RCEES OpenIR  > 土壤环境科学实验室
典型烃类物质的微生物降解过程与机制研究
Alternative TitleStudies on processes and mechanisms of microbial degradation of hydrocarbons
陈松灿
Subtype博士
Thesis Advisor朱永官 ; 段桂兰
2019-06
Degree Grantor中国科学院生态环境研究中心
Place of Conferral北京
Degree Name理学博士
Degree Discipline环境科学
Keyword烃类物质,稳定同位素探针,纳米二次离子质谱,多重组学,荧光原 位杂交
Abstract

    烃类化合物是一类由碳与氢原子构成的有机物。烃类物质及其衍生物在环境中分布广泛且污染形势日趋严峻。研究烃类物质的微生物降解过程对开发、优化生物技术修复有机污染环境、减缓全球气候变化具有重要的意义。好氧和厌氧生物降解是去除自然环境中烃类有机物的重要途径。目前,大多数研究集中在好氧降解微生物的分离培养以及降解机制。相反,对复杂环境中烃类有机物的原位降解过程以及厌氧微生物的降解机制研究较少。
    本文利用实时定量 PCR(real-time  PCR,RT-PCR)、核酸稳定同位素探针技术(  DNA-stable  isotope  probing,  DNA-SIP)和纳米二次离子质谱技术(nanoscale secondary ion mass  spectrometery,NanoSIMS)等原位表征技术研究并可视化了土壤环境中芳香烃的微生物降解过程,并通过荧光原位杂交技术(fluorescence in  situ hybridization,FISH)、宏基因组学(metagenomics)、宏蛋白组学(metaproteomics)和宏代谢组学( metabolomics)等技术在分子水平揭示了烷烃的厌氧微生物氧化机制。具体研究内容以及结果如下:
1)采用  RT-PCR 和克隆文库技术研究了农田土壤和多环芳烃(  Polycyclic aromatic hydrocarbons,PAHs)污染的工业土壤中功能微生物种群和功能基因对芘污染的动态响应。两种土壤在添加芘培养   35 天后,超过  80%的芘被降解。qPCR结果显示芘降解功能微生物以及芘降解功能基因在培养过程中的丰度显著增加,包括编码环羟基化双加氧酶的革兰氏阳性菌(PAH-RHDα  GP)、分支杆菌(Mycobacterium)、nidA和   nidA3。 这些结果表明对应的功能微生物以及功能基因参与了土壤芘的原位降解过程。两种土壤在添加芘后,降解菌的丰度以及多样性存在明显差异,表明土壤类型、理化性质及污染程度可能影响芘的原位生物降解过程。
2)通过   13C-芘培养、核酸稳定同位素探针技术和高通量测序技术,鉴定了农田土壤以及工业土壤中代谢活跃的芘降解微生物。在培养  35 天后,13C-芘在两种土壤发生显著矿化,其中农田土壤中芘的矿化速率比工业土壤较慢。DNA-SIP 结果表明,假诺卡氏菌属(Pseudonocardia)是农田土壤中芘降解的主要参与者,该菌属的相对丰度在培养过程中增加了  3  个数量级。在工业土壤中,节杆菌属(Arthrobacter)的微生物为芘降解的主要类群,而假诺卡氏菌属未被检测。分支杆菌(Mycobacterium)作为一类重要的芘降解微生物,参与了两种土壤中芘的降解。
3)利用  13C-联苯标记实验结合 NanoSIMS技术在单细胞成像水平上研究了四种多氯联苯( polychlorinated  biphenyls,PCBs)污染土壤中具有联苯代谢功能的微生物种群。对土壤微宇宙顶空   13CO2的测定表明联苯在4天的培养过程中发生了显著的矿化。通过对  NanoSIMS  离子图像的分析表明,联苯在土壤中的降解由少数代谢活性极强的微生物介导,具有联苯同化功能的微生物占土壤总细胞的 5%-15%。根据联苯降解菌细胞中  13C的富集程度,计算了原位降解细胞的碳同化速率。四种土壤中联苯降解微生物的相对丰度以及细胞的碳代谢活性存在明显的差异。
4)通过富集培养、荧光原位杂交、宏基因组、宏蛋白组以及宏代谢组技术揭示了能够厌氧氧化乙烷的古菌。研究所用的共培养体系能够在硫还原条件下将乙烷完全氧化为二氧化碳。古菌为该富集体系的主导微生物菌群,将其命名为 Candidatus  Argoarchaeum ethanivorans;另外也包括具有硫酸盐还原功能的�-变形菌。Ca.  Argoarchaeum 的基因组中含有编码甲基辅酶   M 还原酶(methyl-
coenzyme  M reductase)的所有基因,且这些基因的产物在宏蛋白组中均被检测。同时,利用傅里叶回旋共振质谱以及液相色谱 -质谱确认了中间代谢产物乙基辅酶  M(ethyl-coenzyme M)的存在。这些结果表明  Ca.  Argoarchaeum  通过合成乙基辅酶 M来催化乙烷的活化,这与近期报道的  Ca. Syntrophoarchaeum厌氧丁烷氧化的机制相似。蛋白质基因组学分析显示乙烷氧化生成的中间产物乙酰辅酶 A通过  Wood Ljungdahl途径被氧化为二氧化碳。
    本文围绕烃类物质的微生物降解过程和机制展开研究,首先表征了烃类物质在复杂环境中的原位降解过程。利用  qPCR,DNA-SIP 和  NanoSIMS  技术,发现了对土壤芳香烃降解起关键作用的未培养微生物及降解途径,可视化了原位环境中代谢活跃的降解微生物。其次,本文探索了烃类物质的厌氧微生物氧化机制。结合 FISH和多重组学技术,首次报道了具有乙烷厌氧氧化功能的古菌,并揭示了其氧化烷烃的机理。相关研究结果为准确认知复杂环境中烃类物质的微生物降解过程提供了方法学基础,拓展了人们对烃类物质微生物降解机制的理解。

Other Abstract

     Hydrocarbons  are  a  class  of  compounds  that  consist  entirely  of  carbon  and hydrogen.    Typical    hydrocarbons    and    their    derivatives,    including    aromatic hydrocarbons  and alkanes,  are  widespread  in  the environments,  leading  to  serious problems   worldwide.    Understanding   the   microbial    processes   responsible   for degradation   of   hydrocarbons   can   assist   in  developing   efficient   strategies   for bioremediation  of  organic  pollutants  and  mitigating  global  climate  changes. Both aerobic   and  anaerobic   microorganisms  play   an   important  role   for  removal   of hydrocarbons under natural settings. Microbes and the underlying reactions catalyzing biodegradation of  organic pollutants  under aerobic  conditions have  been intensively studied since several decades.  However, the in situ degradation  processes occurred in natural complex environments like  soil and sediments, as well as  the microbiology of anaerobic hydrocarbon degradation remain elusive.

    In this study,  we applied real-time PCR  (RT-PCR), DNA-stable isotope probing (DNA-SIP) and  nanoscale secondary ion  mass spectrometry (NanoSIMS)  to identify and  visualize  the  active  microorganisms  responsible for  in  situ  biodegradation  of aromatic   hydrocarbons  in   soil  environments.   Furthermore,   fluorescence  in   situ
hybridization (FISH), metagenomics, metaproteomics and metabolomics were utilized to   reveal   the   microorganisms   and    underlying   reactions   catalyzing   biological consumption of alkanes  under anoxic conditions. The main  content and results are  as followings:
1)  qPCR  and  clone  library   were  used  to  monitor  the  dynamic  response   of functional microbes and  genes to pyrene  contamination in an agricultural  soil and an industrial soil.  Over  80% of  added pyrene  was dispersed  in both  soils  after 35-day incubation.   Concurrently,  gene   abundances   targeting  pyrene-degrading   bacteria,
including  Gram-positive   bacteria  harboring  PAH-ring  hydroxylation   dioxygenase (PAH-RHDα  GP) and  Mycobacterium,  were dramatically  enriched. In  addition,  the abundances   of   pyrene    dioxygenase   encoded   by   nidA    and   nidA3,   increased significantly  during   pyrene  incubation,  suggesting  that   two  underlying  catabolic pathways are responsible for pyrene degradation in both soils.  The different behaviors of  two soil  microbial  communities  in  response to  pyrene  stress  indicates  that soil conditions may affect in situ processes of pyrene biodegradation.
2)   To   identify   active   pyrene-degrading   bacteria,    we   applied    13C-pyrene incubations, DNA stable isotope probing (DNA-SIP)  and high-throughput sequencing in an  agricultural soil and  an industrial soil.  After 35-day incubation,  pyrene in both soil was mineralized, with industrial soil showed higher rates than agricultural soil. As revealed  by   DNA-SIP,  the   uncultivated   members  of   Pseudonocardia  were   the dominant pyrene degraders in agricultural  soil; their relative abundances increased by more than 3 orders of magnitude. In contrast, Arthrobacter sp. was found as  the major pyrene-degrading  populations  in  industrial  soil,  whereas  Pseudonocardia  was  not detected. In  both soils,  Mycobacterium was  found to be  actively involved  in pyrene
degradation.
3)  By  applying  13C-biphenyl  incubation  followed by  NanoSIMS  analysis,  we visualized  the biphenyl-degrading  populations in  four  PCB-contaminated sediments and  soils.  13CO2  measurement in  headspace  of microcosms  indicated  that biphenyl was  significantly mineralized  during  4-day incubation.  NanoSIMS  analysis  on soil
microorganisms  showed  that  biphenyl-degrading  microbial  populations  comprised 5%-10%  of  the  whole  communities.   These  results  emphasize  that  the  numerous inconspicuous   microbes  play   a   key   role   in  biphenyl/PCB   degradation   in   the environments. Using  13C/12C ratio, the carbon assimilation rate was estimated for each metabolically  active  cell.  The  relative  abundances  and  catabolic  activities   of  the biphenyl-degraders differ significantly among four sediments/soils.
4) We reveal  hitherto unknown ethane-utilizing archaea via  specific enrichment, fluorescence  in situ  hybridization,  metagenomic  and  metaproteomic and  metabolic analyses. The enrichment culture, which coupled ethane oxidation  to sulfate reduction,was  predominated by  an  archaeon named  Candidatus  Argoarchaeum  ethanivorans;
other  members  include  sulfate-reducing   Deltaproteobacteria.  The  genome  of  Ca. Argoarchaeum  encodes   all  genes  for   a  methyl-coenzyme   M  reductase  with   all subunits detected  in protein extracts.  Consistently, ethyl-coenzyme  M was identified as an  intermediate by  liquid chromatography/mass  spectrometry. This  indicated that Ca. Argoarchaeum activates ethane by  forming ethyl-CoM, which is analogous to the anaerobic  butane-oxidizing  archaea  Ca.  Syntrophoarchaeum. Further  metagenomic and proteomic analysis suggest oxidation  of acetyl-CoA to CO2 through the oxidative Wood-Ljungdahl pathway.
This  study  expanded  our knowledge  of  microbial  processes  and  mechanisms involved   in   hydrocarbon   degradation.   The   identification   and   visualization   of environmentally-relevant  microorganisms  responsible   for  degradation  of  aromatic hydrocarbons will have a potential impact in defining future bioremediation strategies. Furthermore, we  revealed  the microorganisms  and reactions  that catalyze  anaerobic oxidation of ethane. These  results fill a gap in  our knowledge of microorganisms that specifically oxidize members of homologous alkanes series without oxygen.
 

Pages132
Language中文
Document Type学位论文
Identifierhttp://ir.rcees.ac.cn/handle/311016/42184
Collection土壤环境科学实验室
Recommended Citation
GB/T 7714
陈松灿. 典型烃类物质的微生物降解过程与机制研究[D]. 北京. 中国科学院生态环境研究中心,2019.
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