王銀華,馬悅欣,劉長發(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)殖重點實驗室,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)器載體生物膜細菌多樣性的研究

王銀華1,馬悅欣1,劉長發(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)殖重點實驗室,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)

采用細菌16S rRNA通用引物1055F/1392R-GC獲得的PCR產(chǎn)物進行變性梯度凝膠電泳(DGGE),分析了兩個厭氧氨氧化反應(yīng)器在不同運行時間其載體生物膜上的細菌多樣性。結(jié)果表明,兩個反應(yīng)器在不同運行時間其細菌種群多樣性有一定差異。DGGE優(yōu)勢條帶序列系統(tǒng)發(fā)育分析結(jié)果表明,反應(yīng)器載體生物膜上的細菌類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Chloroflexi、綠彎菌門Acidobacteria及不可培養(yǎng)細菌。當反應(yīng)器運行208 d時,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)。針對厭氧氨氧化細菌16S rRNA基因引物Pla46F/Amx368R-GC獲得的PCR產(chǎn)物,采用DGGE技術(shù)對載體生物膜上的厭氧氨氧化細菌進行了檢測。DGGE優(yōu)勢條帶序列分析結(jié)果表明,在反應(yīng)器中富集得到的厭氧氨氧化細菌分別與Planctomycete KSU-1、Candidatus Jettenia asiatica的相似性均為96%,可以認為它們是反應(yīng)器內(nèi)起厭氧氨氧化作用的主要細菌。

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

1 材料與方法

1.1 試驗裝置及運行參數(shù)

試驗采用兩個上流式生物濾池(UBF)反應(yīng)器,編號為1號、2號,其反應(yīng)裝置見圖1。反應(yīng)器為圓柱形,有效體積為6 L。1號反應(yīng)器以聚乙烯塑料小球為載體,2號反應(yīng)器以無紡布為載體。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)℃,水力停留時間(HRT)為1 d,試驗過程中充氮氣除氧。1號、2號反應(yīng)器于2009年3月5日啟動,1號反應(yīng)器中直接培養(yǎng)厭氧氨氧化菌,2號反應(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)指標的測定 氨氮、亞硝態(tài)氮采用德國SEAL公司生產(chǎn)的流動注射分析儀(Bran Luebbe Auto Analyzer 3)測定。氨氮或亞硝態(tài)氮去除率= [(進水氨氮或亞硝態(tài)氮濃度-出水氨氮或亞硝態(tài)氮濃度)/進水氨氮或亞硝態(tài)氮濃度]×100%。

1.2.2 生物膜總DNA的提取和PCR擴增 于反應(yīng)器運行第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]描述的細菌16S rRNA基因通用引物1055F/1392R-GC對1號、2號反應(yīng)器運行第88天和第208天的樣品總DNA進行PCR擴增;采用厭氧氨氧化菌特異引物Pla46F/ Amx368R-GC對1號、2號反應(yīng)器運行第208天的樣品總DNA進行PCR擴增[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)程序為: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,共進行28個循環(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通用突變檢測系統(tǒng)對PCR產(chǎn)物進行DGGE分析。將PCR反應(yīng)產(chǎn)物(質(zhì)量分數(shù)為8%)直接上樣于含體積分數(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,并用凝膠成像儀進行觀察。

1.2.4 克隆、測序及系統(tǒng)發(fā)育分析 切下DGGE優(yōu)勢條帶浸泡于50 μL無菌水中,4℃下過夜。取2 μL作為模板,細菌、厭氧氨氧化菌分別用引物1055F/1392R、Pla46F/Amx368R進行PCR擴增,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行檢測,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)細胞中,在含有X-gal、IPTG和Amp的LB平板上培養(yǎng)16 h(37℃);將藍白斑篩選陽性轉(zhuǎn)化子接種于LB培養(yǎng)基中,37℃下振蕩過夜,以培養(yǎng)的菌液為模板,用引物RV-M/MB -47進行PCR反應(yīng)檢測陽性克隆,將陽性克隆產(chǎn)物送北京天根生化科技有限公司測序。將所測DNA序列在GenBank數(shù)據(jù)庫中進行檢索,從Blast比對結(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)庫中獲得的相似序列,用系統(tǒng)發(fā)生推斷軟件包Mega 4.0進行統(tǒng)計和聚類分析[22]。采用鄰接法(neighbor-joining method)獲得分支系統(tǒng)樹,并通過自舉分析(boostrap)進行置信度檢測,自舉數(shù)據(jù)集為1 000次。

2 結(jié)果

2.1 總DNA的提取和PCR擴增

從圖2可見,反應(yīng)器中樣品總DNA條帶較亮,

無拖帶現(xiàn)象。

圖2 1號、2號反應(yīng)器運行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分別對總DNA進行PCR擴增,將擴增產(chǎn)物用10 g/L的瓊脂糖凝膠電泳進行檢測,結(jié)果見圖3和圖4,通過與Marker對比,可知目標片段長度分別約為350 bp和370 bp,其條帶較亮,無拖帶現(xiàn)象,說明PCR擴增效果良好。

圖3 1號、2號反應(yīng)器運行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、2號反應(yīng)器運行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

兩個反應(yīng)器載體生物膜樣品的DGGE結(jié)果見圖5、圖6?？梢钥闯?反應(yīng)器運行88 d,生物膜中的細菌多樣性(DGGE條帶數(shù))較高(圖5-a),但兩個反應(yīng)器中的細菌種群組成有一定差異,此時出水氨氮和亞硝態(tài)氮雖然能同時被去除(1號反應(yīng)器氨氮和亞硝態(tài)氮的去除率分別為35%、27%,2號的為25%、7%),但去除量少且兩者去除量之比沒有規(guī)律。反應(yīng)器運行208 d(圖5-b),生物膜中的細菌多樣性有所減少,同一反應(yīng)器不同時間段的細菌區(qū)系也有所不同,這時1號反應(yīng)器氨氮和亞硝態(tài)氮的去除率分別為86%、99%,2號的為82%、90%,兩者去除率之比約為1.1。通過厭氧氨氧化菌的檢測(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菌。

對DGGE優(yōu)勢條帶序列的Blast分析結(jié)果見表1。將所測序列與已知的相似序列用Mega 4.0軟件進行聚類分析,采用鄰接法構(gòu)建了生物膜中優(yōu)勢細菌的系統(tǒng)發(fā)育樹(圖7)。DGGE優(yōu)勢條帶序列系統(tǒng)發(fā)育分析結(jié)果表明,反應(yīng)器載體生物膜上的細菌類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、綠彎菌門及不可培養(yǎng)的細菌。在反應(yīng)器中富集得到的厭氧氨氧化細菌分別與Planctomycete KSU-1和Candidatus Jettenia asiatica的相似性均為96%。

3 討論

圖5 1號、2號反應(yīng)器運行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、2號反應(yīng)器運行208 d的生物膜樣品總DNA用引物Pla46F/Amx368R-GC獲得的PCR擴增產(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,啟動上流式生物

濾池反應(yīng)器。反應(yīng)器運行88 d時,載體生物膜中的細菌多樣性較高,但兩個反應(yīng)器中的細菌種群有一定差異,可能與啟動方法不同有關(guān);反應(yīng)器運行208 d時,生物膜中的細菌多樣性有所減少,同一反應(yīng)器不同時間段的細菌區(qū)系也有所不同,可能是隨著反應(yīng)器運行時間的推移,某些種類的細菌不適應(yīng)反應(yīng)器的運行條件而死亡。這時從反應(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)。通過檢測發(fā)現(xiàn),反應(yīng)器內(nèi)確實存在厭氧氨氧化細菌。

圖7 根據(jù)所測序列和Blast結(jié)果構(gòu)建的反應(yīng)器載體生物膜細菌系統(tǒng)發(fā)育樹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)勢條帶序列分析結(jié)果表明:條帶A1為1號反應(yīng)器特有,與外硫紅螺旋菌科細菌Su4的相似性為98%;條帶A2、A8、B6與不可培養(yǎng)的綠彎菌門細菌克隆的相似性為94%～100%。研究表明:綠彎菌門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)類似綠彎菌門的絲狀細菌[25];秦玉潔等[18]用傳統(tǒng)方法從試驗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%,該菌屬于反硝化細菌。DGGE和16S rRNA基因文庫分析結(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菌。Egli等[29]結(jié)合16S rDNA克隆文庫、熒光原位雜交(FISH)和斑點雜交技術(shù)研究分析了無有機碳富氨廢水的生物轉(zhuǎn)盤生物膜的微生物組成,發(fā)現(xiàn)鞘氨醇單胞菌、氨氧化細菌Nitrosomonas和厭氧氨氧化細菌Candidatus Kuenenia stuttgartiensis等的存在。條帶B2與不可培養(yǎng)的酸桿菌門細菌克隆JAB SHC 22的相似性為91%。Li等[16]用構(gòu)建浮霉狀菌門的特定16S rRNA基因文庫分析了厭氧氨氧化反應(yīng)器中的細菌多樣性,發(fā)現(xiàn)反應(yīng)器中除了有厭氧氨氧化細菌外,還存在酸桿菌門等非浮霉狀菌門的細菌。

條帶C1與Planctomycete KSU-1的相似性為96%,KSU-1菌株被鑒定為以無紡布為載體的生物膜厭氧氨氧化反應(yīng)器中的優(yōu)勢菌種[30]。條帶C2與已知厭氧氨氧化細菌Candidatus Jettenia asiatica的相似性為96%。Quan等[14]通過建立浮霉狀菌目Planctomycetales的特定16S rRNA基因文庫分析了粒狀(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菌,并命名為Candidatus Jettenia asiatica。其余條帶均與數(shù)據(jù)庫中主要來源于環(huán)境樣品的不可培養(yǎng)細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(phylotypes)。

本研究結(jié)果表明,反應(yīng)器運行穩(wěn)定時,厭氧氨氧化細菌和其它異養(yǎng)細菌共同存在于生物膜中,這些共同存在的異養(yǎng)細菌的具體生理作用和功能目前還不清楚,有待進一步研究。

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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

國家“863”計劃項目(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