彭蘭生,關(guān)孟欣,黃 魁,夏 慧,桑春雷

蚯蚓攝食污泥對其腸道功能區(qū)微生物種群及耐藥基因的影響

彭蘭生,關(guān)孟欣,黃 魁*,夏 慧,桑春雷

(蘭州交通大學(xué)環(huán)境與市政工程學(xué)院,甘肅 蘭州 730070)

以赤子愛勝蚓()為實(shí)驗(yàn)蚓種,研究蚯蚓消化污泥前后,其腸道各功能區(qū)微生物種群結(jié)構(gòu)及耐藥基因(ARGs)豐度的變化情況.為表征活體功能性微生物,所取新鮮樣品均采用疊氮溴化丙錠(PMA)預(yù)處理后,再進(jìn)行DNA相關(guān)分析.結(jié)果表明:蚯蚓消化污泥5d后,細(xì)菌16S rDNA和真核生物18S rDNA的豐度在其胃中分別顯著增加了28.2倍和42.2倍(<0.05),而在砂囊和后腸中均顯著性降低(<0.05).此外,蚯蚓消化污泥使其砂囊中的優(yōu)勢菌門由變形菌門變?yōu)檐洷诰T,胃中的優(yōu)勢菌門由擬桿菌門變?yōu)楹癖诰T,而對后腸微生物種群結(jié)構(gòu)的影響較小.對于ARGs而言,大環(huán)內(nèi)酯類耐藥基因F、四環(huán)素類耐藥基因X和磺胺類耐藥基因2在蚯蚓胃中的豐度分別顯著升高了1.9×102倍、8.4×105倍和25.9倍(<0.05),而在其砂囊和后腸中ARGs總豐度分別顯著下降了11.0倍和45.2倍(<0.05).基于網(wǎng)絡(luò)分析結(jié)果顯示,蚯蚓攝食污泥可影響其腸道內(nèi)ARGs宿主細(xì)菌的結(jié)構(gòu)多樣性.

污泥資源化；蚯蚓堆肥；抗性基因；腸道微生物；生物污染物

隨著我國污水處理能力的快速提升,污泥產(chǎn)量亦逐年增加.截止2019年底,我國污泥年產(chǎn)量達(dá)3904萬t(以含水率80%計(jì))[1],污水污泥的處理處置已成為水處理行業(yè)亟需解決的難題[2].脫水污泥作為污水處理的終端副產(chǎn)物,積蓄了豐富的新興污染物如雌激素、微塑料、耐藥基因(ARGs)及病毒等[3].研究證實(shí)污泥是ARGs最豐富的儲庫之一[4].由于污泥具有較高的微生物群落多樣性,致使促進(jìn)ARGs可通過質(zhì)粒、整合子、轉(zhuǎn)座子等可移動遺傳元件在不同微生物之間發(fā)生水平轉(zhuǎn)移[5-6].ARGs一旦轉(zhuǎn)移到人類致病菌中形成超級細(xì)菌,將給人類疾病的治療帶來很大的困難.現(xiàn)有的研究表明傳統(tǒng)污泥資源化方法難以有效去除污泥中的ARGs[7-8],給污水污泥的處理處置帶來了新的挑戰(zhàn).

蚯蚓堆肥是利用蚯蚓和微生物共同作用的環(huán)境友好型污泥資源化技術(shù)[9].蚯蚓腸道消化行為被認(rèn)為是影響污泥蚯蚓堆肥效率的關(guān)鍵因素[10].蚯蚓砂囊、胃及后腸共同組成了其完整的消化系統(tǒng)鏈.砂囊是蚯蚓腸道消化系統(tǒng)的前端,它主要通過機(jī)械研磨作用破碎食物和微生物[11],蚯蚓胃部能夠分泌淀粉酶和蛋白酶,促使食物進(jìn)一步被消化.蚯蚓后腸可以分泌多種酶類消化有機(jī)物,并吸收轉(zhuǎn)化為自身的營養(yǎng)物質(zhì),已被認(rèn)為是蚯蚓消化系統(tǒng)中最關(guān)鍵的部位.先前的研究表明,蚯蚓堆肥可顯著去除污泥中四環(huán)素和氟喹諾酮類ARGs[12-13],且去除效果與蚯蚓腸道消化過程及其定植微生物結(jié)構(gòu)多樣性密切相關(guān).因此,針對蚯蚓腸道各功能區(qū)消化過程對污泥ARGs的影響研究能揭示蚯蚓腸道削減ARGs的關(guān)鍵機(jī)制.

本研究以污泥在蚯蚓腸道消化過程為研究對象,比較蚯蚓腸道消化污泥前后其各功能區(qū)微生物的種群演化及ARGs豐度的變化特征,旨在為污泥蚯蚓堆肥削減ARGs提供理論參考.

1 材料與方法

1.1 實(shí)驗(yàn)材料

采用健康有明顯環(huán)帶的赤子愛勝蚓()為供試蚓種,單體重約0.5g,為本實(shí)驗(yàn)室飼養(yǎng).采用底部開孔的長方體塑料箱(46cm×17cm×13cm)作為堆肥反應(yīng)器.本實(shí)驗(yàn)所使用的脫水污泥取自蘭州市安寧污水處理廠脫水車間的新鮮污泥.污泥的理化性質(zhì)見表1.

表1 實(shí)驗(yàn)所用污泥理化性質(zhì)

1.2 實(shí)驗(yàn)方法

為防止堆肥體系缺氧,先將脫水污泥制成粒徑為5mm的污泥顆粒,之后將已過夜清腸的400條健康的赤子愛勝蚓接種至3kg新鮮污泥顆粒中.蚯蚓堆肥實(shí)驗(yàn)在生化培養(yǎng)箱恒溫20℃下進(jìn)行,實(shí)驗(yàn)設(shè)置3組重復(fù).所有反應(yīng)器用帶孔的保鮮膜覆蓋,每隔3d噴灑少量水分保持濕度在60%～70%左右.蚯蚓消化5d后,分別從每個反應(yīng)器中隨機(jī)挑選3～5條健康的赤子愛勝蚓,將其迅速放進(jìn)裝有無水乙醇的培養(yǎng)皿中殺死.然后在無菌超純水中清洗3次,去除蚯蚓表層微生物的影響.致死后的蚯蚓按照環(huán)節(jié)數(shù)分區(qū)后,立即在無菌操作臺上用鑷子和刀片進(jìn)行解剖并獲取蚯蚓砂囊、胃及后腸組織樣品.每3～5條蚯蚓組織作為一個樣品.

1.3 測試方法

1.3.1 理化指標(biāo) 理化測試方法參考黃魁等[14]方法進(jìn)行.簡述如下:含水率及有機(jī)質(zhì)含量均采用恒重法.將研磨后的風(fēng)干樣與去離子水(干樣:水=1:50kg/L)混勻后測定pH值(雷磁PHS-3C,上海)和電導(dǎo)率(雷磁DDS-307,上海).氨氮采用納氏試劑分光光度計(jì)法(HJ 535-2009)[15].溶解性有機(jī)碳(DOC)為上述混合液稀釋10倍后,過0.45μm濾膜,用碳氮分析儀(耶拿MULTI N/C,2100,德國)進(jìn)行測定.

1.3.2 疊氮溴化丙錠(PMA)處理及DNA提取 將蚯蚓砂囊、胃與后腸組織樣品分別放入無菌研缽,加入2mL磷酸鹽緩沖液(pH=7.4)研磨成液體狀.隨后將組織樣品收集到無菌離心管中,加入5μL的25μmol/L PMA,于4℃靜置10min.然后置于LED燈(EM200,Takara)下照射20min,每隔5min將離心管取出搖晃一次,以確保PMA能充分與死菌DNA結(jié)合.采用DNeasy PowerSoil Kit(QIAGEN,德國)試劑盒分別提取蚯蚓砂囊、胃及后腸組織總DNA.并用1%的瓊脂糖凝膠電泳檢測濃度,最后將DNA樣品于-20℃冰箱保存?zhèn)溆?

1.3.3 熒光定量PCR 熒光定量PCR反應(yīng)在Thermal Cycler Dice Real Time System Lite TP700 (Takara,大連)儀器上進(jìn)行.定量反應(yīng)為25μL體系: 12.5μL TB GreenⅡ(Takara,大連),20μmol/L上下游引物各0.5μL,1μL DNA模板以及10.5μL DNA-free超純水.采用攜帶目的基因的大腸桿菌(,)質(zhì)粒,經(jīng)連續(xù)10倍稀釋作為定量PCR的標(biāo)準(zhǔn)品.用DNA-free超純水作為陰性對照,每個樣品做3次重復(fù).所用引物由生工生物工程(上海)股份有限公司合成,引物序列及反應(yīng)條件見表2.

1.3.4 質(zhì)粒的制備 目的基因進(jìn)行PCR擴(kuò)增后,切膠回收與純化,利用T-A克隆將其連接至 pMD20- T載體(Takara,大連).而后轉(zhuǎn)化至DH5α感受態(tài)細(xì)胞內(nèi),Luria-Bertani固體培養(yǎng)基中培養(yǎng)16h后,篩選白色陽性克隆子進(jìn)行培養(yǎng)富集.使用質(zhì)粒提取試劑盒(Takara,大連)提取質(zhì)粒DNA.利用Nanodrop (Thermo,美國)檢測質(zhì)粒DNA濃度.

表2 耐藥基因引物及其PCR反應(yīng)條件

1.3.5 PCR和高通量測序 采用細(xì)菌通用引物341F(5'-CCTAYGGGRBGCASCAG-3')和806R (5'- GGACTACNNGGGTATCTAAT-3')對細(xì)菌16S rDNA的V3-V4區(qū)進(jìn)行擴(kuò)增.使用Phusion High- Fidelity PCR Master Mix with HF Buffer(M0531NEB)高保真酶進(jìn)行擴(kuò)增.PCR反應(yīng)體系為30μL.PCR擴(kuò)增條件為:95℃預(yù)變性1min;30個循環(huán)包括98℃變性10s,50℃退火30s,72℃延伸30s;72℃終延伸5min.使用2%的瓊脂糖凝膠電泳對產(chǎn)物進(jìn)行檢測后,利用GeneJET膠回收試劑盒(Thermo Scientific)對產(chǎn)物進(jìn)行純化.使用TruSeq DNA PCR-Free Sample Preparation Kit對文庫進(jìn)行定量,接頭效率檢測,合格后使用NovaSeq6000平臺進(jìn)行測序(諾禾致源生物信息科技有限公司,北京).測序完成后,使用FLASH (v1.2.7)對序列進(jìn)行拼接,測序結(jié)果使用QIIME(1.9.1)軟件對序列進(jìn)行質(zhì)控過濾后,將獲得高質(zhì)量的tags序列使用VSEARCH二進(jìn)制文件(https://github. com/torognes/vsearch/)與物種注釋數(shù)據(jù)庫進(jìn)行比較,去除嵌合序列.隨后使用Uparse軟件(Uparse v7.0.1001)將相似度97%OTUs (Operational Taxonomic Units)進(jìn)行聚類.而后對OTUs序列進(jìn)行物種注釋,通過MUSCLE 3.8.31與Silva132數(shù)據(jù)庫(http://www.arb-silva.de/)進(jìn)行對比,獲得各OTU的分類學(xué)信息.測序結(jié)果已上傳至NCBI數(shù)據(jù)庫,上傳序列號為:SAMN19321836～SAMN19321856.

1.4 統(tǒng)計(jì)分析

使用SPSS 26統(tǒng)計(jì)軟件對各測試指標(biāo)在各組之間的差異進(jìn)行單因素方差分析,顯著性水平為0.05.使用Heml 1.0進(jìn)行熱圖繪制并進(jìn)行聚類.圖1、2、4均用OriginPro 2018(version 9.5)繪制.微生物與耐藥基因之間的關(guān)系網(wǎng)絡(luò)圖用Gephi(0.9.2)繪制.

2 結(jié)果與討論

2.1 蚯蚓腸道功能區(qū)活體微生物數(shù)量及種群結(jié)構(gòu)變化

2.1.1 活體微生物數(shù)量的變化 如圖 1 所示,蚯蚓攝食污泥后其砂囊和后腸中活體細(xì)菌豐度分別顯著性降低了1.1×102倍和2.9倍(<0.05),而胃中活體細(xì)菌豐度則顯著性增加了28.2倍(<0.05).對于真核生物而言,其豐度在蚯蚓砂囊和后腸中分別顯著性降低了1.6×103倍和1.3×102倍,但在胃中顯著性增加了42.2倍.由此可見,受攝食污泥的影響,蚯蚓腸道各功能區(qū)中細(xì)菌與真核生物的數(shù)量均表現(xiàn)出相近的變化趨勢.蚯蚓砂囊中活體微生物數(shù)量顯著降低,可能是因?yàn)槲⑸飻?shù)量在砂囊區(qū)域存在瓶頸效應(yīng)[16-18].Hu等[11]研究發(fā)現(xiàn)蚯蚓攝食牛糞或污泥,在其砂囊和后腸區(qū)域細(xì)菌豐度均顯著性降低,但砂囊區(qū)域降低幅度大于后腸.這與本研究的結(jié)果相近.另外,蚯蚓胃中活體微生物數(shù)量顯著增加歸因于蚯蚓胃作為內(nèi)源性微生物和外源性微生物交互的場所,致使大量的微生物群落在蚯蚓胃中集聚.

RS代表原始污泥,IG和FG分別代表原始蚯蚓砂囊和消化后蚯蚓砂囊,IS和FS分別代表原始蚯蚓胃和消化后蚯蚓胃,IH和FH分別代表原始蚯蚓后腸和消化后蚯蚓后腸;同種功能區(qū)(如IG和FG)之間不同字母表示其之間具有顯著性差異(<0.05),相同字母表示沒有顯著性差異(>0.05),不同功能區(qū)之間沒有比較意義,下同

表3 蚯蚓攝食污泥前后蚯蚓功能區(qū)細(xì)菌種群的Shannon指數(shù)與Simpson指數(shù)

注:同種功能區(qū)(如IG和FG)之間相同字母表示沒有顯著性差異(>0.05),不同功能區(qū)之間沒有比較意義.

圖2 蚯蚓攝食污泥前后腸道功能區(qū)細(xì)菌門水平種群結(jié)構(gòu)

圖3 蚯蚓攝食污泥前后蚯蚓腸道功能區(qū)細(xì)菌優(yōu)勢菌屬的豐度熱圖

2.1.2 活體微生物種群結(jié)構(gòu)的變化 由表3可見,蚯蚓攝食污泥后其砂囊和后腸中細(xì)菌種群的Shannon指數(shù)和Simpson指數(shù)均有所降低,但不具有顯著性(>0.05).該結(jié)果說明蚯蚓消化污泥會降低其砂囊和后腸中活體細(xì)菌種群的豐富度和均勻度.相似研究發(fā)現(xiàn),蚯蚓吞食牛糞或污泥,其砂囊區(qū)域細(xì)菌種群多樣性和均勻度均顯著下降[11].本研究中蚯蚓后腸中降低的活體細(xì)菌多樣性和豐富度,可能是由于蚯蚓后腸近乎厭氧的微環(huán)境[19]致使攝入的好氧微生物出現(xiàn)死亡所致.同時,蚯蚓胃中細(xì)菌種群的Shannon指數(shù)和Simpson指數(shù)分別增加了16.7%和3.2%,這種增加的趨勢與微生物數(shù)量的變化類似.

圖4 蚯蚓攝食污泥前后腸道功能區(qū)ARGs的絕對豐度

由圖2可得,蚯蚓攝食污泥前,其砂囊中定殖的活體細(xì)菌菌門主要有變形菌門(64.2%)、厚壁菌門(13.8%)、擬桿菌門(10.5%)和放線菌門(5.2%);而攝食后砂囊中軟壁菌門占絕對優(yōu)勢,其豐度由1.5%激增至55.7%.已有研究表明軟壁菌具有較強(qiáng)的核酸降解能力[20],其豐度的劇增表明蚯蚓砂囊可能是降解污泥中耐藥基因的主要場所.而砂囊中變形菌門的豐度顯著(<0.05)降低了60.4%.變形菌門豐度經(jīng)蚯蚓砂囊顯著下降一方面可能是由于蚯蚓常以γ-變形菌門和δ-變形菌門下屬微生物為食[11,21],另一方面可能是被砂囊中分泌的一些抗菌物質(zhì)所抑制[22].此外,陳景陽等[23]研究發(fā)現(xiàn)四環(huán)素會抑制變形菌門的豐度,且這種抑制與其劑量無關(guān),這也可能是造成變形菌門豐度下降的原因.由圖2可見,蚯蚓攝食污泥前,蚯蚓胃中擬桿菌門(42.3%)、厚壁菌門(19.8%)、放線菌門(16.9%)與變形菌門(14.5%)為細(xì)菌優(yōu)勢菌門,而攝食后胃中優(yōu)勢菌門轉(zhuǎn)變?yōu)楹癖诰T(31.9%)、變形菌門(25.4%)、擬桿菌門(17.0%)、放線菌門(11.3%)與綠彎菌門(3.0%).相比而言,攝食污泥致使蚯蚓胃中變形菌門與厚壁菌門的相對豐度分別顯著(<0.05)增加75.2%和61.1%,可能與其攝食污泥的微生物種群結(jié)構(gòu)有關(guān).同時,蚯蚓攝食污泥后,后腸中軟壁菌門和擬桿菌門的相對豐度分別顯著(<0.05)增加了402.3%和170.4%.這是由于擬桿菌門是大分子有機(jī)物的主要降解者并且軟壁菌門對氮、磷等元素的循環(huán)有重要的驅(qū)動作用[20,24],其二者豐度的激增表明蚯蚓后腸是污泥中大分子有機(jī)物的降解場所.Wang等[22]也研究發(fā)現(xiàn)有機(jī)物的分解消化主要發(fā)生在蚯蚓后腸.本研究發(fā)現(xiàn),與原始污泥相比,蚯蚓消化污泥后的蚓糞中有機(jī)質(zhì)含量下降了13.4%,電導(dǎo)率升高了2.0%,表明蚯蚓腸道是加速有機(jī)物的礦化和降解的重要場所.

由圖3可知,蚯蚓攝食前,砂囊中優(yōu)勢菌屬為、、、、、和,其中的豐度占絕對主導(dǎo)地位.而蚯蚓消化污泥后,砂囊中占主導(dǎo)地位,且顯著增加了37.1倍(<0.05).同時,蚯蚓攝食污泥后胃中與的豐度分別增加了28.9倍和10.8倍(<0.05).屬于蟲原體屬,可利用碳水化合物磷酸轉(zhuǎn)移酶發(fā)酵葡萄糖,同時具有水解精氨酸的能力[25],其在砂囊和胃中豐度的劇增,表明其參與了大量的能量和物質(zhì)的代謝.是屬于β-變形菌門的一種蚯蚓體內(nèi)的共生菌.研究顯示其豐度的增加能夠促進(jìn)蚯蚓更早的性成熟,而且孵化成繭的成功率更高[26].相比而言,蚯蚓攝食前后,其后腸中細(xì)菌屬水平結(jié)構(gòu)的變化較小,未呈現(xiàn)出顯著性差異(>0.05).有研究發(fā)現(xiàn)食物來源會影響蚯蚓腸道微生物種群結(jié)構(gòu),但是與蚯蚓腸道相關(guān)的核心微生物種群基本保持不變[27].Hu等[11]分別以污泥和牛糞為食物飼養(yǎng)蚯蚓,發(fā)現(xiàn)相比于污泥,蚯蚓攝食牛糞后其腸道微生物種群結(jié)構(gòu)變化較小,這與本研究相似.王洪濤等[28]研究了含砷土壤對4種蚯蚓腸道微生物種群的影響,發(fā)現(xiàn)變形菌門、厚壁菌門、綠彎菌門、放線菌門和酸桿菌門是土壤中的優(yōu)勢菌群,占比高達(dá)73.4%.而4種蚯蚓腸道內(nèi)的優(yōu)勢菌基本一致,主要是放線菌、厚壁菌和變形菌等,它們的相對豐度總和為76.6%.這說明底物與蚯蚓腸道微生物種群間既相互影響,又相互共存.由聚類分析(圖3)可見,IG組和FG組、IS組和FS組之間種群結(jié)構(gòu)存在較大差異,而IH組和FH組種群結(jié)構(gòu)差異較小,表明蚯蚓攝食污泥對后腸中微生物種群結(jié)構(gòu)影響較小.這可能是因?yàn)轵球竞竽c特殊的生境構(gòu)建了能夠滿足其生理需求的功能性微生物庫,該庫能夠較好地抵抗外源微生物菌群與環(huán)境條件變化的脅迫,其形成的過程與腸道內(nèi)微生物不斷適應(yīng)新環(huán)境與重金屬、抗生素等污染物有一定的關(guān)聯(lián)[29-30].

2.2 蚯蚓腸道功能區(qū)ARGs豐度變化

紫色節(jié)點(diǎn)代表可移動遺傳元件,藍(lán)色節(jié)點(diǎn)代表ARGs,黃色節(jié)點(diǎn)代表微生物;節(jié)點(diǎn)之間的連線表示節(jié)點(diǎn)之間顯著性的相互關(guān)系,紅色連線代表正相關(guān),綠色連線代表負(fù)相關(guān);連線的粗細(xì)與節(jié)點(diǎn)間的顯著性成正比

如圖4a所示,蚯蚓攝食污泥后,I1(整合子基因)豐度在其砂囊中略有增加(>0.05),而其在胃中顯著增加了4.4倍(<0.05),但其在后腸中顯著降低了3.0倍(<0.05).I1作為一種攜帶ARGs的遺傳元件,生物介質(zhì)中較高的細(xì)菌數(shù)量和多樣性會增加I1水平傳播的可能性[6,31].李建輝等[32]通過宏基因組學(xué)研究了牛糞蚯蚓糞、蔬菜蚯蚓糞和污泥蚯蚓糞中的微生物組成結(jié)構(gòu),發(fā)現(xiàn)含有較高微生物豐度和多樣性的污泥蚯蚓糞中整合子所占比例也較高.本研究中蚯蚓胃和后腸中I1的增減與胃和后腸中微生物數(shù)量及多樣性的變化有密切關(guān)聯(lián).

對于ARGs而言(圖4b～f),蚯蚓攝食污泥后,砂囊中除F(大環(huán)內(nèi)酯類ARGs)豐度顯著升高了10.7倍(<0.05)外,X、M(四環(huán)素類耐藥基因ARGs)、1和2(磺胺類ARGs)均有所降低.其中X顯著下降了18.9倍(<0.05),2顯著下降了87.4倍(<0.05),耐藥基因總豐度顯著下降11.0倍(<0.05).蚯蚓胃中除1外,F、X、M和2豐度在攝食污泥后均有不同程度升高,其中F、X、和2分別顯著升高了1.9×102倍、8.4×105倍和25.9倍(<0.05).對于蚯蚓后腸,除F外,X、M、1和2在攝食污泥后均有不同程度的降低,其中X、1和2分別顯著降低了3.4×102倍、8.4×105倍和45.0倍(<0.05),耐藥基因總豐度顯著下降45.2倍(<0.05).以上結(jié)果說明蚯蚓胃會對污泥中的ARGs具有選擇性富集作用,而蚯蚓砂囊和后腸對ARGs具有一定的抑制作用.這主要是因?yàn)樵谖钢邪l(fā)生了微生物種群的聚集效應(yīng),使得聚集的細(xì)菌群落成為了ARGs的受體[33-34],而蚯蚓后腸中的黏液具有選擇性殺菌作用,可導(dǎo)致某些ARGs的宿主細(xì)菌死亡,如糞產(chǎn)堿菌()、假單胞菌屬()及抗壞血酸克呂沃爾氏菌()等變形菌門中的細(xì)菌[35-36].而變形菌門可同時攜帶四環(huán)素類和磺胺類ARGs[37],從而致使后腸中四環(huán)素類和磺胺類ARGs豐度降低.值得注意的是,蚯蚓攝食污泥后,腸道總ARGs豐度較攝食前仍顯著增加了3.3倍(<0.05),這可能是污泥中較高含量的ARGs所致.污泥中抗生素的脅迫也會影響腸道ARGs的產(chǎn)生與傳播[38].例如,長期暴露于抗生素下導(dǎo)致蜜蜂腸道中四環(huán)素類ARGs中的8種亞型發(fā)生了富集[39]. Ding等[40]對長期施用污泥及雞糞肥土壤蚯蚓腸道中的ARGs進(jìn)行研究發(fā)現(xiàn),污泥和雞糞的施用顯著增加了蚯蚓腸道中ARGs的豐度和多樣性,在9種主要的ARGs中共檢測到98個亞型.雖然F與1的豐度在蚯蚓腸道中表現(xiàn)出下降趨勢,但蚯蚓作為污泥蚯蚓堆肥的關(guān)鍵主導(dǎo)者,其蚯蚓腸道總ARGs豐度的增加說明蚯蚓腸道是可移動的ARGs儲蓄庫,這亦是污泥蚯蚓堆肥難以完全削減污泥中的ARGs的重要原因之一.

2.3 ARGs與活體微生物的網(wǎng)絡(luò)分析

依據(jù)ARGs與活體微生物的共線性,利用網(wǎng)絡(luò)分析可揭示ARGs的潛在宿主細(xì)菌[41-42].如圖5所示,ARGs與活細(xì)菌之間構(gòu)建的網(wǎng)絡(luò)共包括16個節(jié)點(diǎn)和60條邊.蚯蚓攝食污泥前(圖5a),蚯蚓腸道內(nèi)有6種細(xì)菌菌屬與I1呈正相關(guān),說明其為I1潛在的宿主細(xì)菌.相比而言,F潛在的宿主細(xì)菌有3種,X和M潛在的宿主細(xì)菌均有7種,而1和2潛在的宿主細(xì)菌都為5種.蚯蚓攝食污泥后(圖5b),I1潛在的宿主細(xì)菌變?yōu)?種,F潛在的宿主細(xì)菌變?yōu)?種,X和M潛在的宿主細(xì)菌分別為6種和7種,而1和2潛在的宿主細(xì)菌分別為5種和4種.這些結(jié)果表明蚯蚓攝食污泥可影響其腸道內(nèi)ARGs宿主細(xì)菌菌群的結(jié)構(gòu)多樣性.不僅如此,蚯蚓堆肥還可顯著影響超級細(xì)菌的種群結(jié)構(gòu)多樣性[43].有研究表明,環(huán)境中的重金屬銅與大環(huán)內(nèi)酯類ARGs之間能夠形成共選擇作用,從而激活大環(huán)內(nèi)酯類ARGs的表達(dá)[44].因此可推測本研究中及等細(xì)菌從攝食前的非F宿主變?yōu)閿z食后F宿主,可能與污泥中的重金屬等污染物激活了F的表達(dá)有關(guān).

3 結(jié)論

3.1 蚯蚓攝食污泥會增加其胃中活體微生物的數(shù)量、多樣性和均勻度,而對其砂囊和后腸則表現(xiàn)出相反的結(jié)果.

3.2 蚯蚓攝食污泥后,蚯蚓腸道總ARGs豐度略有增加.蚯蚓砂囊和后腸具有削減耐藥基因的作用,而蚯蚓胃對ARGs具有一定的富集作用.

3.3 蚯蚓攝食污泥可影響其腸道內(nèi)ARGs宿主細(xì)菌的結(jié)構(gòu)多樣性.

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Effects of excess sludge fed by earthworms on microbial community and antibiotic resistance genes in their intestinal functional area.

PENG Lan-sheng, GUAN Meng-xin, HUANG Kui*, XIA Hui, SANG Chun-lei

(School of Environmental and Municipal Engineering, Lanzhou Jiaotong University, Lanzhou 730070, China)., 2022,42(1)：465～473

This study aimed to investigate the changes of microbial community structure and their antibiotics resistance genes (ARGs) abundances in the intestinal tract of, via detecting the sludge passing through different functional areas in earthworms’ gut. Prior to the DNA extraction, all fresh samples were pretreated with propidium monoazide (PMA) to characterize active functional microorganisms. The results showed that the abundances of bacterial 16S rDNA and eukaryotic 18S rDNA significantly increased (<0.05) by 28.2 and 42.2 times in stomach, and they significantly decreased (<0.05) in the gizzard and hindgut, after 5 days of earthworm digestion. In addition, the sludge digested by earthworms changed dominant proteobacteria to tenericutes in gizzard, and modified dominant bacteroidetes to firmicutes in stomach. However, the digestion process of earthworms had little effect on bacterial community structure in hindgut. For the ARGs, the abundances ofF,X and2 significantly increased (< 0.05) by 1.9×102, 8.4×105and 25.9 times in stomach of earthworm, respectively. While the total abundances of ARGs in gizzard and hindgut significantly decreased (<0.05) by 11.0 and 45.2 times, respectively. Moreover, network analysis revealed that the feeding behavior of earthworms for sludge could indicate effects on the structural diversity of host bacteria of ARGs in their intestine.

sludge recycling；vermicomposting；resistance genes；gut microbiota；biological pollutants

X172

A

1000-6923(2022)01-0465-09

彭蘭生(1995-),男,甘肅蘭州人,蘭州交通大學(xué)碩士研究生, 主要研究方向?yàn)槲勰噘Y源化.發(fā)表論文1篇.

2021-5-25

國家自然科學(xué)基金資助項(xiàng)目(51868036;52000095);蘭州交通大學(xué)百人計(jì)劃(第二批);甘肅省青年博士基金項(xiàng)目(2021-QB051);甘肅省優(yōu)秀研究生“創(chuàng)新之星”項(xiàng)目(2021CXZX-629)

* 責(zé)任作者, 副教授, huangk1199@hotmail.com