魏楓沂,徐俊杰,陳 進(jìn),李建輝,黃 魁,2*,董夕琳,夏 慧

基于活體微生物揭示蚯蚓對污泥耐藥基因轉(zhuǎn)歸的影響

魏楓沂1,徐俊杰1,陳 進(jìn)1,李建輝1,黃 魁1,2*,董夕琳3,夏 慧1

(1.蘭州交通大學(xué)環(huán)境與市政工程學(xué)院,甘肅 蘭州 730070；2.甘肅省黃河水環(huán)境重點(diǎn)實(shí)驗(yàn)室,甘肅 蘭州 730070；3.長春水務(wù)集團(tuán)有限公司,吉林 長春 130000)

為削減污泥蚯蚓堆肥產(chǎn)物中耐藥基因(ARGs),以無蚯蚓組為對照,采用疊氮溴化丙錠對蚯蚓堆肥樣品進(jìn)行預(yù)處理,探究蚯蚓對污泥中活微生物種群及其ARGs的影響.結(jié)果顯示,與對照組相比,蚯蚓堆肥產(chǎn)物中有機(jī)物礦化度與降解量分別顯著提升82.5%與5.2%(<0.05).并且接種蚯蚓使其產(chǎn)物中放線菌門的豐度顯著增加了65.6%(<0.05),而厚壁菌門和擬桿菌門的豐度分別顯著降低了74.7%和34.6%(<0.05).相較于對照組,蚯蚓堆肥致使1、2、F和M基因豐度分別顯著減少了66.5%、82.8%、72.8%和77.6%(<0.05),但B的豐度顯著增加了5.7倍(<0.05).蚯蚓堆肥產(chǎn)物中I1基因豐度比對照組顯著降低了67.2%(<0.05),蚯蚓處理后ARGs總絕對豐度為4.19×1013copies/g,ARGs總?cè)コ蕿?2.6%,比對照組高45.4%.研究表明,蚯蚓可通過改變活細(xì)菌種群結(jié)構(gòu),減少ARGs潛在活體宿主的豐度,進(jìn)而降低其傳播擴(kuò)散的潛在風(fēng)險(xiǎn).

堆肥；微生物種群；抗性基因；蚯蚓糞；污泥資源化

隨著我國城市污水處理量的逐年增加,剩余污泥產(chǎn)量也與日俱增[1].然而,當(dāng)前多數(shù)污水廠剩余污泥穩(wěn)定化處理能力不足,污泥處理處置形勢非常嚴(yán)峻[2].此外,污泥成分極為復(fù)雜,既含有碳、氮、磷等資源性物質(zhì),也含有重金屬、持久性有機(jī)污染物、ARGs、微塑料等污染性物質(zhì)[3].其中ARGs是一種新型的生物污染物,在環(huán)境中具有較強(qiáng)的擴(kuò)散傳播能力[4].研究表明,剩余污泥中ARGs種類多樣,總豐度高達(dá)1015copies/g[5].同時(shí),污泥中多元的微生物亦為ARGs的水平轉(zhuǎn)移起到促進(jìn)作用,增加人畜共患抗生素抗性致病細(xì)菌的傳播風(fēng)險(xiǎn)[5-6].因此,如何消減污泥處理過程中的ARGs,已成為污泥處理處置所要解決的關(guān)鍵問題[7].

蚯蚓堆肥通過蟲體與微生物的相互作用完成有機(jī)物的生物降解與穩(wěn)定,具有工藝簡單、能耗低、蚓糞肥效高等優(yōu)點(diǎn)[8-9],被認(rèn)為是一種生態(tài)環(huán)境友好的綠色技術(shù).由于污泥來源及性質(zhì)復(fù)雜多樣,堆肥處理后的蚯蚓糞中亦含有大量的ARGs.雖有研究表明蚯蚓堆肥可減少污泥中ARGs總豐度,但各研究結(jié)果存在爭議性[10-11].例如相關(guān)研究發(fā)現(xiàn)蚯蚓堆肥能有效降低污泥中磺胺類、四環(huán)素類抗性基因和整合子的豐度[12-13],陳景陽等[15]也證實(shí)蚯蚓堆肥可以顯著降低整合子的豐度,控制ARGs的傳播.但另有研究報(bào)道污泥蚯蚓糞中2、G及C的豐度在堆肥后顯著增加[14-15].前人的研究均基于總DNA,未區(qū)分堆肥體內(nèi)的活死菌可能是造成以上結(jié)果存在差異的原因之一.雖然死菌編碼的ARGs仍有傳播潛力,但環(huán)境中ARGs只有在活體微生物內(nèi)才能進(jìn)行代謝和轉(zhuǎn)錄等生命活動[16].因此,活體微生物體內(nèi)ARGs的豐度及轉(zhuǎn)移機(jī)制更為重要.然而目前關(guān)于ARGs的研究極少關(guān)注活體微生物.

疊氮溴化丙錠(PMA)可在強(qiáng)光下進(jìn)入膜受損細(xì)胞,形成不能PCR擴(kuò)增的修飾DNA[17],已廣泛應(yīng)用于細(xì)菌、病毒等各種微生物的活性檢測[18].因此,本研究采用PMA對堆肥樣品進(jìn)行預(yù)處理,結(jié)合熒光定量PCR和高通量測序技術(shù),分析蚯蚓處理污泥過程中活體微生物及其ARGs的影響,旨在為削減污泥蚯蚓糞中ARGs提供科學(xué)依據(jù).

1 材料和方法

1.1 供試材料

選用赤子愛勝蚓()作為堆肥蚓種,實(shí)驗(yàn)前蚯蚓用脫水污泥飼養(yǎng)馴化.堆肥實(shí)驗(yàn)反應(yīng)器選用帶蓋的矩形塑料箱(60cm×40cm×30cm).供試污泥取自蘭州市安寧區(qū)七里河污水廠脫泥車間(含水率73%),供試污泥的理化性質(zhì)見表1.

1.2 實(shí)驗(yàn)方法

將10kg新鮮脫水污泥投加至反應(yīng)器中,塊狀污泥稍加分散,堆體厚度為10cm.之后接種1200條單體重約0.5g活躍的赤子愛勝蚓作為蚯蚓處理開始堆肥實(shí)驗(yàn),以不添加蚯蚓的污泥作為對照處理.每個(gè)處理設(shè)3個(gè)重復(fù),實(shí)驗(yàn)在室溫(20～25℃)下進(jìn)行30d.為保持水分、濕度和有氧條件,在各反應(yīng)器上覆蓋帶孔保鮮膜,每隔一周手動翻堆減少污泥顆粒積壓團(tuán)聚,翻堆后噴灑少量自來水.堆肥20d后,蚯蚓處理組中污泥全部轉(zhuǎn)化為顆粒狀蚯蚓糞,將蚯蚓從堆肥反應(yīng)器中取出,堆肥產(chǎn)物繼續(xù)腐熟10d.隨后從各反應(yīng)器中取樣,一式兩份.一份自然風(fēng)干后研磨,過60目篩,置于4℃冰箱中保存,用于理化性質(zhì)分析;另一份新鮮樣品直接經(jīng)過PMA處理后提取DNA,并置于-20℃冰箱中冷凍保存,用于DNA相關(guān)分析.

1.3 測定方法

1.3.1 理化性質(zhì)分析 采用灼燒減量法測定樣品有機(jī)質(zhì)(HJ 761-2015)[19].將風(fēng)干研磨樣品與去離子水混勻(干樣:水=1:50;質(zhì)量濃度),磁力攪拌30min后測定混合液的pH值(雷磁,PHS-3C,上海)和電導(dǎo)率(雷磁,DDS-307,上海).將上述混合液稀釋10倍后,經(jīng)0.45μm薄濾膜抽濾,采用碳氮分析儀(耶拿,MULTI N/C2100,德國)測定溶解性有機(jī)碳.硝酸鹽氮采用紫外分光光度法(HJ/T 346-2007)[20],氨氮采用納氏試劑分光光度法(HJ 535-2009)[21],總氮采用堿性過硫酸鉀消解紫外分光光度法(HJ 636-2012)[22],總磷采用鉬酸銨分光光度法(GB 11893-89)[23]測定.具體理化測試參照黃魁等[24]方法進(jìn)行.

1.3.2 PMA處理與DNA提取 對采集樣品的PMA預(yù)處理參考Van Frankenhuyzen等[25]的方法進(jìn)行,簡述如下.將1g新鮮樣品加入200mL無菌超純水和2mL磷酸緩沖鹽溶液(0.01mol/L,pH=7.4)中,充分混勻后以300r/min磁力攪拌30min.隨后取2mL混勻液,加入5μL PMA(25μmol/L),將其充分均勻后,在4℃下靜置10min.隨后將溶液在發(fā)光二極管光解裝置(Takara,EM200,日本)中光解20min.每隔5min將離心管取出搖勻一次,以確保PMA能充分與死菌DNA結(jié)合.取上述經(jīng)PMA處理后的樣品用DNeasy?Power Soil?Kit(QIAGEN,德國)試劑盒提取DNA,并用1%瓊脂糖凝膠電泳檢測其濃度.

1.3.3 熒光定量PCR 采用熒光定量PCR儀(Takara,TP700,日本)對細(xì)菌16S rDNA(V3～V4區(qū))、四環(huán)素類抗性基因(M)、大環(huán)內(nèi)酯類抗性基因(B、F)、磺胺類抗性基因(1、2)以及移動遺傳元件(MGEs)整合酶基因(I1)進(jìn)行定量.引物序列及PCR擴(kuò)增條件參照文獻(xiàn)[14]的方法進(jìn)行.所用引物均購置于生工生物工程(上海)股份有限公司.25μL的定量PCR反應(yīng)體系為:TB Green II(Takara,日本)12.5μL,20μmoL上下游引物各0.5μL, DNA模板1μL,DNA-free超純水10.5μL.利用TB Green II與雙鏈 DNA結(jié)合發(fā)出的強(qiáng)烈熒光信號來監(jiān)測整個(gè)擴(kuò)增過程,擴(kuò)增效率控制在90%～110%.然后通過Ct值(擴(kuò)增循環(huán)次數(shù))和標(biāo)準(zhǔn)曲線對樣品中DNA的起始濃度進(jìn)行定量檢測.其中繪制標(biāo)準(zhǔn)曲線的標(biāo)準(zhǔn)品為攜帶目的基因的質(zhì)粒(Takara,pMD20-T,大連),詳細(xì)制備過程見文獻(xiàn)[26].

1.3.4 PCR和高通量測序 采用帶有Barcode堿基信息的細(xì)菌通用引物341F(5'-CCTACGGGAGGC- AGCAG-3')和806R(5'-GGACTACVSGGGTATCT- AAT-3')對16S rDNA的V3～V4區(qū)進(jìn)行擴(kuò)增.PCR擴(kuò)增使用Phusion? High-Fidelity PCR Master Mix with GC Buffer(New England Biolabs)的高效高保真酶進(jìn)行,其反應(yīng)條件為:98℃預(yù)變性30s;30個(gè)循環(huán)包括98℃變性15s,58℃退火15s,72℃延伸15s;72℃終延伸1min.所得擴(kuò)增產(chǎn)物使用2%濃度瓊脂糖凝膠進(jìn)行電泳檢測,并利用Thermo Scientific公司GeneJET 膠回收試劑盒對其進(jìn)行純化.使用Illumina公司TruSeq DNA PCR-Free Library Preparation Kit建庫試劑盒進(jìn)行文庫構(gòu)建,經(jīng)過Qubit定量和文庫檢測合格后,使用NovaSeq 6000平臺進(jìn)行上機(jī)測序(諾禾致源生物信息科技有限公司,北京).所得序列使用FLASH(v1.2.7)進(jìn)行拼接,而后使用QIIME(v1.9.1)進(jìn)行質(zhì)控過濾.Tags序列通過(https://github.com/ torognes/vsearch/)與物種注釋數(shù)據(jù)庫進(jìn)行比對檢測嵌合體序列,去掉嵌合體,得到有效Clean reads.使用Uparse(v7.0.1001)對序列進(jìn)行聚類,隨后通過MUSCLE 3.8.31與Silva 138數(shù)據(jù)庫(http://www.arb- silva.de/)比對分類,最終得到有效的測序數(shù)據(jù).測序結(jié)果已上傳至NCBI數(shù)據(jù)庫,序列號為SAMN21235626～21235634.

1.4 統(tǒng)計(jì)方法

使用Statistica 10.0軟件對樣品的理化性質(zhì)、ARGs數(shù)量在各組之間的差異進(jìn)行單因素方差分析(One-way ANOVA)和相關(guān)性分析,顯著性水平為0.05.ARGs的豐度圖和活微生物細(xì)菌群落豐度堆積圖使用OriginPro 2018(version 9.5)繪制.用HemI 1.0軟件繪制熱圖.用Canoco 4.5軟件對環(huán)境因子、微生物和ARGs之間的關(guān)系進(jìn)行冗余分析.

2 結(jié)果與討論

2.1 堆肥前后理化性質(zhì)的變化

電導(dǎo)率和有機(jī)質(zhì)的變化常用來表征蚯蚓堆肥過程中有機(jī)物降解與轉(zhuǎn)化的程度.由表1可知,實(shí)驗(yàn)結(jié)束后,蚯蚓堆肥組的電導(dǎo)率較對照組相比顯著增加了0.82倍(<0.05),而有機(jī)質(zhì)下降了1.40%.電導(dǎo)率的增加可能是由于蚯蚓提升了污泥中有機(jī)物的礦化作用,釋放出礦物鹽和無機(jī)離子等[27].在蚯蚓與微生物的共同作用下,有機(jī)質(zhì)的降解速率提升.溶解性有機(jī)碳(DOC)可以作為判斷堆肥腐熟的指標(biāo),相關(guān)研究[28]表明堆肥產(chǎn)物中DOC含量為4g/kg時(shí)即可認(rèn)為腐熟.相比對照組,蚯蚓組DOC減少了10.42%.這可能是由于蚯蚓的取食、破碎等刺激作用促進(jìn)了微生物量的增長[29],加速了堆肥基質(zhì)中有機(jī)質(zhì)的分解,進(jìn)而加快了蚯蚓對DOC的利用[30],產(chǎn)物蚯蚓糞的穩(wěn)定化程度較好.

表1 供試污泥及不同處理堆肥產(chǎn)物的理化性質(zhì)

注:同列指標(biāo)后字母不同表明其兩兩之間具有顯著性差異(<0.05),同行字母之間沒有比較意義,下同.

表1結(jié)果顯示,堆肥結(jié)束后兩處理組硝酸鹽氮的含量均顯著增加(<0.05),且蚯蚓組比對照組顯著提升了4.26倍(<0.05),表明蚯蚓能提升堆肥過程中的硝化作用.吳穎等[31]研究發(fā)現(xiàn)蚯蚓堆肥可顯著增加氨氧化古菌和氨氧化細(xì)菌的數(shù)量.同時(shí),蚯蚓活動增加了污泥內(nèi)部的孔隙率,為硝化細(xì)菌提供充足的氧氣,促進(jìn)硝化反應(yīng)的進(jìn)行,從而提高了有機(jī)物的轉(zhuǎn)化速率.對總氮而言,污泥蚯蚓堆肥產(chǎn)物比對照組增加了13.47%,但二者并不顯著.這可能是在蚯蚓活動過程中,蚯蚓排泄物及其體壁分泌的黏液所致[32].本文蚯蚓堆肥組總磷相比于對照組增加了17.39%,可能是因?yàn)槲⑸餄B出有機(jī)酸、磷酸酶的活化導(dǎo)致的有機(jī)磷礦化[33].Busato等[34]人認(rèn)為磷能富集在蚯蚓的糞便中,并向可利用的形態(tài)轉(zhuǎn)化.以上結(jié)果表明,蚯蚓堆肥可顯著提高有機(jī)物降解轉(zhuǎn)化的速率,使堆肥產(chǎn)物更加穩(wěn)定,其產(chǎn)物蚯蚓糞含大量營養(yǎng)元素,具有很大的農(nóng)用潛力.

2.2 堆肥前后活細(xì)菌群落的變化

由表2可知,與對照組相比,蚯蚓堆肥組活細(xì)菌群落的Shannon指數(shù)和Simpson指數(shù)分別增加了1.25%和0.34%.可見,蚯蚓能增加堆肥產(chǎn)物中活細(xì)菌的豐富度和均勻度,促進(jìn)微生物的生長繁殖.此前黃魁等[24]基于總DNA的污泥蚯蚓堆肥實(shí)驗(yàn)中,蚯蚓堆肥細(xì)菌群落的Shannon指數(shù)為7.40,相較于無蚯蚓組減小了2.63%.蚯蚓堆肥活細(xì)菌多樣性的增加,可能是由于蚯蚓的攝食和掘穴活動增加了體系孔隙率,促進(jìn)了好氧微生物的生長,同時(shí)蚯蚓黏液和蚓糞中富含的多種可生物利用的營養(yǎng)組分[35],也可以刺激某些微生物的生長.

表2 供試污泥與堆肥產(chǎn)物中微生物群落的Shannon和Simpson指數(shù)

圖1 供試污泥與不同處理堆肥產(chǎn)物中活細(xì)菌門和屬水平相對豐度

由圖1(a)可知,變形菌門(32.6%)、厚壁菌門(19.4%)、擬桿菌門(8.6%)與放線菌門(6.2%)是原始污泥活細(xì)菌的優(yōu)勢菌門.堆肥結(jié)束后,對照產(chǎn)物中活細(xì)菌的優(yōu)勢菌門依次為變形菌門(43.7%)、擬桿菌門(14.2%)和放線菌門(12.8%).而蚯蚓堆肥產(chǎn)物活細(xì)菌種群中變形菌門(35.2%)占比最大,其次是放線菌門(21.2%)和擬桿菌門(5.6%).上述結(jié)果表明,與對照組相比,蚯蚓堆肥后活細(xì)菌種群在門水平結(jié)構(gòu)上發(fā)生了顯著改變,其中放線菌門豐度增加了65.6%,而厚壁菌門和擬桿菌門豐度分別減少了74.6%和34.6%.先前針對總細(xì)菌DNA的研究[24]結(jié)果顯示,接種蚯蚓致使堆肥產(chǎn)物中變形菌門與厚壁菌門減少,而擬桿菌門和放線菌門顯著增加(<0.05).放線菌門常被認(rèn)為是堆肥腐熟的指示菌門[36],蚯蚓堆肥產(chǎn)物中較高的活放線菌豐度表明蚯蚓堆肥可產(chǎn)生更穩(wěn)定的污泥蚯蚓糞.擬桿菌門能將污泥中有機(jī)碳、有機(jī)氮化合物轉(zhuǎn)化為相對穩(wěn)定的產(chǎn)物[13],經(jīng)過蚯蚓腸道轉(zhuǎn)運(yùn)會降低擬桿菌門的豐度[37].也可能是本文采用了PMA預(yù)處理對活死細(xì)菌進(jìn)行區(qū)別,而活細(xì)菌中的擬桿菌門豐度較小.

圖1(b)為活細(xì)菌屬水平復(fù)雜熱圖,對照組中活體微生物(6.1%)、(3.8%)、(2.7%)、(1.9%)和(1.8%)等菌屬豐度占比較高,而蚯蚓堆肥產(chǎn)品中優(yōu)勢活體菌屬為(6.9%)、(4.1%)、(3.8%)、(1.9%)和(1.7%).結(jié)果顯示,與對照組相比,蚯蚓組中和的豐度分別顯著增加了2.6%和1.9%(<0.05).其中貝氏谷氨酸桿菌()屬于農(nóng)業(yè)益生菌[38],進(jìn)一步說明蚯蚓污泥堆肥產(chǎn)物的農(nóng)用潛力高.放線菌屬()占比的增加很可能與堆肥過程中抗生素類物質(zhì)有 關(guān).

2.3 堆肥前后ARGs和MGEs的豐度變化

兩處理組堆肥前后各ARGs絕對豐度的變化如圖2所示,對照組和蚯蚓組堆肥后1、F和M的豐度變化呈相近的下降趨勢.相較于供試污泥,對照組和蚯蚓組中1、F和M的豐度分別顯著降低了46.3%～82.1%、48.3%～86.0%和66.1%～ 92.4%(<0.05).其中蚯蚓堆肥對1、F和M的去除率均顯著高于對照組(<0.05),見圖2(a、d、e).陳景陽等[15]的研究證實(shí),蚯蚓可以在污泥蚯蚓堆肥過程中去除部分四環(huán)素抗性基因.先前的研究結(jié)果表明,與對照處理相比,蚯蚓堆肥產(chǎn)物中1和F分別降低了24.6%和69.4%[11].這表明蚯蚓堆肥可以減少污泥活細(xì)菌中1、F和M的豐度,ARGs的選擇性減少可能歸因于蚯蚓的腸道消化過程[26].此外,與原始污泥相比,堆肥后對照組中2的豐度顯著增加5.5倍(<0.05). Qian等[39]在對牛糞進(jìn)行堆肥后也發(fā)現(xiàn)其中2豐度增加了21.4～30.8倍.相比而言,蚯蚓堆肥產(chǎn)物中2的豐度比對照組顯著降低了82.8%(<0.05),說明蚯蚓能降低污泥中2的豐度,見圖2(b).對B來說,其豐度在兩處理組中均低于原始污泥.但與對照組相比,蚯蚓堆肥產(chǎn)物中B的豐度顯著增加了5.74倍(<0.05).在此前好氧堆肥實(shí)驗(yàn)中發(fā)現(xiàn),污泥中B豐度可減少23.9%～99.3%[40].由此推斷,在堆肥體內(nèi)添加蚯蚓能增加B基因的豐度.同時(shí)B和F豐度的不同變化趨勢可能與其抗性機(jī)制不同有關(guān).

IS代表供試污泥,C代表對照,E代表蚯蚓堆肥,下同

圖3 蚯蚓堆肥前后ARGs的總絕對豐度

大量可移動遺傳元件(質(zhì)粒、整合子、轉(zhuǎn)座子)在細(xì)菌群落ARGs的水平轉(zhuǎn)移中發(fā)揮著重要作用[41],因此本文分析了一類整合子I1在堆肥過程中的豐度變化,見圖2(f).與原始污泥相比,I1的絕對豐度在對照組中增加了91.1%,但在蚯蚓組中顯著減少了37.3%(<0.05).該結(jié)果進(jìn)一步表明蚯蚓能夠降低堆肥產(chǎn)物中ARGs水平轉(zhuǎn)移的風(fēng)險(xiǎn).Cui等[26]研究新鮮蚯蚓排泄糞便發(fā)現(xiàn),經(jīng)PMA處理后蚯蚓糞便中I1的絕對豐度較原始污泥降低了82.1%,進(jìn)而證實(shí)蚯蚓腸道短消化對I1的豐度具有顯著削減作用.而本實(shí)驗(yàn)蚯蚓堆肥產(chǎn)物中I1豐度的減少率低于其在蚯蚓糞便當(dāng)中,可能是由于經(jīng)過腸道短消化后,I1的豐度在堆肥產(chǎn)物活微生物體內(nèi)再次增加.另外,兩組中2與I1的豐度變化具有顯著正相關(guān)性(<0.05),表明2的增多可能與堆肥產(chǎn)物中I1的豐度有關(guān)[42].

如圖3所示,堆肥結(jié)束后對照組與蚯蚓組中代表性ARGs的總豐度均顯著降低(<0.05).兩處理組對ARGs總?cè)コ史謩e為37.2%和82.6%.與對照組相比,蚯蚓堆肥后的污泥活體微生物中ARGs的總豐度顯著減少了72.3%(<0.05).可見,蚯蚓堆肥可顯著降低堆肥產(chǎn)物中ARGs的絕對豐度[43],減輕污泥蚯蚓糞后續(xù)農(nóng)用的潛在生物風(fēng)險(xiǎn).先前針對總細(xì)菌DNA的研究[11]結(jié)果顯示,蚯蚓堆肥減少了整合子的類型和豐度,堆肥后污泥中的ARGs總豐度比對照組降低了41.5%.Cui等[26]研究發(fā)現(xiàn)蚯蚓腸道中的厭氧環(huán)境可能會使部分?jǐn)y帶ARGs的優(yōu)勢需氧細(xì)菌難以生存,蚯蚓可在一定程度上降低堆肥產(chǎn)物中ARGs的絕對豐度.說明蚯蚓活動對活體微生物的群落變化影響更大.同時(shí)這種微生物的群落變化和可移動遺傳元件的減少可能是蚯蚓堆肥過程中ARGs豐度減少的主要原因.在蚯蚓組樣品中絕對豐度占比最高的ARGs為F(2.88×1013copies/g),豐度最低的為M(3.30×108copies/g).此前的研究[44]顯示蚯蚓堆肥產(chǎn)物中2豐度最高,其次為X.說明活體微生物中ARGs多樣性與之前總DNA中有差別.

2.4 環(huán)境因子、微生物和ARGs之間的關(guān)系

圖4顯示活細(xì)菌群落及環(huán)境因子對堆肥過程中ARGs豐度變化的貢獻(xiàn),其中活細(xì)菌群落前10個(gè)菌門可以解釋驅(qū)動ARGs變化的62.1%,表明ARGs的豐度受活細(xì)菌群落的影響較大.其中蚯蚓堆肥樣品中高含量的EC及NO3-,表明蚯蚓可以促進(jìn)污泥中有機(jī)物的降解與礦化.同時(shí),EC對F豐度的增加有顯著的積極影響(<0.05).以上結(jié)果表明本文所研究的理化性質(zhì)會導(dǎo)致ARGs的豐度發(fā)生改變,控制這些關(guān)鍵的環(huán)境因子有助于去除堆肥體中的ARGs.

圖4 ARGs、活細(xì)菌種群和環(huán)境因子的冗余分析

先前研究表明放線菌門是抗生素的主要生產(chǎn)者,可以攜帶和傳播ARGs[45].本文蚯蚓堆肥前后Actinobacteria(放線菌門)豐度顯著增加了2.8倍,但蚯蚓堆肥產(chǎn)物中ARGs的總絕對豐度卻顯著降低.造成這一結(jié)果的原因可能是放線菌門產(chǎn)生的抗生素可以殺死污泥中的ARGs,使大部分ARGs存在于游離態(tài)DNA中.本文研究的是堆肥體系中活體微生物的菌群結(jié)構(gòu),而此前的研究并未區(qū)分活死微生物,導(dǎo)致宿主體內(nèi)可表達(dá)的ARGs定量不準(zhǔn)確.此外,I1與Proteobacteria(變形菌門)和Bacteroidetes (擬桿菌門)存在顯著正相關(guān)性(<0.05),這一結(jié)果顯示蚯蚓堆肥過程中Proteobacteria和Bacteroidetes可能是I1的潛在攜帶菌.進(jìn)一步相關(guān)分析表明I1菌屬水平的宿主為和,該宿主在堆肥產(chǎn)物中較高的豐度可能會促進(jìn)ARGs的水平傳播[46].以上相關(guān)性結(jié)果表明,在蚯蚓組中,變形菌門的和是B的潛在宿主,M、F和1的共同潛在宿主屬于厚壁菌門,推斷它們之間可能存在共生互惠關(guān)系.可見,蚯蚓能通過控制堆肥產(chǎn)物中Proteobacteria和Bacteroidetes的豐度來降低參與ARGs水平轉(zhuǎn)移的I1基因的增殖,減小ARGs傳播擴(kuò)散的潛在風(fēng)險(xiǎn).

冗余分析結(jié)果顯示pH值、DOC、EC及NO3-等環(huán)境因子可以調(diào)控ARGs的豐度,且活細(xì)菌群落變化對堆肥過程中ARGs的變化有一定的影響[14].因此,環(huán)境因子對ARGs豐度的影響主要取決于它們對其潛在宿主細(xì)菌種群結(jié)構(gòu)的影響[11-47].本文結(jié)果顯示,蚯蚓可通過改變污泥中環(huán)境因子及活體微生物種群結(jié)構(gòu),進(jìn)而對蚯蚓堆肥產(chǎn)物中ARGs的分布和豐度產(chǎn)生影響[48].

3 結(jié)論

3.1 污泥蚯蚓堆肥使活體細(xì)菌群落的豐富度和均勻度顯著增加,污泥蚯蚓糞中變形菌門(35.2%)、放線菌門(21.2%)、擬桿菌門(5.6%)為優(yōu)勢菌門.

3.2 蚯蚓堆肥后的污泥活細(xì)菌中總ARGs和I1的絕對豐度分別顯著減少了72.3%和37.3% (< 0.05),污泥蚯蚓堆肥能減輕ARGs在環(huán)境中的傳播風(fēng)險(xiǎn).

3.3 蚯蚓通過改變堆體環(huán)境,影響活細(xì)菌群落演替,減少活微生物中ARGs潛在宿主菌的豐度,是削減污泥蚯蚓糞中ARGs的主要原因.

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Effects of earthworms on the antibiotic resistance genes of vermicompost from dewatered sludge revealed by active microbes.

WEI Feng-yi1, XU Jun-jie1, CHEN Jin1, LI Jian-hui1, HUANG Kui1,2*, DONG Xi-lin3, XIA Hui1

(1.School of Environmental and Municipal Engineering, Lanzhou Jiaotong University, Lanzhou 730070, China；2.Key Laboratory of Yellow River Water Environment in Gansu Province, Lanzhou 730070, China；3.Changchun Water Group Co. Ltd, Changchun 130000, China)., 2022,42(7)：3425～3433

To eliminate the abundances of ARGs in sludge vermicompost, this study aimed to reveal the underlying effects of earthworms on the active bacterial community structure and their ARGs involved in vermicomposting systems for sludge recycling. For this, vermicomposting with and without earthworms was set up in parallel. Moreover, the dyeing pretreatment for samples with propidium monoazide (PMA) was adopted to screen out the DNA of active bacteria. The results showed that the electrical conductivity of sludge vermicompost was significantly increased by 82.5% (<0.05), the degradation rate of organic matter was increased by 5.2% (<0.05). Compared with the control treatment, the abundance of Actinobacteria significantly increased by 65.6% (<0.05), while the abundance of Firmicutes and Bacteroidetes significantly decreased by 74.7% and 34.6%, respectively. Meanwhile, vermicomposting led to the selected ARGs abundances ofM,1,2,B andF significantly decreased by 66.5%, 82.8%, 72.8% and 77.6% (<0.05), while the abundance ofB significantly increased by 5.7times (<0.05) in active bacteria, compared to the counterpart. The abundance ofI1gene in vermicompost products significantly reduced by 67.2% compared with the control treatment. The total absolute abundance of ARGs was 4.19×1013copies/g, and the total removal rate of ARGs was 82.6%, 45.4% higher than that of the counterpart. This study suggests that earthworms can reduce the abundance of dominant hosts of ARGs by modifying the active microbial community structure of sludge, thus reducing the associated dissemination risks of the spread of ARGs.

composting；microbial community；resistance genes；vermicompost；sludge recycling

X171.5

A

1000-6923(2022)07-3425-09

魏楓沂(1997-),女,甘肅白銀人,蘭州交通大學(xué)碩士研究生, 主要研究方向?yàn)樯镂廴疚餁w趨與控制.發(fā)表論文1篇.

2021-12-06

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;甘肅省青年博士基金資助項(xiàng)目(2021-QB051);甘肅省科技計(jì)劃(20JR2RA002);甘肅省優(yōu)秀研究生“創(chuàng)新之星”項(xiàng)目(2021CXZX-629)

* 責(zé)任作者, 教授, huangkui@mail.lzjtu.cn