丁麗飛,李海燕,白敏冬*,鄭 武,郭 楓,張芝濤(.廈門大學(xué)環(huán)境與生態(tài)學(xué)院,海洋生物資源開發(fā)利用協(xié)同創(chuàng)新中心,福建 廈門 3602；2.大連海事大學(xué)環(huán)境工程研究所物理系,遼寧 大連 6026)

羥基自由基快速殺滅典型水華藻的研究

丁麗飛1,李海燕1,白敏冬1*,鄭 武1,郭 楓1,張芝濤2*(1.廈門大學(xué)環(huán)境與生態(tài)學(xué)院,海洋生物資源開發(fā)利用協(xié)同創(chuàng)新中心,福建 廈門 361102；2.大連海事大學(xué)環(huán)境工程研究所物理系,遼寧 大連 116026)

以典型水華藻銅綠微囊藻、針桿藻和四尾柵藻為研究對(duì)象,利用大氣壓強(qiáng)電離放電高效生成的羥基自由基(?OH)對(duì)3種藻進(jìn)行殺滅.采用熒光染色、流式細(xì)胞儀和光合活性等生物學(xué)方法,確定?OH殺滅的閾值濃度和時(shí)間,并觀察細(xì)胞形態(tài)變化.結(jié)果表明,當(dāng)混合藻中銅綠微囊藻、針桿藻和四尾柵藻的初始藻密度分別為19.5×104、21.8×104和4.90×104cells/mL時(shí), ?OH殺滅的閾值濃度為1.07mg/L,致死時(shí)間為4.5s;形態(tài)觀察結(jié)果表明,處理后各種藻的形態(tài)是完整的,無(wú)內(nèi)溶質(zhì)溢出.因此,采用?OH可實(shí)現(xiàn)高效快速殺滅水華藻,有效保障飲用水安全.

羥基自由基；水華藻；致死閾值；暴露時(shí)間

藻華是世界性的水環(huán)境問題,而我國(guó)的諸多湖泊與河流庫(kù)區(qū)的高藻爆發(fā)形勢(shì)日益嚴(yán)峻.這些湖庫(kù)水是我國(guó)飲用水水源的重要組成部分,高藻爆發(fā)時(shí),藻密度、化學(xué)需氧量、氨氮等水質(zhì)指標(biāo)超過地表水Ⅲ類標(biāo)準(zhǔn),不僅會(huì)增加自來(lái)水廠的生產(chǎn)成本,甚至引起減產(chǎn)或停產(chǎn).常規(guī)飲用水處理工藝難以解決藻類毒素、嗅味物質(zhì)等高藻引發(fā)的水質(zhì)超標(biāo)問題,嚴(yán)重威脅城市供水和飲用水安全[1].

目前,采用化學(xué)氧化法控制藻華已成為全世界范圍內(nèi)的研究熱點(diǎn).Daly等[2]研究氯法殺滅密度為30×104cells/mL的銅綠微囊藻,當(dāng)CT值(氧化劑的暴露總量)為7～29(mg·min)/L時(shí),所有藻細(xì)胞失去活性,但產(chǎn)生三氯甲烷、鹵乙酸等鹵代有機(jī)消毒副產(chǎn)物[3].Zhou等[4]研究了二氧化氯法殺滅密度為100×104cells/mL的銅綠微囊藻,作用時(shí)間為10min時(shí),藻細(xì)胞去除率達(dá)到91.5%,但產(chǎn)生的副產(chǎn)物 ClO2-和 ClO3-具有較大的潛在毒性[5]. Huo等[6]采用濃度為0～60mg/L的H2O2殺滅銅綠微囊藻,作用時(shí)間為3h時(shí),致死率達(dá)到99%,然而H2O2濃度大,殺滅高藻的成本過高[7], H2O2在使用和運(yùn)輸過程中存在爆炸隱患.Coral等[8]研究臭氧法致死細(xì)胞密度為25×104和150×104cells/ mL的銅綠微囊藻,在CT值小于0.2mg·min/L時(shí),所有細(xì)胞失活,若水中Br-和Cl-濃度較高,O3氧化會(huì)生成 HOBr等消毒副產(chǎn)物[9],且臭氧法的一次性設(shè)備投資、運(yùn)行成本高,設(shè)備及附屬設(shè)備龐大.Li等[10]利用水利空化產(chǎn)生羥基自由基技術(shù),處理初始葉綠素含量為 0.17mg/m3的銅綠微囊藻,當(dāng)羥基濃度為 2.03μmol/L,3d內(nèi)對(duì)藻的去除率可達(dá)91.2%.顧雨辰等[11]利用高壓脈沖氣液混合放電殺滅銅綠微囊藻,結(jié)果表明在優(yōu)化條件下,該方法在5d時(shí)間內(nèi),對(duì)葉綠素含量為0.15mg/m3的銅綠微囊藻的致死率可達(dá) 99%以上.因此采用氯氣、二氧化氯、H2O2和O3等常規(guī)氧化劑去除水中藻細(xì)胞時(shí),存在生成具有潛在毒性的消毒副產(chǎn)物、反應(yīng)時(shí)間長(zhǎng)、投資和運(yùn)行成本高、安全性差、設(shè)備龐大等多種問題.而采用電離放電法除藻成為當(dāng)今熱點(diǎn)[12].

本文針對(duì) 3種典型的水華藻,即銅綠微囊藻、針桿藻和四尾柵藻,在大氣壓條件下采用強(qiáng)電離放電高效制備?OH溶液,開展了?OH對(duì)上述3種典型水華藻的殺滅研究.結(jié)合SYTOX Green熒光染色技術(shù)、流式細(xì)胞儀法和光合活性參數(shù)Fv/Fm值,分別確定了?OH對(duì)3種典型水華藻的致死閾值和時(shí)間,為高藻水高效安全處理提供新方法,為高藻爆發(fā)時(shí)期飲用水處理工藝的改進(jìn)提供新思路.

1 材料與方法

1.1 材料

實(shí)驗(yàn)所用的銅綠微囊藻(Microcystis aeruginosa, FACHB-905)、針桿藻(Synedra sp., FACHB-843)和 四 尾 柵 藻 (Scenedesmus quadricuauda, FACHB-44)購(gòu)自中科院武漢水生所.銅綠微囊藻屬藍(lán)藻門色球藻科微囊藻屬,細(xì)胞球形、圓球形,直徑3～5μm左右,細(xì)胞淡藍(lán)色或綠色,常聚集生長(zhǎng),群體具無(wú)色柔和溶解性的膠被,以二分裂形式進(jìn)行繁殖.針桿藻屬硅藻門羽紋綱無(wú)殼縫目,細(xì)胞長(zhǎng)桿形,長(zhǎng)10μm左右,殼面披針形,中部寬,從中間到兩端逐漸狹窄;四尾柵藻屬綠藻門柵藻科柵藻屬,細(xì)胞為長(zhǎng)圓形、圓柱形,長(zhǎng)15～20μm,常由4個(gè)細(xì)胞構(gòu)成,群體中的各個(gè)細(xì)胞以其長(zhǎng)軸相互平行、其細(xì)胞壁彼此緊密排列在一個(gè)平面上,互相平齊,群體兩側(cè)細(xì)胞的上下兩端各具 1刺,刺長(zhǎng) 10～13μm.3種藻的培養(yǎng)基依次為BG11、Erdschreiber和SE,培養(yǎng)條件均為(25±1)℃, pH=(7.1±0.1),光照 2000lux,光:暗=12h:12h.實(shí)驗(yàn)過程中取對(duì)數(shù)期的藻細(xì)胞完成殺滅實(shí)驗(yàn).

實(shí)驗(yàn)配水是由純水機(jī)(Millipore Milli-Q,美國(guó))制取,用于實(shí)驗(yàn)系統(tǒng)配制試劑.

1.2 實(shí)驗(yàn)系統(tǒng)

?OH快速殺滅典型水華藻的實(shí)驗(yàn)系統(tǒng)如圖1所示,待處理高藻水流量為 4L/min,由泵泵入管路中,O2(純度為99.9%)的流量為0.5L/min,通入到氧等離子體發(fā)生器中,在大氣壓下微輝光與微流注交替促成的強(qiáng)電離放電作用下,O2被電離、離解生成O2+、O(1D)、O、O2-、O2(a1?g)、O3等氧活性粒子,通過高壓射流空化效應(yīng)高傳質(zhì)效率地注入到水中,生成以?OH為主的氧自由基溶液,其中還包括H2O2、HO2ˉ、O2?ˉ、O3?ˉ、HO3?等自由基[10-11],統(tǒng)稱為總氧化劑 TRO.在管路中?OH對(duì)藻細(xì)胞進(jìn)行快速高效的殺滅,取樣點(diǎn)的殺滅時(shí)間分別為0.4s、1.3s、2.1s、2.9s、3.7s、4.5s.

圖1 ?OH處理高藻水系統(tǒng)Fig.1 ?OH treating system of algae-laden water

1.3 實(shí)驗(yàn)方法

1.3.1 總氧化劑TRO濃度檢測(cè) TRO是以?OH為主,包括H2O2、HO2ˉ、O2?ˉ、O3?ˉ、HO3?和O2+H2O等氧自由基的總氧化劑濃度,由在線監(jiān)測(cè)儀(ATi Q45H,美國(guó))檢測(cè),同時(shí)依據(jù)USEPA 330.5標(biāo)準(zhǔn)中的 DPD(N,N-二乙基對(duì)苯二胺)分光光度法進(jìn)行測(cè)定[13].

1.3.2 藻細(xì)胞活性分析 熒光顯微鏡計(jì)數(shù)法,染色劑為SYTOX?Green(Life Technologies,美國(guó))核酸染色劑是帶 3個(gè)正電荷的不對(duì)稱花青染色,與表面帶正電荷的活體藻細(xì)胞互相排斥,并且由于其大分子染色結(jié)構(gòu),它不能穿透活細(xì)胞膜.相反,?OH影響藻細(xì)胞膜的通透性,允許 SYTOX Green核酸染色劑進(jìn)入失去活性的藻細(xì)胞內(nèi),與核酸結(jié)合,在488nm藍(lán)激發(fā)光激發(fā)下,呈現(xiàn)綠色熒光;活細(xì)胞呈現(xiàn)葉綠素的自體紅色熒光[14-15].通過觀察紅色熒光和綠色熒光清晰地辨別藻細(xì)胞死活.采用徠卡DM6000B全自動(dòng)熒光顯微鏡,放大400倍,在自然光下找到藻細(xì)胞,分別在綠色激發(fā)光和藍(lán)色激發(fā)光下判別死活、計(jì)數(shù),以 100格為一個(gè)計(jì)數(shù)單位,按1mL記錄.

流式細(xì)胞儀(Accuri C6,BD,美國(guó))檢測(cè),加入適量的染色劑(SYTOX?Green)于 1mL樣品中,避光,置于渦旋振蕩后靜置7min,經(jīng)60μm篩絹過濾,發(fā)出的綠色熒光在 FL1(525nm)通道被收集,發(fā)出的紅色熒光在FL3(620nm)通道被收集,數(shù)據(jù)通過FlowJo 7.6分析處理[16-17].

1.3.3 藻細(xì)胞的光合能力分析 光合活性參數(shù)Fv/Fm表示藻細(xì)胞光合反應(yīng)中心PSII的最大光量子產(chǎn)量,反應(yīng)了植物的最大潛在光合能力,該值越大說(shuō)明光和潛力越大.樣品經(jīng)15min的暗適應(yīng),使用葉綠素?zé)晒鈨x脈沖儀(PHYTO-PAM Walz,德國(guó))和Photo win v2.13(ED)操作軟件測(cè)定,其計(jì)算公式為:

2 結(jié)果與討論

2.1 采用熒光染色法確定?OH殺滅閾值

基于?OH致死混合藻的實(shí)驗(yàn),銅綠微囊藻、針桿藻和四尾柵藻的初始藻濃度分別為19.5×104、21.8×104和 4.90×104cells/mL,總藻為46.2×104cells/mL,總氧化劑TRO濃度設(shè)置為0、0.51、0.79、0.96、1.07、1.24和1.31mg/L,反應(yīng)時(shí)間為4.5s.TRO致死混合藻的關(guān)系曲線如圖2所示,隨著TRO濃度的增高,活藻密度急劇下降,?OH殺滅3種藻的閾值濃度為1.07mg/L.

圖2 TRO對(duì)3種藻致死閾值的影響Fig.2 Effect of TRO on the lethal threshold of 3algae species

圖3 ?OH致死3種藻前后的熒光顯微鏡觀察結(jié)果Fig.3 Light and fluorescence microscope of 3kinds of algae before and after ?OH inactivation

采用顯微鏡分析原藻細(xì)胞及閾值(1.07mg/L)致死藻細(xì)胞形態(tài)的變化,如圖3所示.自然光下觀察,原藻細(xì)胞(圖A、B、C)通體周圓,細(xì)胞壁光滑完好,胞內(nèi)結(jié)構(gòu)分布清晰,顏色鮮亮稠密;處理后(圖A1、B1、C1),藻細(xì)胞外形基本沒有變化,但顏色暗淡,胞內(nèi)分布模糊.在熒光下觀察,原藻細(xì)胞(圖a、b、c)發(fā)出紅色的葉綠素自體熒光;處理后(圖a1、b1、c1)細(xì)胞核發(fā)出強(qiáng)烈的綠色熒光,證明細(xì)胞死亡,染色劑通過細(xì)胞膜進(jìn)入細(xì)胞,與細(xì)胞核中的 DNA結(jié)合,使細(xì)胞核染色.根據(jù)藻細(xì)胞形態(tài)學(xué)分析,閾值濃度下細(xì)胞形態(tài)完整,無(wú)觀察到內(nèi)溶質(zhì)溢出.

2.2 采用光合能力確定?OH殺滅閾值

圖4 TRO對(duì)藻的光合參數(shù)Fv/Fm的影響Fig.4 Effect of TRO on photosynthetic parameter Fv/Fmof algae

2.3 采用流式細(xì)胞儀確定?OH殺滅閾值

圖5 ?OH致死3種藻的流式細(xì)胞儀檢測(cè)結(jié)果Fig.5 Flow cytometry results of ?OH inactivation of 3algae species Control為對(duì)照組;A:銅綠微囊藻;B:針桿藻;C:四尾柵藻

?OH致死 3種藻的流式細(xì)胞儀檢測(cè)結(jié)果如圖5所示,橫坐標(biāo)反應(yīng)了SYTOX Green染色劑與細(xì)胞核酸結(jié)合后發(fā)出的綠色熒光強(qiáng)度,通過FL1-H通道收集;縱坐標(biāo)反應(yīng)了葉綠素的自發(fā)紅色熒光強(qiáng)度,通過FL3-H通道收集;3種藻分布在不同區(qū)域,其中A、B、C依次為銅綠微囊藻、針桿藻和四尾柵藻,3部分總和占總藻 99.8%(如圖Control),隨著TRO濃度增高,A、B、C部分的藻細(xì)胞數(shù)量逐漸減少,并向右邊偏移;當(dāng) TRO濃度為0.51mg/L時(shí),A、B、C部分的藻細(xì)胞占34.8%;當(dāng)TRO濃度為0.96mg/L時(shí),A、B、C部分的藻細(xì)胞占16.4%;當(dāng)TRO濃度為閾值濃度1.07mg/L時(shí),3部分的藻細(xì)胞數(shù)量為 0,且在熒光顯微鏡下觀察,所有藻細(xì)胞發(fā)綠色熒光,藻細(xì)胞結(jié)構(gòu)完整,由此表明,強(qiáng)氧化劑?OH可能對(duì)藻細(xì)胞中的DNA造成損傷,進(jìn)而致死藻細(xì)胞.

2.4 ?OH殺滅3種藻的時(shí)間效應(yīng)

在致死閾值濃度1.07mg/L時(shí),將初始藻密度為46.2×104cells/mL的混合藻液注入到實(shí)驗(yàn)系統(tǒng)中(圖1),以氧活性粒子注入到管路中的高壓射流器處為0s,分別依次于不同作用時(shí)間(分別為0.3、2.1、2.9、3.7、4.5s)的取樣口取樣,同時(shí)用過量的飽和硫代硫酸鈉終止反應(yīng),測(cè)定樣品的光合活性參數(shù)Fv/Fm,結(jié)果如圖6所示.由圖6可知,隨著作用時(shí)間的增加,藻細(xì)胞光合活性呈現(xiàn)明顯下降趨勢(shì).Fv/Fm值在4.5s內(nèi)快速由0.65降至0(儀器顯示為不能檢出),表明?OH能快速進(jìn)入細(xì)胞,在4.5s內(nèi)使藻細(xì)胞的光合系統(tǒng)損傷而失去光合活性.藻細(xì)胞形態(tài)分析結(jié)果顯示,閾值濃度下細(xì)胞結(jié)構(gòu)沒有變化,無(wú)內(nèi)溶質(zhì)溢出,細(xì)胞形態(tài)完整.

圖6 ?OH致死藻時(shí)間對(duì)光合參數(shù)Fv/Fm的影響Fig.6 Effect of ?OH inactivation time for algae on photosynthetic parameter Fv/Fm

據(jù) Zhou等[4]研究二氧化氯法殺滅密度為100×104cells/mL 的銅綠微囊藻,在 ClO2為 1.0mg/L,作用時(shí)間 10min時(shí),藻細(xì)胞去除率達(dá)91.5%,但藻的細(xì)胞膜破裂,產(chǎn)生的副產(chǎn)物ClO2-和具有較大的潛在毒性[5];Coral等[8]研究臭氧法殺滅細(xì)胞密度為150×104cells/mL的銅綠微囊藻,在O3為4.0mg/L,作用時(shí)間10min時(shí),所有細(xì)胞失活,內(nèi)溶質(zhì)溢出;據(jù)Huase等[22]研究表明高錳酸鉀法殺滅密度為390×104cells/mL的銅綠微囊藻,在KMnO4為5mg/L,當(dāng)作用2h時(shí),Fv/Fm由0.45降至0.07,藻細(xì)胞去除率達(dá)84.6%,大量?jī)?nèi)溶質(zhì)溢出;Zhou等[23]研究過氧化氫法和硫酸銅法殺滅密度為400×104cells/mL的銅綠微囊藻,當(dāng)作用4h時(shí),在0.5mmol/L(即17mg/L)H2O2作用下,藻細(xì)胞的光合參數(shù)由 0.42降至 0.05;在 2.5μmol/L(即0.4mg/L)CuSO4作用下,藻細(xì)胞的光合參數(shù)由0.42降至0.04,但細(xì)胞膜破裂,藻毒素MC-LR外溢.常規(guī)氧化劑與藻細(xì)胞長(zhǎng)時(shí)間接觸作用是導(dǎo)致藻細(xì)胞失活和破損的主要原因.而?OH法可實(shí)現(xiàn)快速殺滅,這是由于?OH具有非常高的反應(yīng)速率常數(shù)(109mol/L·s),是其它氧化劑的107倍以上,反應(yīng)速度極快,可在數(shù)秒內(nèi)完成整個(gè)生化反應(yīng)過程.

3 結(jié)論

3.1 采用?OH開展致死銅綠微囊藻、針桿藻和四尾柵藻混合藻的實(shí)驗(yàn)研究,當(dāng)初始藻密度分別為19.5×104、21.8×104和4.90×104cells/mL時(shí),混合藻的致死 TRO閾值為 1.07mg/L,致死時(shí)間為4.5s.

3.2 在低劑量致死閾值條件下,?OH氧化銅綠微囊藻、針桿藻和四尾柵藻后,藻細(xì)胞失去活性,且藻細(xì)胞形態(tài)完整,細(xì)胞未發(fā)生破裂.

3.3 本研究規(guī)模化制備的?OH為高藻水的高效快速安全處理提供了新方法.

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Research on the rapid inactivation of typical algae blooms by hydroxyl radical.

DING Li-fei1, LI Hai-yan1, BAI min-dong1*, ZHENG Wu1, GUO Feng1, ZHANG Zhi-tao2*(1.College of Environment &Ecology, Xiamen University, Xiamen 361102, China；2.Environmental Engineering Institute, Dalian Maritime University, Dalian 116026, China). China Environmental Science, 2017,37(7)：2633～2638

Algae blooming in water sources breaks out increasingly and seriously threatened the water supply safety. Bench scale tests were conducted to study the effects of ?OH generated from strong ionization discharge and high pressure water jet cavitation on cell density, cell integrity and photosynthetic capacity of 3kinds of typical freshwater algae. Algae species including Microcystis aeruginosa, Synedra sp., and Scenedesmus quadricuauda were respectively prepared at concentrations of 19.5×104, 21.8×104and 4.90×104cells/mL, and the cell integrity was assessed by flow cytometry. Results suggested that the?OH lethal threshold of the algae was 1.07mg/L within the exposure time of 4.5s. The cell morphological observation results showed that all the cells were integral and no cytoplasm composition spilled. Hence, large-scale production of ?OH is a novel method to inactive typical algae species efficiently and to protect drinking water safety simultaneously.

hydroxyl radical；algae blooms；lethal threshold；exposure time

X52

A

1000-6923(2017)07-2633-06

丁麗飛(1990-),女,福建寧德人,碩士研究生,主要從事水污染防控研究.

2016-11-01

國(guó)家科技支撐計(jì)劃資助項(xiàng)目(2013BAC06B01, 2013BAC06B02);國(guó)家重大科研儀器研制項(xiàng)目(61427804)

* 責(zé)任作者, 白敏冬, 教授, mindong-bai@163.com; 張芝濤, 教授, newzhangzhitao@163.com