刘梦帅1, ?,,
陈帅民1,
王新珍1,
王仕琴1,
胡春胜1,
刘彬彬1,
王凤花1,,
1.中国科学院遗传与发育生物学研究所农业资源研究中心/中国科学院农业水资源重点实验室/河北省土壤生态学重点实验室 石家庄 050022
2.中国科学院大学 北京 100049
基金项目: 国家重点研发计划项目2016YFD0800100
详细信息
通讯作者:王凤花, 主要研究方向为微生物分子生态学。E-mail:fhwang@sjziam.ac.cn
?同等贡献者: 赵会成, 主要研究方向为微生物分子生态学, E-mail:hczhao@sjziam.ac.cn;刘梦帅, 主要研究方向为微生物分子生态学, E-mail:lms9095@163.com中图分类号:S154
计量
文章访问数:310
HTML全文浏览量:7
PDF下载量:399
被引次数:0
出版历程
收稿日期:2020-06-29
录用日期:2020-09-01
刊出日期:2021-01-01
Isolation, identification, and functional characterization of denitrifiers from the deep vadose zone and aquifer in the North China Plain
ZHAO Huicheng1, 2, ?,,LIU Mengshuai1, ?,,
CHEN Shuaimin1,
WANG Xinzhen1,
WANG Shiqin1,
HU Chunsheng1,
LIU Binbin1,
WANG Fenghua1,,
1. Center for Agricultural Resources Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences/Key Laboratory of Agricultural Water Resources, Chinese Academy of Sciences/Hebei Key Laboratory of Soil Ecology, Shijiazhuang 050022, China
2. University of Chinese Academy of Sciences, Beijing 100049, China
Funds: the National Key Research and Development Program of China2016YFD0800100
More Information
Corresponding author:WANG Fenghua, E-mail:fhwang@sjziam.ac.cn
?Equivalent contributors摘要
HTML全文
图
参考文献
相关文章
施引文献
资源附件
访问统计
摘要
摘要:华北平原农田由于长期过量施用氮肥，造成了土壤硝酸盐累积，导致地下水硝酸盐污染日趋严重。微生物的反硝化作用可将土壤中累积的硝酸盐或亚硝酸盐还原为气态产物，是消减厚包气带土壤累积的硝酸盐的重要途径。因此筛选高效反硝化微生物资源，对人工强化厚包气带土壤反硝化脱氮，阻控地下水硝酸盐污染具有重要作用。基于此，本研究采集位于华北平原的中国科学院栾城农业生态系统试验站长期施氮[施氮量为600 kg（N）?hm-2?a-1]定位试验0~150 m农田厚包气带及含水层土壤样品，从中筛选到62株细菌。16S rRNA基因序列分析表明这62株菌株与变形菌门（Proteobacteria）、放线菌门（Actinobacteria）、厚壁菌门（Firmicutes）中的9个属具有较高的同源性。根据系统发育树的结果，挑选7株亲缘关系较远的菌株进行反硝化潜势试验，结果表明，菌株L71、L13和L103具备反硝化产气能力。电镜观察结果表明，这3株菌均为无鞭毛的杆状细菌，其长度分别为1.0 μm、1.5 μm和1.5 μm，只有L103具有运动能力。此外，菌株L103具有完全反硝化能力，且脱氮能力受到pH的影响，在本试验条件下，菌株L103的反硝化速率高达1.62~2.36 g（KNO3）?d-1?L-1，具备实际应用潜力。本研究表明华北平原厚包气带土壤中存在完全反硝化微生物，并可为人工强化治理厚包气带土壤硝酸盐污染提供菌种资源和理论依据。
Abstract:The long-term excessive use of nitrogen (N) fertilizer in agricultural regions has increased soil nitrate and residual N accumulation and poses a threat to groundwater quality. Nitrate leaching into the vadose zone is becoming a global concern as the N stock in this habitat comprises a significant portion of N budgets. The vadose zone is also an essential channel for the conversion and reduction of nitrate. Therefore, the elimination of nitrate accumulation in the vadose zone is significant for maintaining groundwater safety. Microbial denitrification is the reduction of nitrogen nitrate (NO3--N) to gaseous nitric oxide (NO), nitrous oxide (N2O), or dinitrogen (N2). This mechanism is essential for removing excess nitrate in the subsoil before it leaches into the groundwater and saturates deep soil zones or discharges into ground aquifers through subsurface drainage. Therefore, isolating and screening bacteria with strong denitrification abilities may strengthen the vadose zone and aquifer microbial denitrification process, preventing groundwater nitrate pollution. In this study, 62 microbial denitrifiers were isolated from the 0-150 m vadose zone in a position experiment of long-term nitrogen application in the Agricultural Ecosystem Experimental Station of Luancheng, Chinese Academy of Sciences, located the North China Plain. 16S ribosomal RNA (16S rRNA) gene sequence analysis showed that the isolated denitrifiers had high homology with nine genera, belonging to the phyla Proteobacteria, Actinobacteria, and Firmicutes. Of the 62 denitrifiers, seven strains (L37, L71, L96, L103, L104, L133, and L13) were selected for denitrification potential experiments based on the phylogenetic tree results. Gas kinetics under anoxic incubations showed that three strains (L71, L13, and L103) could reduce nitrate substrates to nitrous oxides, such as N2O and N2, in anaerobic conditions. Electron microscopy showed that the denitrifying strains were 1.0 μm (L71), 1.5 μm (L13), and 1.5 μm (L103) long rod-shaped bacteria. Strain L103 had motile and complete denitrification abilities, and the denitrification rate was between 1.62 and 2.36 g(KNO3)?d-1?L-1, indicating a high potential for use in agricultural practices. Furthermore, the denitrification ability of strain L103 was inhibited in acidic conditions, suggesting that pH also affects the microbial denitrification potential. Bacterial denitrifiers that reduce nitrate to N2 in hypoxic/anoxic conditions exist in the deep vadose zone of the North China Plain. The denitrification potential of these strains is important for understanding how microorganisms contribute to the soil nitrate accumulation self-remediation process.
?Equivalent contributors
注释:
1) ?同等贡献者: 赵会成, 主要研究方向为微生物分子生态学, E-mail:hczhao@sjziam.ac.cn;刘梦帅, 主要研究方向为微生物分子生态学, E-mail:lms9095@163.com
HTML全文
图1基于16S rRNA基因序列的筛选得到62株潜在反硝化菌株的系统进化树
Figure1.Phylogenetic tree of screened 62 potential denitrifying strains based on 16S rRNA gene sequences analysis
下载: 全尺寸图片幻灯片
图2菌株L71、L13和L103的反硝化潜势
Figure2.Denitrification potentials of the selected strains L71, L13 and L103
下载: 全尺寸图片幻灯片
图3菌株L71、L13和L103的细菌形态特征
Figure3.Morphological characteristics of the selected strains L71, L13 and L103
下载: 全尺寸图片幻灯片
图4菌株L103在pH 7.4和pH 6.6下的反硝化潜势
Figure4.Denitrification potential of the strain L103 at pH 6.6 and pH 7.4
下载: 全尺寸图片幻灯片表1菌株L71、L13和L103的生物学特性
Table1.Biological features of the selected strains L71, L13 and L103
| 菌株 Strain | 运动性 Motile | 形状 Shape | 长度 Length (μm) | 鞭毛 Flagellum |
| L71 | 无 No | 杆状 Rod | 1.0 | 无 No |
| L13 | 无 No | 杆状 Rod | 1.5 | 无 No |
| L103 | 有 Yes | 杆状 Rod | 1.5 | 无 No |
下载: 导出CSV表2菌株L103在不同pH下将KNO3还原成N2所需时间及最大反硝化速率
Table2.Time consumed to reduce KNO3 to N2 and max denitrification rate of the strain L103 at different pH
| 7.8 | 7.4 | 7.0 | 6.6 | 6.2 | 5.8 | |
| 时间Time (h) | 6 | 6 | 10 | 16 | — | — |
| 最大反硝化速率 Max denitrification rate [g(KNO3)?d–1?L–1] | 2.36 | 2.02 | 1.62 | 0.77 | — | — |
| “—”表示菌株L103不能在该pH条件下生长。“—” indicates strain L103 can not grow under this pH condition. | ||||||
下载: 导出CSV参考文献
| [1] | ERISMAN J W, GALLOWAY J N, SEITZINGER S, et al. Consequences of human modification of the global nitrogen cycle[J]. Philosophical Transactions of the Royal Society B:Biological Sciences, 2013, 368(1621):20130116 doi: 10.1098/rstb.2013.0116 |
| [2] | 胡春胜, 张玉铭, 秦树平, 等.华北平原农田生态系统氮素过程及其环境效应研究[J].中国生态农业学报, 2018, 26(10):1501-1514 http://www.ecoagri.ac.cn/zgstny/ch/reader/view_abstract.aspx?file_no=2018-1009&flag=1 HU C S, ZHANG Y M, QIN S P, et al. Nitrogen processes and related environmental effects on agro-ecosystem in the North China Plain[J]. Chinese Journal of Eco-Agriculture, 2018, 26(10):1501-1514 http://www.ecoagri.ac.cn/zgstny/ch/reader/view_abstract.aspx?file_no=2018-1009&flag=1 |
| [3] | JU X T, XING G X, CHEN X P, et al. Reducing environmental risk by improving N management in intensive Chinese agricultural systems[J]. Proceedings of the National Academy of Sciences of the United States of America, 2009, 106(9):3041-3046 doi: 10.1073/pnas.0813417106 |
| [4] | 梁慧雅, 王仕琴, 魏守才.华北山前平原典型厚包气带硝态氮分布累积规律[J].土壤, 2017, 49(6):1179-1186 https://www.cnki.com.cn/Article/CJFDTOTAL-TURA201706017.htm LIANG H Y, WANG S Q, WEI S C. Nitrate distribution and accumulation in deep unsaturated zone soils in piedmont of North China Plain[J]. Soils, 2017, 49(6):1179-1186 https://www.cnki.com.cn/Article/CJFDTOTAL-TURA201706017.htm |
| [5] | 马洪斌, 李晓欣, 胡春胜.中国地下水硝态氮污染现状研究[J].土壤通报, 2012, 43(6):1532-1536 https://www.cnki.com.cn/Article/CJFDTOTAL-TRTB201206046.htm MA H B, LI X X, HU C S. Status of nitrate nitrogen contamination of groundwater in China[J]. Chinese Journal of Soil Science, 2012, 43(6):1532-1536 https://www.cnki.com.cn/Article/CJFDTOTAL-TRTB201206046.htm |
| [6] | 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 |
| [7] | HENRY S, BRU D, STRES B, et al. Quantitative detection of the nosZ gene, encoding nitrous oxide reductase, and comparison of the abundances of 16S rRNA, narG, nirK, and nosZ genes in soils[J]. Applied and Environmental Microbiology, 2006, 72(8):5181-5189 doi: 10.1128/AEM.00231-06 |
| [8] | CANFIELD D E, GLAZER A N, FALKOWSKI P G. The evolution and future of earth's nitrogen cycle[J]. Science, 2010, 330(6001):192-196 doi: 10.1126/science.1186120 |
| [9] | CICERONE R J. Analysis of sources and sinks of atmospheric nitrous oxide (N2O)[J]. Journal of Geophysical Research:Atmospheres, 1989, 94(D15):18265-18271 doi: 10.1029/JD094iD15p18265 |
| [10] | 王海涛, 郑天凌, 杨小茹, 等.土壤反硝化的分子生态学研究进展及其影响因素[J].农业环境科学学报, 2013, 32(10):1915-1924 https://www.cnki.com.cn/Article/CJFDTOTAL-NHBH201310004.htm WANG H T, ZHENG T L, YANG X R, et al. Molecular ecology research progress for soil denitrification and research status for its influencing factors[J]. Journal of Agro-Environment Science, 2013, 32(10):1915-1924 https://www.cnki.com.cn/Article/CJFDTOTAL-NHBH201310004.htm |
| [11] | 方晶晶, 马传明, 刘存富.反硝化细菌研究进展[J].环境科学与技术, 2010, 33(6E):206-210 https://www.cnki.com.cn/Article/CJFDTOTAL-FJKS2010S1056.htm FANG J J, MA C M, LIU C F. The advance of study on denitrifying bacteria[J]. Environmental Science & Technology, 2010, 33(6E):206-210 https://www.cnki.com.cn/Article/CJFDTOTAL-FJKS2010S1056.htm |
| [12] | 王惠芬, 温展, 张秋玲, 等.土壤中反硝化细菌的分离与脱氮性能初步研究[J].环保科技, 2018, 24(6):19-24 https://www.cnki.com.cn/Article/CJFDTOTAL-GZHB201806004.htm WANG H F, WEN Z, ZHANG Q L, et al. A preliminary study on the screening of denitrifiers and their denitrification performance in soil[J]. Environmental Protection and Technology, 2018, 24(6):19-24 https://www.cnki.com.cn/Article/CJFDTOTAL-GZHB201806004.htm |
| [13] | 陈寒玉, 靳慧征, 靳慧霞.一株好氧反硝化细菌的筛选及对土壤脱氮效果研究[J].中国农学通报, 2018, 34(20):55-62 https://www.cnki.com.cn/Article/CJFDTOTAL-ZNTB201820009.htm CHEN H Y, JIN H Z, JIN H X. Screening of an aerobic denitrification strain and its nitrogen removal characteristics for soil[J]. Chinese Agricultural Science Bulletin, 2018, 34(20):55-62 https://www.cnki.com.cn/Article/CJFDTOTAL-ZNTB201820009.htm |
| [14] | FALK S, LIU B B, BRAKER G. Isolation, genetic and functional characterization of novel soil nirK-type denitrifiers[J]. Systematic and Applied Microbiology, 2010, 33(6):337-347 |
| [15] | LIU B B, MORKVED P T, FROSTEG?RD ?, et al. Denitrification gene pools, transcription and kinetics of NO, N2O and N2 production as affected by soil pH[J]. FEMS Microbiology Ecology, 2010, 72(3):407-417 |
| [16] | QU Z, WANG J G, ALM?Y T, et al. Excessive use of nitrogen in Chinese agriculture results in high N2O/(N2O+N2) product ratio of denitrification, primarily due to acidification of the soils[J]. Global Change Biology, 2014, 20(5):1685-1698 doi: 10.1111/gcb.12461 |
| [17] | ?IMEK M, COOPER J E. The influence of soil pH on denitrification:Progress towards the understanding of this interaction over the last 50 years[J]. European Journal of Soil Science, 2010, 53(3):345-354 doi: 10.1046/j.1365-2389.2002.00461.x |
| [18] | LYCUS P, B?THUN L, BERGAUST L, et al. Phenotypic and genotypic richness of denitrifiers revealed by a novel isolation strategy[J]. The ISME Journal, 2017, 11(10):2219-2232 |
| [19] | BAKER G C, SMITH J J, COWAN D A. Review and re-analysis of domain-specific 16S primers[J]. Journal of Microbiological Methods, 2003, 55(3):541-555 |
| [20] | TAMURA K, DUDLEY J, NEI M, et al. MEGA4:Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0[J]. Molecular Biology and Evolution, 2007, 24(8):1596-1599 |
| [21] | MOLSTAD L, D?RSCH P, BAKKEN L R. Robotized incubation system for monitoring gases (O2, NO, N2O, N2) in denitrifying cultures[J]. Journal of Microbiological Methods, 2007, 71(3):202-211 |
| [22] | CHEN S M, WANG F H, ZHANG Y M, et al. Organic carbon availability limiting microbial denitrification in the deep vadose zone[J]. Environmental Microbiology, 2018, 20(3):980-992 |
| [23] | ONYENWOKE R U, BRILL J A, FARAHI K, et al. Sporulation genes in members of the low G+C Gram-type-positive phylogenetic branch (Firmicutes)[J]. Archives of Microbiology, 2004, 182(2):182-192 http://bioinformatics.oxfordjournals.org/external-ref?access_num=10.1007/s00203-004-0696-y&link_type=DOI |
| [24] | STONE M M, KAN J J, PLANTE A F. Parent material and vegetation influence bacterial community structure and nitrogen functional genes along deep tropical soil profiles at the Luquillo Critical Zone Observatory[J]. Soil Biology and Biochemistry, 2015, 80:273-282 http://www.sciencedirect.com/science/article/pii/S0038071714003666 |
| [25] | 马林, 柏兆海, 胡春胜.科技部"十三五"农业面源和重金属污染农田综合防治与修复技术研发重点专项"农田氮磷淋溶损失污染与防控机制研究"项目正式启动[J].中国生态农业学报, 2016, 24(11):1575-1576 http://www.ecoagri.ac.cn/zgstny/ch/reader/view_abstract.aspx?file_no=20161116&flag=1 MA L, BAI Z H, HU C S. Research on mechanisms of nitrogen and phosphorus leaching loss and control of farmland[J]. Chinese Journal of Eco-Agriculture, 2016, 24(11):1575-1576 http://www.ecoagri.ac.cn/zgstny/ch/reader/view_abstract.aspx?file_no=20161116&flag=1 |
| [26] | SAMESHIMA-SAITO R, CHIBA K, MINAMISAWA K. Correlation of denitrifying capability with the existence of nap, nir, nor and nos genes in diverse strains of soybean bradyrhizobia[J]. Microbes and Environments, 2006, 21(3):174-184 |
| [27] | MINAMIKAWA K, NISHIMURA S, SAWAMOTO T, et al. Annual emissions of dissolved CO2, CH4, and N2O in the subsurface drainage from three cropping systems[J]. Global Change Biology, 2010, 16(2):796-809 http://europepmc.org/abstract/AGR/IND44311406 |
| [28] | PHILIPPOT L, HALLIN S, SCHLOTER M. Ecology of denitrifying prokaryotes in agricultural soil[J]. Advances in Agronomy, 2007, 96:249-305 http://europepmc.org/abstract/AGR/IND44035527 |
| [29] | LIU M S, CHEN S M, WANG S Q, et al. High-quality draft genome sequence of Pseudomonas songnenensis L103, a denitrifier isolated from a 100-meter-deep aquifer in a heavily nitrogen-fertilized agricultural area[J]. Microbiology Resource Announcements, 2019, 8(36):e00373-19 |
| [30] | GUO J H, LIU X J, ZHANG Y, et al. Significant acidification in major Chinese croplands[J]. Science, 2010, 327(5968):1008-1010 http://www.cabdirect.org/abstracts/20103070770.html |
| [31] | STEVENS R J, LAUGHLIN R J, MALONE J P. Soil pH affects the processes reducing nitrate to nitrous oxide and di-nitrogen[J]. Soil Biology and Biochemistry, 1998, 30(8/9):1119-1126 http://europepmc.org/abstract/AGR/IND21969752 |
| [32] | RICHARDSON D, FELGATE H, WATMOUGH N, et al. Mitigating release of the potent greenhouse gas N2O from the nitrogen cycle-could enzymic regulation hold the key?[J]. Trends in Biotechnology, 2009, 27(7):388-397 http://www.ncbi.nlm.nih.gov/pubmed/19497629 |
