1.1 The widespread use of nanomaterials. The enhanced strength, durability, flexibility, and performance associated with nanomaterials have been exploited in a multitude of applications. Engineered nanomaterials (ENPs) are already being used in sporting goods, tires, stain-resistant clothing, sun-screens, cosmetics, and electronics and are being increasingly utilized in medical devices (Nel et al., 2006). By some estimates, the production of ENPs is expected to increase to 58,000 tons by 2020 (Maynard et al., 2006). The impacts of engineered nanomaterials on the environment are two-sided: on one hand, technological advances in nanotechnology have undoubtedly brought great potential for innovative environmental remediation and monitoring applications (Gao & Wang, 2014). On the other hand, the potential harmful effects of engineered nanomaterials on humans and the environment, particularly the natural aquatic environment are of great interest. A recent study estimated that 10-30%, 3-17% and 4-19% NPs are discarded into water bodies in Asia, Europe and North America respectively (Keller et al., 2013). Wastewater treatment plants (WWTP) are among the last barriers prior to their release to the environment and the presence of nanomaterials in wastewater effluent has already been reported (Kunhikrishnan et al., 2015).

1.2 Objectives. The focus of this review is on the effects of various ENPs on nitrification and it is arranged as follows: 1). a brief introduction to nitrification, 2). a review of important issues that impact NP dynamics at full scale operations of wastewater treatment plants 3). effects of NPs on nitrification including observations from pure and enriched cultures, activated sludge and annamox processes; 4) proposed toxicity mechanisms, and 5) concluding remarks. Nitrification is a critical, microbially-driven process needed as part of most modern municipal water pollution control facilities, and this article discusses its susceptibility to NP-induced inhibition.

2.1. Introduction. Nitrification is the chemical conversion of oxidation of ammonia into nitrate. Ammonia must be removed from wastewater in order to help prevent eutrophication of receiving waters, and regulatory agencies have responded to this need by promulgating strict (e.g. < 0.5 mg NH₃-N/L) effluent NH₃-N limits. Such limits can be achieved by incorporating nitrification into the wastewater treatment process. Typically, wastewater treatment plants operate in a manner that supports the microbial communities that carry out nitrification, and there are now a wide range of suspended growth or fixed film configurations available for successful implementation. Nitrification is also, in many cases, coupled with denitrification to convert NO₃-N to nitrogen gas and achieve complete removal of soluble nitrogen. Nitrification is widely-practiced, and one of the most important components of modern water quality infrastructure.

2.2. Microbiology and Biochemistry. Nitrification in wastewater treatment is primarily mediated by the cooperative action of two distinct groups of chemoautotrophic bacteria: ammonia-oxidizing bacteria (AOB) for oxidation of ammonia (NH₃) to nitrite (NO₂^-) and nitrite-oxidizing bacteria (NOB) for oxidation of NO₂^- to nitrate (NO₃^-) (Dionisi et al., 2002). AOB are distinguished by their gram-negative multilayered cell walls and they are motile by means of flagella. There are five genera of AOBs that belong to two phylogenetically distinct groups, β- and γ-subclass ofProteobacteria . The β-subclass consists of four genera, includingNitrosomonas , Nitrosospira , Nitrosovibrio andNitrosolobus ; the γ-subglass contains Nitrosococcus(Madigan et al, 2000). AOBs are obligate chemolithotrophs that derive energy from the oxidation of NH₃ to nitrite (Arp et al, 2002). NOBs belong to the alpha- , beta -, andgamma-proteobacteria , including Nitrobacteria sp.(alpha ), Nitrotoga sp. (beta ), Nitrococcus sp. (gamma ). Nitrospina sp. and Candidatus Nitromaritima belong to the Nitrospinae sp. phyla andNitrospira sp. belong to the Nitrospirae phyla (Daims et al., 2016). Nitrotoga sp. and Nitrobacteria sp are commonly detected at WWTPs (Ge et al, 2015).

Ammonia oxidation is the first and rate-limiting step of the nitrification process. Ammonia oxidation in AOB takes places in three steps: ammonia is first oxidized to hydroxylamine by ammonia monooxygenase (AMO), hydroxylamine is then oxidized to nitric oxide (NO) by hydroxylamine oxidoreductase (HAO), and NO is further oxidized to nitrite by as-yet-unidentified nitric oxide oxidoreductase (NOO), likely NcyA (Stein 2019). AMO is an integral membrane metalloenzyme that uses Cu as a cofactor whereas HAO is located in the periplasm and is a α-trimer of 60-kDa polypeptide each containing eight hemes (Arp & Stein, 2003). In the reaction carried out by AMO:

NH₃ + O₂ + 2e^- + 2H⁺ NH₂OH + H₂O (Eq.1)

Two exogenous electrons must be supplied to AMO to reduce one atom of O₂ to water; these electrons are provided from the oxidation of hydroxylamine by HAO:

2NH₂OH + 1.5O₂ 2NO +3H₂O + 3e^- (Eq.2)

The oxidization of NO to nitrite is carried out by NOO/NcyA:

NO + 0.5O₂ + e^-NO₂^- (Eq.3)

The subsequent oxidation of nitrite to nitrate is carried out by NOB. The key enzyme involved in this one-step oxidation process is nitrite oxidoreductase (NXR).

NO₂^- + 1.5 O₂NO₃^- (Eq.4)

The membrane-bound NXR consists of α-, β-, and γ-subunits (Pester et al., 2014).

2.3. Inhibition. AOB and NOB activities are susceptible to inhibition through direct effects on the cell wall, essential enzymes, or on components involved in electron transport. Both AOB and NOB are slow-growing bacteria and sensitive to environmental factors (e.g., pH, dissolved oxygen, temperature) (Gu et al., 2012, Zhang et al., 2014, Fitzgerald et al., 2015). They are also inhibited by a large number of common wastewater constituents, including:

Since many nanoparticles are designed to inhibit or prevent biological activity, nanoparticles retained in the activated sludge flocs are expected to alter the interactions among activated sludge bacterial populations and impact the effectiveness of ammonia oxidation. This review is focused on the effects of ENPs on nitrification because of the unique and well-documented history of nitrifier inhibition. For convenience, summaries of ENP-related inhibition studies are provided in tabular form in Supplemental Materials (Tables S1 – S4).

Studies with pure and enriched cultures can guide activated sludge research by identifying relevant inhibition mechanisms. There are few pure culture ENP studies with AOB and there are no relevant pure culture NOB studies in published literature. Pure culture studies with N. europaea demonstrated that ZnO NP can reduce cell growth and ammonia removal by causing severe damage to the cell membrane and by interfering with AMO and HAO gene expression (Wu et al., 2018). The threshold ZnO NP concentration responsible for nitrification inhibition appears to be between 1 – 20 mg ZnO NP/L (Wu et al., 2018), and likely depends on the population density and the availability of stress response mechanisms. In the case of AgNPs, inhibition may be caused by the dissolution of Ag+, which in turn leads to ROS production and membrane cell membrane damage (Arnaout & Gunsch, 2012). The threshold inhibition AgNP concentration appears to be not more than 2 mg/L, and depends on how the NP is coated (Arnaout & Gunsch, 2012). None of these pure culture studies demonstrated that AOB can recover following NP-induced inhibition.

Enriched AOB cultures contain relatively small populations of non-AOB, and such cultures have been exposed to AgNP (Michels et al., 2017, Alito & Gunsch, 2013), TiO₂ NP (Luo et al 2015), and ZnO NP (Luo et al., 2015). In each case nitrification was reduced in the presence of NP concentrations that exceeded the inhibition threshold. Additionally, phylogenetic data revealed that inhibition coincided with reductions in both the diversity and abundance of AOBs (Luo et al, 2015). Alito and Gunsch (2013) demonstrated that enriched nitrifying bioreactors can recover within 3 to 5 days following inhibition caused by exposure to influent pulses containing 0.2 mg Ag NP/L. This result implied that stress response mechanisms may attenuate toxicity. Michels et al (2017) also demonstrated that specific nitrite production of an enriched AOB culture can recover following inhibition caused by a shock load of magnetic NPs.