李 冬,何永平,張肖靜,梁瑜海,張玉龍,范 丹(.北京工業(yè)大學(xué)水質(zhì)科學(xué)與水環(huán)境恢復(fù)工程北京市重點(diǎn)實(shí)驗(yàn)室,北京 004；.哈爾濱工業(yè)大學(xué)城市水資源與水環(huán)境國(guó)家重點(diǎn)實(shí)驗(yàn)室,黑龍江 哈爾濱 50090)

MBR系統(tǒng)CANON工藝的快速啟動(dòng)及微生物種群特征

李 冬1*,何永平1,張肖靜2,梁瑜海1,張玉龍1,范 丹1(1.北京工業(yè)大學(xué)水質(zhì)科學(xué)與水環(huán)境恢復(fù)工程北京市重點(diǎn)實(shí)驗(yàn)室,北京 100124；2.哈爾濱工業(yè)大學(xué)城市水資源與水環(huán)境國(guó)家重點(diǎn)實(shí)驗(yàn)室,黑龍江 哈爾濱 150090)

為了考察CANON工藝的快速啟動(dòng)策略及功能微生物的種群特征,在常溫MBR反應(yīng)器內(nèi)接種普通活性污泥后間歇運(yùn)行.啟動(dòng)策略為以調(diào)控曝氣時(shí)間和曝氣量作為主要方法,首先在限氧條件下啟動(dòng)亞硝化,之后進(jìn)一步降低DO啟動(dòng)CANON工藝.在CANON工藝啟動(dòng)成功后,通過調(diào)整曝氣時(shí)間和無機(jī)碳源濃度提高了總氮去除負(fù)荷,并采用PCR-DGGE技術(shù)分析了穩(wěn)定運(yùn)行的CANON工藝內(nèi)功能微生物的種群特征.結(jié)果表明,CANON工藝經(jīng)36d成功啟動(dòng),NH4+-N去除率和總氮去除率最終穩(wěn)定在99%和84%左右,氮去除負(fù)荷達(dá)到0.41kg/(m3·d).DGGE測(cè)序結(jié)果表明,Nitrosomonas和Candidatus Kuenenia stuttgartiensis是反應(yīng)器內(nèi)的優(yōu)勢(shì)菌種,兩種微生物協(xié)同作用,共同在MBR內(nèi)完成了高效的自養(yǎng)脫氮.

MBR；普通活性污泥；CANON；啟動(dòng)；PCR-DGGE；微生物

基于亞硝化的全程自養(yǎng)脫氮(CANON)工藝具有脫氮效率高、耗氧量低、無需外加碳源且污泥產(chǎn)量低、經(jīng)濟(jì)環(huán)保等優(yōu)點(diǎn),是近年來受到廣泛關(guān)注的一種新型生物脫氮工藝.在CANON工藝中,氨氧化菌(AOB)在限氧條件下,以氧作為電子受體將部分NH4+-N氧化為NO2--N,厭氧氨氧化(Anammox)菌以AOB產(chǎn)生的NO2--N為電子受體,與剩余NH4+-N反應(yīng),生成N2并釋放,達(dá)到脫氮目的.

現(xiàn)階段對(duì)CANON工藝的研究多在生物濾柱[1]或SBR[2-3]中進(jìn)行.但生物濾柱多采用火山巖等硬性填料,造成其堵塞問題一直無法有效解決,而SBR污泥易流失,導(dǎo)致生物量減少,去除負(fù)荷低,且由于AOB和Anammox菌均是自養(yǎng)菌,生長(zhǎng)緩慢,尤其是Anammox菌,倍增時(shí)間為11d[4],導(dǎo)致CANON工藝啟動(dòng)時(shí)間長(zhǎng).因此尋找合適的反應(yīng)器類型對(duì)CANON工藝的啟動(dòng)及穩(wěn)定運(yùn)行意義重大.膜生物反應(yīng)器(MBR)依靠膜滲透原理,將所有微生物截留在反應(yīng)器內(nèi)部,可防止污泥流失,提高反應(yīng)器內(nèi)生物濃度,特別適用于AOB菌和Anammox菌這類生長(zhǎng)緩慢,倍增時(shí)間長(zhǎng)的微生物生長(zhǎng).因此,若將MBR應(yīng)用于CANON工藝可以有效解決污泥流失,處理負(fù)荷低等問題,從而加快啟動(dòng)時(shí)間.

目前,國(guó)內(nèi)外關(guān)于CANON工藝的接種污泥多采用具有亞硝化或厭氧氨氧化活性的特種污泥[5-8],而這些污泥相對(duì)稀缺,不易獲得且價(jià)格昂貴,相比之下普通活性污泥分布廣泛,容易得到且價(jià)格低廉.可見,若能以普通活性污泥為種泥來啟動(dòng)CANON,可以說是為該工藝的啟動(dòng)提供了既方便又經(jīng)濟(jì)的污泥源.

因此,本研究在常溫條件下,接種普通活性污泥,采用MBR反應(yīng)器,以間歇運(yùn)行方式啟動(dòng)CANON,同時(shí)通過PCR-DGGE、克隆等分子生物學(xué)技術(shù)研究反應(yīng)器內(nèi)功能微生物的種群特征,以期為縮短CANON工藝的啟動(dòng)時(shí)間和微生物特性提供理論依據(jù).

1 材料與方法

1.1 試驗(yàn)裝置

試驗(yàn)裝置采用有機(jī)玻璃制成的圓柱形MBR反應(yīng)器,如圖1所示.圓柱內(nèi)徑13cm,高度40cm,有效容積3L,內(nèi)置聚偏氟乙烯(PVDF)中空纖維膜組件,膜孔徑為0.1μm,有效膜面積為0.2m2,膜通量36L/h.反應(yīng)器底部設(shè)置曝氣環(huán),采用鼓風(fēng)曝氣,曝氣量由轉(zhuǎn)子流量計(jì)控制,中間設(shè)有攪拌機(jī),用于基質(zhì)和O2均勻擴(kuò)散,外部設(shè)置水浴套筒,由溫度控制儀控制反應(yīng)器內(nèi)溫度.

1.2 接種污泥

試驗(yàn)接種污泥取自北京市某污水處理廠普通活性污泥.接種前,污泥先經(jīng)自來水和蒸餾水各清洗3遍,去除其中的雜質(zhì),之后接種至MBR反應(yīng)器內(nèi).接種時(shí)MLSS為12.9g/L,MLVSS為10.6g/L,接種量為1L.

圖1 試驗(yàn)裝置示意Fig.1 Schematic of the experimental reactor

1.3 試驗(yàn)用水與試驗(yàn)方法

試驗(yàn)用水采用人工配水,分別以(NH4)2SO4和NaHCO3作為NH4+-N和堿度的來源,NH4+-N濃度和堿度固定不變.進(jìn)水中額外添加MgSO4·5H2O、CaCl2、KH2PO4和營(yíng)養(yǎng)液Ⅰ、Ⅱ作為營(yíng)養(yǎng)物質(zhì),營(yíng)養(yǎng)液Ⅰ包括EDTA 5000mg/L和FeSO45000mg/L．營(yíng)養(yǎng)液Ⅱ(mg/L)包括EDTA 15000、ZnSO4·7H2O 430、CoCl2·6H2O 240、MnCl2·4H2O 990、CuSO4·5H2O 250、Na2MoO4· 2H2O 220、NiCl2·6H2O 190、Na2SeO4·10H2O 210和H3BO414.試驗(yàn)用水水質(zhì)見表1.

表1 試驗(yàn)用水水質(zhì)Table 1 Key water quality of the influent

試驗(yàn)在常溫下(23～25℃)采用間歇運(yùn)行方式,每個(gè)周期包括:瞬時(shí)進(jìn)水,曝氣反應(yīng),曝氣完成后,膜抽吸出水.換水比83.3%,一個(gè)周期完成后進(jìn)入下一個(gè)周期.CANON工藝的啟動(dòng)采用先啟動(dòng)亞硝化富集AOB,再限氧富集Anammox,啟動(dòng)CANON.試驗(yàn)主要分為3個(gè)階段:階段Ⅰ,亞硝化啟動(dòng);階段Ⅱ, CANON工藝啟動(dòng);階段Ⅲ,CANON工藝負(fù)荷提高.不同階段主要運(yùn)行條件見表2.

表2 不同階段主要運(yùn)行條件Table 2 Operation conditions at different stages

1.4 分析項(xiàng)目與方法

NH4+-N、NO2--N、NO3--N、MLSS、MLVSS等指標(biāo)均采用國(guó)家規(guī)定的標(biāo)準(zhǔn)方法測(cè)定[9]; NO2--N積累率(NAR)可按式(1)計(jì)算;DO、pH值及溫度測(cè)定分別采用EUTECH DO2000PPG多功能溶解氧在線測(cè)定儀,WTW pH296 型在線測(cè)定儀.

式中:[NO2--N]eff為出水NO2--N濃度; [NO3--N]eff為出水NO3--N濃度.

1.5 DNA提取,PCR-DGGE,克隆和測(cè)序

1.5.1 基因組DNA的提取 在CANON工藝的穩(wěn)定期,從MBR反應(yīng)器內(nèi)采集混合液.用UNIQ-10柱式細(xì)菌基因組DNA抽提試劑盒(上海生工)提取基因組DNA,具體操作按說明書進(jìn)行.所提取的基因組DNA用0.8wt%(質(zhì)量分?jǐn)?shù))的瓊脂糖凝膠電泳檢測(cè),以備PCR用.

1.5.2 PCR擴(kuò)增及DGGE電泳 采用巢式PCR方法,分別擴(kuò)增β-proteobacteria菌門的AOB, Planctomycetales菌門的Anammox菌.為擴(kuò)增AOB的16S rDNA,第一輪擴(kuò)增使用CTO189fA/B和CTO189fC混合引物(體積比2:1)作為正向引物,反向引物采用CTO654r.之后以第一輪PCR擴(kuò)增產(chǎn)物為模板,使用通用引物對(duì)F338(帶GC夾)/R518,進(jìn)行第二輪PCR擴(kuò)增.對(duì)于Anammox菌的特異性片段的擴(kuò)增,第一輪先以引物對(duì)Pla46F/630R進(jìn)行浮霉球菌擴(kuò)增.之后以第一輪PCR擴(kuò)增產(chǎn)物為模板,使用引物對(duì)Amx368f(帶GC夾)/Amx820r,進(jìn)行第二輪PCR擴(kuò)增.PCR 反應(yīng)體系為25μL,其中包含2.5μL 10×Ex Taq buffer (Mg2+Plus ),2.0μL DNTP,1.0μL BSA,1.0μL引物,0.125μL, TaKaRa Ex Taq酶,模板DNA約1.0ng,用無菌水補(bǔ)齊至25μL.引物堿基序列及反應(yīng)條件見表3.

表3 PCR常用引物對(duì)應(yīng)程序Table 3 Corresponding programs of commonly used PCR primers

PCR擴(kuò)增產(chǎn)物用1.5wt%的瓊脂糖凝膠進(jìn)行電泳檢測(cè).采用Sanprep柱式DNA膠回收試劑盒(上海生工)進(jìn)行PCR 產(chǎn)物的純化回收,具體操作按說明書進(jìn)行.對(duì)PCR產(chǎn)物進(jìn)行DGGE 分析:聚丙烯酰胺質(zhì)量分?jǐn)?shù)8wt%,變性梯度為30%～60%,電壓120V,電泳時(shí)間5h,電泳在Dcode Universal MutationDetection System儀器上進(jìn)行.電泳結(jié)束后按Bassam等[14]的方法對(duì)凝膠進(jìn)行銀染,并對(duì)凝膠拍照.

1.5.3 克隆和測(cè)序 切取DGGE 圖譜中的目的條帶溶于150μL T E(pH 8.0)溶液中,4℃過夜,以此為模板,以不含GC夾的引物進(jìn)行PCR擴(kuò)增,并對(duì)PCR 產(chǎn)物進(jìn)行純化.按照pMD19-T plasmid vector system說明書進(jìn)行基因片段與載體的連接后,轉(zhuǎn)化到大腸桿菌DH5α感受態(tài)細(xì)胞中,通過藍(lán)白斑法篩選陽性克隆子,過夜培養(yǎng)后并進(jìn)行測(cè)序.采用BLAST對(duì)測(cè)序結(jié)果和基因庫(kù)中已知序列進(jìn)行相似性分析.并將所獲得的序列提交至Gentbank,授權(quán)序列號(hào)為:KF171345～KF171352 (AOB),KF442618～KF442619(Anammox).

2 結(jié)果與討論

2.1 亞硝化的啟動(dòng)及穩(wěn)定運(yùn)行

亞硝化的實(shí)現(xiàn)是成功啟動(dòng)CANON的基礎(chǔ),該階段的目的是富集AOB并抑制NOB的活性.此階段,以限氧方式啟動(dòng)亞硝化,初始進(jìn)水NH4+-N濃度為200mg/L,曝氣量為0.3L/min, DO為0.3mg/L, 曝氣時(shí)間為5h.由圖2可見,在第1d,反應(yīng)器內(nèi)就有NO2--N積累,這說明在限氧反應(yīng)器內(nèi),NOB的活性就已經(jīng)受到抑制,不能及時(shí)氧化NO2--N,從而造成了NO2--N積累,NAR在30%左右.在前6d, NAR整體呈上升趨勢(shì),第6d NAR達(dá)到50%左右,認(rèn)為亞硝化啟動(dòng)成功.但是在第5d,第6d, NH4+-N去除率(ARR)有所波動(dòng)且較低,所以第7d曝氣時(shí)間由原來的5h延長(zhǎng)為7h,使更多的NH4+-N被氧化,提高ARR.曝氣時(shí)間延長(zhǎng)后,DO為0.3mg/L,ARR提高并持續(xù)上升,到第25d達(dá)到70%左右,此后至第30d穩(wěn)定在75%左右.NAR也一直升高,最終穩(wěn)定在90%左右,總氮去除率(TNR)維持在2%(總氮損失造成).因?yàn)閱?dòng)亞硝化的目的是為了啟動(dòng)CANON,因此NAR無需上升并穩(wěn)定在95%以上.而且,ARR也沒有必要達(dá)到100%,反應(yīng)器內(nèi)留有NH4+-N既可以抑制NOB[15]還可以為Anammox菌快速提供基質(zhì).

亞硝化快速啟動(dòng)并穩(wěn)定運(yùn)行的主要原因是:①DO控制得當(dāng).AOB氧飽和常數(shù)為(0.2～0.4)mg/L,NOB 為(1.2～1.5)mg/L[16],本研究中DO控制在0.3mg/L,有效的抑制了NOB的活性.②殘留NH4+-N的影響.反應(yīng)器內(nèi)一直殘留部分NH4+-N,也可以抑制NOB的活性.從而使AOB快速富集,成功啟動(dòng)了亞硝化.

圖2 階段Ⅰ反應(yīng)器運(yùn)行性能Fig.2 Performance of the reactor during stageⅠ

2.2 CANON工藝的啟動(dòng)及穩(wěn)定運(yùn)行

反應(yīng)器運(yùn)行至第31d,降低DO,啟動(dòng)CANON.曝氣量由0.3L/min下降到0.2L/min, DO為0.15～0.2mg/L,曝氣時(shí)間延長(zhǎng)為9h.從圖3可知,此階段ARR在經(jīng)過3d的平緩期后持續(xù)上升并穩(wěn)定在80%左右,NAR下降并穩(wěn)定在73%左右,反應(yīng)器內(nèi)開始出現(xiàn)明顯的總氮損失現(xiàn)象,TNR和氮去除負(fù)荷(NRR)最高分別為35%和0.22kg/(m3·d).由圖3可知,出水NO2--N濃度急劇下降,而出水NO3--N濃度增長(zhǎng)卻較緩慢,說明減少的NO2--N并沒有全部被NOB氧化為NO3--N,并且出水NH4+-N濃度不斷減小,推測(cè)反應(yīng)器內(nèi)發(fā)生了厭氧氨氧化反應(yīng),Anammox菌將NH4+-N和NO2--N轉(zhuǎn)化為少量NO3--N和N2,導(dǎo)致出水NH4+-N和NO2--N濃度同時(shí)下降,出水NO3--N濃度輕微上升.此外,總氮開始有損失,TNR不斷上升,而配水中不含有機(jī)物,總氮損失不可能是由反硝化細(xì)菌造成的,并且ΔNO3--N/ΔNH4+-N已接近于0.11,更進(jìn)一步論證了反應(yīng)器內(nèi)發(fā)生了Anammox反應(yīng),建立了CANON工藝.同時(shí)2.4節(jié)中DGGE圖譜結(jié)果也證實(shí)此階段AOB菌和Anammox菌共存于反應(yīng)器內(nèi).降低DO后,僅經(jīng)過1d就出現(xiàn)了氮去除現(xiàn)象,說明前期活性污泥中含有Anammox菌,之所以沒有表現(xiàn)活性,是受外界條件(基質(zhì)、DO等)和自身的影響.Strous等[17]的研究表明Anammox菌細(xì)胞濃度>1010～1011個(gè)/mL時(shí)活性才能顯現(xiàn)出來.本研究中,調(diào)節(jié)DO后,Anammox菌立即顯現(xiàn)出來.同時(shí)ARR和TNR在31～34d較后面幾天增勢(shì)緩慢,說明CANON啟動(dòng)初期,Anammox菌活性較低,隨著反應(yīng)的進(jìn)行,Anammox菌逐漸適應(yīng),后期活性不斷提高.在第36d時(shí),NRR達(dá)到0.1kg/(m3·d)以上,認(rèn)為CANON工藝啟動(dòng)成功,圖中顯示,本研究在常溫下僅經(jīng)歷36d就完成了MBR內(nèi)CANON工藝的啟動(dòng).相比于國(guó)內(nèi)外其他研究,本研究?jī)H接種普通活性污泥,就能在短時(shí)間成功啟動(dòng)CANON,說明利用MBR反應(yīng)器可以縮短CANON工藝的啟動(dòng)時(shí)間(表4).

CANON工藝快速啟動(dòng)的原因分析如下:①反應(yīng)器內(nèi)較低的DO,在保證AOB需氧量的前提下,低DO會(huì)有效抑制NOB,并且對(duì)Anammox菌的影響較小,有利于富集AOB和Anammox菌;②較高的NH4+-N濃度有利于AOB和Anammox菌的生長(zhǎng),同時(shí)亞硝化啟動(dòng)后,殘存的NH4+-N及生成的NO2--N也會(huì)誘導(dǎo)Anammox菌的活性;③由于膜的抽吸作用,使膜絲表面附著少量活性污泥逐漸形成泥餅層且由于氧傳質(zhì)限制,在泥餅層內(nèi)部會(huì)形成局部缺氧微環(huán)境,有利于Anammox菌的生長(zhǎng)繁殖;④因MBR反應(yīng)器能有效的進(jìn)行固液分離,將全部污泥截留在反應(yīng)器內(nèi),沒有污泥流失,從而使反應(yīng)器內(nèi)微生物數(shù)量不斷增多,特別適用于AOB和Anammox菌的生長(zhǎng)繁殖.這一點(diǎn)是其他反應(yīng)器不具備的,也是最關(guān)鍵的一點(diǎn).

圖3 階段Ⅱ反應(yīng)器運(yùn)行性能Fig.3 Performance of the reactor during stage Ⅱ

2.3 CANON工藝負(fù)荷提高

階段Ⅲ為CANON工藝負(fù)荷提高階段.階段Ⅱ完成后,NH4+-N和NO2--N還有一定量的剩余,為進(jìn)一步降低出水NH4+-N濃度,提高TNR,將曝氣時(shí)間延長(zhǎng)為10h.從圖4可以看出,ARR和TNR迅速上升,直至第55d分別為99.78%和69.04%,這說明延長(zhǎng)曝氣時(shí)間,能夠使更多的NH4+-N被氧化,同時(shí)Anammox菌活性也沒有受到抑制.此時(shí)出水中NH4+-N濃度幾乎為0,但還殘留NO2--N,說明Anammox菌活性有待提高. Yang等[22]通過研究證明在厭氧氨氧化工藝中添加足夠的無機(jī)碳濃度可以提高NRR.為此將堿度由1600提高到2000mg/L,并將曝氣時(shí)間縮短至9h,減少AOB氧化NH4+-N的量,提高Anammox菌的活性.之后出水NH4+-N濃度雖有短暫升高但后來一直降低, 第59d再次降低到0左右并維持穩(wěn)定.出水NO2--N呈下降趨勢(shì),最終接近0.第56至76d,ARR和TNR分別穩(wěn)定在99%、84%左右, ΔNO3--N/ΔNH4+-N及ΔNO3--N/ΔTN分別穩(wěn)定在0.12和0.14,接近于0.11和0.127[23].氮去除負(fù)荷最高可達(dá)0.41kg/(m3·d),平均氮去除負(fù)荷為0.38kg/(m3·d).表明MBR反應(yīng)器內(nèi)高效穩(wěn)定的CANON工藝已經(jīng)實(shí)現(xiàn).

表4 CANON反應(yīng)器啟動(dòng)時(shí)間總結(jié)Table 4 Summary of the start-up time for CANON reactors

圖4 階段Ⅲ反應(yīng)器運(yùn)行性能Fig.4 Performance of the reactor during stage Ⅲ

2.4 DGGE分析

圖5 CANON工藝穩(wěn)定期AOB和Anammox的DGGE圖譜Fig.5 DGGE profiles of AOB and Anammox in stable phase of CANON process

由圖5(a)可見,AOB有8個(gè)條帶(1～8),說明反應(yīng)器內(nèi)AOB種類還是較多的.對(duì)這8條主要條帶進(jìn)行切割、溶解、回收、擴(kuò)增,通過對(duì)DNA序列測(cè)序分析表明所有的AOB均屬于βproteobacteria(表5),它們與Nitrosomonas的相似度都在97%及以上,表明Nitrosomonas是反應(yīng)器內(nèi)的優(yōu)勢(shì)菌種,且較穩(wěn)定,這與Thomas F. Ducey等[24]的研究是一致的.并且Liu等[6]認(rèn)為Nitrosomonas相對(duì)于Nitrosospira更適于在CANON反應(yīng)器中生長(zhǎng),而Liu等[25]也發(fā)現(xiàn)Nitrosomonas是許多水生態(tài)系統(tǒng)中最常見的氨氧化菌類型.

圖5(b)顯示, Anammox有2個(gè)條帶,均屬于Planctomycetia(表5),其中條帶9與Candidatus Kuenenia stuttgartiensis的相似度高達(dá)99%,條帶10與anaerobic ammonium-oχidizing planctomycete的相似度也高達(dá)99%,這與Jing[26]等的研究結(jié)果是一致的.研究表明Candidatus Kuenenia stuttgartiensis是一種存在于淡水環(huán)境中的Anammox[27],而且在污水脫氮系統(tǒng)中常見[28]. DGGE圖譜顯示,本研究中Nitrosomonas和Candidatus Kuenenia stuttgartiensis 是CANON反應(yīng)器內(nèi)的優(yōu)勢(shì)菌種,共同完成脫氮過程.

表5 DGGE條帶上AOB和Anammox的DNA序列比對(duì)結(jié)果Table 5 Results of sequence allignment between DNA sequences of AOB and Anammox from DGGE band

3 結(jié)論

3.1 首先,在限氧條件下啟動(dòng)亞硝化,控制DO為0.3mg/L,經(jīng)過6d,NO2--N積累率達(dá)到50%左右,亞硝化啟動(dòng)成功.之后,降低DO至0.15～0.2mg/L,由亞硝化階段向CANON工藝轉(zhuǎn)變,經(jīng)歷36d實(shí)現(xiàn)了MBR內(nèi)CANON工藝的快速啟動(dòng).

3.2 通過調(diào)整曝氣時(shí)間和添加無機(jī)碳源提高氮去除負(fù)荷,經(jīng)過76d,氮去除負(fù)荷最高可達(dá)0.41kg/(m3·d),平均氮去除負(fù)荷為0.38kg/(m3·d).實(shí)現(xiàn)了MBR內(nèi)CANON工藝的高效穩(wěn)定運(yùn)行.

3.3 DGGE圖譜顯示在CANON工藝穩(wěn)定期, Nitrosomonas和Candidatus Kuenenia stuttgartiensis是反應(yīng)器內(nèi)的優(yōu)勢(shì)菌種,共同完成脫氮過程.

參考文獻(xiàn)：

[1] 岳尚超,王 馨,王啟山,等.上流式生物濾池中CANON工藝的恢復(fù)啟動(dòng)方法 [J]. 天津大學(xué)學(xué)報(bào)(自然科學(xué)與工程技術(shù)版), 2013,(7):641-647.

[2] Sliekers A O, Derwort N, Gomez J L C, et al. Completely autotrophic nitrogen removal over nitrite in one single reactor [J]. Water Research, 2002,36(10):2475-2482.

[3] Vázquez-Padín J R, Pozo M J, Jarpa M, et al. Treatment of anaerobic sludge digester effluents by the CANON process in an air pulsing SBR [J]. J. Hazard. Mat., 2009,166(1):336-341.

[4] Strous M, Heijnen J J, Kuenen J G, et al. The sequencing batch reactor as a powerful tool for the study of slowly growing anaerobic ammonium-oxidizing microorganisms [J]. Applied Microbiology and Biotechnology, 1998,50(5):589-596.

[5] Sliekers A O, Third K A, Abma W, et al. CANON and Anammox in a gas-lift reactor [J]. FEMS Microbiology Letters, 2003,218(2):339-344.

[6] Liu S, Yang F, Gong Z, et al. Assessment of the positive effect of salinity on the nitrogen removal performance and microbial composition during the start-up of CANON process [J]. Applied Microbiology and Biotechnology, 2008,80(2):339-348.

[7] Gong Z, Yang F, Liu S, et al. Feasibility of a membrane-aerated biofilm reactor to achieve single-stage autotrophic nitrogen removal based on Anammox [J]. Chemosphere, 2007,69(5):776-784.

[8] Pynaert K, Smets B F, Beheydt D, et al. Start-up of autotrophic nitrogen removal reactors via sequential biocatalyst addition [J]. Environ. Sci. Technol., 2004,38(4):1228-1235.

[9] 國(guó)家環(huán)境保護(hù)總局.水和廢水監(jiān)測(cè)分析方法 [M]. 2版.中國(guó)環(huán)境科學(xué)出版社, 2002.

[10] Kowalchuk G A, Stephen J R, DeBoer W, et al. Analysis of ammoniaoxidizing bacteria of the beta subdivision of the class Proteobacteria in coastal sand dunes by denaturing gradient gel electrophoresis and sequencing of PCR-amplified 16S ribosomal DNA fragments [J]. Applied and Environmental Microbiology, 1997,63(4):1489-1497.

[11] Muyzer G, Dewall E C, Uitterlinden A G, et al. Profilin of complex microbial-populations by denaturing gradient gel-electrophoresis analysis of polymerase chain reactionamplified genes-coding for 16s ribosomal-RNA[J]. Applied and Environmental Microbiology, 1993,59(3):695-700.

[12] Neef A, Amann R, Schlesner H, et al. Monitoring a widespread bacterial group:in situ detection of planctomycetes with 16S rRNA-targeted probes [J]. Microbiology-UK, 1998,144(12):3257-3266.

[13] Schmid M, Walsh K, Webb R, et al. Candidatus "Scalindua brodae", sp. nov., Candidatus "Scalindua wagneri", sp. nov., two new species of anaerobic ammonium oxidizing bacteria [J]. Systematic and Applied Microbiology, 2003,26(4):529-538.

[14] Bassam B J, Caetano-Anolles G, Gresshoff P M, et al. Fast and sensitive silver staining of DNA in polyacrylamide gels [J]. Analytical Biochemistry, 1991,196(1):80-83.

[15] Anthonisen A C, Loehr R C, Prakasam T B S, et al. Inhibition of nitrification by ammonia and nitrous acid [J]. Journal of the Water Pollution Control Federation, 1976,48(5):835-852.

[16] Laanbroek H, Gerards S. Competition for limiting amounts of oxygen between Nitrosomonas europaea and Nitrobacter winogradskyi grown in mixed continuous cultures [J]. Archives of Microbiology, 1993,159(5):453-459.

[17] Strous M, Fuerst J A., Kramer E H M., et al. Missing lithotroph identified as new planctomycete [J]. Natrue, 1999a,400:446-449.

[18] Cho S, Fujii N, Lee T, et al. Development of a simultaneous partial nitrification and anaerobic ammonia oxidation process in a single reactor [J]. Bioresource Technology, 2011,102(2):652-659.

[19] 王 芳,楊鳳林,宮 正,等.炭管膜曝氣生物膜反應(yīng)器SNAD脫氮研究 [J]. 環(huán)境工程學(xué)報(bào), 2009(03):405-408.

[20] Third K A, Paxman J, Schmid M, et al. Treatment of nitrogen-rich wastewater using partial nitrification and Anammox in the CANON process [J]. Wat. Sci. Technol., 2005,52(4):47-54.

[21] Pynaert K, Smets B F, Beheydt D, et al. Start-up of Autotrophic Nitrogen Removal Reactors via Sequential Biocatalyst Addition [J]. Environ. Sci. Technol., 2004,38(4):1228-1235.

[22] Yang J, Zhang L, Fukuzaki Y, et al. High-rate nitrogen removal by the Anammox process with a sufficient inorganic carbon source [J]. Bioresource Technology, 2010,101(24):9471-9478.

[23] Paredes D, Kuschk P, Mbwette T S A, et al. New aspects of microbial nitrogen transformations in the context of wastewater treatment -a review [J]. Engineering in Life Sciences, 2007,7(1): 13-25.

[24] Ducey T F, Vanotti M B, Shriner A D, et al. Characterization of a microbial community capable of nitrification at cold temperature [J]. Bioresource Technology, 2010,101(2):491-500.

[25] Liu T, Dong L, Jie Z, et al. Phylogenetic and microbial community analysis based on amoA Gene and 16SrDNA in nitrosification biofilm reactor [J]. Environmental Biotechnology and Materials Engineering, 2011,183:1051-1056.

[26] Jing X, Dong L, Hai Y, et al. Performance and microbial community of completely autotrophic nitrogen removal over nitrite (CANON) process in two membrane bioreactors (MBR) fed with different substrate levels [J]. Bioresource Technology, 2014,152(0):185-191.

[27] Hu B, Zheng P, Tang C, et al. Identification and quantification of anammox bacteria in eight nitrogen removal reactors [J]. Water Research, 2010,44(17):5014-5020.

[28] Schmid M, Twachtmann U, Klein M, et al. Molecular evidence for genus level diversity of bacteria capable of catalyzing anaerobic ammonium oxidation [J]. Systematic and Applied Microbiology, 2000,23(1):93-106.

The fast start-up of CANON process in MBR system and the characterization of microbes.

LI Dong1*, HE Yong-ping1, ZHANG Xiao-jing2, LIANG Yu-hai1, ZHANG Yu-long1, FAN Dan1(1.Key Laboratory of Beijing for Water Quality Science and Water Environment Recovery Engineering, Beijing University of Technology, Beijing 100124, China；2.State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology, Harbin 150090, China). China Environmental Science, 2014,34(11)：2788～2795

To investigate the fast start-up of CANON process and characterization of functional microbes, conventional activated sludge was inoculated to an MBR and the reactor was operated intermediately at ambient temperature. In the launch strategy, the regulation of aeration and aeration time was used as the main method. First, partial nitrification was applied under oxygen-limited condition. Secondly, DO had to be decreased further to achieve CANON process. Finally, aeration time and inorganic carbon concentration needed to be adjusted to improve the total nitrogen removal rate. Besides, the characterization of functional microbes in stable CANON process was analyzed using PCR-DGGE techniques. The results showed that the CANON process launched successfully after 36 days, the ammonium removal rate and total nitrogen removal rate were kept at around 99% and 84% and the maxinum nitrogen removal rate can reach 0.41kg/(m3·d). DGGE profiles showed Nitrosomonas and Candidatus Kuenenia stuttgartiensis were predominant microbes in the reactor and they worked synergetically to form an efficient autotrophic nitrogen removal process within the MBR.

MBR；conventional activated sludge；CANON；start-up；PCR-DGGE；microbes

X172

A

1000-6923(2014)11-2788-08

李 冬(1976-),女,遼寧丹東人,教授,博士,主要從事水質(zhì)科學(xué)與水環(huán)境恢復(fù)關(guān)鍵技術(shù)研究.發(fā)表論文100余篇.

2014-01-22

國(guó)家自然科學(xué)基金(51222807);國(guó)家重大科技專項(xiàng)水專項(xiàng)(2012ZX07202-005)

* 責(zé)任作者, 教授, lidong2006@bjut.edu.cn