Characteristics Of Nitrogen Removal Genes And Its Application In Aerobic RBC By Alcaligenes Faecalis NR

Function bacteria of heterotrophic nitrification aerobic denitrification have potential application on wastewater treatment.To date,however,the mechanism and pathway of hetertrophic nitrification aerobic denitrification is unclear.In this reach,heterotrophic nitrification aerobic denitrification microbe Alcaligenes faecalis NR(GenBank No.FJ151629)was used to lucubrate the key enzymes of nitrification and denitrification process,and aerobic rotating biological contactor(ARBC)biofilm was used to investigate the nitrogen removal efficiency which strengthen by A.faecalis NR.The main results were as follows:The A.faecalis NR with the amoA,hao,nirK,nor and nos genes in the genome DNA,but without the nirS gene.And the results of bioinformatics analysis showed that the length of AmoA,Hao,NirK,Nor and Nos proteins sequences were 346 aa,222 aa,381 aa,759 aa and 634 aa,respectively.And the molecular weight was 36.7 kDa,25.03 kDa,41.47 kDa,85.72 kDa and 70.10 kDa,respectively.AmoA and Nor was alkaline hydrophobic protein,these two exist transmenbrane areas.Hao,NirK,and Nos were acidic hydrophilic protein,and do not exist transmembrane area or signal peptide.To further uncover the mechanism of heterotrophic nitrification aerobic denitrification the tertiary structure model of AmoA,NirK and Nos were built by using the method of homolgous modeling or threading method.AmoA was a transmembrane protein and embedded in the cell membrane and contains a nickel atom binding sites.NirK was a trimer which contains three type I copper binding sites and three type II copper binding sites.Nos was a dimer which contains two CuA binding sites and two CuZ binding sites.A.faecalis NR can effectively strengthen the ARBC nitrogen removal.The TN concentration and removal rate of rbc 1(inoculated A.faecalis NR)was 1.5 ~ 13.5 mg/L and 88.6 ~ 99.1 %,respectively.The TN concentration and removal rate of rbc 2(noninoculated A.faecalis NR)was 18.2-32.8 mg/L and 70.4-83.6 %.However,the difference of TN concentration was caused by NO3--N and NO2--N,the concentration of NO3--N and NO2--N in rbc 1 was < 2.0 mg/L and < 0.9 mg/L,respectively.Nevertheless,the concentration of NO3--N and NO2--N in rbc 2 was 10.3 ~ 31.3 mg/L and 5.4 ~ 18.5 mg/L,respectively.To further study the strength nitrogen removal efficiency of A.faecalis NR the water feeding pattern was set to continuous influent.When TN,NH4+-N and COD loads(B)were 1.10,0.88 and 22.01 g/(m2?d),the COD,TN,NH4+-N and NO3--N removal efficiency were 97.97 %?93.93 %?97.12 % and 98.55%.And 58.48 % of the inflow nitrogen was assimilated into biomass,38.63 % was transformed to nitrogen gaseous.When TN,NH4+-N and COD loads(C)were 0.77,0.66 and 11.01 g/(m2?d),the COD,TN and NH4+-N removal efficiency were 98.91%,67.21 % and 99.44 %,respectively.However,the NO3--N accumulated seriously with the concention of 21.51 mg/L.moreover,36.72 % of the inflow nitrogen was assimilated into biomass,35.27 % was converted to nitrogen gaseous.MiSeq high-throughput sequencing was used to analyse the biological community structure of ARBC,when the effluent of ARBC was stable.The results indicate that the biodiversity relationship was Con 1(activated sludge)> C(operating condition C)> B(operating condition B)> Con 2(contral group).In genus level the biology community structure of biological film(Con 2,B,C)and activated sludge(Con 1)were distinctly different.the dominants of Con 1 were Chloroflexus,Sphingobacterium,Nitrospira and Thauera et al.The dominant of Con were Paracocccus,Pseudomonas,Azoarcus and Meganema et al.The dominants of B were Meganema,Azoarcus and Pseudomonas et al.The dominants of C were Meganema,Sphaerotilus and Hydrogenophaga et al.The genus capble of heterotrophic nitrification aerobic denitrification were Alcaligenes,Pseudomonas,Shinella,Paracocccus and a small number of Acinetobacter,Zoogloea,Comamonas and Chryseobacterium.Furthermore,Alcaligenes was detected with the relative abundance of 0.38 % and 2.36 % in B and C,which was sequence identical(> 97 % similarity)with A.faecalis NR,indicating ARBC biofilm can effectively fixe A.faecalis NR.