王銀華,馬悅欣,劉長(zhǎng)發(fā),朱學(xué)惠,朱瑩

(1.大連海洋大學(xué)農(nóng)業(yè)部北方海水增養(yǎng)殖重點(diǎn)實(shí)驗(yàn)室,遼寧大連116023;2.大連海洋大學(xué)海洋科技與環(huán)境學(xué)院,遼寧大連116023)

厭氧氨氧化反應(yīng)器載體生物膜細(xì)菌多樣性的研究

王銀華1,馬悅欣1,劉長(zhǎng)發(fā)2,朱學(xué)惠2,朱瑩1

(1.大連海洋大學(xué)農(nóng)業(yè)部北方海水增養(yǎng)殖重點(diǎn)實(shí)驗(yàn)室,遼寧大連116023;2.大連海洋大學(xué)海洋科技與環(huán)境學(xué)院,遼寧大連116023)

采用細(xì)菌16S rRNA通用引物1055F/1392R-GC獲得的PCR產(chǎn)物進(jìn)行變性梯度凝膠電泳(DGGE),分析了兩個(gè)厭氧氨氧化反應(yīng)器在不同運(yùn)行時(shí)間其載體生物膜上的細(xì)菌多樣性。結(jié)果表明,兩個(gè)反應(yīng)器在不同運(yùn)行時(shí)間其細(xì)菌種群多樣性有一定差異。DGGE優(yōu)勢(shì)條帶序列系統(tǒng)發(fā)育分析結(jié)果表明,反應(yīng)器載體生物膜上的細(xì)菌類(lèi)群主要是陶厄氏菌屬Thauera、鞘氨醇單胞菌屬Sphingomonas,外硫紅螺旋菌科Ectothiorhodospiraceae、酸桿菌門(mén)Chloroflexi、綠彎菌門(mén)Acidobacteria及不可培養(yǎng)細(xì)菌。當(dāng)反應(yīng)器運(yùn)行208 d時(shí),水體中氨氮和亞硝態(tài)氮的去除率維持較高水平,兩者去除率之比為1.1,表明反應(yīng)器內(nèi)發(fā)生了厭氧氨氧化反應(yīng)。針對(duì)厭氧氨氧化細(xì)菌16S rRNA基因引物Pla46F/Amx368R-GC獲得的PCR產(chǎn)物,采用DGGE技術(shù)對(duì)載體生物膜上的厭氧氨氧化細(xì)菌進(jìn)行了檢測(cè)。DGGE優(yōu)勢(shì)條帶序列分析結(jié)果表明,在反應(yīng)器中富集得到的厭氧氨氧化細(xì)菌分別與Planctomycete KSU-1、Candidatus Jettenia asiatica的相似性均為96%,可以認(rèn)為它們是反應(yīng)器內(nèi)起厭氧氨氧化作用的主要細(xì)菌。

厭氧氨氧化反應(yīng)器;生物膜;PCR-DGGE;細(xì)菌多樣性;厭氧氨氧化細(xì)菌

1 材料與方法

1.1 試驗(yàn)裝置及運(yùn)行參數(shù)

試驗(yàn)采用兩個(gè)上流式生物濾池(UBF)反應(yīng)器,編號(hào)為1號(hào)、2號(hào),其反應(yīng)裝置見(jiàn)圖1。反應(yīng)器為圓柱形,有效體積為6 L。1號(hào)反應(yīng)器以聚乙烯塑料小球?yàn)檩d體,2號(hào)反應(yīng)器以無(wú)紡布為載體。將大連凌水污水處理廠生化池活性污泥接種于反應(yīng)器中,進(jìn)水為人工模擬廢水,采用NaOH調(diào)節(jié)pH為7.5～8.5。循環(huán)水浴溫度為(33±0.5)℃,水力停留時(shí)間(HRT)為1 d,試驗(yàn)過(guò)程中充氮?dú)獬酢?號(hào)、2號(hào)反應(yīng)器于2009年3月5日啟動(dòng),1號(hào)反應(yīng)器中直接培養(yǎng)厭氧氨氧化菌,2號(hào)反應(yīng)器中先采用反硝化生物膜培養(yǎng)而后富集培養(yǎng)厭氧氨氧化菌。

模擬廢水有以下兩種:1)反硝化培養(yǎng)的模擬廢水包括KH2PO40.03 g/L,KHCO30.5 g/L, MgSO4·7H2O 0.2 g/L,FeCl30.1 g/L,CaCl20.03 g/L,以NaNO3和CH3COONa分別作為電子受體和電子供體;2)厭氧氨氧化培養(yǎng)的模擬廢水包括

KHCO30.5 g/L,KH2PO40.027 g/L,MgSO4·7H2O 0.3 g/L,CaCl20.18 g/L,微量元素Ⅰ、Ⅱ各1.25 mL/L,以NaNO2和NH4Cl分別提供電子受體和電子供體[4]。其中微量元素Ⅰ包括EDTA 5 g/L, FeSO45 g/L;微量元素Ⅱ包括EDTA 15 g/L, ZnSO4·7H2O 0.43 g/L,CoCl2·6H2O 0.24 g/L, MnCl2·4H2O 0.99 g/L,CuSO4·5H2O 0.25 g/L, Na2MoO4·2H2O 0.22 g/L,NiCl2·6H2O 0.19 g/L,Na2SeO4·10H2O 0.21 g/L,H3BO40.014 g/L[19]。

圖1 UBF反應(yīng)器的結(jié)構(gòu)Fig.1 Structure of the UBF reactor

1.2 方法

1.2.1 化學(xué)指標(biāo)的測(cè)定 氨氮、亞硝態(tài)氮采用德國(guó)SEAL公司生產(chǎn)的流動(dòng)注射分析儀(Bran Luebbe Auto Analyzer 3)測(cè)定。氨氮或亞硝態(tài)氮去除率= [(進(jìn)水氨氮或亞硝態(tài)氮濃度-出水氨氮或亞硝態(tài)氮濃度)/進(jìn)水氨氮或亞硝態(tài)氮濃度]×100%。

1.2.2 生物膜總DNA的提取和PCR擴(kuò)增 于反應(yīng)器運(yùn)行第88天和第208天,從生物膜表層5～10 cm處取約4 g的樣品加入10 mL無(wú)菌水中,用力震蕩20 min。取振蕩液加入1.5 mL微量離心管中,離心,棄上清液,使用SDS裂解法抽提沉積物中的DNA。用Ferris等[20]描述的細(xì)菌16S rRNA基因通用引物1055F/1392R-GC對(duì)1號(hào)、2號(hào)反應(yīng)器運(yùn)行第88天和第208天的樣品總DNA進(jìn)行PCR擴(kuò)增;采用厭氧氨氧化菌特異引物Pla46F/ Amx368R-GC對(duì)1號(hào)、2號(hào)反應(yīng)器運(yùn)行第208天的樣品總DNA進(jìn)行PCR擴(kuò)增[13,21],兩者的反應(yīng)體系和反應(yīng)程序相同。50 μL反應(yīng)體系包括:5 μL 10× PCR Buffer,4 μL dNTPs,各2 μL(20 mmol/L)引物,2.5 U Taq酶,2 μL DNA模板,34 μL ddH2O。反應(yīng)程序?yàn)?94℃下預(yù)變性2 min;94℃下變性30 s,57℃下退火1 min,72℃下延伸1 min,共進(jìn)行28個(gè)循環(huán);最后在72℃下延伸10 min。用10 g/L的瓊脂糖凝膠電泳檢測(cè)PCR產(chǎn)物的大小。

1.2.3 DGGE分析 采用Bio-Rad公司D-code通用突變檢測(cè)系統(tǒng)對(duì)PCR產(chǎn)物進(jìn)行DGGE分析。將PCR反應(yīng)產(chǎn)物(質(zhì)量分?jǐn)?shù)為8%)直接上樣于含體積分?jǐn)?shù)為35%～60%(第88天樣品)或35%～50%(第208天樣品)梯度變性劑聚丙烯酰胺凝膠中,在TAE中電泳5 h(溫度為60℃,電壓為200 V),然后采用銀染方法染色,并用凝膠成像儀進(jìn)行觀察。

1.2.4 克隆、測(cè)序及系統(tǒng)發(fā)育分析 切下DGGE優(yōu)勢(shì)條帶浸泡于50 μL無(wú)菌水中,4℃下過(guò)夜。取2 μL作為模板,細(xì)菌、厭氧氨氧化菌分別用引物1055F/1392R、Pla46F/Amx368R進(jìn)行PCR擴(kuò)增,反應(yīng)體系和反應(yīng)程序同“1.2.2”。PCR產(chǎn)物用15 g/L的瓊脂糖凝膠電泳進(jìn)行檢測(cè),并切膠純化。純化后的PCR產(chǎn)物與pMD18-T載體連接,并轉(zhuǎn)化到E.coli DH5α感受態(tài)細(xì)胞中,在含有X-gal、IPTG和Amp的LB平板上培養(yǎng)16 h(37℃);將藍(lán)白斑篩選陽(yáng)性轉(zhuǎn)化子接種于LB培養(yǎng)基中,37℃下振蕩過(guò)夜,以培養(yǎng)的菌液為模板,用引物RV-M/MB -47進(jìn)行PCR反應(yīng)檢測(cè)陽(yáng)性克隆,將陽(yáng)性克隆產(chǎn)物送北京天根生化科技有限公司測(cè)序。將所測(cè)DNA序列在GenBank數(shù)據(jù)庫(kù)中進(jìn)行檢索,從Blast比對(duì)結(jié)果中取相似性最近的序列。將DGGE條帶序列與從GenBank數(shù)據(jù)庫(kù)中獲得的相似序列,用系統(tǒng)發(fā)生推斷軟件包Mega 4.0進(jìn)行統(tǒng)計(jì)和聚類(lèi)分析[22]。采用鄰接法(neighbor-joining method)獲得分支系統(tǒng)樹(shù),并通過(guò)自舉分析(boostrap)進(jìn)行置信度檢測(cè),自舉數(shù)據(jù)集為1 000次。

2 結(jié)果

2.1 總DNA的提取和PCR擴(kuò)增

從圖2可見(jiàn),反應(yīng)器中樣品總DNA條帶較亮,

無(wú)拖帶現(xiàn)象。

圖2 1號(hào)、2號(hào)反應(yīng)器運(yùn)行88 d(1A、2A)和208 d (1B、2B)的樣品總DNA的瓊脂糖凝膠電泳Fig.2 Agarose gel electrophoresis of DNA extraction from samples collected on 88th(1A,2A)and 208th(1B,2B)from reactor 1 and reactor 2

使用通用引物1055F/1392R-GC和厭氧氨氧化菌特異性引物Pla46F/Amx368R-GC分別對(duì)總DNA進(jìn)行PCR擴(kuò)增,將擴(kuò)增產(chǎn)物用10 g/L的瓊脂糖凝膠電泳進(jìn)行檢測(cè),結(jié)果見(jiàn)圖3和圖4,通過(guò)與Marker對(duì)比,可知目標(biāo)片段長(zhǎng)度分別約為350 bp和370 bp,其條帶較亮,無(wú)拖帶現(xiàn)象,說(shuō)明PCR擴(kuò)增效果良好。

圖3 1號(hào)、2號(hào)反應(yīng)器運(yùn)行88 d(1A、2A)和208 d (1B、2B)的樣品總DNA用引物1055F/1392RGC獲得的PCR擴(kuò)增產(chǎn)物的瓊脂糖凝膠電泳Fig.3 Agarose gel electrophoresis of PCR products amplified with primers 1055F/1392R-GC from samples collected on 88th(1A,2A)and 208th(1B,2B)from reactor 1 and reactor 2

圖4 1號(hào)、2號(hào)反應(yīng)器運(yùn)行208 d的樣品用Pla46F/ Amx368R-GC獲得的PCR產(chǎn)物瓊脂糖凝膠電泳(1C,2C)Fig.4 Agarose gel electrophoresis of PCR products amplified with primers Pla46F/Amx368R-GC from samples collected on 208th(1C,2C) from reactor 1 and reactor 2

2.2 DGGE分析

兩個(gè)反應(yīng)器載體生物膜樣品的DGGE結(jié)果見(jiàn)圖5、圖6。可以看出,反應(yīng)器運(yùn)行88 d,生物膜中的細(xì)菌多樣性(DGGE條帶數(shù))較高(圖5-a),但兩個(gè)反應(yīng)器中的細(xì)菌種群組成有一定差異,此時(shí)出水氨氮和亞硝態(tài)氮雖然能同時(shí)被去除(1號(hào)反應(yīng)器氨氮和亞硝態(tài)氮的去除率分別為35%、27%,2號(hào)的為25%、7%),但去除量少且兩者去除量之比沒(méi)有規(guī)律。反應(yīng)器運(yùn)行208 d(圖5-b),生物膜中的細(xì)菌多樣性有所減少,同一反應(yīng)器不同時(shí)間段的細(xì)菌區(qū)系也有所不同,這時(shí)1號(hào)反應(yīng)器氨氮和亞硝態(tài)氮的去除率分別為86%、99%,2號(hào)的為82%、90%,兩者去除率之比約為1.1。通過(guò)厭氧氨氧化菌的檢測(cè)(圖6),結(jié)果發(fā)現(xiàn),反應(yīng)器內(nèi)有3種起厭氧氨氧化作用的細(xì)菌。

對(duì)DGGE優(yōu)勢(shì)條帶序列的Blast分析結(jié)果見(jiàn)表1。將所測(cè)序列與已知的相似序列用Mega 4.0軟件進(jìn)行聚類(lèi)分析,采用鄰接法構(gòu)建了生物膜中優(yōu)勢(shì)細(xì)菌的系統(tǒng)發(fā)育樹(shù)(圖7)。DGGE優(yōu)勢(shì)條帶序列系統(tǒng)發(fā)育分析結(jié)果表明,反應(yīng)器載體生物膜上的細(xì)菌類(lèi)群主要是陶厄氏菌屬Thauera、鞘氨醇單胞菌屬Sphingomonas,外硫紅螺旋菌科、酸桿菌門(mén)、綠彎菌門(mén)及不可培養(yǎng)的細(xì)菌。在反應(yīng)器中富集得到的厭氧氨氧化細(xì)菌分別與Planctomycete KSU-1和Candidatus Jettenia asiatica的相似性均為96%。

3 討論

圖5 1號(hào)、2號(hào)反應(yīng)器運(yùn)行88 d(a)和208 d(b)的生物膜樣品總DNA用引物1055F/1392R-GC獲得的PCR擴(kuò)增產(chǎn)物的DCGE圖譜Fig.5 DGGE profile of PCR amplified 16S rRNA gene fragments of biofilm samples with primers 1055F/ 1392R-GC collected on 88th(a),and 208th(b) from reactor 1 and reactor 2

圖6 1號(hào)、2號(hào)反應(yīng)器運(yùn)行208 d的生物膜樣品總DNA用引物Pla46F/Amx368R-GC獲得的PCR擴(kuò)增產(chǎn)物的DCGE圖譜Fig.6 DGGE profile of PCR amplified 16S rRNA gene fragments of biofilm samples with primers Pla46F/ Amx368R-GC collected on 208thfrom reactor 1 and reactor 2

表1 DGGE條帶序列的Blast結(jié)果Tab.1 The blast analysis of the sequences of DGGE bands

本研究中采用污水處理廠的污泥作為接種物,通過(guò)直接培養(yǎng)厭氧氨氧化菌,以及先培養(yǎng)反硝化生物膜后再富集培養(yǎng)厭氧氨氧化菌,啟動(dòng)上流式生物

濾池反應(yīng)器。反應(yīng)器運(yùn)行88 d時(shí),載體生物膜中的細(xì)菌多樣性較高,但兩個(gè)反應(yīng)器中的細(xì)菌種群有一定差異,可能與啟動(dòng)方法不同有關(guān);反應(yīng)器運(yùn)行208 d時(shí),生物膜中的細(xì)菌多樣性有所減少,同一反應(yīng)器不同時(shí)間段的細(xì)菌區(qū)系也有所不同,可能是隨著反應(yīng)器運(yùn)行時(shí)間的推移,某些種類(lèi)的細(xì)菌不適應(yīng)反應(yīng)器的運(yùn)行條件而死亡。這時(shí)從反應(yīng)器的外觀看,反應(yīng)器內(nèi)污泥的顏色由最初的黑色變?yōu)榧t褐

色,充分表明厭氧氨氧化逐漸成為反應(yīng)器的主導(dǎo)反應(yīng)。通過(guò)檢測(cè)發(fā)現(xiàn),反應(yīng)器內(nèi)確實(shí)存在厭氧氨氧化細(xì)菌。

圖7 根據(jù)所測(cè)序列和Blast結(jié)果構(gòu)建的反應(yīng)器載體生物膜細(xì)菌系統(tǒng)發(fā)育樹(shù)Fig.7 Phylogenetic tree of the bacteria in boifilms associated with media of two anammox bioreactors based on the results of Blast of DGGE bands and the sequences

DGGE圖譜和優(yōu)勢(shì)條帶序列分析結(jié)果表明:條帶A1為1號(hào)反應(yīng)器特有,與外硫紅螺旋菌科細(xì)菌Su4的相似性為98%;條帶A2、A8、B6與不可培養(yǎng)的綠彎菌門(mén)細(xì)菌克隆的相似性為94%～100%。研究表明:綠彎菌門(mén)是厭氧污泥消化器中的主要微生物[23],普遍存在于生物廢水處理廠的活性污泥中[24],也出現(xiàn)于圓柱形上流式厭氧反應(yīng)器中[16];在上流式顆粒床厭氧氨氧化反應(yīng)器中也發(fā)現(xiàn)類(lèi)似綠彎菌門(mén)的絲狀細(xì)菌[25];秦玉潔等[18]用傳統(tǒng)方法從試驗(yàn)的兩套厭氧氨氧化反應(yīng)器中分離到一種綠彎菌。本研究中,條帶A5的序列與陶厄氏菌Thauera sp.240 bp序列的相似性為99%,該菌屬于反硝化細(xì)菌。DGGE和16S rRNA基因文庫(kù)分析結(jié)果表明,在去除喹啉的反硝化反應(yīng)器中,陶厄氏菌屬的細(xì)菌起著重要作用[26]。Mao等[27]從焦化廢水處理廠的反硝化生物反應(yīng)器中也可分離到若干陶厄氏菌。條帶B1與鞘氨醇單胞菌Sphingomonas sp.V1-2的相似性為99%。Lu等[28]用培養(yǎng)方法從一厭氧/好氧SBR反應(yīng)器中分離到鞘氨醇單胞菌屬的細(xì)菌。Egli等[29]結(jié)合16S rDNA克隆文庫(kù)、熒光原位雜交(FISH)和斑點(diǎn)雜交技術(shù)研究分析了無(wú)有機(jī)碳富氨廢水的生物轉(zhuǎn)盤(pán)生物膜的微生物組成,發(fā)現(xiàn)鞘氨醇單胞菌、氨氧化細(xì)菌Nitrosomonas和厭氧氨氧化細(xì)菌Candidatus Kuenenia stuttgartiensis等的存在。條帶B2與不可培養(yǎng)的酸桿菌門(mén)細(xì)菌克隆JAB SHC 22的相似性為91%。Li等[16]用構(gòu)建浮霉?fàn)罹T(mén)的特定16S rRNA基因文庫(kù)分析了厭氧氨氧化反應(yīng)器中的細(xì)菌多樣性,發(fā)現(xiàn)反應(yīng)器中除了有厭氧氨氧化細(xì)菌外,還存在酸桿菌門(mén)等非浮霉?fàn)罹T(mén)的細(xì)菌。

條帶C1與Planctomycete KSU-1的相似性為96%,KSU-1菌株被鑒定為以無(wú)紡布為載體的生物膜厭氧氨氧化反應(yīng)器中的優(yōu)勢(shì)菌種[30]。條帶C2與已知厭氧氨氧化細(xì)菌Candidatus Jettenia asiatica的相似性為96%。Quan等[14]通過(guò)建立浮霉?fàn)罹縋lanctomycetales的特定16S rRNA基因文庫(kù)分析了粒狀(0.6～1.0 mm)污泥厭氧氨氧化反應(yīng)器中厭氧氨氧化細(xì)菌的多樣性,發(fā)現(xiàn)了一種在反應(yīng)器中占50%的新的厭氧氨氧化細(xì)菌,并命名為Candidatus Jettenia asiatica。其余條帶均與數(shù)據(jù)庫(kù)中主要來(lái)源于環(huán)境樣品的不可培養(yǎng)細(xì)菌克隆序列有較高的相似性(97%～100%),這意味著厭氧氨氧化反應(yīng)器中可能存在大量新的種類(lèi)(phylotypes)。

本研究結(jié)果表明,反應(yīng)器運(yùn)行穩(wěn)定時(shí),厭氧氨氧化細(xì)菌和其它異養(yǎng)細(xì)菌共同存在于生物膜中,這些共同存在的異養(yǎng)細(xì)菌的具體生理作用和功能目前還不清楚,有待進(jìn)一步研究。

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The bacterial diversity in boifilms associated with media of anammox bioreactors

WANG Yin-hua1,MA Yue-xin1,LIU Chang-fa2,ZHU Xue-hui2,ZHU Ying1
(1.Key Laboratory of North Mariculture,Ministry of Agriculture,Dalian Ocean University,Dalian 116023,China;2.College of Marine Science and Environment,Dalian Ocean University,Dalian 116023,China)

The bacterial diversity was studied in boifilms associated with media of two anammox bioreactors running at different time by PCR-DGGE using bacterial 16S rRNA universal primers 1055F/1392R-GC.Results showed that the bacterial diversity was different in two reactors at different sampling time.Phylogenetic analysis of sequences from prevalent DGGE bands revealed that the bacteria were found to be Thauera,Sphingomonas,Ectothiorhodospiraceae,Chloroflexi,Acidobacteria and uncultured bacteria.When the bioreactors were operated for 208 days,the removal rates of ammonia and nitrite nitrogens were kept high with the ratio of ammonia nitrogen level to nitrite nitrogen level=1.1,indicating that the anammox reaction took place in the bioreactors.Anammox bacteria in the biofilms associated with media of bioreactors were detected by PCR-DGGE using anammox bacteria specific primers Pla46F/Amx368R-GC.Analysis of sequences from prevalent DGGE bands showed anammox bacteria had 96%similarity to Planctomycete KSU-1 and Candidatus Jettenia asiatica,respectively.It is suggested that these bacteria remove nitrogen by anaerobic ammonium-oxidation in reactors.

anaerobic ammonium oxidation reactor;biofilm;PCR-DGGE;anaerobic ammonium oxidation bacterium;anammox bacterium

X172

A

2095-1388(2011)06-0500-07

2011-01-20

國(guó)家“863”計(jì)劃項(xiàng)目(2007AA10Z410)

王銀華(1985-),女,碩士研究生。E-mail:414795838@qq.com

馬悅欣(1963-),女,博士,教授。E-mail:mayuexin@dlou.edu.cn