吳戈輝,趙 輝,萬琪琪,徐向前,曹瑞華,黃廷林,文 剛

紫外滅活水中3種致病性曲霉的效能及其光復活控制

吳戈輝,趙 輝,萬琪琪,徐向前,曹瑞華,黃廷林,文 剛*

(西安建筑科技大學環(huán)境與市政工程學院,西北水資源與環(huán)境生態(tài)教育部重點實驗室,陜西省環(huán)境工程重點實驗室,西安 710055)

以飲用水中常見的3種曲霉(黑曲霉、黃曲霉、煙曲霉)為研究對象,紫外線作為消毒手段,研究其滅活效能與機制及紫外控制曲霉光復活的效果.結果表明,由于孢子尺寸、孢子色素、疏水性的不同,滅活結果存在差異,3種曲霉孢子對紫外的抗性為:黃曲霉>黑曲霉>煙曲霉.三者的滅活速率常數(shù)()符合Chick-Watson模型,黑曲霉,黃曲霉,煙曲霉的值分別為0.027,0.026,0.031cm2/mJ,大小順序與滅活難度一致.紫外線通過穿透真菌孢子的細胞壁和細胞膜,阻斷DNA復制和轉錄,最終造成細胞膜損傷以及胞內(nèi)ROS水平的升高,滅活后的孢子呈現(xiàn)出明顯凹陷并且表面表現(xiàn)出褶皺.3種曲霉孢子的光復活率和最大存活率:黑曲霉>黃曲霉>煙曲霉,其光復活差異是由于胞內(nèi)的光解酶的數(shù)量和活性差異引起.

紫外；真菌孢子；曲霉；滅活機制；光復活

近年來,水環(huán)境的真菌爆發(fā)嚴重威脅了供水水質(zhì)安全[1-2].研究表明,在全球各地的飲用水水源中分離出了眾多的真菌屬[3-7].部分真菌還可能在飲用水中造成肉眼可見顆粒物,從而利于微生物形成生物膜,產(chǎn)生嗅味,加速管道腐蝕等問題[8-9].

紫外線(UV)由于其在消毒過程中對各種病原體具有廣譜性、形成消毒副產(chǎn)物(DBPs)少以及操作簡易,普遍應用于水處理工藝中[10-11].紫外線(200～ 280nm)可以直接穿透病原微生物的細胞膜而直接對遺傳物質(zhì)DNA與RNA造成損傷,如當UV作用于DNA時,微生物細胞中的DNA鏈上會形成環(huán)丁烷嘧啶二聚體(CPDs)和6-4嘧啶二聚體(6- 4PPs)[12-13],從而阻止DNA的轉錄和復制,最終導致細胞死亡[14].

Nourmoradi等[15]研究了低壓紫外(LPUV)滅活3種曲霉時發(fā)現(xiàn),孢子濃度為1000CFU/mL的煙曲霉、黑曲霉及黃曲霉滅活率達到99.99%所需紫外劑量分別僅為12.45,16.6,20.75mJ/cm2,但沒有對LPUV滅活3種曲霉的滅活機理及光復活控制進行研究.此外,與大腸桿菌等細菌相比,從地下水中分離的真菌孢子更耐紫外線照射,主要源于真菌孢子中存在細胞核,使紫外線照射更難到達和破壞DNA,從而導致真菌孢子具有高的抗紫外線性[16].Oliveira等[17]通過紫外發(fā)光二極管(UV-LED)滅活煙曲霉、黑曲霉和土曲霉,推斷出更大的孢子會吸收更多的輻射,從而減少到達細胞核的輻射.在4種不同屬(木霉、枝頂孢霉、青霉和枝孢霉)的真菌中,體積較大且有色的真菌孢子要比體積較小且透明的真菌孢子對紫外線照射的耐受性更強[18],但對曲霉屬的滅活規(guī)律還沒有探究.據(jù)報道,許多微生物具有2種主要的修復系統(tǒng)來修復紫外線(UV)消毒帶來的損傷,包括光復活和暗修復[12].光復活這一現(xiàn)象的存在,使得紫外線消毒的最終效果下降.

本研究選擇3種曲霉(黑曲霉、黃曲霉和煙曲霉)作為地下水中具有代表性的致病性曲霉物種,研究紫外對于3種曲霉的控制效能及光復活的控制,并采用流式細胞儀(FCM)評估滅活過程中細胞的損傷程度,探討其滅活機制.

1 材料與方法

1.1 培養(yǎng)基的配制

曲霉的培養(yǎng)基采用孟加拉紅培養(yǎng)基(DRBC),稱取31.6g固體DRBC培養(yǎng)基于燒杯中,加入1L超純水,攪拌均勻之后轉入1L的錐形瓶中,封口.將配制好的DRBC培養(yǎng)基置于121 ℃高壓蒸汽滅菌鍋中30min,取出后搖晃均勻,倒入直徑70mm的無菌培養(yǎng)皿中,待其凝固冷卻之后使用[19].

1.2 曲霉的培養(yǎng)及孢子懸液的制備

曲霉的接種在超凈工作臺中進行,使用被酒精燈燒過的接種環(huán)將曲霉菌絲轉移至新DRBC培養(yǎng)基上,置于28℃生化培養(yǎng)箱中,培養(yǎng)2～3周,待其生長至穩(wěn)定期.

使用移液槍吸取5mL高溫滅菌后的磷酸鹽緩沖液(PBS)加入到穩(wěn)定期的曲霉菌落表面,然后輕輕刮下曲霉孢子,將其倒入鋪入3層無菌濾紙的漏斗中進行過濾.將得到的曲霉孢子過濾液,4℃, 8000r/ min離心10min,倒掉上清液,重復3次,最后得到適宜濃度的曲霉孢子懸浮液.通過光學顯微鏡(BX51, Olympus,日本)進行孢子計數(shù),控制最終的孢子濃度為108,存于4℃冰箱中待用[20].

1.3 紫外滅活以及光復活實驗

紫外滅活試驗通過準平行光束儀來完成,采用低壓汞燈(UVC 254nm),實驗前開啟紫外反應器預熱30min至紫外燈穩(wěn)定.紫外劑量通常通過公式(1)計算.

式中:為紫外線劑量,mJ/cm2;為平均紫外光強度,mW/cm2;為紫外輻照時間,s.其中紫外光強度()采用KI-KIO3光量子法測定[21].本研究中紫外光強度測定為0.120mW/cm2.

滅活實驗在直徑為9cm的玻璃圓盤中進行,將1mL的孢子儲備液(108CFU/mL)加入99mL PBS中,搖晃均勻之后,取1mL的樣品作為空白對照樣品,放入紫外反應器中.在不同的時間間隔取樣,經(jīng)稀釋之后,將0.1mL樣品均勻涂布在DRBC培養(yǎng)基上,放在28℃的培養(yǎng)箱中培養(yǎng)2～3d后對曲霉進行計數(shù),單位為CFU/mL.滅活實驗均在室溫、pH=8.0下進行.

光復活實驗在滅活實驗后隨即進行[16],裝置中有2根熒光燈(UVA365, 8W, 15W),熒光燈的輻照強度由紫外輻照計(UVA365,欣寶科儀)測得.UVA的強度為0.10mW/cm2.實驗前,開啟裝置預熱15min使熒光燈達到穩(wěn)定.

UV滅活曲霉孢子2-log之后,放入光復活反應器中,開始光復活實驗,分別在5, 25, 45, 60, 120, 180, 240, 360, 480min時取樣.其他操作步驟同上.

1.4 流式細胞儀分析

采用流式細胞儀(Accuri C6,BD,USA)檢測消毒過程中曲霉孢子DNA含量變化、膜完整性、酯酶活性以及胞內(nèi)ROS的變化[22].使用的流式細胞儀檢測范圍為103～105CFU/mL,如有必要,將樣品懸浮液用超純水稀釋.每一樣品進行2次測定,取其平均值.

1.5 滅活效果評價

1.5.1 滅活動力學

式中:0和N分別是在失活時間0和時間處曲霉孢子的濃度,CFU/100mL.

滅活動力學通過Chick-Watson模型進行擬合[23],表達了滅活效率與暴露時間之間的線性關系.

式中:斜率為準一階滅活速率常數(shù),cm2/mJ;為紫外線通量率,mW/cm2;為滅活時間,s.

1.5.2 光復活動力學

式中:0是紫外線滅活前的孢子濃度,CFU/100mL;r是光復活一定時間后的孢子濃度,CFU/100mL.采用飽和型一階反應模型[24]預測光復活過程:

式中:1為一階光復活速率常數(shù);0為紫外線失活后的存活率;為光復活時間時的存活率;m是光活化過程中的最大存活率.使用相關系數(shù)(2)評估擬合優(yōu)度.

滅活結果的顯著性分析通過SPSS軟件進行.

2 結果與討論

2.1 紫外滅活效果及動力學

由圖1可知,隨著紫外劑量增加,3種曲霉孢子的滅活數(shù)也隨之增加,表明單獨紫外可以有效滅活3種致病性曲霉孢子.3種曲霉的紫外抗性:黃曲霉>黑曲霉>煙曲霉,黃曲霉和黑曲霉的紫外抗性相近.通過Chick-Watson動力學模型擬合紫外滅活黑曲霉、黃曲霉和煙曲霉過程中的滅活速率常數(shù)(),分別為0.027,0.026,0.031cm2/mJ,黃曲霉的滅活速率常數(shù)()最低,煙曲霉的滅活速率常數(shù)最高.

圖1 紫外對3種曲霉孢子的滅活曲線

初始孢子濃度為: (1～3)×106CFU/mL, pH=8.0

紫外滅活3種曲霉存在差異性可能的原因如下(表1):第一,3種曲霉孢子的尺寸存在差異性,可能導致其對于紫外滅活表現(xiàn)出不同的抗性,具體表現(xiàn)為孢子尺寸越大其抗性越強[3,17].煙曲霉在3種曲霉孢子中的尺寸最小,因此對紫外的抗性也就最小;濃度為 (1～3) × 106CFU/mL的3種曲霉孢子在PBS中會表現(xiàn)出不同的顏色,分別為黑色、淡黃色、淺灰色.有研究表明,真菌孢子細胞壁中的色素具有抗氧化特性,可以保護真菌免受消毒劑的損傷[25].真菌孢子中的色素可能與紫外線發(fā)生反應,從而阻止紫外穿透孢子,影響最終的滅活結果.黃曲霉孢子中抗性色素相比于黑曲霉更多,對消毒劑的抗性也更強[26].經(jīng)測定,黑曲霉、黃曲霉和煙曲霉孢子的疏水性分別為61.97%、77.00%和21.38%,而UV滅活曲霉的速率常數(shù)為煙曲霉(0.031cm2/mJ)>黑曲霉(0.027cm2/ mJ)>黃曲霉(0.026cm2/mJ),說明真菌滅活的難易程度與疏水性呈負相關,即孢子疏水性越大,滅活效果越差.這主要是由于疏水性在微生物表面粘附和相互聚集中起作用[27].不同的孢子具有不同的疏水特性[28-30],而疏水性越強的孢子之間具有更大的親和力[31-32],越容易形成聚集.真菌孢子聚集體對消毒劑的抗性會隨孢子聚集程度的增加而增加,與單分散孢子相比,聚集孢子的滅活常數(shù)會降低4～6倍[33].

表1 3種曲霉的生理特性差異

飲用水消毒[15]規(guī)定紫外線劑量為40mJ/cm2, Lehtola等[34]研究表明,在40mJ/cm2的UV254照射下,飲用水中99%以上的細菌可以被滅活,而對木霉、頂孢霉、青霉和枝孢霉滅活率達到99%時的紫外劑量分別為45, 50, 65, 130mJ/cm2. Wen等[35]發(fā)現(xiàn)青霉、木霉、頂孢霉和枝孢霉的紫外滅活速率常數(shù)()分別為0.062, 0.070, 0.065, 0.019cm2/mJ,滅活的難易程度與真菌孢子的表面積和體積有關.對于較大的真菌孢子在UV照射到達細胞核之前,其細胞質(zhì)對紫外線的吸收較高,導致了滅活效率較低[36].

2.2 光復活特征及動力學

如圖2所示,3種曲霉孢子呈現(xiàn)出不同程度的光復活,前100min,黃曲霉表現(xiàn)出更高的光復活率,但隨著時間延長,黑曲霉的光復活率高于黃曲霉和煙曲霉,這與3種曲霉的紫外抗性基本一致.曲霉的紫外抗性越弱,紫外線造成的損傷越嚴重,光復活率也就越低.

圖2 3種曲霉孢子經(jīng)2-log滅活后的光復活曲線

初始孢子濃度為: (1～3)×106CFU/mL, pH= 8.0,=25.0℃

初始孢子濃度為: (1～3)×106CFU/mL, 復活光UVA輻照強度為0.10mW/cm2,pH= 8.0,=25.0℃

由圖3(a)可知,480min的光復活后,3種曲霉孢子中黑曲霉表現(xiàn)出了最高的光復活最大存活率.由圖3(b)可知,黃曲霉光復活速率常數(shù)最小,煙曲霉與黑曲霉光復活速率常數(shù)相同.此外,曲霉孢子的光復活程度與胞內(nèi)的光解酶相關,3種曲霉孢子的光復活結果的差異性可能是光解酶數(shù)量和活性不同所導致的[37].

2.3 紫外滅活三種曲霉的機理分析

圖4 紫外滅活3種曲霉孢子過程中經(jīng)SG染色后的二維點圖

初始孢子濃度為: (1～3)×106CFU/mL, pH=8.0

2.3.1 紫外滅活對曲霉DNA含量的影響 由圖4可以發(fā)現(xiàn),隨著紫外劑量的增加,高FL1熒光強度的孢子越來越小,越來越多的孢子呈現(xiàn)低熒光強度,根據(jù)SG與胞內(nèi)雙鏈 DNA 結合發(fā)出綠色熒光的原理可知,真菌孢子在紫外照射的過程中DNA受到損傷,故而呈現(xiàn)出比初始孢子更低的熒光值,表現(xiàn)為圖5中3種曲霉孢子的DNA含量逐漸下降,即紫外劑量為140mJ/cm2時,黑曲霉,黃曲霉和煙曲霉的DNA含量分別下降40.67%,38.63%以及68.06%,這與3種曲霉孢子的抗性一致,說明紫外照射直接破壞了胞內(nèi)DNA,導致孢子死亡.

圖5 紫外滅活3種曲霉孢子過程中孢子DNA含量的變化

初始孢子濃度為: (1～3)×106CFU/mL, pH=8.0

2.3.2 紫外滅活對曲霉膜通透性的影響 由圖6可知,隨著紫外劑量從0mJ/cm2增加到140mJ/cm2,3種曲霉孢子的膜受損孢子數(shù)均隨之增加.黑曲霉和黃曲霉對紫外抗性較高,因此膜受損孢子數(shù)未有明顯升高;而煙曲霉的紫外抗性最弱,細胞膜損傷也最為嚴重,膜受損孢子數(shù)增加75.70%.圖7反映了滅活過程中曲霉孢子的膜受損孢子數(shù)變化,3種曲霉的膜完整孢子數(shù)為:黃曲霉>黑曲霉>煙曲霉,這與曲霉孢子的紫外抗性一致.

圖6 紫外滅活3種曲霉孢子過程中膜受損孢子變化

初始孢子濃度為: (1～3)×106CFU/mL, pH=8.0

圖7 紫外滅活3種曲霉孢子過程中經(jīng)SG/PI染色后的二維點圖

初始孢子濃度為: (1～3)×106CFU/mL, pH=8.0

2.3.3 紫外滅活對曲霉胞內(nèi)ROS的影響 曲霉的胞內(nèi)ROS在代謝活動中形成,作為調(diào)節(jié)真菌發(fā)育過程和生理反應的信號分子,也可作為代謝指標[38].當曲霉孢子休眠時,胞內(nèi)ROS水平維持在較低的水平,在曲霉孢子受到來自外界的刺激后,胞內(nèi)ROS水平迅速升高,出現(xiàn)氧化應激反應以抵抗外界壓力.曲霉孢子的胞內(nèi)相應防御機制能力存在限制,當胞內(nèi)ROS水平超出限值時,會引起曲霉孢子出現(xiàn)損傷[39-40].由圖8和圖9可知,隨著紫外劑量從0mJ/cm2增加到140mJ/cm2,3種曲霉孢子的胞內(nèi)高ROS水平也逐漸增加.其中,黑曲霉的胞內(nèi)高ROS水平僅增加了5.30%,而黃曲霉和煙曲霉則分別增加了41.90%和24.25%.這表明UV照射對于黑曲霉的影響相對較小.UV照射刺激黃曲霉胞內(nèi)高ROS水平升高,但胞內(nèi)ROS氧化能力并未超過黃曲霉的胞內(nèi)防御機制能力,因此黃曲霉表現(xiàn)出最強的紫外抗性.這是由于細胞內(nèi)ROS水平的變化與真菌細胞內(nèi)的基因和酶有關,如谷胱甘肽過氧化物酶(Gpx),最終導致紫外線照射后這些真菌孢子內(nèi)ROS水平不同[39].

圖8 紫外滅活3種曲霉孢子過程中胞內(nèi)高ROS水平變化

初始孢子濃度為: (1～3)×106CFU/mL, pH=8.0

圖9 紫外滅活3種曲霉孢子經(jīng)DHE染色后的直方圖

初始孢子濃度為: (1～3)×106CFU/mL, pH=8.0

2.3.4 紫外滅活前后曲霉形態(tài)的變化 由圖10可知,對于黑曲霉,在160mJ/cm2劑量的紫外照射之后,其整體呈橢圓狀,表面光滑,未有明顯性狀變化.對于黃曲霉,其整體呈橢圓狀,且在滅活前表面即有一定程度褶皺,與黑曲霉光滑的表面形成鮮明對比,而在滅活結束之后其表面也并未發(fā)生明顯的變化.而煙曲霉在滅活之前,呈橢圓狀,表明光滑,未見褶皺.但是,在經(jīng)過了160mJ/cm2劑量的紫外照射之后,其表面出現(xiàn)了明顯凹陷并且表面表現(xiàn)出褶皺.相比于煙曲霉,黑曲霉和黃曲霉滅活前后的SEM圖像并未發(fā)生明顯變化,可能是由于黑曲霉和黃曲霉的孢子尺寸大于煙曲霉,導致紫外更難直接作用于DNA.

圖10 紫外滅活3種曲霉孢子的SEM圖像(30000倍)

初始孢子濃度為: (1～3)×106CFU/mL, pH=8.0, UV劑量=160mJ/cm2

2.4 能耗分析

E,N(kWh/m3)定義為UV滅活真菌孢子1-log時的能量消耗,如下式(6)所示:

式中:為UV輻照表面積,cm2;N為滅活1-log真菌孢子時所需的紫外線劑量,mJ/cm2;W與kW、s與h、mL與m3之間的換算系數(shù)為3.6×103;為樣品體積,mL;為UV的轉換效率;WF是水因子[41-43].

經(jīng)計算可得,黑曲霉,黃曲霉和煙曲霉的E,N值分別為0.052,0.052,0.026kWh/m3.煙曲霉每滅活1-log真菌孢子所需的E,N最低,而黑曲霉和黃曲霉滅活1-log時所需的紫外劑量均為60mJ/cm2.但3種真菌孢子的E,N值均遠高于大腸桿菌(0.006kWh/ m3)、銅綠假單胞菌(0.011kWh/m3)和嗜肺軍團菌(0.006kWh/m3),且與HAdV2(0.057kWh/m3)和噬菌體Qb(0.049kWh/m3)等病毒相似[44-46].相比于細菌和病毒,真菌具有完整的細胞核和由幾丁質(zhì)組成的細胞壁,阻礙了紫外穿透細胞破壞遺傳物質(zhì)[47];真菌復雜的細胞結構可以使其在受到外界不良刺激時具有更強的應激防御機制;此外,病毒尺寸一般為30～200nm,細菌尺寸一般為0.4～2μm,而真菌尺寸一般為10～100μm,從而造成了真菌較細菌對紫外線的抗性更強.

3 結論

3.1 紫外滅活3種曲霉孢子的難度為:黃曲霉>黑曲霉>煙曲霉,三者的滅活速率常數(shù)大小順序與滅活難度相同,其滅活的差異可能是由于孢子尺寸、孢子色素、疏水性而引起的.

3.2 紫外滅活2-log10之后的3種曲霉孢子的光復活率和最大存活率:黑曲霉>黃曲霉>煙曲霉,其光復活速率常數(shù)分別為0.006 ,0.004 ,0.006min-1.3種曲霉的光復活差異是由于其胞內(nèi)的光解酶的數(shù)量和活性差異引起.

3.3 隨著紫外劑量的增加,3種曲霉孢子的DNA含量逐漸下降,表明紫外會穿透真菌孢子的細胞壁和細胞膜,直接損壞3種曲霉孢子DNA,造成孢子死亡.

3.4 紫外滅活對3種曲霉孢子會造成不同程度的膜損傷,其中對煙曲霉的損傷最嚴重,膜受損孢子數(shù)占比增加75.80%,對黑曲霉和黃曲霉的膜損傷程度較低.3種曲霉的膜受損孢子數(shù)占比與對紫外的滅活抗性表現(xiàn)出一致性.

3.5 紫外滅活3種曲霉孢子會導致其胞內(nèi)高ROS不同水平的升高,黑曲霉的胞內(nèi)高ROS僅增加了5.30%,而黃曲霉和煙曲霉則分別增加了41.90%和24.25%.結合滅活結果,3種曲霉中,黃曲霉具有最強的胞內(nèi)防御機制,煙曲霉的胞內(nèi)防御機制最弱.

3.6 黑曲霉、黃曲霉和煙曲霉分別被紫外滅活,其滅活前后的SEM圖像顯示黑曲霉和黃曲霉的表面性狀未出現(xiàn)明顯變化,煙曲霉則出現(xiàn)了表面凹陷,表明黑曲霉和黃曲霉相對煙曲霉具有更強的滅活抗性.

3.7 相比于黑曲霉和黃曲霉,煙曲霉每滅活1-log真菌孢子所需的E,N最低,但3種真菌孢子的E,N值均遠高于一些細菌,這是因為真菌孢子與細菌對紫外線的抗性不同所導致的.

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Inhibit the photoreactivation of three pathogenicspores in water by UV: kinetics and mechanism.

WU Ge-hui,ZHAO Hui, WAN Qi-qi, XU Xiang-qian, CAO Rui-hua, HUANG Ting-lin, WEN Gang*

(Key Laboratory of Northwest Water Resource, Environment and Ecology, Ministry of Education, Shaanxi Key Laboratory of Environmental Engineering, School of Environmental and Municipal Engineering, Xi’an University of Architecture and Technology, Xi’an 710055, China)., 2022,42(3)：1173～1181

Three kinds ofspores (,and) commonly found in drinking water were used as research objects, and UV was used as the disinfection method to study their inactivation efficiency and mechanism. Control of photoreactivation for threespores inactivated by UV was also evaluated. The different inactivation degree was due to the different size, pigment and hydrophobicity. The resistance of the threespores inactivated by UV was:>>. In addition, the inactivation rate constantsof the threespores were consistent with the Chick-Watson model, in which theof,andwere 0.027, 0.026 and 0.031 cm2/mJ, respectively. The order of the inactivation rate constantof the threespores was the same as the difficulty of inactivation. UV penetrates cell wall and cell membrane, which blocks DNA replication and transcription, ultimately causing membrane damage and the increase of intracellular high ROS level. After inactivation, the spores showed obvious sinking and wrinkle on the surface. Photoreactivation rate constant and maximum survival ratio of the threespores:>>. The discrepancy of photoreactivation among the threespecies was caused by the difference of the number and activity of intracellular photolyase.

UV；fungal spores；；disinfection mechanism；photoreactivation

X523

A

1000-6923(2022)03-1173-09

吳戈輝(1998-),女,陜西西安人,西安建筑科技大學博士研究生,主要從事飲用水水質(zhì)安全保障研究.

2021-08-10

國家自然科學基金(51978557);陜西省杰出青年科學基金(2018JC-026);陜西省重點研發(fā)計劃(2020ZDLSF06-05,2019ZDLSF06- 03);陜西高校青年創(chuàng)新團隊

*責任作者, 教授, hitwengang@163.com