石墨相氮化碳光催化滅活水中多重耐藥菌研究

齊 菲,孫迎雪*,常學明,殷秀峰,陸松柳,胡洪營

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石墨相氮化碳光催化滅活水中多重耐藥菌研究

齊 菲1,孫迎雪1*,常學明1,殷秀峰1,陸松柳2,胡洪營3

(1.北京工商大學環境科學與工程系,北京 100048；2.啟迪水務集團有限公司,上海 200072；3.清華大學環境學院,環境模擬與污染控制國家重點聯合實驗室,國家環境保護環境微生物利用與安全控制重點實驗室,北京 100084)

研究了光輻照和基于石墨相氮化碳(g-C3N4)光催化對二級出水中1株四環素和氨芐西林抗性多重耐藥菌CGMCC 1.1595的滅活效果.結果表明,汞燈輻照功率(100/300/500W)和輻照強度越高,其滅活效率相對越高,在500W汞燈60min輻照條件下,紫外長波(UVA)-可見光(300~579nm)對該多重耐藥菌的滅活率為0.41log;基于g-C3N4的光催化對其滅活率為1.31log.相比未加g-C3N4催化劑的光輻照滅菌,在UVA-可見光條件下g-C3N4對其滅活率的貢獻為61%~69%;在可見光條件下g-C3N4對其滅活率的貢獻達到60%~79%.在UVA-可見光g-C3N4光催化滅活CGMCC 1.1595反應體系中,活性氧自由基和電子空穴的活躍程度為:?OH>?O2->H2O2>h+>1O2,?OH為該光催化體系的主要活性物質,其次是?O2-和H2O2.

光照；石墨相氮化碳(g-C3N4)；多重耐藥菌；消毒；可見光

抗生素的大量生產和廣泛使用,導致我國每年約有5.40萬t抗生素隨污水進入城市污水處理廠[1-4],污水中相對較高抗生素水平導致細菌產生抗生素抗性[5-6],有些抗生素抗性菌(ARB)含有多種抗性基因(即多重耐藥細菌),將嚴重破壞水環境中微生物群落結構和功能,對水生生態系統安全甚至人類健康構成嚴重的威脅[7-8].

消毒工藝是飲用水處理、污水再生處理或排放水體控制微生物安全的重要環節,對滅活ARB發揮著重要作用[9-10].太陽光消毒由于利用清潔能源,近年來引起了消毒領域的廣泛關注[11-12],其機理主要是利用熱量和紫外光子的協同作用[13].對可見光響應的石墨相氮化碳(g-C3N4)是強化太陽光利用的新型半導體光催化材料,研究發現基于g-C3N4的光催化氧化可有效去除水中有機污染物以及使細菌和病毒失活[14-16].g-C3N4為二維層狀結構,具有獨特的電子能帶結構,層內C原子和N原子以sp2雜化方式結合在一起,形成類苯環的雙層結構,具有大量的π鍵存在[17-18].g-C3N4的導帶為-1.12eV,價帶為1.57eV[19],具有2.7eV的禁帶寬度,存在于最高占據分子軌道(HOMO)和最低未占分子軌道(LUMO)之間,可以吸收波長約小于475nm的太陽光[20-21]. g-C3N4還具有優良的化學穩定性和熱力學穩定性、結構和性能易于調控等優勢[22-23],在水處理領域有良好的應用前景.

本研究選取1株對四環素和氨芐西林有多重耐藥性的大腸桿菌為研究對象,研究光輻照和基于g-C3N4的光催化對該菌株的滅活效果和耐藥性的影響,并探討其光催化消毒機理.

1 材料與方法

1.1 實驗水樣

實驗用多重耐藥性大腸桿菌CGMCC 1.1595 (簡稱CGMCC 1.1595),購自中國普通微生物菌種保藏管理中心,該菌株攜帶質粒,且質粒上攜帶四環素耐藥基因和內酰胺類耐藥基因bla,可表達四環素和氨芐西林抗性[24].

1.2 光催化反應樣品的制備

將該多重耐藥菌接種于含有16mg/L四環素和32mg/L氨芐西林[25]的50mL營養肉湯培養基中(g/L:蛋白胨10,牛肉粉3,氯化鈉5,pH=7.2),37℃過夜培養16h[26].將所得菌液離心(6000r/min,10min,4℃),倒掉上清液,用無菌磷酸鹽緩沖溶液(PBS,pH=7.4)渦旋振蕩,充分去除培養基和抗生素,反復洗滌2次,最后將菌液懸浮于150mLPBS中,用麥氏比濁法獲得1.5×108CFU/mL(0.5麥氏)的菌液.加入無菌保存的g-C3N4光催化劑,菌液中的催化劑濃度為5g/L,在無菌黑暗條件下超聲20min,使催化劑均勻懸浮于菌液中,以在光催化劑表面達到吸附-解吸平衡[27-28],然后分裝到已滅菌的反應試管中,每只無菌試管的反應體系為20mL.

1.3 g-C3N4的制備與性能

基于三聚氰胺(C3H6N6)的g-C3N4常用作模擬可見光條件下的光催化劑.以C3H6N6和三聚氰酸(C3H3N3O3)混合物為原料制備g-C3N4[27]:將5g三聚氰胺和5g三聚氰酸(質量比1:1)粉末放入帶蓋氧化鋁坩堝中,使用馬弗爐進行加熱,以2.3℃/min升溫至550℃(過程約4h),保持4h后自然冷卻.冷卻后放入研缽中充分研磨,得到淡黃色粉末,待用.通過紫外-可見漫反射光譜分析得到該g-C3N4光響應波長范圍約為477nm以下,禁帶寬度為2.61eV;BET比表面積為42.3m2/g;掃描電鏡(SEM)和透射電鏡(TEM)表征如圖1所示,g-C3N4光催化劑是具有多孔和較為清晰的多層層狀結構.

圖1 g-C3N4光催化劑的SEM和TEM圖

1.4 光輻照反應

使用光化學反應儀(XPA-7,南京胥江機電廠)進行光輻照實驗,通過循環水和排風扇進行溫度控制,反應溫度設為25℃,反應儀中心部位配有汞燈(功率:100/300/500W;波長范圍:265.2~579nm).配備300和400nm濾波片用于模擬UVA.利用紫外輻照計(UV-A,北京師范大學機電廠,測定范圍:0.1~199.9× 103μW/cm2)和可見光輻照計(FZ-A,北京師范大學機電廠,測定范圍:0.1~199.9×103μW/cm2)測定光輻照強度.將準備好的樣品放入光化學反應儀外圍的固定裝置中進行光催化實驗,并進行磁力攪拌.反應時間為60min,每隔10min取樣一次.

1.5 耐藥菌的檢測和滅活率的計算

采用平板法測定樣品中的耐藥菌.首先在培養皿(Ф90mm)中加入25mL的營養瓊脂培養基(g/L:蛋白胨10,瓊脂15,氯化鈉5,牛肉膏粉3,pH=7.2),待凝固.將光輻照反應后的菌液進行梯度稀釋,取100μL梯度稀釋后的菌液進行平板涂布,每組3個平行樣品,然后將其倒置放于37℃培養箱中培養24h,計菌落數.用單位體積水樣的菌落形成單位(CFU/mL)表示.

滅活率= log10(0/N) (1)

式中,0為光輻照前水樣中耐藥菌的菌落數;N為光輻照后水樣中耐藥菌的菌落數.

1.6 耐藥性分析

抗生素耐藥性是指細菌在抗生素存在條件下的生存和生長能力[29],根據美國臨床和實驗室標準協會制定的抗菌藥物敏感性試驗執行標準,采用藥敏紙片擴散法考察該多重耐藥菌對四環素和氨芐西林耐藥性的變化[25].

上述的營養瓊脂平板上挑取單菌落懸浮于生理鹽水中,充分震蕩,用麥氏比濁法獲得1.5× 108CFU/ mL(0.5麥氏)的菌液.吸取100μL菌液加入到制備好的麥康凱瓊脂培養基(g/L:酪蛋白水解物17.5,淀粉1.5,牛肉浸粉5,瓊脂12.5,pH=7.2)平板表面,并涂布均勻.蓋上皿蓋,置于室溫無菌條件下干燥3~5min,稍干后再用無菌鑷子分別將四環素和氨芐西林藥敏紙片貼放于瓊脂平板表面,每組3個平行樣品.(35±2)℃孵育16~18h,用游標卡尺量取四環素和氨芐西林藥敏紙片周圍的抑菌圈直徑,根據抑菌圈直徑的大小,判斷CGMCC 1.1595對四環素和氨芐西林的耐藥、中介和敏感程度.腸桿科細菌對四環素耐受性的判定標準是:抑菌圈直徑>15mm為敏感,抑菌圈直徑=12~14mm為中介,抑菌圈直徑<11mm為耐藥;對氨芐西林耐受性的判定標準是:抑菌圈直徑>17mm為敏感,抑菌圈直徑=14~16mm為中介,抑菌圈直徑<13mm為耐藥[25].

2 結果與討論

2.1 光輻照對四環素和氨芐西林抗性大腸桿菌的消毒效果

圖2 光輻照對四環素和氨芐西林抗性大腸桿菌的影響

實驗采用不同功率(100,300,500W)的汞燈,利用300nm截止濾波片得到UVA-可見光(波長介于300~579nm),測得其中UVA光強分別為3.73,7.70, 15.70mW/cm2,可見光強分別為13.40,20.90, 39.20mW/cm2,UVA-可見光對該多重耐藥菌的滅活效果如圖2(a,b)所示.可以看出,隨著輻照時間的延長,該多重耐藥菌的濃度逐漸降低,光輻照60min時,100,300,500W汞燈對該多重耐藥菌的滅活率分別為0.23,0.34,0.41log,菌液濃度由1.45×108, 1.68×108,1.68×108CFU/mL降至8.39×107,7.52×107, 6.45×107CFU/mL.且當500W汞燈(UVA光強和可見光強分別為15.70,39.20mW/cm2),輻照60min時對該多重耐藥菌的滅活率最高.

采用400nm截止濾波片分析可見光(波長介于400~579nm)輻照對該多重耐藥菌滅活率的影響.100,300,500W汞燈輻照得到可見光光強分別為15.20,22.34,36.66mW/cm2,同時測得UVA光強分別為0.51,1.03,1.32mW/cm2,可見光輻照對該多重耐藥菌的滅活效果如圖2(c,d)所示,光輻照反應60min時,100,300,500W汞燈對該多重耐藥菌的滅活率分別為0.07,0.13,0.14log,菌液濃度由1.78×108, 1.78× 108,1.82×108CFU/mL分別降至1.51×108, 1.31×108, 1.33×108CFU/mL.在可見光輻照條件下,功率較高的500W汞燈(可見光強為36.66mW/cm2)光輻照60min,對該多重耐藥菌相對較高,達到0.14log.

UVA-可見光輻照在100,300,500W條件下對該多重耐藥菌的滅活率是可見光輻照的3.29、2.62和2.93倍.100,300,500W汞燈UVA-可見光輻照下的UVA光強度分別是可見光輻照下的7.31、7.48和11.89倍,可以判斷UVA-可見光輻照對該多重耐藥菌的滅活率UVA起主要作用,在UVA-可見光輻照60min時,100,300,500W汞燈的UVA對該多重耐藥菌的滅活率的貢獻分別為70%、79%和66%.相比可見光,UVA能夠穿透細菌細胞膜和細胞質,使細菌DNA相鄰的2個胸腺嘧啶共價結合形成二聚體,破壞DNA和RNA中的嘧啶與嘌呤堿基[30],從而破壞DNA和RNA的構型,使DNA損傷干擾其正常復制,導致細菌死亡[31].此外,UVA還會對細胞產生生物學效應[32].因此,UVA可直接破壞抗性基因滅活耐藥菌.

2.2 基于石墨相g-C3N4的光催化消毒效果

實驗采用不同功率(100,300,500W)的汞燈,反應體系中加入g-C3N4光催化劑(劑量為5g/L[27]),光催化對該多重耐藥菌的滅活效果如圖3(a,b)所示.在0~60min的反應過程中,隨著光催化時間的延長,該多重耐藥菌的濃度逐漸降低,反應60min時,100,300, 500W汞燈UVA-可見光對該多重耐藥菌的滅活率分別為0.75,0.87,1.31log,菌液濃度由1.98×108, 1.88×108,1.90×108CFU/mL降至3.51×107, 2.44×107, 9.33×106CFU/mL.且UVA-可見光光催化反應60min時,較高功率的500W汞燈對該多重耐藥菌的滅活率達到最高.

采用400nm截止濾波片分析可見光光催化對多重耐藥菌的滅活率,其實驗結果如圖3(c,d)所示,在該光催化條件下,反應60min時,100,300,500W汞燈對該多重耐藥菌的滅活率分別為0.33,0.32, 0.42log,菌液濃度由1.57×108,1.82×108,1.57× 108CFU/mL降至7.20×107,8.63×107,6.10×107CFU/ mL,且汞燈功率和光強越高,對該多重耐藥菌的滅活率相對越高.

圖3 光催化對四環素和氨芐西林抗性大腸桿菌的影響

在g-C3N4光催化消毒體系中,UVA-可見光(100、300和500W)對該多重耐藥菌的滅活率是可見光滅活的2.27、2.72和3.19倍,且在光催化反應60min時,UVA在100,300,500W條件下對該多重耐藥菌滅活率的貢獻分別為56%、63%和69%;可見光的貢獻為44%、37%和31%.相比不加g-C3N4催化劑的光輻照滅活體系,在UVA-可見光(100、300和500W)光催化條件下,g-C3N4對該多重耐藥菌滅活率的貢獻分別為69%、61%和69%;在可見光光催化條件下,g-C3N4對其滅活率的貢獻分別為79%、60%和66%.

加入g-C3N4光催化劑促進了光輻照對該多重耐藥菌的消毒效果.這是由于g-C3N4的2.61eV的帶隙使其能夠響應吸收波長小于475nm的藍紫光能量[33],受可見光激發可以產生電子躍遷,在催化劑內部生成光生電子和空穴,能夠在催化劑表面發生氧化還原反應.其中光生空穴具有極強的氧化能力[34].同時,還能與水反應生成活性氧(ROS),ROS可通過破壞細菌的細胞壁和細胞膜導致細菌喪失呼吸活性和細菌細胞內的成分泄漏,從而達到除菌的效果[35].ROS具有的強氧化能力,可以誘導DNA氧化損傷[36].而空穴在光照條件下能夠氧化破壞細菌細胞,并且其較大的表面積有利于與ARB充分接觸,增加滅活幾率,提高滅活率[22].

2.3 消毒前后的耐藥性分析

抑菌圈直徑越大,抗生素耐藥菌對該抗生素的耐藥性越低.圖4為光輻照和光催化對四環素和氨芐西林抗性大腸桿菌0~60min的耐藥性影響.

圖4 光輻照/光催化對四環素和氨芐西林抗性大腸桿菌的耐藥性影響

從圖4(a)中可以看出,不同功率(100,300,500W) UVA-可見光輻照條件下,不同輻照時間該多重耐藥菌四環素的抑菌圈直徑無明顯變化,且均小于12mm,氨芐西林藥敏紙片周圍沒有形成抑菌圈,這說明該光輻照條件不能影響四環素和氨芐西林抗性大腸桿菌的耐藥性.

從圖4(b)中可以看出,不同功率(100,300,500W)的UVA-可見光g-C3N4光催化消毒條件下,該多重耐藥菌四環素的抑菌圈直徑也無明顯變化,且均小于12mm,氨芐西林藥敏紙片周圍沒有形成抑菌圈,這說明該光催化條件不能影響四環素和氨芐西林抗性大腸桿菌的耐藥性,相關深入研究有待后續開展.

2.4 活性氧(ROS)對四環素和氨芐西林抗性大腸桿菌的影響

在光反應體系加入g-C3N4光催化劑后,能夠產生電子空穴(h+)和ROS,ROS通常包括:超氧陰離子自由基(?O2-)、羥基自由基(?OH)、單線態氧(1O2)和過氧化氫(H2O2)[37],是激發氧化反應的必要條件[38].由于ROS可與2',7'-二氯二氫熒光素(DCFH,非熒光)快速反應生成2',7'-二氯熒光素(DCF,熒光),通過測定DCF的熒光強度確定光催化劑ROS的濃度,其濃度越高,光催化劑性能越好[39].為了考察g-C3N4光催化對該多重耐藥菌的滅活機理,在光催化體系中分別添加50mmol/L叔丁醇(TBA)、50mmol/L草酸銨(AO)、50mmol/LL-組氨酸(L-H)、10U/mL超氧化物歧化酶(SOD)和1000U/mL過氧化氫酶(CAT),TBA、AO、L-H、SOD和CAT分別為?OH、h+、1O2、?O2-、和H2O2的淬滅劑,用抑制率(未加活性組分時耐藥菌的降解量0減去加入活性組分抑制后耐藥菌的降解量a,然后乘以100/0;%)表示該活性組分的在多重耐藥菌的滅活過程中的活躍程度[27,40].

在UVA-可見光(汞燈100/300/500W)g-C3N4光催化劑體系中,反應時間分別為10,60min的ROS和電子空穴的淬滅實驗結果如圖5(a,b)所示.加入TBA淬滅?OH后,反應時間10,60min,對該多重耐藥菌滅活程度的抑制率分別為32.28%~38.73%和31.59%~39.58%;加入AO淬滅h+后,對其滅活程度的抑制率分別為18.24%~24.46%和22.28%~27.51%;加入L-H淬滅1O2后,對其滅活程度的抑制率分別為17.75%~18.88%和20.69%~24.17%;加入SOD淬滅?O2-后,對其滅活程度的抑制率分別為21.88%~ 31.84%和24.03%~34.10%;加入CAT淬滅H2O2后,對其滅活程度的抑制率分別為23.79%~30.12%和20.55%~29.53%.

綜上可知,ROS和電子空穴對該多重耐藥菌滅活過程的活躍程度為:?OH>?O2->H2O2>h+>1O2,這是因為光激發能引起導帶上電子與價帶上空穴的空間電荷分離,產生光生電子空穴對.電子具有還原性,空穴具有氧化性,空穴與水中的OH-離子生成氧化性很高的?OH,?OH能夠氧化損傷細菌DNA結構中的鳥嘌呤[41].鳥嘌呤易于氧化形成鳥嘌呤基團,其不穩定并且易于與空氣中的O2和H+結合形成R- OOH并產生8-羥基-2'-脫氧鳥苷[42],由于?OH的作用,8-羥基-2'-脫氧鳥苷被氧化成CO2和H2O,細菌失去活性[43].同時,?OH還具有破壞微生物屏障、蛋白質酶和核酸的能力,導致細菌死亡[44].

圖5 活性物質對光催化反應的抑制率

Fig.5 Inhibitory rate of active substances on photocatalytic reaction

3 結論

3.1 光輻照(汞燈100,300,500W)對具有四環素和氨芐西林抗性多重抗性的大腸桿菌的滅活效率隨著汞燈功率和強度的增大而提高.UVA-可見光(300~579nm)光輻照60min時,100,300,500W汞燈(其中UVA光強分別為3.73,7.70,15.70mW/cm2,可見光強分別為13.40,20.90,39.20mW/cm2)對其滅活率分別達0.23,0.34,0.41log,其中UVA對滅活率的貢獻為66%~79%,可見光的貢獻率僅為21%~34%.

3.2 基于石墨相g-C3N4的光催化消毒對該多重耐藥菌的滅活率有明顯提高,UVA-可見光在光催化反應60min時,100,300,500W汞燈對該多重耐藥菌的滅活率分別為0.75,0.87,1.31log,其中可見光對其滅活效率的貢獻達到31%~44%.相比未加g-C3N4催化劑的光輻照滅菌,在UVA-可見光條件下g-C3N4對滅活率的貢獻為61%~69%,在可見光條件下g-C3N4對滅活率的貢獻達到60%~79%.

3.3 在光催化反應中,ROS和電子空穴對CGMCC 1.1595滅活過程中的活躍程度為:?OH> ?O2->H2O2>h+>1O2,?OH為主要催化活性物質,對該多重耐藥菌的滅活起主要作用,淬滅?OH后,對反應的抑制率最高(31.59%~39.58%).

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Graphite carbon nitride (g-C3N4) photocatalytic disinfection on a multidrug resistantstrain from secondary effluent.

QI Fei1, SUN Ying-xue1*, CHANG Xue-ming1, YIN Xiu-feng1, LU Song-Liu2, HU Hong-ying3

(1.Department of Environmental Science and Engineering, Beijing Technology and Business University, Beijing 100048, China；2.Tus-Water Group Limited, Shanghai 200072, China；3.State Key Joint Laboratory of Environmental Simulation and Pollution Control, State Environmental Protection Key Laboratory of Microorganism Application and Risk Control, School of Environment, Tsinghua University, Beijing 100084, China)., 2018,38(10)：3767~3774

The inactivation effects of multi-drug resistant bacteriumCGMCC 1.1595against tetracycline and ampicillin from secondary effluent by light irradiation and photocatalysis with graphite carbon nitride (g-C3N4) were studied. The results showed that the higher irradiation power of the mercury lamp (100/300/500W) with higher irradiation intensity could lead to higher inactivation efficiency. Under the inactivation by 500W mercury lamp irradiation of 60min, the inactivation rate ofCGMCC 1.1595was 0.41log by ultraviolet A (UVA)-visible light (300~579nm) irradiation, and the inactivation rate was up to 1.31log by g-C3N4photocatalysis. The contribution of g-C3N4to the UVA-visible light inactivation was 61%~69% compared to that without g-C3N4catalyst, while the contribution of g-C3N4to the visible light inactivation was 60%~79%. The significance of the reactive oxygen species (ROS) and hole (h+) for the g-C3N4photocatalytic inactivation ofCGMCC 1.1595were also investigated, with the activity order as ?OH>?O2->H2O2>h+>1O2. Hydroxyl radical (?OH) was a leading contributor to the irradiation, followed by ?O2-and H2O2.

light irradiation；graphite carbon nitride (g-C3N4)；multidrug resistant bacteria；disinfection；visible light

X505

A

1000-6923(2018)10-3767-08

齊 菲(1993-),女,北京人,北京工商大學碩士研究生,主要研究方向為水污染控制理論與技術.發表論文1篇.

2018-03-16

國家自然科學基金資助項目(21306003)

* 責任作者, 教授, sunyx@th.btbu.edu.cn