Dynamics of a Bacterial Community in the Anode and Cathode of Microbial Fuel...

[Full title: Dynamics of a Bacterial Community in the Anode and Cathode of Microbial Fuel Cells under Sulfadiazine Pressure]

Citation: Yang, Z.; Li, H.; Li, N.; Sardar, M.F.; Song, T.; Zhu, H.; Xing, X.; Zhu, C. Dynamics of a Bacterial Community in the Anode and Cathode of Microbial Fuel Cells under Sulfadiazine Pressure Int. J Environ. Res. Public Health 2022 , 19 , 6253. https://doi.org/10.3390/ ijerph 19106253 Academic Editor: Paul B. Tchounwou Received: 19 April 2022 Accepted: 19 May 2022 Published: 20 May 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations Copyright: © 2022 by the authors Licensee MDPI, Basel, Switzerland This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/) International Journal of Environmental Research and Public Health Article Dynamics of a Bacterial Community in the Anode and Cathode of Microbial Fuel Cells under Sulfadiazine Pressure Zhenzhen Yang 1 , Hongna Li 1, *, Na Li 2 , Muhammad Fahad Sardar 1 , Tingting Song 1 , Hong Zhu 3 , Xuan Xing 4 and Changxiong Zhu 1 1 Institute of Environment and Sustainable Development in Agriculture, Chinese Academy of Agricultural Sciences, Beijing 100081, China; yangzhenz@126.com (Z.Y.); fahadsardar 16@yahoo.com (M.F.S.); songtingting 0505@163.com (T.S.); zhuchangxiong@caas.cn (C.Z.) 2 Department of Engineering Physics, Tsinghua University, Beijing 100084, China; liyana 1207@163.com 3 College of Bioscience and Resources Environment, Beijing University of Agriculture, Beijing 100096, China; zhuhong 80@bua.edu.cn 4 College of Life and Environmental Science, Minzu University of China, Beijing 100081, China; xingxuanpku@163.com * Correspondence: lihongna 828@163.com; Tel.: +86-10-8210-9561 Abstract: Microbial fuel cells (MFCs) could achieve the removal of antibiotics and generate power in the meantime, a process in which the bacterial community structure played a key role. Previous work has mainly focused on microbes in the anode, while their role in the cathode was seldomly mentioned Thus, this study explored the bacterial community of both electrodes in MFCs under sulfadiazine (SDZ) pressure. The results showed that the addition of SDZ had a limited effect on the electrochemical performance, and the maximum output voltage was kept at 0.55 V. As the most abundant phylum, Proteobacteria played an important role in both the anode and cathode. Among them, Geobacter (40.30%) worked for power generation, while Xanthobacter (11.11%), Bradyrhizobium (9.04%), and Achromobacter (7.30%) functioned in SDZ removal. Actinobacteria mainly clustered in the cathode, in which Microbacterium (9.85%) was responsible for SDZ removal. Bacteroidetes, associated with the degradation of SDZ, showed no significant difference between the anode and cathode. Cathodic and part of anodic bacteria could remove SDZ efficiently in MFCs through synergistic interactions and produce metabolites for exoelectrogenic bacteria. The potential hosts of antibiotic resistance genes (ARGs) presented mainly at the anode, while cathodic bacteria might be responsible for ARGs reduction. This work elucidated the role of microorganisms and their synergistic interaction in MFCs and provided a reference to generate power and remove antibiotics using MFCs Keywords: air-cathode microbial fuel cell; sulfadiazine; anodic bacteria; cathodic bacteria; synergistic interaction 1. Introduction The misuse and overuse of antibiotics has not only caused environmental pollution, but it has also stimulated the selection of antibiotic resistance genes (ARGs) [ 1 ]. Hence, exploring economical and effective treatments to eliminate these pollutants has become a hot topic A microbial fuel cell (MFC) is regarded as a promising alternative treatment to realize waste resource utilization as well as the removal of antibiotics [ 2 , 3 ]. It includes anodic reactions with complex organic compounds as electron donors and cathodic reactions with O 2 , nitrate and nitrite as electron acceptors [ 2 , 3 ]. The mechanism of MFC to remove antibiotics is the combined effect of anaerobic biodegradation and electrical stimulation. Persistent electrical stimulation stimulates the microbial metabolism by transmitting electrons to bacterial cells, and stimulated microorganisms rapidly metabolize antibiotics by secreting enzymes [ 4 ]. Antibiotics, including sulfadiazine (SDZ), sulfamethoxazole (SMX), Tetracycline (TC), Oxytetracycline (OTC), and Chloramphenicol (CAP), could be removed in Int. J. Environ. Res. Public Health 2022 , 19 , 6253. https://doi.org/10.3390/ijerph 19106253 https://www.mdpi.com/journal/ijerph

Int. J. Environ. Res. Public Health 2022 , 19 , 6253 2 of 14 two dual chamber MFCs [ 4 – 6 ], and the voltage output was virtually not affected. Different from two dual chamber MFCs, single-chamber MFCs (mainly air-cathode MFCs) could directly use O 2 in the air as an electron acceptor without the cathode chamber, so they could increase the mass transfer effective and reduced cost, and O 2 diffusion from air to the cathode would help the removal of nitrogen without aeration. Therefore, air-cathode MFCs also showed excellent performance in removing antibiotics [ 4 , 7 , 8 ]. Bacterial community structure played a key role during the power output and contaminant removal in MFCs. Therefore, it is necessary to understand the microbial status in the MFCs to clarify the relationship between power generation and pollutant removal. Previous research revealed that the dominant phylum in MFCs were Proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria. The bacteria affiliated with these phyla were classified into two groups (degradation related and exoelectrogenic) [ 9 ]. The mechanisms of electroactive microorganisms have been summarized in several recent reviews [ 10 – 12 ]. These cases of research mainly focused on anodic bacteria, but the growth of bacteria in the cathode is inevitable for air-cathode MFCs, which was an important factor reducing the electrochemical performance [ 13 , 14 ]. Conversely, cathode-biofilm also could prevent the diffusion of O 2 in the cathode side to the anode chamber to increase the power generation of MFCs [ 15 ]. Therefore, the role of cathodic bacteria is controversial. There is limited literature on cathodic bacteria of air-cathode MFCs. To gain insight into anodic and cathodic communities, Daghio et al. firstly operated a single chamber MFC to investigate microbial communities. It was found that degradation-related bacteria were enriched in the cathode, but electricigens or closely related microbes were clustered in the anode to attribute chlorinated herbicide removal or power generation in soil MFCs [ 16 , 17 ]. Yuan et al. found that the COD/N of wastewater would affect the bacteria, both in the anode and cathode of MFCs [ 18 ]. The distribution trend of nitrifiers and denitrifiers in cathodes varied with the cathode-biofilm depth. These studies demonstrated that the cathode played an important role in power generation and contaminants removal in air-cathode MFCs; however, the research on the degradation of antibiotics in wastewater by MFCs mostly focused on anode biofilm, and the function of cathodic bacteria was seldom mentioned. Therefore, it is necessary to clarify the roles of both the anode and cathode in power generation, antibiotic degradation, and ARG propagation This work investigated the efficacy of air-cathode MFCs in wastewater treatment, with SDZ as the representative antibiotic. SDZ is reported to be one of the most common sulfonamides and is used frequently in veterinary medicine [ 19 ]. The electrochemical and physicochemical performance of MFCs under SDZ pressure was studied. Especially, the structures and interactions between anodic and cathodic microorganisms were mainly analyzed. Finally, the occurrence of ARGs and integrons in the anode and the cathode are discussed. The study will elucidate the role of microorganisms in both electrodes and provide reference for the further application of air-cathode MFCs in pollution control and power generation 2. Materials and Methods 2.1. Chemicals and Reagents Analytical reagent sulfadiazine was purchased from Biotopped Science & Technology Co., Ltd. (Shanghai, China). A graphite fiber brush and carbon cloth were acquired from Cetech CO., Ltd. (Suzhou, China). The catalyst (20% Pt/C) was obtained from Johnson Matthey Co., Ltd. (Shanghai, China). The concentration of stock solution for sulfadiazine was 5 g/L and preserved in a 4 ◦ C refrigerator for further use. Other chemicals were purchased from Sinopharm chemical regent Co., Ltd. (Shanghai, China) 2.2. MFC Start-Up and Operation The MFC reactors were constructed with a fiber brush anode and carbon cloth cathode (Supplementary Materials). They were inoculated with granule sludges collected from a wastewater plant in Shangdong, China. The composition of the anolyte is shown in

Int. J. Environ. Res. Public Health 2022 , 19 , 6253 3 of 14 Table S 1. The external resistance was fixed at 1000 Ω , and titanium wire ( Φ 1 mm) was used as wires connected to the carbon cloth. The reactors were stabilized for two weeks in a biochemical incubator at 35 ◦ C. The medium was changed every 48 h during acclimation; After that, the reactors were operated with the sequencing batch, and the medium was changed every 24 h. The whole experiment was carried out at ambient temperature in the dark. The reactors were designed MFCs and open-circuit control with three replicates of each. A total of 40 mg/L of SDZ was added in each reactor after the output voltage reached stabilization 2.3. Analytical Methods 2.3.1. Determination of Physicochemical Parameters and SDZ Because there were many suspended solids in the MFCs effluent, it was easy to cause deviation due to uneven sampling. The samples were filtrated by a 0.45- µ m filter (Tianjin jinteng experimental equipment Co., Ltd., Tianjin, China) and detected. The concentrations of chemical oxygen demand (COD), ammonium-nitrogen (NH 4 + -N), and total dissolved nitrogen (TDN) were analyzed according to the standard method (See Supplementary Materials). The pH and conductivity were measured with a pH-meter and conductivity gauge. The concentration of SDZ was detected by high-performance liquid chromatography (Ultimate 3000, Thermofisher, Waltham, MA, USA) after filtering by a syringe filter (0.22 µ m), The samples were collected by an automatic sampling device and injected into C 18 liquid chromatography (0.46 × 25 cm, 5 µ m, Thermofisher, Waltham, MA, USA) During the elution of the mobile phase, a UV detector was used for analysis and detection at 270 nm. The mobile phase comprised acetonitrile/3‰ acetic acid ( v / v = 25/75), the flow rate was 1 mL/min, and the injection volume was 20 µ L 2.3.2. Electrochemical Measurement The external voltages were collected using a data acquisition board (PISO-813, ICP DAS Co., Ltd., Shanghai, China) via online monitoring and recording every 30 min. The power density was normalized by the projected surface area of one side of the cathode. A standard three-electrode system was employed for electrochemical tests. The anode was the working electrode, while the cathode was the counter electrode, and Ag/AgCl was used as the reference electrode. Cyclic voltammetry (CV) was carried out on the electrochemical workstation (CHI 660 E, CH Instrument Ins.) by sweeping the cell potential from − 0.8 V to +0.6 V at a rate of 5 mV/s. Electrochemical impedance spectroscopy (EIS) measurements were conducted at the open circuit voltage (OCV) of the MFCs with the amplitude set to 5 mV and using a frequency range of 100 kHz–0.1 Hz, with the anode chamber filled with a fresh anolyte (Table S 1) 2.3.3. DNA Extraction and qPCR of ARGs The samples were collected from the MFCs anode and cathode biofilms in triplicate The shape of the carbon brush and carbon cloth were different, and for the convenience of the comparison, weight was used for quantification (1/4 of carbon brush and 1/6 of carbon cloth was used for DNA extraction). DNA extraction and real-time quantitative PCR (qPCR) were performed according to methods used in a previous study [ 20 ]. DNA samples were stored at − 20 ◦ C until use. Each qPCR was repeated three times. The genes of 16 S rRNA, intI 1, intI 2, sul 1, sul 2, sul 3, and sul A were tested, and the primers were set in Table S 2 2.3.4. Bacterial Community Analysis High throughput sequencing was performed at Beijing Allwegene Technology Co Ltd., Beijing, China. See Supplementary Materials for details.

Int. J. Environ. Res. Public Health 2022 , 19 , 6253 4 of 14 2.3.5. Data Analysis The basic statistical calculations and analysis were performed using SPSS 23.0 (IBM, Chicago, IL, USA) and Origin 9.1 (Origin Lab, San Diego, CA, USA) p < 0.05 was considered statistically significant. The changes of the bacterial community at the phylum-level made use of Circos-0.67–7 software ( http://circos.ca/ , accessed on 8 March 2021), and the networks were performed using Networkx software ( http://networkx.github.io/ , accessed on 20 August 2021) ( p < 0.05), according to the relative contents of each genu after classification 3. Results and Discussion 3.1. Performance of Air-Cathode MFCs under Sulfadiazine Pressure The removal ratio of COD, NH 4 + -N, and TDN was 86.55%, 45.15%, and 45.64% in the MFCs, respectively, and it was 83.46%, 9.56%, and 11.27% in the open circuit treatment, respectively (Figure 1 a). The degradation of SDZ was gradually accelerated with the process (Figure 1 b). Twelve cycles later, over 50% of SDZ could be removed within a cycle, which suggests that the microbes in the MFCs gradually acquired the ability for SDZ degradation. Compared with the open circuit, MFCs reduced the increase of pH and conductivity (Figure S 2) to keep a suitable environment for microorganisms to degrade pollutants, even under SDZ pressure. In air-cathode MFCs, oxygen diffused from the air into the biofilm from the cathode, which played a role in nitrification and mono-oxygenation reactions for the removal of SDZ [ 21 , 22 ]. Compared with other research (13.39–80%) [ 6 , 23 ], our experiment was conducted in the dark, which might have caused a relatively low removal ratio of SDZ (58.72%) Int. J. Environ. Res. Public Health 2022 , 19 , x 4 of 16 2.3.4 Bacterial Community Analysis High throughput sequencing was performed at Beijing Allwegene Technology Co Ltd., Beijing, China See Supplementary Materials for details 2.3.5 Data Analysis The basic statistical calculations and analysis were performed using SPSS 23.0 (IBM, Chicago, IL, USA) and Origin 9.1 (Origin Lab, San Diego, CA, USA) p < 0.05 was consid ‐ ered statistically significant The changes of the bacterial community at the phylum ‐ level made use of Circos ‐ 0.67–7 software (http://circos.ca/, accessed on 8 March 2021.), and the networks were performed using Networkx software (http://networkx.github.io/, accessed on 20 August 2021) ( p < 0.05), according to the relative contents of each genu after classi ‐ fication 3. Results and Discussion 3.1. Performance of Air ‐ Cathode MFCs under Sulfadiazine Pressure The removal ratio of COD, NH 4+ ‐ N, and TDN was 86.55%, 45.15%, and 45.64% in the MFCs, respectively, and it was 83.46%, 9.56%, and 11.27% in the open circuit treatment, respectively (Figure 1 a) The degradation of SDZ was gradually accelerated with the pro ‐ cess (Figure 1 b) Twelve cycles later, over 50% of SDZ could be removed within a cycle, which suggests that the microbes in the MFCs gradually acquired the ability for SDZ deg ‐ radation Compared with the open circuit, MFCs reduced the increase of pH and conduc ‐ tivity (Figure S 2) to keep a suitable environment for microorganisms to degrade pollu ‐ tants, even under SDZ pressure In air ‐ cathode MFCs, oxygen diffused from the air into the biofilm from the cathode, which played a role in nitrification and mono ‐ oxygenation reactions for the removal of SDZ [21,22] Compared with other research (13.39–80%) [6,23], our experiment was conducted in the dark, which might have caused a relatively low re ‐ moval ratio of SDZ (58.72%) Figure 1. Performance of the air ‐ cathode system: ( a ) Contents of COD, NH 4+ ‐ N, and DTN in MFCs; ( b ) Removal rate of SDZ; ( c ) Output voltage; ( d ) CV curves Figure 1. Performance of the air-cathode system: ( a ) Contents of COD, NH 4 + -N, and DTN in MFCs; ( b ) Removal rate of SDZ; ( c ) Output voltage; ( d ) CV curves The maximum output voltage was 0.55 V (Figure 1 c), and the addition of SDZ had no drastic effect on the output voltage of MFCs. Previous studies have demonstrated that the output of MFCs would be enhanced rather than be inhibited under antibiotic pressure [ 7 , 8 , 24 ]. An obvious peak was at − 0.4 V vs. Ag/AgCl in CV curves (Figure 1 d), which was close to the formal potential of outer membrane cytochromes of G. sulfurreducens

Int. J. Environ. Res. Public Health 2022 , 19 , 6253 5 of 14 ( − 0.398 V vs. Ag/AgCl) [ 25 ]. It was consistent with a previous study that concluded that some antibiotics could increase the permeability of exoelectrogens membranes and then facilitate the direct electron transfer [ 7 ]. In addition, the maximum output power (Figure S 3 a) and Nyquist plots (Figure S 3 b) demonstrate that the addition of SDZ showed little influence on the electrochemical activity of MFCs 3.2. Bacterial Community Shift in the Anode and Cathode under Sulfadiazine Pressure The goods coverage of all tested samples was 99%, which indicated that the sizes of all libraries were enough to cover the bacterial communities. As shown in Figure 2 , the bacterial community showed a lower Chao 1 index and a higher Shannon index in the cathode compared with that of the anode. This indicated that although the bacteria community in the cathode was not rich, it had a higher diversity. The principal coordinate analysis (PCoA) based on UniFrac distances showed that the sample had a good reproducibility (Figure S 4 a) The distance between samples further validated the difference of the bacterial community structures between the anode and cathode. The unique species also sharply reduced in the anode and cathode (Figure S 4 b), which will be discussed in detail below. These indicated that the bacteria in the inocula were selectively enriched in the anode and cathode Int. J. Environ. Res. Public Health 2022 , 19 , x 5 of 16 The maximum output voltage was 0.55 V (Figure 1 c), and the addition of SDZ had no drastic effect on the output voltage of MFCs Previous studies have demonstrated that the output of MFCs would be enhanced rather than be inhibited under antibiotic pressure [7,8,24] An obvious peak was at − 0.4 V vs Ag/AgCl in CV curves (Figure 1 d), which was close to the formal potential of outer membrane cytochromes of G.sulfurreducens ( − 0.398 V vs Ag/AgCl) [25] It was consistent with a previous study that concluded that some anti ‐ biotics could increase the permeability of exoelectrogens membranes and then facilitate the direct electron transfer [7]. In addition, the maximum output power (Figure S 3 a) and Nyquist plots (Figure S 3 b) demonstrate that the addition of SDZ showed little influence on the electrochemical activity of MFCs 3.2. Bacterial Community Shift in the Anode and Cathode under Sulfadiazine Pressure The goods coverage of all tested samples was 99%, which indicated that the sizes of all libraries were enough to cover the bacterial communities As shown in Figure 2, the bacterial community showed a lower Chao 1 index and a higher Shannon index in the cathode compared with that of the anode This indicated that although the bacteria com ‐ munity in the cathode was not rich, it had a higher diversity The principal coordinate analysis (PCoA) based on UniFrac distances showed that the sample had a good repro ‐ ducibility (Figure S 4 a) The distance between samples further validated the difference of the bacterial community structures between the anode and cathode The unique species also sharply reduced in the anode and cathode Figure S 4 b), which will be discussed in detail below These indicated that the bacteria in the inocula were selectively enriched in the anode and cathode Figure 2. The microbial diversity of samples: ( a ) Chao 1 index; ( b ) Shannon index Firmicutes, Ignavibacteriae, Chloroflexi, Lentisphaerae, and Euryarchaeota were mainly enriched in the anode, while Actinobacteria mainly clustered in the cathode; Pro ‐ teobacteria and Bacteroidetes existed both in the anode and cathode (Figure 3) Among these phyla, Proteobacteria, Bacteroidetes, and Actinobacteria were three dominant phyla with the relative abundance of 88.6% (anode) and 97.1% (cathode), respectively The func ‐ tion of these bacteria determined their selective enrichment in the anode and cathode, which will be discussed in more detail at follow ‐ up Figure 2. The microbial diversity of samples: ( a ) Chao 1 index; ( b ) Shannon index Firmicutes, Ignavibacteriae, Chloroflexi, Lentisphaerae, and Euryarchaeota were mainly enriched in the anode, while Actinobacteria mainly clustered in the cathode; Proteobacteria and Bacteroidetes existed both in the anode and cathode (Figure 3 ). Among these phyla, Proteobacteria, Bacteroidetes, and Actinobacteria were three dominant phyla with the relative abundance of 88.6% (anode) and 97.1% (cathode), respectively. The function of these bacteria determined their selective enrichment in the anode and cathode, which will be discussed in more detail at follow-up Proteobacteria was most abundant in the anode (70.7%) and cathode (62.6%). The difference was further explicated at the class-level (Figure 4 a). The most abundant class in anodic community was Deltaproteobacteria (40.91%), for which almost all the detected sequences belonged to the genus Geobacter (Figure 4 b) Geobacter was the dominant genus in the MFCs anode, with the relative abundance of 40.3% as Gram-negative and electroactive bacteria [ 26 , 27 ]. Geobacter is strictly anaerobic and could adapt to the low surface potentials of the anode [ 26 , 27 ]; therefore, they mainly colonized in the anode and were responsible for power generation by using acetate Xanthobacter (11.11%, p < 0.01), Bradyrhizobium (9.04%, p < 0.01), and Mesorhizobium (2.68%, p < 0.001) affiliated with Alphaproteobacteria were enriched in the cathode (Figure 4 b). Xanthobacter was dominant in the microbial electrolysis cells (MECs) but was scarcely reported in MFCs. They accumulated in the cathodic community in this experiment, possibly due to the fact that Xanthobacter thrives in micro-oxygen environments [ 18 ], as well as that they could degrade the aromatic structure of SDZ in the

Int. J. Environ. Res. Public Health 2022 , 19 , 6253 6 of 14 cathode [ 28 ]. Similarly, Bradyrhizobium could degrade antibiotics via co-metabolism with acetate [ 29 ]. Mesorhizobium is an aerobic bacterium using oxygen as the terminal electron acceptor to respire. The possible role of Mesorhizobium was to remove acetate and SDZ degradation products because it could metabolize amino salts, nitrates, and various amino acids using various carbohydrates and organic acid as the carbon source [ 30 ]. Azospirillum (1.88%, p < 0.01) affiliated with Alphaproteobacteria was enriched in the anode, which have been reported to be resistant to antibiotics using a wide range of carbon sources [ 31 ]. It was reported that Bradyrhizobium , Mesorhizobium , and Azospirillum could produce extracellular polysaccharides, which supported their metabolism [ 30 , 31 ]. Achromobacter (7.30%, p < 0.001) and Castellaniella (2.24%, p < 0.01) affiliated with Betaproteobacteria were enriched in the cathode (Figure 4 b). Achromobacter was able to degrade sulfonamides [ 32 ], and Castellaniella could degrade pyrene under denitrifying conditions [ 33 ], so they clustered in the cathode to degrade the aromatic structure of SDZ Azospira (5.45%, p < 0.01) affiliated with Betaproteobacteria was enriched in the anode as denitrification organisms, which could denitrify under anaerobic conditions and could use the electrode as the electron acceptor [ 34 ]. They colonized in the anode for denitrification and power generation Hydrogenophaga , an autotrophic H 2 -oxidizing bacteria, could utilize hydrogen as the energy source [ 35 ]. Comamonas as facultative anaerobic microorganisms have been isolated from MFCs, which contributed to the power generation with acetate as the electron donor [ 36 ]. They belong to Betaproteobacteria, with lower abundance in the anode and the cathode ( Hydrogenophaga (0.49% vs. 2.28%, p = 0.114) and Comamonas (0.78% vs. 0.05%, p = 0.403)) Pseudomonas (4.58%, p < 0.01), which belong to Gammaproteobacteria, were enriched in the anode (Figure 4 b). As electroactive bacteria, Pseudomonas were also able to degrade sulfonamides, which were the dominant bacterial genus in the anodic chamber of MFCs [ 37 ], so they accumulated in the anode for SDZ removal and power generation Stenotrophomonas (1.59% vs. 2.68%, p = 0.186) and Dokdonella (1.20% vs. 2.18%, p < 0.01) affiliated with Gammaproteobacteria were enriched in both the anode and cathode Stenotrophomonas is able to degrade many xenobiotic aromatic compounds [ 38 ]. Dokdonella is usually found in the aerobic biological systems to remove nitrogen and degrade aromatic hydrocarbons simultaneously [ 39 ], so they colonized both the anode and cathode, possibly for the degradation of SDZ Int. J. Environ. Res. Public Health 2022 , 19 , x 6 of 16 Figure 3. Phylum ‐ level microbial communities Proteobacteria was most abundant in the anode (70.7%) and cathode (62.6%) The difference was further explicated at the class ‐ level (Figure 4 a) The most abundant class in anodic community was Deltaproteobacteria (40.91%), for which almost all the detected sequences belonged to the genus Geobacter (Figure 4 b) Geobacter was the dominant genus in the MFCs anode, with the relative abundance of 40.3% as Gram ‐ negative and electro ‐ active bacteria [26,27] Geobacter is strictly anaerobic and could adapt to the low surface potentials of the anode [26,27]; therefore, they mainly colonized in the anode and were responsible for power generation by using acetate Xanthobacter (11.11%, p < 0.01), Brady ‐ rhizobium (9.04%, p < 0.01), and Mesorhizobium (2.68%, p < 0.001) affiliated with Alphapro ‐ teobacteria were enriched in the cathode (Figure 4 b) Xanthobacter was dominant in the microbial electrolysis cells (MECs) but was scarcely reported in MFCs They accumulated in the cathodic community in this experiment, possibly due to the fact that Xanthobacter thrives in micro ‐ oxygen environments [18], as well as that they could degrade the aro ‐ matic structure of SDZ in the cathode [28] Similarly, Bradyrhizobium could degrade anti ‐ biotics via co ‐ metabolism with acetate [29] Mesorhizobium is an aerobic bacterium using oxygen as the terminal electron acceptor to respire The possible role of Mesorhizobium was to remove acetate and SDZ degradation products because it could metabolize amino salts, nitrates, and various amino acids using various carbohydrates and organic acid as the carbon source [30] Azospirillum (1.88%, p < 0.01) affiliated with Alphaproteobacteria was enriched in the anode, which have been reported to be resistant to antibiotics using a wide range of carbon sources [31] It was reported that Bradyrhizobium , Mesorhizobium , and Azospirillum could produce extracellular polysaccharides, which supported their metabo ‐ lism [30,31] Achromobacter (7.30%, p < 0.001) and Castellaniella (2.24%, p < 0.01) affiliated with Betaproteobacteria were enriched in the cathode (Figure 4 b) Achromobacter was able to degrade sulfonamides [32], and Castellaniella could degrade pyrene under denitrifying conditions [33], so they clustered in the cathode to degrade the aromatic structure of SDZ Azospira (5.45%, p < 0.01) affiliated with Betaproteobacteria was enriched in the anode as denitrification organisms, which could denitrify under anaerobic conditions and could use the electrode as the electron acceptor [34] They colonized in the anode for denitrifica ‐ tion and power generation Hydrogenophaga , an autotrophic H 2 ‐ oxidizing bacteria, could utilize hydrogen as the energy source [35] Comamonas as facultative anaerobic microor ‐ ganisms have been isolated from MFCs, which contributed to the power generation with Figure 3. Phylum-level microbial communities.

Int. J. Environ. Res. Public Health 2022 , 19 , 6253 7 of 14 Int. J. Environ. Res. Public Health 2022 , 19 , x 8 of 16 Figure 4. The relative abundance of ( a ) class-level microbial communities, ( b ) genera belonging to Proteobacteria, and ( c ) genera belonging to Actinobacteria and Bacteroidetes (Analysis of difference between anode and cathode: * p < 0.05, ** p < 0.01, *** p < 0.001).

Int. J. Environ. Res. Public Health 2022 , 19 , 6253 8 of 14 Actinobacteria were widely used in water treatment field, as they can use glucose, starch, and cellulose as carbon sources [ 40 ]. The abundance of Actinobacteria in the cathode (19.68%) was higher than that in the anode (7.65%, p < 0.01) (Figure 4 a), due to the anaerobic condition that inhibited Actinobacteria [ 41 ]. Microbacterium (9.85%, p < 0.05) and Pseudoclavibacter (2.78%, p < 0.001), which belong to Actinobacteria, clustered in the cathode (Figure 4 c). Microbacterium is an important degradation-related microorganism, which showed a higher degradation rate for sulfonamides, especially for SDZ [ 42 ]; therefore, it was most likely responsible for the degradation of SDZ in the cathode Pseudoclavibacter is a Gram-positive and aerobic genus; although this genus has not been reported in the wastewater treatment field, it was reported to be responsible for the biodesulfurization of organic sulfur [ 43 ]; therefore, they might cluster in the cathode, helping the removal of sulfur in SDZ Rhodococcus (2.69% vs. 1.99%), Mycobacterium (1.03% vs. 0.43%), and Gordonia (1.56% vs. 3.69%, p < 0.01) affiliated with Actinobacteria existed both in the anode and the cathode (Figure 4 c). Rhodococcus degraded SDZ and regulated biofilm thickness in both the anode and cathode, for they could degrade various aromatic compounds via a ring-cleavage pathway under anaerobic conditions [ 44 ]. They could also improve MFC performance via controlling the biofilm thickness on the anode surface [ 45 ]. Mycobacterium and Gordonia were beneficial to degrade SDZ, for they could degrade polycyclic aromatic hydrocarbons (PAHs) into phthalate, CO 2 , and other chemicals by decarboxylation, dioxygenation, hydrolysis, and ring-cleavage reactions [ 6 , 46 ]. Gordonia is an aerobic bacterial genus, but it was found in the anode in previous studies [ 46 , 47 ], which is possibly due to less attention being paid to microorganisms in the cathode of MFCs Bacteroidetes were enriched both in the anode (10.28%) and cathode (14.86%) without a significant difference between each other Proteiniphilum (2.02%, p < 0.05) and Petrimonas (1.83%, p < 0.05) affiliated with Bacteroidetes mainly accumulated in the anode (Figure 4 c), possibly because they are typical fermenters Proteiniphilum and Petrimonas could degrade complex substrates, such as PAHs, to simple organic compounds by syntrophic metabolism [ 48 , 49 ], which indicates that they could degrade the product of SDZ in anode There was no significant difference for the relative abundance of Chryseobacterium between the anode (1.27%) and the cathode (1.17%) (Figure 4 c). Considering their ability to degrade aromatic compounds [ 50 ], they might degrade SDZ in both electrodes Generally, Proteobacteria played an important role both in the anode and cathode, without a significant difference as the predominant phylum: Deltaproteobacteria mainly accumulated in the anode, represented by the genus of Geobacter ; it was responsible for power generation. Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria colonized both the anode and cathode and were associated with the degradation of SDZ; Actinobacteria mainly clustered in the cathode, which were responsible for the removal of SDZ; Bacteroidetes showed no significant difference between the anode and cathode, and they were associated with the degradation of SDZ. The nature (aerobic/anaerobic) and function of bacterial genera decided their distribution in the anode and cathode 3.3. Potential Bacterial Roles in SDZ Degradation The microorganisms in air-cathode MFCs can be functionally categorized into the following two groups: exoelectrogenic bacteria ( Geobacter , Pseudomonas , Azospira , and Comamonas ) and degradation-related bacteria ( Xanthobacter , Bradyrhizobium , Achromobacter , Azospirillum , Microbacterium , Pseudoclavibacter , Mycobacterium , Castellaniella , Dokdonella , Rhodococcus , Mycobacterium , Gordonia , Proteiniphilum , Petrimonas , and Chryseobacterium ) Based on the variation of bacterial communities in the biofilms, the ecological model of SDZ biodegradation in air-cathode MFCs was proposed (Figure 5 a). Substrates (acetate and SDZ) diffused into the biofilm both in the anode and cathode, while oxygen diffused from the cathode side. In the anodic biofilms, Geobacter was mainly responsible for power generation by the metabolizing acetate, and SDZ degradation products were further oxidized by fermentation bacteria (e.g., Proteiniphilun and Petrimonas ) or Gordonia to low molecular organics. Nitrification bacteria used the O 2 from cathode. The oxygen-utilizing of cathodic

Int. J. Environ. Res. Public Health 2022 , 19 , 6253 9 of 14 bacteria can create anoxic zones, offering a favorable zone for denitrifying bacteria and exoelectrogenic bacteria. Almost all cathodic bacteria and part of anodic bacteria were involved in nitrification and denitrification Int. J. Environ. Res. Public Health 2022 , 19 , x 10 of 16 from the cathode side In the anodic biofilms, Geobacter was mainly responsible for power generation by the metabolizing acetate, and SDZ degradation products were further oxi ‐ dized by fermentation bacteria (e.g., Proteiniphilun and Petrimonas ) or Gordonia to low mo ‐ lecular organics Nitrification bacteria used the O 2 from cathode The oxygen ‐ utilizing of cathodic bacteria can create anoxic zones, offering a favorable zone for denitrifying bacte ‐ ria and exoelectrogenic bacteria Almost all cathodic bacteria and part of anodic bacteria were involved in nitrification and denitrification Figure 5. ( a ) Ecological model of SDZ biodegradation in air ‐ cathode MFCs; ( b ) Scheme of potential degradation mechanisms [6,42,51]. (A ‐ n: the genus mainly accumulated in the anode; C ‐ n: the genus mainly clustered in the cathode; AC ‐ n: the genus clustered both in the anode and cathode; I and II: Possible SDZ biodegradation pathway) Based on published literature, the possible degradation pathway of SDZ is shown in Figure 5 b. Furthermore, the role of microorganisms in the degradation of SDZ and their Figure 5. ( a ) Ecological model of SDZ biodegradation in air-cathode MFCs; ( b ) Scheme of potential degradation mechanisms [ 6 , 42 , 51 ]. (A-n: the genus mainly accumulated in the anode; C-n: the genus mainly clustered in the cathode; AC-n: the genus clustered both in the anode and cathode; I and II: Possible SDZ biodegradation pathway) Based on published literature, the possible degradation pathway of SDZ is shown in Figure 5 b. Furthermore, the role of microorganisms in the degradation of SDZ and their relationship in air-cathode MFCs was explained according to specific functions. It is noteworthy that the initial electrophilic attack by oxygenases of aerobic bacteria is often a rate-limiting step and the first of a chain of reactions which is responsible for the biodegradation of many organic compounds [ 52 ]. This might be the main reason why

Int. J. Environ. Res. Public Health 2022 , 19 , 6253 10 of 14 degradation bacteria were mostly distributed in the cathode. In the pathway I, SDZ obtained electrons in the presence of O 2 to form dihydroxyl SDZ (I- 1 ). As mentioned in Section 3.2 , Mycobacterium might work in the process by deoxygenation. Moreover, Microbacterium was able to degrade SDZ into 2-aminopyrimidine, which was initiated by NADH-dependent ipso-hydroxylation [ 53 ], corresponding to the S-N bond hydrolysis, (I- 2 ) or (II- 1 ). The p -anilinesulfonic acid was further hydrolyzed to generate aniline and sulfate (I- 3 ), or N atoms were bio-consumed to form benzenesulfinic acid (II- 2 ), then leading to the formation of benzene and sulfate. During this process, Pseudoclavibacter was responsible for biodesulfurization. The benzene and catechol can be decomposed into acetate and pyruvate by Proteiniphilun , Petrimonas , Rhodococcus , and Castellaniella (I- 6 , II- 4 ). 2-amino-4,6-dihydro-pyrimidine contains more electron-donating functional groups, which render the molecules more prone to electrophilic attack by oxygenases of aerobic bacteria (mainly cathodic bacteria); then, it was decomposed into N 2 , formic acid, and acetate by nitrifiers/denitrifiers (I- 7 , II- 6 ) [ 52 ]. Finally, formic acid, acetate, and pyruvate can be used by a Geobacter for power generation. The degradation of SDZ was the result of the synergistic reaction of anodic and cathode bacteria in air-cathode MFCs. O 2 was harmful to power generation in MFCs, but it was conducive to remove contaminants Therefore, the role of O 2 in air-cathode MFCs should be reconsidered All the reactions in air-cathode MFCs can be summarized as follows: (1) Exoelectrogenic bacteria were responsible for power generation in the anode of air-cathode MFCs; (2) The electrons provided by exoelectrogenic bacteria could increase the metabolic reaction of degradation-related bacteria located in the anode and cathode; (3) Degradation-related bacteria might contribute to the power generation by producing metabolites, which could be used as electron donors by the electroactive bacteria. These functional bacteria could effectively remove pollutants and generate power via complex synergistic interactions 3.4. ARGs in the Anode and Cathode under Sulfadiazine Pressure As shown in Figure 6 a, 16 S rRNA gene, intI 1, intI 2, sul 1, and sul 2 were detected in the anode and cathode. This is consistent with previous work that showed that sul 3 and sul A were not detected in any samples [ 54 ]. The integrons, intI 1 and intI 2, are natural mobile genetic elements that can capture, integrate, and express resistance gene cassettes with the help of integrase genes. Integrase genes, being important players in ARG transfer, were the driving force for bacterial evolution. Integrons have been reported to be involved in the occurrence of new resistant and pathogenic species [ 8 ]. The resistance of bacteria to sulfonamides are mutated of the enzyme dihydropteroate synthase (DHPS) or acquired alternative DHPS gene, among which sul 1 and sul 2 belong to the latter [ 55 ]. The absolute abundance of 16 S rRNA gene, intI 1, intI 2, sul 1, and sul 2 in the anode was higher than that of the cathode (Figure 6 a), and a significant difference was observed between the anode and cathode. Moreover, the relative abundance of these ARGs in the anode was also higher than that in the cathode, except for sul 2 (Figure 6 b). The relative abundance of sul 1 and sul 2 ranged from 6.59 × 10 − 4 to 3.43 × 10 − 2 , and sul 2 > sul 1 in the cathode. Similar to other studies, the abundance of sul 1 was usually lower than sul 2 [ 56 ]. A network analysis was conducted in order to further explore the relationship of the bacterial community, integrons, and ARGs in the air-cathode MFCs (Figure 6 c). intI 1 showed a positive correlation with Geobacter and presented a negative correlation with Bradyrhizobium , Mesorhizobium , Achromobacter , and Hydrogenophaga . It indicated that Geobacter might be the host bacteria of intI 1, while the proliferation of others might be effective to reduce intI 1 Geobacter mainly clustered in the anode, and others were enriched in the cathode, which explains why the abundance of intI 1 in the anode was higher than that in the cathode. The sul 1 presented a positive correlation with Proteiniphilun , Petrimonas , and Azospirillum and showed a negative correlation with Microbacterium , Dokdonella , Stenotrophomonas , and Castellaniella Proteiniphilun , Petrimonas , and Azospirillum were mainly enriched in the anode, and Microbacterium and Castellaniella mainly clustered in the cathode Though Stenotrophomonas and Dokdonella were both enriched in anode and cathode, the

Int. J. Environ. Res. Public Health 2022 , 19 , 6253 11 of 14 abundance of these bacteria in the cathode was higher than that in the anode. All of these were consistent with the abundance of sul 1 in the anode being higher in the cathode The sul 2 showed a positive correlation with Hydrogenophaga and a negative correlation with Geobacter . In addition, the relative abundance of Geobacter in the anode was substantially higher than the abundance of Hydrogenophaga in the cathode. This was consistent with the relative abundance of sul 2 being lower in the anode than that of the cathode. The results showed that many anodic bacteria were potential hosts of the tested ARGs, while the cathodic bacteria might play a role in the reduction of these ARGs. The findings were consistent with previous research that aerobic conditions showed better removal capacities of ARGs than anaerobic conditions [ 57 ]. Int. J. Environ. Res. Public Health 2022 , 19 , x 12 of 16 Figure 6. ( a ) Absolute abundance of ARGs; ( b ) Relative abundance of ARGs (Analysis of difference between anode and cathode: * p < 0.05, ** p < 0.01); ( c ) Network analysis of ARGs and the bacterial community in MFCs Species with p < 0.05 were shown by default according to Spearman’s rank analysis Node size was weighted according to the degree The orange color represents the positive correlation, and the blue represents the negative correlation A network analysis was conducted in order to further explore the relationship of the bacterial community, integrons, and ARGs in the air ‐ cathode MFCs (Figure 6 c) intI 1 showed a positive correlation with Geobacter and presented a negative correlation with Bradyrhizobium , Mesorhizobium , Achromobacter , and Hydrogenophaga It indicated that Geo ‐ bacter might be the host bacteria of intI 1, while the proliferation of others might be effective to reduce intI 1 Geobacter mainly clustered in the anode, and others were enriched in the cathode, which explains why the abundance of intI 1 in the anode was higher than that in the cathode The sul 1 presented a positive correlation with Proteiniphilun , Petrimonas , and Azospirillum and showed a negative correlation with Microbacterium , Dokdonella , Steno ‐ trophomonas , and Castellaniella Proteiniphilun , Petrimonas , and Azospirillum were mainly enriched in the anode, and Microbacterium and Castellaniella mainly clustered in the cath ‐ ode Though Stenotrophomonas and Dokdonella were both enriched in anode and cathode, the abundance of these bacteria in the cathode was higher than that in the anode All of these were consistent with the abundance of sul 1 in the anode being higher in the cathode The sul 2 showed a positive correlation with Hydrogenophaga and a negative correlation with Geobacter In addition, the relative abundance of Geobacter in the anode was substan ‐ tially higher than the abundance of Hydrogenophaga in the cathode This was consistent with the relative abundance of sul 2 being lower in the anode than that of the cathode The results showed that many anodic bacteria were potential hosts of the tested ARGs, while the cathodic bacteria might play a role in the reduction of these ARGs The findings were consistent with previous research that aerobic conditions showed better removal capaci ‐ ties of ARGs than anaerobic conditions [57] Figure 6. ( a ) Absolute abundance of ARGs; ( b ) Relative abundance of ARGs. (Analysis of difference between anode and cathode: * p < 0.05, ** p < 0.01); ( c ) Network analysis of ARGs and the bacterial community in MFCs. Species with p < 0.05 were shown by default according to Spearman’s rank analysis. Node size was weighted according to the degree. The orange color represents the positive correlation, and the blue represents the negative correlation 4. Conclusions In summary, the addition of SDZ has a limited effect on the electrochemical performance of air-cathode MFCs, with the maximum output voltage kept at 0.55 V. Anodic bacteria were mainly responsible for power generation, and part of them could remove contaminants. Cathodic bacteria were responsible for pollutants removal. The nature (aerobic/anaerobic) and function of bacteria decided their distribution in the anode and cathode. The electrons provided by exoelectrogenic bacteria could increase the metabolic reaction of degradation-related bacteria, and degradation-related bacteria might contribute to the power generation by producing secondary metabolites. They could remove SDZ and generate power through synergistic interactions. The potential hosts of ARGs mainly presented in anodic bacteria, while cathodic bacteria possibly played a role in ARG reduc-

Int. J. Environ. Res. Public Health 2022 , 19 , 6253 12 of 14 tion. Regardless, the spread risk of antibiotic resistance should be seriously concerned for both electrodes Supplementary Materials: The following supporting information can be downloaded at https: //www.mdpi.com/article/10.3390/ijerph 19106253/s 1 . Figure S 1: Construction of the air-cathode MFC reactor used in this study; Figure S 2: The physicochemical performance of air-cathode MFCs: (a) pH; (b) EC; Figure S 3: The electrochemical performance of air-cathode MFCs: (a) power density curves; (b) EIS polt; Figure S 4. (a) PCoA on the similarity of bacterial communities in samples; (b) Venn diagram for the unique/shared OTUs; Table S 1: The ingredients of anolyte; Table S 2: q-PCR primers used in this study. References [ 54 , 58 – 61 ] are cited in the Supplementary Materials Author Contributions: Conceptualization, Z.Y. and H.L.; Data curation, Z.Y. and H.L.; Formal analysis, Z.Y. and H.L.; Funding acquisition, H.L. and C.Z.; Investigation, Z.Y.; Methodology, Z.Y. and T.S.; Project administration, H.L. and C.Z.; Resources, H.L. and C.Z.; Software, Z.Y., T.S., and X.X.; Supervision, Z.Y.; Validation, Z.Y. and H.L.; Visualization, Z.Y.; Writing—original draft, Z.Y.; Writing—review and editing, H.L., N.L., M.F.S., and H.Z. All authors have read and agreed to the published version of the manuscript Funding: This research was funded by the National Key Research and Development Program, grant number 2021 YFC 3201503; the Outstanding Agricultural Scientist Sponsorship Program (2021– 2026); the Top-Notch Young Talents Program of China; and the Agricultural Science and Technology Innovation Program of China Institutional Review Board Statement: Not applicable Informed Consent Statement: Not applicable Data Availability Statement: Not applicable Conflicts of Interest: The authors declare no conflict of interest References 1 Qiao, M.; Ying, G.; Singer, A.C.; Zhu, Y. Review of antibiotic resistance in China and its environment Environ. Int 2018 , 110 , 160–172. [ CrossRef ] 2 Logan, B.E.; Hamekers, B.; Rozendal, R.; Schroder, U.; Keller, J.; Freguia, S.; Aelterman, P.; Verstraete, W.; Rabaey, K. Microbial fuel cells: Methodology and technology Environ. Sci. Technol 2018 , 40 , 5181–5192. 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