土壤环境科学实验室知识库

      硝化作用是氮循环的重要环节，在土壤生态系统中广泛存在。硝化作用不仅直接影响着植物对氮素的利用效率，还与土壤酸化、温室气体 N2O产生、硝酸盐淋失造成水体污染等密切相关。一百多年来，硝化作用被认为是由氨氧化过程（NH3转变为  NO2−-N）和亚硝化过程（NO2−-N氧化成  NO−3 -N）两个反应步骤组成，其中氨氧化过程由氨氧化细菌（AOB）和氨氧化古菌（AOA）催化完成；亚硝化过程由亚硝酸盐氧化细菌（NOB）催化完成，二者密切合作才能完成整个硝化过程。2015年，研究发现亚硝酸盐氧化菌中的一些硝化螺菌可将氨直接氧化为硝酸盐，即全程氨氧化（Comammox，complete ammonia oxidation）,Comammox的发现打破了我们对微生物驱动的硝化作用的传统认知。但目前有关 Comammox Nitrospira在土壤环境中的分布特征、功能活性及其与  AOA、AOB对土壤硝化作用的相对贡献还不清楚。
      本论文以代表不同土地利用方式的农田、草地、灌丛、灌丛-自然林间过渡带和自然林土壤为研究对象，利用 qPCR、克隆测序、稳定性同位素示踪（SIP）等技术，研究了不同土地利用方式下土壤中 Comammox Nitrospira、AOA和AOB的分布特征及其功能活性，取得了以下主要结果：
（1）不同土地利用方式下土壤的理化性质有明显差异，五种土壤均偏酸性，pH 4.99~6.50；农田和草地土壤有效氮含量较高（P<0.05），且主要以硝态氮形式存在；灌丛，过渡带和自然林土壤中有效氮含量相对较低，且主要以铵态氮形式存在。
（2）不同氨氧化微生物类群的丰度在不同土壤中差异显著，农田土壤中氨氧化微生物的丰度为 AOB > AOA > Comammox Nitrospira，在其他土壤中均为Comammox Nitrospira > AOA > AOB。AOA的丰度在过渡带土壤中最高，为  1.2×10copies•g-1 干土，在农田、草地、灌丛和自然林土壤间无显著差异；AOB的丰度在农田土壤中最高，为 8.51×106 copies•g-1干土，在草地、灌丛、过渡带和自然林土壤无显著差异；Comammox Nitrospira clade A在过渡带土壤最高，为 1×10 7 copies•g干土，clade    B在灌丛中最高，为 8.91×106  copies•g干土；clade A和  clade B均在农田土壤中最低。

（3）对农田土壤（pH5.80），过渡带土壤（pH6.07）和自然林土壤（pH4.99）进行13CO2稳定同位素标记研究结果表明，农田土壤中硝化作用比过渡带和自然林土壤活跃。在  30天的培养过程中，农田土壤中    AOA，AOB和 Comammox Nitrospira  clade B的丰度显著上升；过渡带土壤中仅  AOA的丰度显著上升；自然林土壤中 Comammox Nitrospira的   clade A和  clade B均显著上升。对 13/12CO2标记的DNA样品进行超高速离心分离和分析发现，农田和过渡带土壤中AOA可通过化能自养作用同化13CO2到其DNA中，而  AOB和  Comammox  Nitrospira 没有检测到，表明 AOA为酸性土壤中活跃的氨氧化微生物类群，农田土壤中这些活性的   AOA主要为     Nitrososphaera（91.9%）,    Nitrosopumilus（3.6%）和Nitrosotalea（4.5%）三个簇，而过渡带土壤中活跃的 AOA主要为   Nitrososphaera簇。
    综上，Comammox Nitrospira在五种土壤中均有分布且丰度高于  AOA和AOB，预示其对土壤硝化过程的潜在作用，通过 SIP研究发现  AOA是酸性农田土壤中活跃的氨氧化微生物，没有检测到有活性的 Comammox Nitrospira，可能与其活性较弱有关。同时我们的研究也再次证明了之前酸性土壤中 AOA起主要作用的结论，并且发现其群落组成会受土地利用方式的影响。

     Nitrification  is  an important  component  of  the global  nitrogen  (N)  cycle  and widely takes place in soil  ecosystems. Nitrification could directly affect the efficiency of  plants  in  nitrogen  utilization,  and  is  closely  related  to  soil  acidification,  N2O emissions  and  water pollution  through  nitrate  leaching.  For  more  than 100  years,nitrification was recognized  as a two-step process  including oxidation of ammonia to nitrite  catalyzed   by  ammonia-oxidizing   bacteria   (AOB)  and   ammonia-oxidizing archaea (AOA), and oxidation of nitrite  to nitrate by nitrite-oxidizing bacteria (NOB),and  could  only be  completed  by  the  cooperation  between  ammonia-oxidizers  and NOB. At the year of  2015, this long-held concept was  challenged by the discovery of Comammox  (Complete ammonia  oxidation)  Nitrospira, being  capable  of oxidizing ammonia to nitrate in  a single organism. However, the  distribution and the functional activity  of  Comammox  Nitrospira  and  the  different  contributions  of  Comammox Nitrospira, AOA and AOB to nitrification were still rarely known in soils.In this study, soil  samples under diffent land uses, including  cropland, grassland, bushland,  forest  land  and  transition  land  between  bushland  and  forest  land  were collected.  Quantitative  PCR, clone  library,  DNA-SIP and  clone  library  sequencing were   employed   to   study   the  distribution   pattern   and   functional   activities   of ammonia-oxidizers，including AOA, AOB and Comammox Nitrospira. The obtained results were listed below.

    (1) Significant  differences in  soil basic  properties were observed  in soils  under different land uses. All five soils were acidic with pH  ranging from 4.99 to 6.50; there were  significant  differences  in  the  available  nitrogen  concentrations  of  the  soils. Cropland  and   grassland   soils  had   relatively  higher   concentrations   of  available nitrogen with  mainly nitrate form  than bushland, transition  land and forest  land with mainly ammonium form.

(2) Significant  differences in the  abundance of ammonia-oxidizers  were found in soils under different land uses. The abundance relationship of ammonia-oxidizers in cropland  is  AOB  >  AOA  >   Comammox  Nitrospira  while  that  in  other   soils  is Comammox  Nitrospira >  AOA  > AOB.  The  highest abundance  of  AOA could  be observed in  transition land soils at  8.51×106 copies•g-1 dry soil while no significant difference in AOA abundance  in other soils; the  highest abundance of AOB  could be observed  in  cropland  soils  at  1.2×107  copies•g-1  dry  soil  while   no  significant difference in  AOA abundance  in  other soils;  the highest  abundance of  Comammox
Nitrospira clade A was in transition land  soils at 1×107 copies•g-1 dry soil, and that of Comammox Nitrospira clade  B was in bushland soils at  8.91×106 copies•g-1 dry soil; both clade A and clade B had the lowest abundance in cropland soils.
(3) In  three lower  pH soils, cropland  soil with  pH at  5.80, transition land  soil with pH  at 6.07 and  forest soil with pH  at 4.99, the  results of incubation  with    13CO2 was  that the  nitrification acitivity  was  more active  in cropland  soils  than transition land soils and forest land soils. After 30 days incubation, in cropland  soil, AOA, AOB and   Comammox   Nitrospira   abundance    had   increased   significantly;   only   the abundance of AOA increased significantly  in transition land soil while the  abundance of  Comammox  Nitrospira  both  clades  increased  significantly   in  forest  land  soil.However, the results from    CO2 labled DNA showed that it is AOA, rather than AOB and  Comammox  Nitrospira,  that  could  be  able  to  assimilate  CO2  by  autotrophic pathway  in cropland  (at  least  10%) and  transition  land  (at least  25%)  soils  which means that AOA was  the active ammonia oxidizer. The  active AOA in cropland soils affiliated  with  the clusters  of  Nitrososphaera  (91.9%),  Nitrosopumilus  (3.6%)  and Nitrosotalea  (4.5%),  but that  in  transition  land only  affiliated  with  the  clusters of Nitrososphaera. 
    In  summary,   Comammox  Nitrospira  could   be  detected   in  five  soils   under different  land  uses,   and  is  the   most  abundant  ammonia-oxidizer,  indicating   its potential role  in nitrification  of acidic  soils. Besides, in  DNA-SIP study,  AOA was identified  as  the   main  active  nitrifier   in  acidic  agricultural  soil   and  no  active Comammox   Nitrospira   was  detected,   partly   because   of   the   low   activity  of
Comammox Nitrospira.  Our study proved the  previous conclusion that  AOA play a predominant role in nitrification  of acidic soils and we  also found that different land uses could affect the community composition of AOA in the soils.