Impact of metallic and metal oxide nanoparticles on wastewater treatment and anaerobic digestion

Abstract

Metallic and metal oxide nanomaterials have been increasingly used in consumer products (e.g. sunscreen, socks), the medical and electronic industries, and environmental remediation. Many of them ultimately enter wastewater treatment plants (WWTPs) or landfills. This review paper discusses the fate and potential effects of four types of nanoparticles, namely, silver nanoparticles (AgNPs), nano ZnO, nano TiO₂, and nano zero valent iron (NZVI), on waste/wastewater treatment and anaerobic digestion. The stabilities and chemical properties of these nanoparticles (NPs) result in significant differences in antimicrobial activities. Analysis of published data of metallic and metal oxide NPs suggests that oxygen is often a prerequisite for the generation of reactive oxygen species (ROS) for AgNPs and NZVI, while illumination is necessary for ROS generation for nano TiO₂ and nano ZnO. Furthermore, such nanoparticles are capable of being oxidized or dissolved in water and can release metal ions, leading to metal toxicity. Therefore, AgNPs and nano TiO₂ are chemically stable NPs that have no adverse effects on microbes under anaerobic conditions. Although the toxicity of nanomaterials has been studied intensively under aerobic conditions, more research is needed to address their fate in anaerobic waste/wastewater treatment systems and their long-term effects on the environment.

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Environmental impact

This review article discusses the fate and potential effects of metallic and metal oxide nanoparticles on waste/wastewater treatment and anaerobic digestion. The review addresses the possible chemical dissolution and oxidation reactions of these nanoparticles in waste/wastewater treatment systems. The antimicrobial activity of these nanoparticles is related to their stability and chemical properties. The mechanisms of microbial growth inhibition by nanoparticles are proposed, with an emphasis on the release of heavy metal ions and the role of oxygen and illumination in the generation of reactive oxygen species (ROS). The information summarized from the recent literature will help better predict the behaviors of nanoparticles and control their negative impacts in engineered systems and the environment.

1. Introduction

Nanoparticles (NPs) have been increasingly used in the manufacture of consumer products as well as new materials in industries such as medical devices and diagnostics, construction, and electronics.^1–3 Among these NPs, the major metallic elements are silver, titanium and zinc, which represent 50%, 10%, and 10% in commercial nanoproducts, respectively.² These NPs have been incorporated in cosmetics and clothing products (e.g., nanosilver embedded in the fabric, nano TiO₂ in sunscreens, and nano ZnO as a UV-absorber in lotion).^4–6 Therefore, they can be released from the consumer products to wastewater treatment plants (WWTPs) through washing.^5,7 Ultimately, NPs and the nanotechnology-enhanced products are placed in landfills at the end of their life time.⁴ Nano zero valent iron (NZVI), which is increasingly used for environmental remediation and waste/industrial wastewater treatment, also raises concerns about its potential toxicity and other biological effects.^8,9

Silver nanoparticles (nanosilver or AgNPs) are recognized as one of the NPs with the fastest growing demand.² Silver-containing biocidal products have been registered in the U.S. since 1954, and more than 50% of these silver products may contain nanosilver.¹⁰ Nanosilver can enter WWTPs through daily washing of silver-containing plastics and textiles.^5,11,12 With a concentration factor (CF, defined as the mass ratio of the chemical in the solid phase to that in the liquid phase) of around 100, silver ions and nanosilver in wastewater are mostly accumulated in sludge.^13–15 Furthermore, nanosilver can be adsorbed to sludge and embedded in sludge to form new products such as Ag₂S.^16,17 Ultimately, landfills are likely the final destination of the disposed sludge or discarded nanosilver products.¹⁶ In a recent model simulation, an average of 4.77 tons AgNPs per year may be dumped into landfills.¹⁸ The concentration of AgNPs is estimated to be 21 ng L⁻¹ and 1.55 mg kg⁻¹ in the effluent and sludge of WWTPs, respectively.¹⁹ As they have shown strong antimicrobial activities against a variety of microbes, there are increasing concerns about the potential negative impact of AgNPs on waste and wastewater treatment performance.^20–23

Nano zinc oxide (nano ZnO) has many unique properties, including semiconducting, pyroelectric, piezoelectric, photocatalytic, and desirable biocompatibility.^24–26 Thus, nano ZnO has gained more and more applications in the industry, such as sensors and transducers, medical care, and ceramic processing.^24–27 As one common component of sunscreens, nano ZnO can easily enter the water when it comes into contact with water through washing, bathing and swimming.^27,28 The other popular metal oxide NP, nano TiO₂, is heavily used in manufacturing, cosmetic applications (as a UV-absorber), food products, and environmental remediation.^29–31 The estimated global annual production of nano TiO₂ could be above 10 000 tons per year from 2011–2014.³² When it is released to water bodies, the estimated concentration of nano TiO₂ ranges from 0.7 to 16 μg L⁻¹.³³

NZVI has broad applications in environmental remediation due to its excellent capability for electron donation to reduce a wide range of pollutants, such as trichloroethylene (TCE), nitro aromatic compounds (NACs) – nitrobenzene, and heavy metal ions such as arsenate.^34–36 Recent research also suggests that NZVI coupled with electrochemical means can increase the rate of anaerobic granulation and organic removal in an upflow anaerobic sludge blanket (UASB) reactor treating coking wastewater.³⁷ However, due to its fast hydrogen production and its potential impact on wastewater treatment and anaerobic sludge digestion,^9,38,39 the application of NZVI in contaminated site remediation and wastewater treatment needs to be carefully evaluated.

This review summarizes recent research findings along with theoretical analysis of these metallic and metal oxide NPs to better understand the impact of AgNPs, NZVI, nano ZnO, and nano TiO₂ on waste/wastewater treatment and anaerobic digestion. First, we describe that their potential toxicity can be linked to their stability and chemical properties under aerobic and anaerobic conditions. We then discuss the potential inhibitory effects of these NPs in engineering systems, with an emphasis on wastewater treatment and anaerobic digestion.

2. Synthesis of metallic and metal oxide nanoparticles

Commonly used methods of nanoparticle synthesis are summarized in Table 1. Among the approaches for metallic nanoparticle production, chemical reduction techniques have been widely used,⁴⁰ since this method does not require any special instruments and can be done easily in a regular wet lab.⁴¹ The production of AgNPs or NZVI is mainly through chemical reduction of Ag⁺ or Fe²⁺/Fe³⁺.^34,40,42 In addition, top down methods may be an alternative way for NZVI production, which is accomplished by milling or etching large iron particles (e.g., micro size).^41,43

Table 1 Common methods for synthesis of metallic and metal oxide NPs

Type of NPs	Method	Descriptions	Size (nm)	Reference
AgNPs	Chemical reduction	Reduction of Ag^+ by NaBH4	19 ± 5	50
		Reduction of Ag^+ by H2	58 ± 11	50
	Photo reduction	Reduction of Ag^+ by PVP/UV	4–6	51
		Reduction of Ag^+ by sodium dodecyl sulfate/UV	23–67	52
	Microbial reduction	Reduction of Ag^+ by bacteria, Enterobacteriaceae	52.5	53
		Reduction of Ag^+ by fungus, Penicillium brevicompactum WA 2315	58 ± 18	54
NZVI	Top-down	Milling on micro iron particle	∼50	41, 43
	Bottom-up	Chemical reduction of Fe^2+/Fe^3+	1–100	34
Nano TiO2	Sol–gel	Acid solution drip into titanium isopropoxide solution (precursor)	5.9–14	55
Nano ZnO	Sol–gel	NaOH solution drip into ZnCl2 solution	21 ± 5	48

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Enormous efforts have been made to synthesize nano TiO₂ materials, such as sol–gel, sol, hydrothermal/solvothermal, chemical vapor methods, and so on.⁴⁴ Among these ways, the sol–gel method is one of the most popular methods for nano TiO₂ synthesis, which only requires hydrolysis of a titanium precursor and subsequent condensation.^45,46 Similarly, nano ZnO can also be easily produced in a sol–gel method along with others.^47–49

3. Chemical stability of metallic and metal oxide nanoparticles

Nanoparticles have high surface/volume ratios because of their small particle sizes (1–100 nm).⁵⁶ Thus, nanotoxicity in cells is associated with the size of NPs, because the smaller fraction of NPs could enter into the cell and lead to cell membrane disruption.^57,58 Beside the size dependent nanotoxicity, AgNPs and NZVI can be oxidized by oxygen, resulting in release of metal ions and generation of oxidative stress (e.g., reactive oxygen species or ROS).⁵⁸ Nano TiO₂ and ZnO can initiate light-induced redox activities and generate ROS.^59–62 When it dissolves in the neutral pH solution, nano ZnO produces Zn²⁺ and Zn(OH)⁺.^60,63 Therefore, the physical and chemical property changes of NPs may eventually affect their toxicity in the following aspects: (i) NP size change due to aggregation or complexation, which affects particle adsorption to the cell surface and transport across the cell membrane, (ii) generation of ROS by oxidation or light-induced redox activities, and (iii) release of metal ions by dissolution, which are thiol-reactive toxicants by complexing with the thiol groups of enzymes and proteins.^56,60,64 Details about their physical/chemical properties and changes are discussed in the following section.

3.1. Oxidative dissolution of AgNPs and NZVI

The oxidation of metallic NPs can lead to the release of metal ions in the solution, which are directly linked to their toxicity.⁵⁶ Under aerobic conditions, nanosilver can be oxidized by oxygen with a negative change in the standard Gibbs free energy as shown in Table 2, during which ROS may be produced.^64,65 ROS can induce oxidative stress on cells, destroy important cell components such as nucleic acids, proteins/enzymes, lipids, and finally cause cell death.^62,66 Therefore, the antimicrobial activity of nanosilver is related to the oxidative dissolution of AgNPs resulting in continuous release of silver ions and ROS production under aerobic conditions.^66,67 However, there is no silver ion release under anaerobic conditions, suggesting that dissolved oxygen (DO) is essential to the process of oxidative dissolution to convert nanosilver to Ag⁺ ions.^68–70

Table 2 Standard redox potential and change in Gibbs free energy (adapted from ref. 71)

Half reduction reaction	E^o (V, at 25 °C, 1 atm)
O2 + 4H^+ + 4e^− ↔ 2H2O	1.229
Ag^+ + e^− ↔ Ag	0.800
Fe^3+ + e^− ↔ Fe^2+	0.771
2H^+ + 2e^− ↔ H2	0
Fe^3+ + 3e^− ↔ Fe	−0.037
Fe^2+ + 2e^− ↔ Fe	−0.447

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Oxidation reactions	Standard Gibbs free energy change
4Ag + O2 + 4H^+ ↔ 4Ag^+ + 2H2O
Fe + 2H^+ ↔ Fe^2+ + H2
4Fe + 3O2 + 12H^+ ↔ 4Fe^3+ + 6H2O

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Iron is a more reactive metal than copper and silver. NZVI can therefore reduce various chemicals, such as chlorinated solvents, nitro aromatic compounds (NACs) – nitrobenzene, and oxidized heavy metals.^34–36 In deoxygenated aqueous solutions, water can oxidize NZVI to ferrous ions and generate hydrogen gas,⁴¹ as shown by the negative standard Gibbs free energy change (Table 2). Under aerobic conditions, water leads to rapid surface oxidation of NZVI and dissolved oxygen further converts Fe²⁺ to Fe³⁺.⁵⁷ In the NZVI–H₂O system, hydrogen ions and DO compete for available electrons donated by NZVI. Thus the reduction efficiency of NZVI is affected by pH and DO concentration.^72,73

3.2. Photoactivity and dissolution of nano ZnO and nano TiO₂

Physical and chemical property changes of nano ZnO and TiO₂ could induce their toxic effects on macro and microorganisms.⁷⁴ Nano ZnO and nano TiO₂ can generate ROS after absorbing photons.^56,75 Under illumination, electrons excited by photon absorption diffuse to the surface, and react with oxygen to form the super oxide anions (O₂˙⁻); while electron holes diffuse to the surface and react with water to form hydroxyl radicals (OH˙).^75,76 Therefore, illumination is required for nano ZnO and TiO₂ to generate ROS such as O₂˙⁻ and OH˙.^61,77

Nano TiO₂ is almost insoluble but nano ZnO can dissolve in water.⁷⁸ The dissolution of nano ZnO can release free zinc ion (Zn²⁺) and zinc hydroxide (Zn(OH)⁺),⁵⁶ which are the dominate zinc species under neutral pH and moderate alkalinity conditions⁵⁶ and are highly toxic to aquatic organisms.^79,80 Dissolution of nano ZnO (particle size = 70 nm) at a total concentration of 100 mg L⁻¹ was observed in nanopure water producing about 7 mg L⁻¹ dissolved Zn, while moderately hard water exhibited lower Zn solubility, likely because of Zn carbonate complexation and precipitation.⁸¹

3.3. Aggregation of nanoparticles

Aggregation of metallic and metal oxide NPs in solutions could significantly affect their activity and toxicity.⁸² These aggregated particles with an increased size will inevitably reduce their ion release rate, the potential to be delivered to the cell surface, and thereby lower their antimicrobial activities.^83,84 Therefore, polymers and surfactants are commonly used to stabilize NP suspensions as capping agents.⁸⁵ In a recent study, three types of AgNP suspensions were prepared based on the use of various capping agents: (a), uncoated AgNPs (H₂–AgNPs), (b) electrostatically stabilized (citrate and NaBH₄–AgNPs, branched polyethyleneimine (BPEI)–AgNPs), (c) sterically stabilized (polyvinylpyrrolidone (PVP)–AgNPs and polyvinyl alcohol (PVA)–AgNPs).^50,86 These AgNPs had different stabilities with changes in ionic strength and solution pH. For instance, high ionic strengths (100 mM NaNO₃) and/or low pH (3.0) conditions led to the aggregation of uncoated AgNPs (H₂–AgNPs), the citrate and NaBH₄-coated AgNPs.⁵⁰ In contrast, sterically stabilized PVP–AgNPs did not aggregate at the pH range of 2 to 11 in the 0.1 M NaNO₃ solution,⁵⁰ indicating that ionic strength, pH and electrolyte type (Cl⁻, NO₃⁻) have minimal impact on aggregation of sterically stabilized AgNPs. However, even with the protection by capping agents, NP aggregation can occur in biological media. For instance, aggregation of AgNPs in the presence of planktonic bacteria results in a particle size increase by a factor of 15–40.⁸⁷

DO affects metallic nanoparticle aggregation. The oxidative dissolution of AgNPs triggered 3–8 times faster aggregation than those without DO.^69,88 Metal complexing agents or ligands such as chloride and sulfide also play an important role in the stability of NP suspensions.^89,90 As they are also the key factors that affect antimicrobial activities, more discussion about their roles and function is described earlier in section 3.1.

NZVI particles are more active and less stable than AgNPs and their stability can be visually evaluated by gravitational sedimentation.⁹¹ Without capping agents, NZVI will quickly aggregate in the form of clusters.⁹² In order to prepare stable NZVI slurry with high activity, polymers are therefore often applied as a supporting skeleton. The commonly used polymers include starch, polyacrylic acid (PAA) and carboxymethyl cellulose (CMC).^92,93 Among them, NZVI with PAA/CMC has showed much better stability than NZVI modified with starch alone.^92,94 One of the most important mechanisms of NZVI aggregation is the magnetic attractive force generated between iron particles while there is no magnetic interaction between AgNPs.⁹⁵ As a result, increasing particle concentration and saturation magnetization (e.g. intrinsic magnet moment) may lead to faster aggregation.⁹⁵ Still, adsorbed polymers can decrease the aggregation rate by electrostatic and/or electrosteric mechanisms.⁹⁶ Electrosteric stabilization provides the best stability for NZVI, which could strengthen repulsive force among particles and reduce the magnetic force between particles as well.^96,97

Nano ZnO and nano TiO₂ suspensions also easily aggregate in aqueous solutions. At a total concentration of 80 mg L⁻¹, nano ZnO formed larger aggregates in seawater than ZnO powder, while nano ZnO had a solubility of 3.7 mg L⁻¹ in seawater compared to that of ZnO powder with only 1.6 mg L⁻¹.²⁷ Rapid aggregation of nano TiO₂ was also observed in both cell culture media and physiological buffers,³⁰ increasing the particle size to more than 1000 nm.⁹⁸

4. Impact of metallic and metal oxide NPs on wastewater treatment

4.1. Fate and transport of metallic and metal oxide NPs in activated sludge treatment systems

Metallic and metal oxide NPs in cosmetics and clothing products can easily enter the sewer systems and WWTPs.^4–6 Though information is limited about the fate of NPs in WWTP by onsite investigation, pilot studies and model estimation still provide useful knowledge about their transport and transformation in simulated wastewater treatment processes.^17,99,100 Related studies have focused on the following questions: (i) to what extent do metallic and metal oxide NPs remain in the effluent? (ii) will these NPs be precipitated out or completely adsorbed to the biosolids? (iii) how does the metal speciation change in the presence of metallic and metal oxide NPs in WWTPs?

4.1.1. Fate of AgNPs in activated sludge systems. AgNPs can be easily removed from water by chemical transformation, adsorption and precipitation in an activated sludge system.^17,99 A recent study with a 12 h shocking load of AgNPs in a Modified Ludzack–Ettinger (MLE) activated sludge treatment system suggests that more than 90% of AgNPs were associated with biomass and the total silver concentration in effluent wastewater was below 0.05 mg L⁻¹.⁹⁹ Another study in a pilot WWTP after spiking AgNPs indicates the transformation of nanosilver to Ag₂S in less than 2 h in a non-aerated tank, while most of the AgNPs were sorbed to the sludge.¹⁷ Sulfide plays an important role in the oxysulfidation of nanosilver, which requires dissolved oxygen. As a result, AgNPs react with dissolved sulfide species (H₂S, HS⁻) to produce silver sulfide nanostructures.⁶⁸ The strong complexation reaction limits their bioavailability and reduce the toxicity to microorganisms (stability constant K to form AgS₂ = 10^50.1).^68,90

4.1.2. Fate of nano ZnO and nano TiO₂ in activated sludge systems. Most of the nano ZnO and nano TiO₂ are adsorbed by the sludge/biomass,¹⁰¹ indicating that activated sludge wastewater treatment processes can effectively remove these NPs. Based on model simulation results, about 1.75 μg L⁻¹ nano TiO₂ and 0.3 μg L⁻¹ nano ZnO may be discharged from the WWTP effluent.¹⁹ The predicted concentrations of nano ZnO and nano TiO₂ are 0.3 mg kg⁻¹ and 23.2 mg kg⁻¹ in the sludge, respectively.¹⁹ However, concentrations appeared to be higher in real WWTPs. The raw sewage may contain 100 to nearly 3000 μg Ti per L and the effluent Ti ranged from <5 to 15 μg L⁻¹.¹⁰¹ Since sludge containing nano ZnO and nano TiO₂ could be finally dumped into landfill sites,¹¹ it is estimated that about ¾ of the total nano TiO₂ entering WWTPs would finally end up in landfills.³³

4.1.3. Fate of NZVI in wastewater treatment. Rapid oxidation of NZVI under aerobic conditions results in the transformation of elemental iron to ferrous and ferric ions and related precipitates.¹⁰² The life time of NZVI is highly dependent on many factors including solution pH and temperature.⁷³ At pH 8.9, NZVI had a half-life of 90–180 days in a Fe(OH)₂–H₂O system.⁷³ When pH decreased from 8.9 to 6.5, H₂ evolution rate increased from 0.008 to 0.22 day⁻¹.⁷³ For comparison, in a sludge digestion study, the half-life of NZVI was about three days at pH 7.2 based upon the measurement of hydrogen production rate.¹⁰³ Since these results are obtained in batch experiments, more research is needed to explore the fate of NZVI in continuous wastewater treatment systems.¹⁰⁴

4.2. Inhibition of microbial activities by metallic and metal oxide NPs in wastewater treatment

4.2.1. Toxicity of AgNPs in activated sludge system. The antimicrobial activity of AgNPs against aerobic microorganisms has been well studied.^20,66,105 The antimicrobial function of nanosilver is attributed to the continuous release of Ag⁺ ions by nanosilver dissolution under aerobic conditions,^105,106 ROS production, and transport of smaller NPs across the cell membrane by a Trojan-horse type mechanism.^107,108 Small size AgNPs (<10 nm) may enter the cells directly to release silver ions, inactivate cellular enzymes and DNA, generate ROS, and lead to growth inhibition and eventually cell death.^20,99,109

Batch test results have shown size dependent nanosilver inhibition on nitrifying bacteria,⁶⁶ which have slower growth rate and higher sensitivity to environmental perturbations than heterotrophs.^66,110 The fraction of AgNPs less than 5 nm is correlated with inhibition to nitrifying organisms.⁶⁶ Moreover, intracellular ROS fraction instead of photocatalytic ROS correlates well with the inhibitory effect by AgNPs.⁶⁶ In a continuous activated sludge system, nitrification inhibition can last for more than one month after a 12 h shock load of AgNPs to reach a final peak silver concentration of 0.75 mg L⁻¹ in the system.⁹⁹ Therefore, nanosilver appears to be very toxic to nitrifiers. Silver ions released to soil can lead to a significant reduction in denitrification activity, and this inhibition could prolong to 90 days.¹¹¹ Thus, the release of Ag⁺ from AgNPs in wastewater treatment might also affect denitrification at high silver concentrations.

4.2.2. Impact of nano ZnO and nano TiO₂ on wastewater treatment. The presence of nano ZnO in wastewater treatment systems can strongly influence the effluent water quality. In a recent sequencing batch reactor study, nano ZnO decreased the total nitrogen removal efficiencies from 81.5% (control) to 75.6% and 70.8% at the concentrations of 10 mg L⁻¹ and 50 mg L⁻¹, respectively.¹¹² Furthermore, nano ZnO at concentrations of 10 mg L⁻¹ and above induced the release of phosphorus from sludge, and thereby decreased the phosphorus removal efficiencies.¹¹² Although nano TiO₂ is considered less toxic because of its extremely low solubility, its impact on wastewater treatment has not been well investigated. The antimicrobial activities of nano ZnO and nano TiO₂ are generally attributed to their nano size effect and their ability of generating ROS.⁷⁵ More importantly, nano ZnO can release zinc ions in water which contribute to its antimicrobial activity.^56,78 The toxicity of nano ZnO may not be directly linked to the ROS generation, since illumination did not enhance the antimicrobial activity.^113,114

4.2.3. Impact of NZVI on wastewater treatment. Despite its widespread applications in environmental remediation, little is known about the effect of NZVI on bacterial growth and activities.^57,115 A previous study examined the effectiveness of ZVI to remove viruses φX 174 and MS 2 either by inactivation or absorption.¹¹⁶ The inactivation efficiency on MS2 was higher in air saturated solutions than that in deaerated aqueous solutions, suggesting that a stronger oxidation of NZVI under aerobic conditions can cause more severe inactivation.¹⁰² Bacteria have different responses to NZVI. For instance, NZVI rapidly inactivated Escherichia coli by inducing reductive stress and disrupting the cell membrane in aqueous solutions, likely due to the oxidation of NZVI by oxygen or intracellular oxygen, or hydrogen peroxide present in cells.⁵⁷ Interestingly, a higher amount of NZVI is required to inhibit bacterial growth under aerobic conditions, as the dissolved oxygen might lead to the corrosion and surface oxidation of NZVI, reducing its “redox” activity.^57,117 The inhibitory effect by NZVI is further complicated by other factors such as polyelectrolyte coatings, natural organic matter (NOM) and humic acids.^118,119 Although batch studies have been conducted to use NZVI in industrial wastewater treatment, aiming at reducing chlorinated compounds, 2,4,6-trinitrotoluene (TNT), petroleum refinery wastewater, and textile dyes,^{34,72,120,121} it is still unclear about the potential effect of NZVI in activated sludge wastewater treatment systems.

5. Impact of metallic and metal oxide NPs on anaerobic digestion

Sludge and municipal solid waste (MSW) need to be stabilized by removing organic solids under anaerobic conditions, during which a series of biological reactions occur. These include: (1) hydrolysis, where large organic molecules are broken down into simple sugars, amino acids, and fatty acids; (2) acidogenesis, where these compounds are further broken down into simple organic acids, ammonia, carbon dioxide, hydrogen and other byproducts; (3) acetogenesis, where carbon dioxide, hydrogen and the organic acids from acidogenesis are converted into acetic acid; (4) methanogenesis, which includes hydrogenotrophic methanogenesis (4H₂ + CO₂ → CH₄ + 2H₂O) and acetoclastic methanogenesis (CH₃COO⁻ + H⁺ → CO₂ + CH₄), resulting in methane production.¹¹⁰

While anaerobic sludge digestion is most commonly used for sludge stabilization, landfilling is the most common method of MSW disposal. Landfill bioreactors are operated through controlled leachate recirculation, which have changed the traditional landfill technology from a storage/containment concept into a process-based organic waste degradation approach.¹²² Compared to a traditional landfill, the bioreactor landfill process offers benefits such as rapid waste degradation, and generally improved landfill gas recovery.^123–125 As NPs released from different nanomaterials end up in wastewater sludge and landfills, there is an urgent need to determine their fate and toxicity in anaerobic digestion.

5.1. Impact of metallic and metal oxide NPs on anaerobic sludge digestion

Heavy metals such as Zn, Cr, Cu, and Cd inhibit anaerobic microbial activities in the digestion processes.^126,127 Because of their smaller size and higher specific surface area, the metallic and metal oxide NPs may exhibit different physicochemical and toxicological behaviors from bulk metal species under anaerobic conditions. Although soluble metal ions released from the NPs and ROS production are related to bacterial growth inhibition,^66,110 the presence of oxygen is a prerequisite of ROS generation for AgNPs and NZVI.^64,65,128 For metal oxide NPs, spontaneous ROS generation by TiO₂/ZnO occurs when excited electrons diffuse to the surface of NPs and react with O₂ to form O₂˙⁻ or electron holes diffuse to the surface and react with water to generate OH˙.^75,129,76,60 Theoretically, in the absence of oxygen, the UVA radiation on TiO₂ can still trigger the generation of OH˙ by reaction between electron holes and water, along with simultaneous reduction of Ti⁴⁺ by excited electrons as shown in the following reactions:¹³⁰

TiO₂ + hv → h⁺_vb + e⁻_cb

h⁺_vb + H₂O → H⁺ + ˙OH

h⁺_vb + OH⁻ → ˙OH

e⁻_cb + Ti⁴⁺ → Ti³⁺

However, the generation of ROS is limited under anaerobic conditions,¹³¹ likely because of low solubility of TiO₂.⁷⁸ Other studies have shown that no ROS was detected when Pseudomonas stutzeri was exposed to quantum dots in the absence of oxygen.¹³² Therefore, regardless of the uncertainty of ROS generation from metal oxide NPs, ROS cannot be produced¹³³ in dark anaerobic digestion due to the absence of oxygen and illumination.^65,131

5.1.1. Potential impact of AgNPs on anaerobic sludge digestion. Although the antimicrobial activity of AgNPs against anaerobic microorganisms is relatively less studied, we recently found that there was no significant difference in biogas or methane production between the sludge treated with AgNPs at 40 mg Ag per L and the control in ambient (22 °C) and mesophilic (37 °C) sludge digestion.⁷⁰ There were no silver ions released from nanosilver under anaerobic conditions.⁷⁰ Our results and other research findings suggest that dissolved oxygen is essential to oxidative dissolution of AgNPs.^68–70 The absence of oxygen leads to the non-release of silver ions from AgNPs, and thus nanosilver does not inhibit methanogenic activities in anaerobic sludge digestion.⁷⁰

5.1.2. Potential effect of nano ZnO and nano TiO₂ in anaerobic sludge digestion. Metal oxide NPs may exhibit different physicochemical and toxicological behaviors than bulk metal species, as indicated by several recent studies.¹³⁴ For instance, the half maximal effective concentration (EC50) used to inhibit methane production by nano ZnO was 57.4 mg Zn per L, while the EC50 concentration was 101 mg Zn per L for bulk ZnO.¹³⁴ Other researchers found nano ZnO inhibited methane production by 18.3% and 75.1% at the concentrations of 30 and 150 mg ZnO per g-TSS (total suspended solids), respectively.

The presence of nano TiO₂ did not affect methane generation at concentrations of 6 to 150 mg per g-TSS.¹³⁵ On the contrary, under illumination, nano TiO₂ can enhance hydrogen gas production after dark fermentation and ammonia removal by using a photofermentation technique.¹³⁶ Nano TiO₂ at 100 mg L⁻¹ increased hydrogen generation efficiency by 46.1%.¹³⁶ The improvement of hydrogen production was attributed to the ability of nano TiO₂ to facilitate the removal of organic matter, promote the growth of photosynthetic bacteria, and inhibit the activity of hydrogen-uptake enzymes.¹³⁶

5.1.3. Impact of NZVI on anaerobic digestion. Under anaerobic conditions, NZVI can produce hydrogen gas and thus stimulate sulfate reducers and methanogenic populations in trichloroethylene contaminated aquifers.¹³⁷ In one study, at a concentration of 1 g L⁻¹, NZVI significantly increased the methane production from 58 ± 5 (NZVI free control) to 275 ± 2 μmol.¹³⁸ Other researchers, however, found that NZVI at 1 g L⁻¹ resulted in the inactivation of sulfate reducing bacteria (SRB) by 75.6%, which could be due to the coating of cells by FeO(OH) precipitates.¹³⁹ Similarly, at concentrations higher than 0.3 g L⁻¹, NZVI inhibited biological sulfate reduction.¹⁴⁰

To determine the effect of NZVI on anaerobic sludge digestion, we have conducted a series of batch tests recently. At concentrations of 1 mM and above, NZVI reduced methane production.¹⁰³ A dose of 30 mM NZVI led to fast hydrogen production, significant increase in soluble chemical oxygen demand (SCOD), volatile fatty acids (VFA) accumulation and slow growth of methanogens.¹⁰³ The rapid dissolution of NZVI in sludge resulted in a high concentration of soluble ferrous iron, which might be harmful to methanogens. In contrast, ZVI powders are beneficial to methanogenesis due to their slow release of hydrogen gas.^103,141

5.2. Impact of metallic NPs on bioreactor landfill operation

Much less research has been done to determine the impact of metallic NPs on landfill operations. In a new generation of landfills, a bioreactor landfill typically employs anaerobic processes for waste stabilization. However, a bioreactor landfill has a microaerobic period during the initial stage of landfilling.¹⁴² Hence, MSW decomposition starts with hydrolysis under microaerobic conditions,¹⁴³ during which the oxygen content in the landfill biogas ranges from 0.1 to 1%.¹⁴⁴ As oxygen is gradually depleted, CO₂ becomes the primary component in the biogas,¹⁴³ while bioreactors change from oxic to anoxic/anaerobic conditions.¹⁴⁵ Therefore, the trapped oxygen may lead to different inhibitory behaviors by metallic NPs in landfill bioreactors, compared with a well-mixed anaerobic sludge digestion system.

AgNPs released from industrial activities and consumer products may be disposed of directly or indirectly in sanitary landfills. In our recent study, nanosilver at a concentration of 1 mg Ag per kg did not cause any change in the cumulative biogas volume or gas production rate compared to the control bioreactor. However, landfill solids exposed to AgNPs at 10 mg kg⁻¹ resulted in a reduced biogas production, accumulation of volatile fatty acids (including acetic acid), and a prolonged period of low leachate pH (between 5 and 6).¹⁴⁶ Quantitative polymerase chain reaction (PCR) results targeting the 16S rRNA gene of methanogens indicated a lower methanogenic population in the bioreactor treated with 10 mg AgNPs per kg, compared to the groups of control and 1 mg AgNPs per kg.¹⁴⁶ The results suggest that the transition period from microaerobic to anaerobic conditions in landfills uniquely caused nanosilver inhibition on methanogenesis.

To differentiate the effect of AgNPs and silver ions, we also conducted another bench-scale landfill bioreactor study to treat the solid waste samples at silver concentrations of 10 mg kg⁻¹ solids in the form of AgNPs and AgNO₃.¹⁰³ There was no significant difference in the cumulative methane volume or methane production rate between the groups of 10 mg kg⁻¹ silver ions and control. However, the bioreactor exposed to 10 mg AgNPs per kg resulted in reduced methane production and the accumulation of volatile fatty acids (including acetic acid).¹⁰³ The results suggest that nanosilver is more toxic than its counterpart resulting in reduced methane production and methanogenic assemblages, likely because of higher silver concentrations available in the leachate due to the slow nanosilver dissolution under landfill operations. More studies are needed to understand the impact of other metallic and metal oxide NPs on landfill operations. Moreover, caution should be taken in the disposal of nano ZnO, which can be dissolved in aqueous solutions to inhibit anaerobic digestion.^56,147

6. Summary and open questions

All NPs may have a nano-size effect, and can be sorbed onto cell surfaces and thereby alter the membrane properties (e.g., increase in membrane permeability) and result in membrane disruption.^58,148 Beside this factor, the effect of metallic and metal oxide NPs on waste/wastewater treatment and sludge digestion is highly dependent on aerobic and anaerobic conditions. Under aerobic conditions, AgNPs, nano TiO₂, nano ZnO, and NZVI can generate ROS and cause oxidative stress to the cell. More importantly, some of them release metal ions (Ag⁺ and Zn²⁺) and thereby cause metal toxicity.^56,60,64 Under dark deoxygenated conditions, no ROS could be generated while the toxicity of such NPs is mainly associated with the release of metal ions.^75,132 On the other hand, NZVI produces hydrogen gas uniquely and rapidly, which might be harmful to anaerobic digestion.¹⁰³ Based upon this discussion, the possible antimicrobial mechanisms may fall into three categories as shown in Table 3.^56,58,70

Table 3 Possible antimicrobial mechanisms of metallic and metal oxide NPs

Nanoparticles	Aerobic conditions			Dark anaerobic conditions
	Membrane property change	Oxidative stress (ROS)	Ion release	Membrane property change	Oxidative stress (ROS)	Ion release
AgNPs	✓	✓	Ag^+	Possible	None	None
NZVI	✓	✓	Fe^2+, Fe^3+	Possible	None	Fe^2+
TiO2	✓	✓	None	Possible	None	None
ZnO	✓	✓	Zn^2+, Zn(OH)^+	Possible	None	Zn^2+

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On the beneficial side, metallic and metal oxide NPs can be applied to water treatment for disinfection purposes, such as enhanced inactivation of viruses and biofouling control.^58,116 Nano ZnO, nano TiO₂, and NZVI can also be applied in industrial wastewater treatment.^{34,56,72,120,121} Future research is needed to strike a balance between the beneficial use of such NPs and their potential toxicity, which will help accelerate their large-scale applications in various areas.

Since many studies focus on the antimicrobial function of metallic and metal oxide NPs under aerobic conditions, more research about the fate and transport of NPs is needed to explore their effect under anaerobic conditions such as those in sludge digestion and landfill operations. Moreover, the fate of NPs in landfills will vary greatly with factors such as the source of solid waste, recirculation rate, and even packing methods. Therefore, these factors should be well considered to better understand the fate and toxicity of metallic and metal oxide NPs in wastewater treatment and anaerobic digestion.

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