Study On The Denitrification Characteristics Of A Denitrifier DNF409 And Its NarG Gene Disruption

The deterioration of aquarium water has become an important limiting factor of national aquiculture development in our country. And the nitrite pollution has become an usual harmful factor which is difficult to be prevented in aquiculture. Biodenitrification is one of the effective methods to remove nitrite from water in theory.In this study, we researched the denitrification characteristics of the denitrifier named DNF409, which has been isolated in the laboratory, in order to get the parameters, which could be worthy in its application to the biodenitrification; and the gene-knockout protocol was used to construct narG gene disruption strain, which encodes the catalytic a subunit of the respiratory nitrate reductase, to improve the characteristics of denitrifier DNF409. The results were showed as below:1. Denitrifier DNF409 was identified as Bacillus sp. by physiological and biochemical test and the comparative 16S rDNA sequences analysis. The optimum pH value of its growth was 6～7 and the optimum pH value of its denitrification was 7～8. It possessed highly effective denitrification during its whole growth period, and ethanol was the most effective carbon source as the electron donor.2. Denitrifier DNF409 could possess highly effective denitrification in the aquarium water. The data showed that when the C/N ratio = 8 and the bacterial concentration reached 10~8cfu/L, the highest degradation efficiency of nitrate and nitrite was 94.79% and 99.94% respectively, which demonstrated the extensive application of DNF409 to biodenitrification in the future.3. The vector of narG gene targeting pEGude was constructed from shuttle plasmid pEG491. It had a temperature-sensitive replicon,chloramphenicol resistance gene and two fragments which were 1.1Kb,2.1Kb respectively matching the sequence of narG gene. And the erythromycin resistance gene, which could be expressed in denitrifier DNF409, was inserted in to the two fragments.4. The vector of narG gene targeting pEGude was introduced into denitrifier DNF409 by electrotransformation. An Erm~r Cm~s isolate DNF409EF was obtained and narG gene disruption was confirmed by PCR.5. The denitrification analysis indicated that strain DNF409EF did not lose the capability of nitrate reduction. But compared with strain DNF409, both the nitrate reduction and the nitrite reduction of strain DNF409EF were delayed to some degree in the temporal, which demonstrated that there might be some relationship between the nitrate reduction and the nitrite reduction in denitrification pathway. The result doesn't accord with the anticipation and the reason remains to elucidate.