江晓影,刘泳汐,秦 艳,高 品

聚丙烯微塑料对污泥厌氧消化效能作用影响

江晓影,刘泳汐,秦 艳,高 品*

(东华大学环境科学与工程学院,上海 201620)

以聚丙烯(PP)微塑料为研究对象,考察不同浓度PP微塑料对污泥厌氧消化产CH4和产酸效能的作用影响,同时采用荧光定量PCR方法定量检测了乙酸激酶()和基因在不同PP微塑料作用下的丰度变化. 结果表明,PP微塑料对污泥厌氧消化产CH4和产酸效能具有促进影响,CH4和乙酸累计产量随PP微塑料投加量的增大而升高,当PP微塑料投加量为0.2g/g VSS时,CH4和乙酸累计产量与空白对照相比分别提高148.2%和15.2%,达227.1mL/g VSS和1291.2mg/L. 相应地,基因丰度随之提高98.2%,表明PP微塑料对产甲烷菌的生长和繁殖具有促进作用,进而强化污泥厌氧消化产CH4效能.

微塑料；聚丙烯；污泥厌氧消化；基因

微塑料是指尺寸小于5mm的塑料颗粒,已被发现广泛存在于不同环境介质中,如海洋[1-3]、河流[4-5]、湖泊[6]、土壤[7-10]、大气[11-12]等,甚至在饮用水[13-14]和人体粪便[15]中也检出一定数量的微塑料.微塑料不仅可以通过摄食作用对生物体产生物理性伤害[16],同时其自身所含添加剂的溶出及表面吸附的有毒物质也会对生态环境造成毒性影响[17-18],并可通过食物链进行传递[19],对人体健康造成潜在威胁[20].因此,微塑料污染已成为国内外环境领域研究者的关注焦点和研究热点.

有研究表明,城市污水处理厂是环境中微塑料的重要污染源[21].尽管微塑料在污水处理过程中能够被有效去除,但仍有相当数量的微塑料通过出水被排入环境水体[22].在污水处理过程中被去除的微塑料大部分都被截留在污泥中,其浓度水平可达1.5×103～24×103粒/kg[23],然而现有的污泥处理工艺对微塑料的削减作用较小,部分微塑料可能会在聚合物降解菌的作用下被断链分解为尺寸更小的塑料颗粒.有研究报道,微塑料对污泥厌氧消化处理过程会产生抑制作用,如聚氯乙烯(PVC)微塑料溶出双酚A[24]、聚乙烯(PE)微塑料诱导产生活性氧物质(ROS)[25]、聚对苯二甲酸乙二酯(PET)微塑料抑制胞外聚合物生成[26]等.尽管如此,目前关于微塑料对污泥厌氧消化效能影响方面还较少[27],针对聚丙烯(PP)微塑料的影响也尚未见有报道.基于此,本文主要考察不同浓度PP微塑料对污泥厌氧消化产CH4和产酸效能的过程影响,采用荧光定量PCR (qPCR)方法对关键功能酶基因丰度进行定量分析,探讨PP微塑料对污泥厌氧消化效能的影响机制,以期为微塑料对污泥厌氧消化过程的影响及调控提供数据支撑.

1 材料与方法

1.1 厌氧消化反应器的建立和运行

接种污泥取自上海松江污水处理厂的剩余污泥,运回实验室静置24h,沉降污泥挥发性悬浮固体(VSS)质量浓度约为10.0g/L.厌氧消化反应器体积为1L,有效容积为0.6L,VSS平均浓度为8.3g/L,共设置平行2批,每批4组,分别标记为AD-0、AD-0.002、AD-0.02和AD-0.2,其中AD-0为空白对照组,AD-0.002、AD-0.02和AD-0.2反应器中分别投加质量浓度为0.002, 0.02和0.2g/g VSS的PP微塑料,微塑料颗粒粒径为150μm,呈白色球状.反应器在密闭前采用纯度为99.9%氮气进行吹脱,以排出残留氧气,然后置于恒温摇床培养箱(SPH-2012C,上海世平实验设备有限公司),控制温度为(37±1)℃,转速为110r/min.厌氧消化反应周期为15d,每隔2d采样分析挥发性脂肪酸(VFAs),每隔5d采样分析功能酶基因丰度,每组样品分析检测设置3个平行.

1.2 CH4和VFAs的测定

污泥厌氧产生物气采用带有标准刻度的针筒进行收集,并记录生物气累计产量.生物气中CH4采用7900型气相色谱测定(上海天美科学仪器公司),配置氢火焰离子化检测器(FID)和TM-FFAP毛细管柱(0.5μm×0.32mm×30m),载气为氮气,流速为60mL/min,分流比10:1,进样口和检测器温度均为200℃,柱温为80℃.采用CH4与N2混合标准气进行定量分析.

厌氧消化液VFAs同样采用FID检测器的气相色谱检测,包括乙酸、丙酸、正丁酸、异丁酸、正戊酸和异戊酸[28].检测载气为氮气,流速为60mL/min,分流比10:1,进样口温度均为200℃,检测器温度为250℃,初始柱温为50℃,采用程序升温模式,在起始温度保持1min,再以30℃/min速率升至110℃,然后以10℃/min速率升至190℃/min并维持3min,总测定时长14min.

1.3 污泥DNA提取

取一定量厌氧消化泥水混合物,在4℃和6000r/min条件下离心10min,倒去上清液,收集沉淀物,采用TIANamp Soil DNA Kit (TIANGEN)进行DNA提取,具体提取方法参照试剂盒说明书.每次取样设置3个平行样,将所提取的DNA进行均质混合,其纯度和浓度分别采用1%琼脂糖凝胶电泳和Qubit 2.0核酸蛋白测定仪进行检测[29].

1.4 目标功能基因检测

为了考察PP微塑料对污泥厌氧消化产乙酸和产甲烷过程的作用影响,本研究以乙酸激酶()和基因作为研究对象,其中,是乙酸生成过程的关键酶基因,控制着乙酸的最终生成;基因编码甲基辅酶M还原酶的α亚基,是甲烷生成过程的关键酶基因,存在于所有已知的产甲烷菌中[30].

目标基因采用罗氏LightCycler®96型qPCR进行定量检测分析,扩增引物序列、扩增子大小和退火温度见表1.qPCR反应体系总体积为20μL,包括FastStart Essential DNA Green Master 10μL,浓度为4μmol/L的上下游引物1.5μL,DNA模板1μL,以及ddH2O 6μL.qPCR扩增热循环反应程序如下:95℃预变性10min,95℃变性10s,共40个循环,退火20s,72℃延伸30s,同时利用熔解曲线(每隔0.5℃进行读数)分析扩增产物的特异性,qPCR反应扩增效率为92%～100%.每组样品设置3个平行样,并使用无菌水作为阴性对照.

表1 qPCR所使用的基因引物信息

1.5 数据分析

采用Microsoft EXCEL 2016和Origin 8.5进行数据分析,使用IBM SPSS 22.0软件进行统计分析,计算因变量和自变量之间的皮尔逊相关系数和显著性水平值,若<0.05,则认为具有显著相关性,反之则认为相关性不显著.

2 结果与讨论

2.1 PP微塑料对污泥厌氧消化产气过程的作用影响

在厌氧消化反应体系中,甲烷和生物气生成量通常被用作评价厌氧消化效能的关键性指标[33].由图1(a)可以看出,各反应器生物气累计产量随反应时间的延长而不断升高,在反应后期均逐渐趋于稳定,其中AD-0累计产量约为137mL/g VSS.随着PP微塑料投加量的增加,生物气累计产量呈递增趋势.当PP微塑料投加量为0.002g/g VSS时,生物气累计产量变化不显著(= 0.69),但当PP微塑料投加量增大至0.02g/g VSS时,生物气累计产量显著升高(= 0.02),厌氧反应15d时生物气累计产量达212mL/g VSS,相比AD-0反应器提高了54.7%,当继续增大PP微塑料投加量至0.2g/g VSS时,生物气累计产量显著升高至340mL/g VSS(=0.001).由此可见,低浓度(如0.002g/g VSS)PP微塑料对污泥厌氧消化产气效果影响不大,但较高浓度(>0.02g/g VSS)PP微塑料对生物气生成具有显著促进作用.类似地,PP微塑料对污泥厌氧消化CH4累计产量的影响过程同样呈现随其投加量的增大而升高的变化趋势(图1b),与AD-0相比(91.5mL/g VSS),当PP微塑料投加量为0.2g/g VSS时,CH4累计产量显著升高(= 0.0004),厌氧反应15d时为227.1mL/g VSS,增幅达148.2%,表明一定浓度的PP微塑料能够促进污泥厌氧产CH4过程的进行.Wei等[24]研究同样发现,低浓度PVC微塑料(10粒/g TSS,粒径为1mm)能够略微促进CH4的生成,但当其投加量高于20粒/g TSS时,CH4累计产量显著下降,这可能与PVC微塑料在厌氧过程中溶出的双酚A显著抑制污泥水解酸化过程有关.类似地,低浓度PE微塑料(10～60粒/g TSS,粒径为40μm)对污泥厌氧产CH4过程影响不显著,但当其投加量高于100粒/g TSS时,产CH4过程受到显著抑制,这可能与PE微塑料诱导产生胞内活性氧(ROS)有关,导致细胞死亡,抑制污泥水解、酸化和产甲烷过程[25].Li等[34]研究发现,聚酯(PES)微塑料(1～200粒/g TS,粒径为200μm)对污泥厌氧消化产CH4效能同样具有抑制作用.此外,Fu等[35]报道了聚苯乙烯(PS)纳米塑料对污泥和秸秆混合厌氧消化CH4产量和产CH4速率具有抑制影响.本研究发现较高浓度的PP微塑料对污泥厌氧消化产CH4过程具有显著促进作用,这可能与PP微塑料本身性质有关.相比PE和PVC,PP被认为是一种无害的聚合物形式,有研究报道较大尺寸(25～200μm)的PP微塑料不会产生细胞毒性[36].在污泥厌氧消化反应器中,PP微塑料可以作为微生物生长载体,强化污泥消化功能微生物的富集,从而促进其产CH4效能.Chen等[37]同样发现,10粒/g TS浓度的聚酰胺6(PA6)微塑料能够使污泥厌氧消化CH4累计产量提高39%.

2.2 PP微塑料对污泥厌氧消化产酸过程的作用影响

污泥厌氧消化甲烷化效能的高低主要依赖于前期污泥水解和酸化的进行程度.如图2(a)所示,在厌氧消化反应第2d时,PP微塑料对污泥消化液中VFAs(包括乙酸、丙酸、丁酸和戊酸)的生成具有一定促进作用,其生成量随着PP微塑料投加量的增加而呈现递增趋势,但与空白对照AD-0反应器相比差别并不显著.当PP微塑料投加量分别为0.002, 0.02和0.2g/g VSS时,各反应器中VFAs浓度与AD-0相比分别提高约2.0%、2.6%和8.7%,这可能是因为PP微塑料有利于污泥的水解和酸化过程,进而也促进了后期CH4的生成(图1b).前期研究同样报道,在污泥厌氧体系中加入10粒/g TSS浓度的PVC微塑料能够强化VFAs生成,生成量可提高约4.9%[24],与本研究结果类似.随着污泥厌氧消化反应的进行,各反应器中VFAs浓度迅速降低,反应8d后基本趋于稳定,这主要是由于产甲烷菌对VFAs(如乙酸)消耗所导致的,到反应后期逐渐达到平衡,CH4产量也趋于稳定(图1b).

从图2(b)可以看出,在整个厌氧消化反应过程中,不同反应器中乙酸浓度的变化趋势与VFAs基本一致,随着厌氧反应的持续进行呈现降低趋势,到反应8d后趋于稳定.在反应第2d时,厌氧消化液中乙酸浓度达到峰值,其中AD-0中乙酸浓度约为1120.9mg/L,PP微塑料的加入能够促进乙酸生成,当投加量分别为0.002, 0.02和0.2g/g VSS时,AD- 0.002、AD-0.02和AD-0.2中乙酸浓度分别为1164.1, 1181.0和1291.2mg/L,比AD-0分别提高了约3.9%、5.4%和15.2%.由此可见,一定量的PP微塑料有利于污泥厌氧酸化进程,对污泥厌氧消化产乙酸具有促进作用,且随着投加量的增加而呈现增强趋势,这也可能是反应器中VFAs生成量提高的主要原因.

有研究表明,不同类型的微塑料在污泥厌氧消化过程中会溶出对功能微生物具有毒性的物质,如PVC微塑料能够溶出双酚A,进而削弱反应体系中关键酶的反应活性,抑制污泥水解、酸化和产CH4过程[24],而PE微塑料在厌氧转化过程中会产生ROS,同样会对污泥厌氧消化过程产生抑制作用[25].此外,PA6微塑料在厌氧过程中会溶出己内酰胺,低浓度时可提高关键酶活性,促进酸化和甲烷化过程,但高浓度时反而会抑制酶活性[36].这些已有研究均表明,低浓度微塑料对污泥厌氧消化过程影响较小,有时具有一定的促进作用,但高浓度微塑料则会显著抑制污泥厌氧消化效能.相比之下,本研究结果显示,当PP微塑料投加量在0.002～0.2g/g VSS范围时,污泥厌氧消化效能随着PP投加量的增大而呈现增强趋势,这可能是因为PP本身生物毒性较小[36],且PP微塑料(150μm)相对较大的比表面积能够与污泥微生物紧密结合,增强功能微生物反应活性,进而强化污泥厌氧消化效能.

2.3 PP微塑料对污泥厌氧消化过程中关键酶基因的作用影响

污泥厌氧消化过程中关键酶基因的丰度和活性高低会直接影响其消化效能,而酶基因丰度与其反应活性之间通常密切相关[38].在本研究中,是乙酰辅酶A转化为乙酸的关键酶基因,而是甲烷生成的关键酶基因,可作为污泥厌氧消化产CH4效能的良好标记[39].如图3所示,PP微塑料对污泥基因丰度具有削减作用,当PP微塑料投加量分别为0.002, 0.02和0.2g/g VSS时,各反应器中基因丰度分别为4.00×102, 2.71×102和2.93×102copies/ g VSS,与空白对照AD-0反应器(4.55×102copies/g VSS)相比,分别下降12.1%、40.4%和35.6%.由图1(b)可知,PP微塑料能够强化污泥厌氧消化产CH4效能,在污泥厌氧消化初始阶段,PP微塑料的投加可以促进污泥水解和酸化,促使VFAs生成量升高(图2),但随着污泥厌氧反应过程的进行,反应器中底物浓度逐渐降低,污泥厌氧酸化程度减弱,从而抑制基因的合成和表达,由于PP微塑料一定程度上能够促进污泥有机物的厌氧分解,其在反应后期对基因合成的抑制作用加强,从而造成基因的相对丰度低于空白对照组(图3).相比之下,PP微塑料对污泥基因丰度具有促进作用,当PP微塑料投加量分别为0.002, 0.02和0.2g/g VSS时,各反应器中基因丰度分别为6.51×104, 7.57×104和7.61× 104copies/g VSS,与AD-0反应器相比分别提高69.0%、96.4%和98.2%,表明PP微塑料能够促进产甲烷菌中基因的合成和表达,进而强化污泥厌氧消化产CH4效能,这与前述CH4生成量的变化趋势是一致的(图1b).

图3 污泥厌氧消化过程中PP微塑料对关键酶基因丰度的影响

目前,关于微塑料对污泥厌氧消化过程功能酶基因影响方面的研究还很少,有研究报道微塑料本身在厌氧过程中所溶出的添加剂及其表面吸附的有毒物质会削弱和420基因的反应活性,进而影响污泥厌氧酸化和产CH4效能[24,27].本研究结果表明,PP微塑料对基因具有促进作用,这可能是因为PP微塑料本身对微生物细胞不会产生毒性作用[36],一定程度上有利于产甲烷菌的生长和繁殖,但其内在作用机制过程还有待进一步探究.尽管如此,Feng等[40]研究发现,随着PS纳米塑料浓度的升高,其对基因的抑制作用越显著,表明PS纳米塑料对产甲烷菌具有毒性作用,这与微塑料尺寸效应密切相关,微塑料尺寸越小,其比表面积就越大,其本身所含有的有毒添加剂越易溶出,也易产生氧化应激[41-42],进而导致其对细菌微生物的毒性作用越显著.基于此,在后续研究中应考虑微塑料尺寸效应对污泥厌氧消化效能的影响过程和内在机制.

3 结论

3.1 PP微塑料能够强化污泥厌氧消化产气效能,生物气和CH4生成量随着PP微塑料投加量的增加而升高,当PP微塑料投加量为0.2g/g VSS时,生物气和CH4生成量分别可达340和227.1mL/g VSS.

3.2 PP微塑料有利于污泥厌氧酸化过程的进行,VFAs和乙酸生成量随着PP微塑料投加量的增加而呈现递增趋势,当PP投加量为0.2g/g VSS时,乙酸生成量提高约15.2%,可达1291.2mg/L.

3.3 由污泥厌氧消化过程功能酶基因丰度变化可知,PP微塑料对基因的富集具有促进作用,表明其有利于产甲烷菌的生长和繁殖,强化污泥厌氧消化产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

江晓影(1996-),女,浙江台州人,东华大学硕士研究生,主要从事微塑料对污泥厌氧消化效能及微生物群落的影响研究.

2020-09-30

国家自然科学基金资助项目(51978136)

*责任作者, 教授, pingao@dhu.edu.cn