| Summary: | 碩士 === 東吳大學 === 微生物學系 === 93 === Nitrification is one of the most important step is the nitrogen cycle, this series of reactions are responsible for chemoautotrophic nitrobacteria, therefore the chemoautotrophic nitrobacteria play an important role in nitrogen cycle. Nitrobacteria grow in a slow rate, and during their growth, they produce some acidic and toxic metabolites and that will inhibit their growth too. Because of the slow growth rate, that increases the contaminant opportunity by other micro organisms, for these reason it risen the difficulty of culturing and purifying the nitrobacteria, and limit the counting method.
According to those limitations, this study would like to use the molecular biology method to enumerate the nitrobacteria in the sample fast and precisely. We use fluorescent in situ hybridization method (FISH). First, we choose ammonia- oxidizing bacteria and nitrite- oxidizing bacteria 16S rDNA from the gene bank, then alignment by GCG SeqWeb software, to find out the the genus specific region sequences to be the FISH probe Nso1225, and Nit3. And to make sure the specificity of the probe, we use the probe as one side primer to do the PCR test. The result shows no cross reaction with other species of bacteria.
The FISH result shows that the optima conditions of different probes are Eub338: 0% formamide, 0.9M NaCl, Nso1225: 35% formamide, 0.08MNaCl, Nit3: 40% formamide, 0.056M NaCl.
Our study detects the ammonia- oxidation rate and the nitrite- oxidation rate of the sample, to find out the linkage of nitrification rate and the numbers of the ammonia- oxidizing bacteria and the nitrite- oxidizing bacteria. According to the results, the generation time of the ammonia- oxidizing bacteria in the medium are 9 hours, and the generation time of the nitrite- oxidizing bacteria in the medium is 12 hours. The ammonia-oxidation rate of the ammonia- oxidizers is 2.58 × 10-7 μmol/cell.h, and the nitrite-oxidation rate of the nitrite-oxidizers is 6.13 × 10-8 μmol/cell.h.
We also applied this experiment in soil sample, the ammonia- oxidation rate in the soil is 1.69 × 10-7 μmol/cell.h, and the nitrite-oxidation rate is 4.17 × 10-8 μmol/cell.h. The amount of ammonium-oxidizers and nitrite-oxidizers in the soil are counted by Flow Cytometry after hybridized by specific probes. The ratio of ammonium -oxidizers and nitrite-oxidizers are 14.57% and 12.65%.
According to our results, use both FISH and Flow Cytometry method to calculate the numbers of nitrifying bacteria and their nitrification rates in different samples more precisely.
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