王 宇,厲 巍,2*,崔長征,2,劉勇弟,2

sp. AD-3強(qiáng)化活性污泥耐鹽降解菲效能

王 宇1,厲 巍1,2*,崔長征1,2,劉勇弟1,2

(1.華東理工大學(xué)資源與環(huán)境工程學(xué)院,工業(yè)廢水無害化與資源化國家工程研究中心,國家環(huán)境保護(hù)化工過程環(huán)境風(fēng)險評價與控制重點實驗室,上海 200237；2.上海污染控制與生態(tài)安全研究院,上海 200092)

基于嗜鹽菌sp. AD-3優(yōu)配市政活性污泥搭建耐鹽高效降解菲的生物反應(yīng)器,在3.0 %鹽度,進(jìn)水菲濃度20mg/L的運行條件下,菲去除率高達(dá)97 %.批次實驗證明,鹽度為3.0 %,pH值為7.5～8.5,底物濃度為20～200mg/L是菲降解的最佳環(huán)境條件,此時污泥比活性高于1.0mg/(gVSS×h).各共存底物的受試濃度下,酵母提取物和苯酚促進(jìn)菲降解,鎘和氰化物則抑制該過程,乙酸鈉、銅、鉻對該過程沒有明顯影響.16S rRNA基因高通量測序結(jié)果表明AD-3菌在反應(yīng)器內(nèi)具有長期穩(wěn)定性,其相對豐度維持在1.5 %.、和是反應(yīng)器中的優(yōu)勢菌,其相對豐度分別為20.7 %,15.1 %和11.9 %.RT-qPCR結(jié)果顯示接種AD-3菌后,編碼PAHs雙加氧酶RHDa的功能基因差異倍數(shù)從2.1提升至11.7.

耐鹽活性污泥；菲降解；環(huán)境條件；微生物群落結(jié)構(gòu)；功能基因；高通量測序

菲(PHE)是一種具有3個環(huán)狀結(jié)構(gòu)的多環(huán)芳烴(PAHs),在工業(yè)廢水處理設(shè)施和環(huán)境中常被檢出[1-4],因此也被作為模式污染物研究PAHs的降解過程[5-7].目前,高鹽有機(jī)廢水中PAHs的去除技術(shù)主要包括納米材料吸附[8]、膜過濾[9]、芬頓氧化[10]、臭氧氧化[11]等物理化學(xué)過程.但上述技術(shù)大多存在運行維護(hù)成本高、污染物礦化程度低、易產(chǎn)生二次污染等問題.通過微生物的作用將PAHs完全降解成為CO2是一種高效且經(jīng)濟(jì)的廢水處理方式.然而傳統(tǒng)活性污泥對于PAHs的降解能力有限,總PAHs去除率通常小于50%[12],且運行負(fù)荷低于2.4mg/(m3×d)[13].目前,關(guān)于高鹽條件下依然能高效降解PAHs的活性污泥系統(tǒng)鮮有報道.近些年,陸續(xù)有報道稱從自然界中篩分出一些可以耐鹽且高效降解PAHs的功能菌如[14],[15],[16]等,將這些功能菌定植到傳統(tǒng)活性污泥中以強(qiáng)化PAHs降解能力是突破這一技術(shù)瓶頸的有效方法.

焦化、煉油工業(yè)廢水成分復(fù)雜,運行的環(huán)境條件也時常發(fā)生改變.溫度、鹽度、pH值、底物濃度的不穩(wěn)定可能對微生物降解PAHs過程產(chǎn)生不良影響[17-19],工業(yè)廢水中常見的酚類、氰化物、重金屬等物質(zhì)[20-22]可能對微生物產(chǎn)生毒害作用,進(jìn)而削弱PAHs的降解效能.大多數(shù)已報道的活性污泥系統(tǒng)有效降解PAHs的良好效能只能維持在特定環(huán)境條件下[23-24].

本研究以課題組前期分離的嗜鹽PAHs降解功能菌sp. AD-3[16]優(yōu)配傳統(tǒng)活性污泥構(gòu)建耐鹽活性污泥體系,考察其在不同底物濃度、鹽度、pH值以及各類共存底物的條件下對PHE的降解效能.最后通過16S rRNA基因高通量測序和實時熒光定量PCR(RT-qPCR)技術(shù),識別關(guān)鍵功能菌群和功能基因.

1 材料與方法

1.1 反應(yīng)器運行

試驗用活性污泥由中度嗜鹽菌sp. AD-3[16]和上海市長橋污水處理廠曝氣池活性污泥[25]組成.根據(jù)前期研究結(jié)果[26],本研究以AD-3菌和活性污泥質(zhì)量比為1.0%(以VSS計)構(gòu)建耐鹽(3.0% NaCl)活性污泥SBR體系(R1).同時設(shè)置只接種活性污泥SBR反應(yīng)器(R2)作為對照實驗組.以PHE為研究PAHs降解的模式污染物,在序批式反應(yīng)器(SBR)中進(jìn)行連續(xù)實驗.SBR反應(yīng)器的有效工作體積2L,污泥濃度約為3.0g/L,運行溫度為(25±1) ℃,pH值維持在7.5,每個運行周期包含:進(jìn)料5min,曝氣反應(yīng)225min,沉淀5min,出水5min,體積交換比65%,水力停留時間約為5.7h,曝氣階段的曝氣量為0.5L/min.根據(jù)實際工業(yè)廢水處理系統(tǒng)污染物在水相和固相的總濃度[3-4],本研究進(jìn)水含PHE 20mg/L和乙酸鈉100mg/L,其它無機(jī)鹽成分和微量元素組成見參考文獻(xiàn)[27].定期(每周)檢測進(jìn)水和出水中的PHE濃度以及COD. PHE的提取和檢測方法見參考文獻(xiàn)[27], COD的檢測通過哈希試劑和DR6000紫外可見分光光度計完成.在反應(yīng)進(jìn)行至90d時提取R1和R2中污泥樣品的DNA,進(jìn)行后續(xù)的菌群結(jié)構(gòu)和功能基因分析.

1.2 環(huán)境條件影響批次試驗

從反應(yīng)器中取用具有活性的污泥至100mL的無機(jī)鹽培養(yǎng)液,使污泥濃度約為1.5g/L.以PHE作為唯一碳源,PHE降解比活性(以PHE/VSS計)為指標(biāo),計算公式如式(1),依次進(jìn)行底物濃度、pH值、鹽度(NaCl)、不同共存底物對PHE降解影響的批次實驗,期間保持溫度(25±1) ℃,搖床轉(zhuǎn)速150r/min.PHE初始濃度設(shè)置為5, 10, 20, 50, 100, 200, 300, 400mg/L;pH值設(shè)置為6.5, 7.5, 8.5, 9.5;鹽度設(shè)置為0.5%, 1.0%, 2.0%, 3.0%, 4.0%, 5.0%, 6.0%;以焦化行業(yè)廢水中常見的物質(zhì)及其濃度為根據(jù)[20-22],共存底物選取乙酸鈉、酵母提取物作為易降解COD,選取苯酚、Cu2+(硫酸銅)、Cd2+(硫酸鎘)、Cr3+(氯化鉻)、氰化鈉作為難降解COD或有毒物質(zhì)分別進(jìn)行試驗,重金屬的濃度以離子質(zhì)量濃度計量.

(1)

式中:PHE降解比活性,mg/(gVSS×h);0為初始底物濃度,mg/L;為剩余底物濃度,mg/L;為活性污泥的VSS濃度,mg/L;為反應(yīng)時間,h.

1.3 DNA提取、PCR擴(kuò)增和高通量測序

使用FastDNASPIN試劑盒(MP Biomedicals,美國)提取污泥樣品中的DNA.選用引物338F/ 806R(5'-ACTCCTACGGGAGGCAGCA-3和5'-G- GACTACHVGGGTWTCTAAT-3')擴(kuò)增樣品中的16S rRNA基因[28],引物5'-TTGACTTCCTCGA- CAAGGGC-3'和5'-ATCTTGCGCACCTGATCCT- C-3'用于擴(kuò)增PAHs雙加氧酶功能基因RHDa[26],引物5'-CGCTTAGATCCGGTCAGTCC-3'和5'-AGA- CGTCATATAGCGCACCG-3'用于擴(kuò)增萘雙加氧酶(也作用于PHE)功能基因[26].委托上海美吉生物醫(yī)藥科技有限公司進(jìn)行RT-qPCR試驗以及16S rRNA基因高通量測序,并完成微生物的物種鑒定分析.

1.4 統(tǒng)計分析

方差分析(ANOVA)用于進(jìn)行實驗組與對照組之間差異的顯著性檢驗,當(dāng)<0.05時視為具有顯著差異.

2 結(jié)果與討論

2.1 反應(yīng)器效能

如圖1所示,前3周(未添加AD-3菌)R1和R2的PHE去除率為4.9%～15.0%,COD去除率為35.7%～44.1%,PHE和COD的去除效能較低.在第4周加入AD-3菌后,R1的PHE的去除率提升至88.0%,并在隨后持續(xù)11周的實驗中維持在90%以上,最高可達(dá)97%;相應(yīng)地,COD去除率提升至80%以上,說明在微生物的作用下PHE可能被完全降解,促進(jìn)了COD的去除;而未添加AD-3菌的R2的PHE和COD去除效能分別為4.9%～21.0%和35.7%～ 55.5%.以上結(jié)果說明接種AD-3菌能夠有效提升傳統(tǒng)活性污泥的PAHs去除效能,并且這種強(qiáng)化效應(yīng)具有長期穩(wěn)定性.

對比其他活性污泥體系的PAHs去除效果(表1),耐鹽且能高效降解PAHs的活性污泥體系十分罕見.本研究在3%鹽度的條件下實現(xiàn)了對進(jìn)水20mg/LPHE的高效去除.實際工業(yè)廢水的PAHs常見濃度約在5～20mg/L范圍內(nèi)[4,6],在試驗條件下本研究的耐鹽活性污泥可以滿足廢水處理需要.

圖1 SBR反應(yīng)器運行效能

a: PHE去除效能;b: COD去除效能;R1第23d(點劃線標(biāo)識)添加AD-3菌,R2全程不添加AD-3菌

表1 各活性污泥系統(tǒng)對PAHs去除效果的對比

注:N.A.表示數(shù)據(jù)缺失.

2.2 環(huán)境條件對PHE降解的影響

在鹽度為3%,pH=7.5的條件下(圖2a),初始PHE濃度為5, 10mg/L時污泥比活性分別為0.22和0.50mg/(gVSS×h);初始PHE濃度為20, 50, 100, 200mg/L時污泥比活性分別為1.13, 1.25, 1.24, 1.21mg/(gVSS×h),達(dá)到峰值;之后隨著初始PHE濃度升高,污泥比活性出現(xiàn)下降趨勢,說明過高的底物濃度對微生物降解PHE過程產(chǎn)生了抑制作用,而這與之前的報道相符[31].在初始PHE濃度為20mg/L,鹽度為3%的條件下(圖2b),pH值為6.5, 7.5, 8.5, 9.5時污泥的比活性分別為0.88, 1.08, 1.12, 0.98mg/(gVSS×h),弱堿性條件對微生物降解PHE有利.PHE的微生物代謝過程會產(chǎn)生如1-羥基-2-萘甲酸等有機(jī)酸物質(zhì),過低的pH值可能會導(dǎo)致反應(yīng)平衡不利于正向進(jìn)行[32],因此表觀上的降解速率會減緩;而pH值過高則會影響微生物的正常代謝功能.在初始PHE濃度為20mg/L,pH=7.5的條件下(圖2c),鹽度為0.5%, 1.0%, 2.0%, 3.0%, 4.0%, 5.0%, 6.0 %時污泥比活性分別為0.22, 0.45, 0.65, 1.07, 0.69, 0.54, 0.32mg/(gVSS×h),說明微生物對鹽度的變化較為敏感,降解PHE的最適鹽度為3.0%.以PHE初始濃度20mg/L為試驗條件,在鹽度2.0%～3.0%和pH值為7.5～8.5的范圍內(nèi)PHE降解污泥比活性大于0.9mg/ (gVSS×h),為較高水平(圖2d).在所有受試條件下,污泥比活性均高于0.2mg/(gVSS×h).以連續(xù)流反應(yīng)器為例預(yù)測本研究的耐鹽活性污泥除PHE性能,當(dāng)水力停留時間為30h,污泥濃度為3g/L,進(jìn)水PHE濃度為20mg/L時,即使在污泥比活性為0.2mg/(gVSS×h)的受試條件下PHE的降解率均能達(dá)到90%.因此本研究的耐鹽活性污泥系統(tǒng)能夠適應(yīng)實際廢水處理的復(fù)雜環(huán)境條件.

a:初始PHE濃度; b: pH值; c:鹽度; d:鹽度和pH值的復(fù)合影響

圖3 共存底物對PHE降解的影響

*表示實驗組與對照組具有顯著差異(<0.05),對照組只添加PHE; 初始PHE濃度20mg/L,pH=8.5,鹽度3.0 %; 各底物濃度為:乙酸鈉100mg/L、酵母提取物50mg/L、苯酚200mg/L、銅10mg/L、鉻2mg/L、鎘2mg/L、氰化物5mg/L

如圖3所示,對照組PHE降解比活性為1.19mg/ (gVSS×h),乙酸鈉、銅、鉻對PHE降解無明顯影響.酵母提取物和苯酚可以促進(jìn)PHE的降解(<0.05),當(dāng)作為共存基質(zhì)時兩組試驗的PHE降解比活性分別為1.40和1.29mg/(gVSS×h),有研究表明苯酚與PHE結(jié)合形成穩(wěn)定的苯酚-PHE物質(zhì),能夠增大PHE在水中的溶解度,從而增加了PHE的生物可利用性[33],同時苯酚的存在可能刺激微生物分泌相關(guān)的代謝酶系,引發(fā)PAHs共代謝機(jī)制[34].鎘和氰化物的存在會明顯抑制PHE的降解活性(<0.05),當(dāng)二者作為共存基質(zhì)的試驗組降解比活性分別為0.97和0.70mg/(gVSS×h).氰化物對PHE降解的抑制作用更明顯,有研究表明當(dāng)氰化物濃度超過2mg/L時,細(xì)胞的呼吸速率會受到明顯抑制[35],因此在開發(fā)廢水處理工藝時需要考慮氰化物的抑制效應(yīng).

2.3 菌群結(jié)構(gòu)和功能基因

16S rRNA基因測序及鑒定結(jié)果顯示R1中相對豐度占前3的細(xì)菌分別來自、和屬,三者的相對豐度分別為20.7%, 15.1%, 11.9%(圖4a).其中,[36]和[37]已被報道具有降解PAHs的能力,它們可能參與到PHE降解過程中.此外,在反應(yīng)器初期時以質(zhì)量比1%接種的AD-3菌經(jīng)過15周的實驗后依然保有1.5%的相對豐度,說明該菌可以長期在反應(yīng)器中持留,并且維持穩(wěn)定的相對豐度,而這也解釋了反應(yīng)器PHE降解效能的長期穩(wěn)定性.事實上,降解PAHs的微生物之間存在營養(yǎng)缺陷互補的現(xiàn)象[38],即由于部分功能基因缺失導(dǎo)致單靠一株細(xì)菌難以執(zhí)行PAHs徹底礦化的全過程,多種功能菌分別負(fù)責(zé)PAHs的上游或下游降解過程,從而建立了穩(wěn)定的協(xié)同關(guān)系[39].

RHDa是多環(huán)芳烴雙加氧酶a亞基的縮寫, RHD負(fù)責(zé)催化PAHs的起始雙加氧反應(yīng)[40],該酶的a亞基被認(rèn)為是PAHs降解功能單元[41].編碼RHDa的基因,按照同源性分類,(萘雙加氧酶),(菲雙加氧酶)等基因的同源性高且序列高度相似,它們與,,等基因的同源性較低[42].現(xiàn)有研究報道,AD-3菌攜帶有與高度同源的RHDa的基因[43].如圖4b所示,在接種和未接種AD-3菌的活性污泥樣品中基因的差異倍數(shù)分別為1.5和1.3,表明接種AD-3菌沒有直接提升類功能基因在活性污泥系統(tǒng)中的豐度.然而接種AD-3菌后活性污泥樣品中編碼RHDa基因的差異倍數(shù)從2.1提升至11.7,是未接種AD-3菌的活性污泥樣品的約6倍.這說明AD-3菌可能激活了活性污泥土著菌潛在的非類RHDa的基因的表達(dá),從而提升PAHs降解能力.至于具體的互作激活機(jī)制,還需要利用宏基因組學(xué)耦合宏轉(zhuǎn)錄組學(xué)等手段進(jìn)一步去研究.

圖4 關(guān)鍵功能菌群和功能基因

Fig.4 Key microbial community and functional gene

a: R1的菌群結(jié)構(gòu); b:功能基因的RT-qPCR結(jié)果

3 結(jié)論

3.1 以嗜鹽菌AD-3和市政活性污泥構(gòu)建耐鹽活性污泥體系,在水力停留時間為5.7h,進(jìn)水PHE濃度20mg/L,污泥濃度約3g/L,鹽度為3 %的運行條件下,連續(xù)11周PHE降解率維持在90 %以上.

3.2 批次實驗證明PHE濃度為20～200mg/L,pH值為7.5～8.5,鹽度為3.0%時污泥的PHE降解比活性最大,高于1.0mg/(gVSS×h).酵母提取物和苯酚促進(jìn)PHE降解,鎘和氰化物則抑制PHE降解,乙酸鈉、銅、鉻對該過程沒有明顯影響.

3.3 微生物菌群結(jié)構(gòu)分析表明AD-3菌能在反應(yīng)器中長期持留,且相對豐度維持在1.5%.RT-qPCR結(jié)果顯示接種AD-3菌后活性污泥的phnA基因的差異倍數(shù)增幅較小,RHDa基因的差異倍數(shù)顯著升高.

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sp. AD-3 enhanced the degradation of phenanthrene in a halotolerant activated sludge system.

WANG Yu1, LI Wei1,2*, CUI Chang-zheng1,2, LIU Yong-di1,2

(1.National Engineering Research Center of Industrial Wastewater Detoxication and Resource Recovery, State Environmental Protection Key Laboratory of Environmental Risk Assessment and Control on Chemical Process, School of Resources and Environmental Engineering, East China University of Science and Technology, Shanghai 200237, China；2.Shanghai Institute of Pollution Control and Ecological Security, Shanghai 200092, China)., 2022,42(9)：4226～4231

A halotolerant bioreactor with high efficiency degrading phenanthrene was built based on inoculating halophilicsp. AD-3 with municipal activated sludge. At the salinity of 3.0%, phenanthrene removal ratio reached 97% with influent concentration of 20mg/L. Batch experiments showed that salinity of 3.0%, pH of 7.5～8.5 and initial concentrations of 20～200mg/L are the optimal conditions for phenanthrene degradation, under which the specific activity of the sludge exceeded 1.0mg/(gVSS×h). Yeast extract and phenol can promote the degradation of phenanthrene while cadmium and cyanide would inhibit the process. Sodium acetate, copper and chromium had no obvious effect on phenanthrene degradation. 16S rRNA gene high-throughput sequencing showed that the relative abundance of strain AD-3 had long-term stability in the reactor.,andwere the dominant bacteria with their relative abundances of 20.7%, 15.1% and 11.9% respectively. RT-qPCR results showed that after inoculation with strain AD-3, the fold change of PAHs dioxygenase functional gene encoding RHDaincreased from 2.1 to 11.7.

halotolerant activated sludge；phenanthrene degradation；environmental conditions；microbial community；functional gene；high-throughput sequencing

X703

A

1000-6923(2022)09-4226-06

2022-02-18

國家重點研發(fā)計劃(2019YFC0408202);國家自然科學(xué)基金資助項目(52170076);教育部基本科研業(yè)務(wù)費(JKB01221710)

*責(zé)任作者, 副教授, wei_li@ecust.edu.cn

王 宇(1996-),男,貴州遵義人,華東理工大學(xué)碩士研究生,主要從事工業(yè)廢水生物處理相關(guān)研究.