过表达亚硝酸盐还原酶的重组大肠杆菌辅助污水短程硝化辛玉峰

ActaMicrobiologicaSinica2017,57(12):1898-1907:10.13343/j.cnki.wsxb.20170068ResearchArticle(ZR2012CQ011)(XJ201108)*[e-mail]@126.com2017-02-162017-04-232017-04-27**273165-OD600=2.02h1mmol/LAcinetobactersp.YF1412h(36.0±7.4)%4h37%(OD600=1.0)12h(31.0±5.7)%[(6.40.7)mg/L][1–2][2–3][2]20-[4–6]-|,2017,57(12)1899[7]pH[6,8][8][9]pH[10–11]--(NO)N2(NitritereductaseNIR)(NO)(EC1.7.1.2)(1)[12]NO2–++2H+→NO↑++H2O[13](1)NIRNONIR-Acinetobactersp.YF14[14]11.1E.coliBL21(DE3)LB37°CAcinetobactersp.YF14LB[14]SigmaAldrichpH7.0100mmol/LHEPES(1.0–1.5mmol/L)1.2(OMEGAUSA)PseudomonasaeruginosaPAO1Phusion(LifetechnologyUSA)PCRPaNirPaNirF15′-CACACAGGAAACAGCTATGCCATTTGGCAAG-3′R15′-TTCCATTCGCCATTCATCAGTACACGTCGTGC-3′pBBR1MCS2PCRF25′-TGAATGGCGAATGGAAATTGTAAG-3′R25′-AGCTGTTTCCTGTGTGAAATTGTTATC-3′In-fusionE.coliBL21(DE3)BL21(PaNIR)pBBR1MCS2BL21(DE3)BL21(C)1900YufengXinetal.|ActaMicrobiologicaSinica,2017,57(12)[e-mail]1.3LB1%100mLOD6000.60.5mmol/LIPTG30°C4h(5000r/min5min)pH7.0100mmol/LHEPES2OD6002.0(13000r/min2min)1.42(1)-Acinetobactersp.YF14[14](2)[14]:10025OD600=1.0BL21(PaNIR)BL21(C)2100mL25mL25°C100r/minIPTGYF14(1.3)100-mL25mLOD600=2.0YF14(1.5mmol/L)OD600=2.0IPTGBL21(PaNIR)5min25°C(50100200r/min)(DO4.50.66.40.77.80.9mg/L)2h(NH4+-N)1.5[14]NONONO2–Griess(SigmaUSA)OD540NO2–NONO2–1:1NO(DO)RDO(ThermoScientificUSA)1.6GraphPadPrism5.0NCBIBlastP(=blastp&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome)PCR22.1BL21(DE3)Pseudomonasaeruginosa(PaNIR:NP_249210.1)P.aeruginosaNIRPaNIRPCR()pBBR1MCS2BL21(DE3)BL21(DE3)(1)|,2017,57(12)1901(□):NOconcentrationofBL21(C);(○):NOconcentrationofBL21(PaNIR);(■):NitriteconcentrationofBL21(C);(●):NitriteconcentrationofBL21(PaNIR).Datarepresenttheaverageofthreeindependentexperiments.Errorbarsindicatestandarddeviations.2.2NOBL21(PaNIR)OD600=2.0(1)30min50%NO2h1mmol/LNO[0.64±0.70mmol/(L·h)][0.72±0.10mmol/(L·h)]BL21(C)NO[15]BL21(PaNIR)2.3YF14YF14-[14]BL21(PaNIR)YF14OD6002.0YF14BL21(PaNIR)[BL21(C)]BL21(PaNIR)YF14(2-abc)12hOD6001.0(2-a)2.0(2-b)4.0(2-c)YF14(24.0±3.5)%(36.0±7.4)%(42.0±8.3)%BL21(C)YF14(2-abc)[15]BL21(C)BL21(PaNIR)NIROD6001.02.04.011.9%22.3%28.8%YF14[14](2-def)4hOD6001.0(2-d)2.0(2-e)4.0(2-f)BL21(PaNIR)YF1418.5%37.0%56.9%-YF14(2-ghi)(1)1902YufengXinetal.|ActaMicrobiologicaSinica,2017,57(12)[e-mail](a),(d),(g),(j)and(m)showedtheseveralchangesofthemixtureofOD600of1.0BL21(C)orBL21(PaNIR)andOD600of2.0YF14;Thefigures(b),(e),(h),(k)and(n)showedthemixtureofOD600of2.0BL21(C)orBL21(PaNIR)andOD600of2.0YF14;Andthefigures(c),(f),(I),(l)and(o)showedthemixtureofOD600of4.0BL21(C)orBL21(PaNIR)andOD600of2.0YF14;○:YF14alone;∆:YF14andBL21(C);□:YF14andBL21(PaNIR).Errorbarsrepresentsthestandarddeviations.|,2017,57(12)1903(2)OD600(2-jkl)12hOD600[14]DO(2-mno)2.4BL21(PaNIR)NO(3-A)BL21(PaNIR)12h(31.0±5.7)%BL21(C)BL21(PaNIR)()NIR2DO(3-B)3.Figure3.RecombinantE.colicontributetothedenitrificationofsludge.Theaminonitrogen(A)anddissolvedoxygen(B)wereshowed.○:Sludgealone;∆:SludgeandBL21(C);□:SludgeandBL21(PaNIR).Errorbarsrepresentsthestandarddeviations.2.5OD600=2.0BL21(PaNIR)(4)YF14(4-A)(4-B)6.40.7mg/L2h19%28%(7.80.9mg/L)1904YufengXinetal.|ActaMicrobiologicaSinica,2017,57(12)[e-mail]ate(%)wereshowedinthemixtureofYF14andBL21(PaNIR)(A)orsludgeandBL21(PaNIR)(B).Errorbarsrepresentsthestandarddeviations.325%[16]pHpH[16]-[17–20][21–22][23]YF14(2)(3-A)(1)NONON2[9]NOYF14(YF14)(23)(YF14)YF14|,2017,57(12)1905[24]IPTGNIRSBRSBR[16][5](23)3(1)(2)(3)[1]HaoXD,ZhangZJ.Nitrogenpollutionofwateranditscontrol.EnvironmentalPollution&Control,1990,12(4):25–27,47.(inChinese),..,1990,12(4):25–27,47.[2]LiXY,XuF.Progressofbiologicaltreatmenttechnologyofmicro-pollutedwaterwithlowconcentrationofammonia-nitrogen.PollutionControlTechnology,2012,25(4):31–34.(inChinese),..,2012,25(4):31–34.[3]WangH,LiHB.Studyanddevelopmentofbiotechnologyofammoniaremovalinwastewater.JournalofHenanNormalUniversity(NaturalScience),2008,36(5):97–100,103.(inChinese),..(),2008,36(5):97–100,103.[4]YuanLJ,PengDC,WangZY.Biologicalnitrogenremovalbyshortcutnitrification-denitrification.ChinaWater&Wastewater,2000,16(2):29–31.(inChinese),,.-.,2000,16(2):29–31.[5]ZhuGB,PengYZ,GuoJH.Biologicalnitrogenremovalwithnitrificationanddenitrificationvianitritepathway.JournalofHarbinInstituteofTechnology,2008,40(10):1552–1557.(inChinese),,..,2008,40(10):1552–1557.[6]LiuCX,HuHY,PengDC,WangZY,QianY.Short-cut1906YufengXinetal.|ActaMicrobiologicaSinica,2017,57(12)[e-mail]ificationfortreatmentofhighammoniacontentwastewaterfromcokingplant.ChinaWater&Wastewater,2003,19(8):11–14.(inChine