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Ma, T.; Ma, H.; Wu, J.; Yu, Y.; Chen, T.; Yao, Y.; Liao, W.; Feng, L. Engineered Nano-ZnO in the Biological Nitrogen Removal Process. Encyclopedia. Available online: https://encyclopedia.pub/entry/53410 (accessed on 02 July 2024).
Ma T, Ma H, Wu J, Yu Y, Chen T, Yao Y, et al. Engineered Nano-ZnO in the Biological Nitrogen Removal Process. Encyclopedia. Available at: https://encyclopedia.pub/entry/53410. Accessed July 02, 2024.
Ma, Teng-Fei, Hong-Xi Ma, Jin Wu, Yi-Chang Yu, Ting-Ting Chen, Yuan Yao, Wei-Ling Liao, Li Feng. "Engineered Nano-ZnO in the Biological Nitrogen Removal Process" Encyclopedia, https://encyclopedia.pub/entry/53410 (accessed July 02, 2024).
Ma, T., Ma, H., Wu, J., Yu, Y., Chen, T., Yao, Y., Liao, W., & Feng, L. (2024, January 04). Engineered Nano-ZnO in the Biological Nitrogen Removal Process. In Encyclopedia. https://encyclopedia.pub/entry/53410
Ma, Teng-Fei, et al. "Engineered Nano-ZnO in the Biological Nitrogen Removal Process." Encyclopedia. Web. 04 January, 2024.
Engineered Nano-ZnO in the Biological Nitrogen Removal Process
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This entry is adapted from the peer-reviewed paper 10.3390/w16010017

Engineered nano-ZnO is extensively utilized in both production and daily life, leading to its inevitable entry into the wastewater treatment system through various pathways. Nitrogen removal microorganisms in wastewater treatment systems are highly susceptible to environmental impacts. The antibacterial properties of nano-ZnO can impede the biological nitrogen removal (BNR) process and adversely affect the nitrogen removal performance. A comprehensive understanding of the inhibitory effect and mechanism of nano-ZnO on the BNR process is crucial in devising appropriate countermeasures to ensure optimal nitrogen removal performance. 

nano-ZnO inhibition nitrification denitrification anammox wastewater treatment

1. Effect of Nano-ZnO on BNR Process in WWTSs

Owing to the widespread utilization of nanomaterials in various industries and daily life, their discharge into WWTSs in the form of industrial or domestic wastewater is inevitable during their production, usage, transportation, and disposal processes. Research has indicated that nanomaterials are primarily trapped in WWTSs, with biological treatment processes capable of capturing 80–90% of NPs [1]. Nano-ZnO, as one of the most commonly employed nanomaterials, has been detected in WWTSs for a long time. In 2008, the concentration of nano-ZnO in wastewater treatment plant sludge was approximately 17.1 mg/kg and 23.2 mg/kg in Europe and the United States, respectively.
In recent years, the impact of nano-ZnO on the BNR process in wastewater treatment has emerged as a research hotspot. Nevertheless, current research mainly focuses on laboratory studies, specifically on the impact of the concentration and exposure duration of nano-ZnO on the BNR process. The following sections summarize and discuss the effects of the concentration and exposure mode of nano-ZnO on the BNR process.

1.1. Effect of the Nano-ZnO Concentration on the BNR Process

Most studies have shown that the inhibitory effect of the nano-ZnO concentration increases in the lower concentration range [2]. For instance, in a study conducted on pure cultured ammonia-oxidizing bacteria Nitrosomonas europaea, it was discovered that the ammonia nitrogen removal efficiency of N. europaea decreased by approximately 22.9% after being treated with 10 mg/L of ZnO NPs for 6 h [3]. Furthermore, when the concentration of ZnO NPs was increased to 20 mg/L, the ammonia nitrogen removal efficiency of Nitrosomonas europaea at the fourth hour decreased by roughly 67% [4]. In nano-ZnO exposure experiments with pure cultured aerobic denitrification bacteria Pseudomonas stutzeri PCN-1, the total nitrogen removal of P. stutzeri PCN-1 decreased from 100% to 1.7% as the concentration of ZnO NPs increased from 1 mg/L to 128 mg/L [5]. Similar phenomena were also observed in a study of activated sludge systems and granular sludge systems. For instance, Zhang et al. found that the removal efficiencies of TN decreased from 75.6% to 70.8% when the concentrations of added ZnO NPs in the reactor increased from 10 mg/L to 50 mg/L [6]. Similarly, Song et al. observed that the specific anammox activity of anammox granular sludge exhibited a significantly decreasing trend with the increase in ZnO NPs’ concentration within the range of 5 mg/(gVSS) to 20 mg/(gVSS). When the concentration of ZnO NPs surpassed 20 mg/(gVSS), the specific anammox activity of anammox granular sludge was almost entirely lost [7].
It is also reported that the introduction of high concentration (450–2000 mg/L) ZnO NPs can result in a significant reduction in the removal efficiency of ammonia nitrogen in the activated sludge system [8]. However, as the concentration of ZnO NPs increases, the removal efficiency of ammonia nitrogen does not experience a further decline, and the decrease in ammonia nitrogen removal efficiency remains within the range of 13–14% [8]. Due to the limited research on the BNR exposed to a high concentration of nano-ZnO, the underlying mechanisms behind this phenomenon require further investigation. It is speculated that this may be related to the state of high-concentration ZnO NPs in wastewater, such as their aggregation, distribution, and surface electrical properties.

1.2. Effects of the Exposure Mode of Nano-ZnO on BNR Process

Most studies on the impact of engineered nanomaterials on BNR indicate that the acute inhibitory effect of nanomaterials on BNR typically occurs at the beginning of exposure, but this system can usually recover after long-term operation [9]. As the system operates for a longer period of time, the added nanomaterials are adsorbed and enveloped by microbial extracellular substances in the system, which reduces their inhibitory effect. At the same time, the toxicity of metal and metal oxide nanomaterials released by metal ions also decreases or loses their toxicity due to coordination reactions and/or adsorption [10]. Meanwhile, the BNR performance of the system gradually recovers. However, in real scenarios, there are also cases where wastewater contains nanomaterials for a long time, so the long-term exposure of the BNR process to nanomaterials cannot be ignored.
In studies investigating the impact of nano-ZnO on the BNR process, both short-term and long-term exposure may produce different inhibitory effects. For example, in short-term exposure experiments (6 h), ZnO NPs at concentrations of 1 mg/L, 25 mg/L, and 50 mg/L all showed dose-dependent inhibitory effects on the TN removal performance of activated sludge but had no inhibitory effect on ammonia nitrogen removal [11]. However, after long-term exposure to 10 mg/L of ZnO NPs for 45 days, the reactor showed an almost complete loss of TN and the ammonia nitrogen removal performance [12]. Similarly, in the study of aerobic granular sludge, He et al. found that short-term exposure to 10 mg/L of ZnO NPs resulted in a decrease of 16.4% and 5.7% in the removal efficiencies of TIN and ammonia nitrogen, respectively, compared to the control group [13], while long-term exposure further reduced the removal rates of TIN and ammonia nitrogen, with a decrease of about 25.2% and 7.4%, respectively, compared to the control group [14].
Furthermore, it is noteworthy that the intermittent exposure mode has the potential to augment the resistance and/or tolerance capacity of the BNR system to nano-ZnO [15], thereby enhancing this system’s ability to counteract the inhibitory impact of nanomaterials. This is discussed later.

2. Inhibition Mechanism of Nano-ZnO to the BNR Process

The antibacterial mechanism of nano-ZnO, which is detailed in a prior review [16], typically encompasses the release of zinc ions, the generation of reactive oxygen species (ROS), and contact-mediated impairment. Nonetheless, these inhibitory mechanisms are primarily grounded in pure culture investigations of bacterial strains that are relevant to human health. Certain studies have been undertaken to explore the inhibitory effects of nano-ZnO on the BNR process. The following sections primarily delineate the inhibitory mechanism of the nitrification process, the denitrification process, and the anammox process.

2.1. Nitrification Process

The nitrification process refers to the gradual oxidation of ammonium (NH4+) to nitrate salts (NO3−) via microorganisms through the action of ammonia monooxygenase (AMO), hydroxylamine oxidoreductase (Hao), and nitrite oxidoreductase (NXR). This process is divided into the following two stages: ammonia oxidation (NH4+-NO2−) and nitrite oxidation (NO2−-NO3−), which are completed using ammonia-oxidizing microorganisms and nitrite-oxidizing bacteria (NOB), respectively. Ammonia-oxidizing microorganisms include ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea. AOB and NOB are collectively referred to as nitrifying bacteria. From the perspective of the types of microorganisms involved, current research has mainly focused on nitrifying bacteria. These studies can be broadly divided into the following three directions: microbial community structure, cell physiology, and gene expression, depending on the research direction.
In terms of the microbial community structure, AOB, as a type of environmentally sensitive microbe, may experience a decrease in relative abundance with the addition of a certain concentration of nano-ZnO [2][17]. The main reason for this is that AOB growth and activity are inhibited by nano-ZnO, while other bacterial groups can be promoted. For example, Wang et al. [17] found that, when activated, sludge in an SBR was exposed to 20 mg/L of ZnO NPs while the growth of some typical AOB, such as Nitrosococcus sp. and Nitrosomonas sp., were inhibited, while some types of denitrifying bacteria, such as Nitratiruptor sp. and Pseudomonas sp., were promoted.
In terms of cell physiology, nano-ZnO can cause damage to cell morphology, cell density, cell membrane integrity, and key enzyme activity, thereby inhibiting nitrification processes. When the model AOB strain Nitrosomonas europaea was exposed to 10 mg/L of nano-ZnO for 6 h, Yu et al. found that these cells underwent significant deformation, most of the multi-layer membranes of the cells could not be distinguished, cavities appeared inside the cells, and the cell density decreased by 34.7 ± 7.0% compared to the control group. The integrity of the cell membrane decreased by about 6% and was accompanied by physiological damage, while the strain’s removal rate of ammonia nitrogen decreased by 22.9 ± 0.7% [3]. Similarly, Wang et al. observed damage to cell membrane integrity when N. europaea was exposed to 20 mg/L and 50 mg/L of nano-ZnO for 4 h [17]. Damage to bacterial cell walls and cell membrane integrity was also frequently observed in the study of other metal and metal oxide nanomaterials [18][19][20][21]. The possible reason for this is that NPs accumulate on the outer surface of the cell membrane, neutralize the surface potential of the cell membrane, increase surface tension, and depolarize the membrane, ultimately causing changes and rupture to the cell membrane structure and damage to cell wall/membrane integrity [18][19][22]. In addition, the uneven surface structure and rough edges of ZnO nanomaterials can also wear down the cell membrane [23][24]. The addition of nano-ZnO also leads to the inhibition of key enzyme activity in the nitrification process. Daraei et al. found that 1–50 mg/L of ZnO NPs inhibited the activity of AMO and NXR in activated sludge, and the inhibition increased with the increase in the ZnO NP concentration [25]. Similarly, Wang et al. found that 20 and 50 mg/L of ZnO NPs significantly inhibited the activities of AMO and NXR in traditional activated sludge, with the highest inhibition rates reaching 39.24% and 44.84%, respectively [17].
In order to further understand the inhibitory mechanism of nano-ZnO on nitrification processes, researchers studied the impact of nano-ZnO exposure on the genetic level of typical nitrification processes. Yu et al. [3] monitored the global transcriptional expression of the model strain N. europaea under short-term exposure to nano-ZnO using the whole-genome microarray technique. They found that the most abundant differentially downregulated genes were those related to cell wall/membrane biogenesis and post-translational modification, protein turnover, and chaperone encoding. The downregulation of cell wall/membrane biogenesis genes may partially explain why the impairment of the cell membrane was not repaired in time. At the same time, an up-regulation of genes related to ammonia oxidation processes (such as amoA1, amoB1, amoC1, and amoC2) was observed, which could be a response of the bacteria to the inhibition of AMO. Ye et al. [26] found that after the long-term exposure of activated sludge to 10 mg/L of ZnO NPs, the abundance of amoA and nxrA genes in the system decreased significantly compared to the control group. Phan et al. [27] enriched the nitrifying bacterial community in activated sludge and exposed it to 1, 5, and 10 mg/L nano-ZnO. They found that after 6 h of exposure, the transcription levels of amoA and hao genes in the community were significantly decreased compared to the control group, indicating that the presence of nano-ZnO can directly or indirectly affect the expression of these key genes in nitrification processes.

2.2. Denitrification Process

The denitrification process refers to the process in which nitrates are transformed into nitrogen gas through a series of reduction reactions under microbial action, which is an important part of the nitrogen cycle. Currently, there are numerous research reports on the effects of various nanomaterials on microbial denitrification processes. However, research on the impact of nano-ZnO on denitrification processes is relatively limited.
Like many other nanomaterials reported, nano-ZnO can also affect denitrification processes by altering the microbial community structure. ZnO NPs can reduce the diversity of denitrifying bacteria communities and change the dominance of some denitrifying bacteria. For example, Chen et al. found that under low and high concentrations of ZnO NPs, the abundance of the nirS gene in denitrifying bacteria decreased by 83.8% and 95.8%, respectively, indicating a significant decrease in the abundance of denitrifying bacteria in the community. At the same time, the relative abundance of some bacterial genera in the denitrifying bacterial community also changed, such as an increase in the relative abundance of Pseudomonas and a decrease in the relative abundance of Bacillus [28]. Similarly, Chen et al. also reported the inhibitory effect of high doses of ZnO NPs (5 mg/L) on denitrifying bacterial genera in activated sludge, including Diaphorobacter species, Thauera species, and those in the Sphaerotilus Leptothrix group [29]. Cheng et al. also found that the relative abundance of denitrifying bacteria in the denitrifying granular sludge system significantly decreased after being exposed to 2.5 mg/L ZnO NPs, with the relative abundance of the dominant denitrifying bacterium Castellaniella decreasing from 51.0% to 8.0% [30]. These studies indicate that the presence of nano-ZnO can affect the microbial community’s structure, reduce the relative abundance of denitrifying bacteria in the microbial community, and thus affect the denitrification process.
The inhibition of crucial denitrification enzyme activities in denitrifying bacteria is a significant mechanism through which nano ZnO impedes the bacterial denitrification process. The bacterial denitrification process primarily depends on four denitrifying enzymes, namely nitrate reductase (NAR), nitrite reductase (NIR), nitric oxide reductase (NOR), and nitrous oxide reductase (NOS). These enzymes are encoded by the gene clusters of narGHJI (encoding respiratory nitrate reductase), napABC (encoding periplasmic nitrate reductase), nirSECF, norCBQDEF, and nosRZD, respectively. Nano-ZnO can affect bacterial denitrification by inhibiting the activity of key denitrification enzymes [26][31]. For example, Ma et al. found that exposure to 0.75 mM ZnO NPs significantly inhibited the activity of key enzymes in aerobic denitrifying bacteria, with NAR activity decreasing by 38.8% and NIR activity decreasing by 56.4% [32]. Similarly, Chen et al. observed that, under the action of 5 mg/L ZnO NPs, the highest decrease in NIR activity in the system could reach 32.8% [28]. The inhibition of these key denitrification enzyme activities may be related to the ROS generated by nanomaterials [33]. For example, Chen et al. found a good linear relationship between the activity of NAR and NIR in the denitrifying strain P. stutzeri PCN-1 and the amount of ROS generated under the action of ZnO NPs, indicating that the decrease in the enzyme’s activity could be related to the generation of ROS [5]. In addition, as one of the important mechanisms of the antibacterial effect of nano-ZnO, the dissolution and release of zinc ions can also inhibit key denitrification enzymes, such as NAR [34] and NOS [35], thereby inhibiting bacterial denitrification [31].
In addition, nanomaterials have excellent adsorption capabilities due to their high specific surface area. They may also adsorb trace metal elements such as Fe, Mo, or Cu, which are required by denitrifying bacteria. These metal elements are essential for denitrifying enzymes [36][37][38], and their reduction has been shown to lead to a decrease in key enzyme activity in model strains [39][40][41]. Therefore, the adsorption of trace metal elements by nano-ZnO may also be one of the reasons for the inhibition of denitrifying bacterial activity [31].

2.3. Anammox Process

The anammox process uses ammonium nitrogen as an electron donor to reduce nitrite nitrogen to nitrogen gas, thereby achieving denitrification [42]. Due to its advantages, such as no need for a carbon source, low sludge production, and no need for aeration, anammox technology is considered a more economical and promising BNR technology than traditional nitrification–denitrification technology [43][44]. However, the practical application of anammox still faces challenges due to the long generation cycle of anammox bacteria and their sensitivity to environmental stimuli. In recent years, the impact and mechanism of zinc oxide nanoparticles on the anammox process have gradually received attention as a nanomaterial that has been shown to affect traditional BNR processes. Similar to the mechanism of biological toxicity on nitrification and denitrification processes, zinc oxide nanoparticles have adverse effects on the anammox process by disrupting cell membrane integrity, inhibiting key enzyme activity, and disrupting metabolic processes. The main mechanism includes zinc ion release and the production of ROS.
The production and accumulation of ROS caused by nanomaterials is one of the mechanisms that inhibit anammox bacteria [45][46]. The accumulation of ROS produced by ZnO NPs and the death of anammox bacteria caused by the ROS have been reported [47]. However, some studies found that ZnO NPs do not cause oxidative damage to anammox [15][48]. This could be related to the fact that most studies operate anammox reactors under light-avoiding conditions [49]. Under anaerobic and dark conditions, ZnO NPs in the reactor may not produce ROS or produce very limited ROS, leading to a decrease in oxidative toxicity. Further research is needed to investigate the production and accumulation of ROS in anammox reactors to clarify the inhibitory mechanism of ROS on anammox bacteria.

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Subjects: Environmental Sciences
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Teng-Fei Ma
,
Hong-Xi Ma
,
Jin Wu
,
Yi-Chang Yu
,
Ting-Ting Chen
,
Yuan Yao
,
Wei-Ling Liao
,
Li Feng
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Revisions: 2 times (View History)
Update Date: 05 Jan 2024
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