厭氧氨氧化耦合脫氮系統(tǒng)中反硝化細(xì)菌研究

王春香,劉常敬,鄭林雪,李 軍 (北京工業(yè)大學(xué)建筑工程學(xué)院,北京 100124)

厭氧氨氧化耦合脫氮系統(tǒng)中反硝化細(xì)菌研究

王春香,劉常敬,鄭林雪,李 軍*(北京工業(yè)大學(xué)建筑工程學(xué)院,北京 100124)

采用聚合酶鏈?zhǔn)椒磻?yīng)、分子克隆等分子生物學(xué)方法,通過構(gòu)建nirS克隆文庫(kù)研究了厭氧氨氧化耦合異養(yǎng)反硝化脫氮系統(tǒng)中的反硝化微生物.進(jìn)行同源性分析和系統(tǒng)發(fā)育樹構(gòu)建,結(jié)果表明,從nirS克隆文庫(kù)中隨機(jī)挑選的100個(gè)克隆子中有51個(gè)陽(yáng)性克隆子,共分為12個(gè)操作單元.經(jīng)比對(duì)發(fā)現(xiàn)這些微生物主要為變形菌門細(xì)菌和未知菌類,其中 β-proteobacteria占據(jù)絕對(duì)優(yōu)勢(shì)且有較多種類,少部分屬于γ-proteobacteria.

厭氧氨氧化；反硝化菌；聚合酶鏈?zhǔn)椒磻?yīng)(PCR)；nirS克隆文庫(kù)

厭氧氨氧化(ANAMMOX)工藝是由荷蘭Delft大學(xué)開發(fā)的生物脫氮新技術(shù)[1],厭氧氨氧化反應(yīng)器對(duì)氮的去除主要是利用厭氧氨氧化細(xì)菌作用,以氨氮為電子受體,將亞硝酸鹽氮還原為氮?dú)?反硝化作用的概念是由Gayon等[2]于 1882 年提出的,是指硝酸根和亞硝酸根通過微生物的異養(yǎng)還原作用,變成一氧化氮,一氧化二氮,或最終還原成氮?dú)獾倪^程,在廢水處理系統(tǒng)中反硝化作用已經(jīng)發(fā)展成為一個(gè)有效去除氮素的步驟.異養(yǎng)的反硝化過程需要碳源,研究發(fā)現(xiàn),不但小分子的有機(jī)物,如甲醇、甲醛等可以作為反硝化過程的能量物質(zhì),一些復(fù)雜的大分子有機(jī)物也能夠被反硝化細(xì)菌所利用.根據(jù)這一特性,可以利用反硝化細(xì)菌來(lái)降解廢水中的復(fù)雜有機(jī)物.反硝化作用主要是由反硝化細(xì)菌引起的,反硝化細(xì)菌具有降解各種有機(jī)污染物的能力,包括芳香族和脂肪烴類[3].然而大部分細(xì)菌是不可培養(yǎng)或難以培養(yǎng)的,使得分離培養(yǎng)方法研究反硝化細(xì)菌往往不夠全面,利用分子生物學(xué)方法來(lái)認(rèn)識(shí)反硝化菌將更具有科學(xué)性.

反應(yīng)器以苯酚作為有機(jī)碳源,酚等有機(jī)物雖然對(duì)微生物有抑制作用,但可以作為電子供體用于反硝化[4-6],反硝化降解酚類有機(jī)物就會(huì)解除其毒性抑制作用.Toh等[7]在用ANAMMOX工藝處理焦化廢水的研究中證實(shí)了反硝化菌的存在.

本實(shí)驗(yàn)利用PCR、分子克隆等分子生物學(xué)方法,研究了以苯酚為電子供體的厭氧氨氧化耦合異養(yǎng)脫氮系統(tǒng)內(nèi)的反硝化菌的種類.

1 材料與方法

1.1 采樣

污泥取自厭氧氨氧化耦合異養(yǎng)反應(yīng)器底部.試驗(yàn)采用 UASB生物膜反應(yīng)器,有效容積為10L,反應(yīng)器內(nèi)溫度維持在 25℃左右,水力停留時(shí)間為1.5h,進(jìn)水pH值控制在7.8左右,反應(yīng)器長(zhǎng)期穩(wěn)定運(yùn)行.反應(yīng)器的進(jìn)出水指標(biāo)如圖 1所示.-N、-N去除率高達(dá) 100%,TN去除率為 87.51%,異養(yǎng)反硝化對(duì) TN去除貢獻(xiàn)率達(dá)到13.28%.

圖1 進(jìn)、出水-N、-N和-N含量變化Fig.1 Variation of -N, -N and -N during the operation

1.2 細(xì)菌總DNA提取

泥樣在1.5mL離心管中靜沉10min后舍去上清液,15000r/min高速離心 5min后,去除上層清液,取0.3g用于總DNA提取,剩余污泥冷凍備用.采用Ezup柱式基因組DNA提取試劑盒(上海生工)提取后,取 5μL用 1.2%瓊脂糖凝膠檢測(cè).其余置于-20℃儲(chǔ)存?zhèn)溆?

1.3 聚合酶鏈?zhǔn)椒磻?yīng)(PCR)

將提取的細(xì)菌總 DNA作為 PCR的模板,PCR反應(yīng)在 MyiQ Real-time PCR擴(kuò)增儀(美國(guó),Bio-Rad)上進(jìn)行,采用反硝化菌特異性引物對(duì)nirS1F和nirS6R[8].nirS1F和nirS6R引物序列分別為:

nirS1F:5’-CCT A(C/T)T GGC CGC C (A/G)C A (A/G)T-3’

nirS6R:5’-CGT TGA ACT T (A/G)C CGG T-3’

采用25μL反應(yīng)體系:10x PCR Buffer, 2.5μL;dNTPs (各 2.5mmol/L),1.0μL; MgCl2(25mmol/L),1.5μL;nirS1F(20μmol/L),0.5μL;nirS6R(20μmol/L),0.5μL;Taq酶(5U,Takara)0.3μL; DNA 1.0μL,加滅菌超純水到25μL.

PCR 反應(yīng)條件:95℃預(yù)變性 6min;95℃變性30s,60℃退火40s,72℃延伸1min,10個(gè)循環(huán);95℃變性 30s,55℃退火 40s,72℃延伸 1min,25個(gè)循環(huán);72℃延伸7min;4℃保持.

1.4 克隆、轉(zhuǎn)化和nirS克隆文庫(kù)的建立

PCR產(chǎn)物采用 SanPrep 柱式 DNA 膠回收試劑盒(上海生工)進(jìn)行純化后,與pMD18-T載體(Takara)連接.連接體系(總體積 10μL):PCR 產(chǎn)物3μL;pMD18-T Vector 1μL;Solution I 5μL;ddwater 1μL.將連接產(chǎn)物轉(zhuǎn)入 DH5α感受態(tài)細(xì)胞中,用含X-Gal、IPTG、Amp的LB培養(yǎng)基37℃培養(yǎng)16h后,進(jìn)行藍(lán)白班篩選.從克隆文庫(kù)中隨機(jī)挑選 100個(gè)白斑,用pMD18-T載體引物RV-M和M13-47作為PCR引物,驗(yàn)證篩選的克隆插入的片段大小是否正確.

M13-47:5’-GAGCGGATAACAATTTCAC ACAGG-3’;

RV-M:5’-CGCCAGGGTTTTCCCAGTCAC GAC-3’.

片段大小正確的PCR產(chǎn)物用 Hha I (Takara,Japan)限制性內(nèi)切酶進(jìn)行酶切.Hha I 酶切體系(10μL)為:Hha I,0.5μL;10×PCR Buffer,1μL;PCR產(chǎn)物5μL;ddwater,3.5μL.酶切反應(yīng)于37℃進(jìn)行4h,酶切產(chǎn)物用 3%的瓊脂糖凝膠電泳檢測(cè),然后在凝膠成像系統(tǒng)中成像,并根據(jù)凝膠成像結(jié)果中的酶切帶型劃分操作單元(OTU).基因文庫(kù)中每個(gè)OTU選擇一個(gè)代表菌株送往上海生工生物公司測(cè)序.

2 結(jié)果與討論

2.1 細(xì)菌總DNA提取

污泥樣品提取的總 DNA經(jīng)瓊脂糖凝膠電泳檢測(cè),DNA提取電泳圖如圖 2,可見所提取的 DNA片段分子量大小在 23kbp左右,屬于比較完整的細(xì)菌總 DNA,適用于下一步PCR擴(kuò)增.

圖2 細(xì)菌總DNA提取電泳Fig.2 Electrophoresis of the DNA extracts

2.2 PCR擴(kuò)增及產(chǎn)物純化

PCR擴(kuò)增結(jié)果如圖 3所示.用反硝化細(xì)菌nirS基因特異性引物擴(kuò)增所產(chǎn)生的DNA片段大小為890bp,符合預(yù)期的長(zhǎng)度,但有非特異性擴(kuò)增,需要進(jìn)行切膠純化.圖4為PCR擴(kuò)增產(chǎn)物切膠純化后的結(jié)果.

圖3 反硝化細(xì)菌PCR擴(kuò)增電泳圖Fig.3 Electrophoresis of the PCR productions

圖4 反硝化細(xì)菌PCR擴(kuò)增產(chǎn)物純化Fig.4 Electrophoresis of the PCR productions after purification

2.3 反硝化細(xì)菌nirS克隆文庫(kù)

在構(gòu)建的反硝化細(xì)菌nirS克隆文庫(kù)中,隨機(jī)挑選100個(gè)進(jìn)行PCR,共得到51個(gè)陽(yáng)性克隆子.圖5為部分結(jié)果.

圖5 克隆子的PCR電泳Fig.5 Electrophoresis of the PCR results of clones

2.4 反硝化菌的酶切和序列測(cè)定

采用Hha I限制性內(nèi)切酶對(duì)51個(gè)陽(yáng)性克隆子進(jìn)行酶切分型,部分酶切結(jié)果如圖5所示.共得到12個(gè)OTUs,每個(gè)OUT所包含的克隆子數(shù)量不一樣,從1到21個(gè)不等.

圖6 HhaI酶切圖譜Fig.6 Restriction patterns of denitrifying bacteria digested by Hha I

對(duì)12個(gè)OUTs的代表性克隆子進(jìn)行克隆測(cè)序,將測(cè)序所得基因序列輸入到NCBI網(wǎng)站,利用BLAST程序與數(shù)據(jù)庫(kù)中已有的序列進(jìn)行比對(duì)鑒定,發(fā)現(xiàn)大部分克隆子比對(duì)后與最近物種的相似度為 77%～94%.這是由于 nirS基因序列較長(zhǎng)(達(dá)900bp左右).因此比對(duì)到的與最相似物種的相似度并沒有像普通 16SrDNA比對(duì)結(jié)果那么高(200bp左右的16SrDNA比對(duì)的相似度一般都高達(dá) 99%左右.根據(jù)莫旭華等[9]的群落結(jié)構(gòu)計(jì)算方法和系統(tǒng)學(xué)分析,該文庫(kù)的庫(kù)容大小為 90%,Shannon–Weiner指數(shù)為 2.02, Simpson指數(shù)為0.79,豐富度為3.05.

表1 樣品克隆測(cè)序比對(duì)結(jié)果Table 1 Results of the best march to known species of the clones

表1所示為克隆序列測(cè)序比對(duì)后的信息.從表中可以看出比對(duì)的結(jié)果主要為變形菌門細(xì)菌和未知菌類.其中β-proteobacteria占據(jù)絕對(duì)優(yōu)勢(shì)且有較多種類,其 OTUs和克隆豐富度分別為58.3%和 62.7%.少部分屬于 γ-proteobacteria的Pseudomonas stutzeri(惡臭假單胞菌屬)和鹽單胞菌屬(Halomonas sp.),其 OTUs和克隆豐富度分別為16.7%和7.8%.說(shuō)明變形桿菌在該脫氮系統(tǒng)中是最主要的菌群[10],還有部分未培養(yǎng)的環(huán)境樣品,其OTUs和克隆豐富度分別為16.7%和9.8%,證實(shí)了傳統(tǒng)純化分離、培養(yǎng)等微生物學(xué)分析測(cè)定技術(shù)難于真實(shí)準(zhǔn)確的反映出反應(yīng)器運(yùn)行過程中微生物的群落構(gòu)成及數(shù)量,分子生物學(xué)技術(shù)可以檢測(cè)出未培養(yǎng)菌屬和未知菌屬,優(yōu)勢(shì)顯而易見.此外,還有一個(gè)克隆在數(shù)據(jù)庫(kù)中比對(duì)后沒有找到已知的相近物種,未能得到詳細(xì)信息,相關(guān)研究也得出類似結(jié)論[11-12].

鄭平等[13]和Heylen等[14-15]指出,各種環(huán)境中最常見的反硝化細(xì)菌都屬于變形菌綱.在污水中,nirS反硝化菌屬多是 β-proteobacteria和γ-proteobacteria[16].在本研究中,比對(duì)得到的菌屬大 部 分 歸 屬 于 Proteobacteria 門,βproteobacteria綱數(shù)量上占有絕對(duì)優(yōu)勢(shì),包括Comamonadaceae、Azoarcus tolulyticus、Thauera aromatica和 Cupriavidus.少部分屬于γ-proteobacteria,包括 Pseudomonas stutzeri 和Halomonas sp.,研究結(jié)果具有一致性.這些細(xì)菌不僅具有反硝化作用,而且為異養(yǎng)型細(xì)菌,對(duì)有機(jī)碳源苯酚的降解有重要作用.對(duì)于苯酚生物降解的研究,國(guó)外起步比較早,己經(jīng)分離得到的許多苯酚降解菌中就包括 Pseudomonas[17-21].β-proteobacteria綱中的 Azoarcus和 Thauera屬的大多數(shù)成員都與反硝化條件下芳香族化合物[22-23]的降解有關(guān).Anders等[24]在1995年發(fā)現(xiàn),在厭氧條件下 Thauera aromatica能夠利用phenol、benzoate等作為唯一碳源生長(zhǎng)來(lái)降解污染物.此外,Manefield等[25]使用RNA-SIP 技術(shù)證明 Thauera sp.和Acidovorax[26]是苯酚的主要降解菌.1997年,Hinteregger等[27]發(fā)現(xiàn)在鼓泡塔反應(yīng)器中Halomonas sp.可完全降解鹽含酚廢水中的苯酚.Comamonadaceae屬細(xì)菌具有降解芳香族化合物的能力也已被報(bào)道[28-29].本研究中,苯酚濃度 18.82mg/L的條件下,耦合反應(yīng)器能長(zhǎng)期穩(wěn)定運(yùn)行.異養(yǎng)反硝化對(duì) TN去除貢獻(xiàn)率達(dá)到13.28%,可以證明 Proteobacteria對(duì)于系統(tǒng)的脫氮和苯酚的降解有很大貢獻(xiàn).

王弘宇等[30]發(fā)現(xiàn)生物陶粒脫氮反應(yīng)區(qū)內(nèi)假單胞菌(Pseudomonas sp.)對(duì)氮素的去除率達(dá)到 43.90%.李佳霖等[31]研究長(zhǎng)江口鄰近海域沉積物中通過微生物的反硝化途徑去除的氮約為初級(jí)生產(chǎn)消耗無(wú)機(jī)氮量的1/3.以上數(shù)據(jù)說(shuō)明,反硝化細(xì)菌對(duì)于污水生物處理系統(tǒng)內(nèi)氮素的去除有很大的貢獻(xiàn).本研究中,反硝化菌對(duì) TN去除貢獻(xiàn)率達(dá)到 13.28%,與以上研究結(jié)果具有相似性.

2.5 構(gòu)建系統(tǒng)發(fā)育樹

對(duì)獲得的克隆和相應(yīng)的最相近已知物種構(gòu)建系統(tǒng)發(fā)育樹,結(jié)果如圖7所示.從系統(tǒng)發(fā)育樹可以看出,大部分的陽(yáng)性克隆子和已知的反硝化細(xì)菌具有較高的相似性,也就說(shuō)明了在本研究中的反硝化菌基本由普通的反硝化菌構(gòu)成.同時(shí)也有大量分離的克隆子不與目前已知的 nirS 基因相似,屬于未知菌屬,說(shuō)明水處理中的反硝化群落需要進(jìn)一步研究.

圖7 反硝化細(xì)菌系統(tǒng)發(fā)育分析Fig.7 The phylogenetic analysis results of the denitrifying bacteria

3 結(jié)論

3.1 反應(yīng)器所得克隆子比照后菌屬主要為變形菌門細(xì)菌和未知菌類.其中 β-proteobacteria占據(jù)絕對(duì)優(yōu)勢(shì)且有較多種類,其OTUs和克隆豐富度分別為58.3%和62.7%.少部分屬于 γ-proteobacteria,其OTUs和克隆豐富度分別為16.7%和7.8%.說(shuō)明變形桿菌在該脫氮系統(tǒng)中是最主要的菌群.還有部分未培養(yǎng)的環(huán)境樣品,其OTUs和克隆豐富度分別為16.7%和9.8%.

3.2 反應(yīng)器中的優(yōu)勢(shì)反硝化菌為變形菌綱細(xì)菌,對(duì)于系統(tǒng)的氮的去除和苯酚的降解有很大貢獻(xiàn),對(duì)TN去除貢獻(xiàn)率達(dá)到13.28%.

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Denitrifying bacteria of anaerobic ammonium-oxidizing denitrifying system.

WANG Chun-xiang, LIU Changjing, ZHENG Lin-xue, LI Jun*(Key Laboratory of Beijing for Water Quality Science and Water Environment Recovery Engineering, Beijing University of Technology, Beijing 100124, China). China Environmental Science,2014,24(7)：1878～1883

Denitrifying microorganisms in the anaerobic ammonium oxidation coupling heterotrophic denitrification system were investigated. The methods used were PCR and molecular cloning. By building nirS clone libraries, homology analysis and constructing system Phylogenetic tree, 51clones were assigned to 12 OTUs. By BLAST alignment found these microbes were mainly proteobacteria and uncultured environment samples. Proteobacteria were mainly β-Proteobacteria and γ-Proteobacteria.

anaerobic ammonium-oxidizing (ANAMMOX)；denitrifying bacteria；polymerase chain reaction (PCR)；nirS clone libraries

X172

A

1000-6923(2014)07-1878-06

2013-10-25

國(guó)家自然科學(xué)基金(51078008);北京市自然科學(xué)基金(8122005);北京市教委重點(diǎn)項(xiàng)目(KZ201110005008)

* 責(zé)任作者, 教授, jglijun@bjut.edu.cn

王春香(1988-),女,山東菏澤人,北京工業(yè)大學(xué)碩士研究生,研究方向污水生物處理.