不同植物对沟渠沉积物反硝化速率及功能基因的影响研究
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摘要
植物是影响沉积物反硝化作用的重要因素之一,国内外已有不少研究探讨了植物对河口或湖泊沉积物反硝化速率的影响,但关于植物类型对自然沟渠沉积物反硝化速率及其相应功能基因的影响研究不多.因此,本文以7种常见沟渠植物为研究对象,通过室内盆栽试验,利用改进的乙炔抑制法和实时荧光定量PCR技术研究了不同植物对自然沟渠沉积物反硝化速率和相应功能基因(nirS和nirK)拷贝数的影响.结果表明,培养至第180 d时,不同植物生长条件下沉积物反硝化速率在2.85～13.20μg·m~(-2)·h~(-1)之间,不同植物间反硝化速率差异显著,且大型挺水植物>浮水植物>小型挺水植物.不同植物之间沉积物中nirS基因拷贝数在2.70×10~8～5.02×10~8 copies·g~(-1)之间,nirK基因拷贝数在3.97×10~5～6.91×10~5 copies·g~(-1)之间,与培养初期相比,培养180 d后沉积物中nirK、nirS基因拷贝数明显增多.7种植物中,美人蕉沉积物中的反硝化功能基因拷贝数较高,狐尾藻较低,整体来看,基因拷贝数大小顺序为:挺水植物>浮水植物.nirS基因拷贝数与NO~-_3-N含量、TN含量及反硝化速率之间均呈显著性的正相关关系(p<0.05),但nirK基因拷贝数与沉积物碳氮含量及反硝化速率之间相关性不显著.
The influence of plant on the denitrification of sediment in the estuary or lake have been reported by many studies. However, the effects of plant species on the denitrification of sediment and function genes in the natural ditches is still not clear. Here, we explored the effects of seven ditch plants on denitrification and functional genes(nirS, nirK) in ditch sediment via pot experiments by using the chloramphenicol-amended acetylene inhibition procedure and real time-PCR. The results show that the denitrification rates of the sediment under different plant species ranged from 2.85 μg·m~(-2)·h~(-1) to 13.2 μg·m~(-2)·h~(-1) after 180 days. The denitrification rates were as follows: Macrophytes> floating plants> small emerged plants, and there was a significant difference among different plant species. The gene abundances of nirS ranged from 2.70×10~8 copies·g~(-1) to 5.02×10~8 copies·g~(-1), and the gene abundances of nirK ranged 3.97×10~5 copies·g~(-1) to 6.91×10~5 copies·g~(-1). Furthermore, the gene abandunces of nirK and nirS increased significantly after 180 days of incubation. The denitrification functional gene copies was the highesin the sediment of Canna indica, while the lowest in Myriophyllum spicatum. The gene abundances of emerged plants were higher than that of floating plants. Moreover, pearson correlation analysis showed that there was a significant positive correlation between nirS and NO~-_3-N content, TN content, and denitrification rate(p<0.05), but the correlation between nirK and sediment physicochemical properties, denitrification rate were not significant.
The influence of plant on the denitrification of sediment in the estuary or lake have been reported by many studies. However, the effects of plant species on the denitrification of sediment and function genes in the natural ditches is still not clear. Here, we explored the effects of seven ditch plants on denitrification and functional genes(nirS, nirK) in ditch sediment via pot experiments by using the chloramphenicol-amended acetylene inhibition procedure and real time-PCR. The results show that the denitrification rates of the sediment under different plant species ranged from 2.85 μg·m~(-2)·h~(-1) to 13.2 μg·m~(-2)·h~(-1) after 180 days. The denitrification rates were as follows: Macrophytes> floating plants> small emerged plants, and there was a significant difference among different plant species. The gene abundances of nirS ranged from 2.70×10~8 copies·g~(-1) to 5.02×10~8 copies·g~(-1), and the gene abundances of nirK ranged 3.97×10~5 copies·g~(-1) to 6.91×10~5 copies·g~(-1). Furthermore, the gene abandunces of nirK and nirS increased significantly after 180 days of incubation. The denitrification functional gene copies was the highesin the sediment of Canna indica, while the lowest in Myriophyllum spicatum. The gene abundances of emerged plants were higher than that of floating plants. Moreover, pearson correlation analysis showed that there was a significant positive correlation between nirS and NO~-_3-N content, TN content, and denitrification rate(p<0.05), but the correlation between nirK and sediment physicochemical properties, denitrification rate were not significant.
引文
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Braker G,Zhou J,Wu L,et al.2000.Nitrite reductase genes (nirK and nirS) as functional markers to investigate diversity of denitrifying bacteria in pacific northwest marine sediment communities[J].Applied and Environmental Microbiology,66(5):2096-2104
Canfield D,Glazer A,Falkowski P.2010.The evolution and future of earth′s nitrogen cycle[J].Science,330:192-196
Chen Y N,Zhou W,Li Y P,et al.2014.Nitrite reductase genes as functional markers to investigate diversity of denitrifying bacteria during agricultural waste composting[J].Applied Microbiology and Biotechnology,98:4233-4243
程建华,窦智勇,孙庆业.2016.铜陵市河流沉积物中硝化和反硝化微生物分布特征[J].环境科学,37(4):1362-1370
陈斌泽.2013.长期施肥条件下青藏高原高寒草甸土壤中反硝化细菌丰度和群落结构的季节性变化研究[D].兰州:兰州大学
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Dang H Y,Wang C,Li J,et al.2008.Diversity and distribution of sediment nirS-encoding bacterial assemblages in response to environmental gradients in the Eutrophied Jiaozhou Bay,China[J].Microbial ecology,58(1):161-169
Francis C A,OMullan G D,Cornwell J C,et al.2013.Transitions in nirS-type denitrifier diversity,community composition,and biogeochemical activity along the Chesapeake Bay estuary[J].Frontiers in Microbiology,4:237
Guan X Y,Zhu L L,Li Y X.2014.Composition and variation of sediment bacterial and nir S-harboring bacterial communities at representative sites of the Bohai Gulf coastal zone,China[J].World Journal of Microbiology and Biotechnology,30:1291-1300
郭丽芸,时飞,杨柳燕.2011.反硝化菌功能基因及其分子生态学研究进展[J].微生物学通报,38(4):583-590
Heylen K,Gevers D,Vanparys B,et al.2006.The incidence of nirS and nirK and their genetic heterogeneity in cultivated denitrifiers [J].Environmental Microbiology,8(11):2012-2021
贺纪正,张丽梅.2013.土壤氮素转化的关键微生物过程及机制[J].微生物学通报,40(1):98-108
Kalscheur K N,M Rojas,C G Peterson,et al.2012.Algal exudates and stream organic matter influence the structure and function of denitrifying bacterial communities[J].Microbial Ecology,64:881-892
Kong A,Hristova K,Scow K,et al.2010.Impacts of different N management regimes on nitrifier and denitrifier communities and N cycling in soil microenvironments[J].Soil Biology and Biochemistry,42:1523-1533
Knops J M H,Bradley K L,Wedin D A.2002.Mechanisms of Plant Species Impacts on Ecosystem Nitrogen Cycling [J].Ecology Letters,5(3):454-466
Lefebvre S,Marmonier P,Peiry J L.2006.Nitrogen dynamics in rural streams:differences between geomorphologic units[C]//Annales de Limnologie-International Journal of Limnology[J].EDP Sciences,42(1):43-52
刘杏认,赵光昕,张晴雯,等.2018.生物炭对华北农田土壤N2O通量及相关功能基因丰度的影响[J].环境科学,39(8):3816-3824
鲁如坤.1999.土壤溶液化学分析方法,第一版[M].北京:中国农业科技出版社,308-311
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李凤娥.2017.黄河入海口沉积物反硝化细菌分布特征及其影响因素研究[D].济南:山东农业大学
李筱宛,张亚平,万宇,等.2016.上覆水不同C/N比条件下沉积物环境反硝化及氨氧化功能基因丰度变化特征[J].南京大学学报(自然科学),52(1):27-35
Mathieu N K,Zhu B,Diana K M.2017.Assessing the influence of different plant species in drainage ditches on mitigation of non-point source pollutants (N,P,and sediments) in the Purple Sichuan Basin[J].Environmental Monitoring and Assessment,189:267
McCrackin M L,Elser J J.2012.Denitrification kinetics and denitrifier abundances in sediments of lakes receiving atmospheric nitrogen deposition (Colorado,USA) [J].Biogeochemistry,108 (1/3):39-54
Mosier A C,Francis C A.2010.Denitrifier abundance and activity across the San Francisco Bay estuary[J].Environmental Microbiology Reports,2:667-676
Mulholland P,Helton A,Poole G,et al.2008.Stream denitrification across biomes and its response to anthropogenic nitrate loading[J].Nature,452 (7184):202-205
Page A L,Miller R H,Keeney D R.1982.Methods of soil analysis:part 2,Chemical and microbiological properties [M].Madison,Wisconsin:American Society of Agronomy and Soil Science Society of America,539-579
Pierobon E,Castaldelli G,Mantovani S,et al.2013.Nitrogen removal in vegetated and unvegetated drainage ditches impacted by diffuse and point sources of pollution[J].Clean-Soil Air Water,41:24-31
任光前,刘守江,王彦,等.2016.几种沟渠植物对村镇生活污水氮、磷去除效率的对比研究[J].资源开发与市场,32(5):599-603
Roley S S,Tank J L,Williams M A,et al.2012.Hydrologic connectivity increases denitrification in the hyporheic zone and restored floodplains of an agricultural stream [J].Journal of Geophysical Research:Biogeosciences,117:0148-0227
Rysgaard S,Risgaard P N,Nielsen L P,et al.1993.Nitrification and denitrification in lake and estuarine sediments measured by the 15N dilution technique and isotope Pairing[J].Applied and Environmental Microbiology,59(7):2093-2098
Seitzinger S,Harrison J,Bohlke J,et al.2006.Denitrification across landscapes and waterscapes:asynthesis[J].Ecological Applications,16(6):2064-2090
Senzia M A,Mashauri D A,Mayo A W.2003.Suitability of constructed wetlands and waste stabilization ponds in waste water reatment:nitrogen transformation and removal[J].Physics and Chemistry of the Earth,28:1117-1124
Smith C J,Nedwell D B,Dong L F,et al.2007.Diversity and abundance of nitrate reductase genes (narG and napA),nitrite reductase genes (nirS and nirK),and their transcripts in estuarine sediments[J].Applied and Environmental Microbiology,73:3612-3622
Throback I N,Enwall K,Jarvis A,et al.2004.Reassessing PCR primers targeting nirS,nirK and nosZ genes for community surveys of denitrifying bacterica with DGGE[J].Fems Microbiology Ecology,49(3):401-417
汪涛,龙虹竹,赵原,等.2016.川中丘陵区自然沟渠水体氮磷污染状况评价[J].山地学报,34(2):150-156
王博,叶春,杨勋,等.2009.腐解黑藻生物量对高硝态氮水体氮素的影响[J].环境科学研究,22(10):1198-1203
王超,单保庆.2012.子牙河水系水和沉积物好氧氨氧化微生物分布特征[J].环境科学学报,32(12):2943-2950
武姣娜,魏晓东,李霞,等.2018.植物氮素利用效率的研究进展[J].植物生理学报,54(9):1401-1408
徐红灯,席北斗,翟丽华.2007.沟渠沉积物对农田排水中氨氮的截留效应研究[J].农业环境科学学报,26(5):1924-1928
谢佳,马晓航,代嫣然,等.2017.有机质对城市湿地微生物丰度的影响[J].生物技术通报,33(10):217-224
Yin G Y,Hou L J,Liu M,et al.2017.Effects of multiple antibiotics exposure on denitrification process in the Yangtze Estuary sediments[J].Chemosphere,171:118-125
于洪刚,杨希晨,孟琦,等.2017.富营养化水体中附植生物夏季反硝化脱氮作用研究[J].绿色科技,20:36-37
杨柳青.2017.石灰性潮土N2O产生过程及相关功能基因丰度和表达[D].北京:中国农业大学
Zhang X L,Agogué H,Dupuy C,et al.2014.Relative abundance of ammonia oxidizers,denitrifiers,and anammox bacteria in sediments of hyper-nutrified estuarine tidal flats and in relation to environmental conditions [J].Clean-Soil,Air,Water,42(6):815-823
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Braker G,Fesefeldt A,Witzel K P.1998.Development of PCR primer stems for amplification of nitrite reductase genes(nirK and nirS) to detect denitrifying,bacteria in environment samples[J].Applied &Environmental Microbiology,64:3769-3775
Braker G,Zhou J,Wu L,et al.2000.Nitrite reductase genes (nirK and nirS) as functional markers to investigate diversity of denitrifying bacteria in pacific northwest marine sediment communities[J].Applied and Environmental Microbiology,66(5):2096-2104
Canfield D,Glazer A,Falkowski P.2010.The evolution and future of earth′s nitrogen cycle[J].Science,330:192-196
Chen Y N,Zhou W,Li Y P,et al.2014.Nitrite reductase genes as functional markers to investigate diversity of denitrifying bacteria during agricultural waste composting[J].Applied Microbiology and Biotechnology,98:4233-4243
程建华,窦智勇,孙庆业.2016.铜陵市河流沉积物中硝化和反硝化微生物分布特征[J].环境科学,37(4):1362-1370
陈斌泽.2013.长期施肥条件下青藏高原高寒草甸土壤中反硝化细菌丰度和群落结构的季节性变化研究[D].兰州:兰州大学
陈登,蔡启佳,田翠翠.2018.3种沉水植物根际对沉积物中典型氮循环微生物功能基因丰度的影响[J].云南农业大学学报(自然科学),33(2):314-323
Dang H Y,Wang C,Li J,et al.2008.Diversity and distribution of sediment nirS-encoding bacterial assemblages in response to environmental gradients in the Eutrophied Jiaozhou Bay,China[J].Microbial ecology,58(1):161-169
Francis C A,OMullan G D,Cornwell J C,et al.2013.Transitions in nirS-type denitrifier diversity,community composition,and biogeochemical activity along the Chesapeake Bay estuary[J].Frontiers in Microbiology,4:237
Guan X Y,Zhu L L,Li Y X.2014.Composition and variation of sediment bacterial and nir S-harboring bacterial communities at representative sites of the Bohai Gulf coastal zone,China[J].World Journal of Microbiology and Biotechnology,30:1291-1300
郭丽芸,时飞,杨柳燕.2011.反硝化菌功能基因及其分子生态学研究进展[J].微生物学通报,38(4):583-590
Heylen K,Gevers D,Vanparys B,et al.2006.The incidence of nirS and nirK and their genetic heterogeneity in cultivated denitrifiers [J].Environmental Microbiology,8(11):2012-2021
贺纪正,张丽梅.2013.土壤氮素转化的关键微生物过程及机制[J].微生物学通报,40(1):98-108
Kalscheur K N,M Rojas,C G Peterson,et al.2012.Algal exudates and stream organic matter influence the structure and function of denitrifying bacterial communities[J].Microbial Ecology,64:881-892
Kong A,Hristova K,Scow K,et al.2010.Impacts of different N management regimes on nitrifier and denitrifier communities and N cycling in soil microenvironments[J].Soil Biology and Biochemistry,42:1523-1533
Knops J M H,Bradley K L,Wedin D A.2002.Mechanisms of Plant Species Impacts on Ecosystem Nitrogen Cycling [J].Ecology Letters,5(3):454-466
Lefebvre S,Marmonier P,Peiry J L.2006.Nitrogen dynamics in rural streams:differences between geomorphologic units[C]//Annales de Limnologie-International Journal of Limnology[J].EDP Sciences,42(1):43-52
刘杏认,赵光昕,张晴雯,等.2018.生物炭对华北农田土壤N2O通量及相关功能基因丰度的影响[J].环境科学,39(8):3816-3824
鲁如坤.1999.土壤溶液化学分析方法,第一版[M].北京:中国农业科技出版社,308-311
龙虹竹,汪涛,田琳琳,等.2016.川中丘陵区农业源头沟渠反硝化速率特征及其影响因素[J].重庆师范大学学报(自然科学版),33(4):166-172
李博超.2017.辽河口沉积物反硝化相关功能基因丰度和多样性分析[D].大连:大连海事大学
李凤娥.2017.黄河入海口沉积物反硝化细菌分布特征及其影响因素研究[D].济南:山东农业大学
李筱宛,张亚平,万宇,等.2016.上覆水不同C/N比条件下沉积物环境反硝化及氨氧化功能基因丰度变化特征[J].南京大学学报(自然科学),52(1):27-35
Mathieu N K,Zhu B,Diana K M.2017.Assessing the influence of different plant species in drainage ditches on mitigation of non-point source pollutants (N,P,and sediments) in the Purple Sichuan Basin[J].Environmental Monitoring and Assessment,189:267
McCrackin M L,Elser J J.2012.Denitrification kinetics and denitrifier abundances in sediments of lakes receiving atmospheric nitrogen deposition (Colorado,USA) [J].Biogeochemistry,108 (1/3):39-54
Mosier A C,Francis C A.2010.Denitrifier abundance and activity across the San Francisco Bay estuary[J].Environmental Microbiology Reports,2:667-676
Mulholland P,Helton A,Poole G,et al.2008.Stream denitrification across biomes and its response to anthropogenic nitrate loading[J].Nature,452 (7184):202-205
Page A L,Miller R H,Keeney D R.1982.Methods of soil analysis:part 2,Chemical and microbiological properties [M].Madison,Wisconsin:American Society of Agronomy and Soil Science Society of America,539-579
Pierobon E,Castaldelli G,Mantovani S,et al.2013.Nitrogen removal in vegetated and unvegetated drainage ditches impacted by diffuse and point sources of pollution[J].Clean-Soil Air Water,41:24-31
任光前,刘守江,王彦,等.2016.几种沟渠植物对村镇生活污水氮、磷去除效率的对比研究[J].资源开发与市场,32(5):599-603
Roley S S,Tank J L,Williams M A,et al.2012.Hydrologic connectivity increases denitrification in the hyporheic zone and restored floodplains of an agricultural stream [J].Journal of Geophysical Research:Biogeosciences,117:0148-0227
Rysgaard S,Risgaard P N,Nielsen L P,et al.1993.Nitrification and denitrification in lake and estuarine sediments measured by the 15N dilution technique and isotope Pairing[J].Applied and Environmental Microbiology,59(7):2093-2098
Seitzinger S,Harrison J,Bohlke J,et al.2006.Denitrification across landscapes and waterscapes:asynthesis[J].Ecological Applications,16(6):2064-2090
Senzia M A,Mashauri D A,Mayo A W.2003.Suitability of constructed wetlands and waste stabilization ponds in waste water reatment:nitrogen transformation and removal[J].Physics and Chemistry of the Earth,28:1117-1124
Smith C J,Nedwell D B,Dong L F,et al.2007.Diversity and abundance of nitrate reductase genes (narG and napA),nitrite reductase genes (nirS and nirK),and their transcripts in estuarine sediments[J].Applied and Environmental Microbiology,73:3612-3622
Throback I N,Enwall K,Jarvis A,et al.2004.Reassessing PCR primers targeting nirS,nirK and nosZ genes for community surveys of denitrifying bacterica with DGGE[J].Fems Microbiology Ecology,49(3):401-417
汪涛,龙虹竹,赵原,等.2016.川中丘陵区自然沟渠水体氮磷污染状况评价[J].山地学报,34(2):150-156
王博,叶春,杨勋,等.2009.腐解黑藻生物量对高硝态氮水体氮素的影响[J].环境科学研究,22(10):1198-1203
王超,单保庆.2012.子牙河水系水和沉积物好氧氨氧化微生物分布特征[J].环境科学学报,32(12):2943-2950
武姣娜,魏晓东,李霞,等.2018.植物氮素利用效率的研究进展[J].植物生理学报,54(9):1401-1408
徐红灯,席北斗,翟丽华.2007.沟渠沉积物对农田排水中氨氮的截留效应研究[J].农业环境科学学报,26(5):1924-1928
谢佳,马晓航,代嫣然,等.2017.有机质对城市湿地微生物丰度的影响[J].生物技术通报,33(10):217-224
Yin G Y,Hou L J,Liu M,et al.2017.Effects of multiple antibiotics exposure on denitrification process in the Yangtze Estuary sediments[J].Chemosphere,171:118-125
于洪刚,杨希晨,孟琦,等.2017.富营养化水体中附植生物夏季反硝化脱氮作用研究[J].绿色科技,20:36-37
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