碳源和氧对设施菜田土壤N_2O排放的影响
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摘要
利用在线自动监测培养系统(Robot系统),研究不同氧分压、碳源投入以及不同氧分压和碳源投入组合下,添加硝化抑制剂双氰胺(DCD)对设施菜田土壤N_2O排放的影响。结果表明:随着土壤氧分压的升高,N_2O排放量呈指数下降(P<0.001),土壤氧分压大于等于3%O_2后,N_2O排放量不足于无氧和微量氧(1%氧)处理的30%。添加碳源降低了有氧条件下土壤N_2O和N_2产生量,显著增加了微量氧下异养反硝化途径对N_2O的贡献量(P<0.01)。在微量氧和3%O_2下,与未添加DCD的处理相比,无碳源添加且施用DCD后,N_2O的排放分别降低了64.4%和88.8%,同时N_2排放分别降低了23.4%和18.6%。从微量氧至3%O_2,虽然无碳源添加的处理硝化细菌反硝化作用对N_2O排放的贡献从17.2%增加至42.6%,但由于排放总量的急剧下降,硝化细菌反硝化作用对设施菜田土壤N_2O排放的贡献较小。本研究所用土壤pH较高,且添加DCD的处理培养前后硝酸盐基本平衡,异养的同步硝化-反硝化过程可能很弱。总之,设施菜田土壤N_2O排放主要发生在无氧和微量氧条件下。异养反硝化菌对土壤N_2O排放的直接贡献最大,尤其是在碳源较为充足的条件下。
【Objective】 In order to explore effects of oxygen level(0%, 1%, 3%, 10% and 21%)and carbon input on major sources of N_2 O emission in greenhouse vegetable soil, an on-line robotized monitoring and incubation system was used to automatically monitor real-time dynamics of O_2, N_2 O and N_2 in greenhouse vegetable soil after a crop of tomato was harvested. In addition, dicyandiamide(DCD), a kind of nitrification inhibitor, was added to investigate contribution of nitrification to N_2 O emission from the soil relative to carbon source and oxygen level.【Method】 A certain amount of soil sample collected from a vegetable field under greenhouse after a crop of tomato was harvested, was washed with deionized water and divided into two groups, labeled as T1 and T2, separately. T2 was spiked with glutamate, whereas T1 was not. NH_4 NO_3 was added as nitrogen source for both groups. Then the pretreated soil samples were put into 120 mL vials, 10.0 g in dry soil weight in each. Deionized water or solution containing NH_4 NO_3,C.H8 NO.Na·H_2 O or DCD was sprayed onto the soil surface to adjust moisture content of the soil up to 250 g·kg~(-1) in line with the requirement of the treatment, T1 or T2. Then the soils in the sealed vials were placed in a thermostatic(20℃) waterbath trough for incubation. Concentrations of O_2, N_2 O, N_2 and CO_2 in the headspace of a vial were monitored online at intervals of 6 h, and pure O_2 was supplemented with a hermetic syringe in the light of the monitoring results of the gases in the headspace to maintain an approximately constant oxygen concentration(1%, 3%, 10% or 21%(v/v)) in the sealed vials. 【Result】Results show that N_2 O emission declined exponentially with rising soil oxygen partial pressure(OPP)(R2=0.82, P<0.001).It peaked when OPP was 0% or 1%, and fell below 30% of the peak when OPP got equal to or higher than3%. Addition of available carbon into the vial reduced N_2 O and N_2 production in the soil under aerobic conditions, while significantly increasing the contribution of the process of heterotrophic denitrification in the soil to N_2 O emission under near-anaerobic conditions(P<0.01), which suggests that this kind of soil is highly capable of triggering heterotrophic denitrification, and anaerobicity and near-anaerobicity is more favorable to N_2 O emission. Compared with Group T1, Group T2 was 64.4% and 88.8% lower in N_2 O emission, 23.4% and 18.6% lower in N_2 emission, and 14.5% and 62.3% lower in N_2 O/(N_2 O+N_2)index(IN_2 O), respectively, when OPP was 1% and 3% and no carbon supplemented. However, when carbon was supplemented, the two groups did not vary much in N_2 O and N_2 emissions and IN_2 O, which suggests that strong nitrification occurs in the soil with no carbon supplemented and N_2 O comes mainly from heterotrophic denitrification process(HD) under aerobic conditions in the soil with sufficint carbon supply.DCD would lower the accumulation of NO_2-, a substrate of nitrification-coupled denitrification(NCD),which is considered to be an important contributor to N_2 O emission under aerobic conditions; nevertheless,the process of NCD is still a heterotrophic one in nature. That is to say, DCD would reduce N_2 O emission from nitrification processes, and from heterotrophic denitrification processes, too. With OPP rising from1% to 3%, N_2 O emission from nitrifier-induced denitrification(ND) increased from 17.2% to 42.6%,however, the contribution of ND to N_2 O emission in the vegetable soil was still quite low due to the drastic reduction in total N_2 O emission. Moreover, the commonly used dual isotopic labeling method would lead to overestimation of the contribution of ND to N_2 O emission because this method may rule partial NCD or HD processes into ND. As the soil in this study was quite high in pH and remained almost unchanged in nitrate content after the treatment with DCD, simultaneous heterotrophic nitrification-denitrification processes might be very weak. 【Conclusion】Soil N_2 O emission mainly occurs under anaerobic and near-anaerobic conditions(OPP=1%). Heterotrophic denitrifiers make the biggest direct contribution to soil N_2 O emissions,especially when carbon sources are abundant. NO_2-is an important substrate of the process of nitrificationcoupled denitrification.
【Objective】 In order to explore effects of oxygen level(0%, 1%, 3%, 10% and 21%)and carbon input on major sources of N_2 O emission in greenhouse vegetable soil, an on-line robotized monitoring and incubation system was used to automatically monitor real-time dynamics of O_2, N_2 O and N_2 in greenhouse vegetable soil after a crop of tomato was harvested. In addition, dicyandiamide(DCD), a kind of nitrification inhibitor, was added to investigate contribution of nitrification to N_2 O emission from the soil relative to carbon source and oxygen level.【Method】 A certain amount of soil sample collected from a vegetable field under greenhouse after a crop of tomato was harvested, was washed with deionized water and divided into two groups, labeled as T1 and T2, separately. T2 was spiked with glutamate, whereas T1 was not. NH_4 NO_3 was added as nitrogen source for both groups. Then the pretreated soil samples were put into 120 mL vials, 10.0 g in dry soil weight in each. Deionized water or solution containing NH_4 NO_3,C.H8 NO.Na·H_2 O or DCD was sprayed onto the soil surface to adjust moisture content of the soil up to 250 g·kg~(-1) in line with the requirement of the treatment, T1 or T2. Then the soils in the sealed vials were placed in a thermostatic(20℃) waterbath trough for incubation. Concentrations of O_2, N_2 O, N_2 and CO_2 in the headspace of a vial were monitored online at intervals of 6 h, and pure O_2 was supplemented with a hermetic syringe in the light of the monitoring results of the gases in the headspace to maintain an approximately constant oxygen concentration(1%, 3%, 10% or 21%(v/v)) in the sealed vials. 【Result】Results show that N_2 O emission declined exponentially with rising soil oxygen partial pressure(OPP)(R2=0.82, P<0.001).It peaked when OPP was 0% or 1%, and fell below 30% of the peak when OPP got equal to or higher than3%. Addition of available carbon into the vial reduced N_2 O and N_2 production in the soil under aerobic conditions, while significantly increasing the contribution of the process of heterotrophic denitrification in the soil to N_2 O emission under near-anaerobic conditions(P<0.01), which suggests that this kind of soil is highly capable of triggering heterotrophic denitrification, and anaerobicity and near-anaerobicity is more favorable to N_2 O emission. Compared with Group T1, Group T2 was 64.4% and 88.8% lower in N_2 O emission, 23.4% and 18.6% lower in N_2 emission, and 14.5% and 62.3% lower in N_2 O/(N_2 O+N_2)index(IN_2 O), respectively, when OPP was 1% and 3% and no carbon supplemented. However, when carbon was supplemented, the two groups did not vary much in N_2 O and N_2 emissions and IN_2 O, which suggests that strong nitrification occurs in the soil with no carbon supplemented and N_2 O comes mainly from heterotrophic denitrification process(HD) under aerobic conditions in the soil with sufficint carbon supply.DCD would lower the accumulation of NO_2-, a substrate of nitrification-coupled denitrification(NCD),which is considered to be an important contributor to N_2 O emission under aerobic conditions; nevertheless,the process of NCD is still a heterotrophic one in nature. That is to say, DCD would reduce N_2 O emission from nitrification processes, and from heterotrophic denitrification processes, too. With OPP rising from1% to 3%, N_2 O emission from nitrifier-induced denitrification(ND) increased from 17.2% to 42.6%,however, the contribution of ND to N_2 O emission in the vegetable soil was still quite low due to the drastic reduction in total N_2 O emission. Moreover, the commonly used dual isotopic labeling method would lead to overestimation of the contribution of ND to N_2 O emission because this method may rule partial NCD or HD processes into ND. As the soil in this study was quite high in pH and remained almost unchanged in nitrate content after the treatment with DCD, simultaneous heterotrophic nitrification-denitrification processes might be very weak. 【Conclusion】Soil N_2 O emission mainly occurs under anaerobic and near-anaerobic conditions(OPP=1%). Heterotrophic denitrifiers make the biggest direct contribution to soil N_2 O emissions,especially when carbon sources are abundant. NO_2-is an important substrate of the process of nitrificationcoupled denitrification.
引文
[1]Shi X Z,Hu H W,Xia Z,et al.Nitrifier-induced denitrification is an important source of soil nitrous oxide and can be inhibited by a nitrification inhibitor3,4-dimethylpyrazole phosphate.Environmental Microbiology,2017,19(12):4851-4865
[2]Butterbach-Bahl K,Baggs E M,Dannenmann M,et al.Nitrous oxide emissions from soils:how well do we understand the processes and their controls?Philosophical Transactions of the Royal Society,2013,368(1621):20130122
[3]Kool D M,Dolfing J,Wrage N,et al.Nitrifier denitrification as a distinct and significant source of nitrous oxide from soil.Soil Biology and Biochemistry,2011,43(1):174-178
[4]Zhu X,Burger M,Doane T A,et al.Ammonia oxidation pathways and nitrifier denitrification are significant sources of N2O and NO under low oxygen availability.Proceedings of the National Academy of Sciences,2013,110(16):6328-6333
[5]Ju X T,Lu X,Gao Z,et al.Processes and factors controlling N2O production in an intensively managed low carbon calcareous soil under sub-humid monsoon conditions.Environmental Pollution,2011,159(4):1007-1016
[6]Cui F,Yan G,Zhou Z,et al.Annual emissions of nitrous oxide and nitric oxide from a wheat-maize cropping system on a silt loam calcareous soil in the North China Plain.Soil Biology and Biochemistry,2012,48(4):10-19
[7]Sutka R L,Ostrom N E,Ostrom P H,et al.Distinguishing nitrous oxide production from nitrification and denitrification on the basis of isotopomer abundances.Applied and Environmental Microbiology,2006,72(1):638-644
[8]Hu H W,Chen D,He J Z.Microbial regulation of terrestrial nitrous oxide formation:Understanding the biological pathways for prediction of emission rates.FEMS Microbiology Reviews,2015,39(5):729-749
[9]Yan Z,Chen S,Li J,et al.Manure and nitrogen application enhances soil phosphorus mobility in calcareous soil in greenhouses.Journal of Environmental Management,2016,181:26-35
[10]Saito T,Ishii S,Otsuka S,et al.Identification of novel Betaproteobacteria in a succinate-assimilating population in denitrifying rice paddy soil by using stable isotope probing.Microbes&Environments,2008,23(3):192-200
[11]Morley N,Baggs E M.Carbon and oxygen controls on N2O and N2 production during nitrate reduction.Soil Biology and Biochemistry,2010,42:1864-1871
[12]Yang X,Wang S,Zhou L.Effect of carbon source,C/N ratio,nitrate and dissolved oxygen concentration on nitrite and ammonium production from denitrification process by Pseudomonas stutzeri D6.Bioresource Technology,2012,104(1):65-72
[13]Maharjan B,Venterea R T.Nitrite intensity explains N management effects on N2O emissions in maize.Soil Biology and Biochemistry,2013,66(11):229-238
[14]Fan Z B,Lin S,Zhang X M,et al.Conventional flooding irrigation causes an overuse of nitrogen fertilizer and low nitrogen use efficiency in intensively used solar greenhouse vegetable reduction.Agricultural Water Management,2014,144(2):11-19
[15]Molstad L,Dorsch P,Bakken L R.Robotized incubation system for monitoring gases(O2,NO,N2O,N2)in denitrifying cultures.Journal of Microbiological Methods,2007,71(3):202-211
[16]鲍士旦.土壤农化分析.3版.北京:中国农业出版社,1999:25-111Bao S D.Soil agrochemical analysis(In Chinese).3rd ed.Beijing:China Agriculture Press,1999:25-111
[17]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.Global Change Biology,2014,20(5):1685-1698
[18]Wu D,Dong W,Oenema O,et al.N2O consumption by low-nitrogen soil and its regulation by water and oxygen.Soil Biology and Biochemistry,2013,60(1):165-172
[19]杨艳菊,蔡祖聪,张金波.氧气浓度对水稻土N2O排放的影响.土壤,2016,48(3):539-545Yang Y J,Cai Z C,Zhang J B.Effect of oxygen concentration on N2O emission from paddy soil(In Chinese).Soils,2016,48(3):539-545
[20]Diao T,Xie L,Guo L,et al.Measurements of N2Oemissions from different vegetable fields on the North China Plain.Atmospheric Environment,2013,72(2):70-76
[21]Yamazaki T,Hozuki T,Arai K,et al.Isotopomeric characterization of nitrous oxide produced by reaction of enzymes extracted from nitrifying and denitrifying bacteria.Biogeosciences,2014,11(10):2679-2689
[22]Morkved P T,Dorsch P,Bakken L R.The N2Oproduct ratio of nitrification and its dependence on long-term changes in soil pH.Soil Biology and Biochemistry,2007,39(8):2048-2057
[23]Lim N Y N,Frostegard A,Bakken L R.Nitrite kinetics during anoxia:The role of abiotic reactions versus micr o b ial reduct io n.S o il Bio logy and Biochemistry,2018,119:203-209
[24]Third K A,Burnett N,Cord-Ruwisch R.Simultaneous nitrification and denitrification using stored substrate(PHB)as the electron donor in an SBR.Biotechnology and Bioengineering,2003,83(6):706-720
[25]Zhang J,Müller C,Cai Z.Heterotrophic nitrification of organic N and its contribution to nitrous oxide emissions in soils.Soil Biology and Biochemistry,2015,84:199-209
[26]毛新伟,程敏,徐秋芳,等.硝化抑制剂对毛竹林土壤N2O排放和氨氧化微生物的影响.土壤学报,2016,53(6):1528-1540Mao X W,Cheng M,Xu Q F,et al.Effects of nitrification inhibitors on soil N2O emission and community structure and abundance of ammonia oxidation microorganism in soil under extensively managed Phyllostachys edulis stands(In Chinese).Acta Pedologica Sinica,2016,53(6):1528-1540
[27]陈浩,李博,熊正琴.减氮及硝化抑制剂对菜地氧化亚氮排放的影响.土壤学报,2017,54(4):938-947Chen H,Li B,Qiong Z Q.Effects of N reduction and nitrification inhibitor on N2O emissions in intensive vegetable field(In Chinese).Acta Pedologica Sinica,2017,54(4):938-947
[28]孙海军,闵炬,施卫明,等.硝化抑制剂影响小麦产量、N2O与NH3排放的研究.土壤,2017,49(5):876-881Sun H J,Min J,Shi W M,et al.Effects of nitrification inhibitor application on wheat grain yield,N2O emission and NH3 volatilization(In Chinese).Soils,2017,49(5):876-881
[29]Wrage N,Velthof G L,van Beusichem M L,et al.Role of nitrifer denitrifcation in the production of nitrous oxide.Soil Biology and Biochemistry,2001,33(12/13):1723-1732
[30]Lee A,Winther M,PrieméA,et al.Hot spots of N2O emission move with the seasonally mobile oxicanoxic interface in drained organic soils.Soil Biology and Biochemistry,2017,115:178-186
[31]Bergstrom DW,Tenuta M,Beauchamp E G.Increase in nitrous oxide production in soil induced by ammonium and organic carbon.Biology&Fertility of Soils,1994,18(1):1-6
[32]Peterson S O,Henriksen K,Blackburn T H.Coupled nitrification-denitrification associated with liquid manure in a gel-stabilized model system.Biology&Fertility of Soils,1991,12(1):19-27
[33]Kool D M,van Groenigen J W,Wrage N.Source determination of nitrous oxide based on nitrogen and oxygen isotope tracing dealing with oxygen exchange.Methods in Enzymology,2011,496(16):139-160
[34]Bakken L R,Frostegard ASources and sinks for N2O,can microbiologist help to mitigate N2Oemissions?Environmental Microbiology,2017,19(12):4801-4805
[2]Butterbach-Bahl K,Baggs E M,Dannenmann M,et al.Nitrous oxide emissions from soils:how well do we understand the processes and their controls?Philosophical Transactions of the Royal Society,2013,368(1621):20130122
[3]Kool D M,Dolfing J,Wrage N,et al.Nitrifier denitrification as a distinct and significant source of nitrous oxide from soil.Soil Biology and Biochemistry,2011,43(1):174-178
[4]Zhu X,Burger M,Doane T A,et al.Ammonia oxidation pathways and nitrifier denitrification are significant sources of N2O and NO under low oxygen availability.Proceedings of the National Academy of Sciences,2013,110(16):6328-6333
[5]Ju X T,Lu X,Gao Z,et al.Processes and factors controlling N2O production in an intensively managed low carbon calcareous soil under sub-humid monsoon conditions.Environmental Pollution,2011,159(4):1007-1016
[6]Cui F,Yan G,Zhou Z,et al.Annual emissions of nitrous oxide and nitric oxide from a wheat-maize cropping system on a silt loam calcareous soil in the North China Plain.Soil Biology and Biochemistry,2012,48(4):10-19
[7]Sutka R L,Ostrom N E,Ostrom P H,et al.Distinguishing nitrous oxide production from nitrification and denitrification on the basis of isotopomer abundances.Applied and Environmental Microbiology,2006,72(1):638-644
[8]Hu H W,Chen D,He J Z.Microbial regulation of terrestrial nitrous oxide formation:Understanding the biological pathways for prediction of emission rates.FEMS Microbiology Reviews,2015,39(5):729-749
[9]Yan Z,Chen S,Li J,et al.Manure and nitrogen application enhances soil phosphorus mobility in calcareous soil in greenhouses.Journal of Environmental Management,2016,181:26-35
[10]Saito T,Ishii S,Otsuka S,et al.Identification of novel Betaproteobacteria in a succinate-assimilating population in denitrifying rice paddy soil by using stable isotope probing.Microbes&Environments,2008,23(3):192-200
[11]Morley N,Baggs E M.Carbon and oxygen controls on N2O and N2 production during nitrate reduction.Soil Biology and Biochemistry,2010,42:1864-1871
[12]Yang X,Wang S,Zhou L.Effect of carbon source,C/N ratio,nitrate and dissolved oxygen concentration on nitrite and ammonium production from denitrification process by Pseudomonas stutzeri D6.Bioresource Technology,2012,104(1):65-72
[13]Maharjan B,Venterea R T.Nitrite intensity explains N management effects on N2O emissions in maize.Soil Biology and Biochemistry,2013,66(11):229-238
[14]Fan Z B,Lin S,Zhang X M,et al.Conventional flooding irrigation causes an overuse of nitrogen fertilizer and low nitrogen use efficiency in intensively used solar greenhouse vegetable reduction.Agricultural Water Management,2014,144(2):11-19
[15]Molstad L,Dorsch P,Bakken L R.Robotized incubation system for monitoring gases(O2,NO,N2O,N2)in denitrifying cultures.Journal of Microbiological Methods,2007,71(3):202-211
[16]鲍士旦.土壤农化分析.3版.北京:中国农业出版社,1999:25-111Bao S D.Soil agrochemical analysis(In Chinese).3rd ed.Beijing:China Agriculture Press,1999:25-111
[17]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.Global Change Biology,2014,20(5):1685-1698
[18]Wu D,Dong W,Oenema O,et al.N2O consumption by low-nitrogen soil and its regulation by water and oxygen.Soil Biology and Biochemistry,2013,60(1):165-172
[19]杨艳菊,蔡祖聪,张金波.氧气浓度对水稻土N2O排放的影响.土壤,2016,48(3):539-545Yang Y J,Cai Z C,Zhang J B.Effect of oxygen concentration on N2O emission from paddy soil(In Chinese).Soils,2016,48(3):539-545
[20]Diao T,Xie L,Guo L,et al.Measurements of N2Oemissions from different vegetable fields on the North China Plain.Atmospheric Environment,2013,72(2):70-76
[21]Yamazaki T,Hozuki T,Arai K,et al.Isotopomeric characterization of nitrous oxide produced by reaction of enzymes extracted from nitrifying and denitrifying bacteria.Biogeosciences,2014,11(10):2679-2689
[22]Morkved P T,Dorsch P,Bakken L R.The N2Oproduct ratio of nitrification and its dependence on long-term changes in soil pH.Soil Biology and Biochemistry,2007,39(8):2048-2057
[23]Lim N Y N,Frostegard A,Bakken L R.Nitrite kinetics during anoxia:The role of abiotic reactions versus micr o b ial reduct io n.S o il Bio logy and Biochemistry,2018,119:203-209
[24]Third K A,Burnett N,Cord-Ruwisch R.Simultaneous nitrification and denitrification using stored substrate(PHB)as the electron donor in an SBR.Biotechnology and Bioengineering,2003,83(6):706-720
[25]Zhang J,Müller C,Cai Z.Heterotrophic nitrification of organic N and its contribution to nitrous oxide emissions in soils.Soil Biology and Biochemistry,2015,84:199-209
[26]毛新伟,程敏,徐秋芳,等.硝化抑制剂对毛竹林土壤N2O排放和氨氧化微生物的影响.土壤学报,2016,53(6):1528-1540Mao X W,Cheng M,Xu Q F,et al.Effects of nitrification inhibitors on soil N2O emission and community structure and abundance of ammonia oxidation microorganism in soil under extensively managed Phyllostachys edulis stands(In Chinese).Acta Pedologica Sinica,2016,53(6):1528-1540
[27]陈浩,李博,熊正琴.减氮及硝化抑制剂对菜地氧化亚氮排放的影响.土壤学报,2017,54(4):938-947Chen H,Li B,Qiong Z Q.Effects of N reduction and nitrification inhibitor on N2O emissions in intensive vegetable field(In Chinese).Acta Pedologica Sinica,2017,54(4):938-947
[28]孙海军,闵炬,施卫明,等.硝化抑制剂影响小麦产量、N2O与NH3排放的研究.土壤,2017,49(5):876-881Sun H J,Min J,Shi W M,et al.Effects of nitrification inhibitor application on wheat grain yield,N2O emission and NH3 volatilization(In Chinese).Soils,2017,49(5):876-881
[29]Wrage N,Velthof G L,van Beusichem M L,et al.Role of nitrifer denitrifcation in the production of nitrous oxide.Soil Biology and Biochemistry,2001,33(12/13):1723-1732
[30]Lee A,Winther M,PrieméA,et al.Hot spots of N2O emission move with the seasonally mobile oxicanoxic interface in drained organic soils.Soil Biology and Biochemistry,2017,115:178-186
[31]Bergstrom DW,Tenuta M,Beauchamp E G.Increase in nitrous oxide production in soil induced by ammonium and organic carbon.Biology&Fertility of Soils,1994,18(1):1-6
[32]Peterson S O,Henriksen K,Blackburn T H.Coupled nitrification-denitrification associated with liquid manure in a gel-stabilized model system.Biology&Fertility of Soils,1991,12(1):19-27
[33]Kool D M,van Groenigen J W,Wrage N.Source determination of nitrous oxide based on nitrogen and oxygen isotope tracing dealing with oxygen exchange.Methods in Enzymology,2011,496(16):139-160
[34]Bakken L R,Frostegard ASources and sinks for N2O,can microbiologist help to mitigate N2Oemissions?Environmental Microbiology,2017,19(12):4801-4805