冉治霖,李紹峰,黃君禮,崔崇威,袁一星 (.哈爾濱工業(yè)大學(xué)市政環(huán)境工程學(xué)院,黑龍江 哈爾濱 50090；2.深圳職業(yè)技術(shù)學(xué)院建筑與環(huán)境工程系,廣東 深圳 58055)

氯氣滅活飲用水中隱孢子蟲的影響因素

冉治霖1,李紹峰2*,黃君禮1,崔崇威1,袁一星1(1.哈爾濱工業(yè)大學(xué)市政環(huán)境工程學(xué)院,黑龍江 哈爾濱 150090；2.深圳職業(yè)技術(shù)學(xué)院建筑與環(huán)境工程系,廣東 深圳 518055)

應(yīng)用熒光活體染色法研究 Cl2在水體中殺滅隱孢子蟲的效果,并探討投加量、作用時(shí)間、渾濁度、pH值、溫度、有機(jī)物含量等對(duì)Cl2滅活隱孢子蟲效果的影響規(guī)律,找出最佳投加量和作用時(shí)間.結(jié)果顯示,隱孢子蟲濃度1×106個(gè)/mL,溫度22℃,pH7.0,濁度為1.0NTU,氯氣投加量大于6.3mg/L,反應(yīng)時(shí)間360min,隱孢子蟲的滅活率可以達(dá)到預(yù)期滅活效果(滅活率＞99.0%).隱孢子蟲的滅活率與氯氣投加量和作用時(shí)間成正相關(guān);滅活率隨著渾濁度增加逐漸下降,渾濁度0.1～20.0NTU范圍內(nèi),氯氣投加量為6.3mg/L,作用時(shí)間大于900min,即可保證隱孢子蟲的滅活率符合預(yù)定要求;在弱酸性條件下氯氣滅活隱孢子蟲能力強(qiáng)于堿性條件,反應(yīng)溫度(5.0～35.0℃)范圍內(nèi),隱孢子蟲的滅活率與溫度成正相關(guān);HA濃度0～10.0mg/L,作用時(shí)間為360min時(shí),滅活率隨有機(jī)物濃度增加而降低.當(dāng)作用時(shí)間為900min時(shí),水中隱孢子蟲的滅活率均大于99.0%.

氯氣；滅活；隱孢子蟲；影響因素

隱孢子蟲(Cryptosporidium)是一種常見的腸道原蟲病原體,具有廣泛的脊椎動(dòng)物宿主,包括哺乳動(dòng)物、嚙齒動(dòng)物、鳥類、爬行動(dòng)物和魚類等[1].受感染的宿主可排放出大量卵囊污染廢水、地表水和地下水[2-3].如果水處理不充分,飲用水中的隱孢子蟲就會(huì)對(duì)人類造成嚴(yán)重威脅,特別是免疫功能缺陷者如嬰兒,老人以及艾滋病患者等,被感染后會(huì)引起腹瀉,甚至危及生命[4].近年來水源性隱孢子蟲病不斷爆發(fā),水中隱孢子蟲的污染及其消毒問題備受關(guān)注[5-6].據(jù)美國疾病控制預(yù)防中心估計(jì),每年約有250萬人感染隱孢子蟲病[7].而我國于1987年在南京首次發(fā)現(xiàn)了人隱孢子蟲病病例,之后在江蘇、重慶、安徽、內(nèi)蒙、福建、山東和湖南都有相關(guān)病例報(bào)道[8].

現(xiàn)有研究去除隱孢子蟲的水處理工藝和技術(shù)有光催化[9]、紫外[10]、臭氧[11]等.本文使用自制Cl2對(duì)飲用水中隱孢子蟲進(jìn)行滅活,探討投加濃度、滅活時(shí)間、溫度、pH值、濁度、有機(jī)物等因素的影響,以期獲取影響 Cl2滅活水中隱孢子蟲效果的最佳條件.

1 材料與方法

1.1 材料

Cl2由啟普發(fā)生器制取,多參數(shù)水質(zhì)分析儀(Merck NOVE 60)測(cè)定氯濃度.隱孢子蟲(Cryptosporidium)采于患病猴,經(jīng)過篩、硫酸鋅漂浮和蔗糖梯度離心等步驟,得到濃度為 1.0×107個(gè)/mL隱孢子蟲樣品.以2.5%重鉻酸鉀懸浮保存于4℃冰箱.

試劑:4,6-二脒基-2-苯基-吲哚(DAPI, Sigma),普匹碘胺(PI,Sigma USA),HBSS平衡鹽溶液(Sigma USA).

1.2 熒光活體染色

取 0.5mL PBS(磷酸緩沖溶液)保存樣品,加入1mL HBSS平衡鹽溶液漂洗 2次;沉淀于 160μL HBSS平衡鹽溶液中,加入20μL DAPI, 20μL PI儲(chǔ)備液,37℃溫浴1h;溫浴后加入1mL HBSS 清洗3次,洗去未染上顏色的DAPI和PI;涂片,熒光顯微鏡下鏡檢,各樣品分別取200個(gè)孢囊鏡檢.

1.3 實(shí)驗(yàn)方法

啟普發(fā)生器產(chǎn)生的 Cl2通入一棕色瓶,內(nèi)裝有1L 0.01mol/L的磷酸鈉緩沖液,調(diào)節(jié)pH值.使用德國Merck公司生產(chǎn)的 NOVE 60多參數(shù)水質(zhì)分析儀測(cè)定氯濃度.取若干支 10mL比色管分別加入稀釋后不同濃度的 Cl2緩沖溶液,迅速加入1×106個(gè)/mL 隱孢子蟲懸浮液,搖床轉(zhuǎn)速為100r/min,避光反應(yīng).首先,檢測(cè)不同作用時(shí)間對(duì)隱孢子蟲的滅活效果,同時(shí)檢測(cè)反應(yīng)體系中 Cl2的剩余濃度,找出最佳 Cl2投加量和作用時(shí)間.接著探討在最佳滅活條件下不同渾濁度、溫度、pH值、有機(jī)物濃度等對(duì)滅活效果的影響.

2 結(jié)果與討論

2.1 隱孢子蟲活性評(píng)價(jià)

本實(shí)驗(yàn)采用熒光活體染色法評(píng)價(jià)隱孢子蟲活性,圖 1為同一視野中隱孢子蟲經(jīng)過PI染色,在自然光和綠光下的照片.由圖 1a可見,自然光下隱孢子蟲周圍有淡綠色光環(huán).綠光照射(圖 1b)隱孢子蟲發(fā)出耀眼紅光為PI+型,PI-型不發(fā)光.其中PI+型為已死的隱孢子蟲.

圖1 熒光活體染色圖片F(xiàn)ig.1 Photo of Cryptosporidium detected by fluorescence staining method

2.2 Cl2投加量和作用時(shí)間對(duì)滅活率的影響

取10mL比色管,分別加入初始濃度為10.1, 8.2,6.3,3.1,1.5,0.6mg/L的氯氣溶液,加入 1×106個(gè) /mL隱孢子蟲懸浮液,溫度20℃, pH7.0,濁度為1.0NTU,避光反應(yīng).由圖 2可見,隨著反應(yīng)時(shí)間的增加,隱孢子蟲的存活率降低,特別是當(dāng)氯氣投量大于6.3mg/L,反應(yīng)360min(水中余氯小于3mg/L,符合國家生活飲用水衛(wèi)生標(biāo)準(zhǔn)對(duì)出水余氯的要求),隱孢子蟲的存活率小于1%,可以達(dá)到預(yù)期滅活效果(滅活率＞99.0%).如果氯氣投加量為3.1mg/L,作用時(shí)間必須大于 900min,方可達(dá)到滅活效果.

氯氣加入水中可轉(zhuǎn)變?yōu)辂}酸和次氯酸.其反應(yīng)式如下:

反應(yīng)生成的次氯酸體積小,具有很強(qiáng)的穿透力,呈電中性,能擴(kuò)散到帶負(fù)電的細(xì)菌表面,并迅速穿過微生物的細(xì)胞膜進(jìn)入生物體內(nèi),破壞其多種酶系統(tǒng)(主要是磷酸葡萄糖去氫酶的巰基被氧化破壞)及染色體系統(tǒng)[12],使之失去活力而死亡.另一方面次氯酸性質(zhì)很不穩(wěn)定,容易釋放出新生態(tài)氧.新生態(tài)氧與銨鹽、硫化氫、氧化亞鐵、亞硝酸鹽及有機(jī)物腐敗后產(chǎn)生的物質(zhì)相結(jié)合,進(jìn)而氧化水中的有機(jī)物和一些無機(jī)物質(zhì),從而抑制了依靠這些物質(zhì)為營養(yǎng)的大部分微生物的生長.

圖2 Cl2投加量和作用時(shí)間對(duì)隱孢子蟲活性的影響Fig.2 Viability of Cryptosporidium effected by different concentrations of Cl2 and contact time

2.3 濁度對(duì)滅活率的影響

由于天然水中含有各種懸浮物及膠體物質(zhì),影響了水體的濁度,而Falabi等[13]研究表明,賈第鞭毛蟲的去除與濁度有顯著關(guān)系,本研究在pH7.0,氯氣投加量為 6.3mg/L,作用時(shí)間分別為360,480,900min,設(shè)置反應(yīng)體系的渾濁度為 0.1, 0.5,1.0,2.0,5.0,10.0,20.0 NTU,檢測(cè)隱孢子蟲滅活率,結(jié)果見圖 3.由圖 3可見,氯氣作用時(shí)間為360min時(shí),隨著渾濁度的增加(0.1～20.0NTU),隱孢子蟲的滅活率下降(99.5%下降至 86.5%),飲用水的濁度越低,氯氣滅活水中隱孢子蟲卵囊越容易.隨著作用時(shí)間的增加(480,900min),水中隱孢子蟲的滅活率逐漸增高,尤其是作用時(shí)間大于900min,渾濁度0.1～20.0NTU范圍內(nèi),滅活率均大于 99%.原因可能是水中的懸浮物或膠體物質(zhì)對(duì)隱孢子蟲有一定的吸附作用,阻礙了氯氣消毒效果,從而影響了滅活率,另一方面,隱孢子蟲暴露時(shí)間的增加本身就有一定數(shù)量的蟲體裂解,從而提高了系統(tǒng)總的滅活率[14].出水廠濁度要求1.0NTU以下,因此實(shí)際生產(chǎn)中,氯氣投加量為6.3mg/L,作用時(shí)間大于 900min,即可保證隱孢子蟲的滅活.

圖3 濁度對(duì)隱孢子蟲滅活效果的影響Fig.3 Effect of different turbidities on the inactivation ratio of Cryptosporidium

2.4 pH值對(duì)滅活率的影響

圖4 不同pH值下隱孢子蟲滅活率的比較Fig.4 Comparison the inactivation ratio of Cryptosporidium under different pH values

飲用水的pH值范圍在6～9,設(shè)置較高或較低的 pH 值實(shí)際應(yīng)用價(jià)值較小[15],因此選取PH6,7,7.5,8,9進(jìn)行實(shí)驗(yàn).氯氣投加量為6.3mg/L,作用時(shí)間360,480min,渾濁度為1.0 NTU時(shí),不同pH值的反應(yīng)體系,氯氣對(duì)隱孢子蟲的滅活作用變化不大(97.3%～99.9%),酸性條件略好于堿性條件(圖4).作用時(shí)間大于480min后,pH值在6～9范圍內(nèi),隱孢子蟲滅活率均大于99%,達(dá)到預(yù)定滅活要求.

酸性條件略好于堿性條件的原因主要是:氯氣溶于水后,部分與水反應(yīng),生成次氯酸和鹽酸.次氯酸是弱電解質(zhì),在水中會(huì)電離成次氯酸根和氫離子,在水中起主要消毒殺菌作用的是次氯酸,而次氯酸根的殺菌能力遠(yuǎn)遠(yuǎn)低于次氯酸.因此,在弱酸性條件下,氯氣的殺滅隱孢子蟲能力較強(qiáng),而在堿性條件下,由于大多數(shù)的次氯酸電離成次氯酸根而使其滅活能力大大降低.

2.5 溫度對(duì)滅活率的影響

在pH7.0,濁度為1.0NTU,氯氣的投加濃度為6.3mg/L,作用時(shí)間 360,480min時(shí),分別設(shè)置反應(yīng)溫度為 5,15,22,30,35℃,檢測(cè)隱孢子蟲的存活率,結(jié)果見圖5.由圖5可見,作用時(shí)間為360min時(shí),反應(yīng)溫度(5.0～35.0℃)范圍內(nèi),隱孢子蟲的滅活率與溫度呈正相關(guān),滅活率從 95.4%上升為 99.8%,當(dāng)時(shí)間增加為480min后,隱孢子蟲滅活率均大于99%,達(dá)到預(yù)定的滅活要求.其原因可能是較低的溫度促使隱孢子蟲進(jìn)入休眠狀態(tài),消毒劑不易滅活隱孢子蟲,較高的溫度對(duì)隱孢子蟲具有一定的滅活作用,從而提高了滅活率.

圖5 不同溫度下隱孢子蟲滅活率的比較Fig.5 Comparison the inactivation ratio of Cryptosporidium under different temperatures

2.6 有機(jī)物對(duì)滅活率的影響

腐殖酸(HA)在自然水體中含量較高,是動(dòng)植物殘?bào)w經(jīng)過復(fù)雜的物理,化學(xué),生物等過程轉(zhuǎn)化而成的一種廣泛存在于水體,土壤中的高分子聚合物,為飲用水中主要的去除對(duì)象.本實(shí)驗(yàn)以HA為有機(jī)物代表成分,分別設(shè)置HA濃度為0,0.5,1.0, 2.0,3.0,5.0,10.0mg/L,氯氣投加量6.3mg/L,作用時(shí)間分別為360,480,900min,考察其對(duì)Cl2滅活隱孢子蟲的影響(圖6).

圖6 不同濃度HA對(duì)隱孢子蟲滅活效果的影響Fig.6 Effect of different HA concentrations on the inactivation of Cryptosporidium

由圖6可見,HA濃度0～10.0mg/L,作用時(shí)間為 360min時(shí),隱孢子蟲的滅活率分別為99.1%,98.0%,95.2%, 86.7%, 75.7%,66.9%,62.1%.滅活率與有機(jī)物濃度呈負(fù)相關(guān).這主要由于可溶性有機(jī)物的存在消耗了一定量的 Cl2,從而降低了Cl2的消毒效果.當(dāng)作用時(shí)間為900min時(shí),水中隱孢子蟲的滅活率均大于99.0%.

3 結(jié)論

3.1 隱孢子蟲1×106個(gè)/mL,溫度22℃,pH7.0,濁度為 1.0NTU,氯氣投加量大于 6.3mg/L,反應(yīng)360min,隱孢子蟲的滅活率＞99.0%.隱孢子蟲的滅活率與氯氣投量和作用時(shí)間成正相關(guān).

3.2 氯氣投加量為 6.3mg/L,氯氣的作用時(shí)間為360min時(shí),隨著渾濁度的增加(0.1～20.0NTU),隱孢子蟲的滅活率下降(由99.5%下降至86.5%),渾濁度 0.1～20.0NTU 范圍內(nèi),氯氣投加量為6.3mg/L,作用時(shí)間大于 900min,即可保證隱孢子蟲的滅活.

3.3 在弱酸性條件下隱孢子蟲的滅活率略好于堿性條件下,反應(yīng)溫度(5.0～35.0℃)范圍內(nèi),隱孢子蟲的滅活率與溫度成正相關(guān),隨著HA濃度的升高,滅活率降低.

3.4 當(dāng)氯氣投加量為 6.3mg/L,作用時(shí)間為900min,在飲用水消毒的渾濁度、溫度、pH值、有機(jī)雜質(zhì)濃度范圍內(nèi),隱孢子蟲的滅活率均在99.0%以上.

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Effect of various factors on chlorine inactivating Cryptosporidium in water.

RAN Zhi-lin1, LI Shao-feng2*, HUANG Jun-li1, CUI Chong-wei1, YUAN Yi-xing1(1.School of Municipal and Environmental Engineering, Harbin Institute of Technology, Harbin 150090, China；2.Department of Building and Environmental Engineering, Shenzhen Polytechnic Institute, Shenzhen 518055, China). China Environmental Science, 2010,30(6)：786～790

In order to study the effect of Cl2inactivating Cryptosporidium in water, different factors as Cl2concentration, contact time, pH, temperature, turbidity and organic content which might influence the inactivation were studied by using fluorescence staining method. With the Cryptosporidium concentration was 1×106/mL, turbidity 1.0NTU, temperature 22℃, pH 7.0, and after 360 min reaction, under the condition that the Cl2concentrations was 6.3mg/L, the inactivation ratio could be more than 99%. The lower turbidity was, the higher inactivating ratio could be

. If the inactivating time achieved 900min, it could meet the presetting inactivation ratio with turbidity 0.1～20.0 NTU. The ability of Cl2inactivating Cryptosporidium was stronger under acidic condition than that in alkali circumstance. With the increasing temperature, the inactivating effect was increased. When the reaction system contained different concentration of organics(0～10.0 mg/L) and the inactivating time 360min, the inactivation ratio reduced with concentration increasing. Besides, when the inactivating time achieved 900min, the inactivation ratio could achieve 99%.

Cl2；inactivating；Cryptosporidium；effect factors

2009-10-29

國家“863”項(xiàng)目(2006AAZ309)

* 責(zé)任作者, 教授, solve28@163.com

X505

A

1000-6923(2010)06-0786-05

冉治霖(1980-),男,河南鄭州人,哈爾濱工業(yè)大學(xué)市政環(huán)境工程學(xué)院博士研究生,主要從事水污染控制及污水資源化技術(shù).發(fā)表論文8篇.