Metabolic Pathway Of Nitrogen By Heterotrophic Nitrification Bacteria Alcaligenes Faecalis Strain NR

Heterotrophic nitrification is a kind of phenomenon that organic nitrogen or ammonium were transformed to hydroxylamine, nitrate and nitrite under the action of heterotrophic nitrification bacteria. But studies have shown that many heterotrophic nitrification bacteria have the ability to transform ammonium into nitrogen gas under aerobic condition which means they can complete a complete biological denitrification process. The process is called heterotrophic nitrogen removal. Alcaligenes faecalis strain NR(Gen Bank accession No. FJ151629) was isolated from a membrane bioreactor which has simultaneous nitrification and denitrification function. Previous studies had confirmed that the bacteria has the ability of heterotrophic nitrogen removal under aerobic condition.In this paper, the intermediate which appeared in the process of metabolism of ammonium by Alcaligenes faecalis strain NR were tested, and the utilization of these intermediate by Alcaligenes faecalis strain NR were reviewed, in combination with the isotope tracer experiment the preliminary nitrogen metabolic pathways was deduced.The ultrasonic cell break method was used to isolate key enzyme which involved in the pathways of nitrogen removal, and then the enzyme activity were testsd to verify the derivation by enzymatic way. And the ammonium sulfate fractionation were used to purify HAO.The products of NH2 OH catalyzed by HAO was analyzed to further confirm the pathways of nitrogen removal of Alcaligenes faecalis strain NR.According to the results of the experiment what intermediate appeared in the process of metabolism of ammonium and the utilization way of intermediate(NH2OH-N、NO2--N、NO3--N)by Alcaligenes faecalis strain NR, in combination with the isotope tracer experiment two kinds of nitrogen removal pathway of Alcaligenes faecalis strain NR were derived: ①It was similar to the traditional biological nitrogen removal pathway, NO2--N as the combination of nitrification and denitrification for biological denitrification, ② Another way was that NH4+-N was transformed into NH2OH-N, and then the generated NH2OH-N was converted into N2 O and N2 directly.Single factor experiments of ultrasonic extraction of key enzyme showed that:when total radiation time was 300 s, ultrasonic power was 300 w, single radiation time was 5s, the concentration of bacteria was OD600=1.535, performs best under these working conditions respectively. The experiment combined RSM showed the bestextraction conditions were ultrasonic extraction power 310 w, radiation time 400 s, the single radiation time 5s, concentration of bacterium OD600=1.930. Based on the ultrasonic extraction the hydroxylamine oxidase, nitrate reductase and nitrite reductase activity were deducded in the crude enzyme liquid, enzyme activity were 23.75, 3.29,and 12.16 m U/mg protein respectively. Combined with previous experimental conclusion it can be concluded that there exsited a heterotrophic nitrification and aerobic denitrification enzyme system which proved that there existed a combination way of heterotrophic nitrification and aerobic denitrification in the pathways of nitrogen removal of Alcaligenes faecalis strain NR.The results of ammonium sulfate fractionation experiment showed that under the gradient 50%~65% the impurity protein could removed effectively.And the HAO specific activity was 53 m U/mg, the purification multiple achieved to 1.907. At the temperature of 30℃、p H=8, the maximum activity of HAO was tested. There were two kinds of productions of enzymatic reaction,one was a liquid phase product NO2--N,and another one was N2 O, so it proved that there existed pathways of nitrogen removal by Alcaligenes faecalis strain NR,NH4+-N�'NH2OH�'N2O�'N2.