Mechanisms of nitrous oxide emission during photoelectrotrophic denitrification by self-photosensitized Thiobacillus denitrificans

Water Research 172 (2020) 115501

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Mechanisms of nitrous oxide emission during photoelectrotrophic denitrification by self-photosensitized Thiobacillus denitrificans Man Chen, Xiaofang Zhou, Xiangyu Chen, Quanhua Cai, Raymond Jianxiong Zeng, Shungui Zhou* Fujian Provincial Key Laboratory of Soil Environmental Health and Regulation, College of Resources and Environment, Fujian Agriculture and Forestry University, Fuzhou, Fujian, 350002, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 13 November 2019 Received in revised form 31 December 2019 Accepted 10 January 2020 Available online 11 January 2020

Photoelectrotrophic denitrification (PEDeN) using bio-hybrids has the potential to remove nitrate (NO
3) from wastewater in an economical and sustainable way. As a gas of global concern, the mechanisms of nitrous oxide (N2O) emissions during this novel process remain unclear. Herein, a self-photosensitized bio-hybrid, i. e., Thiobacillus denitrificans-cadmium sulfide, was constructed and the factors affecting N2O emissions during PEDeN by the bio-hybrids were investigated. The system was sensitive to the input
NO
3 -N and NO2 -N, resulting in changes in the N2O/(N2þN2O) ratio from 1% to 95%. In addition to free nitrous acid (FNA), reactive oxidative species (ROS) were a unique factor affecting N2O emission during PEDeN. Importantly, the N2O reduction step exhibited greater susceptibility to the ROS than nitrate reduction step. The contributions of hydrogen peroxide (H2O2), superoxides (O
 2 ), hydroxyl radicals (OH) and FNA to the inhibition of N2O reduction were >15.0%, >5.4%, 1.3%, and <70.2%, respectively for a reduction of 13.5 mg/L NO
3 -N. A significant down-regulation of the relative transcription of the gene nosZ demonstrated that the inhibition of N2O reductase occurred at the gene level. This finding has important implications not only for mitigating N2O emissions during the PEDeN process but also for encouraging a reexamination process of N2O emissions in nature, particularly in systems in which ROS are present during the denitrification process. © 2020 Elsevier Ltd. All rights reserved.

Keywords: Photoelectrotrophic denitrification Nitrous oxide emission Reactive oxidative species FNA inhibition Nitrate pollutant

1. Introduction Photoelectrotrophic denitrification (PEDeN) using biosemiconductor hybrids is a novel concept (Cheng et al., 2017). Upon irradiation, a non-phototrophic denitrifier uptakes photoexcited electrons, instead of an organic or inorganic electron source, for denitrification (Cheng et al., 2017; Chen et al., 2019a). This pathway avoids the drawbacks associated with traditional electron sources, such as the potential risk of secondary organic pollution, mass sludge production, and transportation difficulty (e.g., H2) (Chen et al., 2019a). Moreover, the use of organic carbons or H2 results in a costly nitrogen removal process (Puig et al., 2012). Solar energy represents a more economical and sustainable way of producing electrons for nitrate (NO
3 ) reduction than heterotrophic or conventional autotrophic denitrification (Chen et al., 2019a). Therefore, denitrification driven by sunlight has the potential to

* Corresponding author. E-mail address: [email protected] (S. Zhou). https://doi.org/10.1016/j.watres.2020.115501 0043-1354/© 2020 Elsevier Ltd. All rights reserved.

achieve nitrogen removal in an economical and sustainable manner. This process is well suited for NO
3 removal from oligotrophic water, such as NO
3 -polluted surface water, secondary effluents of wastewater, or seawater, which lacks an organic carbon source (Chen et al., 2019a). Since the PEDeN system is a hybrid system, in which a semiconductor is coupled with a microorganism, the system is more complex than traditional denitrification systems. Upon irradiation, the semiconductor not only provides photo-electrons to the microorganisms but also produces reactive oxidative species (ROS), including holes (hþ), hydrogen peroxide (H2O2), hydroxyl radicals (OH), superoxides (O
 2 ), etc (Nosaka and Nosaka, 2017). In addi
tion, because NO
3 /NO2 also has photochemical properties, sunlight is absorbed and ROS are produced under irradiation (Mack and Bolton, 1999). In addition to the well-known toxicity of ROS to microorganisms, these oxidative species also create an oxidative environment, thereby possibly affecting the sensitive N2O reductase (Song et al., 2002). In microbial denitrification, the emission of nitrous oxide (N2O) is an ongoing concern because N2O is a potent

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M. Chen et al. / Water Research 172 (2020) 115501

greenhouse gas (Reay et al., 2012). Normally, microbial denitrifi
cation goes through four steps, namely NO
3 / nitrite (NO2 ) / nitric oxide (NO) / N2O / nitrogen (N2), where N2 is the end product (Chen and Strous, 2013). However, due to the sensitivity of N2O reductase, an inhibition of N2O reduction often occurs, resulting in N2O emissions (Qu et al., 2016). N2O emissions can be caused by high oxygen, a low chemical oxygen demand (COD)/N ratio, a low pH, free nitrous acid (FNA) accumulation, or changing operating conditions during wastewater treatment (Law et al., 2012). The phenomenon of FNA accumulation was also observed in the PEDeN process; however, N2O generation does not appear to be solely the result of FNA inhibition (Chen et al., 2019a). When a selfphotosensitized Thiobacillus denitrificans-cadmium sulfide (T. denitrificans-CdS) bio-hybrid was used for denitrification, the maximum accumulated FNA was 0.0007 mg N/L with an input NO
3 -N of 14 mg/L (Chen et al., 2019a). In this case, extremely highpurity N2O (>96%) was produced. The accumulated FNA concentration was much lower than that in the common denitrification process but with a higher percentage of N2OeN (Scherson et al., 2013; Lan et al., 2019). For example, the activity of N2O reductase was inhibited only about 40%e50%, even when the influent FNA was greater than 0.004 mg N/L in sulfide-based autotrophic denitrification (Lan et al., 2019). Besides, the accumulated N2O in the T. denitrificans-CdS-based denitrification process could not be easily reduced to N2 (Chen et al., 2019a). This is in contradiction to the competitive inhibition of N2O reduction by FNA, which is reversible and is alleviated when NO
2 is depleted (Zeng et al., 2003). Therefore, in addition to FNA, it is very likely that other factors such as ROS inhibit N2O reduction during PEDeN. However, the relationship between N2O emissions and ROS has been largely ignored in previous studies, resulting in a lack of an understanding of N2O emissions affected by ROS. In fact, the presence of ROS and in the denitrification process does not exclusively occur in the PEDeN system but is also common in nature (Petasne and Zika, 1987). For example, denitrification process commonly occurs in the ocean and high levels of ROS (H2O2 and O
 2 ) due to photochemical process have been detected (Canfield et al., 2010; Petasne and Zika, 1987). More importantly, even without irradiation, ROS can be generated in the dark in sediments at the oxic/ anoxic interfaces, where denitrification also occurs (Page et al., 2012; Brune et al., 2000). Therefore, the influence of ROS on N2O emission may be a common occurrence. An understanding of the mechanisms of N2O emissions in the PEDeN process provides an insight into N2O emissions and their control during denitrification under laboratory conditions and in nature. The overall goal of this work is to provide an understanding of the mechanisms of N2O emission in PEDeN using bio-hybrids. T. denitrificans-CdS was used as a model bio-hybrid in PEDeN. ROS and FNA were considered the two important factors affecting N2O emissions during the PEDeN process. Different concentration of nitrogenous species were analyzed to determine changes in the N2O/(N2O þ N2) ratio and to ascertain the factors affecting N2O emissions. This method has been frequently used in previous works (Kampschreur et al., 2009). The concentrations of the main ROS (H2O2, O
 2 and OH) and their contributions to the N2O inhibition were determined. The expressions of the denitrifying genes encoding the related denitrifying enzymes were quantified to determine the mechanism of denitrification at the gene level. The findings may have important implications not only for mitigating N2O emissions during the PEDeN process but also for helping reexamine the process of N2O emissions in nature, particularly in systems in which ROS are present during denitrification.

2. Materials and methods 2.1. Development of T. denitrificans-CdS hybrids Thiobacillus denitrificans (DSM 12475) was purchased from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures. The development of the T. denitrificans-CdS hybrids was described in our previous work (Chen et al., 2019a). Briefly, the inoculum was first grown in Medium113 (Table S1) until the optical density (OD600) of the medium was approximately 0.2. Subsequently, sterilized and degassed cysteine and Cd2þ solutions were injected into the medium. When the color of the medium changed from opaque white to bright yellow, the suspension was centrifuged, washed, and injected into 20 mL of Medium113-M1 (Table S1), from which NH4Cl, Na2S2O3, and KNO3 were removed.
Different electron acceptors (NO
3 -N, NO2 -N, or N2OeN) were added based on the requirements. When N2OeN was used as the electron acceptor, the gas in the headspace was first washed with high-purity helium (99.999%). Then a dose of pure N2O (>97%, another composition is helium gas) was injected into the bottle to the required concentration. The reaction temperature was 30  C and the initial pH of the solution was 7.1. Sodium lactate (0.1%, v/v) was added to each anaerobic bottle as the sacrificial reagent. The irradiation was performed using an array composed of 395 ± 5 nm violet LEDs (3.07 ± 0.14 mW cm
2). The kinetic constants of N2OeN or NO
3 -N reduction were calculated following a first-order reaction (Chen et al., 2019a). 2.2. PEDeN experiments The batch-mode PEDeN experiments were performed in Medium113-M1 containing different concentration of NO
3 -N (1.1e13.5 mg/L) or N2OeN as the electron acceptor. The concen
trations of NO
3 -N, NO2 -N, N2OeN, and N2eN during the reaction were analyzed. The continuous-flow-mode PEDeN experiments were performed in Medium113-M1 containing a constant concentration of NO
2 -N as an electron acceptor. Before irradiation, different volumes of a sterilized and degassed NO
2 -N solution were injected into the reaction solutions to ensure that the final concentrations of NO
2 -N were 0, 0.5, 2, 3, 7.5, 15, and 30 mg/L. Then, a high concentration of NO
2 -N stock solution (300 mg/L) was pumped into the bottles via a peristaltic pump (10.9 mL/min, BT100-1L-A, LongerPump, Baoding) to ensure that the NO
2 -N in solution was constant during PEDeN. The concentrations of NO
2 -N, N2OeN, and N2eN during the reaction were analyzed. To investigate the effects of the ROS on N2O reduction or NO
3 reduction, CH3OH (0.05%, v/v) was added to the medium (N2OeN or NO
3 -N as an electron acceptor) to quench OH during PEDeN. Similarly, 10 mg/L catalase was used for quenching H2O2 and 10 mg/ L superoxide dismutase (SOD) was used for O
 2 quenching. The concentration of N2OeN or NO
3 -N during the reaction was analyzed. 2.3. Effects of cysteine on N2O reduction by T. denitrificans The bare T. denitrificans (OD600 approximately 0.2) was first incubated in 0%, 0.01%, 0.05%, 0.1%, 0.2% and 1% (wt%) of cysteine solutions. After a 3-d reaction (similar to the time required for biohybrid formation), the T. denitrificans was centrifuged, washed, and injected into 20 mL of Medium113-M1 with N2OeN as the electron acceptor. Na2S2O3 was used as electron donors. The influence degree (Gn%) of n% cysteine on N2OeN reduction was calculated based on equation (1):

M. Chen et al. / Water Research 172 (2020) 115501

Gn% ¼ (1-kn%/k0)  100%

(1)

where kn% is the kinetic constant with n% cysteine addition and k0 is the kinetic constant without cysteine addition. 2.4. Recovery of rate of N2O reduction from inhibition Test 1 (without quenching reagents): (I) The T. denitrificans-CdS pellets were injected into Medium113-M1 containing 13.5 mg/L NO
3 -N solution and were illuminated for 48-h (the maximum accumulated FNA was ~0.002 mg N/L); (II) the T. denitrificans-CdS pellets were injected into Medium113-M1 containing 11 mg/L NO
2N solution (equal to ~0.002 mg N/L FNA) and were illuminated for 48-h; (III) the T. denitrificans-CdS pellets were injected into Medium113-M1 containing 11 mg/L NO
2 -N solution and were immerging for 48-h in the dark; (IV) (control experiment): T. denitrificans-CdS was immersed in Medium113-M1 for 48-h in the dark. After the 48-h reaction, the T. denitrificans-CdS pellets were centrifuged, washed, and injected into 20 mL of Medium113-M1 to test the N2OeN reduction performance in PEDeN. Test 2 (with quenching reagents): The T. denitrificans-CdS pellets were injected into Medium113-M1 solution containing (I) 13.5 mg/
L NO
3 -N only (light); (II) 13.5 mg/L NO3 -N þ 0.05% CH3OH (v/v)
(light); (III) 13.5 mg/L NO3 -N þ 10 mg/L catalase (light); (IV) 13.5 mg/L NO
3 -N þ 10 mg/L SOD; (V) control experiment: Medium113-M1 (dark). After the 48-h reaction, the T. denitrificans-CdS pellets were centrifuged, washed, and injected into 20 mL of Medium113-M1 to test the N2OeN reduction performance in PEDeN. 2.5. Analytical methods
The concentrations of NO
3 -N and NO2 -N were determined using ion chromatography (ICS 900, Dionex, Thermo Fisher, USA). The ammonium (NHþ 4 -N) concentration was measured using the indophenol blue colorimetry method. The produced N2O and N2 were analyzed using a robotized system; the details of the procedures were described in a previous study (Chen et al., 2019a). The realtime polymerase chain reaction (RT-PCR) method for analyzing the relative transcription of the denitrifying genes was described in our previous work (Chen et al., 2019a). Amplification reactions were performed in a volume of 20 mL and the reaction mixture contained 10 mL of 2  SYBR® Green Real-time PCR Master Mix, 0.6 mL of each primer (Genscript, Nanjing, China), 5 mL of the cDNA templates, and 3.8 mL of RNase-free water (Chen et al., 2019a). The FNA concentration was calculated using the method described by previous work (Anthonisen et al., 1976). The FNA concentration is defined in Equation (2):

  
c NO
2
N cðFNAÞ mgN L
1 ¼ 0 1 @e

2300
270þT

fluorometric analysis (lex ¼ 316.5 nm, lem ¼ 408.5 nm) of the fluorescent dimer formed in the horseradish peroxidase-catalyzed reaction of H2O2 with p-hydroxyphenylacetic acid (POHPA) using a fluorescence spectrophotometer (G9800A, Agilent Technologies, USA) (Bernardini et al., 2010). The standard curves in the measurements of OH and H2O2 have been provided in Fig. S1a and Fig. S1b, respectively.

3. Results and discussion 3.1. Effects of input NO
3 -N concentration on N2O emission during PEDeN A schematic of PEDeN based on T. denitrificans-CdS is shown in Fig. 1. Upon photoexcitation, the excited photoelectrons from the CdS transfer to the denitrifying enzymes and then reduce NO
3 to N2 þ via the intermediates NO
2 , NO, and N2O. The produced holes h in the valence band are quenched by the sacrificial reagents (lactate was used in this work). The effects of the initial NO
3 -N concentration on N2O emission during PEDeN were investigated first. The nitrogenous species
including NO
3 -N, NO2 -N, N2OeN, and N2eN were monitored. As shown in Fig. 2, a timely N2O emission was observed when the input NO
3 -N was 1.1 or 2.3 mg/L, whereas delayed emission of N2O occurred when the input NO
3 -N was higher than 3.4 mg/L, indicating that the rate of NO
3 -N reduction was higher than that of
NO
2 -N reduction. The change in the initial NO3 -N concentration from 1.1 to 13.5 mg/L significantly affected the conversion amount
of NO
3 -N, i.e., a conversion of NO3 -N to N2eN from 97.3% to 2.3% and NO
-N to N OeN from 0.9% to 52.1% after a 68-h reaction time 3 2 (Table S2). The corresponding value of N2O/(N2O þ N2) changed from 1.0% to 95.0%. In traditional heterotrophic or chemoautotrophic denitrification, it was also observed that an increase in the input NO
3 -N resulted in N2O emission. Alinasfi et al. reported that the conversion of NO
3 -N to N2OeN increased from 1% to 5.1% when the input NO
3 -N concentration increased from 57 to 133 mg/L in a heterotrophic denitrifying activated sludge (Alinsafi et al., 2008). In sulfurbased autotrophic denitrification, the value of N2O/(N2O þ N2) increased from 0 to 25% after increasing the input NO
3 -N concentration from 500 to 2500 mg/L (Park et al., 2002). In both studies, NO
2 -N accumulation was considered the reason for the N2O emission (Alinsafi et al., 2008; Park et al., 2002). A higher concentration of NO
2 -N generates more FNA, thereby inhibiting N2O reduction. In this work, a positive correlation was also found be
tween the input NO
3 -N concentration and the accumulated NO2 -N
(Fig. S2). As shown in Fig. S2c, when the initial [NO3 -N] increased from 1.1 to 13.5 mg/L, the maximum NO
2 -N concentration

(2)

A,10pH

where T is the temperature, which is equal to 30  C.(Gao et al., 2016) All experiments were conducted in triplicate. The concentration of OH that was formed during PEDeN was measured using the terephthalic acid method (Barreto et al., 1994). The intensity of fluorescence of the solution after the 48-h reaction was analyzed with a fluorescence spectrophotometer (lex 320 nm, lem ¼ 425 nm, G9800A, Agilent Technologies, USA). The concentration of O
 2 that was formed during PEDeN was measured using the nitro blue tetrazolium (NBT) method (Chen et al., 2019b). The concentration of H2O2 during PEDeN was monitored using

3

Fig. 1. Schematic of photoelectrotrophic denitrification by T. denitrificans-CdS.

4

M. Chen et al. / Water Research 172 (2020) 115501

Fig. 2. Effects of initial concentration of NO
3 -N (a) 1.1, (b) 2.3, (c) 3.4, (d) 4.5, (e) 9.0 and (f) 13.5 mg/L on the N2OeN and N2eN production during the PEDeN using T. denitrificansCdS.

increased from 1.03 to 11.2 mg/L, corresponding to FNA from 0.00017 to 0.0019 mg N/L. N2O was detected even when the maximum FNA concentration was only 0.00017 mg N/L. To determine the role of FNA in N2O emission, a continuous-flow mode process using stable [NO
2 -N] as electron acceptor was performed. 3.2. FNA inhibits N2O reduction in continuous-flow-mode As shown in Table S3, the concentration of NO
2 -N and the pH during PEDeN remained relatively stable, indicating that the FNA concentration in each assay was relatively constant during the reaction. When the FNA concentration increased from 0 to 0.0036 mg N/L, the conversion rate of NO
2 -N to N2eN decreased from 60.7% to 10.8%, whereas that of NO
2 -N to N2OeN increased from 35.9% to 75.7% (Fig. 3a, Table S3). The rate of N2OeN reduction decreased to approximately 50% when the FNA concentration was 0.0003e0.0004 mg N/L (Fig. 3b). This value is slightly lower than

the value reported by Zhou et al. for a denitrifying phosphorus removal process; an FNA concentration of 0.0007e0.001 mg N/L decreased the N2O reduction activity by 50% (Zhou et al., 2008). However, the inhibition of N2OeN reduction seemed to occur at a very low FNA concentration (<0.00004 mg N/L) (Fig. 3b), which was much lower than the reported values in a sulfide-driven autotrophic denitrification system (>0.0002 mg N/L) and a denitrifying phosphorus removal process (>0.0001 mg N/L) (Zhou et al., 2008; Yang et al., 2016). Furthermore, when the concentration of FNA was closed to 0 mg N/L, 38.1% of N2OeN was still generated from NO
2 -N (Table S3, entry 1). It is possible that the N2O reduction by T. denitrificans-CdS is a slow step because N2O reductase was damaged by cysteine during the formation of bio-hybrids, as mentioned in our previous work (Chen et al., 2019a). To verify that,
NO
3 -N, NO2 -N and N2OeN were used as the electron acceptor, respectively. Fig. S3a shows that the kinetic constant of the N2OeN

Fig. 3. (a) Values of N2O/(N2O þ N2) during PEDeN using stable [NO
2 -N] as the electron acceptor at continue-flow mode. (b) Plots of value of N2O/(N2O þ N2) and rates of N2O reduction with the concentration of FNA.

M. Chen et al. / Water Research 172 (2020) 115501

reduction via PEDeN by T. denitrificans-CdS was 0.021 h
1 (R2 > 0.97, following a first-order reaction), which was only 9.5%
1 and 5.8% of the kinetic constant of the NO
2 -N reduction (0.22 h ,
1 2 R2 > 0.99) and NO
-N reduction (0.36 h , R > 0.98), respectively. 3
The much lower reduction rate for N2OeN than for NO
2 -N or NO3 N explained the easy accumulation of N2OeN during the PEDeN process (Fig. 2). Moreover, Figs. S3bec also shows that the kinetic constant of the N2OeN reduction by T. denitrificans greatly decreased from 1.4 h
1 (R2 > 0.99) to 0.0062 h
1 (R2 > 0.99) as the concentrations of cysteine increased from 0% to 1% (wt%), demonstrating the roles of cysteine in N2O reduction. In a typical formation process of T. denitrificans-CdS, 0.1% cysteine (wt%) was required, causing a decrease in the rate of N2OeN reduction G0.1% by approximately 80% (Fig. S3c). Although the N2O reductase of T. denitrificans-CdS was strongly inhibited by cysteine, the bio
hybrids were still capable of completely converting NO
3 -N/NO2 N to N2eN (Figs. 2 and 3). 3.3. Recovery of rate of N2O reduction from inhibition To further investigate the influence of FNA on N2O reduction, recovery experiments were performed under saturated condition of FNA of approximately 0.002 mg N/L (Fig. 3b). As shown in Fig. 4, when the T. denitrificans-CdS pellets were directly soaked in 0.002 mg N/L FNA solution in the dark for 48-h, the obtained T. denitrificans-CdS showed a recovery of 29.8% (from 0.0594 to 0.0177 h
1). The recovery was 6.6 and 5.2 times larger than the recovery (4.5%, from 0.0594 to 0.0454 h
1) after reducing 11 mg/L NO
2 -N (~0.002 mg N/L FNA) and the recovery (5.7%, from 0.0594 to 0.0034 h
1) after reducing 13.5 mg/L NO
3 -N (maximum accumulated FNA ~0.002 mg N/L), respectively (Fig. 4). This result indicates that FNA inhibition is only partly responsible for the inhibition of N2OeN reduction during PEDeN. The contribution of FNA inhibition was <70.2% because the FNA concentration was less than
0.002 mg N/L in the reduction of 13.5 mg/L NO
3 -N or 11 mg/L NO2 N most of the time. In contrast, the recovery of rate of N2OeN reduction from inhibition was 87% (from 0.0594 to 0.0513 h
1) when the initial NO
3 -N concentration was 1.1 mg/L (Fig. S4). Therefore, several conclusions were deduced from these results: first, in addition to FNA inhibition, other factors contributed to the inhibition of N2O reduction during the PEDeN process; Second, these influencing factors resulted in lower recovery of rate of N2O reduction from inhibition; third, the extent of the inhibition of N2O


reduction was related to the input concentration of NO
3 -N/NO2 -N.

3.4. ROS generation during PEDeN The concentrations of the main ROS (OH, H2O2, and O
 2 ) were measured after the 48-h PEDeN reaction. Fig. 5aec shows that the concentrations of OH, H2O2 and O
 2 increased with the input

[NO
3 -N]/[NO2 -N], indicating that a higher concentration of NO3 -N/

 NO
2 -N generated more OH, H2O2, and O2 . In addition, more O2 , OH and H2O2 formed in the reduction of the equivalent amount of
N of NO
2 -N than NO3 -N. In contrast, the amount of generated OH, H2O2, and O
 2 were lowest in the reduction of N2OeN and the concentrations of these ROS were not significantly difference from those of the input [N2OeN]. When the concentrations of the input nitrogenous species were lower than 24 mg N/L, the concentration of OH was in the range of 0.5e0.5 mM, whereas the concentration of H2O2 was in the range of 50e350 mM. The concentration of O
 2 in this system could not be calculated precisely because the product generated by the reaction of O
 2 with NBT was easily adsorbed on the surface of the bio-hybrids (Nosaka and Nosaka, 2017). Therefore, the method of NBT was used for a semi-quantitative calculation of O
 2 . There are three possible pathways for ROS generation in PEDeN system. 1) The photo-absorption of the nitrogenous species produces ROS (Scholes et al., 2019). To verify this, the Medium113-M1 solutions (no bio-hybrids) containing high/low levels of NO
3 -N/ NO
2 -N/N2OeN were irradiated for 48-h and the concentration of the generated OH, H2O2, and O
 2 were determined. However, there was no difference in the OH, H2O2, and O
 2 concentrations compared to the control experiment (medium113-M1 only), indicating that few ROS were generated solely from the nitrogenous species. This result is consistent with that of a previous report, in
which the excitation wavelength of NO
3 -N/NO2 -N was in the UV range (normally <350 nm) (Mack and Bolton, 1999). 2) The higher
concentration of NO
3 -N or NO2 -N facilitated the denitrification process, producing more NADPH and ROS based on equations (3)e(5) (Clifford and Repine. 1982). 3) The higher concentration of
NO
3 -N or NO2 -N, which served as electron donors, may have promoted the charge separation of CdS, producing more e- and holes hþ (equations (6)), and promoting ROS formation (equations (3) and (7)) (Wang et al., 2019). NADPH

O2 þ e
!O
$ 2 SOD


2O
$ 2 !H2 O2 þ e Fe2þ

H2 O2 !$OH þ OH
hv

Fig. 4. Recovery of rate of N2OeN reduction from inhibition (without quenching reagents). N2OeN reduction via PEDeN by T. denitrificans-CdS after (I) reducing 13.5 mg/L
NO
3 -N under irradiation; (II) reducing 11 mg/L NO2 -N under irradiation; (III) immerging in 11 mg/L NO
2 -N solution in the dark; (IV) immerging in Medium113-M1 in the dark.

5

(3) (4) (5)

CdS !hþ þ e


(6)

OH
þ hþ /$OH

(7)

The higher generation of ROS in the NO
2 reduction process than the NO
3 reduction process may have resulted from the faster
photoelectron transfer in the NO
2 -N reduction than the NO3 -N reduction during denitrification process. Although the rate of NO
2N reduction by T. denitrificans-CdS was lower than the rate of NO
3N reduction, the electron-accepting pathways differ for NO
2 -N
reduction and NO
3 -N reduction. In the reduction of NO3 -N, the electrons are first transferred to the quinone pool and then to NO
3 reductase, whereas in the reduction of NO
2 -N, the electrons are transferred to NO
2 reductase via c-type cytochrome (Chen and

6

M. Chen et al. / Water Research 172 (2020) 115501


Fig. 5. Generated ROS with the concentration of input nitrogenous species (NO
3 -N/NO2 -N/N2OeN) during the 48-h PEDeN reaction by T. denitrificans-CdS: (a) OH, (b) H2O2, (c)
O
 2 . (d) Recovery of rate of N2O reduction from inhibition (with quenching reagents). N2OeN reduction via PEDeN by T. denitrificans-CdS after (I) reducing NO3 -N by PEDeN; (II)

reducing NO
-N with 0.05% CH OH addition by PEDeN; (III) reducing NO -N with 10 mg/L SOD addition by PEDeN; (IV) reducing NO -N with 10 mg/L catalase addition by PEDeN; 3 3 3 3
(V) immerging in Medium113-M1 in the dark. Input [NO
3 -N] ¼ 13.5 mg/L. (e) Effects of quenching reagents on the reduction of NO3 -N (~12 mg/L) and N2OeN (~12 mg/L) via PEDeN.

Strous, 2013). The lack of a difference in the ROS concentration compared to that of [N2OeN] might be attributed to the fact that the concentrations of N2OeN in the liquid phase did not exhibit large differences. 3.5. Reactive oxidative species inhibit N2O reduction To verify the influences of the ROS on N2O reduction, different quenching reagents were used to trap the ROS immediately during PEDeN using NO
3 -N as the electron acceptor. The recovery of rate of N2O reduction from inhibition was evaluated. In the presence of CH3OH, SOD and catalase, the recovery of rate of N2O reduction was 5.7% (from 0.0594 to 0.0034 h
1), 9.9% (from 0.0594 to 0.0059 h
1), 19.5% (from 0.0594 to 0.0116 h
1), respectively (Fig. 5d). Catalase decomposes H2O2 to O2 (equation (8)) and SOD decomposes O
 2 to H2O2 (equation (4)). Since the decomposed products O2 and H2O2 are also the inhibitors of N2O reductase, the enhanced recovery of rate of N2O reduction after the addition of catalase and SOD inferred that 1) the impact of H2O2 on N2O reduction was greater than the impact of equivalent amount of O2 and 2) the impact of O
 2 on N2O reduction was greater than the impact of equivalent amount of H2O2. Therefore, the contributions of OH, O
 2 , and H2O2 to the inhibition of N2O reduction were 1.3%, >5.4% and >15.0%, respectively, in comparison to the recovery (4.5%) in the absence of the quenching reagents. catalase

2H2 O2 !O2 þ 2H2 O

(8)

It was reported that H2O2 appears to cause a rearrangement in the catalytic center (tetranuclear Cuz [4CuþS]) of N2O reductase and a loss of a part of its copper (Pauleta et al., 2013; Haltia et al., 2003). Thus, the depletion of N2O reductase may have been the result of

the difficult recovery of the activity of N2O reductase after N2O reductase was exposed to H2O2. The generated OH is also capable of oxidizing Cuz. However, since OH is strongly oxidative, it is easily quenched by organic species (such as sacrificial reagents), which results in lower contributions to the inhibition of N2O reductase than H2O2 or O
 2 . The toxicity of ROS to microbe was widely mentioned in previ et al., 2000). ous work (Clifford and Repine. 1982; Cabiscol Catala Thus, it is possible that the generated ROS weakened the activity of microbe, thereby decreasing the activity of N2O reductase. However, the influence of the ROS on NO
3 -N reduction was significantly different from the influence on N2OeN reduction. As shown in Fig. 5e, the addition of CH3OH/SOD/catalase increased the rate of NO
3 -N reduction by 29%, 29%, and 37.8%, respectively. The effects of the three quenching reagents on NO
3 -N reduction were not significantly different. In comparison, the addition of SOD and catalase increased the rate of N2OeN reduction by 177% and 226%, whereas the addition of CH3OH only increased the rate by 20%; this result is in agreement with the results in Fig. 5d. It implies that the ROS, especially H2O2 and O
 2 , have a larger influence on N2O reductase than on NO
3 -N reductase. It is well known that N2O reductase is the most sensitive enzyme among the four N-oxide reductases involved in denitrification (Qu et al., 2016). Therefore, the inhibition of N2O reduction by ROS resulted from that N2O reduction step exhibited great susceptibility to the ROS, rather than the toxicity of ROS on microbes. It is noteworthy that when N2OeN was used as the input nitrogenous species, no FNA was observed. The addition of SOD and catalase increased the rates of N2OeN reduction 2.7 times (from 0.0165 to 0.0458 h
1) and 3.2 times (from 0.0165 to 0.0597 h
1), respectively (Fig. 5e). In this case, the concentration of H2O2 was approximately 50 mM (Fig. 5b). The ROS had a larger influence on

M. Chen et al. / Water Research 172 (2020) 115501
the N2O reduction in the reduction of NO
3 -N/NO2 -N because more ROS were generated (Fig. 5aec). Since the ROS were immediately generated upon illumination, the inhibition of N2O reduction by the ROS always occurs and is inevitable during PEDeN. Therefore, in some cases, such as under an inhibitory concentration of FNA, the ROS play a dominant role in inhibiting N2O reduction. This is the reason for the larger N2O emissions resulting from a slight increase
in the initial NO
3 -N or NO2 -N concentration at extremely low N levels, for example, an increase in the FNA concentration from 0 to 0.00004 mg N/L (Fig. 3b). Moreover, the inhibition of N2O reduction by the ROS is non-competitive, resulting in accumulated damage of N2O reductase and difficult recovery of rate of N2O reduction from inhibition.

3.6. Mechanism of the inhibition of N2O reduction To confirm the inhibition of N2O reduction occurring at the gene level, real-time PCR was used to analyze the relative transcription of the denitrifying genes. Fig. 6a shows that the relative transcription of gene nosZ was significantly down-regulated from 1.46 ± 0.15 to 1.00 ± 0.05 as the input [NO
3 -N] increased from 2.3 to 9.0 mg/L. Moreover, the up-regulation of the relative transcription of nosZ was three times higher in the batch with the addition of NO
3 -N (2.25  4 mg/L) than in the batch with the one-dose addition (9.0 mg/L), strongly supporting the inhibition of N2O reductase in the high-concentration [NO
3 -N] input (Fig. 6b). The relative transcription of gene narG was significantly upregulated from 1.00 ± 0.08 to 2.05 ± 0.03 as the input [NO
3 -N] changed from 2.3 to 9.0 mg/L (Fig. 6c). It is interesting to find that the relative transcription of the nirB gene, which encodes the enzyme of dissimilatory nitrite reduction to ammonium (DNRA), significantly increased from 1.00 ± 0.16 to 2.49 ± 0.18 as the [NO
3 -N] increased

7

from 2.3 to 9.0 mg/L (Fig. 6d). This result explains the increase in
the production of NHþ 4 -N from 0.9 to 52.1% as the [NO3 -N] increased from 1.1 to 13.5 mg/L (Table S2). The irreversible inhibition of N2O reduction enables the pathway of DNRA and promotes NHþ 4 -N production. This result is consistent with the results of previous reports, which stated that sulfide inhibition of N2O reductase resulted in incomplete denitrification, driving the electron flow to NHþ 4 (Sørensen et al., 1980; Brunet and Garcia-Gil, 1996). In summary, the ROS (Pathway 1) and FNA (Pathway 2) are the main factors affecting the inhibition of N2OeN reduction during PEDeN (Fig. 7). In the reduction of 13.5 mg/L NO
3 -N, recovery of rate of N2O reduction was 4.5%. The contributions of OH, H2O2 and O
 2 to the inhibition of N2O reduction were 1.3%, >5.4%, and >15.0%, respectively. The accumulated 11 mg/L NO
2 -N produced ~0.002 mg N/L FNA, thereby decreasing the activity of N2O reductase <70.2%. Although FNA played the dominant role in the inhibition of N2O reduction at this concentration, the ROS are dominant in the case of an inhibitory concentration of FNA. 3.7. Implications Since PEDeN is a novel concept, the mechanism of N2O emission in this process remain unclear. In addition to the common-sense “FNA inhibition”, the ROS generated during photochemical reaction represents a unique factor affecting N2O emissions during the PEDeN process. The ROS not only generate toxicity to microorganisms but, more importantly, they also have a large impact on N2O reductase, which is an oxygen-sensitive enzyme. Although it is well-known that O2 inhibits N2O reduction (Law et al., 2012), the ROS, especially H2O2 and O
 2 , have a larger influence than O2. The ROS cause oxidative damage to the structure of N2O reductase,

Fig. 6. The relative transcription of gene nosZ (aeb), narG (c) and nirB (d) with different initial [NO
3 -N] after 48-h reaction. The control experiment was T. denitrificans-CdS (dark). An asterisk (*) represents a significant difference P < 0.05, while two asterisks (**) represent a significant difference P < 0.01.

8

M. Chen et al. / Water Research 172 (2020) 115501

Fig. 7. Pathways of inhibition of N2OeN reduction during PEDeN by T. denitrificans-CdS. N2OR represents N2O reductase.

resulting in low recovery of the activity of N2O reductase due to inhibition. Since ROS generation during the PEDeN process is inevitable, N2O gas is easily generated via during this process. N2O purity higher than 95% can be achieved. This process is an economical and sustainable method to recover N2O from wastewater treatment in comparison to technologies using reductive iron(II) salt or the coupled aerobic-anoxic nitrous decomposition operation (CANDO) process, which are costly and result in mass sludge production (Zhang et al., 2019). The purity of N2O depends on the concentrations of ROS. Therefore, higher purity of N2O will be obtained after
enhancing input NO
3 -N/NO2 -N or loading of semiconductors in practice. However, since N2O is a strong greenhouse gas, it is necessary to further evaluate the environmental sustainability, economic viability and engineering feasibility of recovering energy from N2O (Zhang et al., 2019). On the other hand, the N2O emissions during the PEDeN process can be reduced by adding ROS quenchers or using SOD/catalase-like materials as coating for bacteria to decompose O
 or H2O2 immediately, such as metal-organic 2 frameworks as coating for bacteria for cytoprotection (Ji et al., 2018). In this work, the concentration of H2O2 was 0e400 mM (Fig. 5b), which is in the range of the concentration of ROS ([OH] ¼ 6.7e2531 mM and H2O2 is the precursor) generated by sediments oxygenation in the dark (Tong et al., 2016). The 50 mM H2O2 decreased the rate of N2O reduction by 72.4% from 0.0597 to 0.0165 h
1. Therefore, ROS derived from sediments may also have a large influence on N2O reduction and cause N2O emission. It was reported that the rates of H2O2/O
 2 production induced by sunlight were (6.2e41.0)  1011 mol L
1 s
1 and (8.6e22.0)  108 mol L
1 s
1 in coastal seawater (Petasne and Zika, 1987). These concentrations of ROS were much higher than those generated in this work. The level of ROS induced by sunlight depends on the intensity of sunlight but ROS are also generated at the bottom of shallow water, such as the coastal sea, where they participate in denitrification (Canfield et al., 2010; Petasne and Zika, 1987). Therefore, the inhibition of ROS on N2O reduction may be ubiquitous. Since the effect of ROS on N2O reduction has rarely been investigated, natural N2O emissions may have been underestimated. The results of this study encourage a reexamination of the process of N2O emissions in nature, particularly in systems in which ROS are present during the denitrification process. 4. Conclusion In this work, the mechanisms of N2O emission during PEDeN process by bio-hybrids were revealed. The main conclusions are: (1) This work demonstrated in addition to free nitrous acid (FNA), reactive oxidative species (ROS) were a unique factor

affecting N2O emission during PEDeN. The N2O reduction step exhibited great susceptibility to the ROS. (2) The contributions of H2O2, O
 2 , OH and FNA to the inhibition of N2O reduction were >15.0%, >5.4%, 1.3% and <70.2%, respectively for a reduction of 13.5 mg/L NO
3 -N.
(3) The system was sensitive to the input NO
3 -N and NO2 -N, resulting in changes in the N2O/(N2þN2O) ratio from 1% to 95%. (4) A significant down-regulation of the relative transcription of the gene nosZ demonstrated that the inhibition of N2O reductase occurred at the gene level. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments This work was supported by the National Science Fund for Distinguished Young Scholars (41925028), the National Natural Science Foundation of China (41671264) and the Project of Fujian Agriculture and Forestry University Program for Distinguished Young Scholar (No. XJQ2018003). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.watres.2020.115501. References Alinsafi, A., Adouani, N., Beline, F., Lendormi, T., Limousy, L., Sire, O., 2008. Nitrite effect on nitrous oxide emission from denitrifying activated sludge. Process Biochem. 43 (6), 683e689. Anthonisen, A.C., Loehr, R.C., Prakasam, T.B.S., Srinath, E.G., 1976. Inhibition of nitrification by ammonia and nitrous acid. J. Water Pollut. Control Fed. 48 (5), 835e852. Barreto, J.C., Smith, G.S., Strobel, N.H.P., McQuillin, P.A., Miller, T.A., 1994. Terephthalic acid: a dosimeter for the detection of hydroxyl radicals in vitro. Life Sci. 56 (4), PL89ePL96. Bernardini, C., Cappelletti, G., Dozzi, M.V., Selli, E., 2010. Photocatalytic degradation of organic molecules in water: photoactivity and reaction paths in relation to TiO2 particles features. J. Photochem. Photobiol. A Chem. 211 (2), 185e192. Brune, A., Frenzel, P., Cypionka, H., 2000. Life at the oxiceanoxic interface: microbial activities and adaptations. FEMS Microbiol. Rev. 24 (5), 691e710. Brunet, R.C., Garcia-Gil, L.J., 1996. Sulfide-induced dissimilatory nitrate reduction to ammonia in anaerobic freshwater sediments. FEMS Microbiol. Ecol. 21 (2), 131e138. Cabiscol Catal a, E., Tamarit Sumalla, J., Ros Salvador, J., 2000. Oxidative stress in bacteria and protein damage by reactive oxygen species. Int. Microbiol. 3, 3e8. Canfield, D.E., Glazer, A.N., Falkowski, P.G., 2010. The evolution and future of earth’s nitrogen cycle. Science 330 (6001), 192. Chen, J., Strous, M., 2013. Denitrification and aerobic respiration, hybrid electron

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