稳定性同位素DNA-SIP示踪中性紫色土的氨氧化过程
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摘要
研究表明酸性土壤中氨氧化作用主要是由氨氧化古菌(ammonia-oxidizing archaea,AOA)催化进行;而在中性和碱性土壤中则主要是由氨氧化细菌(ammonia-oxidizing bacteria,AOB)主导.虽然AOA在中性土壤中具有很高的丰度,但其对硝化过程的贡献仍不清楚.因此本文选取p H为7. 2的中性紫色土为研究对象,通过稳定性同位素核酸探针技术结合克隆测序探究中性紫色土中活性氨氧化微生物群落组成.结果表明中性紫色土的净硝化速率为9. 68 mg·(kg·d)~(-1),AOA和AOB在中性紫色土中均有较高的丰度且共同推动硝化作用的进行.系统发育分析结果表明培养初期(0d)在数量上占优势的AOB为Nitrosospira Cluster 3a. 1,而Nitrosospira Cluster 3a. 2只占较小的一部分,经过56d的培养后Nitrosospira Cluster 3a. 2替代了Nitrosospira Cluster 3a. 1成为主导氨氧化的活性AOB.培养初期(0d)在数量上占优势的AOA是Nitrososphaera Subcluster 9,但经过培养后变为Nitrososphaera Subcluster 3. 2/3. 3.在培养期间AOA和AOB的群落结构均发生了改变.对~(13)C标记DNA的测序分析证明AOA和AOB在硝化过程中都起着重要作用,主导氨氧化的活性AOA和AOB主要分别隶属于Nitrososphaera Subcluster 3. 2/3. 3和Nitrosospira Cluster 3a. 2.本研究明确了AOA及AOB对中性紫色土氨氧化过程的推动作用并从微生物层面探究硝化作用的发生机制,为进一步研究紫色土中硝化作用提供理论基础.
Increasing evidence suggests that ammonia oxidation in acidic soils is primarily catalyzed by ammonia-oxidizing archaea(AOA),while ammonia-oxidizing bacteria(AOB) drive ammonia oxidation in neutral and alkaline soils in which AOA overwhelmingly outnumber AOB. Therefore,neutral purple soil with a pH of 7. 2 was selected to study the composition of the active ammoxidation microbial community with a stable isotope nucleic acid probe technique combined with cloning sequencing. Results showed that the nitrification rate was 9. 68 mg·(kg·d)~(-1),and AOA and AOB were abundant in neutral purple soils. By using DNA-based stable isotope probing(SIP),we gathered strong evidence of archaeal ammonia oxidation by AOA and AOB. Phylogenetic analysis indicated that the Nitrosospira Cluster 3a. 1 AOB was dominant in terms of quantity at 0 days,and the Nitrosospira Cluster 3a. 2 only accounted for a small part. After 56 days of cultivation,the Nitrosospira Cluster 3a. 2 replaced the Nitrosospira Cluster 3a. 1 as the active AOB that dominated ammonia oxidation. The AOA that predominated quantitatively at day 0 was Nitrososphaera Subcluster 9,but after cultivation this became Nitrososphaera Subcluster 3. 2/3. 3. Thus,the community structure of AOA and AOB changed. Active autotrophic nitrification was found in this neutral purple soil. Sequencing analysis of the ~(13)C-labeled DNA provided robust evidence that both archaea and bacteria played important roles in the nitrification and not all ammonia oxidizers in native soil were active in the nitrification. Phylogenetic analysis clearly showed that the dominant active archaea and bacteria during the incubation were affiliated with Nitrososphaera Subcluster 3. 2/3. 3 within the soil group 1. 1b lineage and Nitrosospira Cluster 3a. 2,respectively,which were different from the dominant ammonia oxidizers at the beginning of the incubation. These results suggest that the community structure of ammonia oxidizers can shift quickly upon changes in the substrate availability in soils.
Increasing evidence suggests that ammonia oxidation in acidic soils is primarily catalyzed by ammonia-oxidizing archaea(AOA),while ammonia-oxidizing bacteria(AOB) drive ammonia oxidation in neutral and alkaline soils in which AOA overwhelmingly outnumber AOB. Therefore,neutral purple soil with a pH of 7. 2 was selected to study the composition of the active ammoxidation microbial community with a stable isotope nucleic acid probe technique combined with cloning sequencing. Results showed that the nitrification rate was 9. 68 mg·(kg·d)~(-1),and AOA and AOB were abundant in neutral purple soils. By using DNA-based stable isotope probing(SIP),we gathered strong evidence of archaeal ammonia oxidation by AOA and AOB. Phylogenetic analysis indicated that the Nitrosospira Cluster 3a. 1 AOB was dominant in terms of quantity at 0 days,and the Nitrosospira Cluster 3a. 2 only accounted for a small part. After 56 days of cultivation,the Nitrosospira Cluster 3a. 2 replaced the Nitrosospira Cluster 3a. 1 as the active AOB that dominated ammonia oxidation. The AOA that predominated quantitatively at day 0 was Nitrososphaera Subcluster 9,but after cultivation this became Nitrososphaera Subcluster 3. 2/3. 3. Thus,the community structure of AOA and AOB changed. Active autotrophic nitrification was found in this neutral purple soil. Sequencing analysis of the ~(13)C-labeled DNA provided robust evidence that both archaea and bacteria played important roles in the nitrification and not all ammonia oxidizers in native soil were active in the nitrification. Phylogenetic analysis clearly showed that the dominant active archaea and bacteria during the incubation were affiliated with Nitrososphaera Subcluster 3. 2/3. 3 within the soil group 1. 1b lineage and Nitrosospira Cluster 3a. 2,respectively,which were different from the dominant ammonia oxidizers at the beginning of the incubation. These results suggest that the community structure of ammonia oxidizers can shift quickly upon changes in the substrate availability in soils.
引文
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[3] Hyman M R,Arp D J.14C2H2-and14CO2-labeling studies of the de novo synthesis of polypeptides by Nitrosomonas europaea during recovery from acetylene and light inactivation of ammonia monooxygenase[J]. The Journal of Biological Chemistry,1992,267(3):1534-1545.
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[9] Ste-Marie C,ParéD. Soil,pH and N availability effects on net nitrification in the forest floors of a range of boreal forest stands[J]. Soil Biology and Biochemistry,1999,31(11):1579-1589.
[10] Cheng Y,Wang J,Mary B,et al. Soil pH has contrasting effects on gross and net nitrogen mineralizations in adjacent forest and grassland soils in central Alberta,Canada[J]. Soil Biology and Biochemistry,2013,57:848-857.
[11] Suzuki I,Dular U,Kwok S C. Ammonia or ammonium ion as substrate for oxidation by Nitrosomonas europaea cells and extracts[J]. Journal of Bacteriology,1974,120(1):556-558.
[12] Gubry-Rangin C,Hai B,Quince C,et al. Niche specialization of terrestrial archaeal ammonia oxidizers[J]. Proceedings of the National Academy of Sciences of the United States of America,2011,108(52):21206-21211.
[13] Hu B L,Liu S,Wang W,et al. pH-dominated niche segregation of ammonia-oxidising microorganisms in Chinese agricultural soils[J]. FEMS Microbiology Ecology,2014,90(1):290-299.
[14] He J Z,Shen J P,Zhang L M,et al. Quantitative analyses of the abundance and composition of ammonia-oxidizing bacteria and ammonia-oxidizing archaea of a Chinese upland red soil under long-term fertilization practices[J]. Environmental Microbiology,2007,9(9):2364-2374.
[15] Nicol G W,Leininger S,Schleper C,et al. The influence of soil pH on the diversity,abundance and transcriptional activity of ammonia oxidizing archaea and bacteria[J]. Environmental Microbiology,2008,10(11):2966-2978.
[16] Prosser J I, Nicol G W. Archaeal and bacterial ammoniaoxidisers in soil:the quest for niche specialisation and differentiation[J]. Trends in Microbiology,2012,20(11):523-531.
[17] Offre P,Prosser J I,Nicol G W. Growth of ammonia-oxidizing archaea in soil microcosms is inhibited by acetylene[J]. FEMS Microbiology Ecology,2009,70(1):99-108.
[18] Gubry-Rangin C,Nicol G W,Prosser J I. Archaea rather than bacteria control nitrification in two agricultural acidic soils[J].FEMS Microbiology Ecology,2010,74(3):566-574.
[19] Di H J,Cameron K C,Shen J P,et al. Nitrification driven by bacteria and not archaea in nitrogen-rich grassland soils[J].Nature Geoscience,2009,2(9):621-624.
[20] Xiang X J,He D,He J S,et al. Ammonia-oxidizing bacteria rather than archaea respond to short-term urea amendment in an alpine grassland[J]. Soil Biology and Biochemistry,2017,107:218-225.
[21] Pester M,Rattei T,Flechl S,et al. Amo A-based consensus phylogeny of ammonia-oxidizing archaea and deep sequencing of amoA genes from soils of four different geographic regions[J].Environmental Microbiology,2012,14(2):525-539.
[22] Wang B Z,Zheng Y,Huang R,et al. Active ammonia oxidizers in an acidic soil are phylogenetically closely related to neutrophilic archaeon[J]. Applied and Environmental Microbiology,2014,80(5):1684-1691.
[23] Radajewski S, Mc Donald I R, Murrell J C. Stable-isotope probing of nucleic acids:a window to the function of uncultured microorganisms[J]. Current Opinion in Biotechnology,2003,14(3):296-302.
[24] Alves R J E,Wanek W,Zappe A,et al. Nitrification rates in arctic soils are associated with functionally distinct populations of ammonia-oxidizing archaea[J]. The ISME Journal,2013,7(8):1620-1631.
[25] Wang X L,Chen H,Zhang J B,et al. Long-term fertilization effects on active ammonia oxidizers in an acidic upland soil in China[J]. Soil Biology and Biochemistry,2015,84:28-37.
[26] Wang Z H, Meng Y, Zhu-Barker X, et al. Responses of nitrification and ammonia oxidizers to a range of background and adjusted p H in purple soils[J]. Geoderma,2019,334:9-14.
[27]王梅,王智慧,石孝均,等.长期不同施肥量对全程氨氧化细菌丰度的影响[J].环境科学,2018,39(10):4727-4734.Wang M,Wang Z H,Shi X J,et al. Long-term fertilization effects on the abundance of complete ammonia oxidizing bacteria(Comammox Nitrospira)in a neutral paddy soil[J].Environmental Science,2018,39(10):4727-4734.
[28] Jiang X J,Hou X Y,Zhou X,et al. pH regulates key players of nitrification in paddy soils[J]. Soil Biology and Biochemistry,2015,81:9-16.
[29]闫小娟.三种紫色土硝化作用及其硝化微生物的研究[D].重庆:西南大学,2016.Yan X J. Research on nitrification and nitrifying microoganisms of three purple soils[D]. Chongqing:Southwest University,2016.
[30] Rotthauwe J H, Witzel K P, Liesack W. The ammonia monooxygenase structural gene amoA as a functional marker:molecular fine-scale analysis of natural ammonia-oxidizing populations[J]. Applied and Environmental Microbiology,1997,63(12):4704-4712.
[31] Francis C A,Roberts K J,Beman J M,et al. Ubiquity and diversity of ammonia-oxidizing archaea in water columns and sediments of the ocean[J]. Proceedings of the National Academy of Sciences of the United States of America,2005,102(41):14683-14688.
[32] Avrahami S,Conrad R. Patterns of community change among ammonia oxidizers in meadow soils upon long-term incubation at different temperatures[J]. Applied and Environmental Microbiology,2003,69(10):6152-6164.
[33] Hu H W,Macdonald C A,Trivedi P,et al. Water addition regulates the metabolic activity of ammonia oxidizers responding to environmental perturbations in dry subhumid ecosystems[J].Environmental Microbiology,2015,17(2):444-461.
[34] Santoro A E,Francis C A,de Sieyes N R,et al. Shifts in the relative abundance of ammonia-oxidizing bacteria and archaea across physicochemical gradients in a subterranean estuary[J].Environmental Microbiology,2008,10(4):1068-1079.
[35] Zhang L M,Hu H W,Shen J P,et al. Ammonia-oxidizing archaea have more important role than ammonia-oxidizing bacteria in ammonia oxidation of strongly acidic soils[J]. The ISME Journal,2012,6(5):1032-1045.
[36] Wang B Z,Zhao J,Guo Z Y,et al. Differential contributions of ammonia oxidizers and nitrite oxidizers to nitrification in four paddy soils[J]. The ISME Journal,2015,9(5):1062-1075.
[37] Pratscher J, Dumont M G, Conrad R. Ammonia oxidation coupled to CO2fixation by archaea and bacteria in an agricultural soil[J]. Proceedings of the National Academy of Sciences of the United States of America,2011,108(10):4170-4175.
[38] Xia W W,Zhang C X,Zeng X W,et al. Autotrophic growth of nitrifying community in an agricultural soil[J]. The ISME Journal,2011,5(7):1226-1236.
[39] Zhao J,Wang B Z,Jia Z J. Phylogenetically distinct phylotypes modulate nitrification in a paddy soil[J]. Applied and Environmental Microbiology,2015,81(9):3218-3227.
[40] Zhong W H,Bian B Y,Gao N,et al. Nitrogen fertilization induced changes in ammonia oxidation are attributable mostly to bacteria rather than archaea in greenhouse-based high N input vegetable soil[J]. Soil Biology and Biochemistry,2016,93:150-159.
[41] Bertagnolli A D, Mc Calmont D, Meinhardt K A, et al.Agricultural land usage transforms nitrifier population ecology[J]. Environmental Microbiology, 2016, 18(6):1918-1929.
[42] Lennon J T,Jones S E. Microbial seed banks:the ecological and evolutionary implications of dormancy[J]. Nature Reviews Microbiology,2011,9(2):119-130.
[43] Kaprelyants A S,Gottschal J C,Kell D B. Dormancy in nonsporulating bacteria[J]. FEMS Microbiology Reviews,1993,10(3-4):271-285.
[2] Ravishankara A R,Daniel J S,Portmann R W. Nitrous oxide(N2O):The dominant ozone-depleting substance emitted in the21st century[J]. Science,2009,326(5949):123-125.
[3] Hyman M R,Arp D J.14C2H2-and14CO2-labeling studies of the de novo synthesis of polypeptides by Nitrosomonas europaea during recovery from acetylene and light inactivation of ammonia monooxygenase[J]. The Journal of Biological Chemistry,1992,267(3):1534-1545.
[4] Treusch A H,Leininger S,Kletzin A,et al. Novel genes for nitrite reductase and amo-related proteins indicate a role of uncultivated mesophilic crenarchaeota in nitrogen cycling[J].Environmental Microbiology,2005,7(12):1985-1995.
[5] Leininger S,Urich T,Schloter M,et al. Archaea predominate among ammonia-oxidizing prokaryotes in soils[J]. Nature,2006,442(7104):806-809.
[6] Jia Z J, Conrad R. Bacteria rather than Archaea dominate microbial ammonia oxidation in an agricultural soil[J].Environmental Microbiology,2009,11(7):1658-1671.
[7] Allison S M, Prosser J I. Urease activity in neutrophilic autotrophic ammonia-oxidizing bacteria isolated from acid soils[J]. Soil Biology and Biochemistry,1991,23(1):45-51.
[8] Bruns M A,Stephen J R,Kowalchuk G A,et al. Comparative diversity of ammonia oxidizer 16S rRNA gene sequences in native, tilled, and successional soils[J]. Applied and Environmental Microbiology,1999,65(7):2994-3000.
[9] Ste-Marie C,ParéD. Soil,pH and N availability effects on net nitrification in the forest floors of a range of boreal forest stands[J]. Soil Biology and Biochemistry,1999,31(11):1579-1589.
[10] Cheng Y,Wang J,Mary B,et al. Soil pH has contrasting effects on gross and net nitrogen mineralizations in adjacent forest and grassland soils in central Alberta,Canada[J]. Soil Biology and Biochemistry,2013,57:848-857.
[11] Suzuki I,Dular U,Kwok S C. Ammonia or ammonium ion as substrate for oxidation by Nitrosomonas europaea cells and extracts[J]. Journal of Bacteriology,1974,120(1):556-558.
[12] Gubry-Rangin C,Hai B,Quince C,et al. Niche specialization of terrestrial archaeal ammonia oxidizers[J]. Proceedings of the National Academy of Sciences of the United States of America,2011,108(52):21206-21211.
[13] Hu B L,Liu S,Wang W,et al. pH-dominated niche segregation of ammonia-oxidising microorganisms in Chinese agricultural soils[J]. FEMS Microbiology Ecology,2014,90(1):290-299.
[14] He J Z,Shen J P,Zhang L M,et al. Quantitative analyses of the abundance and composition of ammonia-oxidizing bacteria and ammonia-oxidizing archaea of a Chinese upland red soil under long-term fertilization practices[J]. Environmental Microbiology,2007,9(9):2364-2374.
[15] Nicol G W,Leininger S,Schleper C,et al. The influence of soil pH on the diversity,abundance and transcriptional activity of ammonia oxidizing archaea and bacteria[J]. Environmental Microbiology,2008,10(11):2966-2978.
[16] Prosser J I, Nicol G W. Archaeal and bacterial ammoniaoxidisers in soil:the quest for niche specialisation and differentiation[J]. Trends in Microbiology,2012,20(11):523-531.
[17] Offre P,Prosser J I,Nicol G W. Growth of ammonia-oxidizing archaea in soil microcosms is inhibited by acetylene[J]. FEMS Microbiology Ecology,2009,70(1):99-108.
[18] Gubry-Rangin C,Nicol G W,Prosser J I. Archaea rather than bacteria control nitrification in two agricultural acidic soils[J].FEMS Microbiology Ecology,2010,74(3):566-574.
[19] Di H J,Cameron K C,Shen J P,et al. Nitrification driven by bacteria and not archaea in nitrogen-rich grassland soils[J].Nature Geoscience,2009,2(9):621-624.
[20] Xiang X J,He D,He J S,et al. Ammonia-oxidizing bacteria rather than archaea respond to short-term urea amendment in an alpine grassland[J]. Soil Biology and Biochemistry,2017,107:218-225.
[21] Pester M,Rattei T,Flechl S,et al. Amo A-based consensus phylogeny of ammonia-oxidizing archaea and deep sequencing of amoA genes from soils of four different geographic regions[J].Environmental Microbiology,2012,14(2):525-539.
[22] Wang B Z,Zheng Y,Huang R,et al. Active ammonia oxidizers in an acidic soil are phylogenetically closely related to neutrophilic archaeon[J]. Applied and Environmental Microbiology,2014,80(5):1684-1691.
[23] Radajewski S, Mc Donald I R, Murrell J C. Stable-isotope probing of nucleic acids:a window to the function of uncultured microorganisms[J]. Current Opinion in Biotechnology,2003,14(3):296-302.
[24] Alves R J E,Wanek W,Zappe A,et al. Nitrification rates in arctic soils are associated with functionally distinct populations of ammonia-oxidizing archaea[J]. The ISME Journal,2013,7(8):1620-1631.
[25] Wang X L,Chen H,Zhang J B,et al. Long-term fertilization effects on active ammonia oxidizers in an acidic upland soil in China[J]. Soil Biology and Biochemistry,2015,84:28-37.
[26] Wang Z H, Meng Y, Zhu-Barker X, et al. Responses of nitrification and ammonia oxidizers to a range of background and adjusted p H in purple soils[J]. Geoderma,2019,334:9-14.
[27]王梅,王智慧,石孝均,等.长期不同施肥量对全程氨氧化细菌丰度的影响[J].环境科学,2018,39(10):4727-4734.Wang M,Wang Z H,Shi X J,et al. Long-term fertilization effects on the abundance of complete ammonia oxidizing bacteria(Comammox Nitrospira)in a neutral paddy soil[J].Environmental Science,2018,39(10):4727-4734.
[28] Jiang X J,Hou X Y,Zhou X,et al. pH regulates key players of nitrification in paddy soils[J]. Soil Biology and Biochemistry,2015,81:9-16.
[29]闫小娟.三种紫色土硝化作用及其硝化微生物的研究[D].重庆:西南大学,2016.Yan X J. Research on nitrification and nitrifying microoganisms of three purple soils[D]. Chongqing:Southwest University,2016.
[30] Rotthauwe J H, Witzel K P, Liesack W. The ammonia monooxygenase structural gene amoA as a functional marker:molecular fine-scale analysis of natural ammonia-oxidizing populations[J]. Applied and Environmental Microbiology,1997,63(12):4704-4712.
[31] Francis C A,Roberts K J,Beman J M,et al. Ubiquity and diversity of ammonia-oxidizing archaea in water columns and sediments of the ocean[J]. Proceedings of the National Academy of Sciences of the United States of America,2005,102(41):14683-14688.
[32] Avrahami S,Conrad R. Patterns of community change among ammonia oxidizers in meadow soils upon long-term incubation at different temperatures[J]. Applied and Environmental Microbiology,2003,69(10):6152-6164.
[33] Hu H W,Macdonald C A,Trivedi P,et al. Water addition regulates the metabolic activity of ammonia oxidizers responding to environmental perturbations in dry subhumid ecosystems[J].Environmental Microbiology,2015,17(2):444-461.
[34] Santoro A E,Francis C A,de Sieyes N R,et al. Shifts in the relative abundance of ammonia-oxidizing bacteria and archaea across physicochemical gradients in a subterranean estuary[J].Environmental Microbiology,2008,10(4):1068-1079.
[35] Zhang L M,Hu H W,Shen J P,et al. Ammonia-oxidizing archaea have more important role than ammonia-oxidizing bacteria in ammonia oxidation of strongly acidic soils[J]. The ISME Journal,2012,6(5):1032-1045.
[36] Wang B Z,Zhao J,Guo Z Y,et al. Differential contributions of ammonia oxidizers and nitrite oxidizers to nitrification in four paddy soils[J]. The ISME Journal,2015,9(5):1062-1075.
[37] Pratscher J, Dumont M G, Conrad R. Ammonia oxidation coupled to CO2fixation by archaea and bacteria in an agricultural soil[J]. Proceedings of the National Academy of Sciences of the United States of America,2011,108(10):4170-4175.
[38] Xia W W,Zhang C X,Zeng X W,et al. Autotrophic growth of nitrifying community in an agricultural soil[J]. The ISME Journal,2011,5(7):1226-1236.
[39] Zhao J,Wang B Z,Jia Z J. Phylogenetically distinct phylotypes modulate nitrification in a paddy soil[J]. Applied and Environmental Microbiology,2015,81(9):3218-3227.
[40] Zhong W H,Bian B Y,Gao N,et al. Nitrogen fertilization induced changes in ammonia oxidation are attributable mostly to bacteria rather than archaea in greenhouse-based high N input vegetable soil[J]. Soil Biology and Biochemistry,2016,93:150-159.
[41] Bertagnolli A D, Mc Calmont D, Meinhardt K A, et al.Agricultural land usage transforms nitrifier population ecology[J]. Environmental Microbiology, 2016, 18(6):1918-1929.
[42] Lennon J T,Jones S E. Microbial seed banks:the ecological and evolutionary implications of dormancy[J]. Nature Reviews Microbiology,2011,9(2):119-130.
[43] Kaprelyants A S,Gottschal J C,Kell D B. Dormancy in nonsporulating bacteria[J]. FEMS Microbiology Reviews,1993,10(3-4):271-285.
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