# Weak electricity stimulates biological nitrate removal of wastewater: Hypothesis and first evidences

Hongbo Liu, Feiyu Ouyang, Zihua Chen, Zhongbing Chen, Eric Lichtfouse

► To cite this version:

Hongbo Liu, Feiyu Ouyang, Zihua Chen, Zhongbing Chen, Eric Lichtfouse. Weak electricity stimulates biological nitrate removal of wastewater: Hypothesis and first evidences. Science of the Total Environment, 2021, 757, pp.1-9. 10.1016/j.scitotenv.2020.143764. hal-03325206

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# Weak electrical stimulation on biological denitrification: insights from the denitrifying enzymes

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Xinyi Dong¹ᵃ, Hongbo Liu¹*ᵃ, Shiping Longᵇ, Suyun Xu*ᵃ, Eric Lichtfouseᶜ

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a School of Environment and Architecture, University of Shanghai for Science and Technology, 516 Jungong Road, 200093, Shanghai, China

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b Chongqing New World Environment Detection Technology Co.LTD, 22 Jinyudadao, 401122, Chongqing, China

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c Aix-Marseille Univ, CNRS, IRD, INRA, Coll France, CEREGE, 13100 Aix en Provence, France

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**Abstract:** In order to improve the denitrification efficiency of low carbon to nitrogen ratio (C/N) wastewater, we conducted continuous flow experiments of weakly electrically stimulated denitrification using a direct current output voltage. The results showed that the best denitrification was achieved at a voltage of 0.2 V. The removal of nitrate and total nitrogen was increased by 20% and the production of intermediate greenhouse gas ($N_2O$) was reduced by 62.6%. We explored the specific pathways involved in the weak electrical stimulated denitrification using enzyme activity as a cut-off point. The enzyme activity analysis and 3D fluorescence spectroscopy revealed that nitrate reductase (NAR) and nitrite reductase (NIR) activities were significantly enhanced by weak electrical stimulation, and the aromatic protein content in extracellular polymers substances (EPS) increased, accelerating electron transfer and promoting the conversion of loosely bound EPS (LB) to tightly bound

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<footnote>* Corresponding author ADD: 516, Jungong Road, 200093, Shanghai, China; Email: Liuhb@usst.edu.cn (H., Liu); xusy@usst.edu.cn (S., Xu) Tel: +86(21)55275979; Fax: +86(21)55275979 1 Xinyi Dong and Hongbo Liu contribute equally to this work</footnote>
EPS (TB). The accelerated electron transfer further increased enzyme activity and the metabolic rate of microorganisms. This study indicates that weak electrical stimulation could improve activities of biological enzymes to enhance denitrification efficiency.

**Keywords:** Weak electrical stimulation; Denitrification; Enzyme activity; EPS; Electron transfer

## 1. Introduction

Increasingly prominent nitrogen pollution of water bodies poses a serious threat to human health and ecological functions[1, 2]. Biological nitrogen removal has become one of the cost-effective methods for nitrogen removal because of its high efficiency and low cost. Biological denitrification is completed by aerobic nitrification ($NH_4^+$-N—$NO_2^-$-N—$NO_3^-$-N) and anoxic denitrification ($NO_3^-$-N—$NO_2^-$-N—$N_2$) using aerobic nitrification bacteria and denitrification bacteria respectively[3]. However, in practical wastewater applications, when the C/N (COD/TN) ratio of wastewater is low, the microorganisms will not be able to perform efficient denitrification due to insufficient carbon source[4]. To solve this issue, researchers have combined microbial fuel cells (MFC) with denitrification as an alternative to additional dosage of carbon sources such as methanol and acetate for the treatment of low C/N wastewater[5, 6]. In this case nitrate and nitrite acted as electron acceptors in the cathode chamber of MFC. Under the catalytic action of denitrifying microorganisms, the cathode electrode electrons could be used for electrochemical denitrification directly while energy could be recovered[7, 8]. Thus in recent years, studies using voltages lower than the hydrolysis voltage (1.23V) to promote microbial processes have attracted wide attention[9].
Studies have shown that electrical stimulation at constant potential shortens the duration of the growth arrest period of denitrifying bacteria cells and significantly increases the denitrification efficiency[10]. At the same time, microbial activity increases with current density[11]. Higher current intensity promotes denitrification at surface of the MFC cathode, increasing denitrifying bacteria activity, and leading to further denitrification[12]. When the current intensity exceeds a certain range, it also has an inhibitory effect on microbial activity, which affects the denitrification reaction[13]. Denitrification is carried out gradually by specific enzymes as shown in equations 1-4.

$$2NO_3^- + 4H^+ + 4e^- \xrightarrow{NAR} 2NO_2^- + 2H_2O \quad (1)$$

$$2NO_2^- + 4H^+ + 2e^- \xrightarrow{NIR} 2NO + 2H_2O \quad (2)$$

$$2NO + 2H^+ + 2e^- \xrightarrow{NOR} N_2O + H_2O \quad (3)$$

$$N_2O + 2H^+ + 2e^- \xrightarrow{NOS} N_2 + H_2O \quad (4)$$

Wang et al.[14] investigated the effect of carbon nanotubes on the denitrification performance of the alkali-producing gene and found that denitrification could be promoted by increasing the activity of NAR, NIR and nitric oxide synthase (NOS). This promotion is directly responsible for the increased nitrate removal efficiency and reduced $N_2O$ production. Previous studies have shown that denitrifying enzyme activity is influenced by electron acceptors[15], C/N[16], pH and other factors[17]. The decrease in C/N ratio affected synthesis of denitrifying enzymes and hindered the growth of denitrification microorganisms. Low pH and high $H^+$ concentration caused unstable enzyme structure and inhibited the NAR activity.

In this study, we used direct current (DC) power supply to study the effect of weak electrical stimulation on the denitrification process by adjusting the C/N ratios and weak
electrical stimulation intensity, and investigate the pathway of weak electrical stimulation and
its effect on denitrification performance from the perspective of denitrifying enzymes.

Components of produced gas during denitrification have been detected together with
denitrification efficiency analysis of the process. The effect of weak electrical stimulation on
microbial metabolism was explored through denitrifying enzyme activity analysis, EPS
content detection and distribution profiling. The mechanism of weak electrical stimulation on
microbial denitrification enhancement was further proposed, providing a new tool improving
denitrification performance under low carbon source water quality conditions.

## 2. Materials and methods

### 2.1 Experimental setup

Fig.1 shows schematic diagram of the experimental setup. The volume of the
experimental set-up in this study was 1.2 L. It was divided into a control group (CK, no
electrical stimulation set-up) and an experimental group (EG, electrical stimulation set-up).
The electrical stimulation voltage values of 0.2 V (0.567mA/cm²), 0.4 V (1.12mA/cm²) and
0.6 V (1.74mA/cm²) were selected according to results of our previous study[18]; while the
corresponding current intensity was set as the output value of the DC power supply. The
experimental electrodes and the usage method are also consistent with the previous study[18].
In this study, 200 mL of anaerobic denitrification sludge and 800 mL of artificial wastewater
were fed to the reactor in a continuous ascending flow with a hydraulic retention time of 12
hours. The C/N ratio was set at two values as 4 (COD and NO₃⁻-N concentrations of 200 and
50 mg/L, respectively) and 3 (COD and NO₃⁻-N concentrations of 150 and 50 mg/L,
respectively).
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**Insert Fig. 1. Continuous flow of weak electrically stimulated denitrification schematic diagram of the experimental setup**

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The main components of the low C/N wastewater are shown in Table 1. The components of the concentrated solution of trace elements are 1.61 g/L $CoCl_2 \cdot 6H_2O$, 1.5g/L $FeSO_4 \cdot 6H_2O$, 0.15 g/L $H_3BO_3$, 0.18 g/L KI, 0.12 g/L $ZnSO_4 \cdot 7H_2O$, 0.12 g/L $MnCl_2 \cdot 4H_2O$, 0.03 g/L $CuSO_4 \cdot 5H_2O$ and 0.06 g/L $Na_2MoO_4 \cdot H_2O$.

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**Insert Table 1. Composition of the artificial simulated wastewater**

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The anaerobic denitrification sludge was obtained from the East Shanghai Wastewater Treatment Plant (31°16′40.50″N, 121°32′39.99″E) and domesticated using the same conditions as the experimental synthetic wastewater. The sludge was 100% returned and replenished with new sludge every three to five days to maintain a sludge concentration of about 5000 mg/L in the reactor.

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## 2.2 Analytical methods

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### 2.2.1 Analysis of nitrogenous pollutants

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Sludge sampling was carried out every 12 hours. After adjusting the equipment current intensity and/or C/N ratios and other parameters, it needs to be stabilized for 3-5 days before sampling. The concentrations of $NH_4^+$-N, $NO_2^-$-N, $NO_3^-$-N and TN were determined by the nano-reagent photometry, N-(1-naphthyl)-ethylenediamine spectrophotometric, ultraviolet spectrophotometry and potassium persulfate to eliminate ultraviolet spectrophotometric determination respectively.

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### 2.2.2 Gas component detection

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Gas production was collected continuously for 7 days at an optimum voltage of 0.2 V to
analyze the effect of weak electrical stimulation on the denitrification process. The hydrogen, nitrogen and nitrous oxide were measured by the Shimadzu GC-14B gas chromatograph (Japan), Thermo TRACE 1300 gas chromatograph (USA) and Agilent 6890N gas chromatograph (USA), respectively. The experimental samples were collected using an E-Switch sampling bag, and a 1 mL syringe was used for the measurement. The injection volume of hydrogen, nitrogen and nitrous oxide were 1.0 mL, 0.1 mL and 1.0 mL respectively.

A single point calibration was performed using the lowest concentration point when the sample concentration was well below the lowest point of the standard series (The standard curve is shown in Fig.S1). The measuring conditions are listed as in Table 2.

<caption>**Insert Table 2. Conditions for determination of hydrogen, nitrogen and nitrous oxide**

**2.2.3 Denitrifying enzymes (NAR, NIR) extraction and activity determination**</caption>

We investigated the effect of weak electrical stimulation on denitrifying microbial activity under weak electrical stimulation by measuring NAR and NIR activities. The mixed liquid suspended solid from different groups was centrifuged at 5000 r for 5 min at 4 °C. The centrifuged precipitate was rinsed with 0.01 M phosphate buffer (PBS) at pH 7.4 for three times, resuspended in 0.01 M PBS buffer at pH 7.4, and sonicated in an ice bath (20 kHz) for 5 min. After sonication, the cell suspension was placed in a centrifuge at 12000r for 15 min at 4°C, and the supernatant was then taken and stored. The reaction system consisted of 1.0 mL of crude enzyme extract with 0.01 M PBS (pH=7.4), 1.0 mM NaNO₃, 1.0 mM methyl violet gum and 5.0 mM Na₂S₂O₄. The reaction system was placed in a thermostat and shaken for 30 min at 30°C, and then the nitrite concentration was measured by an ultraviolet spectrophotometer (UV-2600 Shimadzu, Japan). NAR and NIR enzyme activities were
128 expressed as NO₃⁻-N produced or reduced during the reaction (µg NO₃⁻-N/min--mg protein)[19].
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### 2.2.4 Analysis of extracellular polymers substances (EPS)

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The extracellular polymer substances (EPS) was extracted from the sludge by thermal
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extraction[20] after filtered through a 0.45 µm membrane to remove the particulate matter and
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then analyzed qualitatively using a 3D fluorescence spectrometer F-7000 FL (Hitachi, Japan).
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The excitation wavelength of the 3D fluorescence spectrometer was set from 200 to 420 nm,
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and the emission wavelength was set from 250 to 550 nm in increments of 2 nm. The slit
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widths of both excitation and emission were 2.5 nm, and the scanning speed was 12000
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nm/min. The EPS was also quantified and the protein content in the supernatant of the
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samples was determined by the Komas Brilliant Blue method[21].
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## 3. Results and discussion

### 3.1 Effect of weak electrical stimulation on denitrification efficiency

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Fig.2 shows the variation of NO₃⁻-N, NO₂⁻-N, NH₄⁺-N and TN concentrations under the
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influence of different C/N and microbial weak electrical stimulation voltages. By comparing
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the performances at different voltages in Fig.2, it can be found that the best effect of microbial
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weak electrical stimulation is at 0.2 V under both C/N ratios of 3 and 4. The total nitrogen and
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nitrate in the EG group with the C/N ratio of 3 decreased to about 20 mg/L and 17 mg/L at a
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weak stimulation voltage of 0.2 V, while the total nitrogen and nitrate in the CK group
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decreased to about 24 mg/L and 20 mg/L, respectively. The removal rates of total nitrogen
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and nitrate in the EG group increased both by 8% compared with those in the CK group
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(Fig.2a, b). When the microbial weak electrical stimulation voltage was 0.4 V, there was little
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difference between the degradation in the CK group and the EG group; while at the weak
electrical stimulation voltage of 0.6 V, the nitrate removal rate in the EG group was lower
than that in the CK group. It could be speculated that under relatively low carbon source
conditions, the degradation effect may be negatively correlated with an increase in the weak
electrical stimulation, with 0.6 V corresponding to a weak electrical stimulation intensity that
is detrimental to microbial metabolisms. The above results are comparable to the results of
Prosňanský et al.[22]. The best effect was obtained when the current density was 0.27
mA/cm², and there was no difference between the experimental group and the control group
when the current density is 0.467 mA/cm². The denitrification rate decreased when the current
density was greater than 1.2 mA/cm², and when the current density exceeded 2 mA/cm², the
denitrification effect decreased significantly.

<caption>Insert Fig.2 Removal effect and pH change of weakly electrically stimulated denitrification continuous flow experiment;(a)TN, (b)NO3⁻-N, (c) NO2⁻-N , (d) NH4⁺-N,(e)pH</caption>

Under the condition of an influent C/N of 4, the total nitrogen and nitrate could be
reduced to about 11 mg/L and 9 mg/L in the EG group and about 22 mg/L and 18 mg/L in the
CK group; meanwhile, the total nitrogen and nitrate removal rates in the EG group were
improved by about 12% and 20%, respectively, compared with the CK group (Fig.2a, b).
Microbial heterotrophic denitrification using organic matter or autotrophic denitrification
using hydrogen are both alkali-producing processes, and the reduction of 1 mg of nitrate can
simultaneously produce 3.57 mg of alkalinity. As shown in Fig.2e, the alkalinity of the EG
effluent was higher than that of the CK effluent, indicating that weak electrical stimulation
172 has a positive effect on the denitrification process.

173 The variation curves of nitrite nitrogen and ammonia nitrogen in Fig. 2 c, d showed that 174 nitrite nitrogen in the effluent of the EG group was lower than that of the CK group under the 175 effect of weak electrical stimulation at 0.2 V, 0.4 V and 0.6 V, while the concentration of 176 ammonia nitrogen showed an opposite trend. There are two main pathways of biological 177 nitrogen cycling associated with nitrate in nature, in addition to biological denitrification, and 178 the dissimilatory nitrate reduction to ammonium (DNRA)[23].

$$179 \quad NO_3^- + 8e^- + 10H^+ \rightarrow NH_4^+ + 3H_2O \quad (5)$$

180 NH₄⁺-N is the end product of the DNRA process, and it can be demonstrated from Fig.2d 181 that there was an accumulation of NH₄⁺-N in the EG group under weak electrical stimulation, 182 partly because the high ammonia nitrogen content of the synthetic water after sludge dilution 183 and partly because DNRA was presumed to have occurred during denitrification. Meanwhile, 184 the degree of accumulation in the EG group increased with the increase of voltage compared 185 to the CK group, indicating that electrical stimulation accelerated the DNRA process. 186 According to Fig.4, the NIR activity of the EG group was enhanced by weak electrical 187 stimulation, which reduced the accumulation of nitrite due to insufficient carbon source.

188 The above results showed that the effect of weak microbial electrical stimulation on 189 nitrate-containing wastewater was largely positive at different voltages and C/N ratios. the 190 highest removal efficiencies for both TN and nitrate nitrogen were obtained at 0.2 V. the 191 nitrite nitrogen content was significantly higher in the CK group than in the EG group.

192 **3.2 Variation of gas production & compositions under weak electric stimulation**

193 When the denitrification efficiency reached the highest value with a weak electrical
stimulus of 0.2V, we collected gas production fractions from the EG group at 0.2V for comparison with the CK group. Gas production during the denitrification process varies under different C/N conditions[24]. Fig.3a,b and c demonstrated H₂, N₂ and N₂O contents of the denitrification gas at the optimal weak electrical stimulation intensity (0.2 V) for C/N ratios of 3 and 4. The percentage of H₂ content in the gas produced by both EG and CK groups was at a lower level at C/N ratios of 3 and 4, but percentage of H₂ content in the EG group increased compared to the CK group, presumably because the accelerated electron transfer by the applied voltage promoted the activity of hydrogen-producing bacteria. It is generally believed that autotrophic denitrifying microorganisms use the H₂ produced by cathode electrolysis as an electron supply. H₂ can replace organic carbon as an electron donor, relieving the challenge of insufficient carbon source, and promoting denitrification, as shown in Fig.2. The N₂ content in the produced gas was lower in the EG group than in the CK group since the microbial activity of N₂O reductase synthesis was probably inhibited by DOC depletion in the EG group at the late stage of denitrification due to the low carbon source, while the anaerobic ammonia oxidation process could exist in the CK group at the same time, and NH₄⁺ replaced DOC as an electron donor to undergo redox reactions with NO₂⁻, and then more N₂ was produced[25].

**Insert Fig.3 Denitrification gas production at the optimal weak electrical stimulation intensity of 0.2 V; (a) H₂, (b) N₂, (c) N₂O**

It was controversial that when the C/N ratio is different, the N₂O content changes in the opposite trend. At a C/N ratio of 3, it was evident that lower levels of N₂O were produced with the 0.2 V weak electrical stimulation, representing a more complete denitrification
process. In contrast, at a C/N ratio of 4, the N₂O production was higher with 0.2 V weak electrical stimulation. This may be partly due to the accumulation of nitrite (Fig. 2c), which leads to the production of N₂O[26], and partly due to the weak electrical stimulation, which enhances the denitrifying enzyme activity. Denitrification by microorganisms consists of several stages, such as NO₃⁻→NO₂⁻→NO→N₂O→N₂. The microorganisms and related enzymes NAR, NIR, NOR and N₂OR in each stage are different, so are the effects of weak electrical stimulation on the activities of different microorganisms and enzymes. Interactions between enzymes can affect the electron supply in endogenous denitrification. Restricted electron supply will lead to a significant accumulation of N₂O in the denitrification phase[27]. According to the microbial community detection and analysis results (Fig.5), it can be concluded that weak electrical stimulation plays a selective driving role for autotrophic and heterotrophic denitrifying bacteria. Under 0.2 V weak electrical stimulation, the genus abundance of microbial communities in the EG group was significantly increased compared to the CK group, especially *Thaurea* (8.2% and 10.4%), *Rhodocyclaceae* (8.0% and 9.1%), *Denitratisoma* (3.1% and 3.4%), *Candidatus Competibacter* (1.3% and 1.5%), and other microorganisms involved in the denitrification process. Most *Candidatus Competibacter* can only undergo NO₃⁻-N reduction but not NO₂⁻-N reduction because they form NAR but not NIR[28]. In contrast, *Thauera* was able to determine the reduction of NO₃⁻-N to nitrogen. From this we speculate that it is the competition for electrons between various enzymes during denitrification that affects N₂O accumulation[16],[29], causing both exogenous and endogenous denitrification reactions in different situations. We then tested and analyzed the enzyme activity and EPS of the different groups of sludge.
## 3.3 Effect of enzyme activity on denitrification process

The denitrification performance is related to the activity of four enzymes NAR, NIR, NOR (nitric oxide reductase) and NOS[30], as expressed in formula (6)[31].

$$NO_3^- \xrightarrow{Nar} NO_2^- \xrightarrow{Nir} NO \xrightarrow{Nor} N_2O \xrightarrow{Nos} N_2 \quad (6)$$

Insert Fig.4 Weak electrically stimulated denitrification enzyme activity; (a)NAR activity, (b) NIR activity

The proceeding of denitrification depends to a large extent on the enzymatic activity, which is significantly influenced by the relevant physicochemical factors. We examined and analyzed the activity of two key enzymes (NAR and NIR) involved in the accumulation of nitrite in sludge. Based on Fig.4, compared with the CK group, the activities of NAR and NIR in the EG group were significantly enhanced by weak electrical stimulation at both 0.2 V and 0.4 V. Combined with the results in Fig.2, electrical stimulation can promote denitrification by increasing the activity of NAR and NIR, which is one of the ways to improve $NO_3^-$ removal efficiency and reduce $N_2O$ accumulation. $NO_2^-$-N is the product of the enzymatic reaction of NAR, which in turn is promoted by NIR to reduce $NO_2^-$-N to $N_2$. In other words, an increase in NAR activity promotes the activity of NIR. The weak electrical stimulation also optimized the microbial population structure (Fig.5). The proportion of heterotrophic microorganisms such as *Saccharibacteria* and mutualistic microorganisms such as *Smithella* decreased and the proportion of autotrophic microorganisms such as *Thauera* increased under the weak electrical stimulation of 0.2V, which enhanced the autotrophic denitrification of microorganisms.

The weak electrical stimulation at 0.6 V with low carbon source showed a slight
inhibition of NAR and NIR, which confirms that the lack of carbon source cannot be
remedied by increasing the voltage stimulation alone. At low C/ N ratios, insufficient electron
donors affect the ability of less competitive reductases such as NOR and NOS to compete for
electrons so that expressed genes related to denitrification are weakened. The carbon source
available was unable to synthesize sufficient denitrifying enzymes, which affected the growth
of the organism and the removal of nitrate and nitrite nitrogen. Denitrification enzymes
require a large number of electrons to operate in the reduction reaction. EPS can act as a
transient medium for electron energy transfer (EET) in activated sludge because of the
presence of large amounts of protein, which is closely related to the activity of NAR and NIR.

<caption>269
**Insert Fig.5 Level abundance of microbial population structure species under weak**
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**electrical stimulation**</caption>

271 **3.4 Effect of extracellular polymers on denitrifying enzyme activity**

EPS played crucial roles in the ability of microorganisms to flocculate and in building
and maintaining biofilm structure. We carried out a qualitative and quantitative analysis of the
EPS from the three-dimensional fluorescence spectrogram (Fig.6). It can be observed that the
fluorescence intensity and area of fluorescence region I (Aromatic protein tyrosine) and II
(Aromatic protein tryptophan) of LB three-dimensional fluorescence spectrogram of the EG
group were higher than those of the CK group under the effect of weak electrical stimulation
of microorganisms, while the corresponding TB showed roughly the opposite trend. The
fluorescence intensities and areas of TB and LB in the EG and CK groups were in general
consistent with the trends of protein contents in the corresponding TB and LB. EPS played an
important role in enhancing intercellular communication as well as electron transfer[32].
282 Protein is the main component of EPS, in which aromatic protein-like substances have the effect of accelerating electron transfer[33]. The increase of aromatic protein substances 283 promoted electron transfer between electroactive microorganisms and other microorganisms, 284 further enhancing denitrification efficiency. 285

286 **Insert Fig.6 EPS three-dimensional fluorescence spectrum of weak electrically stimulated denitrification continuous flow experimental sludge** 287

288 The effect of weak electrical stimulation on EPS protein was not only in terms of distribution, but also in terms of content. Fig.7 demonstrated that weak electrical stimulation 289 has a significant effect on the extracellular polymer. It is obvious that the sludge LB protein 290 content in the EG group was lower compared to the CK group, while the TB protein content 291 was higher than the CK group, with a tendency for the total EPS protein to increase. LB-EPS 292 was located in the outer layer, so the increase of its content can increase the zeta potential of 293 the cells and/or flocs. According to the DLVO theory, the electrostatic repulsion between the 294 cells and flocs increases, which hindered the flocculation effect and had an impact on SVI 295 (Sludge Volume Index). TB-EPS is located in the inner layer, which binds tightly to the cell 296 surface, stably adheres to the cell wall, and has little effect on the sludge[34]. The LB of the 297 EG group was much less than that of the CK group, which was consistent with the removal 298 performances of total nitrogen and nitrate nitrogen. With the decrease of LB, the degree of 299 sludge flocculation increases, and the adsorption and removal efficiency of pollutants was 300 better. When the intensity of weak electrical stimulation was 0.6 V, the protein content of LB 301 and TB in the EG group was lower than that of CK, and the total amount of EPS protein 302 decreased, which was obviously inhibited. It was speculated that the appropriate intensity of 303
weak electrical stimulation can transform EPS protein from LB to TB and increase the total amount of EPS protein. Overall, weak electrical stimulation enhanced microbial metabolic activity and protein secretion, and the appropriate intensity of weak electrical stimulation enhanced microbial cell-to-cell communication and electron transfer by affecting the distribution of LB and TB and the protein content in EPS.

Insert Fig.7 EPS protein content of weak electrically stimulated denitrification
continuous flow experimental sludge. (a) COD:TN=3:1, (b) COD:TN=4:1

## 4. Conclusions

Weak electrical stimulation has a selective effect on the dominant autotrophic microorganisms and enhances denitrification of low carbon source wastewater through enhanced autotrophic denitrification. Appropriate weak electrical stimulation effectively enhanced denitrification by affecting the distribution and content of EPS aromatic proteins with electron transfer function and the activity of denitrifying enzymes NAR and NIR, while reducing the production of the intermediate greenhouse gas N₂O. The variation in gas production showed that the various enzymatic activities responded differently to the weak electrical stimuli and that when the microbial weak electrical stimuli were too intense, they inhibited the enzymatic activity and thus affected the denitrification process. Weak electrically stimulated denitrification is a process in which multiple microorganisms work in concert. Based on the pattern of denitrifying enzyme effects, denitrification can be further promoted in the future by detecting and regulating denitrifying bacteria.
325 Acknowledgments

326 The authors have stated that there was no conflict of interest. We gratefully
327 acknowledged the co-funding of this work by the National Natural Science Foundation of
328 China (No.52070130) and the Shuguang Project of Shanghai (Education and Scientific
329 Research Project of Shanghai,18SG45).

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## 432 Figure Captions

433 **Figure.1.** Continuous flow of weak electrically stimulated denitrification schematic diagram
434 of the experimental setup

435 **Figure.2** Removal effect and pH change of weakly electrically stimulated denitrification
436 continuous flow experiment;(a)TN, (b)NO₃⁻-N, (c) NO₂⁻-N, (d) NH₄⁺-N,(e)pH

437 **Figure.3** Denitrification gas production at optimal weak electrical stimulation intensity of 0.2
438 V; (a) H₂, (b) N₂, (c) N₂O

439 **Figure.4** Weak electrically stimulated denitrification denitrifying enzyme activity; (a)NAR
440 activity, (b) NIR activity
441 **Figure.5** Microbial population structure species level abundance under weak electrical

442 stimulation

443 **Figure.6** EPS three-dimensional fluorescence spectrum of weakly electrically stimulated

444 denitrification continuous flow experimental sludge

445 **Figure.7** EPS protein content of weak electrically stimulated denitrification continuous flow

446 experimental sludge. (a) COD:TN=3:1, (b) COD:TN=4:1

447
<table><caption>Table 1. Composition of artificial simulated wastewater</caption><thead><tr><th>Ingredient</th><th>Concentration</th></tr></thead><tbody><tr><td rowspan="2">C<sub>6</sub>H<sub>12</sub>O<sub>6</sub></td><td>0.0703g/L (C/N=3)</td></tr><tr><td>0.0934g/L (C/N=4)</td></tr><tr><td rowspan="2">C<sub>2</sub>H<sub>3</sub>NaO<sub>2</sub>·3H<sub>2</sub>O</td><td>0.1594g/L (C/N=3)</td></tr><tr><td>0.2127g/L (C/N=4)</td></tr><tr><td>NaNO<sub>3</sub></td><td>0.3036g/L</td></tr><tr><td>KH<sub>2</sub>PO<sub>4</sub></td><td>0.0099 g/L</td></tr><tr><td>NaHCO<sub>3</sub></td><td>0.1g/L</td></tr><tr><td>CaCl<sub>2</sub></td><td>0.01g/L</td></tr><tr><td>MgSO<sub>4</sub>·7H<sub>2</sub>O</td><td>0.1g/L</td></tr><tr><td>Trace elements</td><td>1 mL/L</td></tr></tbody></table>
<table><caption>451 Table 2. Conditions for determination of hydrogen, nitrogen and nitrous oxide</caption><thead><tr><th>Composition</th><th>Detector</th><th>Temperature</th><th>Packed column</th><th>Inlet temperature</th><th>Column temperature</th><th>Carrier gas and velocity</th></tr></thead><tbody><tr><td>H<sub>2</sub></td><td>TCD</td><td>120°C</td><td>TDX-02, 2m*2mm</td><td>100°C</td><td>100°C</td><td>99.999%Ar, 30 ml/min</td></tr><tr><td>N<sub>2</sub></td><td>TCD</td><td>200°C</td><td>SH-Rt®-Msieve 5A Capillary column, 30m*0.53mm*5 0µm</td><td>120°C</td><td>120°C</td><td>99.999%Ar, 1.5 ml/min</td></tr><tr><td>N<sub>2</sub>O</td><td>µECD</td><td>100°C</td><td>Propack Q, 2m*2mm</td><td>100°C</td><td>70°C</td><td>99.999%Ar, 1.5 ml/min</td></tr></tbody></table>

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<caption>Figure.1. Continuous flow of weak electrically stimulated denitrification schematic
diagram of the experimental setup</caption>

<figure><img src="image_3.png" /><figcaption>Click here to access/download:Fig 1.jpg</figcaption></figure>
<figure><img src="image_4.png" /></figure>
<figure><img src="image_5.png" /></figure>
<figure><img src="image_6.png" /><figcaption>Figure.4 Weak electrically stimulated denitrification denitrifying enzyme activity: (a)NAR activity, (b) NIR activity</figcaption></figure>
<figure><img src="image_7.png" /></figure>

A.Saccharibacteria; B.Bacteroidetes vadinHA17:C.Flavobacterium;

D.Candidatus-competibacter; E.Parcubacteria; F.Fluviicola;

G.Anaerolineaceae; H.Smithella; I.Bacteria clone OPB95; J.Syntrophaceae;

K.Thauera; L.Rhodocyclaceae; M.Thiobacillus; N.PeM15;

O.Denitratisoma; P.Clostridium sensu stricto 1; Q.Sulfurisoma
<figure><img src="image_8.png" /></figure>
<figure><img src="image_9.png" /><figcaption>Figure.7 EPS protein content of weak electrically stimulated denitrification continuous flow experimental sludge. (a) COD:TN=3:1, (b) COD:TN=4:1</figcaption></figure>
Click here to access/download

**Supplementary material for on-line publication only**
Fig. S1.png