曾 薇,張麗敏,王安其,張 潔,彭永臻,段俊嶺(.北京工業(yè)大學(xué)環(huán)境與能源工程學(xué)院,北京 004;.北京城市排水集團(tuán)有限責(zé)任公司,北京 00044)
污水處理系統(tǒng)中硝化菌的菌群結(jié)構(gòu)和動(dòng)態(tài)變化
曾 薇1*,張麗敏1,王安其1,張 潔1,彭永臻1,段俊嶺2(1.北京工業(yè)大學(xué)環(huán)境與能源工程學(xué)院,北京 100124;2.北京城市排水集團(tuán)有限責(zé)任公司,北京 100044)
研究分析了4種不同工藝類(lèi)型的城市污水處理廠中氨氧化細(xì)菌(AOB)和亞硝酸鹽氧化細(xì)菌(NOB)的豐度及菌群結(jié)構(gòu).實(shí)時(shí)定量PCR結(jié)果表明4種工藝中AOB菌群的豐度范圍為8.56×106~4.46×107cells/gMLSS;NOB菌群的豐度為3.37×108~1.53×109cells/gMLSS.每個(gè)工藝中Nitrospira都是優(yōu)勢(shì)NOB,占NOB菌群的88% 以上. A2O工藝冬季AOB和Nitrospira豐度比夏季均有所降低,這是導(dǎo)致冬季生物脫氮效果變差的主要原因.基于 amoA基因的系統(tǒng)發(fā)育分析結(jié)果顯示所有的序列屬于Nitrosomonas,其中Nitrosomonas oligotropha cluster 占克隆文庫(kù)的60.1%,是 AOB 種群中的優(yōu)勢(shì)菌屬,Nitrosomonas-like cluster和 Nitrosomonas europaea cluster次之,分別占克隆文庫(kù)的29.6%和9.1%.N. europaea cluster只在A2O工藝中出現(xiàn),且在A2O工藝夏季污泥樣品克隆文庫(kù)中達(dá)到44.7%.低DO運(yùn)行使N. europaea cluster成為優(yōu)勢(shì) AOB是 A2O工藝夏季出現(xiàn)較高亞硝酸鹽積累率的主要原因.研究結(jié)果證實(shí)了城市污水處理廠中優(yōu)勢(shì) AOB和 NOB分別為Nitrosomonas和Nitrospira,硝化菌群占總菌群的1%~7%,其豐度、相對(duì)含量和菌群結(jié)構(gòu)是影響硝化效果的主要因素.
氨氧化細(xì)菌(AOB);亞硝酸鹽氧化細(xì)菌(NOB);城市污水處理廠(WWTPs);實(shí)時(shí)定量PCR (QPCR);amoA基因
生物脫氮已廣泛應(yīng)用于城市污水處理廠,硝化作用是生物脫氮的首要環(huán)節(jié),其功能微生物包括將氧化為的氨氧化細(xì)菌(AOB)和將氧化為的亞硝酸鹽氧化細(xì)菌(NOB)[1].AOB菌群主要屬于變形菌綱的β-Proteobacteria和γ-Proteobacteria兩個(gè)亞綱[2],其中β亞綱分為亞硝化單胞菌群(Nitrosomonas)和亞硝化螺菌群(Nitrosospira).NOB主要有硝化桿菌屬(Nitrobacter)、硝化螺菌屬(Nitrospira)、硝化刺菌屬(Nitrospina)及硝化球菌屬(Nitrococus)[3].硝化菌群的豐度和菌群結(jié)構(gòu)直接影響污水處理廠的硝化效果.
近年來(lái),不依賴于純培養(yǎng)的分子生物學(xué)分析方法成為研究污水處理系統(tǒng)硝化菌群的主要技術(shù)[4-6].已有研究證實(shí)絕大多數(shù)的生物反應(yīng)器里Nitrosomonas是AOB中的優(yōu)勢(shì)菌屬,而Nitrosospira只出現(xiàn)在個(gè)別反應(yīng)器里[2,7-8].對(duì)于NOB菌群,以前一直認(rèn)為Nitrobacter是污水生物處理系統(tǒng)中亞硝酸鹽氧化的主導(dǎo)者[9].也有研究者認(rèn)為自然環(huán)境中的 NOB以 Nitrobacter和Nitrospira類(lèi)型為主,其中 Nitrobacter是土壤中NOB的主導(dǎo)類(lèi)型,而Nitrospira在污水生物處理系統(tǒng)中分布較為廣泛[10].后來(lái)的一些研究表明Nitrospira和Nitrobacter均存在于城市污水處理廠[11],但是發(fā)現(xiàn) Nitrospira 在污水生物處理系統(tǒng)中更為常見(jiàn)且數(shù)量高于Nitrobacter[12].由此可見(jiàn),城市污水處理系統(tǒng)中 NOB 的優(yōu)勢(shì)菌群因工藝類(lèi)型和運(yùn)行參數(shù)的不同而具有各自的特征.很多實(shí)際污水處理系統(tǒng)中的硝化細(xì)菌對(duì)環(huán)境因素以及工藝參數(shù)非常敏感,例如溫度、DO、水力停留時(shí)間(HRT)以及抑制劑等因素均可以影響硝化細(xì)菌的組成[13-15].以往對(duì)硝化菌群的定量分析主要集中AOB菌群的研究上[16],關(guān)于NOB的定量分析,尤其是 NOB的兩個(gè)亞屬 Nitrospira和Nitrobacter的定量研究非常有限.本研究通過(guò)對(duì)不同工藝的城市污水處理廠中AOB、NOB的定量分析,考察工藝類(lèi)型及運(yùn)行條件對(duì) AOB和NOB(Nitrospira和Nitrobacter)豐度的影響.
基于16S rRNA和amoA基因的現(xiàn)代分子生物學(xué)方法為復(fù)雜環(huán)境中AOB的多樣性以及菌群結(jié)構(gòu)分析提供了有力的手段[17-19].氨單加氧酶(Ammonia monooxygenase, AMO)是氨氧化菌所特有的一種胞內(nèi)酶,由amoA、amoB和amoC 3個(gè)亞基組成,其中 amoA的基因產(chǎn)物含有該酶的活性位點(diǎn).很多學(xué)者對(duì)不同AOB菌株的amoA基因和16S rRNA基因進(jìn)行測(cè)序和系統(tǒng)發(fā)育樹(shù)分析后,發(fā)現(xiàn)大部分AOB在分別基于amoA基因和16S rRNA基因的系統(tǒng)發(fā)育樹(shù)上的分類(lèi)有著高度的相似性[19-21],但 amoA基因類(lèi)引物的擴(kuò)增特異性更強(qiáng),對(duì) AOB菌群遺傳差異的分辨能力更高[18,22].本研究基于amoA基因建立系統(tǒng)發(fā)育樹(shù),能夠?qū)OB菌群進(jìn)行更為細(xì)致精確的分類(lèi).
本研究選擇具有代表性的大型市政污水處理廠,分析不同工藝的城市污水處理廠活性污泥中AOB和NOB菌群結(jié)構(gòu)和代謝活性的差異,揭示污水處理廠AOB和NOB菌群結(jié)構(gòu)、豐度與工藝運(yùn)行的相關(guān)性.
1.1城市污水處理廠的活性污泥樣品
表1 4個(gè)污水處理系統(tǒng)的進(jìn)出水水質(zhì)指標(biāo)及運(yùn)行參數(shù)Table 1 Influent and effluent characteristics and operation parameters of four WWTPs
實(shí)驗(yàn)所用的 5個(gè)污泥樣品均取自二沉池回流污泥,命名為: A、B、C、D-S(夏季樣品)、D-W(冬季樣品).A、B、C、D-S取樣時(shí)間為夏季7月,D-W為冬季2月份.A和B取自同一水廠不同工藝:A是倒置A2O工藝,缺氧段位于前端,優(yōu)先考慮脫氮效果,即:缺氧-厭氧-好氧方式運(yùn)行;B是AO工藝,采用缺氧-好氧技術(shù);C取自卡魯賽爾氧化溝工藝;D-S和D-W取自同一工藝的夏季和冬季,工藝為傳統(tǒng)的A2O工藝,即厭氧-缺氧-好氧.各個(gè)工藝進(jìn)出水水質(zhì)指標(biāo)及運(yùn)行參數(shù)見(jiàn)表1.
1.2DNA提取、PCR、克隆測(cè)序
用1×PBS清洗污泥樣品3次,14000×g離心2min(離心機(jī):MIKRO 22 R,德國(guó)Hettich),去除上清液,置于-20℃保存.采用試劑盒(Fast DNA Spin kit for soil, MP, USA)對(duì)DNA進(jìn)行提取,提取后的DNA 通過(guò) Nanodrop Spectrophotometer ND-1000 (Thermo Fisher Scientific, USA)測(cè)量核酸濃度及純度.
PCR 反應(yīng)采用試劑盒(Promega GoTaq Green Master Mix, USA),反應(yīng)體系為 25μL:12.5μL GoTaq Green Master Mix,1μL (10mmol/L)正向引物,1μL (10mmol/L)反向引物,0.5~2μL DNA模板,一定量的ddH2O.PCR程序見(jiàn)表2.
表2 PCR擴(kuò)增程序及特異性引物Table 2 Specific primers and PCR programs
amoA的 PCR產(chǎn)物經(jīng)瓊脂糖凝膠電泳(Agarose MS-6,TaKaRa,Japan)檢測(cè),為單一的目的條帶,切膠,用純化試劑盒(Agarose Gel DNA Purification Kit Ver. 2.0TaKaRa, Japan )進(jìn)行純化.純化后的 DNA用試劑盒(Zero Background TA Topoisomerase Cloning Kit, Clonesmarter, USA)進(jìn)行連接轉(zhuǎn)化,連接體系為 10μL,包括 1μL pCloneEZ-TOPO 載體,1μL 10×Enhancer, 0.5~8μLDNA,一定量的用 ddH2O.連接反應(yīng)完成后將產(chǎn)物加入到感受態(tài)細(xì)胞DH5a(中美泰和,國(guó)產(chǎn))中進(jìn)行轉(zhuǎn)化.每個(gè)樣品隨機(jī)挑出50個(gè)amoA基因的陽(yáng)性克隆子進(jìn)行測(cè)序,構(gòu)建克隆文庫(kù).
1.3克隆文庫(kù)建立和系統(tǒng)發(fā)育分析
構(gòu)建文庫(kù)的序列通過(guò)Mothur軟件按照97%相似度進(jìn)行OTU劃分,將每個(gè)OTU的代表序列與NCBI數(shù)據(jù)庫(kù)中利用BLAST下載的相似性最高最具代表性的菌株序列一起進(jìn)行比對(duì).采用MEGA5.0利用鄰接法(Neighbor joining method)進(jìn)行系統(tǒng)發(fā)育分析,通過(guò)自舉分析方法(Bootstrap)檢驗(yàn)系統(tǒng)發(fā)育樹(shù)各分支置信度,重復(fù)1000次.
1.4實(shí)時(shí)定量PCR (QPCR)
采用特異性引物對(duì)AOB amoA功能基因,隸屬于NOB菌群的Nitrospira和Nitrobacter以及全菌的 16S rRNA進(jìn)行 QPCR擴(kuò)增.反應(yīng)在Mx3005P實(shí)時(shí)定量PCR擴(kuò)增儀(Agilent Technologies,American)上進(jìn)行,采用試劑盒( SYBR Premix Ex Taq kit,TaKaRa,Japan)進(jìn)行反應(yīng),體系為25μL包括12.5μL的SYBR緩沖液,正反向引物各 1μL(10mmol/L),0.5μL ROX,DNA模板 2μL,一定量的 ddH2O.采用試劑盒(MiniBEST Plasmid Purification Kit Ver.4.0,TaKaRa,Japan)回收質(zhì)粒.
標(biāo)準(zhǔn)曲線的建立:用 NanoDrop ND-1000(Thermo,American)分光光度計(jì)測(cè)定回收質(zhì)粒的濃度(MiniBEST Plasmid Purification Kit Ver.4.0,TaKaRa,Japan).將純化后的質(zhì)粒以10倍的濃度梯度稀釋用作QPCR標(biāo)準(zhǔn)品.反應(yīng)在Mx3005P實(shí)時(shí)定量PCR擴(kuò)增儀(Agilent Technologies,American)上進(jìn)行,采用試劑盒(SYBR Premix Ex Taq kit,TaKaRa,Japan)進(jìn)行反應(yīng),體系為25μL.
1.5登錄號(hào)
本研究所測(cè)得的AOB序列上傳至GenBank數(shù)據(jù)庫(kù),AOB的序列登錄號(hào)為:KR018127-KR018368.
2.1AOB與NOB的實(shí)時(shí)熒光定量PCR分析
AOB、NOB (Nitrobacter和Nitrospira) 和總菌的實(shí)時(shí)熒光定量PCR的標(biāo)準(zhǔn)曲線的效率都在90%~110%之間,相關(guān)系數(shù)均大于0.998,特異性和擴(kuò)增效率都符合精確定量的要求.標(biāo)準(zhǔn)品分別如下:AOB 為 8.55×101~8.55×108拷貝,Nitrobacter為1.05×102~1.05×109拷貝, Nitrospira 為 1.31×102~1.31×109拷貝,總菌為 4.98×101~4.98×108拷貝.
表3為AOB和NOB絕對(duì)定量和相對(duì)定量的結(jié)果.5個(gè)污泥樣品中總菌豐度在一個(gè)數(shù)量級(jí)上,定量范圍為 1.10×1010(B)~9.27×1010(D-S)cells/gMLSS.硝化菌群在總菌中的百分含量大約為1%-7%.AOB種群數(shù)量AO工藝(B)最低,為8.56×106cells/gMLSS,其它 4個(gè)污泥樣品在1.20×107~4.46×107cells/gMLSS之間.倒置A2O工藝 (A樣品)AOB占總菌比例最高為0.3%,其他3個(gè)工藝在0.02%~0.08%之間.結(jié)合表1的進(jìn)出水水質(zhì)指標(biāo)可以看出倒置A2O工藝的出水氨氮濃度最低(0.72mg/L),硝化效果最好.因此,AOB在總菌中的相對(duì)含量決定了水廠的硝化效果. Nitrospira在 D-S中豐度最高,達(dá)到了 1.53× 109cells/gMLSS,占總菌比例為1.64%,在其他3個(gè)工藝中數(shù)量級(jí)均為 108cells/gMLSS,占總菌比例為 1.36%~6.12%之間.Nitrobacter除了在樣品D-S中豐度為零外,其余在 1.69×107~6.78× 107cells/gMLSS之間,占總菌比例為 0.15%~0.39%之間.D-W中的總菌、AOB、Nitrospira豐度較D-S均有所降低,但Nitrobacter卻由0增加到 1.45×107cells/gMLSS.冬季樣品(D-W)中硝化菌群豐度和百分含量的降低可能是很多城市污水處理廠冬季脫氮效果變差的主要原因.
表3 總菌、AOB和NOB的實(shí)時(shí)定量結(jié)果Table 3 QPCR results of bacteria, AOB and NOB
本研究AOB amoA基因定量分析的數(shù)量級(jí)大致在107~108cells/gMLSS之間,與已有的多數(shù)研究結(jié)果基本一致[7,24-25],但略低于個(gè)別研究[26-27].對(duì) NOB定量結(jié)果顯示本研究中的城市污水處理系統(tǒng)中Nitrospira是NOB菌群中的優(yōu)勢(shì)菌屬,豐度要比Nitrobacter高出一個(gè)數(shù)量級(jí).關(guān)于AOB和NOB豐度,有的研究認(rèn)為AOB和NOB在同一個(gè)數(shù)量級(jí)[16],也有研究發(fā)現(xiàn)AOB豐度比NOB低一個(gè)數(shù)量級(jí)[28].從表3可以看出,本研究的水廠中 AOB種群豐度要比 NOB (Nitrobacter和 Nitrospira)低一個(gè)數(shù)量級(jí).除了樣品 D-S(A2O工藝),其他工藝的 Nitrospira和Nitrobacter均存在,且相差一個(gè)數(shù)量級(jí),只有樣品D-S中Nitrobacter含量為0.結(jié)合表1中D-S出水的和,計(jì)算出亞硝積累率為24%,由此推斷采用A2O工藝的D水廠在夏季可能出現(xiàn)了短程硝化現(xiàn)象,并且在向短程硝化轉(zhuǎn)化的過(guò)程中,Nitrobacter先于 Nitrospira被淘汰.結(jié)合表 1的運(yùn)行參數(shù)發(fā)現(xiàn)D-S的溶解氧要明顯低于其他3個(gè)工藝,低溶解氧可能是造成短程硝化的原因之一.進(jìn)入冬季,由于水溫降低,短程硝化向全程硝化轉(zhuǎn)化,Nitrobacter又出現(xiàn)在D-W樣品中.
2.2基于OTU的AOB系統(tǒng)發(fā)育分析
5個(gè)amoA基因克隆文庫(kù)中共得到242條amoA序列,按照 97%的相似度劃分為 21個(gè)OTUs.5個(gè)污泥樣品構(gòu)建的 amoA基因克隆文庫(kù)的Good覆蓋率、Chao1豐富度估計(jì)、OTU值與估計(jì)的 Chao1值之比、香農(nóng)指數(shù)見(jiàn)表4. 5個(gè)污泥樣品的amoA基因的OTU分布如圖1所示.
表4 amoA基因克隆文庫(kù)的序列多樣性及覆蓋率Table 4 Sequences diversity and Good coverage of amoA gene clone libraries
圖1 5個(gè)污泥樣品中AOB amoA基因的OTU分布Fig.1 Relative distribution of OTUs based on amoA genes in 5samples
如表 4所示,Good覆蓋率最低的是 DS74.47%,但其OTU值與估計(jì)的Chao1值之比達(dá)到89%.OTU值與估計(jì)的Chao1值之比最低的是樣品C75%,其Good覆蓋率達(dá)到了93.88%.這兩組數(shù)據(jù)說(shuō)明這2個(gè)amoA克隆文庫(kù)可以代表2個(gè)污水處理廠AOB的群落組成.樣品A和B的 Good覆蓋率均在 90%以上,OTU值與估計(jì)的Chao1值之比均為 100%.與D-S比較,D-W的OTU值與估計(jì)的Chao1值為87.76%,明顯高于D-S(74.47%),數(shù)據(jù)可信度更高.高景峰等[29]對(duì) 10個(gè)污水處理系統(tǒng)中的 AOB菌群進(jìn)行研究,Good覆蓋率在 65.4%~100%之間,說(shuō)明本研究建立的amoA克隆文庫(kù)可以代表每個(gè)污水處理廠中AOB群落組成.表4計(jì)算了5個(gè)amoA基因克隆文庫(kù)中的香農(nóng)指數(shù),根據(jù)表中結(jié)果發(fā)現(xiàn)C樣品中amoA基因多樣性要低于其他樣品,香農(nóng)指數(shù)只有0.2.另外4個(gè)樣品的香農(nóng)指數(shù)在0.67~2.16之間,對(duì)比其他研究可說(shuō)明本研究的 5個(gè)樣品的AOB 群落多樣性在正常范圍之內(nèi)[24-25,29],其中D-S多樣性最豐富.從圖1和表4可以看出,樣品C僅有3個(gè)OTU,且有2個(gè)OTU各僅含有1條序列,意味著樣品C中的AOB多樣性最不豐富.雖然樣品A和B所含OTUs數(shù)分別為4和3,但從圖1可以看出,每個(gè)樣品所含序列較均勻的分布在各個(gè) OTU,因此具有一定的生物多樣性水平;樣品D-S含有10個(gè)OTUs,多樣性水平最高,與表4的香農(nóng)指數(shù)分析一致. D-W共有6個(gè)OTU,其中有4個(gè)OTU與D-S是重復(fù)覆蓋的,說(shuō)明雖然污水處理系統(tǒng)溫度發(fā)生了變化,OTU分布會(huì)有一定變化,但其優(yōu)勢(shì)OTU得以保留.
圖2 基于amoA基因的AOB系統(tǒng)發(fā)育樹(shù)Fig.2 NJ phylogenetic tree of AOB group based on amoA genes
利用21個(gè)OTUs中的代表序列建立的NJ系統(tǒng)發(fā)育樹(shù)如圖2所示.從圖2可以看出有11個(gè)OTU屬于N. oligotropha,有 6個(gè)OTU 屬于N. europaea cluster,有2個(gè)OTU屬于Nitrosomonaslike cluster,有2個(gè)OTU (僅包括3條序列)屬于N. marina 和N. aestuarii.其中OTU1、OTU2、OTU3分別含有58、47、28條序列,是AOB菌群的優(yōu)勢(shì)OTU,屬于N. oligotropha和Nitrosomonas-like cluster.
2.3AOB的系統(tǒng)發(fā)育樹(shù)分析
5個(gè)污泥樣品中AOB種群分布以及相對(duì)含量如圖3所示.基于 amoA基因的系統(tǒng)發(fā)育分析結(jié)果(圖2)顯示所有的序列屬于Nitrosomonas,沒(méi)有發(fā)現(xiàn)Nitrosospira cluster.AOB的分布從多到少分 別 為 :N. oligotropha、 Nitrosomonas-like cluster、N. europaea cluster、N.matina and N.aestuarii cluster.其中Nitrosomonas oligotropha cluster和Nitrosomonas-like cluster是AOB種群中的兩大優(yōu)勢(shì)菌群,分別占克隆文庫(kù)的60.08%和29.63%,與已有研究結(jié)果一致[2,30].從圖 3可以看出,除了樣品B中N.oligotropha cluster僅為2%,其余4個(gè)樣品都含有N.oligotropha cluster且含量豐富.其中樣品C所有的 AOB amoA序列以及樣品D-W中97.96%的AOB amoA序列都屬于N.oligotropha cluster.其 他2個(gè) 樣 品 中N.oligotropha cluster占克隆文庫(kù)的比例在40.43%~60.42%之間.Nitrosomonas-like cluster分布在3個(gè)樣品中,并且在樣品B中所占比例達(dá)到98%,在樣品A和D-S中所占比例分別為39.58% 和 8.51%,在樣品 C和 D-W 中未檢測(cè)到.N. europaea cluster占克隆文庫(kù)的 9.05%,只在采用A2O工藝的樣品D-S和D-W中發(fā)現(xiàn),這可能與該A2O工藝進(jìn)水氨氮濃度相對(duì)較高有關(guān).而且N. europaea cluster在夏冬兩季樣品中的百分含量發(fā)生明顯的變化,D-S中 N. europaea cluster占AOB總量的 44.68%,是優(yōu)勢(shì) AOB菌屬,但在D-W樣品中降到了2.04%.在其他3個(gè)工藝中并未檢測(cè)到N. europaea cluster.另外,在D-S樣品中發(fā)現(xiàn)了少量的N.matina和N.aestuarii cluster.
比較樣品D-S和D-W的AOB群落結(jié)構(gòu),一個(gè)明顯的變化特征是進(jìn)入冬季后,原本處于優(yōu)勢(shì)的N. europaea cluster明顯減少,而N. oligotropha cluster顯著增多,達(dá)到AOB總量的98%.因此,冬季水廠硝化效果變差的原因除了與硝化菌群豐度降低有關(guān),還可能與上述AOB群落結(jié)構(gòu)的變化有關(guān).如前所述,樣品D-S所在的污水處理廠夏季出現(xiàn)較高的亞硝酸鹽積累率,這可能與夏季樣品中N. europaea cluster的優(yōu)勢(shì)地位有關(guān);冬季隨著短程現(xiàn)象的消失,N. europaea cluster含量減少.有研究認(rèn)為N. europaea 有優(yōu)先利用亞硝酸鹽作為電子受體的能力[30-31],經(jīng)過(guò)低 DO馴化的短程硝化活性污泥中以N. europaea為主[32].從表1可以看出,4種工藝中,D-S樣品所在的A2O工藝 DO值最低,僅為 1.8mg/L,遠(yuǎn)低于其他 3種工藝(3~6mg/L).而且只有A2O工藝(D-S和D-W)中出現(xiàn)了N. europaea,并在樣品D-S克隆文庫(kù)中所占比例達(dá)到44.69%.冬季短程硝化逐漸變?yōu)槿滔趸?,隸屬于NOB的Nitrobacter重新出現(xiàn),同時(shí)系統(tǒng)內(nèi)N. europaea cluster大量消失,在樣品D-W中僅占AOB總量的2%.由此可以推測(cè)低DO運(yùn)行使N. europaea成為優(yōu)勢(shì)AOB可能是D-S出現(xiàn)短程硝化的原因.
圖3 5個(gè)污泥樣品中AOB種群分布以及種群相對(duì)含量Fig.3 Phylogenetic distribution and relative abundance of AOB groups in 5samples
3.1本研究的城市污水處理廠活性污泥樣品中優(yōu)勢(shì) AOB和 NOB分別為 Nitrosomonas和Nitrospira.硝化菌群達(dá)到總菌群的 1%~7%,其豐度,尤其是 AOB在總菌中的相對(duì)含量決定了系統(tǒng)的硝化效果.AOB豐度比NOB豐度低一個(gè)數(shù)量級(jí).NOB菌群中的Nitrospira豐度比Nitrobacter豐度高一個(gè)數(shù)量級(jí),是明顯的優(yōu)勢(shì)NOB.
3.2本研究的活性污泥樣品中的 AOB全部隸屬于Nitrosomonas,其中N. oligotropha cluster是AOB 中的優(yōu)勢(shì)菌屬,占克隆文庫(kù)的 60%. Nitrosomonas-like cluster次之,占克隆文庫(kù)的30%. N. europaea cluster只出現(xiàn)在A2O工藝中. 3.3 A2O工藝冬季AOB和Nitrospira豐度的降低是導(dǎo)致冬季生物脫氮效果變差的原因.A2O工藝夏季污泥樣品中N. europaea cluster占該克隆文庫(kù)的 44.7%,是優(yōu)勢(shì) AOB.低 DO運(yùn)行使 N. europaea cluster成為優(yōu)勢(shì)AOB是A2O工藝夏季出現(xiàn)較高亞硝酸鹽積累率的主要原因.
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Community structures and population dynamics of nitrifying bacteria in activated sludges of wastewater treatmentplants.
ZENG Wei1*, ZHANG Li-min1, WANG An-qi1, ZHANG Jie1, PENG Yong-zhen1, DUAN Jun-ling2(1.College of Environmental and Energy Engineering, Beijing University of Technology, Beijing 100124, China;2.Beijing Drainage Group Limited Liability Company, Beijing 100044, China).
China Environment Science, 2015,35(11):3257~3265
Community structures and population dynamics of nitrifying bacteria determine biological nitrogen removal from municipal wastewater. The population structures and dynamics of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) in four full-scale wastewater treatment plants (WWTPs) were investigated in this study. Quantitative real-time PCR results showed that the abundance of AOB was in a range of 8.56×106~4.46×107cells/gMLSS,while NOB was varying in 3.37×108~1.53×109cells/gMLSS. In each process Nitrospira was the dominant species of NOB. Nitrospira abundance was obviously higher than Nitrobacter, accounting for 88% of total NOB. In the A2O process the abundances of AOB and Nitrospira in winter were less than those in summer, leading to decline of biological nitrogen removal. The phylogenetic analysis of AOB amoA genes indicated that all the sequences were affiliated with genera Nitrosomonas, among which Nitrosomonas oligotropha cluster was the dominant species, accounting for 60% of the clone libraries. The pre-dominant AOB were Nitrosomonas-like cluster and Nitrosomonas europaea cluster, accounting for 29.6% and 9.1% of the clone libraries, respectively. N. europaea cluster was only found in A2O process, and reached 44.7% of total AOB in summer sample, which was a main reason causing high nitrite accumulation during summer operation of A2O process. The outcomes verified that the dominant AOB and NOB in WWTPs was Nitrosomonas and Nitrospira, respectively. Nitrifying bacteria accounted for 1%~7% of total bacteria. The abundances, relative distributions and community structures of nitrifying bacteria significantly influence the performance of biological nitrogen removal.
ammonium oxidizing bacteria (AOB);nitrite oxidizing bacteria (NOB);wastewater treatment systems (WWTPs);real-time quantitative PCR (QPCR);amoA genes
X703
A
1000-6923(2015)11-3257-09
2015-04-03
國(guó)家自然科學(xué)基金項(xiàng)目(51278007,51578016);教育部新世紀(jì)優(yōu)秀人才支持計(jì)劃項(xiàng)目(NCET-11-0891)
* 責(zé)任作者, 教授, zengwei_1@263.net
曾 薇(1974-),女,黑龍江省哈爾濱人,教授,博士,主要從事污水生物處理研究.發(fā)表論文70余篇.