【作者】 赵昌松；

【导师】 刘凡；

【作者基本信息】 华中农业大学 ， 土壤学， 2014， 博士

【摘要】 锰氧化物在环境中广泛存在,反应活性强,对有机物质降解及污染元素的迁移转化起着非常重要的作用。微生物强烈影响着锰氧化物的形成与转化。目前有关锰氧化物生物形成的研究主要以海相锰矿物为对象,用于研究锰氧化物生物形成的微生物也多来自海相和湖相系统,且陆相表生土壤中锰氧化物的形成比海相和湖相系统复杂得多；从微生物生长的热量变化角度探讨锰细菌氧化Mn(Ⅱ)功能的生物学意义的研究尚未报道。本研究综合运用微生物学、分子生物学、微量热和同步辐射等的理论与技术,从我国4种地带性土壤和结核中筛选锰氧化细菌,探讨了生物氧化锰的形成,并对生物形成的锰氧化物进行了表征和分析；应用基因突变和微量热技术研究了锰氧化物生物形成过程中细菌生长及热量释放特点,通过锰氧化菌催化形成生物氧化锰过程中的生长特点和能量利用效率的关系,探讨了锰细菌具有氧化功能的生物学意义。主要结果如下：1.从我国4种地带性土壤中分离到具有较高锰氧化活性的细菌共30株。其中棕壤分离得到20株、黄褐土和黄棕壤分别得到3和2株、红壤分离得到5株。通过对锰氧化菌16SrDNA序列分析和比对可知,30个菌株属于2个发育群,3个门,5个属,11个物种,其中厚壁菌门(G+)有19株,变形菌门(G-)10株,黄杆菌门1株(G-)。通过复筛实验得到变形菌门大肠杆菌属的一株高氧化活性菌MB266,作为生物氧化锰形成菌株进行深入研究。2.MB266在含1mM Mn(Ⅱ)的Leptothrix培养基中生长5天,培养体系中生物氧化锰浓度达0.047mM,相当于4.7%的Mn(Ⅱ)被氧化。对冷冻干燥处理后的培养物进行X射线衍射图谱、透射电镜图谱及电子衍射分析,结果显示生物形成的锰氧化物与方铁锰矿Mn2O3(JCPDS00-002-0896)的特征峰吻合,但本实验所得生物氧化锰衍射峰强度较低,且峰形严重宽化。光电子能谱(XPS)和X射线吸收光谱(XANES)拟合结果显示所得的产物平均氧化度分别为2.76和2.61,这表明本实验得到的生物锰氧化物是弱晶质的三氧化二锰矿物。3.对培养不同时间形成的生物氧化锰进行XPS和XANES拟合,实验中生物氧化锰的形成过程可能是：Mn(Ⅱ)在酶的催化氧化下生成Mn(Ⅲ),部分Mn(Ⅲ)直接沉淀；另外一部分Mn(Ⅲ)与有机物螯合成可溶的复合体,进一步被酶氧化到Mn(Ⅳ),所得的Mn(Ⅳ)氧化物还可以被Mn(Ⅱ)还原,在菌体表面继续生成Mn(Ⅲ)氧化物。4.利用基因突变方法构建了失去锰氧化活性的MB266的突变株MB32,MB98,应用微量热技术,检测了MB266与突变菌株在Leptothrix培养基和含1mM Mn(Ⅱ)的Leptothrix培养基中的生长及热量释放特点,结合Mn(Ⅱ)的氧化量与微生物的生物量,探讨了锰氧化菌的生长特性和能量利用效能。结果表明：野生型菌株由于能够将Mn(Ⅱ)氧化为锰氧化物,降低了Mn(Ⅱ)对菌株的毒害作用,其生物量显著高于突变株；与此同时,Mn(Ⅱ)对野生菌株毒害作用下降,降低了菌株修复细胞内损伤的能量需要,从而降低了细胞的代谢强度,其热量释放显著小于突变株。通过对与Mn(Ⅱ)化学氧化释放的热量与系统释放总热量的比较表明,锰氧化菌在生长过程中主要利用了有机底物分解的能量进行生长,Mn(Ⅱ)氧化过程中释放的热量对锰氧化细菌生长的贡献很小。本实验所检测的锰氧化菌MB266多铜氧化酶基因并非唯一的催化Mn(Ⅱ)氧化的因子,细菌中还存在有其它未知的氧化因子。更多还原

【Abstract】 Manganese oxides are widely distributed in natural environments, and possess high reactive activity, and thus play a key role in the degradation of organic matters, migration and transformation of contamination elements. Environmental microbiologies strongly influence the formation and microbial transformation of manganese oxide minerals. Current studies are mainly focused on manganese oxides formed in marine conditions and mic obial organisms in marine and lacustrine systems which are involved in the formation of biogenic Mn oxides. However the formation mechanisms of continental Mn oxides in soil nodule sediments are much more complex than those of marine and lacustrine systems; Base on the thermodynamics analysis, there was few research on the physiological relevance of Mn(Ⅱ) oxidation by manganese-oxidizing bacteria. In this study, manganese-oxidizing bacteria were screened from four kinds of zonal soils and nodules in China, and then multiple modern techniques including microbiology, molecular biology, microcalorimetry and synchrotron radiation techniques were used to explore the appropriate conditions for the formation of biogenic manganese oxides, and to characterize and analysis the structures and properties of as-obtained these oxides; further the characteristics of the growth of bacteria involved and heat equilibrium during the biogenic oxidation of Mn(Ⅱ) were investigated by mutation and microcalorimetry techniques, aiming to discover the biological role of biogenic Mn(Ⅱ) oxidation during these processes. The main results are as follows:1.30strains with high Mn(Ⅱ) oxidizing activity were isolated from four kinds of zonal soils,20trains of which were screend from the brunisolic soil,3strains were from cinnamon soil,2strains were from yellow brown soil,5strains were from drab soil. Analysis and comparisons of16S rDNA sequences of these manganese-oxidizing bacteria showed that,30strains belonged to2development groups,3phylums,5genera and11species, of which19Firmicutes (G+),10Proteobacteria (G-), and1Flavobacterium (G-). Proteobacteria Escherichia MB266, which had the hightest oxidation acitvity was selected to conduct further studies. 2. When MB266was cultivated for5days at Leptothrix medium containing1mM Mn(Ⅱ), the concentration of biogenic Mn oxide was0.047mM, suggesting that4.7%of the initial Mn(Ⅱ) was oxidized. The products were then cleaned and freez-dried for further analysis. Powder X-ray diffraction, transmission electron microscopy and electron diffraction pattern analysis demonstrated that the biogenic Mn oxide was bixbyite (JCPDS02-0896), with poor crystallinity evienced by the low intensities of the broad diffraction peaks in the powder XRD pattern. Mn average oxidation state (Mn AOS) of the biogenic Mn oxides determined by X-ray photoelectron spectroscopy (XPS) and X-ray absorption spectroscopy (XANES) s are2.76and2.61respectively. This further confirmed that the obtained Mn oxide was bixbyite with poor crystallinity.3. XPS and XANES analysis of the biogenic Mn oxides cultivated for different times were conducted. Based on these results two possible mechanisms for Mn(Ⅱ) biogenic oxidation can be proposed. Firstly, Mn(Ⅱ) was catalytically oxidized by enzymes to Mn(Ⅲ), part of which was then precipitated as bixbyite; Secondly, the other part of Mn(Ⅲ) was complexed by organic ligands and then further oxidized by enzymes to Mn(Ⅳ). Conproportionation of Mn(Ⅳ) and Mn(Ⅱ) then might result in Mn(Ⅲ) on the mineral surfaces.4. Two gene mutant strains, MB32and MB98, were constructed by gene mutation. The growth characteristics and the energy release during culture process were monitored by microcalorimetry when these bacteria were cultured in Leptothrix medium or1mM Mn(II)-amended medium. By combining the amounts of Mn(Ⅱ) oxidized and microbial biomass, the growth characteristics and energy consuming efficiency were discussed. It demonstrated that, in the absence of Mn(Ⅱ), the growth characteristics of wild strain and mutant strains suffered neglectible change, so did energy efficiency. While in the presence of Mn(Ⅱ), the biomass of the wild-type strain was much higher than that of the mutants by oxidizing Mn(Ⅱ) to manganese oxides to decrease the toxic effect of Mn(Ⅱ). As a result, the intracellular damage repair was no longer indispensable for the wild type, which reduced the intensities of metabolisms and energy release. By comparison of the heat directly released by Mn(Ⅱ) oxidation and the total heat release, it showed that Mn(Ⅱ)-oxidizing bacteria mainly consumed the energy from the decomposition of the organic matters, and the heat released during Mn(Ⅱ) oxidation contributed little. In this experiment, the multi-copper oxidase gene was not the unique way for catalyzing Mn(Ⅱ)-oxidation, there should be other unknown factors.更多还原