变性梯度凝胶电泳法研究我国不同土壤氨氧化细菌群落组成及活性

实验选用了我国3种不同土壤研究土壤硝化活性、硝化细菌数量,并使用变性梯度凝胶电泳(DGGE)的方法研究了不同土壤中氨氧化细菌(AOB)区系变化。通过28d的土壤培养实验研究发现,潮土具有最强的硝化势,几乎100%的铵态氮转化为硝态氮;而红壤中的硝化势最弱,只有4.9%的铵态氮转化为硝态氮。对这3种土壤硝化细菌进行计数发现,3种土壤氨氧化菌数量差异显著,而3种土壤亚硝酸氧化菌(NOB)处于一个数量级。采用氨氧化菌功能基因amoA(氨单加氧酶ammoniamonooxygenase)特异PCR结合DGGE的方法对土壤氨氧化菌区系进行分析。红壤有4个氨氧化菌种属,与潮土和黄泥土没有共同的氨氧化菌种属。4个种属中两个是与潮土和黄泥土亲源性比较远的,特有的氨氧化菌种属,这两个种属与已知的Nitrosospira属的cluster3bz97838和Nitrosospira属的cluster3aAF353263亲源性比较近。潮土有5个氨氧化菌种属,潮土与黄泥土有两个共同的氨氧化菌种属,这两个种属中的一个是潮土和黄泥土特有的,与其他氨氧化菌种属亲源性比较远的氨氧化菌种属,这个种属与已知的Nitrosospira属的cluster3bZ97849亲源性比较近。黄泥土有4个氨氧化菌种属,除了与潮土共有的一个种属是两种土壤特有的氨氧化菌种属外,黄泥土还有一个与其他氨氧化菌种属亲源性比较远的,黄泥土特有的种属,与Nitrosospira属的cluster3aAF353263亲源性很近。3种土壤中分离到的硝化细菌表现出不同的硝化能力。实验结果表明,以amoA基因为目标的PCR-DGGE是比以16SrDNA为目标的PCR-DGGE更有效的研究氨氧化菌种群的方法;3种土壤的氨氧化菌种群差异显著,尤其是红壤的氨氧化菌种群与另外两种土壤差异明显,这种差异可能与红壤的低pH条件对氨氧化菌种群的长期选择有关;3种土壤中的硝化活性与土壤中的硝化细菌数量没有显著相关,可能由于3种土壤差异显著的土壤环境对硝化活性的影响造成。因此在对不同土壤硝化细菌进行研究时不仅需要对硝化细菌数量进行研究,还需要研究不同土壤中硝化细菌的种属及不同土壤环境条件对硝化细菌硝化活性的影响。

Ammonia-oxidizing bacteria communities and their influence on the nitrification potential of Chinese soils measured by denaturing gradient gel electrophoresis (DGGE)

Nitrification is an important component of the global nitrogen cycle and the end-product is nitrate and the rate of nitrate production is very different among soil types. Nitrification can lead to different effects ranging from too much nitrate, causing environmental problems, or too little nitrate can lead to reduced crop yields. Differences in physical and chemical properties of soils are often well documented, but much less is known about the soil nitrifying bacterial communities. We studied three soils of China to determine their soil nitrification activities, numbers of nitrifying bacteria, and the ammonia-oxidizing bacterial community. Maximum nitrification potential was found in Fluvoaquic soil with almost 100% of ammonium-N being transformed into nitrate-N after 28 days. Minimum nitrification potential was measured in the Red Earth soil with only a 4.9% conversion rate during the same time period. Numbers of ammonia-oxidizing bacteria, determined by the MPN-Griess method, were significantly different among the three soils. However, numbers of nitrite-oxidizing bacteria determined by the MPN-PCR method were similar. A PCR technique using the ammonia monooxygenase gene (amoA) combined with the new DNA fingerprinting technique, denaturing gradient gel electrophoresis (DGGE), were used to analyze the composition of ammonia oxidizing bacteria in soils. DGGE analysis of the three soils were different from each other; there were 2 similar bands present in DGGE columns from the Fluvo-aquic soil and the Permeable paddy soil, but no similar bands were found in columns from Red Earth soil. Further study of the sequence of amoA indicated that there were differences of ammonia-oxidizing bacteria communities among the three soils. A phylogenetic tree including the amoA studied in this paper and amoA from other environments the database was established. There were four species of AOB found in Red Earth, both were different from the other two soils studied. Further comparison showed that these two species were closely related to Nitrosospira cluster 3b Z97838 and Nitrosospira cluster 3a AF353263, respectively. There were five species of AOB found in Fluvo-aquic soil and two of these were similar to those found in the Permeable paddy soil. Fluvo-aquic soil and Permeable paddy soil had a specific species which was closely related to Nitrosospira cluster 3b Z97849. There were four species of AOB found in Permeable paddy soil, and one of them was specific species for Permeable paddy soil and this was closely related to Nitrosospira cluster 3a AF353263. The nitrification activities of nitrifiers selected from these three soils were different. All the results obtained from this experiment implied that PCR-DGGE based on the amoA gene was a useful method to study AOB communities in soils. Each soil contained specific AOB species different from the others. Furthermore, the AOB of the Red Earth were particularly different from the other two soils, which might be related to the low pH of this soil. There was no relationship between the nitrification potential and nitrifying bacteria numbers of soils and this might be explained by the different physical properties of the soils.