Research On Screening And Immobilization Of Aerobic Nitrate Reducing Bacteria Delftia Tsuruhatensis NF4 And Cloning, Expression Of The Nap Gene

In recent years,nitrogen-containing substances emitted from sewage are the main factors leading to serious pollution of eutrophication of water bodies,which has aroused widespread concern.Nitrogen removal is an important part of wastewater treatment process.The traditional autotrophic nitrification-anaerobic denitrification treatment mode has the problems of slow denitrification process,separation of nitrification and denitrification,high cost and operation difficulty.Aerobic denitrification has become a research hotspot in recent years due to its characteristics of being able to degrade nitrate under aerobic conditions and fast reaction.However,the separation and research of aerobic denitrifying bacteria is still insufficient.It is necessary to enrich the aerobic denitrifying microbial library,which lays an important foundation for the realization of heterotrophic nitrification-aerobic denitrification and high efficiency nitrogen removal.In this paper,the water of Fenghuanghe Ergou Wastewater Treatment System in Chengdu was taken as the research sample.The diversity of bacterial communities in natural water,heterotrophic nitrification medium cultures and aerobic denitrification mediaum cultures were analyzed and compared.One strain Delftia tsuruhatensis NF4 with strong nitrate reduction ability under aerobic conditions was screened out.Its nitrate reductase gene was cloned and expressed in prokaryotic expression system,and the denitrification function of the immobilized strain was studied.The main results are as follows,?1?High-throughput Illumina Miseq sequencing technology was used to study bacterial diversity in the input water,CRI output water and effluent water of sewage water treatment ecosystem.The differences of bacterial diversity between aerobic nitrification,denitrification media and natural water were compared.As a result,509464 bacterial 16 S rRNA sequences were obtained.The dominant phylum of wastewater treatment ecosystem were seperately,Firmicutes,Bacteroidetes,Proteobacteria,et al.In CRI output water,the abundance of Proteobacteria did not change much in natural state,in aerobic nitrification medium cultures and in aerobic denitrification medium cultures.In the medium of aerobic nitrification and denitrification of total influent and effluent of the treatment ecosystem,the abundance of Proteobacteria decreased significantly,while that of Bacteroides increased significantly.Based on the results of principal component analysis?PCA?of OTU,it can be seen that the samples spots of nitrification and denitrification cultures in the same sampling site closed to each other,which was indicated that the flora separated by the two media overlaped to a large extent.These bacteria may have both aerobic nitrification and denitrification functions.The aggregation degree of three samples spots of CRI was higher than that of input and output water of the treatment system.The results showed that the water samples of CRI cultured in aerobic nitrification and denitrification media had more similarity of community composition to that of the natural water.?2?Based on the results of high-throughput sequencing,the aerobic nitrate-reducing bacteria in the output water of CRI were isolated and purified by plate separation technique.The strain NF4 with strong aerobic nitrate reduction ability was isolated,and the nitrate removal rate reached 96.8%.It was Gram-negative.Compared with the biochemical indexes of Delftia acidovorans and Delftia tsuruhatensis,including fermentation of glucose,xylose,lactose,urea,hydrolysis of arginine and reduction of nitrate,strain NF4 had the highest similarity with Delftia tsuruhatensis and the three strains all had nitrate reduction ability.By molecular biological identification analysis with 16 S rDNA,this strain was preliminary identified to be Delftia tsuruhatensis.Delftia tsuruhatensis was originally known as Comamonas.But with the indepth study of its evolutionary branch in recent years,it had been classified into the genus Delftia.?3?Delftia tsuruhatensis NF4 was immobilized by sodium alginate embedding method,and the denitrification function of Delftia tsuruhatensis NF4 and immobilized Delftia tsuruhatensis NF4 were compared.The experimental results showed that both of them could remove nitrate.The nitrate degradation rate,nitrite accumulation rate and total nitrogen removal rate of the immobilized strains were 98.06%,27.46 mg/L and 26.17% respectively under the conditions: initial nitrate concentration of 33 mg/L,inoculation volume of 5%?V/V?,temperature of 30 ?,rotational speed of 140 r/min and initial pH 7.0,whice were 1.5 times higher than those of the free strains.Therefore,using sodium alginate immobilized strain Delftia tsuruhatensis NF4 could improve the denitrification performance of the strain.In addition,the immobilization conditions were optimized.The effects of initial nitrate nitrogen concentration,initial pH value and culture temperature were studied to find the optimal nitrate removal conditions.The results showed that the immobilized Delftia tsuruhatensis NF4 had nitrate removal activity at pH 3—9,temperature 20 ?—45 ?,and initial nitrate nitrate concentration 16.5—165 mg/L.When the initial pH was 7.0,the temperature was 30 ?,the initial concentration of nitrate nitrogen was 16.5 mg/L and 33 mg/L,the removal rate of nitrate is 100%.The nitrate degradation activity increased at 24 h when the initial concentration of nitrate nitrogen was between 16.5 and 165 mg/L,with initial pH 7.0,temperature=30 ?.?4?The nitrate reductase gene sequence?Login number: CP017420.1?of Delftia_tsuruhatensis strain CM13 in NCBI was used as template.The specific primers of nap gene were designed and the recombinant cloning vector pMD19-T-nap was constructed by TA cloning.The pET28a?+?-nap recombinant plasmid vector was constructed using Nde I/Xho I endonuclease hydrolysis and T4 ligase.pET28a?+?-nap recombinant plasmid vector was transferred into E.coli BL21?DE3?to obtain BL21?DE3?-pET28a?+?-nap engineered E.coli.The results of protein sequence comparison showed that the amino acid sequence of nitrate reductase from Delftia tsuruhatensis NF4 had the highest homology with the nitrate reductase?Sequence ID WP₀47325778.1?from Delftia tsuruhatensis?98.94%?.The homology to the nitrate reductase?Sequence ID: WP₀34399787.1,Sequence ID: WP₀34399-787.1?of Delftia acidovorans is also higher than 98%.This protein belongs to the Molybdopterin-Binding superfamily.The molecular weight of the target protein expressed by the recombinant was 101.375 kDa,which was consistent with the predicted protein size.The results of SDS-PAGE and Western Blot showed that the target protein was mainly expressed in the form of inclusion bodies under the induction of 0.2 mM IPTG,while the expression of supernatant was low.The expression was in the form of inclusion bodies under the induction of 0.4 mM IPTG.By comparing of the nitrate reductase activity of pET28a?+?-nap-BL21 and pET28a?+?-BL21,the former was 40.28 U/L and the latter was 21.62 U/L,which indicated that the pET28a?+?-nap-BL21 could express the active nitrate reductase.