Effects of microplastics on wastewater and sewage sludge treatment and their removal: A review

Journal Pre-proofs Review Effects of microplastics on wastewater and sewage sludge treatment and their removal: A review Zhiqi Zhang, Yinguang Chen PII: DOI: Reference:

S1385-8947(19)32365-4 https://doi.org/10.1016/j.cej.2019.122955 CEJ 122955

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Please cite this article as: Z. Zhang, Y. Chen, Effects of microplastics on wastewater and sewage sludge treatment and their removal: A review, Chemical Engineering Journal (2019), doi: https://doi.org/10.1016/j.cej.2019.122955

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Effects of microplastics on wastewater and sewage sludge treatment and their removal: A review

Zhiqi Zhang 1, Yinguang Chen*1, 2

(1 State Key Laboratory of Pollution Control and Resource Reuse, School of Environmental Science and Engineering, Tongji University, 1239 Siping Road, Shanghai 200092, China: 2 Shanghai Institute of Pollution Control and Ecological Security, Shanghai 200092, P.R. China)

*Corresponding author E-mail: [email protected] Tel: 86-21-65981263 Fax: 86-21-65986313

1

ABSTRACT: Microplastics are widely used and inevitably released into the environment, which can be easily enriched in wastewater treatment plants. This review assesses their potential effects on wastewater and sludge treatment and the methods for removing microplastics from wastewater and sludge. Firstly, recent advances of the methods for purification and detection of microplastics in in wastewater and sewage sludge environment were reviewed. Then, the effects of microplastics on wastewater and sludge treatment and the mechanisms were discussed. It can be seen that when the size of microplastics reached the nanometer level, they infiltrated into the biofilm and produce ROS, which showed acute inhibitory effect on microbial community, key enzymes, metabolic intermediates and final products. Due to their large specific surface area and hydrophobic surface, persistent organic pollutants, metals and pathogens could be easily adsorbed on the surface of microplastics. As various additives were added in the production of plastics, the adsorption of environmental micropollutants and the exudation of additives made the mechanism of microplastics affecting sewage and sludge treatment more complicated. Also, the methods for removing microplastics from wastewater and sludge were reviewed and their removal efficiencies were compared. Finally, the problems that need to be addressed in the future were pointed out, and the key points for future investigation were proposed. Keywords: Microplastics; Wastewater treatment plants; Sludge digestion; Removal

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Abbreviations

FTIR

Fourier transform infrared

AD

Anaerobic digestion

GPC

Gel permeation chromatography

AGS

Aerobic granular sludge

HOCs

Hydrophobic organic contaminants

AK

Acetate kinase

HCHs

Hexachlorocyclohexanes

A2O

Anaerobic anoxic oxic

IR

Infrared

ATR

Attenuated total reflection

LTA

Lipoteichoic acid

AFM

Atomic force microscopy

LC50

Half lethal concentration

BOD

Biochemical oxygen demand

LPS

Lipopolysaccharides

BPA

Bisphenol A

MBR

Membrane bioreactor

BAF

Biological active filter

MPs

Microplastics

CAS

Conventional activated sludge

NMR

Nuclear magnetic resonance

COD

Chemical oxygen demand

NR

Nile red

DW

Dry weight

NTU

Nephelometric turbidity unit

DOP

Dissolved organic phosphorus

NIR

Near ifrared

DO

Dissolved oxygen

Na

Not available

DOC

Dissolved organic carbon

NPs

Nanoplastics

DMs

Dynamic membranes

OTUs

Operational Taxonomic Units

DBP

Di-n-butyl phthalate

OURen

Endogenous respiration rate

DH

Digital holography

OEP

Oil extraction protocol

DDTs

Dichloro diphenyl trichloroethanes

PCBs

Polychlorinated biphenyls

EC

Electrocoagulation

PBDEs

Polybrominated diphenyl ethers

EPS

Extracellular polymer substances

PVC

Polyvinyl chloride

FPA

Focal plane array

PA

Polyamide

3

Abbreviations

SEM-EDS

Scanning electron microscope-energy dispersive spectrometer

PES

Polyester

TP

Total phosphorus

PE

Polyethylene

TS

Total solids

PP

Polypropylene

TGA-DSC

Thermogravimetric analyzer-differential

scanning

calorimetry PC

Polycarbonate

TED-GC-MS

Thermal

extraction

desorption-gas

chromatography-mass spectrometry PU

Polyurethane

TDP

Total dissolved phosphorus

PS

Polystyrene

TN

Total nitrogen

PET

Polyethylene terephthalate

UF

Ultrafiltration

Pyr-GC-MS

Pyrolysis-gas

UV

Ultraviolet

chromatography-mass

spectrometry PAM

Polyacrylamide

VS

Volatile solids

PAHs

Polycyclic aromatic hydrocarbons

VSS

Volatile suspended solids

PAEs

Phthalate esters

VFA

Volatile fatty acid

ROS

Reactive oxygen species

WPO

Wet peroxide oxidation

SBR

Sequencing batch reactor

WW

Wet weight

SCOD

Solluted chemical oxigen demand

WAS

Waste activated sludge

SAL

Small anthropogenic litter

WWTPs

Wastewater treatment plants

SAS

Synthetic amorphous silica

4

1. Introduction As a kind of synthetic chemicals, plastics are extensively used in daily life (e.g. packaging, film, covers, bags and containers) [1]. Unlike ordinary pollutants, smaller and smaller particles are formed during biodegradation of plastics. Plastics with particle size smaller than 5mm are defined as microplastics. Nanoplastics are defined as unintentionally generated particles (i.e., from the degradation and manufacture of plastic objects) and exhibit colloidal behavior in a size range of 1 to 1,000 nm [2].Currently, the toxic impacts of MPs are getting more and more attention [3]. On account of high specific surface area as well as their physical and chemical properties, MPs have higher potential in absorbing and desorbing toxicants, like PAHs, PCBs and metals. Many additives, such as PBDEs, PAEs and so on, are added in the production of plastics [4]. The microplastics produced by living or industry have easy access to WWTPs. Similarly, waste plastics shed from equipment, fillers, etc., in WWTPs can be decomposed into microplastics as well. WWTPs, a significant component of urban water systems, are regarded as potential sources of MPs in the environment [5]. Biological removal of COD, nitrogen and phosphorus is designed by current WWTPs, and high retention potential of MPs in WWTPs has been reported [6]. Previous studies have shown that microplastics in biological wastewater treatment system reduce the abundance of nitrifying bacteria, denitrifying bacteria with nitrogen and phosphorus removal function, heterotrophic nitrification-aerobic denitrifying bacteria, and bacteria capable of degrading phenolic compounds, and inhibit the bio-transformation of NH4+-N [7; 8]. It is well known that large amounts of sludge are produced during biological wastewater treatment, which needs to be disposed, and the most common treated method is digestion. It has been reported that in sludge digestion process microplastics affect microbial communities and inhibit sludge hydrolysis, acetic acid accumulation, hydrogen generation and methane production [9; 10]. Until now, however, the review regarding the effects of microplastics on wastewater and sludge treatment has never been made in the literature. There have been many reviews evaluating the detection and identification methods of microplastics in marine and sediment systems, such as NMR, FTIR, Raman and so on [11]. Recently, some achievemnets in detection and 5

identification of microplastics in wastewater and sludge were made. Thus, in this paper the recent advances of microplastic detection methods in wastewater and sludge samples are reviewed firstly. Then, the effects of microplastics on wastewater and sludge treatment and their mechanisms are discussed in detail. Also, the methods for removing microplastics from wastewater and sludge are summarized. Finally, the key issues that need to be addressed in the future are addressed.

2. New progress in analysis of microplastics and nanoplastics in wastewater and sewage sludge environment 2.1 Sample purification Crichton et al. proposed OEP utilizing the lipophilic properties of MPs [12]. OEP is a high recovery, cost-saving and FTIR compatible method for extracting MPs from the environment [12]. The method utilizes the lipophilicity of plastic polymers and improves the detectability of fibers. It separates dense and heavy plastics from organic matter without expensive or harmful reagents. Catalytic WPO is a method of extracting microplastics. Hydroxyl radicals produced during the decomposition of hydrogen peroxide oxidize most natural organic matter to carboxylic acid, aldehyde, CO2 and H2O, but they do not degrade microplastics. Besides, it can be used to extract microplastics from wastewater streams. The presence of catalysts (FeSO4) allows rapid digestion of organic substances under mild conditions. Its specific operation is to add FeSO4 and H2O2 to the sample, and heat the solution at 70 ℃, stirring for 30 minutes or until the reaction is completed [13]. Sludge is a more challenging sample than sediment because it consists of a viscous matrix of organic materials, microorganisms and inorganic particles bound together by biopolymers, with high affinity for most polymer surfaces. At present, the methods of extracting MPs from sludge are time-consuming and require expensive density separation reagents and a large number of oxidants for organic matter removal. Sujathan et al. proposed an optimized technique for MPs extraction from sludge with a density of up to 1.34g/cm3 [14]. For particles > 20 um, the extraction efficiency was 78 (±8%) [14]. Compared with the previously reported methods used for sediment, the thermal bleaching method requires less than 24 hours of treatment time, reducing the amount of density separation 6

solution by 50% and the costs of 100 ml sample treatment by 75% [14]. Hurley et al. confirmed the applicability of Fenton reagent in the extraction of microplastics from solid substrates (sludge, soil, etc.) together with density separation [15]. In order to reduce the decomposition of hydrogen peroxide, ice bath was used at intervals to adjust the reaction temperature and maintain the temperature less than 40 ℃, which would also protect the microplastics greatly [15]. ZnCl2, NaI and CaCl2 can be used to extract microplastics from sludge. They all rely on density-based extraction techniques, which use density differences between sludge and microplastics to separate them. When sludge samples are placed in high density salt solution, plastic particles tend to float to the surface of the solution, while denser sludge materials remain at the bottom of the solution gradient. High density salts such as ZnCl2 and NaI were used to improve the recovery of microplastics [16; 17]. Briefly speaking, their operation is mainly to add the extracted reagent to the samples, settle down statically, and then filter with polycarbonate membrane. The above operations are repeated several times to extract microplastics from samples. The economic principle should be also considered when selecting extraction methods. Table 1 shows the cost analysis of microplastic extraction methods. OEP has the lowest cost per unit of reagent. Table 1. Cost analysis of extraction and purification methods. Sample volume or weight 50g

Pretreatment time [min] 0

Extraction time [min] 90-168

Aperture (Lower limit of size) [μm] 1

References

191.63

Reagent cost per sample [$] 0.96

CaCl2

67.60

75.70

455-957g

720

35-65

55

[18]

NaI

34.40

90.00

50-200g

0

60

0.45

[19]

ZnCl2

40000

922

500-600ml

0

15-1440

0.3

[20]

Reagent selection

Set up cost

Rapeseed oil

[$]

[12]

2.2 Research progress of detection and identification methods of microplastics and nanoplastics Each specific plastic has its own unique physical and chemical properties, which poses a challenge in accurate identification. Visual, FTIR and Raman are the conventional methods for detection of MPs [11]. Here, we mainly introduce new methods for microplastics identification, which have the characteristics of cost-saving and high 7

efficiency. These new methods include FTIR combined with a FPA detector, NR staining, TGA-DSC, TED-GC-MS, etc. The discovery of new analytical instruments and their mutual coupling or the coupling of existing traditional instruments can meet the recent challenges in identifying microplastics. The detection and identification of NPs are major challenges in the studies of microplastics. AFM coupled with IR or Raman spectroscopy has the possibility to analyze nanoplastics [21], which can provide nano-resolution images, and the probe can operate in contact as well as non-contact modes [22]. The AFM and IR spectra of PS MPs have been obtained by AFM-IR coupling technique successfully [23]. Besides, biosensors can be a candidate for NPs detection, using organisms that are sensitive and specific to the substance and the special relation of NPs to cell surface proteins and stress response genes. Therefore, a highly specific biological system on the surface of NPs can be used for determining NPs [24]. The commonly used techniques for sample purification are time-consuming and the results are prone to errors. Therefore, it is a pressing need for a reliable and easy method that can be employed in actual environmental samples. Continuous spectral analysis of water flowing through a portable Raman micro-spectrometer can promote the quick detection of MPs in water without pretreatment. Shim et al. found that 5 mg/L NR (fluorescent dyeing) solution could dye MPs effectively and they could be easily recognized by green fluorescence [25]. In addition to PVC, PA and PES, NR dyeing has been successfully applied to PE, PP, PS, PC, PU and poly (ethylene-vinyl acetate) MPs [25]. This method is directly and rapidly used to identify and quantify MPs samples in laboratory. Rose red dyeing has been studied as well because it has no toxicity and can stain materials other than plastics [26]. Alkali-assisted hot hydrolysis was used by Wang et al. to depolymerize PC and PET both containing ester groups in pentanol or butanol systems [27]. The contents of PC and PET microplastics in environmental samples were quantified through determining the concentrations of BPA and p-phthalic acid [27]. This method has been successfully applied to quantify PC and PET MPs in sludge. Many techniques have been proposed to analyze the concentrations of MPs in water and sludge, but the data are still incomplete. It's necessary to know the purification and detection processes of MPs in WWTPs (Fig. 1.). Table 2 summarizes the methods for MPs detection and 8

identification in wastewater and sludge, which might have reference value for future research.

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Table 2. Microplastics detection methods and their advantages and disadvantages.

Neuston(333μ

Detection and identification method Stereo

Concentrations and sizes of microplastics 0.330-4.75mm;

rivers in

m ); WPO;

microscope,

15520-4721709

compositions; 2.It can

Illinois,

NaCl (6mol/L)

Pyr-GC-MS

MPs/day

identify any plastic additives

Sample

Extraction method

WWTP in nine

Applicability of the identification method

America

Advantages of the identification method

Disadvantages of the identification method

References

1.Identification of sample

Destructive technology

[28]

present at the same time. AFM-IR

1.50-100nm; 2.It can reveal

[23]

the spatial distribution of these polymer components and chemically identify them. 12 WWTPs in

10μm stainless

Lower Saxony, Germany

FPA-ATR-FTIR

MP>500μm:0-50

1.It can identify particles as

steel filter

MPs/m3;MP

small as 20μm; 2. Time

cartridge;

<500μm:

saving; 3.Maintain

Enzymatic

1×101-9×103

maximum resolution.

immersion

MPs/m3;

treatment;

Sludge(DW):

ZnCl2

1.24×109-5.67× 109MPs/(kg ·year)

10

Destructive technology

[16]

Table 2. Microplastics detection methods and their advantages and disadvantages.-Continued Sample

Extraction method

Detection and identification method TED-GC-MS

Concentrations and sizes of microplastics

Applicability of the identification method

Advantages of the identification method

Disadvantages of the identification method

References

Detection of

1.Rapid detection; 2.Quantification of

It cannot provide direct

[29]

unknown organic

polymers; 3.Degradation (aging) state

information about particle

species

doesn’t affect particle detection.

size and quantity.

1.Cost-effective and direct; 2.It can

1.Only PE and PP can be

Karlsruhe's

Sieve (1mm,

TGA-DSC

Neureut

12μm); ZnCl2

determine the mass concentrations of the

clearly identified;

WWTP

solution (ρ=

polymer type; 3.A very small sample

2.Destructive analysis.

1.85g/ml);

size (1-20mg); 4.Simultaneous analysis

H2O2(30%)

of polymer types and additives. SEM-EDS

1.Distinguishment between plastic and

SEM is not used to identify

non-plastic items; 2.It can check and

the types of plastics.

confirm the inorganic chemical composition of MPs and lots of inorganic additives that MPs may contain.

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[30]

[31]

3. Effects of microplastics on wastewater treatment 3.1 Effects of microplastics on primary treatment The primary treatment mainly removes suspended solids from wastewater by physical methods such as precipitation. By means of oxidation, neutralization and other methods, the strong acid, strong alkali and excessively concentrated toxic substances in wastewater have been initially removed to provide suitable water quality conditions for secondary treatment. The gap between rough grille bars is generally 16-25 mm. The gap between fine grille bars is generally 3-10 mm. Because of their small particle size, microplastics will not cause blockage to the coarse grille, but because of their large amount, they may cause blockage to the fine grille. Microplastics may adsorb agents because of their huge surface area and hydrophobicity. In order to achieve the same treatment effect as the non-microplastic system, it is necessary to increase the dosage of reagents. In addition, microplastics can also adsorb toxic substances, affecting preliminary removal of pollutants [32]. 3.2 Effects of microplastics on secondary treatment The secondary treatment of sewage is based on the primary treatment, and further treatment of sewage by biochemical action. Microplastics affect the biological conversion efficiency of inorganic nitrogen. The conversion efficiency of NH4+-N decreased in the treatment of microplastics, especially nanoplastics [8]. The presence of microplastics may inhibit the denitrification process, which leads to the accumulation of ammonium in water [33; 34]. In short, the presence of microplastics altered the microbial-mediated processes that control ammonium production (ammoniation) and reduction (nitrification and denitrification) [35]. Microplastics have a weak negative correlation with phosphorus removal and are positively correlated with nitrogen biotransformation [36]. This may be due to the fact that bacteria associated with nitrogen removal are more sensitive than phosphorus accumulating organisms [37; 38]. Therefore, microplastics usually have more adverse effects on nitrogen removal than phosphorus removal. The presence of microplastics significantly increased TDP and DOP levels [39], but it showed no significant influence on phosphorus removal in the long-term tests [39]. Besides, BOD, DO, TN and TP are

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positively correlated with the concentration of microplastics [40]. Compared with natural microplastics, weathered microplastics could result in an observable increase in BOD value (P < 0.0001) [41]. However, a study has found that the presence of MPs doesn't affect the efficiency of SBR [42]. For BAF, microplastics can provide surfaces for microbial attachment and growth [43]. However, the local head loss of the filter may be greater than the actual water pressure at the end of the filtration stage, which is prone to air resistance phenomenon. In addition, due to the huge specific surface area of microplastics, they are easy to bond with suspended substances in wastewater to form spheres, resulting in uneven distribution of water. Microplastics led to the decline of apparent VS destruction [10]. It is noteworthy that reduced VS destruction is converted into more sludge, which increases the cost of subsequent treatment. It is estimated that long-term exposure to high levels of MPs will increase the amount of waste sludge by 9.1%, thus increasing the corresponding cost of sludge transportation and disposal [9]. 3.3 Effects of microplastics on tertiary treatment In coagulation process, the amount of effective flocculant is reduced because of the interaction between microplastics with negative surface charge and flocculants such as aluminium salt and iron salt [44]. In order to achieve the same effect as the non-microplastic system, additional chemicals are needed, which increases the cost of treatment. In the air flotation stage, the surface properties of microplastics make it easy to adsorb pollutants and agglomerate. The size and density of the newly formed aggregates are different from the original design parameters of the air flotation tank. The designed bubbles can not bring pollutants to the surface of the water. The shape of microplastics will become irregular due to the previous wastewater treatment processes. In the process of UF, reverse osmosis and microfiltration, the membrane is easily worn by irregular microplastics. The phenomenon of reverse osmosis membranes may be more obvious due to the effect of high pressure. It was found that sand wore the reverse osmosis membrane and causes scratches [45]. At present, most WWTPs use polymer membranes [46]. Microplastics are likely to wear this type of membrane and reduce filtration performance.

13

In addition, due to the surface properties of microplastics, membrane fouling may also be caused by blockage or formation of filter cake [47]. Fouling results in increased transmembrane pressure, which in turn requires longer filtration time and leads to greater energy consumption. The fouling of PS latex (0.2-200 μm) on poly (sulfone) membranes has been studied [48]. The fouling mechanism of particles less than 10 μm is clogging of the membrane pores, and that of particles larger than 10 μm is mainly the formation of filter cake. Microplastics may also affect the disinfection process. Microplastics can be suspended in wastewater, hindering chlorine and UV disinfection. Microplastics can be used as protective substrates for bacteria, which can resist disinfection. In addition, ozone can also oxidize microplastics, resulting in the reduction of effective ozone molecules. 3.4 Mechanisms of microplastics affecting biological wastewater treatment Biofilm is considered to be a metabolic hotspot and an important site for DOC degradation [49]. Approaching of different microorganisms coexisting in microplastics biofilm can promote the formation of microaggregates for co-degradation of organic matter. Increased heterotrophic activity observed on MPs may promote the formation of a lean "dead zone", which may have a certain effect on wastewater treatment functions [50]. Microplastics observably reduced the relative abundances of Rhizobiaceae, Xanthobacteraceae and Isosphaeraceae [51]. Crucial microorganisms that lead to nitrogen cycling and organic decomposition are included in these families. According to the prediction of metabolic pathway, the amino acid metabolism of cofactors and vitamins is obviously different between microplastics and surrounding water [52]. These two metabolic pathways are closely related to the degradation of alanine, aspartic acid, glutamic acid and other carbohydrates [53]. Therefore, microplastics, as unique microbial habitats, affect the metabolic performance and potential nutrient metabolism of specific communities in microplastics accumulation areas [54]. Many studies have shown that nanoparticles can inhibit denitrification, phosphorus removal and COD removal processes [38; 55; 56; 57]. As a kind of nanoparticles, nanoplastics may also inhibit these processes. Microplastics can reduce the abundance of functional microorganisms [7; 58]. The beads produced by cosmetics contain antimicrobial compounds used in scrubbing, which inhibit bacterial processes and give rise to changes in the 14

circulation of nutrients like nitrogen [34]. Higher abundances of Pirellulaceae and Phycisphaerales, belonging to Planctomycetes on MPs substrates, were observed and correlated with ammonium oxidation [59]. Plastics had a high inhibition rate on nitrifying bacteria, with a LC50 of 13.39 at the lowest [58]. Furthermore, the presence of microplastics significantly reduced the number of gene copies of nitrogenase reductase (nifH) marker gene and ammonium monooxygenase (amoA) marker gene [60]. As membrane-bound enzymes, crucial enzymes for nitrification and denitrification are embedded in the cell membrane or situated in the cytoplasm. Protective effects though EPS have, the attachment of microplastics to biomolecules on the cell surface adds to the opportunities of contact between the microplastics and crucial enzymes, which may result in the reduction and inactivation of enzyme activity, accordingly affecting the denitrification performance of the wastewater [41]. Both LPS and LTA are amphoteric polymers existing in gram-positive cell walls and gram-negative cell walls, respectively. Microplastics in wastewater and sludge are most likely to be in contact with them first (Fig. 6c). As a matter of fact, cell-associated toxicity is usually caused by damage to proteins and phospholipids. When microplastics reach the nanometer level, because of their small size, they enter the gaps between the biopolymer chains easily, so the contact opportunities of microplastics with proteins and phospholipids are added to (Fig. 2b). The acute inhibition of activated sludge by nanoplastics in the form of endogenous respiration is shown in Fig. 3 [61]. The higher the concentration is, the stronger the acute inhibition is. OURen is an important indicator of activated sludge activity, which is closely related to the efficiency of wastewater treatment. The mean OURen showed a downward trend as the concentrations of microplastics increased [61]. The secondary structure of the protein in EPS was altered by PS nanoplastics, which resulted in bioflocculation of activated sludge [61]. There is a positive correlation between functional group C-(O, N) and bioflocculation of microorganisms [62]. The change of C-(C, H) might be correlated with the acute inhibition of PS nanoplastics on the OURen of activated sludge (Fig. 6f) [61]. The acute inhibition of activated sludge caused by MPs was influenced by surface charge of them. Perhaps owing to the positive charge on the surface of polyethylene particles, polyethylene MPs have high affinity for the 15

activated sludge with negative charge [63]. Nanoplastics can cause cell damage (Fig. 6e), ROS production, pore or channel blockage and DNA damage [64]. Changes in functional properties may lead to latent effects on carbon and nitrogen cycling in biofilms [52].The leaching solution of microplastics can also affect nitrification. BPA can be released from PVC microplastics, which can inhibit the activity of heterotrophic bacteria and nitrifying bacteria [65; 66]. We summarized the latest results of the effect of microplastics on wastewater treatment (Table 3).

4. Effects of microplastics on sewage sludge treatment 4.1 Effects of microplastics on sludge digestion Several studies showed the majority of microplastics in raw sewage were retained in sewage sludge with the retention rate as high as 99% [67; 68]. According to the detection, the concentrations of MPs detected in sludge ranged from 1.5×103-2.4×104MPs/kg [16; 31; 69]. The mean microplastic concentration in plate-frame process was lower than that in centrifugal process, followed by filtration pressure and belt process [70]. Moreover, the operating load rate of WWTPs also affects the concentrations of microplastics in sewage [71]. Overload operation of wastewater treatment plants reduces hydraulic retention time and enhances the flow rate of wastewater, thus shortening the time of biological pollution and microbial degradation during grease removal process, which leads to an increase in the concentration of microplastics [72]. There are many types and colors of microplastics in WWTPs (Fig. 4.), among which fiber and white microplastics are the main ones. Anaerobic digestion is one of the most commonly used methods of sludge stabilization [73]. The first step of anaerobic digestion is to convert the particulate organic matters into soluble substrates. The higher the content of PVC MPs is, the greater the release of SCOD is [65]. This may be due to lipids and nucleic acids discharged from WAS, which indicates that the dissolution of WAS in anaerobic digestion enhances when exposed to PVC MPs. PE MPs do not affect the dissolution of organic matter involved in digestion, either because dissolution is an abiotic process or because PE MPs do not affect microorganisms related to dissolution. The presence of microplastics in WAS adversely affects the hydrolysis of proteins and polysaccharides, which reduces the availability of acidified

16

substrates and thus produces less gas [10; 74]. Microplastics can also reduce the degradation rate of butyrate [9]. Unlike anaerobic digestion systems without nanoplastics, methane production and maximum daily production declined in the presence of nanoplastics [75]. Methane production is the step most being easily inhibited by MPs in the four digestive processes [10]. The start-up time of the mixed anaerobic digestion system was dragged on when exposed to nanoplastics [75]. It was found that the high concentration of PE microplastics (i.e. 100 and 200 particles / g-TS) resulted in a 12.4-27.5% reduction in methane production, with a lower methane potential and hydrolysis coefficient [10]. The cumulative methane production was reduced by 27.5±0.1% in the presence of microplastics （ 200 particles/g-TS ） [10]. Low levels of PVC MPs can enhance methane production in WAS, whereas higher levels of PVC MPs can restrain methane production and WAS hydrolysis [65]. The impacts of methane production by cationic PS nanoplastics (PS-NH2) were greater than that by anionic PS nanoplastics (PS-SO3H), while there was no significant relationship between the concentrations of VFA and the exposure of PS nanoparticles [76]. AGS is a complicated microbial aggregate, which is more resistant to heterobiotic contaminants, due to EPS protection around AGS [77]. When exposed to PS nanoplastics for a short time, the gaseous products of AGS were temporarily inhibited, while VFA production was not affected, which was attributed to methanogens being more sensitive to chemical toxicity than acidogens [78]. VFA is an intermediate product, which is influenced by producers and consumers. The tendency to inhibit or promote VFA may be more complex than the trend of the final product methane. 4.2 Effects of microplastics on microbial community diversity and key microbial abundances The structure and metabolism of microbial communities on the surface of MPs are distinguished from those in the surrounding [79]. Exposure to MPs may influence non-target species through alterations in their microflora, resulting in alterations in isotope and element incorporation, growth and reproduction [80]. MPs biofilms are also observably less diverse than downstream water column and suspended organic matter bacterial combinations [7]. The structure of microbial community is affected by nanoplasticity in the system (Fig. 5a). In a pure anaerobic digestive system, nanoplastics inhibit the growth and metabolism of Acetobacteroides in part. SEM images showed 17

that the membrane of Acetobacteroides hydrogenigenes was colonized by many nanoplastics (Fig. 5b) [75]. Moreover, there were lots of nano-sized holes caused by the diapirism of nanoplastics on the cell membrane of Acetobacterteroides hydrogenigenes (Fig. 6d) [75]. MPs attached to cell membranes will lead to pitting of cell walls. More seriously, accumulation of MPs in cells will result in cell dysfunction. Chloroflexi, Bacteroidetes, Actinobacteria, Proteobacteria and Firmicutes are dominant bacteria for sludge digestion [81]. The abundance of Firmicutes, Actinobacteria, Chloroflexi, Proteobacteria and Bacteroidetes decreased in the presence of microplastics (Fig. 5c) [9; 75]. Microplastics reduce the microbial community sequence [10]. Microplastics reduced the relative abundance of Rhodobacter sp., which was proved to be related to hydrolysis [82]. In addition, the relative abundance of Proteiniclasticum sp. was also reduced, which can use proteins to produce acetic acid [83]. MPs can also reduce the proportion of Euryarchaeota, which are known as Methanogens [10]. Nevertheless, the relative abundances of Methanothrix, which decarboxylated acetate to CH4 and CO2, declined from 34.20% to 30.60% and 29.90% respectively when exposed to PS-SO3H and PS-NH2 [76] . Methanomassiliicoccus, a hydrogen-dependent methanogen, associated with the abundances of Candidatus Cloacamonas, declined with exposure to PS nanoplastics [84]. Bacteria's response to nanoplastics may vary depending on the structure of their cell membranes. There is an inhomogeneous response of microbial communities to the same nanoplastics. When the concentration of nanoplastics was 0.2 g/L, the relative abundances of family Cloacamononae, Porphyromonadaceae, Anaerolinaceae and Gracilibacteraceae showed a downward trend, while those of family Clostridiaceae, Geobacteraceae, Dethiosulfovibrionaceae and Desulfobulbaceae showed an increasing trend [75]. Candidatus Cloacamonas, correlated with the oxidation of syntrophic propionate are considered to be syntroph that may produce hydrogen [85; 86]. As direct producers or syntrophs, Syntrophobacter, Saccharofermentans, Atribacteria-genera-incertae-sedis, Treponema, Lactivibrio, Smithella and Paludibacter use carbohydrates and sugar to produce acetate [87; 88]. Methanothrix, Methanobacterium, Methanolinea and Methanomassiliicoccus are dominant archaea. Candidatus Cloacamonas, Saccharofermentans, Atribacteria-genera-incertae-sedis, Treponema, Lactivibrio, Smithella and 18

Paludibacter declined when exposed to PS nanoplastics, while the presence of PS nanoplastics had no conspicuous effect on archaea [76] .That is to say, PS nanoplastics can simultaneously restrain the formation and consumption of acetate. 4.3 Effects of microplastics on key enzymes and metabolic intermediates McrA and ACAS are two main functional genes of methane production. It was found that nanoparticles restrained anaerobic digestion primarily by inhibiting metabolic intermediates and key enzymes during fermentation process [89]. With the increasing concentrations of PS nanoplastics, their inhibitory effects on mcrA gene were enhanced, indicating that nanoplastics have a negative impact on methanogenic archaea [76]. Compared with other microorganisms in anaerobic digestive system, methanogens are more sensitive to PS nanoplastics [78]. With the increase of PVC concentrations, the relative activity of key enzymes in anaerobic digestion was decreased [65]. Microplastics and the contaminants they absorb or the additives they release may affect the activity of enzymes. The exposure of MPs has been shown to restrain various enzymes and metabolic pathways in invertebrates and vertebrates [3]. 4.4 Mechanisms of microplastics affecting microbial community and enzyme activity EPS are composed of macromolecule polymers secreted by microorganisms, macromolecule polymers produced by cell lysis and organic compounds adsorbed from wastewater. EPS combine with cells through complicated interactions to form a huge network structure containing a large amount of water to protect cells from dehydration and toxic substances [90]. EPS in microbial aggregates have lots of sites for adsorbing metals and organic matter, such as aromatic compounds, fatty compounds in proteins and hydrophobic regions in carbohydrates [77]. Thus an increase in carbohydrate content indicates an increased ability of the microorganism to adhere to the MPs. Functional groups in the EPS, such as carboxyl, phosphoric acid, sulfhydryl, phenol and hydroxyl, can be complexed with other substances [91; 92]. The functional groups related to the interaction between MPs and EPS are carbonyl and amide groups as well as side chains of lipids or amino acids [61]. Microplastics can affect certain gene 19

expression and cellular structure in cells. For example, the contact with plastic particles changed the gene expression of some sugars used in extracellular polysaccharides [93]. In addition, PS nanoplastics can alter the protein secondary structure in EPS (Fig. 2c) [61]. High concentrations of PVC MPs led to the accumulation of soluble proteins [65]. When nanoplastics enter WWTPs, it is expected that nanoplastics will interact with EPS. Research has shown that contaminants mainly react with proteins in EPS during microbial aggregation [94]. Proteins in EPS are the main compounds involved in PS nanoplastics interactions. Bacteria secrete more proteins to neutralize contaminants for protecting cells from toxic chemicals [95]. Functionalized PS nanoplastics can infiltrate EPS matrix into anaerobic granular sludge with a certain concentration [76]. EPS are always negatively charged and can be combined with positively charged contaminants through electrostatic interactions [96]. The adhesion of microplastics to EPS is mainly affected by their surface charge [97]. Microplastics destroy the integrity of the cell wall and cell membrane. Biofilm controls plenty of cellular functions by adjusting the activity of membrane proteins. Membrane properties like thickness, elasticity or transverse heterogeneity determine the separation and function of proteins [98]. MPs may penetrate into cells through endocytosis [61]. As the particle size decreases to the nanometer scale, it is gradually close to the thickness of the biofilm (about 4nm). In case of the polystyrene nanoplastics were in contact with lipids, the nanoplastics penetrate into the membrane within a few microseconds, followed by dissolution of the polystyrene nanoplastics rapidly (Fig. 6b) [99]. Soluble in the core of the membranes, PS nanoplastics change the structure of the membranes [99]. Besides, it observably reduces molecular diffusion and softens the membrane [99]. Alterations in membrane properties and lateral tissue have seriously effects on the activity of membrane proteins, accordingly affecting cell function. Oxidative stress is a mechanism for explaining the toxicity of these emerging contaminants at the cellular level [100]. The production of ROS in cells may be responsible for the toxicity of microplastics (Fig. 6a) [8]. The increase of ROS content in cells will cause lipid peroxidation (Fig. 6g), damaging cell membrane structure and biological 20

macromolecules such as proteins. In addition, it also causes the collapse of cell membrane skeleton, cell distortion, cell membrane permeability, affecting the normal growth and metabolic processes, such as the exchange of energy inside and outside the cell [8]. It also leads to a drop in the efficiency of electron transfer. Typically, most cells can neutralize a certain amount of ROS by enzymatic reactions. The activity of enzymes like superoxide dismutase and catalase in the cells is enhanced to remove ROS. However, this process is limited because of the limited number of bacterial enzyme molecules. When the ROS concentration is too high and exceeds the cell's own repair ability, the cells die. Adsorption between microplastics and cells is also a major cause of toxicity. Microplastics may block the passage of substances into and out of cells, hindering the exchange of matter and energy inside and outside the cell. ROS, including hydrogen peroxide (H2O2), superoxide (O2-) and hydroxyl radical (OH-), can give rise to toxic oxidative stress in cells or organisms, resulting in loss of cell viability [101; 102]. ROS can trigger redox-sensitive signaling pathways (e.g., the MAP kinases and NF-kB cascades) [103]. Oxygen reacts with reduced groups on MPs to produce O2- and H2O2 by disproportionation or Fenton chemistry [102]. Even under anaerobic conditions, ROS can be produced by oxygen at sub-micromolar concentrations retained in the medium [102]. Besides, the interaction of formed H2O2 and reduced iron rich in mitochondria will induce extra ROS (OH·) [104]. Research has shown that with the increase of microplastics, ROS production in cells was observed to increase, with a maximum increase of 23.3 (±1.9%) [10]. Microplastics can also increase the proportion of dead cells. When the concentration of microplastics is 60 particles/g-TS, the proportion of dead cells is as high as 28.9±1.9%. The higher the concentration is, the larger the proportion is [10]. The oxidative stress of positively charged nanoplastics (50 nm modified by amine) is higher than that of negatively charged nanoplastics (55 nm unmodified) [8]. Positively charged MPs bind cells tightly because of electrostatic attraction, while other negatively charged MPs bind cells loosely through van der Waals force, acid-base interaction together with electrostatic force [105]. In other words, positively charged microplastics are highly toxic to cells due to electrostatic attraction, while negatively charged microplastics are mainly aggregated (Fig. 2a). Although EPS play a protective role, nanoplastic-induced intracellular ROS levels may exceed the protective barrier of EPS 21

and subsequently lead to bacterial damage [8]. When MPs reach nanoscale, they may pass through EPS and interact with bacterial membranes [75]. Stress on the cytoplasmic membrane affects bacterial metabolism [8]. Alpha diversity (richness, evenness and diversity) of bacterial communities on microplastics was observably lower than that on natural substrates [52]. Species sorting takes place during the development of microplastic biofilm giving rise to the decline of resistance of microplastic-related biofilm communities to disturbances [106]. Research has reported alterations in oxidative status, regulations of nerve and energy-related enzymes and intestinal damage in aquatic organisms when exposed to microplastics [107]. At the beginning of exposure, bacterial cell growth may be promoted owing to hormetic response, which is because of cell defense as well as adaptive response to stress [108]. It has been reported that the toxic excitation effect could produce biphasic dose response through low-dose stimulation and high-dose inhibition [109]. At 320 mg/L, all the MPs observably restrained cell growth [8]. Plastics also absorb or include metal ions like iron, copper and silver [110]. They participate in molecular transportation and cellular signaling pathways at low concentrations, thus regulating the fundamental cellular processes in all organisms. Nevertheless, excessive ions of these ions might cause adverse effects, like changes in enzyme activity or oxidative stress, leading to the production of free radicals, DNA damage and membrane lipid peroxidation [110]. Other metal ions, like silver and aluminum, may also give rise to apoptosis for they can directly interact with DNA. Hence, the degradation of aging plastics and the release of toxic substances are a major threat to the environment. Potential effects may arise indirectly from the leaching of polymers from plastics-related chemicals. It is well known that a large number of flexible PVC products like pipes, synthetic leather or plastic floor coverings contain xenoestrogens like phthalates and BPA as plasticizers [111]. In addition, certain metalloestrogens, like aluminium as well as copper, were added to the manufacturing processes as catalysts, biocides, UV stabilizers and heat stabilizers [111]. Additives in plastics provide antimicrobial degradability, which improves the properties of plastics and prolongs the life of plastics, enabling them to exist in sludge for a long time. Chemical additives have relatively low molecular weight and may migrate from MPs, resulting in microbial toxicity of MPs. MPs are prone to leach out 22

potentially toxic substances [112]. It has been reported that additives leach continuously from the MPs within a few months, and the leaching rate doesn't decrease observably over time [112]. Although some additives may be absorbed by other particles in activated sludge during leaching process, over time, more additives will be leached due to the increase of microplastics concentration. The amount of additives leached from sludge gradually increased. Bejgarn et al. proved that over one-third of MPs leaching chemical additives had adverse impacts on Daphnia magna [113]. Studies have shown that additives such as DBP and BPA can inhibit sludge digestion [9; 65]. In other words, leachates are one of the reasons for the negative impacts of MPs on sludge treatment. MPs have a huge specific surface area, which increases their potential contact area with microbial cells. The catalytic reaction of free radicals and molecular dioxygen can take place on their surface to produce ROS. In the presence of oxygen or anaerobic conditions, MPs can induce cytotoxicity by producing ROS [101; 104]. ROS level was positively correlated with microplastics concentrations and negatively correlated with particle sizes [101]. The main factors affecting sludge treatment may be the release of additives and ROS alone or their combined effects. If the toxicity of additives is high, the release of additives is the dominant factor. If the particle size of microplastics is small, and then a higher level of ROS is produced, ROS may be the dominant factor. However, it does not exclude the possibility that other conditions may become potential inhibitors, such as polymer materials, tips that cause damage to organisms or exudation of toxic substances adsorbed by MPs. We summarized the latest results of the effect of microplastics on sludge treatment (Table 3).

23

Table 3. Summary of the effect of microplastics on wastewater and sludge treatment. Types of MPs

Concentrations of MPs

Size of MPs

Affected objects

Degree of effect

References

NH2 modified PS

80mg/L

50nm

Cell viability

-13.8%

[8]

NH2 modified PS

160mg/L

50nm

Cell viability

-24.5%

[8]

PS

160mg/L

55nm

Cell viability

-25.0%

[8]

NH2 modified PS

320mg/L

50nm

Cell viability

-34.0%

[8]

PS

320 mg / L

55nm

Cell viability

-32.7%

[8]

NH2 modified PS

320 mg / L

1μm

Cell viability

-16.4%

[8]

PS

80mg/L

55nm

NH4+-N conversion efficiency

Interrupt

[8]

PE

0.045 particles/g (WW)

0.033-5mm

Ammonium concentration

+45.3%(around)

[34]

PE，PP and PS

Na

0.1-5mm

BOD

Positive correlation

[40]

PE，PP and PS

Na

0.1-5mm

TN

Positive correlation

[40]

PE，PP and PS

Na

0.1-5mm

TP

Positive correlation

[40]

Weathered PVC

Na

<0.3mm

BOD

+164.0%(around)

[41]

Various types

17.93particles/m3

<5mm

OTUs of Nitrospira

-73.3%

[7]

Various types

17.93particles/m3

<5mm

OTUs of Hydrogenophaga

-94.0%

[7]

Various types

17.93particles/m3

<5mm

OTUs of Thauera

-87.6%

[7]

Various types

17.93particles/m3

<5mm

OTUs of Zoogloea

-65.7%

[7]

Various types

Na

<5mm

Inhibition rate on nitrifying bacteria

LC50=13.39 at the lowest

[58]

Table 3. Summary of the effect of microplastics on wastewater and sludge treatment. -Continued

24

Types of MPs

Concentrations of MPs

Size of MPs

Affected objects

Degree of effect

References

PE、PET and PVC

Na

<2mm

Nitrogenase reductase (nifH) marker gene

-99.0%

[60]

PE、PET and PVC

Na

<2mm

Ammonium monooxygenase (amoA) marker

-76.0%

[60]

gene PS

0.2g/L

54.8nm

Methane production rate

-14.4%

[75]

PS

0.2g/L

54.8nm

Maximum daily methane production rate

-40.7%

[75]

PS

0.2g/L

54.8nm

The growth of Acetobacteroides hydrogenes

Inhibited for 35 days

[75]

PS

0.1mg / mL

28.2nm-91.3nm

The average OURen

-13.1%

[61]

PS

5mg/mL

28.2nm-91.3nm

The average OURen

Declined significantly (P <0.001)

[61]

PS

13%PS100(consists of

7 nm in

The diffusion coefficient of lipid DL

-69.4%

[99]

100 styrene monomers)

diameter

13%PS100(consists of

7 nm in

The diffusion coefficient of short helix

-85.8%

[99]

100 styrene monomers)

diameter

transmembrane peptide DP

PS-NH2

20μg/ml

<5mm

Methane production

-23.0%

[76]

PS-NH2

20μg/ml

<5mm

The total relative abundances of related species

-35.6%

[76]

PS-SO3H

100μg/ml

<5mm

Methane production

-17.5%

[76]

PS-SO3H

100μg/ml

<5mm

The total relative abundances of related species

-29.5%

[76]

PET

60 particles/g-TS

<5mm

Maximum hydrogen production

-29.3±0.1%

[9]

PET

60 particles/g-TS

<5mm

Protein degradations

-20.3±0.1%(around)

[9]

PS

Table 3. Summary of the effect of microplastics on wastewater and sludge treatment. -Continued 25

Types of MPs

Concentrations of MPs

Size of MPs

Affected objects

Degree of effect

References

PET

60 particles/g-TS

<5mm

Polysaccharides degradations

-13.2±0.1%(around)

[9]

PET

60 particles/g-TS

<5mm

Amino acid degradations

-17.8%

[9]

PET

60 particles/g-TS

<5mm

Monosaccharide degradations

-11.6±2.1%

[9]

PET

60 particles/g-TS

<5mm

The butyrate degradations

-17.3%

[9]

PET

60 particles/g-TS

<5mm

Abundance of Firmicutes

-12.2±0.1%

[9]

PET

60 particles/g-TS

<5mm

Abundance of Actinobacteria

-20.5±0.1%

[9]

PET

60 particles/g-TS

<5mm

Abundance of Bacteroidetes

-29.6±0.2%

[9]

PET

60 particles/g-TS

<5mm

Abundance of Bacteroides sp.

-23.8±0.1%

[9]

PE

100 particles/g-TS

40±2μm

Methane production

-12.4%

[10]

PE

200 particles/g-TS

40±2μm

Methane production

-27.5±0.1%

[10]

PE

200 particles/g-TS

40±2μm

VS destruction

-27.3±0.1%

[10]

PE

200 particles/g-TS

40±2μm

Digested sludge volume

+9.1%

[10]

PE

200 particles/g-TS

40±2μm

The methane potential (B0)

-24.1%

[10]

PE

200 particles/g-TS

40±2μm

The hydrolysis coefficient of WAS (k)

-15.2%

[10]

PE

200 particles/g-TS

40±2μm

Microbial community sequence

-13.2%

[10]

PE

200 particles/g-TS

40±2μm

Relative abundance of Proteobacteria

-12.2%

[10]

PE

200 particles/g-TS

40±2μm

Relative abundance of Rhodobacter sp.

-15.2±0.1%

[10]

PE

200 particles/g-TS

40±2μm

Relative abundance of Proteiniclasticum sp.

-24.1±0.3%

[10]

26

Table 3. Summary of the effect of microplastics on wastewater and sludge treatment. -Continued Types of MPs

Concentrations of MPs

Size of MPs

Affected objects

Degree of effect

References

PE

200 particles/g-TS

40±2μm

Relative abundance of Proteiniborus sp.

-28.2±0.2%

[10]

PE

200 particles/g-TS

40±2μm

Relative abundance of Fonticella sp.

-19.4±0.1%

[10]

PE

200 particles/g-TS

40±2μm

Soluble protein degradation rate

-16.1%

[10]

PE

200 particles/g-TS

40±2μm

Carbohydrate degradation rate

-8.1%

[10]

PE

200 particles/g-TS

40±2μm

Amino acids degradation rate

-9.4%

[10]

PE

200 particles/g-TS

40±2μm

Monosaccharide degradation rate

-11.2%

[10]

PE

100 particles/g-TS

40±2μm

The cumulative methane production

-10.9±0.5%

[10]

PE

200 particles/g-TS

40±2μm

The cumulative methane production

-22.7±0.4%

[10]

PE

200 particles/g-TS

40±2μm

Cell viability

-15.4%

[10]

PVC

20 particles/g

1mm

Methane production

-9.4%

[65]

PVC

40 particles/g

1mm

Methane production

-19.5%

[65]

PVC

60 particles/g

1mm

Methane production

-24.2%

[65]

PVC

60 particles/g

1mm

The methane potential (B0)

-24.7%

[65]

PVC

60 particles/g

1mm

The hydrolysis coefficient of WAS (k)

-18.1%

[65]

PVC

60 particles/g

1mm

Apparent VS destruction (Y0)

-28.7%

[65]

PVC

60 particles/g

1mm

Soluble protein

Accumulation +14.7%

[65]

PVC

60 particles/g

1mm

VFA

16.8±0.2% of the control

[65]

PVC

60 particles/g

1mm

Protease

-12.9%

[65]

27

Table 3. Summary of the effect of microplastics on wastewater and sludge treatment. -Continued Types of MPs

Concentrations of MPs

Size of MPs

Affected objects

Degree of effect

References

PVC

60 particles/g

1mm

AK

-12.8%

[65]

PVC

60 particles/g

1mm

F420

-20.7%

[65]

PVC

60 particles/g

1mm

Abundance of Proteobacteria

-10.8±0.1%

[65]

PVC

60 particles/g

1mm

Abundance of Chloroflexi

-4.3±0.1%

[65]

PVC

60 particles/g

1mm

Abundance of Firmicutes

-10.2±0.1%

[65]

PVC

60 particles/g

1mm

Abundance of Euryarchaeota

-14.7%

[65]

PVC

60 particles/g

1mm

Abundance of Rhodobacter sp.

-13.1%

[65]

PVC

60 particles/g

1mm

Abundance of Proteiniborus sp.

-25.0%

[65]

PVC

60 particles/g

1mm

Abundance of Garciella sp.

-42.1%

[65]

PVC

60 particles/g

1mm

Abundance of Methanosaeta sp.

-16.5%

[65]

28

5. Enhanced removal of microplastics from wastewater and sewage sludge At present, only greatly expensive or inefficient methods are used for removing MPs and trace contaminants, in which either too ineffective and inflexible (flocculation and precipitation) or chemical reagents are bound to add to sewage (depth filtration, microfiltration and UF), which have been proved to be disadvantage to ecosystems, effective secondary recovery or material recovery. In related studies, MBR technology is more effective than CAS process in removing microplastics from wastewater [114]. Many studies have shown that filtration plays an important role in microplastics removal. However, this method has its drawbacks because the mechanical stress generated by the press cloth may cause the MPs to wear away, allowing them to be released into the environment without restriction. Another reason is that small particles take longer to filter and are prone to clogging, resulting in more time and maintenance costs that are passed on to consumers. Some research has demonstrated that BAF have no impact on the reduction of microplastics concentrations [43]. The removal rate of microplastics in the secondary treatment was approximately between 64% and 99.56% [115; 116]. Christian Baresel et al. found that a combination of MBR, UF and granular activated carbon removed microplastics to concentrations below the detection limit [117]. We summarized the efficiency of different methods for removing microplastics (Table 4). 5.1 Sol-gel method It is a new method of removing polymers from wastewater by sol-gel induced agglomeration to make up huge particle agglomerates. Owing to it is cost-saving and chemically stable, SAS is very fit for using as catalyst, carrier and adsorbent. Studies have been conducted to test SAS as an adsorbent for organic and inorganic contaminants in wastewater treatment [118]. Silane is formed by hydrolysis and condensation to make up macromolecular network during sol-gel process. N-alkyl substituted chlorosilanes are employed since they are highly reactive when they get in touch with water [119]. Because of the low stability of silanols and the catalytic effect of hydrochloric acid released during hydrolysis, silanols are condensed and merged to make up siloxane bonds, which avoids the release of ecotoxicological siloxane into the surroundings [120]. Sol-gel process can be carried out under alkaline and acidic catalytic circumstances [120]. Ground on 29

pH-induced sol-gel process, MPs flocculation can be promoted. Subsequently, huge microplastic flocculates can be easier to be isolated from wastewater. Flocculates extracts can be heat recovered and sustainable recycled which embodies environmental friendliness [120]. The aggregates can be isolated from wastewater with ease using separation systems, like sand traps since they are floating. Another advantage is that agglomeration can be completely independent of the types, sizes and amount of trace contaminants and external effects (pH, temperature, pressure). Sustainable removal of contaminants from aquatic environment is on the basis of the construction of polymer inclusion compounds [121]. The Si-OR bond of alkoxy compounds is hydrolyzed and split [122]. In the subsequent condensation of Si-OH groups, siloxane bonds and three-dimensional solid skeleton are formed as the reaction proceeds [122]. By bio-induced alkoxy-silyl, functionalized molecules act as adhesives between microplastics [122]. This can result in MPs to condense and subsequently isolate them from the wastewater. 5.2 Electrocoagulation Electrochemical technologies, like EC, electrode cantation and electroflotation, provide less expensive solutions that doesn't depend on chemical reagents or microorganisms [44]. EC can produce coagulants electrically with metal electrodes, which is easy and realizable [123]. Hence, EC is an effective solution to help sewage get rid of MPs. EC is compatible, cost effective, energy efficient, sludge minimized and highly automated [124]. The most common coagulants for EC are metal ions (generally Fe2+ or Al3+) which react with OH to form metal hydroxides. Metal hydroxide coagulants are produced by ions formed by electrolysis [44]. Coagulants form a sludge layer, which arrests suspended particles and discharges H2 gas during electrolysis, and subsequently elevates the sludge to the surface of wastewater [125]. It was found that when the pH values are in the range of 3 to 10, the effective removal rate of microplastics by EC exceeded 90% [44]. At present, EC has been proved to be effective in removing dyes, heavy metals and clay particles, of which more than 80% of the contaminated particles were removed after treatment [126; 127]. Efficient removal of certain liquid organic compounds has also been confirmed [123]. Compared with Fe-based salts, Al-based salts have higher PE removal efficiency [128]. Besides, water conditions such as ionic strength and turbidity have little effect on the removal rate [129]. 30

The addition of PAM, particularly anionic PAM, plays a significant role in removing PE since it produces positively charged Al-based flocculants under neutral circumstances [129]. PAM with outstanding performance is often used to promote condensation in WWTPs. Some research proved that pH has important implications on the properties of Al-based flocculants [130]. Compared with pH, PAM, particularly PAM oppositely charged against Al-based flocculants, has significant implications on removing MPs due to the promotion of coagulation. Hence, this kind of innovative separation technology which induces particle growth and promotes separation is worth popularizing. 5.3 Dynamic membranes DMs attract much attention recently for they can reduce energy consumption and cost in wastewater treatment [131]. DMs are a newly formed cake layer on the supporting membrane, which is the second layer formed on the surface of the supporting membrane by filtering particles and other dirt in wastewater. DMs are promising technologies for removing low-density, non-degradable particles, like plastics [131]. Since it uses existing pollutants in water to form a filter layer without introducing additional chemicals or other pollutants, DM technology has been widely used in municipal wastewater treatment, petroleum-contaminated wastewater treatment and surface water treatment [132; 133; 134]. 5.4 Other potential approaches An effective method is to improve the removal of MPs with low density during grease removal and to enhance the settlement of MPs with high density during the removal of sand grains in primary clarifiers such as sand sinks [135]. Next, grease and grit need to be treated to prevent MPs removed at this stage from entering the subsequent treatment system. After aeration, the residual MPs in the primary effluent are easily absorbed by sludge flocs. Flocculants (such as ferric sulfate and aluminium sulfate) can be added to the primary sedimentation tank to help the accumulation of MPs [72]. Because of the hydrophobicity of microplastics, hydrophobic magnetic materials can be used to adsorb microplastics and then collect them for removal [136].

31

For membrane treatment, surface modification techniques such as dipping, blending of additives by UV radiation or plasma polymerization and surface grafting can solve the problem of film fouling. This can avoid film fouling caused by microplastics [137]. Then the material of the film should be changed according to the characteristics of microplastics (hydrophobicity, negatively charged surface) such as using hydrophilic, negatively charged and low roughness film. After that, the skimming should be treated separately. Combining these three processes may achieve the purpose of removing microplastics. For coagulation, the coagulant with positive charge after hydrolysis can be selected according to the characteristics of negative charge on the surface of microplastics. For air flotation, the air flotation parameters such as bubble size can be adjusted according to the combination characteristics of microplastics and pollutants. Anaerobic treatment system plays a significant role in the treatment of wastewater containing nanoplastics, like textile dyeing wastewater [76]. Therefore, anaerobic treatment may be a solution. Based on a preliminary study, plastics could be decomposed into biogas in anaerobic digestion tanks [138]. AD, thermal drying and lime stabilization were carried out on sludge samples containing microplastics. The results indicated that the abundances of MPs in AD samples were low, indicating that AD might reduce the abundances of microplastics [31]. However, it is not certain whether anaerobic digesters can cause the breakdown of microplastics. In order to prevent MPs from affecting sludge digestion, they can be removed by pyrolysis technologies such as thermal pyrolysis, microwave-assisted pyrolysis and catalytic pyrolysis in the pretreatment stage [139].

32

Table 4. Removal efficiency of different microplastic removal methods. Microplastic concentrations after treatment 0.435 MPs/L

Removal efficiency

References

A2O

Microplastic concentrations before treatment 29.85MPs/L

98% or higher

[140]

SBR

16.45 MPs/L

0.14 MPs/L

98% or higher

[140]

Media process

13.865 MP/sL

0.28 MPs/L

98% or higher

[140]

Aeration tank

Seyhan:26,555±3175

Seyhan:6999±764

Seyhan:73%；Yüreğir:79%

[141]

MPs/m3;Yüreğir:23,444±4100 MPs/m3

MPs/m3;Yüreğir:4111±318 MPs/m3

UF

0.48 MPs/L

0.28 MPs/L

41.6%

[17]

Reverse osmosis

0.28 MPs/L

0.21 MPs/L

25%

[17]

Tertiary Postfiltration

MP>500μm:5×101/m3;MP

MP>500μm:none;MP <500μm：

MPs>500μm:completely

[16]

unit

<500μm:2×102/m3;Synthetic

1×101/m3;Synthetic fibers:×101/m3

removed;MPs

Processes / Methods

fibers:9×102/m3

<500μm:93%;Synthetic fibers:98%

MBR

6.9 MPs/L

0.005 MPs/L

99.9％

[116]

Rapid sand filter

0.7 MPs/L

0.02 MPs/L

97％

[116]

Dissolved air flotation

2.0 MPs/L

0.1 MPs/L

95％

[116]

Discfilter

0.5-2.0 MPs/L

0.03-0.3 MPs/L

40-98.5％

[116]

Anaerobic membrane reactor

83 SAL/L

0.5 SAL/L

99.4％

[142]

Activated sludge process

134 SAL/L

5.9 SAL/L

95.6％

[142]

Granular sand filtration

92.8 SAL/L

2.6 SAL/L

97.2％

[142]

33

Table 4. Removal efficiency of different microplastic removal methods. -Continued Processes / Methods DMs

Aluminum electrode

Microplastic concentrations before treatment The turbidity of the influent is about 195

Microplastic concentrations after treatment After 20 minutes of filtration, the

NTU

effluent turbidity <1NTU

0.1 g/L(300-355μm)

0.01-7.6×10-4 g/L

electrocoagulation

Removal efficiency

References

99.5%

[131]

More than 90%, the best removal

[44]

efficiency was found to be 99.24% at pH 7.5

Biological treatment + settler

2.5±0.3 MPs/L

0.9±0.3 MPs/L

64%

[115]

Filter and disinfection

0.9±0.3 MPs/L

0.4±0.1 MPs/L

55.6%

[115]

Sand filter

15.70(±5.23) MPs/L

8.70(±1.56) MPs/L

Maximum reduction of 44.59%

[72]

Primary sedimentation tank

8.70(±1.56) MPs/L

3.40(±0.28) MPs/L

33.75%

[72]

Aeration tank + clarifier

3.40(±0.28) MPs/L

0.25(±0.04) MPs/L

20.07%

[72]

CAS Process

57.6(±12.4) MPs/L

1.0(±0.4) MPs/L

98.3%

[114]

Aerated grit chamber + primary

79.9 MPs/L

Na

40.7%

[6]

A2O

Na

Na

16.6%

[6]

Chlorination disfection

Na

28.4 MPs/L

7.7%

[6]

Anaerobic digestion

Na

Na

A lower abundances of MPs indicates

[31]

settling tank

that the process may reduce MPs abundances

34

Table 4. Removal efficiency of different microplastic removal methods. -Continued Microplastic concentrations after treatment 0.7(±0.6)-3.5(±1.3) MPs/L

Removal efficiency

References

BAF

Microplastic concentrations before treatment 1.0(±0.6)-2.0(±0.2) MPs/L

No removal effect

[43]

Oxidation ditch

5.6±0.09 mg / L

0.168±0.02 mg / L

97%

[143]

Processes / Methods

35

6. Conclusions and outlook We comprehensive reviewed the effects of microplastics on wastewater as well as sludge treatment and their enhanced removal methods. Main conclusions are as follows: (1) At present, there are many studies on the purification and detection methods of microplastics. Handling with chemicals may alter the properties of microplastics and possibly even complete dissolve the sample, leading to misidentification. Different methods and the lack of a standard system make it difficult to compare the results of various studies. (2) Studies have shown that the WWTPs can remove some of the microplastics. The MPs in WWTPs are mainly composed of polyester and polyethylene. The main morphology is granular and fibrous. The removal rate of granule in wastewater treatment processes is higher than that of fiber. Population density, economic level, urban greening areas, wastewater treatment process parameters, sludge dehydration and treatment processes can affect the concentrations and behavior of microplastics in various stages of WWTPs. (3) It has been proved that the increase of microplastics levels will have a negative impact on wastewater and sludge treatment. Microplastics have acute inhibitory effects on activated sludge flocs. Microplastics can inhibit the production of methane in sludge and affect its key enzymes and metabolic intermediates. In addition, microplastics reduce the diversity of biological communities and the abundances of key microorganisms. The adsorption of environmental micropollutants and the exudation of additives make the influence mechanism of microplastics on wastewater and sludge treatment more complicated. While this review highlighted some valuable results, there are many issues that need to be addressed in the future. (1) At present, the identification and detection methods of microplastics are incomplete. No standard system has been formed and all kinds of purification detection methods are defective. In addition, there is a lack of identification and detection methods for smaller sizes of microplastics such as nanoplastics. In terms of current technology, the recovery rate is low and the cost is high. In the future, it is necessary to propose purification and detection methods 36

for microplastics with high recovery rate, low cost, time and labor saving and simple operation. (2) Microplastics reduce the efficiency of wastewater and sludge treatment processes and increase sludge volume. If a large number of MPs enter the sludge digestion system, it will need longer sludge retention time or larger digestion tank to achieve the same treatment effect as the non-microplastic system, which will increase the cost of treatment. Therefore, internal modifications of wastewater and sludge treatment processes should be made to protect the water treatment processes. Sewage treatment plants should avoid overload operation. Hydraulic retention time can be increased to ensure the removal of microplastics. In the future, specific MPs targeted treatment units can be designed and applied to full-scale WWTPs to avoid the harm of microplastics to wastewater and sludge treatment systems. Moreover, current WWTPs processes are not specifically designed to remove microplastics and are ineffective in removing fibers. In the future, it is necessary to develop simple, low-cost and high removal rate methods. (3) The research on the effects of microplastics on the process performance of wastewater treatment and the effects of microplastics on wastewater quality is still scarce, and the mechanism is still not clear enough. If the concentrations of toxic substances adsorbed by microplastics are high enough and easy to release, it will probably affect the treatment of wastewater and sludge. Besides, the effects of MP shape (as well as size) on the performance of different treatment processes should be researched in the future. Understanding the influence mechanism of different size microplastics on the performance of wastewater treatment processes can avoid hazards. (4) Solutions to microplastics should focus on source control, repair and cleaning. Reducing or even removing plastic beads from personal care products, reducing the use of garbage bags, advocating green packaging, eliminating excessive packaging, and gradually replacing plastic products with biodegradable materials should be put on the agenda. At the same time, sludge may be the second largest source of MPs pollution. MPs are stable in digested sludge, which may also affect subsequent treatment and disposal. Particularly, European community and American legislation permits land use of sludge [144]. Therefore, a great number of MPs may be released into terrestrial and aquatic ecosystems, in which MPs can cause potential biological toxicity. In the future, consideration 37

should be given to preventing potential secondary MPs pollution, such as pretreatment before sludge digestion through pyrolysis technologies like pyrolysis, microwave-assisted thermal pyrolysis and catalytic pyrolysis. There is also a portion of microplastics that goes into the recycled water, which needs to be considered. Acknowledgment

This work was supported by the National Natural Science Foundation of China (51425802 and 51778454). References

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63-82.

Graphical Abstract

Highlights: 

Microplastics reduce the efficiency of wastewater treatment processes.



Microplastics affect the effluent quality of wastewater treatment plants.



Microplastics inhibit sludge hydrolysis, acidification, and methanogenesis.



Negative impacts of microplastics come from the leaching of toxic additives and ROS production.

Declaration of interests ☐ The authors declare that they have no known competing financial interests or personal relationships that could 53

have appeared to influence the work reported in this paper.

☒The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

Fig. 1. Flow chart of detection steps and techniques for microplastics in WWTPs.

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Fig. 2. (a) A schematic diagram of microplastics with different charges interacting with cell membranes. (b) A schematic diagram of microplastics adhering to microbial biomolecules [97]. (c) Variation of secondary structure of protein in biofilm caused by microplastics [61].

Fig. 3. Interaction between microplastics and activated sludge [61].

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Fig. 4. (a) Pie chart of microplastics form distribution in sludge [70, 72]. (b) Pie chart of microplastics form distribution in sewage [70, 72]. (c) Pie chart of microplastics color distribution in sludge [70, 72]. (d) Pie chart of microplastics color distribution in sewage [70, 72].

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Fig. 5. (a) Changes in the abundance of microbial communities under N0 and N3 conditions (phylogenetic markers and < 1% of the total sequence in each sample were classified as others; N0: no microplastics; N3: microplastics concentration is 0.2g / L; A: phylum levels; B: family levels) [75]. (b) SEM images of Acetobacteroides hydrogenigenes exposed to nanoplastics [75]. (c) Distribution of genus microbial populations in anaerobic digestion systems after addition of microplastics [65].

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Fig. 6. (a) Potential toxic substances produced by microplastics and potential cytotoxicity of microplastics to cells. (b)PS nanoparticles enter the lipid membrane and dissolve (Purple: PS monomer, cyan: lipid model) [99]. (c) Schematic diagram of microplastic adsorption on LPS. (d) Nanopores produced by microplastics attached to the cell membrane leading to cell dialysis. (e) Cell membrane damage caused by microplastics. (f) Changes in cell membrane protein side chains caused by microplastics. (g) Lipid peroxidation caused by excess ROS.

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