權(quán) 躍,吳 昊,尹振浩,郭春雨,尹成日,*

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靜態(tài)磁場(chǎng)對(duì)生物滴濾池強(qiáng)化的研究——以三氯乙烯廢氣的去除及細(xì)菌群落結(jié)構(gòu)研究為例

權(quán) 躍1,2,吳 昊2,尹振浩3,郭春雨2,尹成日2,3*

(1.延邊大學(xué)農(nóng)學(xué)院環(huán)境科學(xué)系,吉林 延吉 133002；2.延邊大學(xué)理學(xué)院化學(xué)系,吉林 延吉 133002；3.延邊大學(xué)分析測(cè)試中心,吉林 延吉 133002)

構(gòu)建了實(shí)驗(yàn)室規(guī)模磁場(chǎng)強(qiáng)化的生物滴濾池(MF-BTF),并設(shè)對(duì)照生物滴濾池(S-BTF),以火山石為載體,接種活性污泥,處理目標(biāo)廢氣三氯乙烯(TCE).研究了不同磁場(chǎng)強(qiáng)度下,2種生物滴濾池在穩(wěn)定運(yùn)行期間對(duì)TCE的去除效果,并采用高通量測(cè)序技術(shù)對(duì)滴濾池中細(xì)菌群落結(jié)構(gòu)與功能進(jìn)行了分析.實(shí)驗(yàn)結(jié)果表明:在好氧條件下,當(dāng)空床停留時(shí)間(EBRT)為202.5s,苯酚濃度為0.20g/L,TCE濃度范圍為53.6~337.1mg/m3時(shí),對(duì)TCE的去除效果依次為MF-BTF(60.0mT)>MF-BTF(30.0mT)>S-BTF (0.0mT)>MF-BTF(130.0mT),其去除率(RE)和最大去除容量(EC)分別為92.2%~45.5%, 2656.8mg/(m3×h); 89.8%~37.2%, 2169.1mg/(m3×h); 89.8%~29.8%, 1967.7mg/(m3×h); 76.0%~20.8%, 1697.1mg/(m3×h).高通量測(cè)序結(jié)果表明:磁場(chǎng)強(qiáng)度為60.0mT時(shí),細(xì)菌群落均勻度(Shannon index, Simpson index)及豐富度(OTUs,Chao1,ACE)均小于0.0mT的情況;但是60.0mT下優(yōu)勢(shì)門、綱、屬為Proteobacteria, Gammaproteobacteria,,豐度明顯大于0.0mT,豐度分別為73.3%,36.8%,34.7%;69.6%,18.2%,10.9%.實(shí)驗(yàn)結(jié)果表明合適的磁場(chǎng)強(qiáng)度可以增大優(yōu)勢(shì)菌群的豐度,提高對(duì)TCE的去除效果.本實(shí)驗(yàn)為提高生物滴濾池的工業(yè)廢氣處理效果提供了有效的借鑒,具有廣泛的應(yīng)用前景.

磁場(chǎng)；生物滴濾池；三氯乙烯；細(xì)菌群落；高通量

生物法凈化處理有機(jī)廢氣(VOCs)具有高效、低能耗、運(yùn)行管理方便等特點(diǎn),目前成為主要的處理方法[1].生物處理技術(shù)中生物轉(zhuǎn)鼓和膜反應(yīng)器對(duì)廢氣處理效果好,但是前者密封要求高,工程放大比較困難;后者的膜材料價(jià)格高,成本高于其他生物反應(yīng)器[2].而生物滴濾技術(shù)具有占地面積小、操作簡(jiǎn)單、壓降低,填料使用壽命長(zhǎng),耐酸代謝產(chǎn)物的優(yōu)勢(shì)能力[3-4].但由于疏水性VOCs的溶解度小,氣液傳質(zhì)受限制,生物滴濾池對(duì)其處理效果差.

近年來出現(xiàn)很多提高生物滴濾池對(duì)疏水性有機(jī)廢氣降解效果的新技術(shù),如添加表面活性劑、構(gòu)建真菌生物滴濾池、組合的生物過濾技術(shù)等.表面活性劑可以增加疏水性VOCs在氣液界面的傳質(zhì)速率及在水相中的溶解度,能有效降低氣液間的傳質(zhì)阻力[5],如皂苷、十二烷基磺酸鈉均能提高正己烷在生物反應(yīng)器中的降解效果[6-7].而海藻酸R810-60未能提高生物濾池降解TCE,四氯乙烯性能[8].表面活性劑容易被微生物降解,產(chǎn)生一些中間副產(chǎn)物,另外表面活性劑具有清洗作用,減少生物量[9].真菌比細(xì)菌對(duì)疏水性VOCs具有很高的去除能力,但是真菌需要特定的生長(zhǎng)條件(pH值、濕度、溫度及營(yíng)養(yǎng)物質(zhì))[10],菌絲的過度生長(zhǎng)會(huì)使反應(yīng)器壓降升高,引起填料堵塞等問題.組合生物過濾技術(shù)中紫外光解技術(shù)作為一種化學(xué)預(yù)處理技術(shù),把疏水性有機(jī)廢氣光解為可溶的小分子化合物,提高生物滴濾池處理效果[11],但是容易產(chǎn)生O3和一些毒性更強(qiáng)的副產(chǎn)物.另外,光催化氧化技術(shù)是在紫外光的照射下,利用半導(dǎo)體如TiO2、ZnO、WO3、FeTiO3和SrTiO3等材料對(duì)有機(jī)污染物進(jìn)行光誘導(dǎo)使其分解成水溶性較高且可生化性強(qiáng)的物質(zhì)[12].但是,光催化氧化技術(shù)費(fèi)用高,易產(chǎn)生很多種有害副產(chǎn)物,而且存在催化劑易失活的缺點(diǎn),使得光解效率下降[13-15].

因此尋求一種高效、經(jīng)濟(jì)環(huán)保的生物滴濾技術(shù)對(duì)提高對(duì)疏水性VOCs的降解效果十分必要.磁技術(shù)強(qiáng)化微生物降解有機(jī)物是近幾年新興的一項(xiàng)生物強(qiáng)化技術(shù),作為一種環(huán)保、無毒、無須額外的能源和藥劑投入的方法[16],可以提高廢水中氨氮、COD、甲醛、苯酚、染料和重金屬的去除效果[17-20].但到目前為止,磁場(chǎng)在廢氣協(xié)同生物降解中未見報(bào)道.三氯乙烯(TCE)作為一種優(yōu)良的溶劑廣泛應(yīng)用于多個(gè)行業(yè),由于不合理的排放和意外泄露,它被大量釋放到環(huán)境中[21].TCE屬于氯代揮發(fā)性持久有機(jī)污染物,在環(huán)境中很難降解,所以探索高效、經(jīng)濟(jì)、環(huán)保的有效治理途徑尤為重要.

基于上述研究背景分析,本研究以TCE為目標(biāo)廢氣,構(gòu)建了實(shí)驗(yàn)室規(guī)模磁場(chǎng)強(qiáng)化的生物滴濾池(MF-BTF)和對(duì)照生物滴濾池(S-BTF),考察穩(wěn)定期運(yùn)行期間,不同磁場(chǎng)強(qiáng)度對(duì)TCE降解效果的影響,并應(yīng)用高通量測(cè)序技術(shù)對(duì)60.0mT、0.0mT、IAS(原始活性污泥)中細(xì)菌群落結(jié)構(gòu)和功能進(jìn)行分析.以期為磁場(chǎng)條件下提高生物滴濾技術(shù)對(duì)疏水性VOCs的處理效果提供有效借鑒.

1 材料與方法

1.1 生物滴濾池的構(gòu)建與啟動(dòng)

本實(shí)驗(yàn)在室溫(21±2)℃條件構(gòu)建了外加磁場(chǎng)MF-BTF反應(yīng)器及其對(duì)照反應(yīng)器S-BTF,如圖1所示.滴濾池材料為有機(jī)玻璃,高度為1000mm,內(nèi)徑為110mm,工作容積可達(dá)2.7L,內(nèi)部填料為粒徑8~10mm的長(zhǎng)白山火山石.磁場(chǎng)是由兩塊磁鐵(長(zhǎng)120mm,寬100mm)產(chǎn)生的異級(jí)磁場(chǎng),磁鐵材料為釹鐵硼,磁場(chǎng)強(qiáng)度由HT20型特斯拉計(jì)(上海亨通磁電技術(shù)有限公司)測(cè)量.

向MF-BTF反應(yīng)器內(nèi)注入已馴化的活性污泥并于24h后排放.反應(yīng)器采用氣液逆流的方式運(yùn)行.即TCE與空氣混合后從滴濾池底部通入,通過質(zhì)量流量計(jì)控制進(jìn)氣濃度和流量,流量設(shè)置為0.8L/min,對(duì)應(yīng)的空床停留時(shí)間(EBRT)為202.5s;無機(jī)鹽營(yíng)養(yǎng)液通過蠕動(dòng)泵每隔8h,由滴濾池頂部噴灑10min,速度為3L/h,每2d更換一次營(yíng)養(yǎng)液,每天都對(duì)營(yíng)養(yǎng)液的pH值進(jìn)行監(jiān)測(cè),并調(diào)節(jié)pH值為7.2左右.

1.2 微生物的培養(yǎng)與接種

好氧活性污泥取自延吉市污水處理廠的曝氣池.取回的活性污泥靜置24h,棄去上清液,加入營(yíng)養(yǎng)液在水桶內(nèi)曝氣培養(yǎng)一周.之后對(duì)其進(jìn)行馴化,即每隔2d排除上清液,添加新的營(yíng)養(yǎng)液,營(yíng)養(yǎng)液中逐漸減少乙酸鈉的含量,相反增大苯酚濃度至0.20g/L,并加入低濃度的TCE溶液,直到培養(yǎng)馴化60d結(jié)束.營(yíng)養(yǎng)液為:0.60g/L乙酸鈉,0.46g/L磷酸氫二鉀,0.38g/L磷酸二氫鉀,0.48g/L硫酸銨,0.40g/L氯化鈉,0.1g的微量元素混合液,pH值調(diào)節(jié)為7.2.微量元素混合液(g/L)的成分有: CuSO4·5H2O,0.01;KI,0.01;CaCl2·2H2O,0.132;Na2MoO4·2H2O,0.02;KAl(SO4)2·12H2O,0.02;MnSO4·H2O,0.045;CoCl2·6H2O,0.05;FeCl3,0.12;H3BO3,0.05; ZnSO4·7H2O,0.075.

1.3 分析方法

TCE濃度由島津GC-2010氣相色譜儀測(cè)定,FID檢測(cè)器,色譜柱型號(hào)為Rtx-1701 (30m× 0.25mm×0.25μm).分析條件為:進(jìn)樣口和檢測(cè)器的溫度為220℃,毛細(xì)管柱的溫度為80℃,載體的氮?dú)饬魉贋?36.3mL/min,柱體流速為1.32mL/ min.采用分流模式,分流比為100.在此條件下,TCE的保留時(shí)間約為2.59min.

圖1 磁場(chǎng)強(qiáng)化生物滴濾池裝置示意及填料掛膜后SEM圖像

1.4 細(xì)菌群落分析

1.4.1 基因組DNA提取 為分析有、無磁場(chǎng)對(duì)生物膜中細(xì)菌群落結(jié)構(gòu)的影響,本實(shí)驗(yàn)選取MF-BTF反應(yīng)器最佳磁場(chǎng)強(qiáng)度為60.0mT(31d, 182.4mg/m3TCE)和S-BTF反應(yīng)器(0.0mT,15d, 171.1mg/m3TCE)下的生物膜.取自延吉市污水處理廠的原始活性污泥(IAS) 作為對(duì)照.按照Power Soil DNA試劑盒的說明提取基因組DNA.提取的DNA在1%的瓊脂糖凝膠電泳上檢測(cè),并測(cè)定DNA濃度,稀釋至5.0ng/μL待用.DNA樣品在進(jìn)行后續(xù)的分析之前貯存在-80℃的溫度下.

1.4.2 PCR擴(kuò)增反應(yīng) 對(duì)細(xì)菌DNA的16S rRNA的V4區(qū)域進(jìn)行擴(kuò)增,引物為515F(5￠- GTGCCAGCMGCCGCGGTAA-3￠)和806R(5￠- GGACTACHVGGGTWTCTAAT-3￠).PCR反應(yīng)采用50.0μL體系,包含:0.3μL Taq DNA聚合酶(上海生工,5.0U/μL), 6.0μL dNTP mix (2.5mmol/L), 0.6μL BSA, 1.2μL 引物(10μmol/L), 6.0μL 10×ExTaq 緩沖液, 1.0μL DNA和43.7μL無菌去離子水.PCR反應(yīng)條件為:94℃預(yù)變性5.0min, 94 ℃下變性1min,55℃復(fù)性30s, 72℃延伸45s,重復(fù)31個(gè)循環(huán),最終在72℃下延伸10.0min.擴(kuò)增產(chǎn)物在加入嗅化乙錠的2%的瓊脂糖凝膠上檢測(cè).使用TaKaRa DNA Fragment Purification Kit Ver.2.0進(jìn)行DNA純化回收.

1.4.3 高通量測(cè)序 使用TruSeq? DNA PCR- Free Sample Preparation Kit建庫(kù)試劑盒進(jìn)行文庫(kù)構(gòu)建,構(gòu)建好的文庫(kù)經(jīng)過Qubit和Q-PCR定量,文庫(kù)合格后,使用HiSeq2500PE250進(jìn)行上機(jī)測(cè)序(北京諾禾致源生物信息科技有限公司).

1.4.4 生物信息學(xué)分析 對(duì)原始序列截去Barcode和引物序列,后使用FLASH軟件對(duì)每個(gè)樣品的reads進(jìn)行拼接,得到的拼接序列為原始Tags數(shù)據(jù)[22];再對(duì)其經(jīng)過嚴(yán)格的過濾處理得到高質(zhì)量的Clean Tags[23].之后參照Qiime的Tags質(zhì)量控制流程進(jìn)一步對(duì)Tags進(jìn)行截取和長(zhǎng)度過濾[24];所得Tags序列通過UCHIME Algorithm[25]與數(shù)據(jù)庫(kù)(Gold database)進(jìn)行比對(duì)檢測(cè)、去除嵌合體序列[26],從而得到最終的有效數(shù)據(jù)(Effective Tags).對(duì)所有樣品的Effective Tags進(jìn)行聚類,以97%的一致性將序列聚類組合為OTUs,該OTUs是基于Uparse軟件的距離矩陣[27].對(duì)OTUs代表序列用Mothur方法與SILVA的SSUrRNA數(shù)據(jù)庫(kù)進(jìn)行物種注釋分析(設(shè)定閾值為0.8-1),獲得分類學(xué)信息并分別在各個(gè)分類水平,統(tǒng)計(jì)各樣本的群落組成.使用Qiime軟件(Version 1.7.0)計(jì)算Chao1,Shannon,Simpson,ACE,Goods-coverage指數(shù),使用R軟件(Version 2.15.3)繪制稀釋曲線等.原始數(shù)據(jù)已被上傳到NCBI數(shù)據(jù)庫(kù), SRA: SRP1104671.

2 結(jié)果與討論

2.1 磁場(chǎng)強(qiáng)度對(duì)生物滴濾池去除性能的影響

磁現(xiàn)象是一種普遍存在的物理現(xiàn)象,人們每時(shí)每刻都生存在地球這個(gè)大磁場(chǎng)之中.有研究表明,外加磁場(chǎng)和生物體自身的磁場(chǎng)均能對(duì)生物體代謝產(chǎn)生影響,即生物磁效應(yīng)[28].低磁場(chǎng)強(qiáng)度對(duì)微生物生長(zhǎng)有正效應(yīng),提高其對(duì)有機(jī)物的降解能力,然而磁場(chǎng)強(qiáng)度過大,起抑制作用[29].因此,為了確定不同磁場(chǎng)強(qiáng)度對(duì)反應(yīng)器性能的影響,本實(shí)驗(yàn)在反應(yīng)器穩(wěn)定運(yùn)行期,以0.20g/L苯酚作為共代謝底物,EBRT為202.5s,探討了0.0,30.0,60.0和130.0mT條件下,兩個(gè)種反應(yīng)器對(duì)TCE的降解效果,進(jìn)氣濃度為53.6~337.1mg/m3條件下,結(jié)果見圖2.

評(píng)價(jià)生物滴濾池性能的主要指標(biāo)之一是有機(jī)物的去除率 (RE),RE是指處理后有機(jī)物濃度占進(jìn)氣濃度的比率.從圖2可以看出,無論是S-BTF還是MF-BTF,隨著TCE濃度的增大,RE都呈現(xiàn)緩慢下降的趨勢(shì).在穩(wěn)定期1~19d, TCE進(jìn)氣濃度控制在53.6~326.8mg/m3,S-BTF(0.0mT)的RE從89.8%下降到29.8%;而在MF-BTF中,磁場(chǎng)強(qiáng)度為30.0mT, RE從89.8%下降到37.2%,由此說明在開始幾天RE相差不多,但隨著時(shí)間的延長(zhǎng),磁場(chǎng)對(duì)提高生物滴濾池降解TCE效果較為明顯,尤其是到19d,MF-BTF的RE降至37.2%,大于S-BTF反應(yīng)器的29.8%.而接著在21~39d, MF-BTF反應(yīng)器中磁場(chǎng)強(qiáng)度為60.0mT,TCE濃度為53.6~326.8mg/m3,RE從92.2%下降到45.5%.由此說明,60.0mT磁場(chǎng)強(qiáng)度下對(duì)TCE的降解效果好于30.0mT和0.0mT.接著增大磁場(chǎng)強(qiáng)度為130.0mT,此時(shí)的TCE濃度控制為58.7~ 337.1mg/m3,RE為76.0%~20.8%,此時(shí)RE最差,甚至小于0.0mT,這說明當(dāng)磁場(chǎng)強(qiáng)度為130.0mT時(shí),磁場(chǎng)對(duì)生物滴濾的TCE降解起到抑制作用.

評(píng)價(jià)生物滴濾池性能的另一個(gè)主要指標(biāo)是有機(jī)物的降解能力(EC),EC是指單位體積濾料在單位時(shí)間內(nèi)有機(jī)物的降解量.進(jìn)氣負(fù)荷(ILR)對(duì)EC的影響見圖3.由圖3可知,EC均表現(xiàn)為相同的趨勢(shì),隨著進(jìn)氣負(fù)荷的增大,EC均表現(xiàn)為先增大,增大到某一值后,均表現(xiàn)為下降趨勢(shì).4種條件下對(duì)EC的影響趨勢(shì)為MF-BTF(60.0mT) > MF- BTF (30.0mT) > S-BTF (0.0mT) > MF-BTF (130.0mT).最大的EC在這4個(gè)階段分別為 2656.8,2169.1,1967.7和1679.1mg/(m3?h),所對(duì)應(yīng)的進(jìn)氣負(fù)荷(ILR)分別為4253.0, 3732.4, 4681.4和4290.8mg/(m3?h).截至目前,利用生物滴濾池去除TCE的文獻(xiàn)報(bào)道僅有一篇[30],G4作為降解菌,陶粒為填料,牛肉湯為營(yíng)養(yǎng)液,苯酚為共代謝底物,EBRT為3.8~9.8min, TCE進(jìn)氣負(fù)荷為492.0~1708.0mg/(m3×h),所獲得的最大的EC僅為1167mg/(m3×h).由此可見,MF-BTF (60.0mT)對(duì)TCE的去除效果較好,其EC值[2656.8mg/(m3×h)]是上述數(shù)值的近2倍.

通過對(duì)上述RE和EC的評(píng)價(jià),表明60.0mT磁場(chǎng)強(qiáng)化下生物滴濾池對(duì)TCE去除效果較好.已有研究表明弱磁場(chǎng)或中等強(qiáng)度的磁場(chǎng)可以促進(jìn)微生物生長(zhǎng),如370mT磁場(chǎng)強(qiáng)度下,通過縮短生長(zhǎng)滯后期和對(duì)數(shù)期,生長(zhǎng)速度提高28%,活性增加10%,苯酚的降解率提高34%[19].在17mT磁場(chǎng)強(qiáng)化下可促進(jìn)等氨氧化菌的生長(zhǎng),提高氨氧化速率,從而促進(jìn)亞硝酸鹽的積累[31].另外磁場(chǎng)可以提高活性污泥的污染物去除效果,外加80mT磁場(chǎng)的活性污泥系統(tǒng)較對(duì)照系統(tǒng)的COD、NH3-N、PO43--P去除率分別提高10.7%、0.4%和6.1%[32].20~ 40mT磁場(chǎng),可以促進(jìn)活性污泥在低溫5℃產(chǎn)生較多的不飽和脂肪酸,提高脫氫酶含量[33].除此之外,磁場(chǎng)條件下可以提高培養(yǎng)液里溶解氧的含量,從而促進(jìn)需氧菌的生長(zhǎng),進(jìn)而提高生物膜中的溶解氧,提高廢水廢氣的微生物降解效果[34].通過以上研究分析,作者認(rèn)為在外加60.0mT磁場(chǎng)下的生物滴濾器對(duì)TCE具有較好的降解效果,分析原因?yàn)樯锬ぶ械娜芙庋鹾吭黾?促進(jìn)好氧菌的生長(zhǎng)速度,生物膜中胞外多糖和蛋白質(zhì)含量增加,功能酶的含量提高;此外,在磁場(chǎng)強(qiáng)化下,微生物抵御不良條件的能力也增強(qiáng).

圖2 0, 30.0, 60.0, 130.0mT下的進(jìn)氣濃度與去除率曲線

圖3 0.0,30.0,60.0,130.0mT下,TCE進(jìn)氣負(fù)荷對(duì)生物滴濾池去除能力的影響

2.2 細(xì)菌群落多樣性分析

本次測(cè)序各樣品原始序列數(shù)分布均勻,基本在160,000條左右,如表1所示.除去識(shí)別錯(cuò)誤以及質(zhì)量差的序列后,有效序列數(shù)在IAS、MF- BTF、S-BTF中分別為161,309、157,078和152, 160條.對(duì)不同樣品的有效序列在97%一致性閾值(148,419)下的Alpha Diversity分析指數(shù)進(jìn)行統(tǒng)計(jì),見表1.物種多樣性有兩種含義:其一是豐富度,指的是物種數(shù)目的多寡;其二是均勻度,指的是各物種個(gè)體數(shù)目分配的均勻程度.OTUs、Chao1和ACE指數(shù)反映著細(xì)菌的豐富度; Shannon index 和Simpson index反映了細(xì)菌的均勻度[35].

由表1可見,細(xì)菌的豐富度和均勻度依次為IAS>MF-BTF>S-BTF.這是由于IAS是直接取自污水處理廠,含有較多的細(xì)菌.而活性污泥經(jīng)過馴化后,通過競(jìng)爭(zhēng)和選擇作用,一些細(xì)菌被淘汰,適應(yīng)性強(qiáng)的細(xì)菌存活下來,致使豐富度和均勻度均變小.而MF-BTF中細(xì)菌的豐富度和均勻度均小于S-BTF,說明一些細(xì)菌不適應(yīng)在磁場(chǎng)條件下生存,磁場(chǎng)的存在對(duì)有些細(xì)菌產(chǎn)生抑制作用,而有些細(xì)菌適合生長(zhǎng)在磁場(chǎng)條件下.所有樣品的測(cè)序深度均為0.99,表明獲取自每一樣品中的序列可以覆蓋細(xì)菌的多樣性.

表1 MF-BTF,S-BTF和IAS中細(xì)菌豐富度和多樣性指數(shù)

稀疏曲線進(jìn)一步解釋了樣品測(cè)序深度的合理性和準(zhǔn)確性,當(dāng)曲線趨向平坦時(shí),說明測(cè)序數(shù)據(jù)讀取量逐漸合理,并間接反映樣品中物種的豐富度.該曲線是從樣品中隨機(jī)抽取一定測(cè)序量的數(shù)據(jù),統(tǒng)計(jì)它們所代表物種數(shù)目(即OTUs數(shù)目),以抽取的測(cè)序量與對(duì)應(yīng)的物種數(shù)來構(gòu)建曲線,如圖4.由圖4可知,MF-BTF中的OTUs數(shù)目始終低于IAS和S-BTF,說明MF-BTF中細(xì)菌豐富度最小;而當(dāng)所讀取的序列數(shù)超過150,000時(shí),曲線趨于平緩,說明測(cè)序達(dá)到了要求,如果繼續(xù)讀取更多的序列數(shù),新產(chǎn)生的物種很少,會(huì)造成浪費(fèi).

圖4 高通量測(cè)序中IAS,S-BTF和MF-BTF細(xì)菌群落稀釋曲線

2.3 細(xì)菌群落結(jié)構(gòu)分析

圖5 細(xì)菌群落層次聚類

根據(jù)所有樣品在屬水平的物種注釋及豐度信息,選取豐度排名前35的屬,根據(jù)其在每個(gè)樣品中的豐度信息,從物種和樣品兩個(gè)層面進(jìn)行聚類,繪制成熱圖,用來直觀的觀察樣品中細(xì)菌群落結(jié)構(gòu)的差別,如圖5所示.3個(gè)樣品被聚為2類,IAS單獨(dú)為一類,2個(gè)反應(yīng)器中細(xì)菌群落被聚為一類,主要原因?yàn)镮AS為污水廠原始活性污泥,而MF-BTF和S-BTF均經(jīng)過苯酚和TCE的馴化,所以被聚為一類.由圖5可見所有細(xì)菌分成A組、B組、C組3大類,群落豐度在3個(gè)樣品中差別明顯.A組細(xì)菌的豐度在IAS中較大,B組細(xì)菌的豐度在S-BTF中較大,C組細(xì)菌在MF-BTF中較大,環(huán)境差別造成了群落豐度的明顯差別.

為了進(jìn)一步明確3個(gè)樣品細(xì)菌群落的差別,對(duì)所有樣品進(jìn)行均一化處理繪制韋恩圖(圖6).從圖6可知,3個(gè)樣品中共有的OTUs為785,所占比例為28.0%;IAS和MF-BTF中共有的OTUs為973,所占比例為48.8%;IAS和S-BTF樣品中共有的1008,所占比例為50.2%,MF-BTF和S-BTF中共有的OTUs為974,占總OTUs的54.3%.由此說明MF-BTF和S-BTF中細(xì)菌群落相似性最大,主要是由于2者所處的環(huán)境相似性大于IAS,而受磁場(chǎng)的影響二者的相似性僅為54.3%,說明磁場(chǎng)對(duì)細(xì)菌群落結(jié)構(gòu)有很大影響.

圖6 高通量測(cè)序中IAS, S-BTF和MF-BTF細(xì)菌群落韋恩

2.4 細(xì)菌在門、綱、屬分類單元的差別

選取每個(gè)樣品在分類水平(門、綱、屬)上最大豐度排名前10的物種,做成物種相對(duì)豐度柱形累加圖,如圖7所示.圖7a中,在IAS、MF-BTF、S-BTF中排名前10門的相對(duì)豐度之和占總細(xì)菌的比例分別為86.9%、87.5%和88.4%. Bacteroidetes和Proteobacteria是其中2個(gè)優(yōu)勢(shì)門.Bacteroidetes的相對(duì)豐度在IAS中為30.7%,而在MF-BTF、S-BTF中明顯下降僅為10.1%, 8.7%.另外Proteobacteria經(jīng)過馴化在MF-BTF和S-BTF中所占豐度為73.3%和69.6%,明顯大于IAS中的46.2%.

圖7 IAS、MF-BTF和S-BTF中細(xì)菌群落在門、綱、屬水平的相對(duì)豐度

這表明馴化環(huán)境抑制Bacteroidetes的生長(zhǎng),而Proteobacteria門的豐度在TCE降解過程中顯著增加,是苯酚共代謝降解TCE的優(yōu)勢(shì)菌門.已報(bào)道的苯酚降解優(yōu)勢(shì)菌門也屬于Proteobacteria,與本研究結(jié)果一致[36-37].

圖7b表明了3個(gè)樣品中豐度較大的前10個(gè)細(xì)菌綱.在IAS、MF-BTF和S-BTF中,前10個(gè)綱的細(xì)菌分別占總細(xì)菌的81.4%,85.9%,86.1%.優(yōu)勢(shì)綱為Gammaproteobacteria,在MF-BTF中最高為36.84%,而在S-BTF和IAS中分別18.2%和10.4%.Gammaproteobacteria作為優(yōu)勢(shì)綱與已報(bào)道的作為苯酚降解菌綱一致[36-37].

圖7c描述了前10個(gè)豐度較大的細(xì)菌屬.在IAS、MF-BTF和S-BTF中,前10個(gè)屬的細(xì)菌分別占總細(xì)菌豐度的20.2%,55.0%和49.2%.其中優(yōu)勢(shì)屬屬于Proteobacteria門, Gammaproteobacteria綱.其相對(duì)豐度MF-BTF (34.7%)中的為S-BTF(10.9%)中的3倍,為IAS(5.2%)中的6倍.這表明在MF-BTF反應(yīng)器中磁場(chǎng)可以促進(jìn)優(yōu)勢(shì)屬生長(zhǎng),明顯提高在細(xì)菌群落中的豐度sp. 已應(yīng)用于多種污染物的生物修復(fù)中,如阿特拉津[38]、鄰苯二甲酸二丁酯[39]、柴油[40]、萘[41]等.近年來,由于對(duì)苯酚具有較高的生物降解活性[42-44],而受到廣泛關(guān)注,被應(yīng)用到TCE的共代謝降解中.另外兩種細(xì)菌屬于和,二者以TCE和苯酚降解過程中的中間體作為碳源.在MF-BTF中,的相對(duì)豐度最大為4.2%,而S-BTF和IAS中僅為1.6%和0.6%.可降解多種有機(jī)氯農(nóng)藥,并利用其作為唯一碳源和能源[45],這意味著他可以降解TCE共代謝過程中的中間產(chǎn)物.sp.被報(bào)道為TCE共代謝的一種菌,另外還可以降解喹啉[46],聚丁二酸丁二醇酯薄膜[47]和有機(jī)酸[48-49]等.sp.的相對(duì)豐度在MF-BTF(3.8%)中高于S-BTF (2.2%),這可能與降解過程中產(chǎn)生的有機(jī)酸以及TCE共代謝降解有關(guān).MF-BTF反應(yīng)器中和的相對(duì)豐度明顯高于S-BTF,表明磁場(chǎng)促進(jìn)功能微生物的生長(zhǎng)和苯酚加氧酶的產(chǎn)生,從而提高TCE去除效果.門、綱和屬水平的細(xì)菌群落分析結(jié)果表明MF-BTF反應(yīng)器中優(yōu)勢(shì)群落豐度大于S-BTF和IAS,從而提高M(jìn)F-BTF反應(yīng)器對(duì)TCE的降解效果.

3 結(jié)論

3.1 0.20g/L苯酚為共代謝底物,EBRT為202.5s,進(jìn)氣濃度為53.6~337.1mg/m3,不同磁場(chǎng)下生物滴濾池對(duì)TCE降解能力為60.0mT>30.0mT> 0.0mT>130.0mT.磁場(chǎng)強(qiáng)度為60.0mT下的生物濾池對(duì)TCE的RE為92.2%~45.5%,最大EC為2656.8mg/(m3×h),而無磁場(chǎng)(0.0mT)條件下的RE和最大EC僅為89.8%~29.8%和1967.7mg/ (m3×h).

3.2 高通量測(cè)序結(jié)果表明磁場(chǎng)對(duì)細(xì)菌群落結(jié)構(gòu)影響明顯,MF-BTF(60.0mT)和S-BTF(0.0mT)中的細(xì)菌群落聚為一類,但菌群相似率僅為54.3%.另外,MF-BTF(60.0mT)中細(xì)菌的豐富度、均勻度均低于S-BTF(0.0mT)和IAS,但是共代謝降解TCE的優(yōu)勢(shì)菌在門、綱、屬水平分別為Proteobacteria、Gammaproteobacteria, MF-BTF(73.3%、36.8%、34.7%)中的相對(duì)豐度遠(yuǎn)大于S-BTF(69.6%、18.2%、10.9%)和IAS(46.2%、10.4%、5.2%).

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The strengthening effect of a static magnetic field on biotrickling filter—Study on trichloroethylene removal and bacterial community.

QUAN Yue1, WU Hao2, YIN Zhen-hao3, GUO Chun-yu2, YIN Cheng-ri2,3*

(1.Department of Environmental Science, Agricultural College, Yanbian University, Yanji 133002, China；2.Department of Chemistry, College of Science, Yanbian University, Yanji 133002, China；3.Analytical and Testing Center, Yanbian University, Yanji 133002, China)., 2018,38(3)：1099~1108

A laboratory-scale biotrickling filter combined with a magnetic field (MF-BTF) and a single BTF (S-BTF) packed with laver rocks were set up to treat trichloroethylene (TCE) gasthrough inoculation of activated sludge. The influences of different MF intensities were investigated andhigh-throughput sequencing was studied to bacteria community and functions. The results showed that in aerobic conditions, with 0.20g/L of phenol, 53.6 to 337.1mg/m3of TCE, and empty bed residence times (EBRT) of 202.5s, the performances followed the order MF-BTF (60.0mT) > MF-BTF (30.0mT) > S-BTF (0.0mT) > MF-BTF (130.0mT), and the removal efficiencies (REs) and maximum elimination capacities (ECs) corresponded to: 92.2%~45.5%, 2656.8mg/(m3×h); 89.8%~37.2%, 2169.1mg/(m3×h); 89.8%~29.8%, 1967.7mg/(m3×h); 76.0%~20.8%, 1697.1mg/(m3×h), respectively. High-throughput sequencing indicated that the bacterial diversity was lower, whereas the relative abundances of predominant bacteria: Proteobacteria, Gammaproteobacteria, Acinetobacter were higher in MF-BTF (60.0mT: 73.3%, 36.8%, 34.7%) than that in S-BTF (0mT: 69.6%, 18.2%, 10.9%). Results confirmed that a proper MF could improve TCE removal performance in BTF.

magnetic field；biotrickling filter；trichloroethylene；bacterial community；high-throughput sequencing

X505

A

1000-6923(2018)03-1099-10

權(quán) 躍(1978-),女,吉林九臺(tái)市人,講師,博士,主要從事有機(jī)污染物生物降解方面的研究.發(fā)表論文50余篇.

2017-08-22

國(guó)家自然科學(xué)基金(21662038)

* 責(zé)任作者, 教授, cryin@ybu.edu.cn