唐琳欽,宿程遠(yuǎn)*,黃 嫻,李汝婷,王安柳,樊翠萍,先云川

PFOA與PFOS對(duì)厭氧氨氧化污泥特性和微生物群落的影響

唐琳欽1,2,宿程遠(yuǎn)1,2*,黃 嫻2,李汝婷2,王安柳2,樊翠萍2,先云川2

(1.廣西師范大學(xué)珍稀瀕危動(dòng)植物生態(tài)與環(huán)境保護(hù)教育部重點(diǎn)實(shí)驗(yàn)室,廣西 桂林 541004；2.廣西師范大學(xué)環(huán)境與資源學(xué)院,廣西 桂林 541004)

通過(guò)序批實(shí)驗(yàn)研究了不同濃度(0.5,1mg/L)與不同類型的全氟化合物(PFCs)對(duì)厭氧氨氧化(anammox)污泥脫氮性能及微生物群落的影響.結(jié)果表明,0.5與1mg/L全氟辛酸(PFOA)和全氟辛烷磺酸鹽(PFOS)對(duì)anammox污泥的脫氮性能無(wú)明顯抑制作用;1mg/L PFOA(OA1)與PFOS(OS1)添加至anammox污泥中后1d,其去除率分別達(dá)到47.68%和92.7%.污泥的X射線光電子能譜(XPS)分析表明OA1、OS1實(shí)驗(yàn)組存在C-F、MgF2、CaF2等官能團(tuán).PFOA和PFOS脅迫下anammox污泥中血紅素c濃度出現(xiàn)降低,OA1與OS1實(shí)驗(yàn)組分別降低了21.05%、7.5%.對(duì)不同實(shí)驗(yàn)組anammox污泥進(jìn)行高通量測(cè)序分析表明,1mg/L PFOA和PFOS的添加會(huì)降低厭氧氨氧化菌但促進(jìn)反硝化細(xì)菌的相對(duì)豐度,OA1、OS1實(shí)驗(yàn)組中屬的相對(duì)豐度分別降低1.08%、0.28%,而不動(dòng)桿菌屬相對(duì)豐度增加1.73%與0.06%.整體而言,PFOA對(duì)anammox污泥的負(fù)面影響更為明顯.

全氟辛酸；全氟辛烷磺酸鹽；厭氧氨氧化；污泥特性；微生物群落

全氟化合物(PFCs)是碳?xì)浠衔镏械臍湓颖环〈纬傻幕衔?全氟辛酸(PFOA)和全氟辛烷磺酸鹽(PFOS)是典型的PFCs,兩者主要產(chǎn)生于涂料、潤(rùn)滑劑、殺蟲(chóng)劑等產(chǎn)品的生產(chǎn)過(guò)程中[1-2].由于PFOA與PFOS含有共價(jià)鍵C-F,因此具有較強(qiáng)的化學(xué)穩(wěn)定性從而在環(huán)境中具有高度持久性與生物累積性[3].由于PFCs的化學(xué)持久性、生物放大作用和潛在毒性,其越來(lái)越受到人們的廣泛關(guān)注.PFOA和PFOS的廣泛應(yīng)用導(dǎo)致兩者不可避免在污水處理廠中積累.目前,全球諸多污水處理廠、地表水中均檢出了PFOA與PFOS[4].

目前PFOA和PFOS主要處理方法有電化學(xué)氧化法、臭氧氧化法、光催化氧化法、吸附法、生物處理法等[3].生物處理法是污水處理廠采用的主流工藝,具有穩(wěn)定、經(jīng)濟(jì)等優(yōu)點(diǎn).Chiavola等[4]的研究表明,使用活性污泥法去除200～4000ng/L的PFOA和PFOS時(shí),兩者在液相中去除率分別達(dá)到59%～68%和66%～96%.Yang等[2]研究發(fā)現(xiàn)好氧顆粒污泥法可去除廢水中32%～36.4%的PFOA,但1mg/L PFOA會(huì)抑制微生物聚羥基鏈烷酸酯與糖原的轉(zhuǎn)化,并降低亞硝化單胞菌屬()、硝化螺旋菌屬()等細(xì)菌的相對(duì)豐度,可見(jiàn)PFCs可被生物處理法去除卻會(huì)對(duì)微生物造成一定的不利影響.Yu等[5]研究了PFOA對(duì)活性污泥工藝的影響,發(fā)現(xiàn)PFOA濃度達(dá)到20mg/L時(shí)會(huì)抑制微生物的生長(zhǎng)從而影響溶解性有機(jī)碳的去除.厭氧氨氧化(anammox)是在厭氧環(huán)境中,微生物以亞硝酸鹽為電子受體,將氨直接氧化為氮?dú)獾淖责B(yǎng)過(guò)程,可節(jié)省碳源與運(yùn)行成本,被認(rèn)為是最有前途的脫氮處理技術(shù)之一[6].然而隨著工業(yè)的發(fā)展,越來(lái)越多的新型污染物諸如納米金屬、抗生素等出現(xiàn)在污水處理廠中,對(duì)anammox工藝的穩(wěn)定運(yùn)行提出新挑戰(zhàn).Zhang等[7]發(fā)現(xiàn)anammox菌暴露在5mg/L CuNPs下,相對(duì)豐度由29.59%下降至17.53%.Du等[8]發(fā)現(xiàn)5～7mg/L的磺胺二甲嘧啶會(huì)抑制anammox菌活性,并使豐度從2.57%降低至0.39%.

本文研究了不同濃度、類型的PFCs對(duì)anammox污泥脫氮性能的影響;同時(shí)采用高效液相色譜-質(zhì)譜法(HPLC-MS)分析了anammox污泥對(duì)PFOA與PFOS的去除效率;并利用X射線光電子能譜(XPS)與三維熒光光譜(3D-EEM)研究了PFOA和PFOS對(duì)anammox污泥官能團(tuán)及胞外聚合物(EPS)組分的影響.最后,利用16S rRNA擴(kuò)增子測(cè)序分析了anammox污泥中微生物群落與功能代謝在PFOA和PFOS脅迫下的演替情況,從而為厭氧氨氧化體系的穩(wěn)定運(yùn)行提供一定的科學(xué)借鑒.

1 材料與方法

1.1 實(shí)驗(yàn)方法

實(shí)驗(yàn)所用anammox污泥取自實(shí)驗(yàn)室運(yùn)行180d的厭氧折流板反應(yīng)器.實(shí)驗(yàn)所用PFOA購(gòu)自Adamas, PFOS購(gòu)自江西國(guó)化化工.所用廢水為人工配置廢水,包括50mg/L NH4Cl、65mg/L NaNO2、58.6mg/L MgSO4·7H2O、840mg/L NaHCO3、73.5mg/L CaCl·2H2O,以及100mg/L Na2SO3作為脫氧劑,此外還加入1.25mL/L微量元素I和微量元素II[9].

序批實(shí)驗(yàn)在5個(gè)500mL錐形瓶中進(jìn)行,錐形瓶?jī)?nèi)均含有150mL anammox污泥及350mL廢水.參考文獻(xiàn)[10],在本實(shí)驗(yàn)確定0.5, 1mg/L的PFOA和PFOS.實(shí)驗(yàn)中對(duì)照組(CT)不添加PFCs,OA0.5、OA1、OS0.5、OS1實(shí)驗(yàn)組分別添加0.5mg/L PFOA、1mg/L PFOA、0.5mg/L PFOS、1mg/L PFOS[3].實(shí)驗(yàn)周期40d,每天進(jìn)水量350mL,水力停留時(shí)間24h, pH值控制在7.5～8.0,原水DO濃度在0.5mg/L以下.實(shí)驗(yàn)中錐形瓶封口處理并置于35℃恒溫震蕩器中,1～3d時(shí)震蕩速度160r/min,在該震蕩速度下硝酸鹽高度積累,為使厭氧氨氧化細(xì)菌處于合適的生存環(huán)境,對(duì)震蕩速度適當(dāng)調(diào)整,4～8d時(shí)震蕩速度100r/min,9～40d時(shí)震蕩速度120r/min[2].每天測(cè)定進(jìn)水與出水氨氮、亞硝酸鹽氮及硝酸鹽氮濃度;每隔4d分析PFOA與PFOS去除情況;此外分別于第0, 40d取出一定量anammox污泥用于EPS、血紅素c的測(cè)定;實(shí)驗(yàn)結(jié)束時(shí)通過(guò)16S rRNA擴(kuò)增子測(cè)序?qū)Σ煌瑢?shí)驗(yàn)組的微生物群落結(jié)構(gòu)進(jìn)行分析.

1.2 分析方法

水樣經(jīng)0.45 μm濾膜過(guò)濾后使用納氏試劑分光光度法,N-(1-萘基)-乙二胺光度法分光光度法及紫外分光光度法分別測(cè)定其氨氮,亞硝酸鹽氮及硝酸鹽氮濃度.X射線光電子能譜儀(Thermo scientific, 250Xi)用于anammox污泥表面官能團(tuán)分析,將實(shí)驗(yàn)結(jié)束后(40d)的污泥于105℃下干燥24h進(jìn)行XPS分析,測(cè)試束斑為500 μm,全譜通過(guò)能100eV,步長(zhǎng)1.0eV,窄譜通過(guò)能30eV,步長(zhǎng)0.06eV[9].污泥胞外聚合物(EPS)的提取采用熱提取法,并將松散型胞外聚合物(LB-EPS)與緊致型胞外聚合物(TB-EPS)用于EEM光譜分析(日立-F7000),儀器發(fā)射波長(zhǎng)和激發(fā)波長(zhǎng)為5nm,掃描速度為2400r/min[11]. Anammox污泥中血紅素c的測(cè)定采用吡啶分光光度法,于波長(zhǎng)549nm處測(cè)定其吸光度并計(jì)算血紅素c濃度[12].

液相色譜串聯(lián)質(zhì)譜儀(Agilent Q-TOF 6545B)用于水樣及anammox污泥中PFOA、PFOS濃度的測(cè)定;LC-MS所用色譜柱為ZORBAX Eclipse plus c18,流動(dòng)相A為乙腈,流動(dòng)相B為5mmol/L乙酸銨; LC-MS掃描時(shí)間為6min,流速0.2mL/min,0～2.5min流動(dòng)相A與B的體積比例為58:42,2.5～4min流動(dòng)相A與B的體積比例為5:95,4～6min流動(dòng)相A與B的體積比例恢復(fù)到58:42.污泥中PFOA、PFOS的提取方法:取一定量anammox泥水混合物于5mL離心管,于6000r/min離心5min棄去上清液,置于2mL甲醇溶液中,超聲破碎1h后于6000r/min離心5min,取上清液進(jìn)行測(cè)定[4].

1.3 16S rRNA擴(kuò)增子測(cè)序

實(shí)驗(yàn)結(jié)束后對(duì)CT、OA1、OS1組的anammox污泥進(jìn)行16S rRNA擴(kuò)增子測(cè)序,使用Mag-Bind土壤DNA試劑盒(EZNATM,OMEGA,美國(guó))提取污泥的DNA,DNA完整性通過(guò)1%瓊脂糖凝膠電泳驗(yàn)證并使用Qubit定量檢測(cè)DNA樣本濃度[13].PCR一輪擴(kuò)增所用引物為V3-V4通用引物341F(CCTACGGGNG- GCWGCAG)和805R(GACTACHVGGGTATCTAA- TCC)[14];第二輪擴(kuò)增引入Illumina橋式PCR兼容引物[15].Illumina Miseq?/Hiseq?得到的原始圖像數(shù)據(jù)文件經(jīng)堿基識(shí)別分析轉(zhuǎn)化為原始測(cè)序序列[16].然后對(duì)各樣本數(shù)據(jù)的質(zhì)量進(jìn)行質(zhì)控過(guò)濾,得到各樣本有效數(shù)據(jù),之后再進(jìn)行OTU聚類分析和物種分類學(xué)分析.此外KEGG(基因和基因組京都百科全書(shū))數(shù)據(jù)庫(kù)被用來(lái)預(yù)測(cè)anammox污泥中相關(guān)代謝功能預(yù)測(cè)分析[14].

2 結(jié)果與討論

2.1 脫氮性能影響

首先分析了不同濃度PFOA與PFOS污染下,anammox污泥對(duì)氨氮、亞硝酸鹽氮去除及硝酸鹽氮生成的情況,結(jié)果如圖1所示.

由圖1A可知,CT對(duì)照組與OA0.5(0.5mg/L PFOA)、OA1(1mg/L PFOA)、OS0.5(0.5mg/L PFOS)、OS1(1mg/L PFOS)實(shí)驗(yàn)組氨氮去除率變化趨勢(shì)基本一致,1～3d時(shí)振蕩器震蕩速度為160r/min,5個(gè)實(shí)驗(yàn)組氨氮去除率均達(dá)86%以上,去除率相對(duì)穩(wěn)定,這表明PFOA與PFOS對(duì)氨氮去除并未產(chǎn)生急性抑制作用.4～8d時(shí),CT、OA0.5、OA1、OS0.5、OS1的氨氮平均去除率降低至51.48%、53.43%、72.65%、63.21%和61.43%,在此階段氨氮去除率均明顯下降,可能是anammox污泥中細(xì)菌活性受到一定抑制導(dǎo)致.9～40d時(shí)振蕩器震蕩速度為120r/min,CT、OA0.5、OA1、OS0.5、OS1對(duì)氨氮去除率基本保持在98%左右,這表明PFOA、PFOS的添加對(duì)anammox污泥除氨氮能力并未產(chǎn)生不利影響.由圖1B可知,CT、OA0.5、OA1、OS0.5、OS1各組對(duì)亞硝酸鹽氮去除率變化趨勢(shì)與氨氮基本一致,1～3d時(shí),5個(gè)實(shí)驗(yàn)組的亞硝酸鹽氮去除率均達(dá)到76%以上;在4～10d時(shí)亞硝酸鹽氮去除率明顯降低,亞硝酸鹽氮平均去除率分別為41.19%、43.88%、51.12%、51.40%、37.41%,以O(shè)S1實(shí)驗(yàn)組降低的最為明顯.而11d以后,對(duì)照組與實(shí)驗(yàn)組對(duì)亞硝酸鹽氮的去除效率逐漸升高, 13～40d時(shí)各組去除率均達(dá)到97%以上.

根據(jù)Strous提出的anammox反應(yīng)方程,硝酸鹽氮/氨氮理論物質(zhì)的量比為0.26[17],對(duì)不同實(shí)驗(yàn)組出水硝酸鹽氮/氨氮與硝酸鹽氮/亞硝酸鹽氮進(jìn)行分析,如圖2所示.硝酸鹽氮/氨氮在1～3d內(nèi)持續(xù)增大,表明震蕩速度為160r/min時(shí),反應(yīng)器內(nèi)的硝酸鹽氮濃度增加,這可能是由于過(guò)高的震蕩速度使得反應(yīng)器中廢水溶解氧含量增加導(dǎo)致.4～8d時(shí)震蕩速度為100r/min,此時(shí)硝酸鹽氮/氨氮明顯下降同時(shí)伴隨著氨氮、亞硝酸鹽去除率大幅下降,對(duì)照組與實(shí)驗(yàn)組的硝酸鹽氮/氨氮最低為0.082. 9～40d時(shí)振蕩器震蕩速度為120r/min,而硝酸鹽氮/氨氮緩慢增加,此階段硝酸鹽氮/氨氮維持在0.77～1.11之間,超過(guò)理論物質(zhì)的量比,這表明出水硝酸鹽氮濃度增加.9～40d時(shí)CT對(duì)照組與4個(gè)實(shí)驗(yàn)組的氨氮、亞硝酸鹽去除率也逐漸升高并達(dá)到穩(wěn)定狀態(tài)是其轉(zhuǎn)化成硝酸鹽氮所致.CT對(duì)照組與4個(gè)實(shí)驗(yàn)組硝酸鹽氮/亞硝酸鹽氮的變化趨勢(shì)與硝酸鹽氮/亞硝酸鹽氮的變化趨勢(shì)基本一致.

圖2 硝酸鹽氮與氨氮、亞硝酸鹽氮的比值

2.2 全氟化合物的去除

PFOA和PFOS分別具有親水性官能團(tuán)羧基和磺酸基,使PFOA和PFOS具有一定的水溶性,其中PFOA、PFOS的溶解度分別為3.4和0.54g/L[4],為更好評(píng)價(jià)PFOA與PFOS的去除情況,本文對(duì)出水及污泥中的PFOA和PFOS進(jìn)行了測(cè)定,結(jié)果如圖3所示.

由圖3可知,第1d時(shí),OA0.5、OA1實(shí)驗(yàn)組對(duì)PFOA的去除率分別為49.86%、47.68%, OA0.5、OA1實(shí)驗(yàn)組PFOA在污泥相的比例為45.67%和46.48%,此外OA0.5、OA1中PFOA的量均有不同程度損失,其比例分別為1.2%,4.19%.而第5d以后PFOA去除率降低并維持在18.43%～29.17%之間,表明添加PFOA的初始,anammox污泥對(duì)PFOA具有一定吸附能力,而持續(xù)添加PFOA會(huì)降低anammox污泥的吸附能力.OS0.5、OS1實(shí)驗(yàn)組在添加PFOS的1d后,PFOS去除率高達(dá)91.5%和92.7%,而第9d后污泥對(duì)PFOS的吸附能力逐漸趨于飽和,此時(shí)OS0.5、OS1實(shí)驗(yàn)組的PFOS去除率降低至49.08%以下.在添加PFOS的17d后,PFOS的去除率相對(duì)平穩(wěn),OS0.5、OS1實(shí)驗(yàn)組的PFOS平均去除率分別為26.94%和24.83%.此外OS0.5、OS1實(shí)驗(yàn)組的PFOS在污泥相的比例高達(dá)88.77%和89.8%,這表明添加PFOS1d后,anammox污泥吸附PFOS的能力較PFOA更強(qiáng).污泥對(duì)PFOA和PFOS的吸附可歸因于陽(yáng)離子架橋作用與疏水作用,污水及污泥中的帶正電的Ca2+與Mg2+作為陽(yáng)離子連接帶負(fù)電的PFOA和PFOS,從而實(shí)現(xiàn)PFCs被污泥吸附[18];同時(shí)PFOA和PFOS具有羧基和磺酸基及較長(zhǎng)的C-F主鏈,溶解在水相的PFCs會(huì)與anammox污泥發(fā)生疏水作用而被吸附.由于PFOS比PFOA具有更多C-F鍵(多2個(gè))故具有更強(qiáng)疏水能力[19],從而與anammox污泥間具有更強(qiáng)的親和力.

圖3 PFOA與PFOS的去除率及分布情況

堆積條形圖為添加PFCs一天后,PFCs的分布比例

2.3 污泥官能團(tuán)分析

為分析PFOA與PFOS對(duì)污泥表面官能團(tuán)的影響,使用XPS光譜對(duì)對(duì)照組與OA1、OS1實(shí)驗(yàn)組anammox污泥中C、N、F元素進(jìn)行分析,結(jié)果如圖4所示.

圖4 污泥的XPS譜圖分析

圖4表明OA1、OS1實(shí)驗(yàn)組污泥中存在多種含氟官能團(tuán),其中688.5～688.9eV處出現(xiàn)的峰為PFOA、PFOS中含有的C-F[20],且OS1組C-F鍵的峰面積明顯大于OA1,這說(shuō)明OS1組anammox污泥中吸附了較多PFOS;685.9～686.2,684.4～685eV處出現(xiàn)的峰分別為MgF2、CaF2[21],這表明anammox污泥中的Mg2+、Ca2+可通過(guò)陽(yáng)離子架橋連接帶負(fù)電荷的anammox污泥及2種PFCs;此外OS1組在683eV處還存在KF官能團(tuán)[22],說(shuō)明PFOS對(duì)anammox污泥的親和力更高.C1s由3～4種官能團(tuán)組成,其中284.8, 286.3,288eV處出現(xiàn)的峰分別為C-C、C-O-C、C=O[2];另外CT、OS1組在289, 289.4eV出現(xiàn)的峰為-COOH[2].在399.9, 401.8～401.9eV附近處存在2個(gè)峰,399.9eV處出現(xiàn)的峰歸因于非質(zhì)子化氨基(-NH),而401.8～401.9eV處的峰與質(zhì)子化的氨基有關(guān)(-NH3+)[23].

2.4 污泥EPS的3D-EEM分析

胞外聚合物(EPS)是微生物產(chǎn)生的由蛋白質(zhì)與多糖等組成的高分子聚合物,具有較強(qiáng)的吸附能力且能保護(hù)細(xì)菌免受有毒物質(zhì)的影響,主要可分為松散型胞外聚合物(LB-EPS)和緊密型胞外聚合物(TB-EPS),兩者空間分布和理化特性不同[24-25].

圖5 不同實(shí)驗(yàn)組污泥LB-EPS和TB-EPS的3D-EEM譜圖

由圖5可知,第0d時(shí),CT、OA1、OS1組的LB- EPS在x/m=280/345～350nm和x/m=420/ 475nm處存在色氨酸(峰A)和輔酶F420(峰B)2個(gè)主峰[26-27].其中CT、OA1、OS1類色氨酸熒光峰強(qiáng)度存在一定差異,分別為62.11,69.11,76.34,可能是由于實(shí)驗(yàn)之初anammox污泥間混合不勻所致.此外在x/m=230/305～310nm、x/m=270/470nm處存在芳香族蛋白質(zhì)(峰C)、黃腐酸(峰D)熒光峰[26].第40d時(shí),CT、OA1、OS1組色氨酸熒光峰強(qiáng)度均有不同程度降低,分別降低14.2%、28.9%、23.52%. TB-EPS的EEM譜圖中主要存在色氨酸峰(峰A)和輔酶F420(峰B)2種熒光峰.第0d時(shí)色氨酸熒光峰強(qiáng)度較高,CT對(duì)照組與OA1、OS1實(shí)驗(yàn)組峰A的熒光強(qiáng)度達(dá)到302.9,332.9,318.1,而到第40d時(shí),CT、OA1、OS1組色氨酸峰強(qiáng)度分別降低21%、27.7%、31.1%.這表明添加PFCs會(huì)降低anammox污泥色氨酸的分泌,且PFOA對(duì)污泥色氨酸的分泌所產(chǎn)生負(fù)面影響更為明顯.污泥的疏水作用主要與污泥中疏水性氨基酸(色氨酸、丙氨酸、苯丙氨酸等)、腐殖酸、富里酸中的芳香族、脂環(huán)族及污泥中的脂質(zhì)有關(guān)[28].各組污泥的EEM譜圖表明,相較于CT對(duì)照組,PFCs的存在使LB-EPS、TB-EPS中色氨酸熒光峰強(qiáng)度降低更顯著,可能是PFCs與EPS中色氨酸發(fā)生疏水作用所導(dǎo)致.

2.5 污泥血紅素c含量

厭氧氨氧化污泥具有紅色特征主要是由于具有細(xì)胞色素c,細(xì)胞色素c包含血紅素c,當(dāng)二價(jià)鐵和三價(jià)鐵間轉(zhuǎn)換時(shí),血紅素c能夠被氧化和還原,因此可通過(guò)確定血紅素c含量來(lái)估算anammox污泥的細(xì)胞色素c含量[29].Shi等[30]研究表明當(dāng)厭氧氨氧化反應(yīng)器中土霉素濃度為2mg/L時(shí),特定厭氧氨氧化活性(SAA)降低81.3%,同時(shí)血紅素c濃度降低50.1%,這說(shuō)明血紅素c的濃度與SAA存在聯(lián)系,因此本文分析了PFOA與PFOS對(duì)anammox污泥中血紅素c含量的影響,結(jié)果如圖6所示.

圖6 PFOA與PFOS對(duì)血紅素c含量的影響

由圖6可知,在未添加PFCs之前,OA1組的血紅素c含量最高,而添加PFCs后的第20d,CT、OA1、OS1的血紅素c含量均有不同程度降低,分別降低了2.77,9.39,5.7 μmol/g.添加PFCs后的第40d,CT對(duì)照組血紅素c含量出現(xiàn)回升,而OA1、OS1實(shí)驗(yàn)組繼續(xù)降低,降低幅度分別為21.05%和7.5%.因此添加PFCs之后會(huì)降低anammox污泥血紅素c的含量,以PFOA最為顯著,說(shuō)明1mg/L的PFCs抑制了厭氧氨氧化污泥的氧化活性.

2.6 微生物群落分析

由圖7可知,變形菌門(mén)(Proteobacteria)、廣古菌門(mén)(Euryarchaeota)、綠彎菌門(mén)(Chloroflexi)、浮霉菌門(mén)(Planctomycetes)在各組微生物群落中占主導(dǎo)地位,另外還存在裝甲菌門(mén)(Armatimonadetes)、擬桿菌門(mén)(Bacteroidetes)、酸桿菌門(mén)(Acidobacteria)等.變形菌門(mén)與綠彎菌門(mén)是反硝化過(guò)程中主要細(xì)菌,據(jù)報(bào)道PFCs可促進(jìn)變形菌門(mén)與綠彎菌門(mén)相對(duì)豐度增加[10,31].添加1mg/L的PFOA與PFOS使變形菌門(mén)相對(duì)豐度分別增加3.92%與0.32%,OA1中綠彎菌門(mén)的相對(duì)豐度增加2.01%,這表明添加PFCs促進(jìn)了反硝化細(xì)菌的生長(zhǎng).此外OA1組變形菌門(mén)與綠彎菌門(mén)的豐度較OS1組增加比例更高,可能歸因于PFOA C-F鍵更少而更好地被利用.擬桿菌屬具有較好的代謝能力,可以適應(yīng)惡劣的環(huán)境,酸桿菌門(mén)是一種有機(jī)物水解細(xì)菌[10].添加1mg/L的PFOA和PFOS后擬桿菌門(mén)、酸桿菌門(mén)相對(duì)豐度分別增加0.49%和0.24%、0.24%與0.06%,這與先前的研究相一致[10,31].目前已知的厭氧氨氧化菌屬于浮霉菌門(mén)[32],PFCs的存在降低了浮霉菌門(mén)相對(duì)豐度,相對(duì)于CT組,OA1、OS1實(shí)驗(yàn)組中浮霉菌門(mén)比例分別降低1.38%、0.52%.

圖7 門(mén)水平與屬水平細(xì)菌的相對(duì)豐度

甲烷絲菌屬()、不動(dòng)桿菌屬()、甲烷桿菌屬 ()在屬水平上為優(yōu)勢(shì)菌群,此外還存在、、等具有脫氮能力的菌群.高豐度的甲烷絲菌屬、甲烷桿菌屬與馴化污泥有關(guān)[9].據(jù)報(bào)道不動(dòng)桿菌屬和屬能進(jìn)行異養(yǎng)硝化和好氧反硝化[33-34].此外是一種多功能氮代謝細(xì)菌,常發(fā)現(xiàn)于自養(yǎng)反硝化與異養(yǎng)反硝化反應(yīng)器中[35],是一種自養(yǎng)反硝化細(xì)菌[36]1mg/L的PFOA與PFOS使不動(dòng)桿菌屬、的相對(duì)豐度增加1.73%與0.06%、0.65%與0.98%,而、分別增加0.55%與0.22%、0.65%和0.33%,這表明添加PFCs會(huì)促進(jìn)反硝化細(xì)菌的相對(duì)豐度.相較于與等厭氧氨氧化細(xì)菌,具有更高的生長(zhǎng)速度與亞硝酸鹽半飽合常數(shù).1mg/L PFOA、1mg/L PFOS的存在使的相對(duì)豐度降低1.08%、0.28%.總體而言,PFCs的存在能夠刺激反硝化細(xì)菌而抑制厭氧氨氧化菌生長(zhǎng).可能是PFCs能被反硝化菌作為碳源而被生物利用,而厭氧氨氧化細(xì)菌由于生長(zhǎng)速度慢且對(duì)環(huán)境因素更敏感,具有生物毒性的PFCs添加至厭氧氨氧化體系中會(huì)使其被PFCs所抑制.

2.7 KEGG分析

由圖8可知,KEGG通路圖顯示對(duì)照組和OA1、OS1實(shí)驗(yàn)組中主要有4個(gè)一級(jí)通路:代謝(41.43%～ 42.03%)、基因信息處理(17.48%～17.73%)、環(huán)境信息處理(12.81%～9.13.48%)和細(xì)胞過(guò)程(3.26%～ 4.02%).代謝主要以氨基酸代謝(9.56%～9.88%)、碳水化合物代謝(10.26%～10.54%)為主.相對(duì)于對(duì)照組,OA1實(shí)驗(yàn)組的氨基酸代謝、碳水化合物代謝、多糖生物合成的豐度分別增強(qiáng)0.015%、0.073%、0.067%,而能量代謝的比例減少0.27%.1mg/L PFOS的添加使得氨基酸代謝和能量代謝能力增強(qiáng)(0.016%和0.109%),而碳水化合物代謝、多糖生物合成和代謝的能力略微降低.氨基酸代謝和碳水化合物代謝是污泥中的主要代謝路徑,這與污泥去除氮素與有機(jī)污染物有直接關(guān)系[37-38].PFCs的添加使OA1、OS1實(shí)驗(yàn)組anammox污泥的氨基酸代謝能力得到增加,但污泥中的血紅素c含量與浮霉菌門(mén)豐度出現(xiàn)了降低,從而解釋了添加有PFCs的OA1、OS1實(shí)驗(yàn)組對(duì)氨氮、亞硝酸鹽氮去除效果和CT對(duì)照組無(wú)明顯差異的現(xiàn)象.

圖8 PFOA與PFOS對(duì)KEGG代謝途徑的影響

3 結(jié)論

3.1 PFOA與PFOS的濃度為0.5與1mg/L時(shí),對(duì)anammox污泥去除氨氮、亞硝酸鹽氮的效率并無(wú)明顯抑制;由于PFOS更高的疏水性,anammox污泥對(duì)PFOS的去除率高于PFOA,其中污泥的吸附作用占有較大比例.

3.2 PFOA和PFOS的添加會(huì)降低污泥C-C、C- O-C等官能團(tuán)的比例,而會(huì)增加C=O的比例;PFOA和PFOS會(huì)降低LB-EPS、TB-EPS中色氨酸峰的強(qiáng)度;同時(shí)PFOA、PFOS脅迫下使污泥中血紅素c的濃度出現(xiàn)降低,PFOA的抑制作用更為明顯.

3.3 PFOA與PFOS脅迫下,厭氧氨氧化菌相對(duì)豐度降低,特別是PFOA影響下,的相對(duì)豐度降低了1.08%,但PFOA與PFOS的存在促進(jìn)反硝化細(xì)菌相對(duì)豐度及氨基酸代謝能力.

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Evaluation of sludge characteristics and microbial community of anammox sludge during exposure to perfluorooctane acid and perfluorooctane sulfonate.

TANG Lin-qin1,2, SU Cheng-yuan1,2*, HUANG Xian2, LI Ru-ting2, WANG An-liu2, FAN Cui-ping2, XIAN Yun-chuan2

(1.Key Laboratory of Ecology of Rare and Endangered Species and Environmental Protection, Ministry of Education, Guangxi Normal University, Guilin 541004, China；2.School of Environment and Resources, Guangxi Normal University, Guilin 541004, China)., 2022,42(1)：194～202

The sequential batch experiments explored the influence mechanism under different concentrations (0.5mg/L and 1mg/L) and types of perfluorinated compounds (PFCs) on the denitrification performance and microbial community of anaerobic ammonia oxidation (anammox) sludge. Results indicated that perfluorooctanoic acid (PFOA) (at 0.5mg/L, 1mg/L) and perfluorooctane sulfonate (PFOS) had no obvious inhibitory effect on the denitrification performance of the anammox sludge. The addition of 1mg/L PFOA (OA1) and PFOS (OS1) to the anammox sludge for one day resulted in removal rates of respectively 47.68% and 92.7%. X-ray photoelectron spectroscopy (XPS) analysis of the sludge showed the presence of C-F, MgF2, CaF2functional groups in the OA1 and OS1 groups. The addition of PFOA and PFOS reduced the concentration of heme c in the anammox sludge by respectively 21.05% and 7.5%. The high-throughput sequencing analysis of the anammox sludge in different experimental groups showed that the addition of 1mg/L PFOA and PFOS could reduce the relative abundance of anammox bacteria and promote the relative abundance of denitrifying bacteria. The relative abundance ofin the OA1 and OS1 groups decreased by respectively 1.08% and 0.28%, while the relative abundance ofincreased by 1.73% and 0.06%. In general, the negative effects of PFOA on the anammox sludge were more significant than PFOS.

perfluorooctanoic acid；perfluorooctane sulfonate；anammox；sludge characteristics；microbial communities

X703.1

A

1000-6923(2022)01-0194-09

唐琳欽(1997-),男,廣西桂林人,廣西師范大學(xué)碩士研究生,研究方向?yàn)樗幚砑夹g(shù).

2021-06-01

國(guó)家自然科學(xué)基金資助項(xiàng)目(52060003);廣西師范大學(xué)科研育人項(xiàng)目(2020YR009)

* 責(zé)任作者, 教授, suchengyuan2008@126.com