飲用水管網生物膜中弧菌數量影響因素研究

史亞,施曉峰,張永靜,柳景青,程東慶*

(1.浙江中醫藥大學醫學技術學院,杭州310053;2.浙江大學建筑工程學院,杭州310058)

飲用水管網生物膜中弧菌數量影響因素研究

史亞1,施曉峰1,張永靜1,柳景青2,程東慶1*

(1.浙江中醫藥大學醫學技術學院,杭州310053;2.浙江大學建筑工程學院,杭州310058)

摘要研究飲用水管網中管材、管齡、管徑等因素對生物膜弧菌數量的影響.采集中國東部某市管網生物膜樣本12份,用硫代硫酸鹽-檸檬酸鹽-膽鹽-蔗糖瓊脂培養基分離培養弧菌,并用弧菌科細菌生化鑒定系統鑒定并計數.結果表明,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)＞不銹鋼復合管(0.66±0.21 CFU/cm2),并且球墨鑄鐵管中的弧菌數量與其他管材間差異有統計學意義(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量有較大的影響,其中,以球墨鑄鐵管中的弧菌數量最多;因此,飲用水管段的鋪設應考慮鑄鐵管的潛在弧菌風險.

關鍵詞飲用水管網;弧菌;管材;管齡;管徑

隨著現代工業的發展,目前居民小區、公共建筑等終端用戶的飲用水以管網系統供水為主.飲用水供水系統作為出廠水到達用戶的最后一個環節,它是城市供水水質安全保障的最后屏障.研究表明,飲用水管網中有大量細菌以生物膜的形式存在[1].管網中的細菌可誘發管壁腐蝕和結垢,降低管網的輸水能力,引起水質惡化,更為嚴重的是病原微生物也易在生物膜中滋生,其隨管網水傳播更會增加飲用水的微生物風險[23].

在供水系統生物膜中存在諸如硝化螺菌屬、浮霉狀菌屬、酸桿菌屬、鞘氨醇單胞菌、假單胞菌屬等細菌[4].弧菌廣泛分布于自然界,以水體中最多.在已發現的76種弧菌中,至少有12種對人類有致病作用,常引起人體嚴重腹瀉和脫水[5].目前,關于飲用水管網中弧菌檢測以及其數量影響因素等研究鮮有報道.本研究以中國東部某市實際飲用水管網中的生物膜為試驗對象,分析管材、管齡、管徑等因素對生物膜中弧菌數量的影響.

1　材料與方法

1.1采樣點及采樣方法

2013年4月—10月,在中國東部某市12個飲用水管網采樣點進行采樣,管道具體參數見表1.截取所需管段,用無菌試管刷對內壁反復刷洗,再用生理鹽水進行沖洗.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移至實驗室低溫保藏,用于后續試驗.

1.2樣品預處理

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目的無菌篩網對樣品進行過濾,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,用于弧菌的培養鑒定.

1.3弧菌的分離培養及鑒定

將預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)瓊脂培養基上,于37℃恒溫培養24～48 h,根據菌落形態特征進行分類并計數.挑取典型菌落進行分離純化,革蘭氏染色,氧化酶試驗和葡萄糖氧化-發酵試驗(O/F試驗).其中,氧化酶陽性、葡萄糖發酵型的菌為疑似弧菌,采用弧菌科細菌生化鑒定系統(GYZ-9V)進一步鑒定.

表1　供水管網各管段相關參數_Table 1 Parameters of the pipes in drinking water distribution system

1.4細菌總數測定

參照《生活飲用水衛生標準檢驗方法》(GBT 5750—2006),將預處理后的樣品逐級稀釋,取1 m L涂布于營養瓊脂培養基上,每個梯度做3個平行,置于(36±1)℃培養箱內培養24 h后進行菌落計數.

1.5數據分析

應用SPSS 19.0軟件的最小顯著差異法對試驗所得數據進行差異顯著性檢驗.

2　結果與分析

2.1不同管材管網生物膜樣品中弧菌數量測定結果

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量明顯多于其他管材(P＜0.05),達到212±39.40 CFU/cm2,其次為鍍鋅管,數量為4.85±1.03 CFU/ cm2,再次為不銹鋼復合管,數量為0.66±0.21 CFU/ cm2;而細菌總數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水平差異有統計學意義.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菌和弧菌數量Fig.1 Amounts of bacteria and Vibrio on different materials

2.2不同管齡管網生物膜樣品中弧菌數量測定結果

選擇球墨鑄鐵管和鍍鋅管進行弧菌數量與管齡關系分析,結果見圖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,管齡與管網生物膜中的弧菌數量未見明顯規律.

圖2　不同管齡球墨鑄鐵管生物膜中細菌和弧菌數量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菌和弧菌數量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菌菌落總數在1 000～10 CFU/cm2之間,11年的數量最多,為2.12×104±5.63×103CFU/cm2;鍍鋅鋼管中細菌總數在1×105CFU/cm2以上,除管齡17年的生物膜細菌總數為346±26.5 CFU/cm2外.

2.3不同管徑管網生物膜樣品中弧菌數量測定結果

由圖4可見:球墨鑄鐵管3個不同管徑生物膜中弧菌數量以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).管徑與管網生物膜中的弧菌數量也未見明顯規律.

從圖4還可以看出:細菌總數與弧菌數量的變化規律基本一致,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菌和弧菌數量Fig.4 Amounts of bacteria and Vibrio on nodular cast iron pipes of different nominal diameters

3　討論

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

供水管道長年運行,隨著管齡增加,其中生物膜細菌數量也發生著變化.MARTINY等[4]研究飲用水供水管網生物膜形成過程中細菌數量及群落組成的變化發現,當管齡超過500 d(約1.3年)后生物膜的群落結構進入穩定期,超過709 d(約2年)后,生物膜中細菌總量進入穩定階段,細菌數量達到最大值.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菌總數和弧菌數量的研究結果顯示,細菌總數和弧菌數量與管齡的關系未見明顯規律.

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,生物膜中細菌數量越少[11].袁一星等[12]認為,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菌的生長,細菌數量越少.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,余氯質量濃度在0.18～0.48 mg/L范圍內.本試驗結果表明,管徑對細菌總數和弧菌數量的影響均不明顯.

管網材質與管壁表面特征對生物膜細菌有重要影響.金屬管材在水環境下易發生腐蝕,增加了管道內壁的粗糙度、含水率和黏滯性,有利于有機物的吸附,為細菌吸附繁殖生成生物膜提供了物質基礎.此外,多孔結構還可保護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而影響細菌生長.付軍等[14]研究發現,金屬管材中的氯胺衰減速率最快,其生物膜中的細菌數量也明顯多于其他管材.我們研究發現,鍍鋅管生物膜細菌數量最多,不銹鋼復合管最少.這是由于鍍鋅管內壁比較粗糙,而不銹鋼復合管內壁較為光滑,細菌附著生長的條件相對較差,營養物質也較難在管壁沉積,因此,細菌數量明顯較低.

此外,不同管材的生物膜細菌群落組成有很大差異.LIU等[15]研究表明,塑料管生物膜細菌群落以酸硫桿狀菌屬(Acidithiobacillus)、水小桿菌屬(Aquabacterium)和Limnobacter為主,鑄鐵管生物膜細菌群落以硫匣菌屬(Thiocapsa)為主.REN等[16]研究發現,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.本研究發現,弧菌數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離子能促進弧菌的生長.關于球墨鑄鐵管有利于弧菌生長的機制有待于進一步研究.

4　結論

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

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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

文獻標志碼A

基金項目:水體污染控制與治理國家科技重大專項(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;網絡出版日期(Published online):2016 11 19 URL:http://www.zjujournals.com/agr/CN/article/download ArticleFile.do?attach Type=PDF&id=10426