淹水土壤中反硝化菌和硝酸还原菌数量、生理类群及其还原特点的研究

【作者】 陈丽敏；

【导师】 殷士学；

【作者基本信息】 扬州大学 ， 植物生理， 2001， 硕士

【摘要】 依据土壤的硝酸异化还原成铵（DNRA）的潜力不同，本文选用两个土壤，一个来自扬州大学试验农场，DNRA潜力较小；一个来自澳大利亚，DNRA潜力较大。两个土壤的管理方式不同，前者实施水稻-小麦-清除秸秆方式，后者实施水稻-秸秆覆盖-休闲方式。本文目的是比较两种土壤的硝酸还原菌区系及其还原特点，试图说明影响DNRA过程的因素。 用系列稀释-杜哈姆发酵管-肉膏蛋白胨培养基（液体）-最或然数法测定扬州土和Griffith土中硝酸还原菌和反硝化菌数量，计得两土壤中反硝化菌分别占硝酸还原菌总数的13.6％和1.8％。这表明，在所有能够还原硝酸的细菌中反硝化菌并不是数量最多的主导区系。 从厌氧条件下培养的平板中挑取全部菌落并纯化，然后逐一研究各菌株还原硝酸的特点，统计分析两种土壤硝酸还原细菌的组成情况。结果表明，扬州土和Griffith土在硝酸异化还原菌的组成上具有很大差别。扬州土中反硝化菌和亚硝酸积累菌基本上各占一半，而DNRA菌几乎没有。与之相反，Griffith土中DNRA菌占到细菌总数的一半，而反硝化菌却极少，即扬州土中反硝化菌比例（43.7％）高于Griffith土的比例（2.9％），这与倒置试管法测得的两个土壤的反硝化比例呈相同趋势。 硝酸异化还原菌的组成与该两种土壤的DNRA能力是相吻合的，即DNRA能力强的Griffith土壤其DNRA细菌比例较高。对这两种土壤中分离出来的所有硝酸还原菌的初步鉴定发现，能够还原NO₃^-的细菌主要是芽孢菌。扬州土和Griffith土中芽孢菌比例分别占硝酸还原菌总数的56.25％和80％。Griffith土的DNRA主导菌是Bacillus sps。 用K₂Cr₂O₇氧化法和培养法测定扬州土和Gfiffith土的活性有机质含量。培养法测定结果为：Griffith土和扬州土中活性有机质含量分别为216.2mgCO₂-Ckg^-1和93.2mg CO₂-Ckg^-1，两者有显著差别。但K₂Cr₂O₇氧化法测定结果为两个土壤之间没有显著差异（Griffith土为0.58％；扬州土为0.54％）。从分析原理来看，培养法更为合理些，因为它是基于生物培养的方法，而 扬州大学硕士学位论文2 KZCr。O，氧化法是基于化学氧化的方法。因此，培养法得到的结果更能反映 土壤活性有机质含量。两种土壤中活性有机质含量的高低与土壤的DNRA 还原能力相关，活性有机质含量高的Griffith土DNRA能力强；土壤活性有 机质含量与土壤管理方式有关，Griffith土实行水稻－秸秆覆盖－休闲，而扬 州土实行水稻－清除秸秆－小麦－清除秸秆；所以认为土壤管理影响土壤活性 有机质含量，进而影响土壤DNRA过程。 为了进一步了解硝酸还原菌株的特点，从分离获得的菌株中挑选出具有 代表性的7个菌株进行纯培养研究。DNRA菌u 和C12)细胞生长量不 随NO3’浓度的增加而增加，说明伴随NO3”的还原产能较低，是典型的DNRA 菌。DNRA菌在其细胞处于活跃的对数生长期时，培养基中有NH4”的积累。 显然，所积累的NH4”是超出细胞生长所需要的，加上细胞在对数生长期死 亡和细胞刀的再矿化可能性很少，因此积累的NH／应该是DNRA过程而不 是硝酸同化还原过程的产物。反硝化菌（AIg＊）和 CZ)细胞生长量与培 养基中NO3’浓度成正相关，表明伴随着NO3”的还原有较多的能量产出，指 示 N O3”的还原为呼吸过程。反硝化菌在其整个培养过程中，培养基中均没 有NH4”的积累。亚硝酸积累菌（C24）也如此。低浓度的NO3－对亚硝酸积 累菌的生长有刺激作用，但当浓度达7mM时，生长量不再增加。B7和Bg 是两个比较特别的菌株，它们即具有DNRA菌的特点，同时又具有反硝化 菌的特点，因此值得进一步研究。 将DNRA菌和反硝化菌接种于同一培养基中，并设置不同比例的接种 量，培养一个星期后，测定培养基中的NH4＼N。NH／－N的积累量与DNRA 菌的接种量比例相吻合，且NH4”－N占原NO3二N的比例与培养基中添加的 NO3＼N的量有关。说明DNRA发生程度与DNRA菌的数量比例及体系中的 C／NO3乙N比相关。 DNRA菌可以产出N。O。NZO的产出受pH影响。所研究的四个菌株在 pH为中性时N。O产出量最大，且随培养时间的延长而增加。而在pH为吕 时，不同类型菌株表现不同。说明pH对不同类型菌株的NZO产出影响不同。 偏酸性条件不利于所研究菌株的生长和NZO的产出。B7和Bg的NZO产出 量高于 CS和 CIZ。更多还原

【Abstract】 34 Abstract Soils used in this paper were selected based on their contrasting potentials for dissimilatory nitrate reduction to ammonium (DNRA). Yangzhou soil, as indicated by the name, was sampled from the experimental farm of Yangzhou University, Jiangsu Province, China. Griffith soil was sampled from Griffith, New South Wales, Australia. Different soil management practices were employed in the two soils. Rice-straw mulching-fallow has been practiced in Griffith soil for at least sever years (high carbon input) while rice-wheat-straw removal has been practiced for many years in Yangzhou soil (low carbon input). The populations of nitrate reducers and denitrifiers in Yangzhou .and Griffith soil were enumerated. The ratio of denitrifiers to total nitrate reducers in Yangzhou soil was 13.6%, but 1.8% in Griffith soil, indicating that the denitrifiers were not the dominant flora among the bacteria that are capable of reducing nitrate in submerged soils. All nitrate reducers from the two soils were purified and studied. The data showed that the composition of nitrate reducers in the two soils differed significantly. In Yangzhou soil, 43.7% of nitrate reducers isolated were denitrifiers while only 6.25% was DNRA bacteria. On the contrary, in Griffith soil, more than half of nitrate reducers were DNRA bacteria, while only 2.9% were denitrifiers. This data situation corresponded to the DNRA ability of the two soils. All isolated bacteria were primarily identified. Most of them were spore-forming groups, which constituted 56.25% and 80% of the total nitrate reducers isolated in Yangzhou soil and Griffith soil respectively. The dominant genus responsible for DNRA in Griffith soil was Bacillus sps0 The amount of labile organic carbon in these two soils was examined. With soil incubation method, it was 216.2 mg C02-C kg?and 93.2 mg C02-C kg? respectively in Griffith soil and Yangzhou soil. But with potassium dichromate oxidation method, there was no notable difference between these two soils. Incubation method is based on the microbial decomposition and is therefore thought to be more appropriate for labile carbon measurement than the chemical 35 oxidation method. The amount of soil labile organic carbon measured by incubation method was closely correlated with DNRA potentials of the soils. Because of carbon input affects soil labile carbon turnover, it was reasoned that soil management affected soil labile carbon content and soil labile carbon content in turn affected DNRA process. Seven isolated bacteria were selected for further study to see their characters of nitrate reduction. Cell yield of two denitrifiers (C2 and A19(2)), as indicated by optical density, was proportional to the amount of nitrate added to media. But cell yield of two DNRA bacteria (C8 and C12) was not. DNRA bacteria converted nitrate into ammonium during their logarithmic growth phase. Ammonium produced as such was in excess of cell growth and cell death during this phase was minimal. These data suggested that nitrate assimilation and re-mineralization of cell materials were not important processes contributing to the ammonium formed. On the contrast, denitrifiers and a nitrite accumula更多还原