| Summary: | 碩士 === 大同大學 === 生物工程研究所 === 91 === For health and environmental protection, municipal and industrial wastewaters have to be treated. Wastewater with ammonia-nitrogen, generally treated with mixed microbial populations, is produced by domestic wastewater, and wastewater from livestock and manufacture about food, ABS plastic, petroleum, and semiconductor at a large amount. The directly determination of the growths and distributions for relative bacteria are less or unsuccessful because of its complexity of populations in activated sludge. Due to the improvement of technology for molecular biology, the amounts and identify of the species can be directly determinated. There are many enzymes involved in denitrification of nitrogen cycle, we choose nirK and nirS these two enzymes to compare the genes sequences to design the primer pairs. These primer pairs with the genomis DNAs of Alcaligenes xylosoxidans BCRC 12838 (nirK type), Pseudomonas fluorescens BCRC 16016 (nirS type), and denitrifying bacteria in wastewater carry out the polymerase chain reaction for the rapidly determination and quantification of denitrifying bacteria.
From the results, we can see that there is only one primer set, nirK3f-nirK5r, can react properly with Alcaligenes xylosoxidans BCRC 12838 and denitrifying bacteria in wastewater among nine sets of primer pairs. And there is also only one primer set, nirS2f-nirS3r, can react properly with Pseudomonas fluorescens BCRC 16016 and denitrifying bacteria in wastewater among sixteen set of primer pairs.
According to the sequences of nirK and nirS between the two specific primer sets, designed new primers to construct and clone the internal controls, cnirK and cnirS, for competitive PCR. There were 3.342108 denitrifying bacteria with nirS, determinated by competitive PCR, per gram of activated sludge in nitrogen compound-treated tank.
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