, Dash RR, Bhunia P, et al. Role of Bacillus cereus GS-5 strain on simultaneous nitrogen and phosphorous removal from domestic wastewater in an inventive single unit multi-layer packed bed bioreactor[J]. Bioresour Technol, 2018, 262: 251-260. doi: 10.1016/j.biortech.2018.04.087 URL
[18]	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
[19]	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
[20]	Su ZP, Li Y, Pan LQ, et al. Nitrogen removal performance, quantitative detection and potential application of a novel aerobic denitrifying strain, Pseudomonas sp. GZWN4 isolated from aquaculture water[J]. Bioprocess Biosyst Eng, 2021, 44(6): 1237-1251. doi: 10.1007/s00449-021-02523-9
[21]	Fang JK, Liao SA, Zhang SS, et al. Characteristics of a novel heterotrophic nitrification-aerobic denitrification yeast, Barnettozyma californica K1[J]. Bioresour Technol, 2021, 339: 125665. doi: 10.1016/j.biortech.2021.125665 URL
[22]	魏渤惠, 罗晓, 吕鹏翼, 等. 高效异养硝化-好氧反硝化菌Glutamicibacter sp. WS1低温下对多种氮源的脱氮特性及氮代谢机制[J]. 环境科学, 2023, 44(9): 5006-5016.
	Wei BH, Luo X, Lü PY, et al. Nitrogen removal characteristics and metabolism mechanism of high-efficiency cold-tolerant heterotrophic nitrification-aerobic denitrification bacterium Glutamicibacter sp. WS1 for various nitrogen sources at low temperature[J]. Environ Sci, 2023, 44(9): 5006-5016.
[23]	Marek K, Pawęska K, Bawiec A. Treatment of wastewater with high ammonium nitrogen concentration[J]. J Ecol Eng, 2021, 22(4): 224-231.
[24]	杜全能, 朱文娟, 兰时乐. 一株异养硝化-好氧反硝化皱褶念珠菌(Diutina rugosa)的分离及脱氮特性[J]. 生物技术通报, 2020, 36(1): 60-65. doi: 10.13560/j.cnki.biotech.bull.1985.2019-0685
	Du QN, Zhu WJ, Lan SL. Isolation and characterization of heterotrophic nitrification-aerboic denitrification Diutina rugosa[J]. Biotechnol Bull, 2020, 36(1): 60-65.
[25]	Yang M, Lu DW, Yang JX, et al. Carbon and nitrogen metabolic pathways and interaction of cold-resistant heterotrophic nitrifying bacteria under aerobic and anaerobic conditions[J]. Chemosphere, 2019, 234: 162-170. doi: S0045-6535(19)31290-1 pmid: 31207421
[26]	Yang L, Wang XH, Cui S, et al. Simultaneous removal of nitrogen and phosphorous by heterotrophic nitrification-aerobic denitrification of a metal resistant bacterium Pseudomonas putida strain NP5[J]. Bioresour Technol, 2019, 285: 121360. doi: 10.1016/j.biortech.2019.121360 URL
[27]	Taylor SM, He YL, Zhao B, et al. Heterotrophic ammonium removal characteristics of an aerobic heterotrophic nitrifying-denitrifying bacterium, Providencia rettgeri YL[J]. J Environ Sci(China), 2009, 21(10): 1336-1341.
[28]	Patureau D, Bernet N, Delgenès JP, et al. Effect of dissolved oxygen and carbon-nitrogen loads on denitrification by an aerobic consortium[J]. Appl Microbiol Biotechnol, 2000, 54(4): 535-542. pmid: 11092629
[29]	Song T, Zhang XL, Li J, et al. A review of research progress of heterotrophic nitrification and aerobic denitrification microorganisms(HNADMs)[J]. Sci Total Environ, 2021, 801: 149319. doi: 10.1016/j.scitotenv.2021.149319 URL
[30]	黄诗玮, 田云, 马述, 等. 异养硝化-好氧反硝化菌氮代谢特性研究进展[J]. 生物学杂志, 2023, 40(1): 91-97.
	Huang SW, Tian Y, Ma S, et al. Research progress on nitrogen metabolism characteristics of heterotrophic nitrification-aerobic denitrification bacteria[J]. J Biol, 2023, 40(1): 91-97.
[31]	Zhang QL, Liu Y, Ai GM, et al. The characteristics of a novel heterotrophic nitrification-aerobic denitrification bacterium, Bacillus methylotrophicus strain L7[J]. Bioresour Technol, 2012, 108: 35-44. doi: 10.1016/j.biortech.2011.12.139 URL
[32]	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