一體化A2/O-MBR系統(tǒng)中抗性基因分布及去除效果研究

李中浤,杜彩麗,陳素華,張列宇,李曉光*,黎佳茜,田振軍

一體化A2/O-MBR系統(tǒng)中抗性基因分布及去除效果研究

李中浤1,2,杜彩麗2,3,陳素華1,張列宇2,李曉光2*,黎佳茜2,田振軍2

(1.南昌航空大學(xué),江西省持久性污染物控制與資源循環(huán)利用重點實驗室,江西 南昌 330063；2.中國環(huán)境科學(xué)研究院,國家環(huán)境保護地下水污染過程模擬與控制重點實驗室,北京 100012；3.同濟大學(xué)環(huán)境科學(xué)與工程學(xué)院,上海 200092)

選取北京市某農(nóng)村一體化A2/O-MBR污水處理系統(tǒng),系統(tǒng)研究了系統(tǒng)中抗生素抗性基因(Antibiotic Resistance Genes, ARGs)及病原菌在全流程各個處理單元中的分布特征,基于宏基因組學(xué)的高通量測序技術(shù)對農(nóng)村生活污水進水、MBR池中污泥和出水樣品中ARGs及病原菌的豐度變化及去除效果進行了系統(tǒng)分析.結(jié)果表明:ARGs廣泛存在于污水處理系統(tǒng)中,共檢測出包括tetracycline類、aminoglycoside類和sulfonamide類在內(nèi)的19類ARGs,進水中ARGs的相對豐度遠遠高于其在出水中的濃度,通過污水處理系統(tǒng)后ARGs相對豐度下降了72.25%,而大多數(shù)的ARGs在污水處理系統(tǒng)并不能得到完全去除.微生物群落結(jié)構(gòu)變化顯示,32種潛在病原體相對豐度下降明顯,但大多數(shù)病原菌也無法得到完全去除.出水中殘留的ARGs和病原菌仍會對受納水體造成一定的潛在污染風險.

一體化A2/O-MBR；宏基因組學(xué)；ARGs；病原菌

抗生素抗性基因(ARGs)作為一種新型污染物[1],可以通過可移動遺傳元件促進抗生素抗性基因在不同物種間基因水平轉(zhuǎn)移[2].致病細菌攜帶各種ARGs后對抗生素產(chǎn)生耐藥性[3],這些抗生素耐藥致病菌(ARB)通過多種途徑從環(huán)境媒介中傳播給人類,進而對人類公眾健康構(gòu)成嚴重威脅[4].目前, ARGs廣泛分布于湖泊[5]、河流[6]和海洋[7]等自然環(huán)境中.污水處理廠(WWTPs)匯集了醫(yī)院廢水、居民生活污水以及畜禽養(yǎng)殖污水等多種污水,因為抗生素的過度使用甚至濫用上述污水中殘留著高濃度抗生素,高濃度抗生素會對ARGs和ARB產(chǎn)生選擇性壓力,促進其繁殖和擴散,是ARGs和ARB的重要儲存庫[8-9],也是環(huán)境中ARGs和ARB的主要來源[10-11].

目前,針對城市污水處理廠中ARGs的分布特征已經(jīng)開展了大量研究[12-13],但是極少開展對農(nóng)村污水處理系統(tǒng)ARGs變化的研究,且研究方法主要采用qPCR的方法.Chen等通過qPCR技術(shù)對4座城市生活污水處理廠和8座農(nóng)村生活污水處理系統(tǒng)中4種四環(huán)素類抗性基因(M、O、Q和W)和2種磺胺類抗性基因(1和2)的處理效果進行評估,結(jié)果顯示城市生活污水處理廠抗生素抗性基因絕對豐度顯著減少(1～3個數(shù)量級),而農(nóng)村生活污水處理系統(tǒng)對ARGs的削減較少[14].Chen等探究人工濕地系統(tǒng)對于農(nóng)村污水中抗生素和ARGs的去除效果,采用qPCR技術(shù)對包括1、2、1、2、3、B/P、M、O、X、B和C在內(nèi)的ARGs進行檢測,結(jié)果表明1、2、M和O是主要的ARGs,人工濕地系統(tǒng)對于ARGs的去除效率>99%[15].基于qPCR的方法需要特定靶基因的序列,只能針對已知序列的單一ARGs進行檢測,無法全面了解污水處理系統(tǒng)中ARGs的分布特征和變化規(guī)律.宏基因組高通量測序技術(shù)(NGS)是研究環(huán)境中微生物ARGs的有效方法,可獲得環(huán)境中所有抗性基因的序列信息,挖掘未知抗性基因的信息,克服定量qPCR檢測技術(shù)引物或探針設(shè)計與選擇的限制[16].因此,急需對污水處理廠中的ARGs和ARB的來源、分布特征及去除效能開展系統(tǒng)性研究.

一體化污水處理設(shè)備是一種新型農(nóng)村污水處理技術(shù),與大型傳統(tǒng)的污水處理系統(tǒng)相比,具有污染物去除率高、出水水質(zhì)良好、抗沖擊負荷能力強、占地面積小和投資運行費用低等優(yōu)點,在農(nóng)村污水處理過程中得到廣泛應(yīng)用.本文采用宏基因組學(xué)技術(shù),開展農(nóng)村生活污水一體化A2/O-MBR設(shè)備處理過程中(進水、污泥、出水)微生物群落結(jié)構(gòu)、ARGs和ARB的分布特征及變化規(guī)律研究,以期為一體化處理設(shè)備在農(nóng)村生活污水應(yīng)用及再生水回灌過程中環(huán)境風險評估提供科學(xué)依據(jù).

1 材料與方法

1.1 樣品采集

選取北京市順義區(qū)某農(nóng)村生活污水一體化A2/O-MBR系統(tǒng)為研究對象(編號為SP),該一體化A2/O-MBR系統(tǒng)日處理生活污水為200t,主體工藝為A2/O-MBR,出水滿足(DB 11/1612-2019)一級B標準.樣品采集時間為2020年4月21日,采樣點為進水、MBR池中污泥和出水.進水和出水樣品分別采集0.2L和1L,所有采集的水、污泥樣品在2h之內(nèi)于-4℃冷藏運輸回實驗室,進水和出水樣品均經(jīng)過0.22μm濾膜過濾后收集微生物,將過濾后進水和出水樣品與污泥一起凍存于-80℃冰箱中用于DNA提取.

1.2 基因組提取

濾膜樣品先用無菌剪刀剪碎,參考標準流程,剪碎的濾膜樣品和污泥樣品采用FastDNA? SPIN Kit for Soil(MP Biomedicals,CA,USA)試劑盒完成基因組DNA抽提.完成DNA抽提后的樣本使用1%瓊脂糖凝膠電泳檢測DNA的質(zhì)量和完整性,并采用Qubit 2.0熒光光譜儀(Thermo Fisher Scientific, MA,USA)檢測PCR擴增后DNA的產(chǎn)量和純度.基因組DNA樣品用干冰保藏并立即送往上海美吉生物醫(yī)藥科技有限公司進行測序,測序平臺為Illumina HiSeq 4000,采用PE150測序策略進行雙端測序.

1.3 生物信息學(xué)分析

使用Trimmonatic[17]對原始測序數(shù)據(jù)進行質(zhì)量控制,去除低質(zhì)量測序數(shù)據(jù),得到clean reads.基于質(zhì)量控制后的clean reads采用Kraken2法[18]對污泥樣品在門、屬兩個層級開展微生物群落組成分析.應(yīng)用USEARCH(accel￡0.5,e-value￡10-5)和BlastX (alignment length 25aa,amino acids380% and evalue 1e-5)與SARG v2.0抗生素抗性基因數(shù)據(jù)庫進行對比注釋[19],對注釋后的數(shù)據(jù)按照SARG抗生素抗性基因數(shù)據(jù)庫分類成不同的抗生素抗性類型以及亞型.

注釋出的ARGs相對豐度結(jié)果基于16S拷貝數(shù)標準化(copies of ARG per copy of 16S rRNA)[20]

式中:ARG-like sequence序列為檢測某一種特定ARG的clean reads數(shù)量,reads為高通量測序中clean reads的長度,本研究中樣本均為150bp,ARG reference sequence為這一種特定ARG參考序列的長度,16S sequence是在高通量測序中識別的16S序列數(shù)量,16S sequence序列是Greengene數(shù)據(jù)庫中16S序列的平均長度1432bp[20-21].計算出的ARGs相對豐度單位為ARG拷貝數(shù)/16S rRNA拷貝數(shù)(簡稱ratio)[20]..

本文采用origin 2018進行數(shù)據(jù)繪圖.使用SPSS 24.0進行spearman相關(guān)性分析.

2 結(jié)果與討論

2.1 抗生素抗性基因變化規(guī)律

基于抗生素抗性基因數(shù)據(jù)庫(SARG v2.0)對農(nóng)村生活污水一體化A2/O-MBR處理系統(tǒng)不同生物單元抗性基因類型進行分析.結(jié)果顯示,該一體化污水處理過程中共檢出19類ARGs,包括tetracycline類、multidrug類、beta-lactam類、macrolide- lincosamide-streptogramin(MLS)類、aminoglycoside類、sulfonamide類、bacitracin類、chloramphenicol類、fosmidomycin類、polymyxin類、trimethoprim類、kasugamycin類、rifamycin類、quinolone類、vancomycin類、fosfomycin類、bleomycin類、carbomycin類和unclassified類ARGs(圖1a和圖1b).不同處理單元采集樣品中主要的ARGs為tetracycline類、multidrug類、beta-lactam類、MLS類、aminoglycoside類、sulfonamide類和bacitracin類,這與已有研究報道一致[22-23].

生活污水進水樣品中檢測出上述19類ARGs,總相對豐度達到了0.8283ratio,其中beta-lactam類、tetracycline類、aminoglycoside類、sulfonamide類、multidrug類及MLS類ARGs是進水樣品中主要的ARGs,其相對豐度分別達到了0.1445ratio、0.1399ratio、0.1184ratio、0.1129ratio、0.0992ratio和0.0956ratio,占進水樣品中總ARGs的85.79%.這6類ARGs是污水處理廠進水中常見的ARGs[24-25]. MBR池中污泥共檢測出16類ARGs,總ARGs相對豐度為0.3302ratio,出水口檢測出18類ARGs,總的ARGs相對豐度為0.2298ratio.其中kasugamycin和bleomycin類ARGs均未在污泥中檢出,而在出水樣品中檢出,但其相對豐度極低,僅為0.0002ratio和0.00008ratio,這可能由于出水口樣品收集時受到污染造成的.MBR池中污泥和出水樣品中主要的ARGs類型基本相同,主要是multidrug類、bacitracin類和MLS類ARGs,在污泥中相對豐度分別達到為0.0725ratio、0.0597ratio和0.0320ratio,出水口中相對豐度分別為0.0526ratio、0.0434ratio和0.0234ratio.與進水口相比,multidrug類和bacitracin類ARGs在MBR池中污泥和出水中占比有所上升,說明農(nóng)村一體化設(shè)備對這兩類ARGs去除率相對較低.

一體化處理設(shè)備處理后,除了不同生物單元水樣中優(yōu)勢ARGs會發(fā)生了一定的變化,部分ARGs類型相對豐度也會顯著下降.在出水口樣品中carbomycin類ARGs的相對豐度為0,經(jīng)過污水處理該ARGs完全被去除,Tetracycline類、beta-lactam類和aminoglycoside類ARGs去除率高,分別達到了90.92%、89.36%和87.51%.但也有個別ARGs相對豐度有所上升,如rifamycin類ARGs在進水口相對豐度為0.0042ratio,而在MBR池污泥中達0.0167ratio.整體而言,與進水口相比,出水口ARGs相對豐度降低了0.5985ratio,去除率達到了72.25%. Du等[26]使用qPCR技術(shù)研究基于A2O-MBR工藝的城市污水處理廠污水處理過程中5種ARGs(GWX1和1)的變化趨勢,結(jié)果表明ARGs在進水和出水中的下降趨勢為1?>1?>X>G?>W.其與基于A2O-MBR的一體化污水處理系統(tǒng)ARGs的變化趨勢相一致,且宏基因組學(xué)反映更多種類ARGs的變化規(guī)律.

2.2 抗生素抗性基因亞型變化規(guī)律

圖2 樣品中共有的和特有的ARGs數(shù)目

樣品經(jīng)生物信息學(xué)分析共注釋出408種ARGs,進水口、污泥和出水口中各注釋出371、210和217種ARGs,其各自特有的基因數(shù)分別為152種、10種和13種,共有的ARGs為157種,占比為38.4%(圖2).Tetracycline類、aminoglycoside類和sulfonamide類ARGs是污水處理廠進水口樣品中相對豐度最高的3種抗性基因.四環(huán)素是目前應(yīng)用最廣泛的一種抗生素,可應(yīng)用于人類治療、畜禽養(yǎng)殖和水產(chǎn)養(yǎng)殖等多個行業(yè)中,研究表明四環(huán)素使用量在中國抗生素中排第一,且難以被人和動物代謝和吸收,其中有75%以上的四環(huán)素可以通過人和動物的排泄物釋放到環(huán)境中[27],.因此醫(yī)療廢水、生活污水和畜禽養(yǎng)殖廢水等中會殘留高濃度的四環(huán)素類抗生素和抗性基因,污水處理廠作為這些廢水處理的中間站,會是四環(huán)素類抗性基因的重要儲存庫.通過生物信息學(xué)分析在進水口樣品中共注釋出37種tetracycline類ARGs,其中C、E、Q36和A是主要ARGs,相對豐度分別為0.0271ratio、0.0250ratio、0.0163ratio和0.0160ratio,總占比為68.99%,MBR池中污泥和出水口分別注釋出27和26種tetracycline類ARGs,主要是V、W、G和M抗性基因.與進水口相比,污泥和出水口中除V和G相對豐度上升外,大部分抗性基因相對豐度都有不同程度的下降,甚至個別抗性基因如34、J、R、S、X1、X5和Y等經(jīng)過處理后已完全被去除(圖3a).其中部分ARGs相對豐度的增加可能由于抗生素的選擇壓力和/或廢水處理過程中廣泛地去除了敏感細菌[28].磺胺類藥物具有高溶解性、持久性和化學(xué)穩(wěn)定性等特點,可在環(huán)境中長期存在[29],因此導(dǎo)致污水處理廠中磺胺類ARGs相對豐度較高.在進水口樣品中共注釋出4種sulfonamide類ARGs,包括1、2、3和44種ARGs,MBR池中污泥和出水口均注釋出3種,1和2是3個樣品中主要ARGs,其都可以通過使抗生素無法作用于目標酶來抑制抗生素[30],1也是最早被發(fā)現(xiàn)質(zhì)粒攜帶的耐藥基因之一[31],總占比分別為98.42%、99.36%和99.34%.通過污水處理廠后,廢水中1相對豐度顯著下降,為87.52%,3完全被去除,但2相對豐度在MBR池中污泥和出水口有所上升.氨基糖苷類抗生素也是一類常常用于醫(yī)療、農(nóng)業(yè)和養(yǎng)殖業(yè)等多領(lǐng)域中的抗生素[32],其中(3)-Ⅰ、(3)- Ⅱ/Ⅵ、(3)-Ⅲ/Ⅳ、(6’) -Ⅱ/Ⅰb、(2”)-Ⅰ、(2”)-Ⅰ等是在污水、廢水、糞便等環(huán)境媒介中最容易頻繁檢出的aminoglycoside類ARGs[33].本研究均檢測這些ARGs,其中在進水口水樣中共注釋出21種aminoglycoside類ARGs,MBR池中污泥和出水口均注釋出17種(圖3b),A是主要ARGs,與環(huán)境菌株整合子相關(guān),并通過質(zhì)粒在糞腸球菌間轉(zhuǎn)移[34].通過一體化A2/O-MBR污水處理設(shè)備后,其去除達到了91.53%.

圖3 一體化A2/O-MBR污水處理設(shè)備中ARGs亞型相對豐度

白色方塊表示相對豐度為0

2.3 菌群及病原菌變化規(guī)律

微生物群落結(jié)構(gòu)是影響污水中ARGs動態(tài)變化的重要因素[35],本文依據(jù)kraken2的分類結(jié)果得到基于了門、屬水平分類上的微生物群落結(jié)構(gòu).如圖4所示,農(nóng)村生活污水中主要的微生物為細菌(99.81%),其余為古菌(0.19%).變形菌門(Proteobacteria)在進水廢水、污泥和出水樣品中相對豐度最高(53.04%～ 82.19%,平均64.50%),其次為放線菌門(Actinobacteria, 2.23%～20.07%,平均值11.82%)和厚壁菌門(Firmicutes,1.34%～5.74%,平均值2.85%),該研究結(jié)果與相關(guān)研究報道相一致[36].農(nóng)村生活污水的微生物多樣性較為單一,變形菌門相對豐度較高,而在污泥和出水樣本中,微生物群落結(jié)構(gòu)發(fā)生了較大的變化,除了變形菌門外,硝化螺旋菌門(Nitrospirae)也是主要微生物菌門.

圖4 一體化A2/O-MBR污水處理設(shè)備中微生物群落變化(門)

為了探究一體化A2/O-MBR污水處理系統(tǒng)中病原菌的相對豐度變化趨勢,通過宏基因組測序?qū)FDB數(shù)據(jù)庫[37]中列出的32個病原菌屬進行了系統(tǒng)分析(圖5).結(jié)果顯示進水、MBR污泥和出水樣品中共檢測到32種潛在病原體,其中進水、污泥和出水樣品中均檢測出這些潛在病原體.進水、MBR污泥及出水樣品中病原菌占總菌屬的相對豐度均超過5%,分別達到23.25%、9.37%和8.52%.進水樣品中主要的病原菌屬為氣單胞菌屬(, 16.57%)、假單胞菌屬(,1.93%)和不動桿菌屬(,1.05%),MBR污泥樣品中主要的病原菌屬為分枝桿菌屬(,2.61%)、假單胞菌屬(,2.45%)和布克氏菌屬(1.34%);出水樣品中主要的病原菌屬為(,2.62%)、布克氏菌屬(, 1.43%)和分枝桿菌屬(,1.24%).進水樣品中紅孢子蟲屬()未檢出,在污泥和出水樣本中志賀氏菌屬()未檢出.

農(nóng)村生活污水經(jīng)過一體化A2/O-MBR系統(tǒng)處理后,32種病原菌屬占總菌屬的相對豐度有所下降,除紅孢子蟲屬和志賀氏菌屬外,主要病原菌屬也出現(xiàn)了變化,其中14種病原菌屬占總菌屬的相對豐度出現(xiàn)了下降,其中紅孢子蟲屬()、不動桿菌屬()和埃希氏桿菌屬()的下降幅度最大,分別達到了15.89%、0.82%和0.81%;16種病原菌屬占總菌屬的相對豐度出現(xiàn)小幅度的上升,在整個污水處理過程中李斯特菌屬()的相對豐度沒有變化.

圖5 一體化A2/O-MBR污水處理系統(tǒng)中病原微生物(屬)相對豐度變化

白色方塊表示相對豐度為0

ARGs廣泛存在于病原菌中[38],病原菌攜帶ARGs尤其是攜帶多種ARGs后將對人體健康的威脅遠高于單種ARGs的威脅,為探究每類ARGs與病原菌之間的相關(guān)性,使用SPSS 24.0軟件對所有ARGs與病原菌進行Pearson相關(guān)性分析,如表1所示.Chloramphenicol類、polymyxin類和tetracycline類ARGs與多種病原菌屬具有顯著的正相關(guān)性(<0.05),這3類ARGs廣泛分布于各種宿主中;作為進水樣品中相對豐度最高的氣單胞菌屬,其與beta-lactam類、chloramphenicol類、polymyxin類和tetracycline類具有顯著的正相關(guān)性,這4類ARGs極有可能共存于一種病原菌中,形成多重耐藥菌的“超級細菌”.羅曉等[39]發(fā)現(xiàn)多種菌屬與ARGs存在相關(guān)性,與本研究一致.病原菌屬在一體化污水處理設(shè)備中并沒有得到完全去除,其可能攜帶的多種ARGs對人類健康存在著風險.

表1 ARGs與病原微生物(屬)的皮爾遜相關(guān)性分析

注:重復(fù)值、無顯著性關(guān)系值的值未列出; *<0.05 **<0.01.

3 結(jié)論

3.1 基于宏基因組學(xué)的高通量測序方法對北京市某農(nóng)村生活污水一體化A2/O-MBR污水處理系統(tǒng)處理工藝各單元樣本ARGs進行檢測,生活污水進水中的總ARGs相對豐度達到0.8283ratio, beta- lactam類、tetracycline類、aminoglycoside類、sulfonamide類、multidrug類和MLS類ARGs為進水中優(yōu)勢的ARGs,相對豐度占85.79%以上.出水中的總ARGs相對豐度達到0.2298ratio,抗生素抗性基因相對豐度下降了72.25%,ARGs沒有得到完全去除,污水處理系統(tǒng)出水對收納水體造成一定的抗性基因污染風險.

3.2 微生物多樣性分析結(jié)果顯示,變形菌門(Proteobacteria)是一體化A2/O-MBR污水處理系統(tǒng)中數(shù)量最多、種類最豐富的細菌類群;農(nóng)村生活污水中存在著大量病原菌,進水中氣單胞菌屬()、假單胞菌屬()和不動桿菌屬()的比例較高,病原菌相對豐度明顯減少,皮爾遜相關(guān)性分析結(jié)果顯示多種病原菌屬可能同時攜帶多種ARGs.

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Study on the distribution and removal effect of resistance genes in integrated system of A2/O-MBR.

LI Zhong-hong1,2, DU Cai-li2,3, CHEN Su-hua1, ZHANG Lie-yu2, LI Xiao-guang2*, LI Jia-xi2, TIAN Zhen-jun2

(1.Key Laboratory of Jiangxi Province for Persistant Pollutants Control and Resources Recycle, Nanchang Hangkong University, Nanchang 330063, China；2.State Key Laboratory of Environmental Criteria and Risk Assessment, Chinese Research Academy of Environmental Sciences, Beijing 100012, China；3.College of Environmental Science and Engineering, Tongji University, Shanghai 200092, China)., 2021,41(9)：4135～4141

The increase of antibiotic resistance genes among microorganisms has become the main transmission source for sewage treatment plants. The purpose of this study was to explore the removal effect of Antibiotic Resistance Genes (ARGs) and pathogenic bacteria in rural domestic sewage treatment process and evaluate the water quality safety. This study selected a integration A2/O-MBR wastewater treatment system in the village of Beijing,systematic study was implemented in integrated system of A2/O-MBR wastewater treatment system to get the distribution law of ARGs and pathogenic bacteria in the each processing unit. Based on macro genomics, the high-throughput sequencing technology was used to analysis the the ability of removing ARGs and pathogenic bacteria in genus through integrated system of A2/O-MBR. Results showed that the ARGs widely existed in sewage treatment system, 19kinds of ARGs including tetracycline class, aminoglycoside class and sulfonamide class were detected, the relative abundance of the ARGs in influent was much higher than its concentration in the effluent, relative abundance of the ARGs fell by 72.25%, but most of the ARGs in sewage treatment system was not fully removed. The changes of microbial community structure showed that the relative abundance of 32potential pathogens decreased significantly, and most pathogenic bacteria could not be completely removed. The residual ARGs and pathogenic bacteria in the water will lead to the potential pollution risk of receiving water body.

integrated system of A2/O-MBR；metagenomics；ARGs；pathogenic bacteria

X703

A

1000-6923(2021)09-4135-07

李中浤(1995-),男,江西南昌人,南昌航空大學(xué)碩士研究生,主要從事污水生物處理工藝研究.

2021-02-25

國家重點研究計劃(2019YFC0409202)

* 責任作者, 副研究員, xgli1982@163.com