聚丙烯微塑料對污泥厭氧消化效能作用影響

江曉影,劉泳汐,秦 艷,高 品

聚丙烯微塑料對污泥厭氧消化效能作用影響

江曉影,劉泳汐,秦 艷,高 品*

(東華大學環(huán)境科學與工程學院,上海 201620)

以聚丙烯(PP)微塑料為研究對象,考察不同濃度PP微塑料對污泥厭氧消化產(chǎn)CH4和產(chǎn)酸效能的作用影響,同時采用熒光定量PCR方法定量檢測了乙酸激酶()和基因在不同PP微塑料作用下的豐度變化. 結(jié)果表明,PP微塑料對污泥厭氧消化產(chǎn)CH4和產(chǎn)酸效能具有促進影響,CH4和乙酸累計產(chǎn)量隨PP微塑料投加量的增大而升高,當PP微塑料投加量為0.2g/g VSS時,CH4和乙酸累計產(chǎn)量與空白對照相比分別提高148.2%和15.2%,達227.1mL/g VSS和1291.2mg/L. 相應地,基因豐度隨之提高98.2%,表明PP微塑料對產(chǎn)甲烷菌的生長和繁殖具有促進作用,進而強化污泥厭氧消化產(chǎn)CH4效能.

微塑料；聚丙烯；污泥厭氧消化；基因

微塑料是指尺寸小于5mm的塑料顆粒,已被發(fā)現(xiàn)廣泛存在于不同環(huán)境介質(zhì)中,如海洋[1-3]、河流[4-5]、湖泊[6]、土壤[7-10]、大氣[11-12]等,甚至在飲用水[13-14]和人體糞便[15]中也檢出一定數(shù)量的微塑料.微塑料不僅可以通過攝食作用對生物體產(chǎn)生物理性傷害[16],同時其自身所含添加劑的溶出及表面吸附的有毒物質(zhì)也會對生態(tài)環(huán)境造成毒性影響[17-18],并可通過食物鏈進行傳遞[19],對人體健康造成潛在威脅[20].因此,微塑料污染已成為國內(nèi)外環(huán)境領域研究者的關注焦點和研究熱點.

有研究表明,城市污水處理廠是環(huán)境中微塑料的重要污染源[21].盡管微塑料在污水處理過程中能夠被有效去除,但仍有相當數(shù)量的微塑料通過出水被排入環(huán)境水體[22].在污水處理過程中被去除的微塑料大部分都被截留在污泥中,其濃度水平可達1.5×103～24×103粒/kg[23],然而現(xiàn)有的污泥處理工藝對微塑料的削減作用較小,部分微塑料可能會在聚合物降解菌的作用下被斷鏈分解為尺寸更小的塑料顆粒.有研究報道,微塑料對污泥厭氧消化處理過程會產(chǎn)生抑制作用,如聚氯乙烯(PVC)微塑料溶出雙酚A[24]、聚乙烯(PE)微塑料誘導產(chǎn)生活性氧物質(zhì)(ROS)[25]、聚對苯二甲酸乙二酯(PET)微塑料抑制胞外聚合物生成[26]等.盡管如此,目前關于微塑料對污泥厭氧消化效能影響方面還較少[27],針對聚丙烯(PP)微塑料的影響也尚未見有報道.基于此,本文主要考察不同濃度PP微塑料對污泥厭氧消化產(chǎn)CH4和產(chǎn)酸效能的過程影響,采用熒光定量PCR (qPCR)方法對關鍵功能酶基因豐度進行定量分析,探討PP微塑料對污泥厭氧消化效能的影響機制,以期為微塑料對污泥厭氧消化過程的影響及調(diào)控提供數(shù)據(jù)支撐.

1 材料與方法

1.1 厭氧消化反應器的建立和運行

接種污泥取自上海松江污水處理廠的剩余污泥,運回實驗室靜置24h,沉降污泥揮發(fā)性懸浮固體(VSS)質(zhì)量濃度約為10.0g/L.厭氧消化反應器體積為1L,有效容積為0.6L,VSS平均濃度為8.3g/L,共設置平行2批,每批4組,分別標記為AD-0、AD-0.002、AD-0.02和AD-0.2,其中AD-0為空白對照組,AD-0.002、AD-0.02和AD-0.2反應器中分別投加質(zhì)量濃度為0.002, 0.02和0.2g/g VSS的PP微塑料,微塑料顆粒粒徑為150μm,呈白色球狀.反應器在密閉前采用純度為99.9%氮氣進行吹脫,以排出殘留氧氣,然后置于恒溫搖床培養(yǎng)箱(SPH-2012C,上海世平實驗設備有限公司),控制溫度為(37±1)℃,轉(zhuǎn)速為110r/min.厭氧消化反應周期為15d,每隔2d采樣分析揮發(fā)性脂肪酸(VFAs),每隔5d采樣分析功能酶基因豐度,每組樣品分析檢測設置3個平行.

1.2 CH4和VFAs的測定

污泥厭氧產(chǎn)生物氣采用帶有標準刻度的針筒進行收集,并記錄生物氣累計產(chǎn)量.生物氣中CH4采用7900型氣相色譜測定(上海天美科學儀器公司),配置氫火焰離子化檢測器(FID)和TM-FFAP毛細管柱(0.5μm×0.32mm×30m),載氣為氮氣,流速為60mL/min,分流比10:1,進樣口和檢測器溫度均為200℃,柱溫為80℃.采用CH4與N2混合標準氣進行定量分析.

厭氧消化液VFAs同樣采用FID檢測器的氣相色譜檢測,包括乙酸、丙酸、正丁酸、異丁酸、正戊酸和異戊酸[28].檢測載氣為氮氣,流速為60mL/min,分流比10:1,進樣口溫度均為200℃,檢測器溫度為250℃,初始柱溫為50℃,采用程序升溫模式,在起始溫度保持1min,再以30℃/min速率升至110℃,然后以10℃/min速率升至190℃/min并維持3min,總測定時長14min.

1.3 污泥DNA提取

取一定量厭氧消化泥水混合物,在4℃和6000r/min條件下離心10min,倒去上清液,收集沉淀物,采用TIANamp Soil DNA Kit (TIANGEN)進行DNA提取,具體提取方法參照試劑盒說明書.每次取樣設置3個平行樣,將所提取的DNA進行均質(zhì)混合,其純度和濃度分別采用1%瓊脂糖凝膠電泳和Qubit 2.0核酸蛋白測定儀進行檢測[29].

1.4 目標功能基因檢測

為了考察PP微塑料對污泥厭氧消化產(chǎn)乙酸和產(chǎn)甲烷過程的作用影響,本研究以乙酸激酶()和基因作為研究對象,其中,是乙酸生成過程的關鍵酶基因,控制著乙酸的最終生成;基因編碼甲基輔酶M還原酶的α亞基,是甲烷生成過程的關鍵酶基因,存在于所有已知的產(chǎn)甲烷菌中[30].

目標基因采用羅氏LightCycler?96型qPCR進行定量檢測分析,擴增引物序列、擴增子大小和退火溫度見表1.qPCR反應體系總體積為20μL,包括FastStart Essential DNA Green Master 10μL,濃度為4μmol/L的上下游引物1.5μL,DNA模板1μL,以及ddH2O 6μL.qPCR擴增熱循環(huán)反應程序如下:95℃預變性10min,95℃變性10s,共40個循環(huán),退火20s,72℃延伸30s,同時利用熔解曲線(每隔0.5℃進行讀數(shù))分析擴增產(chǎn)物的特異性,qPCR反應擴增效率為92%～100%.每組樣品設置3個平行樣,并使用無菌水作為陰性對照.

表1 qPCR所使用的基因引物信息

1.5 數(shù)據(jù)分析

采用Microsoft EXCEL 2016和Origin 8.5進行數(shù)據(jù)分析,使用IBM SPSS 22.0軟件進行統(tǒng)計分析,計算因變量和自變量之間的皮爾遜相關系數(shù)和顯著性水平值,若<0.05,則認為具有顯著相關性,反之則認為相關性不顯著.

2 結(jié)果與討論

2.1 PP微塑料對污泥厭氧消化產(chǎn)氣過程的作用影響

在厭氧消化反應體系中,甲烷和生物氣生成量通常被用作評價厭氧消化效能的關鍵性指標[33].由圖1(a)可以看出,各反應器生物氣累計產(chǎn)量隨反應時間的延長而不斷升高,在反應后期均逐漸趨于穩(wěn)定,其中AD-0累計產(chǎn)量約為137mL/g VSS.隨著PP微塑料投加量的增加,生物氣累計產(chǎn)量呈遞增趨勢.當PP微塑料投加量為0.002g/g VSS時,生物氣累計產(chǎn)量變化不顯著(= 0.69),但當PP微塑料投加量增大至0.02g/g VSS時,生物氣累計產(chǎn)量顯著升高(= 0.02),厭氧反應15d時生物氣累計產(chǎn)量達212mL/g VSS,相比AD-0反應器提高了54.7%,當繼續(xù)增大PP微塑料投加量至0.2g/g VSS時,生物氣累計產(chǎn)量顯著升高至340mL/g VSS(=0.001).由此可見,低濃度(如0.002g/g VSS)PP微塑料對污泥厭氧消化產(chǎn)氣效果影響不大,但較高濃度(>0.02g/g VSS)PP微塑料對生物氣生成具有顯著促進作用.類似地,PP微塑料對污泥厭氧消化CH4累計產(chǎn)量的影響過程同樣呈現(xiàn)隨其投加量的增大而升高的變化趨勢(圖1b),與AD-0相比(91.5mL/g VSS),當PP微塑料投加量為0.2g/g VSS時,CH4累計產(chǎn)量顯著升高(= 0.0004),厭氧反應15d時為227.1mL/g VSS,增幅達148.2%,表明一定濃度的PP微塑料能夠促進污泥厭氧產(chǎn)CH4過程的進行.Wei等[24]研究同樣發(fā)現(xiàn),低濃度PVC微塑料(10粒/g TSS,粒徑為1mm)能夠略微促進CH4的生成,但當其投加量高于20粒/g TSS時,CH4累計產(chǎn)量顯著下降,這可能與PVC微塑料在厭氧過程中溶出的雙酚A顯著抑制污泥水解酸化過程有關.類似地,低濃度PE微塑料(10～60粒/g TSS,粒徑為40μm)對污泥厭氧產(chǎn)CH4過程影響不顯著,但當其投加量高于100粒/g TSS時,產(chǎn)CH4過程受到顯著抑制,這可能與PE微塑料誘導產(chǎn)生胞內(nèi)活性氧(ROS)有關,導致細胞死亡,抑制污泥水解、酸化和產(chǎn)甲烷過程[25].Li等[34]研究發(fā)現(xiàn),聚酯(PES)微塑料(1～200粒/g TS,粒徑為200μm)對污泥厭氧消化產(chǎn)CH4效能同樣具有抑制作用.此外,Fu等[35]報道了聚苯乙烯(PS)納米塑料對污泥和秸稈混合厭氧消化CH4產(chǎn)量和產(chǎn)CH4速率具有抑制影響.本研究發(fā)現(xiàn)較高濃度的PP微塑料對污泥厭氧消化產(chǎn)CH4過程具有顯著促進作用,這可能與PP微塑料本身性質(zhì)有關.相比PE和PVC,PP被認為是一種無害的聚合物形式,有研究報道較大尺寸(25～200μm)的PP微塑料不會產(chǎn)生細胞毒性[36].在污泥厭氧消化反應器中,PP微塑料可以作為微生物生長載體,強化污泥消化功能微生物的富集,從而促進其產(chǎn)CH4效能.Chen等[37]同樣發(fā)現(xiàn),10粒/g TS濃度的聚酰胺6(PA6)微塑料能夠使污泥厭氧消化CH4累計產(chǎn)量提高39%.

2.2 PP微塑料對污泥厭氧消化產(chǎn)酸過程的作用影響

污泥厭氧消化甲烷化效能的高低主要依賴于前期污泥水解和酸化的進行程度.如圖2(a)所示,在厭氧消化反應第2d時,PP微塑料對污泥消化液中VFAs(包括乙酸、丙酸、丁酸和戊酸)的生成具有一定促進作用,其生成量隨著PP微塑料投加量的增加而呈現(xiàn)遞增趨勢,但與空白對照AD-0反應器相比差別并不顯著.當PP微塑料投加量分別為0.002, 0.02和0.2g/g VSS時,各反應器中VFAs濃度與AD-0相比分別提高約2.0%、2.6%和8.7%,這可能是因為PP微塑料有利于污泥的水解和酸化過程,進而也促進了后期CH4的生成(圖1b).前期研究同樣報道,在污泥厭氧體系中加入10粒/g TSS濃度的PVC微塑料能夠強化VFAs生成,生成量可提高約4.9%[24],與本研究結(jié)果類似.隨著污泥厭氧消化反應的進行,各反應器中VFAs濃度迅速降低,反應8d后基本趨于穩(wěn)定,這主要是由于產(chǎn)甲烷菌對VFAs(如乙酸)消耗所導致的,到反應后期逐漸達到平衡,CH4產(chǎn)量也趨于穩(wěn)定(圖1b).

從圖2(b)可以看出,在整個厭氧消化反應過程中,不同反應器中乙酸濃度的變化趨勢與VFAs基本一致,隨著厭氧反應的持續(xù)進行呈現(xiàn)降低趨勢,到反應8d后趨于穩(wěn)定.在反應第2d時,厭氧消化液中乙酸濃度達到峰值,其中AD-0中乙酸濃度約為1120.9mg/L,PP微塑料的加入能夠促進乙酸生成,當投加量分別為0.002, 0.02和0.2g/g VSS時,AD- 0.002、AD-0.02和AD-0.2中乙酸濃度分別為1164.1, 1181.0和1291.2mg/L,比AD-0分別提高了約3.9%、5.4%和15.2%.由此可見,一定量的PP微塑料有利于污泥厭氧酸化進程,對污泥厭氧消化產(chǎn)乙酸具有促進作用,且隨著投加量的增加而呈現(xiàn)增強趨勢,這也可能是反應器中VFAs生成量提高的主要原因.

有研究表明,不同類型的微塑料在污泥厭氧消化過程中會溶出對功能微生物具有毒性的物質(zhì),如PVC微塑料能夠溶出雙酚A,進而削弱反應體系中關鍵酶的反應活性,抑制污泥水解、酸化和產(chǎn)CH4過程[24],而PE微塑料在厭氧轉(zhuǎn)化過程中會產(chǎn)生ROS,同樣會對污泥厭氧消化過程產(chǎn)生抑制作用[25].此外,PA6微塑料在厭氧過程中會溶出己內(nèi)酰胺,低濃度時可提高關鍵酶活性,促進酸化和甲烷化過程,但高濃度時反而會抑制酶活性[36].這些已有研究均表明,低濃度微塑料對污泥厭氧消化過程影響較小,有時具有一定的促進作用,但高濃度微塑料則會顯著抑制污泥厭氧消化效能.相比之下,本研究結(jié)果顯示,當PP微塑料投加量在0.002～0.2g/g VSS范圍時,污泥厭氧消化效能隨著PP投加量的增大而呈現(xiàn)增強趨勢,這可能是因為PP本身生物毒性較小[36],且PP微塑料(150μm)相對較大的比表面積能夠與污泥微生物緊密結(jié)合,增強功能微生物反應活性,進而強化污泥厭氧消化效能.

2.3 PP微塑料對污泥厭氧消化過程中關鍵酶基因的作用影響

污泥厭氧消化過程中關鍵酶基因的豐度和活性高低會直接影響其消化效能,而酶基因豐度與其反應活性之間通常密切相關[38].在本研究中,是乙酰輔酶A轉(zhuǎn)化為乙酸的關鍵酶基因,而是甲烷生成的關鍵酶基因,可作為污泥厭氧消化產(chǎn)CH4效能的良好標記[39].如圖3所示,PP微塑料對污泥基因豐度具有削減作用,當PP微塑料投加量分別為0.002, 0.02和0.2g/g VSS時,各反應器中基因豐度分別為4.00×102, 2.71×102和2.93×102copies/ g VSS,與空白對照AD-0反應器(4.55×102copies/g VSS)相比,分別下降12.1%、40.4%和35.6%.由圖1(b)可知,PP微塑料能夠強化污泥厭氧消化產(chǎn)CH4效能,在污泥厭氧消化初始階段,PP微塑料的投加可以促進污泥水解和酸化,促使VFAs生成量升高(圖2),但隨著污泥厭氧反應過程的進行,反應器中底物濃度逐漸降低,污泥厭氧酸化程度減弱,從而抑制基因的合成和表達,由于PP微塑料一定程度上能夠促進污泥有機物的厭氧分解,其在反應后期對基因合成的抑制作用加強,從而造成基因的相對豐度低于空白對照組(圖3).相比之下,PP微塑料對污泥基因豐度具有促進作用,當PP微塑料投加量分別為0.002, 0.02和0.2g/g VSS時,各反應器中基因豐度分別為6.51×104, 7.57×104和7.61× 104copies/g VSS,與AD-0反應器相比分別提高69.0%、96.4%和98.2%,表明PP微塑料能夠促進產(chǎn)甲烷菌中基因的合成和表達,進而強化污泥厭氧消化產(chǎn)CH4效能,這與前述CH4生成量的變化趨勢是一致的(圖1b).

圖3 污泥厭氧消化過程中PP微塑料對關鍵酶基因豐度的影響

目前,關于微塑料對污泥厭氧消化過程功能酶基因影響方面的研究還很少,有研究報道微塑料本身在厭氧過程中所溶出的添加劑及其表面吸附的有毒物質(zhì)會削弱和420基因的反應活性,進而影響污泥厭氧酸化和產(chǎn)CH4效能[24,27].本研究結(jié)果表明,PP微塑料對基因具有促進作用,這可能是因為PP微塑料本身對微生物細胞不會產(chǎn)生毒性作用[36],一定程度上有利于產(chǎn)甲烷菌的生長和繁殖,但其內(nèi)在作用機制過程還有待進一步探究.盡管如此,Feng等[40]研究發(fā)現(xiàn),隨著PS納米塑料濃度的升高,其對基因的抑制作用越顯著,表明PS納米塑料對產(chǎn)甲烷菌具有毒性作用,這與微塑料尺寸效應密切相關,微塑料尺寸越小,其比表面積就越大,其本身所含有的有毒添加劑越易溶出,也易產(chǎn)生氧化應激[41-42],進而導致其對細菌微生物的毒性作用越顯著.基于此,在后續(xù)研究中應考慮微塑料尺寸效應對污泥厭氧消化效能的影響過程和內(nèi)在機制.

3 結(jié)論

3.1 PP微塑料能夠強化污泥厭氧消化產(chǎn)氣效能,生物氣和CH4生成量隨著PP微塑料投加量的增加而升高,當PP微塑料投加量為0.2g/g VSS時,生物氣和CH4生成量分別可達340和227.1mL/g VSS.

3.2 PP微塑料有利于污泥厭氧酸化過程的進行,VFAs和乙酸生成量隨著PP微塑料投加量的增加而呈現(xiàn)遞增趨勢,當PP投加量為0.2g/g VSS時,乙酸生成量提高約15.2%,可達1291.2mg/L.

3.3 由污泥厭氧消化過程功能酶基因豐度變化可知,PP微塑料對基因的富集具有促進作用,表明其有利于產(chǎn)甲烷菌的生長和繁殖,強化污泥厭氧消化產(chǎn)CH4效能.

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Effect of polypropylene microplastics on the performance of sludge anaerobic digestion.

JIANG Xiao-ying, LIU Yong-xi, QIN Yan, GAO Pin*

(College of Environmental Science and Engineering, Donghua University, Shanghai 201620, China)., 2021,41(5)：2252～2257

Polypropylene (PP) microplastics (MPs) were used to investigate their effects on the production of methane (CH4) and volatile fatty acids (VFAs) in sludge anaerobic digestion. The abundances of acetate kinase () andgenes under stresses of different amounts of PP MPs were determined by quantitative real-time PCR methods. The results showed that production of CH4and VFAs was enhanced by addition of PP MPs. The cumulative productions of CH4and acetic acid increased with increasing dosages of PP MPs. When the addition of PP MPs was 0.2g/g VSS, the cumulative productions of CH4and acetic acid increased by 148.2% to 227.1mL/g and 15.2% to 1291.2mg/L, respectively, compared with the controls. Correspondingly, the abundance ofgenes increased by 98.2%, which indicated that PP MPs had a promoting effect on the growth of methanogens, thereby enhanced CH4production during sludge anaerobic digestion.

microplastics；polypropylene；sludge anaerobic digestion；gene

X703.1

A

1000-6923(2021)05-2252-06

江曉影(1996-),女,浙江臺州人,東華大學碩士研究生,主要從事微塑料對污泥厭氧消化效能及微生物群落的影響研究.

2020-09-30

國家自然科學基金資助項目(51978136)

*責任作者, 教授, pingao@dhu.edu.cn