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水稻土中噬电子菌群落多样性及其 调控因子
Alternative TitleDiversity of electrotrophs in paddy soils and its environmental regulatory factors
李晓敏
Subtype博士
Thesis Advisor朱永官
2020-06
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噬电子菌,微生物电合成,水稻土,长期施肥,地杆菌科 electrotroph, Microbial Electrosynthesis, Paddy Soil, Long-term Fertilization, Geobacteraceae
Abstract

    噬电子菌是一类能以固态可导电的物质(比如铁矿物和电极 作为电子供体,然后 将电子传递到细胞内的微生物。 噬电子菌可以利用各种电子受体获得能量维持自身生长,比如二氧化碳( CO2)和硝酸盐 NO3-)。在自然环境中,噬电子菌独特的代谢方式耦合了多种营养元素的转化,可以影响生物地球化学循环。水稻土是典型的人工湿地系统,其频繁的干湿交替导致的氧化还原梯度和矿物的形成为噬电子菌提供了合适的栖息地。但是关于水稻土中噬电子菌群落结构和多样性,目前还鲜有报道。此外,施肥是农业生产中常用的增产手段之一,长期不同施肥措施对水稻土中 噬电子菌群落结构和多样性的影响也尚不明确。
      因此本文分别选择了我国由南到北5个不同地点的水稻土,以及同一地点的长期不同施肥处理水稻土作为研究对象,构建了微生物电合成( Microbial electrosynthesis system MES)反应器,以 MES中的阴极作为电子供体富集不同水稻土中潜在的噬电子菌,采用高通量测序和高通量定量 PCR HT-qPCR 等 技术分析其群落多样性,揭示调控噬电子菌群落组成的关键环境因子,并探索不同
施肥措施对噬电子菌群落的影响。具体的研究内容以及结果如下:
    1) 向 MES反应器 中接种 5个不同地点的水稻土,以 MES反应器 中的阴极((–0.7 V versus Ag/AgCl 作为电子供体, CO2为电子受体富集噬电子菌,运行一个月后利用高通量测序技术探究不同土壤中 潜在 的噬电子菌群落结构。在 MES运行过程中 阴极上 大约 25%–45%的电子被 潜在的 噬电子菌捕获并还原 CO2为乙酸。在阴极上,厚壁菌门 Firmicutes 是 微生物群落中 最主要的成分,其中的芽孢杆菌属 Bacillus 的相对丰度最高且在 5种不同的土壤中均显著富集。除了厚壁菌门,变形菌门 Proteoacteria 中的微生物也参与了噬电子的过程。来自海伦市、沈阳市和鹰潭市的水稻土中富集的 潜在的 噬电子菌群落结构明显区别于来自安庆市和雷州市的水稻土中的噬电子菌群落,这种差异的主要驱动因子是土壤 pH、无定形铁含量和土壤电导率 ,这 3个因子可解释不同水稻土中潜在的噬电子菌群落结构差异的 33.5%。
    2)从湖南祁阳 县的中国农业科学院红壤实验站 红壤试验站采集长期不同施肥处理的水稻土作为研究对象,施肥措施分别为:未施肥( CK)、仅施加腐熟牛粪(M)、仅施加氮磷钾化肥 NPK)以及牛粪和化肥配施 MNPK)。将这 4种土壤接种至 MES反 应器的阴极室中,以阴极( (–0.4 V vs Ag/AgCl)作为电子供体, NO3-作为电子受体富集了不同施肥土壤中潜在的噬电子菌。采用高通量测序研究了噬电子菌群落组成,利用 HT-qPCR分析了 MES阴极上参与反硝化和异化硝酸盐还原成铵( Dissimilatory nitrate reduce to ammonia DNRA)过程的功能基因。结果显示,与 CK相比, 3种施肥措施均显著增加了 潜在的 噬电子菌群落的分类学 结构 和系统发育 结构的 多样性。在 CK土壤中,放线菌门( Actinobacteria和变形菌门( Proteobacteria)是最主要的噬电子菌群。施加 NPK偏好性地富集属于厚壁菌门( Firmicutes)的噬电子菌,而施加 M和 MNPK有利于富集属于绿弯菌门( Chloroflexi)、异常球菌 -栖热菌门( Deinococcus-Thermus)和芽单胞菌门Gemmatimonadetes)中的微生物。此外,链霉菌属 Streptomyces)、特吕铂菌属( Truepera)和芽孢杆菌属 Bacillus)是所有施肥土壤中均显著富集的潜在噬电子菌。当 NO3-作为电子受体时,这些潜在的噬电子菌可消耗阴极电流耦合 NO3-还原,其中 18%–35%的电子被噬电子菌传递给 NO3-还原成 NH4+。土壤总碳、总氮和溶解性有机碳 的含量 是影响不同施肥土壤中潜在的噬电子菌群落组成的主要因素,对群落结构差异的贡献率为 53%。
    3)以第二部分研究内容中长期不同施肥处理的水稻土为研究对象,采用地杆菌科( Geobacteraceae 16S rRNA基因特异性引物,通过定量 PCR和高通量测序技术原位研究了长期不同施肥措施对水稻土中噬电子菌 Geobacteraceae群落多样性的影响。 Illumina测序结果显示,长期不同施肥处理对 Geobacteraceae的物种丰富度和多样性的影响并不显著。在所有处理中, Geobacteraceae主要包含两种属,分别为 Geobacter属(占总序列数的 90%–95%)和 Geothermobacter属( 5%–10%)。与对照比较,所有的施肥处理均导致 Geobacter属的相对丰度显著下降( P < 0.05),而 Geothermobacter属的相对丰度明显增加。在不同施肥处理中, Geobacteraceae群落基于 Bray-Curtis距离的分类学结 构和基于加权 UniFrac距离的系统发育结构与未施肥处理中的相比存在明显差异,而长期不同施肥处理之间 Geobacteraceae群落的分类学结构和系统发育结构均无明显差异。长期施肥引起的 Geobacteraceae群落的变化主要是由土壤 pH、总碳和总氮引起的。
    以上结果表明,水稻土中的噬电子菌群落组成丰富多样 ,厚壁菌门中的微生物为优势物种,比如芽孢杆菌属 。 长期不同施肥措施会影响噬电子菌的群落组成,除此之外,土壤 pH、碳和氮含量 是影响噬电子 菌 群落结构变化的主要因素 。 噬电子菌的代谢活动可以影响水稻土中碳、氮元素的循环 主要 为 固碳固氮的作用 。该研究对于深入了解水稻土中噬电子菌群落及其影响的元素生物地球化学循环具有重要意义,也为利用噬电子菌调控氮肥利用效率提供了理论基础。

Other Abstract

      Electrotrophs are microbes that can take up electrons into their cells from external solid-phase conductive substrates (i.e., ferrous iron [Fe(II)]-minerals and electrodes) to obtain energy for growth, and transfer the electrons to various terminal electron acceptors, such as carbon dioxide (CO2) and nitrate (NO3-). The electrotrophic metabolism coupling with the transformations of nutrient elements can affect the biogeochemical cycles in natural environments. Paddy soil is one of the typical and artificial wetlands. The frequent alterations of wetting and drying cause the gradient of redox potential and mineral production which could provide a suitable habitat for electrotroph. However, the community and structure of electrotrophs in paddy soils remain poorly understood. Additionally, fertilization is one of the common practices to improve crop yields, but the influences of long-term different fertilization practices on electrotrophic community in paddy soils remain unclear.
       In this study, we selected five paddy soils with various physicochemical properties and one paddy soil receiving different fertilization, respectively. The poised cathodes in microbial electrosynthesis systems (MESs) were used as proxies of electron donors to enrich the electrotrophic communities from these paddy soils. The Illumina sequencing and high-through quantative PCR (HT-qPCR) technology were used to: (1) analysis the communities of potentital electrotrophic microorganisms enriching from paddy soils; (2) examine the contributions of driving soils properties on the variations of electrotrophic communities; (3) explore the responses of electrtrophic communities to long-term different fertilization practies in paddy soils. The main content and results are as followings:
      1) the putative electrotrophs were enriched by cathodes of MESs constructed from five paddy soils with various properties using bicarbonate as an electron acceptor, and identified by 16S rRNA-gene based Illumina sequencing. The electrons were gradually consumed on the cathodes, and 25%–45% of which were recovered to reduce bicarbonate to acetic acid by the potential electrotrophs during MES operation. Firmicutes was the dominant bacterial phylum on the cathodes, and Bacillus genus within this phylum was greatly enriched and was the most abundant population among the detected putative electrotrophs for almost all soils. Furthermore, several other members of Firmicutes and Proteobacteria may also participate in electrotrophic process in different soils. Soil pH, amorphous iron and electrical conductivity significantly influenced the putative electrotrophic bacterial community, which explained about 33.5% of the community structural variation.
      2) The poised cathodes (−0.4 V versus Ag/AgCl) in microbial electrosynthesis systems (MESs) were used as proxies of electron donors and NO3- as an electron acceptor to enrich the electrotrophic communities in four paddy soils receiving different fertilization treatments since 1982, including no fertilization (CK), compost cattle manure only (M), nitrogen, phosphorus, and potassium (NPK) chemical fertilizers, and a combination of M and NPK fertilizers (MNPK). Illumina MiSeq sequencing was used for electrotrophic community analyses and HT-qPCR was performed to explore the genes involving with nitrate reduction. The results showed that compared CK, the soils of M, NPK, and MNPK significantly increased the taxonomic and phylogenetic diversity of the putative electrotrophic microbial community. Actinobacteria and Proteobacteria were the dominant electrotrophic microbes in the unfertilized soil. NPK use favored electrotrophic populations belonging to Firmicutes, whereas M and MNPK use favored Chloroflexi, Deinococcus-Thermus and Gemmatimonadetes populations. Furthermore, Streptomyces, Truepera, or Bacillus genera were putative electrotrophs enriched by all fertilization practices. These putative electrotrophic microbial populations consumed the biocathodic currents coupling with nitrate reduction when nitrate served as an electron acceptor, and 18–35% of electrons were recovered by electrotrophs to reduce nitrate to ammonium. Soil total carbon, nitrogen, and dissolved organic carbon were major factors influencing the compositions of electrotrophic communities in soils receiving different fertilizers, accounting for 53% of structural variations.
      3) To investigate the impacts of long-term fertilization on the community of electrotrophic Geobacteraceae family, the extacted DNA in unfertilized and fertilized treatments of the section 2 was amplified to target the Geobacteraceae 16S rRNA genes using the primer pairs Geo494F and Geo825R for Illumina sequencing. The results showed that the species richness and diversity of Geobacteraceae community were not significantly changed by fertilizer treatments. Geobacteraceae in the treatments consisted of Geobacter (accounting for 90%–95% of total reads) and Geothermobacter genera (5%–10%), and all fertilizer treatments induced a significant (P < 0.05) decline in Geobacter and a marked enrichment of Geothermobacter. The taxonomic (based on Bray-Curtis distance) and phylogenetic structures (weighted-UniFrac distance) of the Geobacteraceae communities in all fertilizer treatments were clearly different from those in the non-fertilizer treatment; however, there were no significant changes among the different fertilization treatments. The variations in the Geobacteraceae community induced by long-term fertilization were mainly determined by changes in soil pH, total carbon, and total nitrogen.
       The above content indicated that the diverisity of potential electrotrophic communitis in paddy soils were mainly influenced by the soil pH and the contents of carbon and nitrogen, and the microbes in Firmicutes and Proteobacteria were the dominate populations. The metabolisms of electrtrophs also influence the cycles of carbon and nitrogen in paddy soils. This study expands our knowledge about the diversity and compositions of electrotrophic communities and their roles on elemental cycles in paddy soils, it also provides theoretical basis for regulating the nitrogen fertilizer efficiency by electrotrophs.

Pages131
Document Type学位论文
Identifierhttp://ir.rcees.ac.cn/handle/311016/43600
Collection土壤环境科学实验室
Recommended Citation
GB/T 7714
李晓敏. 水稻土中噬电子菌群落多样性及其 调控因子[D]. 北京. 中国科学院生态环境研究中心,2020.
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