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Abstract
Wastewater purification requires the removal of phosphate and nitrogen from the water samples before it can be returned to the ocean. The conversion of nitrite to nitrate is done in most part by Nitrospira sp, which are the second most abundant organisms found after Nitrobacter sp. DNA from a bioreactor simulating wastewater was used to isolate DNA, which was amplified using generic bacterial primers 27-F and 1542-R. The product was then ligated into PCR 2.1 vector and transformed into TOP10 cells. The transformed cells were screened using Ntpsa685. Ntspa685 is a probe specific for Nitrospira moscoviensis and Nitrospira marina and was used to detect positive clones using colony blot hybridization. The positive clones were digested with restriction enzymes for verification of an insert, and then sequenced. It was found that a very strong and large signal was generated on the nitrocellulose membrane containing the colonies; however, it was difficult to differentiate which colonies the signal had arisen from due to smearing. Of the five plasmids isolated and purified, only 1 contained an insert, and when sequenced; was an 88% match for Nitrosococcus mobilis and Nitrosomas europaea. It can be concluded that further experimentation would be required in order to optimize the colony blot hybridization parameters before it can be determined whether or not it would be a viable method used for high throughput screening.