辽河口沉积物反硝化过程研究

明红霞; 陈泉睿; 史银银; 苏洁; 于颖; 樊景凤

doi:10.3969/j.issn.0253-4193.2020.12.009

辽河口沉积物反硝化过程研究以反硝化功能基因丰度及nirK型细菌群落结构分析为例

doi: 10.3969/j.issn.0253-4193.2020.12.009

明红霞^1,,
陈泉睿^{1, 3},
史银银^{1, 2},
苏洁¹,
于颖²,
樊景凤^1, ,

1.
国家海洋环境监测中心国家生态环境保护近岸海域生态环境重点实验室，辽宁大连 116023
2.
大连海事大学环境科学与工程学院，辽宁大连 116026
3.
厦门大学海洋与地球学院，福建厦门 361005

基金项目: 国家重点研发计划"全球变化及应对专项"（2016YFA0601401）；国家自然科学基金（41676115）；海洋赤潮灾害立体监测技术与应用国家海洋局重点实验室基金（MATHAB201815）。

详细信息

作者简介:
明红霞（1981-），女，山东省德州市人，主要从事海洋微生物学研究。E-mail: mingtianhx@163.com

通讯作者:
樊景凤（1972-），研究员，主要从事海洋生态学研究。E-mail: jffan@nmemc.org.cn

中图分类号: P736.21
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- 被引次数: 0
出版历程
- 收稿日期: 2019-12-18
- 修回日期: 2020-06-08
- 网络出版日期: 2021-01-06
- 刊出日期: 2020-12-25

Study on denitrification process of sediment in the Liaohe EstuaryAnalysis of the abundance of denitrification functional genes and the community structure of nirK-type bacteria

Ming Hongxia^1
,,
Chen Quanrui^{1, 3},
Shi Yinyin^{1, 2},
Su Jie¹,
Yu Ying²,
Fan Jingfeng^{1
, ,}

1.
State Environmental Protection Key Laboratory of Coastal Ecosystem, National Marine Environmental Monitoring Center, Dalian 116023, China
2.
College of Environmental Science and Engineering, Dalian Maritime University, Dalian 116026, China
3.
College of Ocean and Earth, Xiamen University, Xiamen 361005, China

摘要

摘要: 近年来，辽河口沿岸人类活动频繁，工农业活动发达，大量的氮、磷等营养物质输入至感潮河段继而排放入海，导致辽河口水体富营养化程度加剧。沉积物区域是反硝化作用的重要发生地和微生物富集地。微生物能够将沉积物中的硝酸盐及亚硝酸盐还原为N₂O和N₂释放到大气中，进而减轻河口生态系统氮负荷。本文中的实验采用定量PCR技术测定辽河口表层沉积物反硝化过程功能基因narG、nirK、norB、nosZ的基因丰度，结果表明，主导硝酸盐还原的narG功能基因丰度最高。使用高通量测序技术对nirK型功能基因进行测序，结果显示，在辽河口反硝化细菌中Devosia、Phaeobacter、Alcaligenes、Pseudomonas菌属丰度较高。反硝化功能基因的丰度主要受到沉积物粒径的影响，norB基因丰度与多个环境因子显著相关。nirK型反硝化细菌的群落结构和多样性主要受盐度、pH、溶解氧以及NO₂⁻的影响。研究分析了反硝化功能基因丰度和细菌群落结构及其主要影响因子，其结果为辽河口水体富营养化中氮元素归趋的认知提供理论依据。
- 辽河口 /
- 沉积物 /
- nirK /
- 细菌群落结构 /
- 反硝化功能基因
Abstract: In recent years, the water quality of the Liaohe Estuary has been declining, and a large input of nitrogen has increased the burden on the environment. The denitrification process releases nitrogen from sediments into the atmosphere in the form of N₂O and N₂, which reduces the nitrogen load in the estuarine ecosystem. In this study, quantitative PCR was used to determine the levels of four denitrification functional genes narG, nirK, norB and nosZ present in the surface sediments of the Liaohe Estuary. The narG gene, which dominates nitrate reduction, was found to be the most abundant. The nirK-type denitrification functional gene was sequenced by high-throughput sequencing. The results showed that the genus of denitrifying bacteria in the Liaohe Estuary was mainly Devosia, Phaeobacter, Alcaligenes, Pseudomonas. The abundance of denitrification function genes is mainly affected by sediment properties, except that norB gene is significantly related to many environmental factors. The community structure and diversity of nirK type denitrifying bacteria are mainly affected by salinity, pH, dissolved oxygen and ${\rm {NO}}_2^- $ contents. The effects of environmental factors on the abundance and community structure of denitrifying bacteria were studied. The results provided a theoretical basis for alleviating the fate of nitrogen elements in the eutrophication of Liaohe Estuary.
- Liaohe Estuary /
- sediment /
- nirK /
- bacterial community structure /
- functional genes of denitrifying bacteria

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图 1 辽河口12个采样点站位布设及分组情况

Fig. 1 Distribution and grouping of 12 stations in Liaohe Estuary

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图 2 辽河口narG、nirK、norB、nosZ功能基因丰度

Fig. 2 Functional gene abundance of narG, nirK, norB, and nosZ in the Liaohe Estuary

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图 3 辽河口环境因子与功能基因丰度的皮尔逊相关性

Fig. 3 Pearson correlation between environmental factors and the abundance of functional genes in the Liaohe Estuary

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图 4 nirK型功能基因OTUs分布的韦恩图（a）和花瓣图（b）

Fig. 4 Venn diagram (a) and petal map (b) of the nirK functional gene OTUs distribution

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图 5 nirK型反硝化细菌属水平的群落结构柱状图

Fig. 5 Histogram of community structure at the genus level of nirK-type denitrifying bacterias

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图 6 基于LEfSe分析的辽河口沉积物中nirK型反硝化细菌的丰度柱状图(a)和进化分支图(b)

LDA SCORE是指用线性判别分析（LDA）对数据进行降维和评估差异显著的物种的影响力

Fig. 6 Abundance histogram (a) and evolutionary branch map (b) of nir K-type denitrifying bacteria in the sediments of the Liaohe Estuary based on LEfSe analysis

LDA SCORE means using linear discriminant analysis (LDA) to reduce the dimension of data and evaluate the influence of species with significant differences

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图 7 nirK型反硝化细菌群落与环境因子变量的冗余分析图

横轴和纵轴分别在两个主要维度上解释了微生物群落组成中携带nirK基因的突变量

Fig. 7 Redundancy analysis of nirK-type denitrifying bacteria community and environmental factor variables

The horizontal axils and the vertical axils explained the variation of nirK gene in microbial community composition in two main dimensions

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表 1 辽河口底层水中环境因子理化参数^[11]

Tab. 1 Physical and chemical parameters of environmental factors in bottom water of the Liaohe Estuary^[11]

站位	温度/℃	盐度	电导率/mS·cm⁻¹	pH	DO浓度/mg·L⁻¹	$ {{\rm {NH}}_4^+} $浓度/μmol·L⁻¹	$ {{\rm {NO}}_2^-} $浓度/μmol·L⁻¹	$ {{\rm {NO}}_3^- }$浓度/μmol·L⁻¹	$ {{\rm {PO}}_4^{3-}} $浓度/μmol·L⁻¹
A1	26.2	31.5	47.3	8.00	6.3	1.08	0.24	0.80	0.11
A2	27.0	28.7	44.6	7.99	6.3	1.11	0.32	7.27	0.13
A3	26.9	27.7	43.2	7.98	6.4	1.18	0.39	8.95	0.18
A4	26.7	25.5	40.0	7.94	6.1	1.49	0.48	12.32	0.01
B1	26.5	19.6	31.5	7.86	6.3	5.89	0.37	17.02	0.29
B2	27.0	20.7	33.1	7.92	6.1	5.61	0.35	15.27	0.20
B3	26.8	19.8	32.0	7.88	5.0	5.94	0.40	15.95	0.28
B4	26.5	13.1	21.8	7.72	3.3	9.50	0.61	15.23	0.27
C1	25.9	7.0	13.3	7.62	4.0	9.22	0.60	18.63	0.34
C2	26.0	5.5	9.7	7.57	3.1	14.28	1.02	22.06	0.40
C3	25.9	2.4	4.5	7.54	6.2	13.61	0.94	21.98	0.31
C4	25.7	0.8	15.8	7.54	6.9	11.83	0.93	5.08	0.23

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表 2 辽河口表层沉积物中环境因子理化参数^[11]

Tab. 2 Physical and chemical parameters of environmental factors in surface sediments of the Liaohe Estuary^[11]

站位	TOC含量/%	TP浓度/μg·g⁻¹	TN浓度/μg·g⁻¹	黏土含量/%	粉砂含量/%	砂含量/%
A1	0.82	185.0	34.7	31.18	58.48	10.34
A2	0.43	57.2	40.2	23.53	51.55	24.92
A3	0.53	94.4	65.3	26.24	61.35	12.41
A4	0.56	90.3	60.5	31.01	60.13	8.85
B1	0.49	97.1	131.0	37.00	62.97	0.03
B2	0.70	99.6	298.0	14.57	56.67	28.76
B3	0.60	102.0	136.0	21.46	61.57	16.97
B4	0.14	117.0	76.5	12.60	59.54	27.87
C1	0.34	109.0	87.9	18.01	66.96	15.04
C2	0.34	47.8	38.3	4.64	47.36	47.99
C3	0.46	84.4	90.5	18.47	53.82	27.71
C4	0.34	26.6	30.1	2.67	12.45	84.88

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表 3 反硝化功能基因的引物

Tab. 3 Primers for denitrification genes

基因	引物	5′-3′ 序列	参考文献
nirK	FlaCu	ATC ATG GTS CTG CCG CG	[12]
nirK	R3Cu	GCC TCG ATC AGR TTG TGG TT	[12]
nosZ	nosZ1126F	GGG CTB GGG CCR TTG CA	[13]
nosZ	nosZ1138R	GAA GCG RTC CTT SGA RAA CTTG	[13]
narG	narG571F	CCG ATY CCG GCV ATG TCS AT	[13]
narG	narG773R	GGN ACG TTN GAD CCC CA	[13]
norB	norB1F	CGN GAR TTY CTS GAR CAR CC	[14]
norB	norB3R	CCY TCV ACC CAG ASA TGC AC	[14]

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表 4 辽河口nirK型反硝化细菌丰度及多样性

Tab. 4 Abundance and diversity of nirK-type denitrifying bacteria in the Liaohe Estuary

采样点	OTUs 个数	有效序列数	Coverage /%	Chao1 指数	ACE 指数	Shannon 指数	Simpson 指数
A1	112	37071	99.96	138.25	127.22	2.66	0.146
A2	96	36947	99.99	97.00	97.99	2.28	0.230
A3	121	36164	99.99	121.27	122.16	2.71	0.151
A4	132	30487	99.95	140.67	139.96	2.54	0.201
B1	126	30315	99.94	146.00	135.69	2.52	0.188
B2	136	32816	99.97	138.80	139.67	2.59	0.169
B3	237	37310	99.96	244.09	242.59	3.25	0.122
B4	162	31673	99.94	183.86	172.69	2.78	0.144
C1	236	37745	99.97	245.43	240.57	3.32	0.103
C2	305	37274	99.94	319.62	313.62	3.62	0.077
C3	291	36136	99.97	293.77	293.63	3.60	0.088
C4	160	33334	99.89	248.00	267.54	2.82	0.140

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参考文献(32)

[1]	Smith D P, Thrash J C, Nicora C D, et al. Proteomic and transcriptomic analyses of “Candidatus Pelagibacter ubique”describe the first PII-independent response to nitrogen limitation in a free-living Alphaproteobacterium[J]. mBio, 2013, 4(6): e00133−12.
[2]	Herbert R A. Nitrogen cycling in coastal marine ecosystems[J]. FEMS Microbiology Reviews, 1999, 23(5): 563−590. doi: 10.1111/j.1574-6976.1999.tb00414.x
[3]	Helen D, Kim H, Tytgat B, et al. Highly diverse nirK genes comprise two major clades that harbour ammonium-producing denitrifiers[J]. BMC Genomics, 2016, 17(1): 155. doi: 10.1186/s12864-016-2465-0
[4]	Zumft W G. Cell biology and molecular basis of denitrification[J]. Microbiology and Molecular Biology Reviews, 1997, 61(4): 533−616. doi: 10.1128/.61.4.533-616.1997
[5]	Penton C R, Johnson T A, Quensen J F, et al. Functional genes to assess nitrogen cycling and aromatic hydrocarbon degradation: primers and processing matter[J]. Frontiers in Microbiology, 2013, 4: 279.
[6]	Santoro A E, Boehm A B, Francis C A. Denitrifier community composition along a nitrate and salinity gradient in a coastal aquifer[J]. Applied and Environmental Microbiology, 2006, 72(3): 2102−2109. doi: 10.1128/AEM.72.3.2102-2109.2006
[7]	Jones C M, Hallin S. Ecological and evolutionary factors underlying global and local assembly of denitrifier communities[J]. The ISME Journal, 2010, 4(5): 633−641. doi: 10.1038/ismej.2009.152
[8]	陶怡乐, 温东辉. 细菌硝酸盐异化还原成铵过程及其在河口生态系统中的潜在地位与影响[J]. 微生物学通报, 2016, 43(1): 172−181. Tao Yile, Wen Donghui. Dissimilatory nitrate reduction to ammonium: The potential and impacts in estuarine regions[J]. Microbiology China, 2016, 43(1): 172−181.
[9]	袁晓敏, 杨继松, 刘凯, 等. 盐分对辽河口湿地土壤DOC及CO₂生成的影响[J]. 生态学杂志, 2017, 36(8): 2111−2117. Yuan Xiaomin, Yang Jisong, Liu Kai, et al. Effects of salinity on DOC concentration and CO₂ production of wetland soil in Liaohe Estuarine[J]. Chinese Journal of Ecology, 2017, 36(8): 2111−2117.
[10]	Bu Hongmei, Meng Wei, Zhang Yuan. Nitrogen pollution and source identification in the Haicheng River basin in Northeast China[J]. Science of the Total Environment, 2011, 409(18): 3394−3402. doi: 10.1016/j.scitotenv.2011.05.030
[11]	张慧珍, 常永凯, 陈泉睿, 等. 辽河口沉积物中古菌和细菌群落结构分析[J]. 海洋学报, 2018, 40(6): 113−130. Zhang Huizhen, Chang Yongkai, Chen Quanrui, et al. Community structure analysis of archaea and bacteria in sediments of Liaohe Estuary[J]. Haiyang Xuebao, 2018, 40(6): 113−130.
[12]	Hallin S, Lindgren P E. PCR detection of genes encoding nitrite reductase in denitrifying bacteria[J]. Applied and Environmental Microbiology, 1999, 65(4): 1652−1657. doi: 10.1128/AEM.65.4.1652-1657.1999
[13]	Chen Zhe, Liu Jinbo, Wu Minna, et al. Differentiated response of denitrifying communities to fertilization regime in paddy soil[J]. Microbial Ecology, 2012, 63(2): 446−459. doi: 10.1007/s00248-011-9909-5
[14]	Casciotti K L, Ward B B. Phylogenetic analysis of nitric oxide reductase gene homologues from aerobic ammonia-oxidizing bacteria[J]. FEMS Microbiology Ecology, 2005, 52(2): 197−205. doi: 10.1016/j.femsec.2004.11.002
[15]	Dang Hongyue, Li Jing, Chen Ruipeng, et al. Diversity, abundance, and spatial distribution of sediment ammonia-oxidizing Betaproteobacteria in response to environmental gradients and coastal eutrophication in Jiaozhou Bay, China[J]. Applied and Environmental Microbiology, 2010, 76(14): 4691−4702. doi: 10.1128/AEM.02563-09
[16]	Jiang Xiaoliang, Yao Lu, Guo Laodong, et al. Multi-scale factors affecting composition, diversity, and abundance of sediment denitrifying microorganisms in Yangtze lakes[J]. Applied Microbiology and Biotechnology, 2017, 101(21): 8015−8027. doi: 10.1007/s00253-017-8537-5
[17]	Yang Jisong, Zhan Chao, Li Yunzhao, et al. Effect of salinity on soil respiration in relation to dissolved organic carbon and microbial characteristics of a wetland in the Liaohe River estuary, Northeast China[J]. Science of the Total Environment, 2018, 642: 946−953. doi: 10.1016/j.scitotenv.2018.06.121
[18]	Mahjoubi M, Aliyu H, Cappello S, et al. The genome of Alcaligenes aquatilis strain BU33N: insights into hydrocarbon degradation capacity[J]. PLoS One, 2019, 14(9): e0221574. doi: 10.1371/journal.pone.0221574
[19]	Jiang Yan, Wen Jianping, Bai Jing, et al. Biodegradation of phenol at high initial concentration by Alcaligenes faecalis[J]. Journal of Hazardous Materials, 2007, 147(1/2): 672−676.
[20]	Chen Yong, Zhu Sidong, Lin Danqiu, et al. Devosia Naphthalenivorans sp. nov., isolated from East Pacific Ocean sediment[J]. International Journal of Systematic and Evolutionary Microbiology, 2019, 69(7): 1974−1979. doi: 10.1099/ijsem.0.003410
[21]	Park S, Jung Y T, Kim S, et al. Devosia confluentis sp. nov., isolated from the junction between the ocean and a freshwater lake, and reclassification of two Vasilyevaea species as Devosia enhydra comb. nov. and Devosia mishustinii comb. nov[J]. International Journal of Systematic and Evolutionary Microbiology, 2016, 66(10): 3935−3941. doi: 10.1099/ijsem.0.001291
[22]	Wolińska A, Kuźniar A, Zielenkiewicz U, et al. Metagenomic analysis of some potential nitrogen-fixing bacteria in arable soils at different formation processes[J]. Microbial Ecology, 2017, 73(1): 162−176. doi: 10.1007/s00248-016-0837-2
[23]	Rivas R, Willems A, Subba-Rao N S, et al. Description of Devosia neptuniae sp. nov. that nodulates and fixes nitrogen in symbiosis with Neptunia natans, an aquatic legume from India[J]. Systematic and Applied Microbiology, 2003, 26(1): 47−53. doi: 10.1078/072320203322337308
[24]	Fu Yingnan, Wang Rui, Zhang Zilian, et al. Complete genome sequence of the d-Amino acid catabolism bacterium Phaeobacter sp. Strain JL2886, isolated from deep seawater of the South China Sea[J]. Genome Announcements, 2016, 4(5): e00913−16.
[25]	Gram L, Rasmussen B B, Wemheuer B, et al. Phaeobacter inhibens from the Roseobacter clade has an environmental niche as a surface colonizer in harbors[J]. Systematic and Applied Microbiology, 2015, 38(7): 483−493. doi: 10.1016/j.syapm.2015.07.006
[26]	Trautwein K, Hensler M, Wiegmann K, et al. The marine bacterium Phaeobacter inhibens secures external ammonium by rapid buildup of intracellular nitrogen stocks[J]. FEMS Microbiology Ecology, 2018, 94(10): 1−14.
[27]	Perkins T L, Clements K, Baas J H, et al. Sediment composition influences spatial variation in the abundance of human pathogen indicator bacteria within an estuarine environment[J]. PLoS One, 2014, 9(11): e112951. doi: 10.1371/journal.pone.0112951
[28]	Luo Ling, Zhou Zhichao, Gu Jidong. Distribution, diversity and abundance of bacterial laccase-like genes in different particle size fractions of sediments in a subtropical mangrove ecosystem[J]. Ecotoxicology, 2015, 24(7/8): 1508−1516.
[29]	Zaghmouri I, Michotey V D, Armougom F, et al. Salinity shifts in marine sediment: importance of number of fluctuation rather than their intensities on bacterial denitrifying community[J]. Marine Pollution Bulletin, 2018, 130: 76−83. doi: 10.1016/j.marpolbul.2018.03.020
[30]	Liu Wenzhi, Yao Lu, Jiang Xiaoliang, et al. Sediment denitrification in Yangtze lakes is mainly influenced by environmental conditions but not biological communities[J]. Science of the Total Environment, 2018, 616−617: 978−987. doi: 10.1016/j.scitotenv.2017.10.221
[31]	Zhou Shilei, Huang Tinglin, Zhang Chunhua, et al. Illumina MiSeq sequencing reveals the community composition of NirS-Type and NirK-Type denitrifiers in Zhoucun reservoir—a large shallow eutrophic reservoir in northern China[J]. RSC Advances, 2016, 6(94): 91517−91528. doi: 10.1039/C6RA18017E
[32]	Fan Haoxin, Bolhuis H, Stal L J. Denitrification and the denitrifier community in coastal microbial mats[J]. FEMS Microbiology Ecology, 2015, 91(3): 1−11.