36 2 20174 Vol.36No.2 Apr.2017 70!,,, (, 266590)!:!"#$%&', ()*, -./ 01 * / 2123' % &,45/' %&6789 3(:; AN 1), 3<= >.>? @A BC D E ' %&67FG H IJKL: 3 AN 1 MNO,, P,Q R S TSU V W 3 AN 1%&' 8XY 30,pH 8.0,' %& 8# 78%; 316SrDNA 犛犺犻狀犲犾犪狕狅狅犵犾狅犲狅犻犱犲狊 (GU930756)Z[ 8#(95%) \], ' %& AN 1^_#$% &' 67,`abO `( 犛犺犻狀犲犾犪 ) "#$:' %& ;bo ; * ; %&' :X172 (:A )* :1672 3767(2017)02 0070 06 犐狊狅犾犪狋犻狅狀狅犳犪狀犃犿犿狅狀犻犪 狀犻狋狉狅犵犲狀犇犲犵狉犪犱犻狀犵犅犪犮狋犲狉犻狌犿犪狀犱犐狋狊犚犲犿狅狏犪犾犆犪狆犪犫犻犾犻狋狔 LIU Yaqiao,HAN Xuejun,TAN Haochen,LU Hongsheng (ColegeofChemicalandEnvironmentalEngineering,Shandong UniversityofScienceandTechnology,Qingdao,Shandong266590,China) 犃犫狊狋狉犪犮狋 :Theobjectiveofthisstudyistoisolateandidentifythebacteriawithhighammonia nitrogendegradingca pabilitydegradingandtoapplythebacteriatowastewaterdenitrificationtreatment.12strainsofammonia nitrogen degradingbacteriawereisolatedfromactivatedsludgeandthebacterium withthehighestammonia nitrogendegrad ingcapability(strainan 1)wasselectedanditsmorphology,biochemicalproperties,phylogeneticpositionandam monia nitrogendegradingcapabilityunderdiferentconditionswereexamined.theresultsindicatethatstrainan 1 isagram negative,rhabditiformbacterium,whosecolonyismilkwhite,andbothitscatalaseandoxidaseareposi tive.strainan 1hastheoptimumammonia nitrogendegradationefectwhenthetemperatureis30 andtheph valueis8.0.itsmaximumremovalratecanreach78%.the16srdnasequenceofstrainan 1showsthehighest similarityof95% withitscloselyrelatedbacteriaof 犛犺犻狀犲犾犪狕狅狅犵犾狅犲狅犻犱犲狊 (GU930756).Itisthusconcludedthat strainan 1,withhighammonia nitrogendegradingcapability,isonememberofgenus 犛犺犻狀犲犾犪. 犓犲狔狑狅狉犱狊 :ammonia nitrogendegradingbacterium;genus 犛犺犻狀犲犾犪 ;activatedsludge;isolation 789:; <=>?'@ABC D>E 7 : = F G= F G = F HG= F>5I,J=K NH 4 N GL7 GL7,MJ=KGL7 GL7,N/7O>,-:2016 11 02./01: (20101008); (201201008) : (1991 ),,!,"#$%,&' #$. ()(1972 ),*,!,,-.,,&' /01 #$, 23456.E mail: hslu628@163.com
c3#$' %& 5' 67 H 71 D,P Q%RSTU D789:;>&'V [1] WXY7 :, = F 7 : D> ZQ%R @[,\];^ = F_` E 7>ab,TU <=ce?' _ = F \ ] > # $ [2 8], > = F E F ( 犅犪犮犻犾狌狊 ) F ( 犘狊犲狌犱狅犿狅狀犪狊 ) F( 犅狉犲狏狌狀犱犻犿狅狀犪狊犱犻犿犻狀狌狋犪 ) F( 犃犾犮犪犾犻犵犲狀犲狊犳犪犲犮犪犾犻狊 ) O F( 犈狀狋犲狉狅犫犪犮狋犲狉犪犲狉狅犵犲狀犲狊 ) F ( 犘狉狅狋犲狌狊 ) F ( 犛犲狉狉犪狋犻犪 )0Q F ( 犕犻犮狉狅犮狅犮犮狌狊 ) [9 10] #$ D \ ] 7 a F,_ L % % = 7 a / 0\ \,K (`) D;^ = F>\] /0 < = T U C > 1 23' 1.1 45 6789: \] 7a F> I>= : = _ α, K\ > U: 60g =, 250mL D, 20g = 100mL D, = ( 10g),, K =,,!9,"# = $ %& ' XY > = D, \, I 400mL,$ :( N)& >X* J,-./01D, 2 U:_ 0.5g, 150mL10%3 D,4 I; α 5 0.1g, 50mL 6]7 D,89,M'' 150mL10%3,4 I I I\: ; 1D, I 1.2 ;. 7a F < [11] :(NH 4 ) 2SO 4,2g/L;NaCl,0.3g/L;FeSO 4 7H 2O,0.03g/L;K 2HPO 4, 1g/L ;MgSO 4 7H 2O,0.03g/L;NaHCO 3,1.6g/L;H 2O,1L;pH,7.2 121 <F20min = < U >? (15g/L),@= < U >? (3g/L) 1.3 2 1.3.1 7A 7A BI \ CS(DES,HJ535 2009) A : UC C> E F I G50mL,M\: 1.0mL HI 1.5mL >,;425nm A J C, KUEFLM J N9 O, 50mL,\: HI (1.0mL) (1.5mL), ;425nm A J C, EFLM, OD 7 C 1.3.2 7a F 0\] 10mL E200mL <>PQ1D,30 RSTUV(160r/min) <,W X1d <,I 2 Y G >%, Z[ 7a F>\] < 7d 10mL < J^ _>200mL <D,? XY`53 3 > < 0.03mL am < = bc <X,30 RST <7d,\: D > F? am <3,) =4 >12 F \: @= 7a F <D, I 1.3.3 ;^ 7a F> 0 % %= % LMA _XY =4 >12 F \:I < <,WX1d < I 2 Y, F > 7a, =N >F )&F K AN 1,F AN 1 K 7a N >F $_F AN 1>% %= A, ^
72 36 2 20174 Vol.36No.2 Apr.2017 = V.P G G I A,`5VS 2 [12] % LMA :I <_F AN 1 <, < ph K7, < CK30,UV < (160r/min),WX1d A < J C( 犗犇 560),5 ~J CLM 1.3.4 RS_ 7a > A 1) ph RS_ 7a > )F AN 1^ <D <3d,\: < 5mL ^ ph \:K6.0 7.0 8.0 9.0>195mL <D,30 RSTUV <(160r/min), <5dA < D 7 89, 7a 2) CRS_ 7a > 1.3.4(1)D < 5mL ^ 195 ml <D,\:;10 20 30 40 RSTUV (160r/min) <5d,A < D 789, 7a 1.3.5 F AN 1> \ 1)DNA F AN 1DNA > BI @ (% %R (X ) E UNIQ 10), VS 2)PCR _F 16SrDNA > PCR RBI F16SrDNA 3I R [13], R : R7F:5 CAGAGTTTGATCCTGGCT 3 ; H R1540R:5 AGGAGGTGATCCAGCCGCA 3 3)PCR H /0H RS PCR H (25μL):2.5μL5 Bufer(8 Mg 2 ),0.5μL c DNA,G0.5μL7F(10uM) 1540R (10uM),1μLdNTP(G2.5mM), 25μL PCR H R S:98 3 min;98 25s,55 25s,72 1min,30,M72 10min,PCR > WX % %R A 4) > )A4>16SrDNA DDBJ(DNA DataBankofJapan,htp://www.ddbj.nig.ac.jp/) D> BlastD _, I CustalX2.1 Mega5.0(MolecularEvolutionaryGeneticsAnalysis) S,BI>VSK ^S(Neighbor Joining) [14 17] 2 < 2.1 =>?'@8AB I 2 _ < YA, cea 7 >F $_F = <,NIM <S \], \] <4 12 7a F, D 7a N >F AN 1K F,I > 2.2 CDCE F AN 1;= bc <X%, F K,, Q &FK ( 1) F _ 7a F AN 1> L % %=, 1 2.3 => 犃犖 1C )F AN 1; <D UV <,WX1dA < >J C( 犗犇 560),5 2 > % LM 2 /,F AN 1>", :24h > < _ ; < 72h %R9 N,;[> <: D,%R9
c3#$' %& 5' 67 H 73 K1 => 犃犖 1?CDCELM Tab.1 Physio chemicalpropertiesofstrainan 1 E F AN 1 (Gram stain) ^ (Catalase) = (Oxidase) -(V.P.) (Voges Proskauerreaction) G (Nitratesreducedtonitrites) G (Nitritesreducedtonitrites) (Glucose) &1 FGH=> AN 1'@ IE=J Fig.1 ColonyisolationandpurificationofstrainAN 1 (Amylase) : : EH ; - : H 2.4 NO 狆犎? F )F AN 1\:^ ph K6.0 7.0 8.0 9.0> <D UV <, < CK30, < 5d,A < D 789,\: 7a,5 ph~ 7a LM, 3 /,F AN 1 ;ph8.0 N;> 7a,K78% ph K9.0> <D%,F AN 1>a E47%,pH K6.0 > 7a K50% W[,F AN 1> 7a NN ph ;8.0,pH :; :b 7a &2 => AN 1C Fig.2 GrowthcurveofstrainAN 1 &3 => AN 1PNO ph? F Fig.3 NH4 Nremovalrateofstrain AN 1withdiferentpHvalues 2.5 NOQR? F \:;10 20 30 40 R S _ F AN 1 U V (160r/min) < 5d, < ph K 8.0,W X1dA < D 789, 76 5 CRST> < ~ 76 LM, 4 4 /,F AN 1;30 <, 76 N;, 77% 10 RST <, 76 K20%,40 <RST> 76 K25%, ; 10 <RST> 76 /, F AN 1N!% > CK30, C:; :b,"ab 76
74 36 2 20174 Vol.36No.2 Apr.2017 2.6 FGH=VW' XY : A,F AN 1 > 16SrRNA > @#$K 1398bp 3 : DDBJ(DNA Data BankofJapan,htp://www.ddbj.nig.ac.jp/),% 4 F AN 1 > & ' K AB917468 ; NCBI (htp://www.ncbi.nlm.nih.gov) D, F AN 116SrRNA ; > @, I S CustalX2.1 Mega5.0 ( 5) I NCBI _ F AN 1 > 16S rrna \, F AN 1 犛犺犻狀犲犾犪犽狌犿犿犲狉狅狑犻犪犲 (EF070131) CN;, K96% F AN 1; D>, / Z[ F AN 1 ( F, K 犛犺犻狀犲犾犪 sp.an 1(AB917468) &4 ; QRS=> 犃犖 1 F TU Fig.4 Efectofcultivationtemperatureon NH4 NremovalrateofstrainAN 1 &5 => 犃犖 1( 犃犅 917468) = ( 犛犺犻狀犲犾犪 )% Z=>16 犛狉犇犖犃 [\?]^VW_`a(bc ) Fig.5 Phylogenetictreeof16SrDNAgenesequenceofstrainAN 1anditscloselyrelatedstrain withinthefamily 犛犺犻狀犲犾犪 (Neighbour adjoiningmethodadopted) 3 #$ 4 >( F 犛犺犻狀犲犾犪 sp.an 1(AB917468)cE >6 D 7>, 7 6 K78%,K; 7` <]` D6 7 >F ) *' I > 7` D,,'_c > @A \, _F AN 1 F,' DNA DNA -.,DNA GC 89A?/ \ 5 : [1], 9,,. -.@A >,. [J].>>,2010,34(2):256 261. ZHANGLieyu,RAOBenqiang,XIONGYing,etal.Themicrobialmechanismhorizontalconstructedwetlandusedtotreated
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