Rapid estimation of ammonium-oxidizing bacteria in the environment using a polymerase chain reaction

• Main Authors: JU YOW CHAIN, 朱幼倩
• Other Authors: 趙維良
• Format: Others
• Language: zh-TW
• Published: 1999
• Online Access: http://ndltd.ncl.edu.tw/handle/44822274816235536730

碩士 === 東吳大學 === 微生物學系 === 87 === Ammonium-oxidizing bacteria are the major organisms responsible for the first step in nitrification, and they play an important role in the global cycling of nitrogen. Nitrification is a process that converts ammonia to nitrate. The reaction was highly valued not only in terms of agriculture but also in wastewater treatment. Traditional method used to quantify ammonium-oxidizing bacteria is tedious and time consuming. The organism is very difficult to culture due to its slow growth rate and the subsequent inhibition by its own metabolic product (i.e., nitrite). Recent years our understanding about the genetics of this organism has greatly improved which suggest the possibility of solving this counting problem by way of molecular biology. Ammonia monooxygenase (amo) is the first enzyme involves in ammonia oxidation and is only found in ammonium-oxidizing bacteria with low similarity to the genes of other bacteria. Furthermore, each ammonium-oxidizing bacterium contain constant copies (i.e., 2-3 copies) of this gene. The above characteristics make it ideal to try to enumerate the organisms using quantitative polymerase chain reaction (QPCR). Based on the nucleic acid sequence of amo gene reported by McTavish in 1993, we have designed two pairs of primer. The base number of the first pair are 16-46 and 1369-1384, and of the second pair are 265-287 and 912-931. Nested PCR was done, and the DNA fragment amplified using these primers is about 666 bp. The fragment obtained was then cloned into pGEM-T Easy vector. Clone pGEM-2 and pGEM-38 were used as probe in later studies. The specificity of the primer was confirmed first by carrying out PCR with 20 species common environment bacteria and then southern hybridization was done using the amplified DNA fragment as probe. The results are highly satisfactory, with no detectable signal been observed with these test organisms. QPCR was done using PCR ELISA kit of Beohringer Mannheim GehH Biochemical. The amplified fragment was first transformed into E. coli, and using this organism a standard curve between bacterial load and luminosity units of PCR ELISA was created. The correlation coefficient (R2 value) between 102-104 CFU/ml and 105-109 CFU/ml is 0.8579 and 0.9632, respectively. When using a known amount of transformed E coli to test this standard curve, the result suggest that at high bacterial load (1.16*107 CFU/ml) the estimated number was 9.19*107 CFU/ml which was about 8 times higher. While at low bacterial load (4.27*104 CFU/ml), the estimated number was 2.67*105 CFU/ml which is 6 times higher. In both test the numbers obtained were reasonably close. We have also created a standard curve of ammonium-oxidizing bacteria similarly. When the number of ammonium-oxidizing bacteria is between 102-105 CFU/ml, the correlation coefficient (R2 value) is 0.9547. Based on this curve, we try to estimate the number of ammonium-oxidizing bacteria in soil sample, the result suggest that the ammonium-oxidizing bacterial load is 6.92*1010 CFU/g of fresh soil. However, in our previous experience, the ammonium-oxidizing bacterial load in this particular soil is about 107-108 CFU/g of fresh soil. Enumeration based on growth has long been challenged by various researchers, due to the diverse physiological characteristics of different organisms. We have over estimated the number of ammonium-oxidizing bacteria in soil or there is really that many in the soil remained to be reexamined.