關(guān)孟欣,彭蘭生,陳景陽(yáng),黃 魁,2*,夏 慧

玉米芯生物炭對(duì)污泥蚯蚓糞中微生物種群及ARGs的影響

關(guān)孟欣1,彭蘭生1,陳景陽(yáng)3,黃 魁1,2*,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.金風(fēng)環(huán)保有限公司,北京 102600)

較多的抗生素抗性基因(ARGs)積蓄于剩余污泥中降低了污泥蚯蚓糞的農(nóng)用價(jià)值.為削減污泥蚯蚓糞中的ARGs,向污泥中分別添加1.25%和5%(質(zhì)量比)的玉米芯生物炭(簡(jiǎn)稱玉米芯炭),以無(wú)添加為對(duì)照組,揭示玉米芯炭對(duì)污泥蚯蚓堆肥過(guò)程中微生物種群結(jié)構(gòu)及ARGs的影響.結(jié)果表明:添加高含量玉米芯炭顯著促進(jìn)污泥有機(jī)質(zhì)的礦化,提升蚯蚓堆肥產(chǎn)物的電導(dǎo)率和pH值(<0.05).同時(shí),添加玉米芯炭能增加污泥蚯蚓糞中細(xì)菌16S rDNA和真核生物18S rDNA的豐度,且其豐度均與玉米芯炭添加量呈顯著正相關(guān)性(<0.05).與對(duì)照組相比,高含量玉米芯炭污泥蚯蚓糞中變形菌門、擬桿菌門、放線菌門與浮霉菌門的相對(duì)豐度分別降低了11.8%、7.1%、33.3%和20%,但厚壁菌門的豐度顯著增加了40%(<0.05).此外,添加玉米芯炭蚯蚓糞中大環(huán)內(nèi)酯類抗性基因(F)和四環(huán)素類抗性基因(X)的絕對(duì)豐度較對(duì)照組分別顯著降低了32%～45%和13%～31%(<0.05),但同時(shí)整合子基因(I1)和磺胺類抗性基因(2)的豐度分別顯著增加了47%～135%和9%～42%(<0.05).研究結(jié)果顯示,添加玉米芯炭能增加污泥蚯蚓糞中微生物數(shù)量和種群多樣性,加速有機(jī)質(zhì)礦化,但對(duì)ARGs的削減具有選擇性.

生物質(zhì)炭；蚯蚓堆肥；污泥資源化；抗生素抗性基因；蚯蚓糞有機(jī)肥；生物污染物

隨著我國(guó)污水處理能力的不斷提升,剩余污泥產(chǎn)量越來(lái)越多[1-2],其處理處置已經(jīng)成為我國(guó)生態(tài)文明建設(shè)過(guò)程中急需解決的難題.蚯蚓堆肥是一種蚯蚓協(xié)同微生物共同降解有機(jī)物的污泥肥料化技術(shù)[3],其產(chǎn)物蚯蚓糞富含多種土壤養(yǎng)分及益生菌群[4-5],是一種高質(zhì)量的生物有機(jī)肥.由于我國(guó)城市生活污水和工業(yè)污水管網(wǎng)的分流,市政污泥中的重金屬與有毒有機(jī)物含量已不再是污泥蚯蚓堆肥的瓶頸.但新近研究發(fā)現(xiàn)剩余污泥中積蓄大量抗生素及其抗性基因(ARGs)[6],亦會(huì)造成污泥蚯蚓糞中功能性菌群攜帶有大量ARGs[7].ARGs作為新型生物污染物,具有很強(qiáng)的遺傳性和傳播性,難以在生物介質(zhì)中將其有效去除[8-9].因此,如何有效降低污泥蚯蚓糞中ARGs的環(huán)境風(fēng)險(xiǎn)尤為重要[10].

生物質(zhì)炭具有比表面積大、吸附能力強(qiáng)、穩(wěn)定性高等特點(diǎn),能夠促進(jìn)污泥有機(jī)物礦化、加速污泥穩(wěn)定化、吸附污泥中有害物質(zhì)[11-12].同時(shí),生物質(zhì)炭為微生物的生長(zhǎng)繁殖提供了多尺度的生態(tài)位[13].由于大多數(shù)ARGs鑲嵌在細(xì)菌胞內(nèi)DNA上,其歸趨受生境下細(xì)菌菌群變化的影響[14-15].因此可推測(cè)污泥蚯蚓堆肥體內(nèi)添加生物質(zhì)炭,不僅可加速有機(jī)物的穩(wěn)定,而且會(huì)改變蚯蚓糞中微生物菌群,進(jìn)而影響其ARGs的多樣性和豐度.已有研究表明生物質(zhì)炭會(huì)影響污泥蚯蚓糞中ARGs[16].但關(guān)于不同含量生物質(zhì)炭對(duì)污泥蚯蚓糞中ARGs的影響還鮮有研究.

本實(shí)驗(yàn)選取網(wǎng)狀多孔結(jié)構(gòu)的玉米芯炭為供試生物質(zhì)炭,比較不同含量玉米芯炭對(duì)污泥蚯蚓堆肥產(chǎn)物中微生物種群結(jié)構(gòu)和ARGs的影響,旨在為減少污泥蚯蚓糞中ARGs的風(fēng)險(xiǎn)提供一種新思路.

1 材料和方法

1.1 供試材料

堆肥蚓種為赤子愛(ài)勝蚓(),并經(jīng)脫水污泥馴化.玉米芯炭購(gòu)置于大連九成物產(chǎn)公司,表面為100μm的網(wǎng)狀多孔結(jié)構(gòu),理化性質(zhì)見(jiàn)表1.實(shí)驗(yàn)反應(yīng)器為底部打孔的矩形塑料盒(46cm′17cm′13cm).污泥取自蘭州市安寧七里河污水處理廠脫泥車間的新鮮脫水污泥(含水率64%),理化性質(zhì)見(jiàn)表1.

表1 供試玉米芯炭和供試污泥理化性質(zhì)

1.2 實(shí)驗(yàn)設(shè)計(jì)

設(shè)置3個(gè)處理,以無(wú)玉米芯炭為對(duì)照組(CK組),低含量玉米芯炭(1.25%CL組)和高含量玉米芯炭(5%CH組)為處理組,每組設(shè)3個(gè)重復(fù).新鮮脫水污泥先用5mm網(wǎng)孔的鐵絲網(wǎng)進(jìn)行造粒,隨后將其與玉米芯炭按填加比例分別混勻后,再次造粒作為蚯蚓堆肥基質(zhì)[16].而后將3kg蚯蚓堆肥基質(zhì)分別放入對(duì)應(yīng)的反應(yīng)器中,最后向各基質(zhì)中接種100 條體重約0.5g的赤子愛(ài)勝蚓.蚯蚓投加密度參照之前實(shí)驗(yàn)進(jìn)行[17].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,每周噴灑一次自來(lái)水并進(jìn)行翻堆.所有反應(yīng)器均在室溫(20～25℃)下進(jìn)行.蚯蚓堆肥實(shí)驗(yàn)共進(jìn)行60d.污泥蚯蚓糞樣品取樣時(shí)將蚓卵剔除,每個(gè)反應(yīng)器取2個(gè)平行樣品.所取樣品分兩份保存,一份存放于無(wú)菌塑封袋中,置于-20℃冰箱中冷凍保存,用于DNA相關(guān)分析;另一份風(fēng)干后研磨,過(guò)60目篩,然后置于4℃冰箱中保存,用于理化性質(zhì)分析.

1.3 測(cè)試方法

1.3.1 理化性質(zhì)分析 理化測(cè)試參照黃魁等[17]方法進(jìn)行.將研磨后的風(fēng)干樣與去離子水(干樣:水=1:50;kg/L)混勻后測(cè)定pH值(雷磁PHS-3C,上海)和電導(dǎo)率(雷磁DDS-307,上海).硝酸鹽氮采用紫外分光光度法(HJ/T 346-2007),氨氮采用納氏試劑分光光度法(HJ 535-2009).溶解性有機(jī)碳(DOC)為上述混合液稀釋10倍后,過(guò)0.45μm濾膜,用碳氮分析儀(耶拿MULTI N/C,2100,德國(guó))進(jìn)行測(cè)定.

1.3.2 DNA提取及熒光定量PCR 取約0.25g解凍樣品用DNeasy?Power Soil?Kit (Qiagen,德國(guó))試劑盒提取總DNA,并用1%瓊脂糖凝膠電泳檢測(cè)其濃度.本實(shí)驗(yàn)選取污泥中常見(jiàn)且含量較高的大環(huán)內(nèi)酯類抗性基因(F)、四環(huán)素類抗性基因(MX)和磺胺類抗性基因(1、2)及第一類整合子基因(I1)進(jìn)行檢測(cè).采用熒光定量PCR儀(Takara,TP700,日本)對(duì)細(xì)菌16S rDNA(V3～V4區(qū)),真核18S rDNA(V4區(qū))及上述幾種ARGs進(jìn)行定量.定量反應(yīng)為25μL體系:TB Green II(Takara,日本)12.5μL, 10μmol/L引物各0.5μL,DNA模板1μL,以及DNA- free水10.5μL.所用引物均購(gòu)置于生工生物工程(上海)股份有限公司,引物序列和反應(yīng)條件見(jiàn)表2.標(biāo)準(zhǔn)品為攜帶目的基因的質(zhì)粒,制備過(guò)程見(jiàn)文獻(xiàn)[18].

表2 抗性基因引物及PCR反應(yīng)條件

1.3.3 PCR和高通量測(cè)序 采用帶有Barcode堿基的引物515F(5’-GTGCCAGCCGCGGTAA-3’)和806R(5’-GGACTACHVGGGTWTCTAAT-3’)對(duì)16S rDNA基因序列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擴(kuò)增條件為: 95℃預(yù)變性5min;35個(gè)循環(huán)包括95℃變性30s,58℃退火30s,72℃延伸30s;72℃終延伸10min.所得到的擴(kuò)增產(chǎn)物使用1%的瓊脂糖電泳進(jìn)行檢測(cè),并利用Agencourt AMPure XP 60mL Kit(A63881 Beckman Coulter)對(duì)產(chǎn)物進(jìn)行純化.使用Qubit dsDNA HS Assay Kit (Q32851Life tech)對(duì)文庫(kù)進(jìn)行構(gòu)建,檢測(cè)合格后使用Illumina HiSeq平臺(tái)進(jìn)行上機(jī)測(cè)序(谷禾信息生物有限公司,杭州).測(cè)序結(jié)果使用QIIME(1.8.0)軟件對(duì)序列進(jìn)行質(zhì)控過(guò)濾,去掉嵌合體,得到有效Clean tags.然后使用MEGA7.0軟件(v7.0.1001)將OTUs (Operational Taxonomic Units)進(jìn)行聚類,相似度達(dá)97%的OTU聚為一類.最后與GreenGene(v gg_13_8)和Silva(SILVA128)數(shù)據(jù)庫(kù)進(jìn)行對(duì)比并注釋,獲得各OTU的分類學(xué)信息.測(cè)序結(jié)果已上傳至DDBJ數(shù)據(jù)庫(kù).

1.4 統(tǒng)計(jì)分析

使用STATISTICA 10.0統(tǒng)計(jì)軟件進(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.使用MEGA7.0軟件對(duì)DNA測(cè)序數(shù)據(jù)進(jìn)行聚類,并用HemI1.0軟件繪制熱圖.使用OriginPro 2018(version 9.5)繪圖.用CANOCO 4.5軟件對(duì)環(huán)境因素、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 玉米芯炭對(duì)堆肥產(chǎn)物理化性質(zhì)的影響

由表3可知,與CK組相比,CL和CH兩組蚯蚓糞中電導(dǎo)率分別提升15%和14%(<0.05),添加玉米芯炭能提升污泥蚯蚓堆肥的礦化水平[13]. Gong等[19]研究發(fā)現(xiàn),高溫堆肥中添加竹子生物質(zhì)炭會(huì)提高其產(chǎn)物的電導(dǎo)率.污泥蚯蚓糞中增加的電導(dǎo)率可能與生物質(zhì)炭中較高的電導(dǎo)率供給有關(guān).與電導(dǎo)率相似,CL和CH兩組蚯蚓糞的pH值也顯著提升(<0.05).

如表3所示,CH組硝酸鹽氮的含量比CK組增加12%(<0.05),但CL與CK組無(wú)顯著性差異(> 0.05).這可能是因?yàn)楦吆康挠衩仔咎吭黾恿宋勰嗟目紫堵?致使堆體中氧含量增加,進(jìn)而促進(jìn)了硝化反應(yīng)的進(jìn)行[20].蚯蚓堆肥后各組氨氮含量均有顯著性下降(<0.05),但CL和CH氨氮含量比CK組顯著提高了1.46倍和1.07倍(< 0.05).這一結(jié)果可能與添加玉米芯炭會(huì)促進(jìn)污泥有機(jī)物的氨化反應(yīng)有關(guān)[13].玉米芯炭組中同時(shí)增多的氨氮和硝氮暗示著生物質(zhì)炭能提升污泥蚯蚓糞的氮肥肥效.

由表3所見(jiàn),實(shí)驗(yàn)后各組污泥蚯蚓糞中溶解性有機(jī)碳(DOC)均顯著降低(<0.05),這與其他研究相近[17].與CK組相比,CL和CH組DOC含量呈增加趨勢(shì)(<0.05).研究發(fā)現(xiàn)[21-22],生物質(zhì)炭的添加會(huì)增加土壤中的DOC含量.這可能歸因于生物質(zhì)炭芳香結(jié)構(gòu)中的碳被蚯蚓與微生物分解,從而增加了污泥蚯蚓糞中DOC的含量.本研究中DOC含量與玉米芯炭含量有較顯著的線性關(guān)系(<0.05,=0.96).

表3 不同處理中污泥蚯蚓糞的理化性質(zhì)

注:同列指標(biāo)的兩個(gè)處理組間存在相同字母表明其兩兩之間不具有顯著性差異(>0.05),同行字母之間沒(méi)有比較意義,下同.

2.2 玉米芯炭對(duì)微生物數(shù)量及種群結(jié)構(gòu)的影響

2.2.1 玉米芯炭對(duì)微生物數(shù)量的影響 如圖1所示,與原始污泥相比,蚯蚓堆肥顯著增加了各處理組細(xì)菌16S rDNA和真核生物18S rDNA微生物的豐度(<0.05).與CK組相比,CL和CH組細(xì)菌16S rDNA豐度分別顯著增加了0.24倍和1.13倍(<0.05);對(duì)于真核生物18S rDNA來(lái)說(shuō),CL和CH組的數(shù)量均顯著高于CK組(<0.05).同時(shí),皮爾遜相關(guān)性結(jié)果顯示,細(xì)菌與真核微生物的生物量與玉米芯炭含量均有極顯著的正相關(guān)性(<0.05,=0.96,=0.97).上述結(jié)果說(shuō)明添加玉米芯炭有利于污泥蚯蚓糞中微生物的生長(zhǎng)繁殖,這與Xu等[23]在土壤學(xué)中的研究結(jié)果相似.生物質(zhì)炭較高的比表面積和孔隙度為微生物的生存提供了多樣的環(huán)境,其可利用成分可以直接被微生物所利用[24],進(jìn)而增加了微生物的生物量[25].與細(xì)菌相比,CH組中真核生物數(shù)量急劇性增長(zhǎng),表明高含量玉米芯炭有利于其生長(zhǎng).這可能是因?yàn)楦吆坑衩仔咎慷嗫捉Y(jié)構(gòu)為真核微生物提供了好氧環(huán)境與庇護(hù)所,減少了被蚯蚓捕食的幾率[13].

圖1 蚯蚓堆肥前后污泥中細(xì)菌16S rDNA和真核18S rDNA的絕對(duì)含量

2.2.2 玉米芯炭對(duì)微生物種群結(jié)構(gòu)的影響 由表4可知,與原始污泥相比,各組Shannon指數(shù)和Simpson指數(shù)均出現(xiàn)了增加,且最高值都出現(xiàn)在CH組,說(shuō)明添加高含量玉米芯炭會(huì)增加堆肥產(chǎn)物微生物的豐富度和均勻度.這與最近在豬糞高溫堆肥的研究[26]結(jié)果相似.微生物均勻度的增加,可能是由于生物質(zhì)炭多孔結(jié)構(gòu)提供了多樣的生境,有利于功能性微生物的生長(zhǎng)繁殖[25].

由圖2可見(jiàn),原始污泥中優(yōu)勢(shì)菌門為變形菌門(25%)、擬桿菌門(13%)、厚壁菌門(13%).蚯蚓堆肥后各處理組中變形菌門、放線菌門、浮霉菌門均顯著增加(<0.05),而厚壁菌門顯著減少(<0.05).與CK組相比,CL組細(xì)菌菌群結(jié)構(gòu)無(wú)顯著性變化.但CH組中厚壁菌門較CK組顯著增長(zhǎng)了40%,這與黃家慶等[26]所得到的結(jié)果一致.厚壁菌門的主要功能為降解大分子有機(jī)物如蛋白質(zhì)、脂肪類、纖維素等[17],因此可推斷高含量玉米芯炭會(huì)促進(jìn)有機(jī)物質(zhì)的轉(zhuǎn)化.而與CK組相比,CH組放線菌門和變形菌門分別減少了33.3%和11.8%.Cui等[27]也發(fā)現(xiàn)在雞糞高溫堆肥中添加5%的稻草生物質(zhì)炭會(huì)降低變形菌門的比例.這說(shuō)明高含量玉米芯炭會(huì)抑制堆肥體中變形菌門和放線菌門的生長(zhǎng).CH組中浮霉菌門和擬桿菌門與CK組相比也有明顯減少.以上結(jié)果表明,添加高含量玉米芯炭顯著影響了堆肥產(chǎn)物中門水平的細(xì)菌群落組成.

表4 不同處理中微生物Shannon與Simpson指數(shù)

注:IS為原始污泥.

圖2 堆肥前后污泥中細(xì)菌門水平結(jié)構(gòu)變化

圖3 堆肥前后污泥中細(xì)菌屬水平相對(duì)豐度

如圖3所示,原始污泥中優(yōu)勢(shì)菌屬為、、Blvii28.而堆肥后3個(gè)處理組中Unclassified-1、、、、、SWB02、、、、、、和均有不同程度的增加.其中,、、、和從無(wú)到有,這說(shuō)明蚯蚓堆肥可以顯著增加細(xì)菌種群多樣性[17].與CK組相比,CL組Unclassified-1和Nocardioides增加了28%和14%,而CH組中Nocardioides降低了36%,SWB02降低了34%,、、和降低了40%.結(jié)果顯示玉米芯炭的含量對(duì)堆肥產(chǎn)物細(xì)菌群落組成有較大的影響.聚類分析可見(jiàn),CK與CL組種群結(jié)構(gòu)差異性較小,而CH組與其余兩組種群結(jié)構(gòu)有較大差異性,這說(shuō)明高含量玉米芯炭對(duì)細(xì)菌屬水平結(jié)構(gòu)影響較大.

2.3 玉米芯炭對(duì)ARGs的影響

如圖4(a)所示,與原始污泥相比,CK組中I1的數(shù)量顯著降低了0.48倍(<0.05),顯示出蚯蚓堆肥可以降低污泥中I1的豐度[17,28].但與CK組比較,添加玉米芯炭使I1的數(shù)量呈現(xiàn)出增長(zhǎng)趨勢(shì),且其豐度與玉米芯炭含量有顯著正相關(guān)性(<0.05,= 0.88).這表明添加玉米芯炭對(duì)I1豐度的增加有促進(jìn)作用.相關(guān)研究證實(shí),環(huán)境中較高的細(xì)菌數(shù)量和多樣性增多了ARGs的潛在宿主及細(xì)菌胞外接觸的可能性,進(jìn)而增大了I1在生物介質(zhì)中水平傳播的可能性[29-30].本研究中,增加的I1豐度與CL和CH組中較高的細(xì)菌數(shù)量和多樣性密切相關(guān),因?yàn)槲勰囹球炯S中的細(xì)菌數(shù)量和I1呈正相關(guān)關(guān)系(<0.05,=0.88).這一結(jié)果暗示著添加生物質(zhì)炭將會(huì)增加污泥蚯蚓糞中ARGs的傳播風(fēng)險(xiǎn).Duan[31]在研究中也發(fā)現(xiàn)生物質(zhì)炭的添加并不會(huì)降低土壤中I1豐度.

圖4 蚯蚓堆肥前后ARGs的絕對(duì)豐度

對(duì)ARGs而言(圖4b～f),各組中F(大環(huán)內(nèi)酯類抗性基因)和M(四環(huán)素類抗性基因)的豐度在堆肥處理后均出現(xiàn)顯著減少(<0.05),其中M與原始污泥相比減少了86%～90%.并且M各組間并無(wú)顯著性差異(>0.05),說(shuō)明添加玉米芯炭對(duì)污泥蚯蚓糞中M的豐度無(wú)顯著影響.但對(duì)F來(lái)說(shuō),隨著玉米芯炭含量的增加,污泥蚯蚓糞中F的豐度逐漸減少,且與玉米芯炭含量有較強(qiáng)的負(fù)相關(guān)性(<0.05,=-0.83).這一現(xiàn)象表明,玉米芯炭可以減少蚯蚓堆肥中F的豐度.Li等[32]也發(fā)現(xiàn)向雞糞高溫堆肥中添加竹子生物質(zhì)炭,F的豐度會(huì)減少.此外,與原始污泥相比,X(四環(huán)素類抗性基因)的豐度在蚯蚓堆肥后會(huì)出現(xiàn)顯著增長(zhǎng)(<0.05),見(jiàn)圖4(f).但與CK組相比,CL和CH組X的豐度顯著下降了13%和31%,且與玉米芯炭含量存在顯著負(fù)相關(guān)(<0.05,=-0.88),說(shuō)明添加玉米芯炭會(huì)降低污泥蚯蚓糞中X的豐度.與之相反,在添加玉米芯炭后,1和2(磺胺類抗性基因)的豐度均出現(xiàn)增加.磺胺類抗性基因的增多可能與污泥蚯蚓糞中I1豐度有關(guān)[31].Chen[33]在I1的保守區(qū)發(fā)現(xiàn)了磺胺類抗性基因,因此I1的增加會(huì)導(dǎo)致1和2的增加.相比而言,CH組中2豐度與CK組相比顯著增加了42%,且其豐度與玉米芯炭含量存在顯著正相關(guān)(<0.05,0.66).Wang等人[34]也發(fā)現(xiàn)高含量玉米秸稈生物質(zhì)炭能增加動(dòng)物糞便高溫堆肥中1和2的豐度.另外,最高的1和2豐度均出現(xiàn)在CH組,暗示著高含量玉米芯炭會(huì)提升蚯蚓堆肥產(chǎn)物中磺胺類抗性基因的豐度.綜上結(jié)果顯示玉米芯炭對(duì)蚯蚓污泥堆肥體系中ARGs的去除具有選擇性,且去除效率與玉米芯炭的含量密切相關(guān).

2.4 環(huán)境因子、微生物和ARGs之間的關(guān)系

圖5冗余分析可知,CK組中總ARGs的豐度最高,EC、pH值以及NH4+對(duì)堆肥體內(nèi)ARGs的總豐度有一定的抑制作用.CH組高含量玉米芯炭及DOC、NO3-的含量與2和M的豐度有顯著正相關(guān)性(<0.05),表明高含量玉米芯炭對(duì)2和M的豐度有積極的影響,這與CH組中高含量2結(jié)果一致.此外,Firmicutes(厚壁菌門)與2和M之間較強(qiáng)的相關(guān)性表明其可能為2和M的潛在宿主細(xì)菌.Sun等[35]在牛糞厭氧消化中添加生物質(zhì)炭也發(fā)現(xiàn)類似結(jié)果.RDA結(jié)果還顯示CL組Planctomycetes (浮霉菌門),Actinobacteria(放線菌門)及Proteobacteria(變形菌門)的豐度較高,表明了在蚯蚓堆肥過(guò)程中添加低含量玉米芯炭會(huì)促進(jìn)上述菌門細(xì)菌的生長(zhǎng),細(xì)菌門水平的變化對(duì)堆肥過(guò)程中ARGs的變化有一定的影響.因此,環(huán)境變量對(duì)ARGs豐度的影響主要取決于它們對(duì)其潛在宿主細(xì)菌的影響[10,36-37].本研究顯示污泥中環(huán)境因子與微生物種群的變化受生物質(zhì)炭含量的影響較大,進(jìn)而對(duì)蚯蚓糞中ARGs分布和豐度產(chǎn)生影響[7,28,38].但由于污泥蚯蚓堆肥體系的復(fù)雜性,玉米芯炭對(duì)污泥蚯蚓堆肥中微生物和ARGs的影響機(jī)制仍須進(jìn)一步研究.

圖5 ARGs、細(xì)菌菌群和環(huán)境因子的冗余分析

3 結(jié)論

3.1 高含量玉米芯炭污泥蚯蚓糞中硝氮含量和電導(dǎo)率分別增加了12%和14%,促進(jìn)了污泥蚯蚓堆肥中有機(jī)物的礦化,提升其產(chǎn)物的穩(wěn)定性.

3.2 高含量玉米芯炭組提升了污泥堆肥產(chǎn)物中微生物種群多樣性,、、Blvii28、、Unclassified-1、、、、、和為生物炭污泥蚯蚓糞的優(yōu)勢(shì)菌屬.

3.3 玉米芯炭降低了污泥中占主導(dǎo)地位的F和X的豐度,但I(xiàn)1和2的豐度在添加玉米芯炭組中分別增加47%～135%和9%～42%,玉米芯炭蚯蚓糞的生態(tài)風(fēng)險(xiǎn)需進(jìn)一步研究.

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Effects of corncob biochar on the microbial communities and ARGs during vermicomposting of dewatered sludge.

GUAN Meng-xin1, PENG Lan-sheng1, CHEN Jing-yang3, HUANG Kui1,2*, 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.Goldwind Environmental Protection Co., LTD, Beijing 102600, China)., 2021,41(6)：2744～2751

The higher content of antibiotics resistance genes (ARGs) accumulated in excess sludge lowers its agricultural value of vermicompost. To eliminate the content of ARGs in sludge vermicompost, this study aimed to reveal the effects of corncob biochars added in sludge on microbial communities and ARGs of vermicomposting. For this, 1.25% and 5% of corncob biochars, were separately added to dewatered sludge, comparing with the counterpart without addition of biochars. The addition high content of corncob biochars significantly (< 0.05) promoted the mineralization of organic matter, thus increasing the conductivity and pH of sludge vermicompost. In addition, the corncob biochars (< 0.05) enhanced the gene abundances of bacterial 16S rDNA and eukaryotic 18S rDNA in the vermicompost, resulting in a significantly (< 0.05) positive correlation between the microbial abundance and biochar concentration. Compared to control, the relative abundance of Proteobacteria, Bacteroidetes, Actinomycetes, and Planctomycetes in sludge vermicompost with high content of corncob biochar decreased by 11.8%, 7.1%, 33.3% and 20%, respectively. However, its abundance of Firmicutes significantly increased by 40% (< 0.05). Besides, the absolute abundance of macrolide resistance genes (F) and tetracycline resistance genes (X) in vermicompost with corncob biochars significantly decreased by 32%～45% and 13%～31% (< 0.05), respectively. But, the abundance of class 1integron (I1) and sulfonamides resistance genes (2) significantly increased by 47%～135% and 9%～42% (< 0.05) in the final vermciompost, respectively. The addition of corncob biochar in sludge can promote the mineralizaiton of sludge by enhancing microbial number and diversity of microbial, but this addition only selectively reduce the ARGs in vermicompost.

biochar；vermicomposting；sludge recycling；antibiotics resistance gene；vermicompost fertilizer；biological pollutants

X171.5

A

1000-6923(2021)06-2744-08

關(guān)孟欣(1996-),男,山西運(yùn)城人,蘭州交通大學(xué)碩士研究生,主要研究污泥資源化技術(shù).發(fā)表論文2篇.

2020-11-13

國(guó)家自然科學(xué)基金資助項(xiàng)目(51868036;52000095);甘肅省高等教師創(chuàng)新能力提升項(xiàng)目(2019A-040);蘭州交通大學(xué)百人計(jì)劃項(xiàng)目

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