飲用水管網(wǎng)生物膜中弧菌數(shù)量影響因素研究

史亞,施曉峰,張永靜,柳景青,程東慶*

(1.浙江中醫(yī)藥大學(xué)醫(yī)學(xué)技術(shù)學(xué)院,杭州310053;2.浙江大學(xué)建筑工程學(xué)院,杭州310058)

飲用水管網(wǎng)生物膜中弧菌數(shù)量影響因素研究

史亞1,施曉峰1,張永靜1,柳景青2,程東慶1*

(1.浙江中醫(yī)藥大學(xué)醫(yī)學(xué)技術(shù)學(xué)院,杭州310053;2.浙江大學(xué)建筑工程學(xué)院,杭州310058)

摘要研究飲用水管網(wǎng)中管材、管齡、管徑等因素對生物膜弧菌數(shù)量的影響.采集中國東部某市管網(wǎng)生物膜樣本12份,用硫代硫酸鹽-檸檬酸鹽-膽鹽-蔗糖瓊脂培養(yǎng)基分離培養(yǎng)弧菌,并用弧菌科細(xì)菌生化鑒定系統(tǒng)鑒定并計(jì)數(shù).結(jié)果表明,弧菌檢出率為50%(6/12).在管齡相近的不同管材管壁生物膜中,弧菌數(shù)量為球墨鑄鐵管(212± 39.40 CFU/cm2)＞鍍鋅鋼管(4.85±1.03 CFU/cm2)＞不銹鋼復(fù)合管(0.66±0.21 CFU/cm2),并且球墨鑄鐵管中的弧菌數(shù)量與其他管材間差異有統(tǒng)計(jì)學(xué)意義(P＜0.05),而在塑料管中未檢出弧菌;而不同管齡的球墨鑄鐵管以及鍍鋅鋼管生物膜中弧菌數(shù)量變化不明顯;在不同管徑(公稱直徑為200、150和300 mm)的球墨鑄鐵管生物膜中,弧菌數(shù)量分別為2.65×103±204、212±39.40和44.20±6.88 CFU/cm2.綜上表明,管材對生物膜弧菌數(shù)量有較大的影響,其中,以球墨鑄鐵管中的弧菌數(shù)量最多;因此,飲用水管段的鋪設(shè)應(yīng)考慮鑄鐵管的潛在弧菌風(fēng)險.

關(guān)鍵詞飲用水管網(wǎng);弧菌;管材;管齡;管徑

隨著現(xiàn)代工業(yè)的發(fā)展,目前居民小區(qū)、公共建筑等終端用戶的飲用水以管網(wǎng)系統(tǒng)供水為主.飲用水供水系統(tǒng)作為出廠水到達(dá)用戶的最后一個環(huán)節(jié),它是城市供水水質(zhì)安全保障的最后屏障.研究表明,飲用水管網(wǎng)中有大量細(xì)菌以生物膜的形式存在[1].管網(wǎng)中的細(xì)菌可誘發(fā)管壁腐蝕和結(jié)垢,降低管網(wǎng)的輸水能力,引起水質(zhì)惡化,更為嚴(yán)重的是病原微生物也易在生物膜中滋生,其隨管網(wǎng)水傳播更會增加飲用水的微生物風(fēng)險[23].

在供水系統(tǒng)生物膜中存在諸如硝化螺菌屬、浮霉?fàn)罹鷮?、酸桿菌屬、鞘氨醇單胞菌、假單胞菌屬等細(xì)菌[4].弧菌廣泛分布于自然界,以水體中最多.在已發(fā)現(xiàn)的76種弧菌中,至少有12種對人類有致病作用,常引起人體嚴(yán)重腹瀉和脫水[5].目前,關(guān)于飲用水管網(wǎng)中弧菌檢測以及其數(shù)量影響因素等研究鮮有報(bào)道.本研究以中國東部某市實(shí)際飲用水管網(wǎng)中的生物膜為試驗(yàn)對象,分析管材、管齡、管徑等因素對生物膜中弧菌數(shù)量的影響.

1　材料與方法

1.1采樣點(diǎn)及采樣方法

2013年4月—10月,在中國東部某市12個飲用水管網(wǎng)采樣點(diǎn)進(jìn)行采樣,管道具體參數(shù)見表1.截取所需管段,用無菌試管刷對內(nèi)壁反復(fù)刷洗,再用生理鹽水進(jìn)行沖洗.將采集的生物膜樣品置于無菌玻璃瓶中,并放入低溫采樣箱中迅速轉(zhuǎn)移至實(shí)驗(yàn)室低溫保藏,用于后續(xù)試驗(yàn).

1.2樣品預(yù)處理

在樣品瓶中加入無菌玻璃珠后置于搖床振蕩15 min,制成菌懸液,使用60目的無菌篩網(wǎng)對樣品進(jìn)行過濾,并用無菌生理鹽水沖洗;離心后棄上清,將沉淀物定容至一定體積,用于弧菌的培養(yǎng)鑒定.

1.3弧菌的分離培養(yǎng)及鑒定

將預(yù)處理后的樣品逐級稀釋,取1 m L接種于硫代硫酸鹽-檸檬酸鹽-膽鹽-蔗糖(thiosulfate-citrate-bile salts-sucrose,TCBS)瓊脂培養(yǎng)基上,于37℃恒溫培養(yǎng)24～48 h,根據(jù)菌落形態(tài)特征進(jìn)行分類并計(jì)數(shù).挑取典型菌落進(jìn)行分離純化,革蘭氏染色,氧化酶試驗(yàn)和葡萄糖氧化-發(fā)酵試驗(yàn)(O/F試驗(yàn)).其中,氧化酶陽性、葡萄糖發(fā)酵型的菌為疑似弧菌,采用弧菌科細(xì)菌生化鑒定系統(tǒng)(GYZ-9V)進(jìn)一步鑒定.

表1　供水管網(wǎng)各管段相關(guān)參數(shù)_Table 1 Parameters of the pipes in drinking water distribution system

1.4細(xì)菌總數(shù)測定

參照《生活飲用水衛(wèi)生標(biāo)準(zhǔn)檢驗(yàn)方法》(GBT 5750—2006),將預(yù)處理后的樣品逐級稀釋,取1 m L涂布于營養(yǎng)瓊脂培養(yǎng)基上,每個梯度做3個平行,置于(36±1)℃培養(yǎng)箱內(nèi)培養(yǎng)24 h后進(jìn)行菌落計(jì)數(shù).

1.5數(shù)據(jù)分析

應(yīng)用SPSS 19.0軟件的最小顯著差異法對試驗(yàn)所得數(shù)據(jù)進(jìn)行差異顯著性檢驗(yàn).

2　結(jié)果與分析

2.1不同管材管網(wǎng)生物膜樣品中弧菌數(shù)量測定結(jié)果

從圖1可以看出:在管齡相近的不同管材管壁生物膜上,在球墨鑄鐵管、鍍鋅管和不銹鋼復(fù)合管中檢出弧菌,而在塑料管中未檢出;在球墨鑄鐵管中弧菌數(shù)量明顯多于其他管材(P＜0.05),達(dá)到212±39.40 CFU/cm2,其次為鍍鋅管,數(shù)量為4.85±1.03 CFU/ cm2,再次為不銹鋼復(fù)合管,數(shù)量為0.66±0.21 CFU/ cm2;而細(xì)菌總數(shù)在不同管材生物膜中的分布為:鍍鋅鋼管(1.67×106±2.43×105CFU/cm2)＞球墨鑄鐵管(2.12×104±5.63×103CFU/cm2)≈塑料管(1.77×104±1.65×103CFU/cm2)＞不銹鋼復(fù)合管(7.17×102±1.35×102CFU/cm2).

柱狀圖上的不同小寫字母表示在P＜0.05水平差異有統(tǒng)計(jì)學(xué)意義.Different lowercase letters above the columns represent statistically significant differences at the 0.05 probability level.圖1 不同管材生物膜中細(xì)菌和弧菌數(shù)量Fig.1 Amounts of bacteria and Vibrio on different materials

2.2不同管齡管網(wǎng)生物膜樣品中弧菌數(shù)量測定結(jié)果

選擇球墨鑄鐵管和鍍鋅管進(jìn)行弧菌數(shù)量與管齡關(guān)系分析,結(jié)果見圖2和圖3.管齡為5年的球墨鑄鐵管生物膜中弧菌數(shù)量為35.4±4.26 CFU/cm2,6年和9年的未檢出弧菌,管齡為11年的生物膜中弧菌數(shù)量最多,為212±39.4 CFU/cm2;管齡為12年的鍍鋅鋼管生物膜中弧菌數(shù)量為4.85±1.03 CFU/ cm2,而在其他管齡(13、17和23年)中均未檢出弧菌.總之,管齡與管網(wǎng)生物膜中的弧菌數(shù)量未見明顯規(guī)律.

圖2　不同管齡球墨鑄鐵管生物膜中細(xì)菌和弧菌數(shù)量Fig.2 Amounts of bacteria and Vibrio on nodular cast iron pipes of different ages

圖3　不同管齡鍍鋅管生物膜中細(xì)菌和弧菌數(shù)量Fig.3 Amounts of bacteria and Vibrio on galvanized pipes of different ages

管齡為5、6和9年的球墨鑄鐵管生物膜中細(xì)菌菌落總數(shù)在1 000～10 CFU/cm2之間,11年的數(shù)量最多,為2.12×104±5.63×103CFU/cm2;鍍鋅鋼管中細(xì)菌總數(shù)在1×105CFU/cm2以上,除管齡17年的生物膜細(xì)菌總數(shù)為346±26.5 CFU/cm2外.

2.3不同管徑管網(wǎng)生物膜樣品中弧菌數(shù)量測定結(jié)果

由圖4可見:球墨鑄鐵管3個不同管徑生物膜中弧菌數(shù)量以200 mm的最多(2.65×103±204 CFU/cm2),顯著多于管徑150 mm(212±39.40 CFU/cm2)和300 mm(44.20±6.88 CFU/cm2) (P＜0.05).管徑與管網(wǎng)生物膜中的弧菌數(shù)量也未見明顯規(guī)律.

從圖4還可以看出:細(xì)菌總數(shù)與弧菌數(shù)量的變化規(guī)律基本一致,即管徑200 mm(8.28×105± 7.87×103CFU/cm2)＞150 mm(2.12×104±5.63× 103CFU/cm2)≈300 mm(1.22×104±1.59×103CFU/cm2).

圖4　球墨鑄鐵管不同管徑生物膜中細(xì)菌和弧菌數(shù)量Fig.4 Amounts of bacteria and Vibrio on nodular cast iron pipes of different nominal diameters

3　討論

細(xì)菌在飲用水管網(wǎng)中的再繁殖方式包括在水體中懸浮生長和在管內(nèi)壁附著生長2種.由于飲用水管網(wǎng)屬于貧營養(yǎng)生長環(huán)境,細(xì)菌在管壁的附著生長更占優(yōu)勢,即形成生物膜[6].生物膜的存在增大了病原菌在管網(wǎng)中滋生的機(jī)會[2-3].弧菌是外環(huán)境水體的定殖菌群,出廠水若消毒不徹底,在輸水過程中外源弧菌可進(jìn)入管道,并且在適宜條件下大量增殖.研究發(fā)現(xiàn),在夏秋季急性腹瀉患者中24.31%由病原性弧菌感染所致[7].此次調(diào)查的12份生物膜樣品中6份檢出弧菌,檢出率為50%,數(shù)量最多的達(dá)到2.65×103±204 CFU/cm2.多種因素(如水剪切力、流速等)皆會導(dǎo)致生物膜從管壁上脫落進(jìn)入水體中[8].研究發(fā)現(xiàn),攝入1×105CFU以上的副溶血弧菌可引起發(fā)病[9].假設(shè)100 cm2的生物膜脫落,則自來水局部弧菌含量很容易達(dá)到人感染所需的菌量,不慎攝入,即可對健康造成極大威脅.另有研究證實(shí),海魚弧菌濃度達(dá)到1×106CFU/m L,可引起傷口感染,并導(dǎo)致多臟器的組織細(xì)胞損傷[10].由此可見,管壁生物膜弧菌對居民健康存在潛在威脅.

供水管道長年運(yùn)行,隨著管齡增加,其中生物膜細(xì)菌數(shù)量也發(fā)生著變化.MARTINY等[4]研究飲用水供水管網(wǎng)生物膜形成過程中細(xì)菌數(shù)量及群落組成的變化發(fā)現(xiàn),當(dāng)管齡超過500 d(約1.3年)后生物膜的群落結(jié)構(gòu)進(jìn)入穩(wěn)定期,超過709 d(約2年)后,生物膜中細(xì)菌總量進(jìn)入穩(wěn)定階段,細(xì)菌數(shù)量達(dá)到最大值.我們選取的管道管齡均在5年以上,對不同管齡的鍍鋅管和球墨鑄鐵管中細(xì)菌總數(shù)和弧菌數(shù)量的研究結(jié)果顯示,細(xì)菌總數(shù)和弧菌數(shù)量與管齡的關(guān)系未見明顯規(guī)律.

供水管道管徑對生物膜的形成亦存在一定影響.在水流流速一致的情況下,管徑越小,生物膜中細(xì)菌數(shù)量越少[11].袁一星等[12]認(rèn)為,管徑會影響余氯衰減速率,管徑愈大,余氯衰減速度越慢,從而使水體中保持較高的氯抑制細(xì)菌的生長,細(xì)菌數(shù)量越少.為了減少水流流速和余氯對生物膜的影響,本文所研究的管段水流流速基本為0.1 m/s,余氯質(zhì)量濃度在0.18～0.48 mg/L范圍內(nèi).本試驗(yàn)結(jié)果表明,管徑對細(xì)菌總數(shù)和弧菌數(shù)量的影響均不明顯.

管網(wǎng)材質(zhì)與管壁表面特征對生物膜細(xì)菌有重要影響.金屬管材在水環(huán)境下易發(fā)生腐蝕,增加了管道內(nèi)壁的粗糙度、含水率和黏滯性,有利于有機(jī)物的吸附,為細(xì)菌吸附繁殖生成生物膜提供了物質(zhì)基礎(chǔ).此外,多孔結(jié)構(gòu)還可保護(hù)生物膜免受水流的沖刷[13].管材也可能通過對消毒劑的衰減進(jìn)而影響細(xì)菌生長.付軍等[14]研究發(fā)現(xiàn),金屬管材中的氯胺衰減速率最快,其生物膜中的細(xì)菌數(shù)量也明顯多于其他管材.我們研究發(fā)現(xiàn),鍍鋅管生物膜細(xì)菌數(shù)量最多,不銹鋼復(fù)合管最少.這是由于鍍鋅管內(nèi)壁比較粗糙,而不銹鋼復(fù)合管內(nèi)壁較為光滑,細(xì)菌附著生長的條件相對較差,營養(yǎng)物質(zhì)也較難在管壁沉積,因此,細(xì)菌數(shù)量明顯較低.

此外,不同管材的生物膜細(xì)菌群落組成有很大差異.LIU等[15]研究表明,塑料管生物膜細(xì)菌群落以酸硫桿狀菌屬(Acidithiobacillus)、水小桿菌屬(Aquabacterium)和Limnobacter為主,鑄鐵管生物膜細(xì)菌群落以硫匣菌屬(Thiocapsa)為主.REN等[16]研究發(fā)現(xiàn),鑄鐵管中以生絲微菌屬(Hyphomicrobium)和脫硫弧菌屬(Desulfovibrio)為主,而在鍍鋅管、不銹鋼管、塑料管中以鞘脂單胞菌屬(Sphingomonas)和假單胞菌屬(Pseudomonas)為主.本研究發(fā)現(xiàn),弧菌數(shù)量在球墨鑄鐵管中最多,可能是球墨鑄鐵管釋放的鐵(Fe)離子有利于弧菌生長.劉連生等[17]研究Fe2+、Fe3+對弧菌生長的影響結(jié)果表明,添加0.1%的Fe2+使弧菌的生長量增加2倍,添加0.1%的Fe3+使弧菌生長量增加25%:說明Fe離子能促進(jìn)弧菌的生長.關(guān)于球墨鑄鐵管有利于弧菌生長的機(jī)制有待于進(jìn)一步研究.

4　結(jié)論

管齡和管徑對生物膜中弧菌數(shù)量的影響無明顯規(guī)律,而管材對生物膜弧菌數(shù)量有較大的影響.在球墨鑄鐵管中弧菌數(shù)量最多,其次為鍍鋅管,再次為不銹鋼復(fù)合管,在塑料管中未檢出弧菌.因此,飲用水管段的鋪設(shè)應(yīng)考慮鑄鐵管的潛在弧菌風(fēng)險.

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SHI Ya1,SHI Xiaofeng1,ZHANG Yongjing1,LIU Jingqing2,CHENG Dongqing1*
(1.College of Medical Technology,Zhejiang Chinese Medical University,Hangzhou 310053,China;2.College of Civil Engineering and Architecture,Zhejiang University,Hangzhou 310058,China)

Summary With the development of modern industries,drinking water in residential area and public building were supplied by water distribution systems.The drinking water distribution system is the final step from water plants to the users,and therefore is essential to assure drinking water safety for customers.Previous studies confirmed that many bacteria in the drinking water distribution systems existed as biofilm,which could induce corrosion and scaling of the pipe walls,reduce the water quality and water-carrying capacity of the pipelines. Moreover,as the pathogens growing in the biofilms transferred in the pipelines,the risks caused by these microorganisms would increase.Vibrio species is an estuarine bacterium widely distributed in the natural aquatic environment around the world.Among the discovered 76 kinds of Vibrio species,at least 12 kinds have pathogenic effect on human,and often result in severe diarrhea and dehydration.We found the Vibrio species in our previoussurvey of pathogenic bacteria in biofilm,indicating that the residents were at potential risk from Vibrio species. Therefore,the effect of pipe materials,pipe ages,and pipe diameters on amount of Vibrio species in biofilm of drinking water distribution system was necessary to investigate.

In this study,12 biofilm samples were collected from the drinking water distribution system in east China. Vibrio was isolated using thiosulfate-citrate-bile salts-sucrose(TCBS)agar medium and was identified according to their biochemical reaction characteristics using biochemical identification kit for Vibrionaceae GYZ-9V.The amounts of total bacteria were determined by a spread-plating method according to the Ministry of Health Standard Examination Methods for Drinking Water(2006).Samples and their dilutions were spread on Petri dishes with nutrient agar medium.Analysis of variance(ANOVA)by a least significant difference test was used to compare Vibrio counts from different pipe materials,ages and diameters.

The results showed that Vibrio species could be detected in nodular cast iron,galvanized steel and stainless steel clad pipes,but not in plastic pipe.The Vibrio species amount on the biofilm attached to the nodular cast iron pipe(212±39.40 CFU/cm2)was significantly higher than those attached to the galvanized steel pipe(4.85±1.03 CFU/cm2)and stainless steel clad pipe(0.66±0.21 CFU/cm2).However,the rank of total bacteria amount was galvanized steel pipe＞nodular cast iron pipe≈plastic pipe＞stainless steel clad pipe.Pipe age had little influence on Vibrio amounts from galvanized steel and nodular cast iron pipes.Nodular cast iron pipe with a nominal diameter of 200 mm had the highest amount of Vibrio species(2.65×103±204 CFU/cm2),followed by 150 mm(212±39.40 CFU/cm2)and 300 mm(44.20±6.88 CFU/cm2),and the tendency of total bacteria amount was similar with Vibrio species.

In conclusion,contamination from Vibrio species with varying levels is observed in nodular cast iron, galvanized steel and stainless steel clad pipes,but not in plastic pipe.The different pipe materials,especially nodular cart iron pipe,have strong influence on Vibrio species amount in biofilm from drinking water distribution system;therefore,the potential risks of Vibrio species from nodular cast iron pipe should be taken into consideration while laying the pipelines.

Key wordsdrinking water distribution system;Vibrio;pipe material;pipe age;pipe diameter

Effects of drinking water distribution system on Vibrio amounts in biofilm.Journal of Zhejiang University(Agric.&Life Sci.),2016,42(6):665- 670

DOI:10.3785/j.issn.1008-9209.2016.01.271

中圖分類號R 123

文獻(xiàn)標(biāo)志碼A

基金項(xiàng)目:水體污染控制與治理國家科技重大專項(xiàng)(2012ZX07403-003).

*通信作者(Corresponding author):程東慶(http://orcid.org/0000-0002-2025-6420),E-mail:B2006069@163.com

第一作者聯(lián)系方式:史亞(http://orcid.org/0000-0001-8433-1584),E-mail:shiya711@126.com

收稿日期(Received):2016 01 27;接受日期(Accepted):2016 04 12;網(wǎng)絡(luò)出版日期(Published online):2016 11 19 URL:http://www.zjujournals.com/agr/CN/article/download ArticleFile.do?attach Type=PDF&id=10426