焦化廢水活性污泥PAH雙加氧酶基因多樣性分析

蒙小俊,李海波,盛宇星,曹宏斌*(1.安康學院旅游與資源環(huán)境學院,陜西 安康 75000；.中國科學院過程工程研究所綠色過程與工程重點實驗室,北京 100190)

焦化廢水活性污泥PAH雙加氧酶基因多樣性分析

蒙小俊1,2,李海波2,盛宇星2,曹宏斌2*(1.安康學院旅游與資源環(huán)境學院,陜西 安康 725000；2.中國科學院過程工程研究所綠色過程與工程重點實驗室,北京 100190)

為分析焦化廢水活性污泥中降解PAH雙加氧酶的多樣性,利用16Sr DNA-PCR-DGGE方法,以實際焦化廢水好氧單元活性污泥總DNA為模板,通過引物對污泥中的雙加氧酶基因進行了克隆表達和多樣性分析.結果表明,以活性污泥總 DNA為模板,利用引物RHD-GN-610F和RHD-GN-916R擴增后有明顯的產物,產物大小約為300bp;DGGE指紋圖譜顯示PCR產物有8條分離條帶,豐度分別為2.99%、8.16%、20.75%、28.50%、8.62%、7.26%、10.62%和13.10%;條帶經切膠回收PCR擴增后出現(xiàn)明顯的擴增產物,TA克隆后成功測試出4條序列,長度分別為305bp,298bp,334bp和294bp,表明焦化廢水活性污泥中存在不同降解PAH的RHD酶.這些結果為焦化廢水中PAHs的風險評估及其潛在生物降解提供理論基礎.

焦化廢水；PAHs；活性污泥；雙加氧酶基因

PAHs是一類由稠合芳環(huán),不含取代基或雜原子組成的有毒有害化合物,這類物質常以混合的形式存在,廣泛分布于不同性質的水環(huán)境介質中,如生活廢水和工業(yè)廢水中.在廢水的處理過程中PAHs會大量積存于初沉池污泥、活性污泥、消化污泥、脫水污泥和外排污泥中,對微生物活性有負效應,因致突、致畸和致癌使該類物質成為最危險的有機污染物之一.因與固體顆粒有很強的親和性,在廢水處理過程中 PAHs很容易被吸附在固體顆粒表面,吸附作用是 PAHs在廢水處理過程中去除的主要途徑[1-3].焦化廠是環(huán)境中PAHs的主要來源之一,焦化排放的PAHs占我國多環(huán)芳烴年排放總量的17.9%,尤其如萘、菲、芘、苯并[a]芘是焦化廢水中PAHs的典型代表[4-6].焦化廢水生化處理過程中PAHs的遷移轉化主要是依賴生物反應器中生物污泥-微生物群落的吸附降解耦合作用來去除,在生物反應器中多環(huán)芳烴遷移轉化的可能途徑主要是生物污泥的吸附,其次是緩慢的生物降解,多環(huán)芳烴的水溶解度隨著分子量的增大而降低,水相遷移能力也隨之變差,多環(huán)芳烴會更容易被懸浮物和沉積物吸附[7-10].焦化廢水中PAHs的最終去除依靠微生物的降解作用,已從焦化廢水活性污泥、土壤或者其他場所鑒定出多株具有降解多環(huán)芳烴能力的菌株,如芘降解菌Pseudomonas sp.[11], Burkholderia sp.[11]和Diaphorobacter sp.[12]等,萘降解菌如 Streptomyces sp.[13]和 Geobacillus sp.[14]等,菲降解菌如Pseudomonas sp.[15]和Sphingomonas sp.[16]等.

PAHs的生物降解需要多種酶系的參與,酶系的生成和不同活性都是由相關基因進行編碼控制,其中重要的關鍵啟動酶為環(huán)羥基雙加氧酶(RHD),它決定和控制后續(xù)的生物降解步驟和速率.RHD由β和α終端雙加氧酶,鐵氧化還原蛋白和鐵氧化還原蛋白酶組成,在 PAH環(huán)裂解過程中另外還需雌二醇雙加氧酶的參與,在不同的染色體或質粒位點上,不同的 PAH降解菌具有不同的 RHD編碼基因[17-18].已從紅杉沉淀物中發(fā)現(xiàn)nidA和ndo (nahAc and phnAc)終端雙加氧酶基因[19-20],利用實時定量 PCR從土壤和沉積物樣品中成功克隆和鑒定出降解 PAHs的環(huán)羥化雙加氧酶基因RHD[21-22].降解PAHs的雙加氧酶基因具有相似性和多樣性,如高效降解芘的放線菌 Mycobacterium sp.雙加氧酶大亞基和小亞基的基因片段與已知降解芘的分枝桿菌的雙加氧酶基因具有高度同源性[23],而降解蒽嗜鹽菌AD-3 的雙加氧酶基因與 Marinobacter sp.NCE312菌株萘雙加氧酶大亞基的部分氨基酸序列同源性相似[24],嗜鹽菌群萘雙加氧酶(ndo)基因有6種基因型[25].實際焦化廢水處理工藝好氧單元多以活性污泥法為主,然而好氧單元活性污泥PAHs雙加氧酶基因多樣性未見報道.對編碼降解PAH啟動基因RHD進行克隆表達和多樣性分析有利于降解PAHs功能微生物的監(jiān)測,風險評估及其PAHs的潛在生物降解.本研究利用16Sr DNA- DGGE技術對采自實際焦化廢水處理廠好氧單元的活性污泥分析其RHD基因的多樣性.

1 材料與方法

1.1 實驗試劑

D5625-01E.Z.N.A.Soil DNA Kit(OMEGA)、Q10212Qubit2.0DNA 檢測試 劑 盒(Life)、Ep0406Taq DNA Polymerase(Thermo) 、SK8192SanPrep柱式DNA膠回收試劑盒(上海生工).

1.2 實驗主要儀器

Pico-2臺式離心機(Thermo Fisher)、GL-88B漩渦混合器(海門市其林貝爾儀器制造有限公司)、 TND03-H-H干式恒溫器(深圳拓能達科技有限公司)、DYY-6C電泳儀電源(北京市六一儀器廠)、DYCZ-21電泳槽(北京市六一儀器廠)、GelDoc-It310凝膠成像系統(tǒng)(美國 UVP)、Qubit?2.0Q32866熒光計(Invitrogen)、T100TMThermal Cyeler PCR儀(BIO-RAD)、DGGE儀(Bio-Rad).

1.3 采樣

活性污泥采自遼寧某焦化廠好氧單元,該焦化廢水生物處理系統(tǒng)采用A-A-O工藝,表1為主要水質指標和好氧單元工藝參數(shù),SRT為15～18d,活性污泥中16種EPA優(yōu)控PAHs單體濃度為18.95～68.50μg/g.

表1 焦化廢水主要水質指標和好氧單元工藝參數(shù)Table 1 Major indices of coking wastewater and operational parameters of aerobic basin

1.4 樣品總DNA提取和PCR擴增

活性污泥基因組 DNA 通過 E.Z.N.A. Soil DNA Kit 提取,Qubit2.0檢測DNA濃度,瓊脂糖凝膠檢測DNA完整性.

所用引物為PAH-RHD,如表2所示[21-22].反應體系50μL:10×Buffer(含2.0mmol/L MgCl2)5μL, dNTP(10mmol/L)1μL,GC(10μmol/L)1μL,NS1(10 μmol/L)1μL,Taq酶(5U/μL)0.25μL,母板 DNA 2μL,ddH2O補至50μL.PCR擴增程序:94℃ 5min預變性;94℃變性 30s,54℃退火 45s,72℃延伸1min,35個循環(huán);延伸7min,4℃保存.取PCR產物各2μL,1.5%瓊脂糖,1×TAE緩沖液,120V穩(wěn)壓電泳 15min,利用凝膠成像系統(tǒng)拍攝電泳圖譜查看PCR的擴增效果.

表2 PCR擴增引物Table 2 PCR amplification primers

1.5 變性梯度凝膠電泳DGGE和條帶回收

利用D-Code突變檢測系統(tǒng)對上述PCR產物(取PCR產物400ng)進行DGGE分析,8%的聚丙烯酰胺凝膠濃度,變性劑濃度梯度 15%～40%,電壓條件為70V,60℃恒溫,1×TAE中電泳13h.電泳結束后,利用超純水將膠沖洗干凈放入含 TE的染液中染色 30min,然后置于凝膠成像系統(tǒng)上拍攝圖譜.待凝膠成像系統(tǒng)拍攝電泳圖譜結束后,用潔凈的手術刀片將預先挑選的代表性目標DGGE條帶完整地切下裝入1.5mL離心管中,按上海生工公司試劑盒方法進行回收,備用.

1.6 PCR擴增、產物回收和目標片段測序

PCR反應體系與程序同上.PCR產物按上海生工測序公司 SK8131試劑盒方法切膠回收.依次經過連接、感受態(tài)細胞制備、連接產物轉化、藍白斑篩選和質粒提取過程后對目標片段進行測序,測序由上海生工完成.

2 結果與討論

2.1 PCR擴增產物和DGGE指紋圖譜分析

提取活性污泥樣品總DNA后,利用表2中的引物以總DNA為模板進行PCR擴增,發(fā)現(xiàn)引物RHD-GN-610F和RHD-GN-916R有明顯的擴增產物,如圖1所示,產物大小為300bp,這與相關研究報道結果一致[21,24],說明 A-A-O處理工藝好氧單元活性污泥中存在降解PAHs的RHD基因酶.

圖1 PCR擴增產物Fig.1 PCR amplification product

圖2 RHD基因DGGE 指紋圖譜分析Fig.2 DGGE analysis of RHD genes

對樣品PCR產物進行DGGE分析,發(fā)現(xiàn)有明顯的分離條帶(圖2),分別編號1、2、3、4、5、6、7、8(圖2),說明焦化廢水活性污泥中RHD基因存在多樣性.RHD基因酶由β和α終端雙加氧酶,鐵氧化還原蛋白和鐵氧化還原蛋白酶組成,在不同的染色體或質粒位點上,不同的PAH降解菌具有不同的 RHD編碼基因[17-18].焦化廢水活性污泥是一個復雜的微生物群落,存在不同的 PAHs降解菌株,如 Burkholderia sp.和 Pseudomonas sp.、Comamonas sp.和Diaphorobacter sp.等[26-28]. 8條條帶的亮度如圖2所示,亮度的大小可反映該條帶的豐富度,表3所示8條帶所占的豐度分別為2.99、8.16、20.75、28.50、8.62、7.26、10.62和13.10%,主要條帶為3和4號條帶,推測3和4號條帶的RHD基因酶是焦化廢水中降解PAHs的主要酶.用潔凈的手術刀片將1～8號目標DGGE條帶完整的切下利用引物 RHD-GN-610F和RHD-GN-916R PCR擴增后進行電泳檢測.

表3 RHD基因的多樣性和豐度Table 3 Diversities and richness of RHD genes

2.2 DGGE條帶PCR產物電泳分析

圖3 DGGE條帶PCR產物電泳圖Fig.3 Electrophoresis of DGGE band PCR products

如圖3所示,對DGGE指紋圖譜中的8條條帶進行回收經PCR擴增后發(fā)現(xiàn)有明顯的擴增產物,進一步證實了活性污泥中存在不同的 RHD基因酶,不同的PAH降解菌具有不同的RHD編碼基因[18],說明焦化廢水活性污泥中存在不同的PAHs降解菌株,萘、菲、芘的代謝過程分別說明

[18],RHD 加氧酶是 PAHs好氧代謝過程中最重要的關鍵酶和啟動酶,決定和控制后續(xù)的生物代謝過程.

2.3 DGGE條帶PCR產物序列分析

為了分析 DGGE條帶 PCR產物的序列,回收產物依次經過連接、感受態(tài)細胞制備、連接產物轉化、藍白斑篩選和質粒提取過程后對目標片段進行測序,成功測試出條帶編號分別為4、5、6和7的序列,序列長度分別為305bp、298bp、334bp、294bp,結果如下所示:

條帶4:305bp

GAGATGCATACCACGTTGGTTGGACGC ACGCATCGTCTTTGCGCTCGGGGCAGTCGA TATTTACTCCTCTTGCGGGCAACGCGATGCT TCCACCCGAAGGCGCGGGCTTGCAAATGAC CAGCAAGTATGGCAGTGGAATGGGCGTATT GTGGGACGCCTACTCCGGTGTCCACAGCGC TGATCTGGTTCCCGAAATGATGGCATTCGGC GGCGCAAAACAGGAAAAGCTCGCCAAAGA AATCGGCGATGTCCGGGCGCGGATTTACCG CAGCCATCTGAACAGCACTATCTTCCCGAA CAACAGC

條帶5:298bp

GAGATGCATACCACGTTGGTTGGACGC ACGCATCGTCTTTGCGCACAGGGCAGTCGA TATTTACCCCTCTTGCGGGCAACGCTATGCC TCCACCCGAAGGCGCGGGCTTACAAGTGA CCAGCAAGTATGGCAGTGGAATGGGCGTAT TGTGGGACGCCTACTCCGGTGTCCACAGCG CAGACCTGGTTCCCGAAATGATGGCATTCG GCGGCGCAAAACAGGAAAAGCTCGCCAAG AAATCGGCGATGTCCGGGCGCGGATTTACC GCAGCCATCGTCGACCTGCAGGCATGCAAG CT

條帶6:334bp

GAGATGCATACCACGTTGGTTGGACGC ACGCATCGTCTTTGCGCTCAGGGCAGTCGA TATTTACCCCTCTTGCGGGCAACGCTATGCC TCCACCCGAAGGCGCGGGCTTACAAGTGA CCAGCAAGTATGGCAGTGGAATGGGCGTAT TGTGGGACGCCTACTCCGGTGTCCACAGCG CTGATCTGGTTGCCGAACTGATGGCATTCG GCGCCGCAAGACAGGAAAAACTCGCCAAG GAAATCGGCGTTGTCCGGGCACAGATTTAC CGCAGCCATCTAAACAGCACTATCTTCCCG AACAACAGCTAACCGCACTATCTTCCCGAA CAACAGCT

條帶7:294bp

AGATGCATACCACGTTGGTTGGACGCA CGCATCGTCTTTGCGCACAGGGCAGTCGAT ATTTACTCCTCTTGCGGGCAACGCTACGCTT CCACCCGAAGGCGCGGGCTTGCAAATGAC CAGCAAGTATGGCAGTGGAATGGGCGTATT GTGGGACGCATACCACGTAGGTTGGACGCA CGCATCGTCTTTGCGCTCAGGGCAGTCGAT ATTTACTCCTCTTGCGGGCAACGCGATGCTT CCACCCGAAGGCGCGGGCTTGCAAATGAC CAGCAAGTATGGCAGTGGAATGGGCGT

利用BLAST在NCBI數(shù)據(jù)庫(http://blast.stva.ncbi.nlm.nih.gov/Blast.cgi)中進行同源性檢索BLAST比對分析顯示:條帶4,條帶5,條帶6和條帶 7分別與 PAH雙加氧酶基因(Accession AM743143.1), (Accession JX297666.1), (Accession AM743159.1),和(Accession AM743143.1)相似,相似度分別為98%,97%,96%和98%,說明克隆表達成功測序的4條的序列基因均為PAH雙加氧酶基因.

3 結論

3.1 提取焦化廢水活性污泥樣品總 DNA,利用引物以總 DNA為模板 PCR擴增,發(fā)現(xiàn)引物RHD-GN-610F和RHD-GN-916R擴增后有明顯產物,對PCR擴增產物進行DGGE分析,發(fā)現(xiàn)有8條條帶,豐度分別為2.99%、8.16%、20.75%、28.50%、8.62%、7.26%、10.62%和13.10%.

3.2 8條條帶經切膠回收PCR擴增后有明顯產物,進一步證實了活性污泥中存在不同的 RHD基因酶.TA克隆后.成功測試出4條序列.長度分別為305bp、298bp、334bp和294bp.

[1] Qiao M, Qi W X, Liu H J, et al. Occurrence, behavior and removal of typical substituted and parent polycyclic aromatic hydrocarbons in a biological wastewater treatment plant [J]. Water Research., 2014,52:11-19.

[2] Yao M, Zhang X W, Lei L C. Polycyclic aromatic hydrocarbons in the centralized wastewater treatment plant of a chemical industry zone: removal, mass balance and source analysis [J]. Science China Chemistry, 2012,55(3):416-425.

[3] Tian W J, Bai J, Liu K K, et al. Occurrence and removal of polycyclic aromatic hydrocarbons in the wastewater treatment process [J]. Ecotoxicology and Environmental Safety, 2012,82: 1-7.

[4] Zhang Y X, Tao S. Global atmospheric emission inventory of polycyclic aromatic hydrocarbons for 2004 [J]. Atmospheric Environment, 2009,43:812-819.

[5] Mu L, Peng L, Cao J J, et al. Emissions of polycyclic aromatic hydrocarbons from coking industries in China [J]. Particuology, 2013,11:86-93.

[6] Ma W, Li Y, Qi H, et al. Seasonal variations of sources of polycyclic aromatic hydrocarbons (PAHs) to a northeastern urban city, China [J]. Chemosphere, 2010,79(4):441-447.

[7] Pavlovich L B, Zhuravleva N V, Bal’tser D V. Polycyclic aromatic hydrocarbons in coke plant wastewater [J].Coke and Chemistry, 2010,53(10):390-395.

[8] Zhang W H, Wei C H, Chai X S, et al. The behaviors and fate of polycyclic aromatic hydrocarbons (PAHs) in a coking wastewater treatment plant [J]. Chemosphere, 2012,88(2):174-182.

[9] Vaessen H A M G, Jekel A A, Wilbers A A M M. Dietary intake of polycyclic aromatic hydrocarbons [J]. Toxicologiacl and Environmental Chemistry, 1988,16:281-287.

[10] 葛晨軍,俞花美.多環(huán)芳烴在土壤中的環(huán)境化學行為 [J]. 中國生態(tài)農業(yè)學報, 2006,14(1):162-165.

[11] Deng L J, Ren Y, Wei C H. Pyrene degradation by Pseudomonas sp. and Burkholderia sp. enriched from coking wastewater sludge [J]. Journal of Environmental Science and Health, part A, 2012, 47:1984-1991.

[12] Klankeo P, Nopcharoenkul W, Pinyakong O, et al. Two novel pyrene-degrading Diaphorobacter sp. and Pseudoxanthomonas sp. isolated from soil [J]. Journal of Bioscience and Bioengineering, 2009,108(6):488-495.

[13] Balachandran C, Duraipandiyan V, Balakrishna K,et al.Petroleum and polycyclic aromatic hydrocarbons (PAHs) degradation and naphthalene metabolism in Streptomyces sp. (ERI-CPDA-1) isolated from oil contaminated soil [J]. Bioresource Technology, 2012,112:83-89.

[14] Zhang J, Zhang X, Liu J, et al. Isolation of a thermophilic bacterium, Geobacillus sp. SH-1, capable of degrading aliphatic hydrocarbons and naphthalene simultaneously and identification of its naphthalene degrading pathway [J]. Bioresource Technology, 2012,124:83-89.

[15] Lin M, Hu X K, Chen W W, et al. Biodegradation of phenanthrene by Pseudomonas sp. BZ-3, isolated, from crude oil contaminated soil [J]. International Biodeterioration & Biodegradation, 2014,94:176-181.

[16] Tao X Q, Lu G N, Dang Z, et al. A phenanthrene-degrading strain Sphingomonas sp. GY2B isolated from contaminated soils [J]. Process Biochemistry, 2007,42:401-408.

[17] Wongwongsee W, Chareanpat P, Pinyakong O. Abilities and genes for PAH biodegradation of bacteria isolated from mangrove sediments from the central of Thailand [J]. Marine Pollution bulletin, 2013,74:95-104.

[18] Peng R H, Xiong A S, Xue Y, et al. Microbial biodegradation of polyaromatic hydrocarbons [J]. FEMS Microbiology Reviews, 2008,32:927-955.

[19] Zhou H W, Guo C L, Wong, Y S, et al. Genetic diversity of dioxygenase genes in polycyclic aromatic hydrocarbon-degrading bacteria isolated from mangrove sediments [J]. FEMS Microbiology Letters, 2006,262:148-157.

[20] Gomes N C M, Borges L R, Paranhos R, et al. Diversity of nod genes in mangrove sediments exposed to different sources of polycyclic aromatic hydrocarbon pollution [J]. Applied Environmental Microbiology, 2007,73:7392-7399.

[21] Cébron A, Norini M P, Beguiristain T, et al. Real-Time PCR quantification of PAH-ring hydroxylating dioxygenase (PAHRHDα) genes from Gram positive and Gram negative bacteria in soil and sediment samples [J]. Journal of Microbiological Methods, 2008,73:148-159.

[22] Ding G C, Heuer H, Zuhlke S, et al. Soil type-dependent responses to phenanthrene as revealed by determining the diversity and abundance of polycyclic aromatic hydrocarbon ring-hydroxylating dioxygenase genes by using a novel PCR detection system [J]. Applied and Environmental Microbiology, 2010,76(14):4765-4771.

[23] 李全霞,范丙全,龔明波,等.降解芘的分枝桿菌M11的分離鑒定和降解特性 [J]. 環(huán)境科學, 2008,29(3):763-766.

[24] 崔長征,馮天才,于亞琦,等.降解蒽嗜鹽菌AD-3的篩選、降解特性及加氧酶基因的研究 [J]. 環(huán)境科學, 2012,33(11):4062-4068.

[25] 何 芬,王立華,寧大亮,等.嗜鹽菌群對菲的降解及萘雙加氧酶基因的表達規(guī)律 [J]. 中國環(huán)境科學, 2012,32(9):1662-1669.

[26] Deng L J, Ren Y, Wei C H. Pyrene degradation by Pseudomonas sp. and Burkholderia sp. enriched from coking wastewater sludge [J]. Journal of Environmental Science and Health, part A, 2012,47: 1984-1991.

[27] Ma Q, Qu Y Y, Shen W L, et al. Bacterial community compositions of coking wastewater treatment plants in steel industry revealed by Illumina high-throughput sequencing [J]. Bioresource Technology, 2015,179:436-443.

[28] Klankeo P, Nopcharoenkul W, Pinyakong O. Two novel pyrene-degrading Diaphorobacter sp. and Pseudoxanthomonas sp. isolated from soil [J]. Journal of Bioscience and Bioengineering, 2009,108:488-495.

Diversity of PAH dioxygenase genes of activated sludge from coking wastewater.

MENG Xiao-jun1,2, LI Hai-bo2, SHENG Yu-xing2, CAO Hong-bin2*
(1.School of Tourism and Environment, Ankang University, Ankang 725000, China；2.Key Laboratory of Green Process and Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China). China Environmental Science, 2017,37(1)：367～372

In order to analyze the biodiversity of PAH dioxygenase in activated sludge from a full-scale coking wastewater, primers were used for dioxygenase gene cloning expression and diversity analysis by 16Sr DNA-PCR-DGGE method using total DNA of aerobic activated sludge as the template. The results showed that significant amplification product was found using primers RHD-GN-610F and RHD-GN-916R, and the product size was 300bp. PCR product was conducted by DGGE analysis and eight separate bands were found, the abundance was 2.99%, 8.16%, 20.75%, 28.50%, 8.62%, 7.26%, 10.62% and 13.10%, respectively. Obvious amplification products further appeared after strips by gel extraction and PCR amplification, and 4sequences were tested successfully by TA cloning, the length of the sequences was 305bp, 298bp, 334bp and 294bp, respectively, indicating the presence of different PAH RHD enzymes in coking activated sludge. These results provide a theoretical basis for the risk assessment and potential biodegradation of PAHs.

coking wastewater；PAHs；activated sludge；dioxygenase gene

X703

A

1000-6923(2017)01-0367-06

蒙小俊(1981-),男,陜西漢中人,博士,主要從事生物法處理工業(yè)廢水研究.發(fā)表論文16篇.

2016-05-10

安康學院高層次人才科研專項經費(2016AYQDZR09);國家自然科學基金項目(31370281);化學工業(yè)廢水處理污泥污染特征與污染風險控制研究(201509053)

* 責任作者, 研究員, hbcao@home.ipe.ac.cn