【作者】 易维洁；

【导师】 曲东；

【作者基本信息】 西北农林科技大学 ， 环境科学， 2011， 博士

【摘要】 微生物Fe(Ⅲ)还原作用能以Fe(Ⅲ)作为电子受体,将有机或无机的电子供体氧化。自然界的厌氧环境几乎都有异化Fe(Ⅲ)还原现象,并有铁还原微生物存在。关于水稻土微生物铁还原过程的知识在最近十年里持续的增加。然而因为铁还原微生物系统发育学上的多样性和铁还原途径的不确定性,使得人们对铁还原微生物的认识仅限于几类典型的物种,如Geobacter和Shewanella。水稻土水旱轮作的耕作方式作为研究氧化还原过程的一种模式系统一直受到人们的重视,其中铁的氧化还原占有重要地位。但对其淹水厌氧状态下微生物群落的分布演替的知识甚少,缺乏对铁还原过程具有贡献率的细菌的相关报道。所以,研究不同碳源富集下水稻土中Fe(Ⅲ)微生物的优势类型,不仅可深化对水稻田微生物生态的认识,而且对于阐明水稻土微生物Fe(Ⅲ)还原机理及群落演替特征具有重要的意义。本文采用厌氧恒温培养方法,在自然和有机质耗竭水稻土中添加无定形氧化铁和不同碳源进行富集驯化,分析铁还原特征;进一步分离筛选出具有高效铁还原能力的菌株,采用16S rDNA-ARDRA分子生物学技术对其多样性进行分析评价;并对典型菌株进行16S rDNA序列测定。直接分离纯化铁还原环境下水稻土中的微生物,通过直接测定其16S rDNA序列,认识其在系统分类学上的归类。比较不同淹水时间及在外加碳源和铁源驯化下微生物群落结构的差异,分析其优势种群的演替特征。提取来源不同植稻区水稻土不同淹水时期的微生物群落,接种于以不同碳源为惟一底物的富铁培养液中,定期测定Fe(II)生成量,pH变化及脱氢酶活性,探索铁还原微生物的活性出现的最大时期及稳定期,以得到铁还原微生物活性恢复最快的时期,为研究不同水稻土微生物铁还原能力差异和影响因素提供基础依据。通过对微生物Fe(Ⅲ)还原过程和脱氢酶活性变化的动力学特征分析,以期阐明脱氢酶活性与微生物Fe(Ⅲ)还原的内在关系,为揭示水稻土中微生物Fe(Ⅲ)还原机理提供必要的理论依据。本论文的主要结果包括以下方面:(1)在有机质耗竭水稻土中添加葡萄糖和丙酮酸盐后,其铁还原反应的趋势跟自然水稻土比较接近,而添加乙酸盐的处理则表现出一定差异。表明淹水水稻土中铁还原反应在初期的快速进行是由利用葡萄糖的发酵产H2型铁还原菌所主导的,淹水后期的铁还原反应则是由利用乙酸盐的铁还原菌所控制。H2-依赖型铁还原菌对水稻田中的铁还原过程的贡献大于乙酸依赖性。(2)铁还原微生物群落组成与结构是影响水稻土中铁还原过程的重要因素。在接种土壤浸提液的混合培养中,不同淹水时期对Fe(Ⅲ)还原特征值Vmax的影响显著,表现为淹水20 d>30 d>12 d>1 d>5 d,水稻土中微生物群落结构变化是导致Fe(Ⅲ)还原能力不同的主要原因。不同淹水时期得到的铁还原微生物群落对碳源的响应具有显著差异,淹水1~12 d的处理以葡萄糖和丙酮酸盐为优势碳源,淹水12 d和20 d的处理能够高效利用乳酸盐,淹水30 d的处理对乙酸盐的利用能力显著增强。(3)柠檬酸铁的生物还原和化学还原具有显著的不同,光照和高温是影响柠檬酸铁化学还原的主要原因,避光和30℃培养条件不会造成柠檬酸铁的化学还原。本试验条件下的接种了微生物的柠檬酸铁还原主要是由微生物引起的。建立了有效筛选铁还原菌株的分离培养方法体系,判定ferrihydrite还原率在50%以上的菌株为高效铁还原菌。在不同碳源富集下,从吉林,四川,湖南,浙江,天津和江西水稻土获得高效铁还原菌株分别为88,54,161,88,33和67个。(4)α多样性指数表明在葡萄糖富集下出现较为集中的优势种群,而在小分子有机酸为碳源的处理中则呈现出较为丰富的多样性。在HN水稻土中,基于16S rDNA-ARDRA分析,其优势种属一共分为7类。不同碳源富集下都均有Paenibacillus spp.和Clostridium.spp.出现;Solibacillus和Lysinibacillus是乙酸盐富集下的优势种群,而Bacillus则是丙酮酸盐富集下的优势种属;除了葡萄糖处理,其他碳源富集及对照中都表现出Azotobacter为其优势物种;而Pseudomonas spp.作为优势物种只出现于葡萄糖富集的群落中。(5)微生物Fe(Ⅲ)还原过程对脱氢酶活性具有明显的影响,不同处理中脱氢酶活性最大值对应的Fe(II)含量为91.0~344.6 mg·L-1,随着铁还原程度的继续增大,脱氢酶活性随之降低。不同处理中脱氢酶活性与微生物Fe(Ⅲ)还原过程具有相关性,脱氢酶活性峰值出现时间与Fe(OH)3还原TVmax具有显著正相关关系,而与Vmax则表现为明显负相关。脱氢酶活性是影响水稻土中铁还原过程的重要因素,推测微生物代谢有机物产H2是水稻土淹水初期铁快速还原的主要原因。(6)自然淹水条件下,水稻土中早期出现的可分离株采用r-策略进行生殖生长,物种多样性相对丰富,可归属于Lysinibacillus,Staphylococcus,Paenibacillus和Azotobacillus的系统发育分支上;演替后期铁还原过程进入平稳阶段且出现相对集中的类群,主要以Bacillus spp.为优势物种代表。在外加碳源和氧化铁富集下,水稻土中可分离株的群落结构发生显著变异。通过本文的研究,对不同碳源富集下水稻土中优势铁还原菌的种属以及铁还原环境下的微生物群落的演替有了更为深入的认识,探讨了脱氢酶活性跟微生物铁还原过程之间的联系,为进一步明确我国水稻土淹水培养后铁还原菌群落结构特征及系统分类、探讨不同水稻土中微生物铁还原能力差异提供基础依据,也对水稻土微生物Fe(Ⅲ)还原机理提出了初步的构想。更多还原

【Abstract】 jMicrobial Fe(III) reduction are capable of oxidizing organic or inorganic electron donors with ferric iron Fe(III) as electron acceptors. Dissimilatory Fe(III) reducing phenomena exist in almost natural anaerobic environment, as well as iron reducing microorganisms. There has been a continuous increase in the knowledge about microbial iron reduction in paddy soil in the last decade. Nevertheless, people only looked at some typical species of iron reducing microorganisms such as Geobacter and Shewanella, because of the uncertainty on phylogenetic diversity of iron reducing microorganisms and iron reducing ways. Iron redox processes play important roles in paddy rice-upland crop rotation systems. The system attracts plenty of attention as a model of redox research. However, little is known about the dynamic distribution of microbial community under flooded anaerobic condition. Furthermore, the data available in present literature fail to prove the contribution rate of bacteria in iron reduction. Therefore, the study on dominant Fe(III) microflora with different carbon sources in paddy soil not only can deepen the understanding of microbial ecology in paddy soil, but also has great significance for clarifying the mechanism of microbial Fe(III) reduction and the characteristics of community succession in paddy soil.In this study, we used anaerobic incubation with a constant temperature, added amorphous iron oxides with different carbon sources enrichment acclimation, analyzed Fe(III) reducing characteristics. Then we used 16S rDNA-ARDRA molecular biology techniques to analyze the diversity of bacterial strains with a high Fe(III) reducing capacity which were isolated and screened from the incubation system. Typical bacterial strains were determined 16S rDNA sequence. Microorganisms in iron reducing environment were isolated and purified directly, then measured 16S rDNA sequence in order to get some information about their categorization in taxonomy. To identify succession on superior dominant population, we compared the differences of microbial community structure with the domestication of amended carbon sources and iron oxides. Microflora extracted from paddy soil of different rice growing region in different flooded time was inoculated with different carbon sources as the only substrate in iron-rich medium. We measured ferrous concentration, pH and dehydrogenase activity regularly, to explore the maximum and stable period of the activity of microbial iron reduction, and get its fastest recovery time. All above can provide the foundation for the research on difference capacity and influencing factors of microbial iron reduction in different paddy soil. We analyzed the dynamic characteristics of microbial Fe(Ⅲ) reduction and dehydrogenase activity, in order to clarify the internal relationship between dehydrogenase activity and microbial Fe(Ⅲ) reduction and provide the necessary theoretical basis for revealing the mechanism of microbial Fe(Ⅲ) reduction in paddy soil.The main results obtained are as following:(1) The iron reduction tendency in the amended glucose and pyruvate paddy soil with organic matter depleted was similar with that in the natural paddy soil; however, there was a difference in the acetate treatment. These results indicate that iron reducing bacteria used H2 from glucose fermentation resulted in a high-speed iron reduction at earlier stage in flooded paddy soil, while iron reducing bacteria relying on H2 made more contribution to iron reduction in paddy soil than those depending acetate.(2) Iron reducing microbial community composition and structure have an important influence on iron reduction in paddy soil. In mixed cultivation with soil extraction, flooding time had a significant impact on Vmax, Fe(Ⅲ) reducing feature value, Vmax decreased in the order 20 d>30 d>12 d>1 d>5 d. The main reason caused different Fe(Ⅲ) reducing ability was the changes of microorganism population structure in paddy soil. There was a significant difference among iron reducing microbial community with carbon sources in different flooding time. Glucose and pyruvate were the superior carbon sources in the flooded 1-12 day treatments; lactate was used efficiently in flooded 12 days and 20 days treatments. There was a significant increase in using acetate in flooded 30-day treatment.(3) There was a significant difference between biological reduction and chemical reduction of iron citrate, because of the effect of illumination and high temperature on iron citrate chemical reduction. Iron citrate chemical reduction will not happen at 30℃and without light. In this experiment, what made this happen was microorganism. However, microorganism able to reduce iron citrate may not reduce ferrihydrite. Hence, we called those bacterial strains that could reduce more than 50% of ferrihydrire as iron reducing bacteria. In enrichment of different carbon sources, we obtained efficient iron reducing strains 88 from JL paddy soil, 54 from SC paddy soil, 161 from HN paddy soil, 88 from ZJ paddy soil, 33 from TJ paddy soil and 67 from JX paddy soil.(4) Fromαdiversity index, we can see that there was concentrative dominant population under glucose enrichment, while there was rich diversity in short organic acid treatment. The dominant population divided into seven kinds in HN paddy soil, according to the analysis of 16S rDNA-ARDRA. Paenibacillus spp. and Clostridium.spp. appeared in all carbon source treatments. Solibacillus and Lysinibacillus were the dominant population in acetate enrichment treatment, as Bacillus in pyruvate enrichment treatment. Azotobacter was the dominant population in all treatment but glucose treatment. Pseudomonas spp. was the dominant population only in glucose treatment.(5) Microbial Fe(Ⅲ) reducing had a significant effect on dehydrogenase activity, ferrous concentration was 91.0~344.6 mg·L-1 corresponding the maximum of dehydrogenase activity in all treatments. Dehydrogenase activity decreased as the level of Fe(Ⅲ) reduction increased. There was a correlation between dehydrogenase activity and level of Fe(Ⅲ) reduction. There was a positive correlation between the time reaching peak value of dehydrogenase activity and TVmax of Fe(OH)3 reduction, while there was a significantly negative correlation between time and Vmax. Dehydrogenase activity was an important influencing factor for iron reduction in paddy soil. We speculate that the main reason for high-speed iron reduction at earlier stage in paddy soil may be the H2 production from organic metabolism of microorganisms.(6) Under natural flooding conditions, isolates from paddy soils appeared in the early successional habitats adopted r-strategists to have growth and reproduction. They had a relatively rich in species diversity which could be attributed to Lysinibacillus, Staphylococcus, Paenibacillus and Azotobacillus phylogenetic branch. Iron reduction reached to a stable stage in late succession and there was a relatively concentrated group, which was predominated as Bacillus spp.. Microbial community structure had a significant variation when the mixed organic carbon sources and ferrihydrite were amended.Through this study, we got further knowledge of dominant iron reducing bacteria species and microbial community succession under different carbon source enrichment in paddy soil. We discussed the relation between dehydrogenase activity and microbial iron reduction, to provide the foundation for further clarifying the iron-reducing bacteria community structure and phyletic classification in flooded paddy soil incubation, as well as the differences of microbial iron reducing capacity. Furthermore, we put forward the initial ideas of microbial Fe(Ⅲ) reducing mechanism in paddy soil.更多还原