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题名: 土壤微生物群落对环境胁迫的响应及其机制
作者: 李晶
学位类别: 博士
答辩日期: 2015-05
授予单位: 中国科学院研究生院
授予地点: 北京
导师: 贺纪正
关键词: 微生物群落结构、微生物多样性、微生物功能、铜胁迫、干湿交替胁 迫、抵抗力, microbial community structure, microbial diversity, microbial function, copper stress, dry-rewetting stress, resistance
其他题名: Responses and the mechanisms of soil microbial community to environmental stresses
学位专业: 生态学
中文摘要:     随着人类活动的加剧,土壤生态系统受到越来越多的人为活动干扰,这些干扰影响到土壤生态系统的功能和稳定性。土壤微生物是土壤生态系统的核心组成,数量巨大且种类丰富,广泛参与元素的生物地球化学循环,对植物生长、气候调节、污染物转化(降解)等陆地生态系统功能起着不可替代的重要作用。土壤微生物群落对暂时性扰动或长期胁迫的抵抗力和恢复力在一定程度上可反映生态系统过程和功能的稳定性。研究土壤微生物群落对外界环境胁迫的响应特征和响应机理,对于认识和预测环境变化对土壤微生物系统及其功能的影响具有重要科学意义,对评估土壤污染或环境变化对土壤生态系统的影响具有重要实用价值。
    本论文采用分子生态学研究方法,如聚合酶链式反应(PCR)、实时荧光定量  PCR、末端限制性片段多态性分析(T-RFLP)、克隆和测序技术等,以及高通量测序(illumina miseq),系统深入地揭示了重金属铜或镍污染梯度对土壤微生物群落结构、多样性和功能的影响,阐释了初次铜胁迫是否能够增强土壤微生物群落对二次铜胁迫的抵抗力、初次铜胁迫是否能够增强土壤微生物群落对干湿交替胁迫的抵抗力,在此基础上探讨了土壤微生物群落对环境胁迫的适应机制。论文的主要研究内容与成果如下:
1.连续两年分别从山东德州(潮土)和湖南祁阳(红壤)采集经历了五年不同铜或镍重金属污染梯度的田间土壤样品,用高通量测序(miseq)方法研究了细菌群落结构、多样性和功能分别随铜或镍重金属浓度梯度的变化特点。两种土壤中的微生物量碳(SMBC)随铜浓度升高显著下降,细菌丰度也基本呈现类似的变化趋势,尽管两种土壤的细菌群落结构有显著差异,但是细菌的多样性均随铜浓度升高而显著下降。结构方程模型(SEM)的结果显示细菌多样性和群落组成的变化决定了 SMBC的变化。与铜胁迫类似,两种土壤中的微生物量碳(SMBC)随镍浓度升高也呈现出显著下降的趋势,尽管两种土壤中细菌群落结构随镍浓度梯度均有显著的变化,但是多样性和丰度变化均未呈现有规律的下降或者上升趋势。本研究还分别找出了铜、镍的敏感型和耐受型微生物群类群,发现放线菌均为铜和镍的耐受型类群,酸杆菌均为铜和镍的敏感型类群,绿弯菌为铜敏感型类群以及浮霉菌为镍敏感型类群。本研究采用大田的实验数据强有力地证明了铜和镍胁迫的确对细菌群落结构和功能都造成了显著的负面影响,但对多样性的影响不同。
2.采用 T-RFLP的技术手段研究了德州  8个铜污染梯度的土壤(浓度范围是0-3200 mg kg-1)中细菌、古菌、氨氧化细菌(AOB)和氨氧化古菌(AOA)的群落结构变化,并测定了硝化潜势(PNR)和土壤呼吸(SIR)等功能指标的变化,及细菌、古菌、AOB和AOA的丰度变化。研究结果发现,PNR、SIR及各微生物的丰度随铜浓度的升高均有显著变化,各微生物的群落结构在高铜浓度和低铜浓度处理也有显著的差异。因此进一步挑选了差异最显著的Cu0和Cu3200处理样品,分别设置了四个处理包括Cu0-C(不添加二次铜胁迫)、Cu3200-C(不添加二次铜胁迫)、Cu0-A(添加二次铜胁迫)、Cu3200-A(添加二次铜胁迫)进行微宇宙培养试验,研究土壤微生物群落对二次铜胁迫的响应。结果发现,PNR和SIR在90天的培养试验中均表现出了恢复的趋势而微生物的丰度则变化趋势不明显。微生物群落的抵抗力大小采用 Bray–Curtis相异性值的大小来量化,Cu0-A处理与  Cu0-C处理相比较微生物群落的  Bray–Curtis相异性值在二次铜胁迫后显著高于胁迫前,而 Cu3200-A处理与Cu3200-C处理相比较微生物群落的 Bray–Curtis相异性值在二次铜胁迫前后差异不大。这表明,初次铜胁迫增强了土壤微生物群落对二次铜胁迫的抵抗力。
3.进一步又研究了初次铜胁迫是否能够增加土壤微生物群落对干湿交替(DW)胁迫的抵抗力,分别选取了潮土中   Cu0(低)、Cu800(中)、Cu3200(高)三个处理设置了 Cu0、Cu0-S(DW胁迫)、Cu800-S(DW胁迫)、Cu3200-S(DW胁迫)四个处理,和红壤中的  Cu0(低)、Cu200(中)、Cu800(高)三处理设置了 Cu0、Cu0-S(DW胁迫)、Cu200-S(DW胁迫)、Cu800-S(DW胁迫)四个处理,干湿交替胁迫共包含 10次干湿交替循环。土壤呼吸的抵抗力(RSSIR)结果表明了在两种土壤中 Cu0-S处理的  RS SIR显著高于其它处理,说明了未受初次铜胁迫的处理抵抗力显著高于经受了初次铜胁迫的处理。对于细菌群落来说,潮土中Cu0-S处理细菌群落的变化速率显著低于Cu800-S和Cu3200-S处理,这说明了细菌群落变化速率快导致经受了初次铜胁迫的处理土壤呼吸的抵抗力低,红壤中细菌群落的变化速率在各处理间虽然没有显著差异,但是由于 Cu800-S处理中的最优势类群 WPS-2在干湿交替胁迫过程中相对丰度大幅增加导致了土壤呼吸的抵抗力显著下降。结构方程模型(SEM)的结果再次印证了细菌群落的变化决定了不同铜胁迫处理抵抗力的不同。此结果有力的证明了初次铜胁迫减弱了土壤微生物群落功能对干湿交替胁迫的抵抗力,这是因为初次铜胁迫处理的细菌群落变化速率或者优势类群的变化较大。
4.综合以上研究结论并综合分析相关文献,提出了土壤微生物群落对环境胁迫可能的响应和适应机制:(1)微生物群落在长期的铜镍胁迫下通过改变群落结构来适应胁迫的环境,铜镍敏感型类群逐渐被铜镍耐受型类群所替代,而耐受型类群之所以能够适应重金属胁迫的环境是通过活跃的重金属外排作用或被重金属螯合物质络合,从而保证胞内重金属浓度限定在一定范围内;(2)微生物的功能并不一定随微生物群落结构的改变而改变,最可能的原因就是微生物群落存在较高程度的功能冗余,即不同的微生物类群可能执行相同的功能,因此虽然微生物的群落结构发生了改变,但是微生物相应的功能并不受显著影响,功能冗余对于维持微生物群落的稳定性有重要作用;(3)初次铜胁迫可以增强微生物群落对二次铜胁迫的抵抗力很可能是因为二次胁迫与初次胁迫类型一致,因此在添加二次铜胁迫后,两次胁迫相似有可能使微生物群落形成协同耐受性从而增强微生物群落的抵抗力。(4)初次铜胁迫降低了微生物功能对其它胁迫如干湿交替胁迫的抵抗力,这是因为经受了初次铜胁迫的土壤微生物群落受到干湿交替胁迫时群落的变化速率快,且优势类群有较大的变化,细菌群落的变化决定了微生物功能对干湿交替胁迫的抵抗力。
    综上所述,本论文通过综合运用各种分子生态学研究手段,从室外场地实验至微宇宙培养实验,揭示了土壤微生物群落对不同环境胁迫的响应特征并分析了其可能机制。
英文摘要:     With   the   intensification    of   anthropogenic   activities,    soil   ecosystem   is increasingly  under  environmental  disturbances   most  related  to  human   activities which seriously  undermine the stability  of soil  ecosystem and affect  the ecosystem services. Microorganisms  living in soil  are abundant  and highly diverse,  which are the  key  players   of  many  soil  functions   such  as  biogeochemical  cycling,   plant productivity,  climate regulation  or  pollutants degradation  and are  essential  for the integrity of terrestrial ecosystems.  The resistance and resilience of microorgnisms  to short  and long  term stresses  can  reflect the  stability  of ecosystem  processes.  It is far-reaching to understand and forecast  the impacts of environmental fluctuations on soil microorganisms and  ecosystem by exploring  the response chracteristics  and the mechanisms of soil microbial community to environmental stresses.
    Various techniques  in moleculer  ecology have  been used,  such as  polymerase chain   reaction    (PCR),   real-time   PCR,    terminal   restriction   fragment    length polymorphism   (T-RFLP),   clone   and  sequencing,   in   addition,   illumina   miseq sequencing has  also been  used. We  deeply studied  the effects  of long-term  copper
and  nickel   gradients  on   soil  microbial   community  composition,   diversity  and function, and  whether initial copper  stress increases the  resistance of soil  microbial community  structure  and   function  to  subsequent  stresses   including  copper  and dry-rewetting  stresses. The  possible  adaption  mechanisms are  also  proposed. The research contents and main findings of this thesis are described as follows:
1.   Soil samples  were collected from two  contrasting soils, that  is, fluvo-aquic soil from Dezhou, Shandong provice and red soil from Qiyang, Hunan province in  in two years, and  they were polluted by copper  and nickel gradients for  about five and six  years,  respectively.  This study  investigated  the  impacts  of  copper and  nickel gradients  on soil  bacterial  community  composition, diversity  and  function in  two agricultural  fields  with contrasting  soil  properties  by  illumina  miseq  sequencing. Consistent  reduction  was  found  in  soil  microbial  biomass  carbon  (SMBC)  with increasing copper  levels  in both  soils,  coupled by  significant declines  in  bacterial abundance  in  most  cases.  Despite  of  contrasting  bacterial  community  structures between  the  two   soils,  the  bacterial  diversity   in  the  copper-contaminated  soils showed  considerably  decreasing  patterns when  copper  levels  elevated.  Structural equation model  (SEM)  analysis ascertained  that  the shifts  of bacterial  community composition and  diversity were  closely related  with the changes  of SMBC  in both soils.   Similar  to   the  copper,   soil   microbial  biomass   carbon  consistently   also decreased along the  nickel gradients in  both soils. Despite the  apparent shifts in  the bacterial  community composition,  no clear  tendency  in the  bacterial diversity  and abundance was identified along the nickel  gradients in either soil. Copper and nickel pollution   selectively  favored   or  impeded   the   prevalence  of   several   dominant bacterial   guilds,   in   particular,    Actinobacteria   showed   both   tolerance,   while Acidobacteria displayed  both sensitivity,  and Chloroflexi  and Planctomycetes  were highly sensitive  to copper  and nickel,  respectively. The  results  provide field-based evidence that copper and nickel contaminations exhibit consistently negative impacts on  soil  bacterial  community  composition  and  function,   however,  the  effects  on bacterial diversity were different.
2.   Terminal restriction  fragment length  polymorphism (T-RFLP)  was used  to
investigate   the   soil  bacterial,   archaeal,   AOB   and   AOA  communities   in   the fluvo-aquic soil  with eight  copper levels ranging  from 0  to 3,200 mg  Cu kg−1 soil. The substrate-induced  respiration (SIR)  and potential  nitrification rate  (PNR) were measured.  Furthermore,  the   abundance  of  bacterial,  archaeal  16S   rRNA  genes,ammonia-oxidizing  bacteria  and  archaea   amoA  genes  were  determined  through quantitative PCR. For the  field samples, the SIR  and PNR as well  as the abundance of  soil   microorganisms  varied   significantly   between  eight   copper  levels.   Soil microbial communities highly differed between the low and high copper  stress. Then,the  soils  with 0  and  3,200  mg  Cu  kg−1  were selected  to  construct  a  microcosm experiment.  Four treatments  were  set up  including  Cu0-C and  Cu3200-C  without further Cu addition, and Cu0-A and Cu3200-A with addition of 57.5 mg Cu kg−1  soil. In the microcosm experiment, the PNR and  SIR both recovered while the abundance of   soil  microorganisms   varied   irregularly  during   the   90-day  incubation.   The differences    of    microbial    communities   measured    by    pairwise    Bray–Curtis dissimilarities between  Cu0-A and  Cu0-C on  day 0  were significantly  higher after subsequent  stress than  before. However,  the differences  of  microbial communities between Cu3200-A and Cu3200-C  on day 0 changed little  between after subsequent stress and before. Therefore, initial copper stress  could increase the resistance of soil microorganisms to subsequent copper stress.
3. It  is   interesting  to  study   whether  initial   copper  stress   could  increase   the resistance  of soil  microorganisms to  subsequent  dry-rewetting (DW)  stress.  Three treatments including  Cu0 (low),  Cu800 (medium),  Cu3200 (high)  were selected  in the  fluvo-aquic soil  to  set up  four  treatments including  Cu0,  Cu0-S  (DW stress),Cu800-S (DW stress), Cu3200-S  (DW stress). As for the red soil,  Cu0 (low), Cu200 (medium), Cu800 (high) treatments were selected  to set up four treatments including Cu0, Cu0-S (DW  stress), Cu200-S (DW stress)  and Cu800-S (DW stress).  The DW stress included ten  dry-rewetting transitions. The  values of resistance  index for SIR
(RS SIR)  in Cu0-S  were higher  than the  other two treatments  in both  soils, which indicated that  RS SIR was  higher in  the non-initial copper  stressed treatments than that in  the initial copper  stressed treatments.  As for the  bactterial communities,  the rates   of  community   variance   were   much   higher  in   Cu800-S   and   Cu3200-S treatments than that in Cu0-S in the fluvo-aquic soil, which indicated that the rates of community variance were higher in the  initial copper stressed treatments than that in the  non-initial copper  stressed treatment.  It  might suggest  that  the higher  rates  of community variance in  copper-stressed treatments lead  to the lower RS  SIR values. In  the  red soil,  although  the  rates  of  community variance  were  not  significantly different between the non-initial and initial copper  stressed treatments, WPS-2 as the most dominant  group in the  bacterial community increased  sharply in the  period of DW  stress  in the  Cu800-S  treatment,  and  the substantial  increase  in  the  relative abundance  might  result  in  the  lower  RS  SIR.  Structural  equation  model  (SEM)
analysis ascertained that  the shifts of bacterial  communities determined the  changes of RS SIR in  both soils.The results provide evidences that initial  copper stress could decrease the resistance of soil microbial function due to subsequent DW stress due to the  higher  microbial  community   variance  rates  and  the  variations   of  dominant
groups.
4.   Based  on  the  above  findings  and comprehensive  analysis  of  the  relevant literatures, we  proposed several  possible mechanisms  of soil  microbial communities in response to environmental stresses: (1) Microbial communities gradually adapted to the  long-term  copper   and  nickel  stresses  by  changing   the  microbial  community structure.  The  shifts  might   be  explained  by  that  some   tolerant  phylotypes  were selected while sensitive ones were obviously  reduced, and the adaption mechanism of these  tolerant groups  was  attributed  to active  metal  efflux transport  systems  or  by complexation with  metal chelating substances  for limiting free  ions of metals  within the cell. (2) Functional redundancy might explain  the phenomenon of little changes in
the activities of  microorganisms despite evident  changes in the  community structure. In  fact, functional  redundancy  in microbial  communities  has  been suggested  to  be high  with  many   species  performing  the  same   functions.  It  was  also   considered important in maintaining microbial community stability.  (3) Exposure to copper stress could increase  the resistance of soil  microorganisms to  the subsequent copper stress. The  nature  of  the  subsequent  stress  was  similar  to  the  first,  therefore,  microbial communities  might   develop  the   co-tolerance   which  increased   the  resistance   to subsequent stress.  (4) Exposure to  copper stress could  decrease the resistance of  soil microbial  function to  the  subsequent  dry-rewetting stress.  The  rates of  community variance were higher  in the initial copper stressed  treatments and dominant groups in the  microbial  community  significantly  changed  in   the  relative  abundance,  which might  explain   the  low   resistance  of   soil  microbial   function  to   the  subsequent dry-rewetting stress.
    Taken   together,   filed   in   combination   with   microcosm   experiments   were employed   with   molecular   methods   in   this   thesis   to   decipher   the   ecological characteristics and  microbial mechanisms  of soil microbial  communities in  response to different environmental stresses.
内容类型: 学位论文
URI标识: http://ir.rcees.ac.cn/handle/311016/34339
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Recommended Citation:
李晶. 土壤微生物群落对环境胁迫的响应及其机制[D]. 北京. 中国科学院研究生院. 2015.
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